Ph. D. Thesis
Ph. D. Program: Fundamental Chemistry,
Organic Chemistry, 1997-1999.
Surface plasmon resonance as a tool in the
functional analysis of an immunodominant site
in foot-and-mouth disease virus
Paula Alexandra de Carvalho Gomes
Department of Organic Chemistry, Faculty of Chemistry.
Division of Experimental and Mathematical Sciences.
University of Barcelona, 2000
Department of Organic Chemistry, Faculty of Chemistry.
Division of Experimental and Mathematical Sciences.
University of Barcelona, 2000
Dissertation presented by
Paula Alexandra de Carvalho Gomes
to apply for the degree of Doctor in Chemical Sciences
at the University of Barcelona
and revised by
Dr. David Andreu
En primer lugar, me gustaría agradecer al Dr. David Andreu, supervisor de la presente tesis, y al Dr.
Ernest Giralt por la calurosa acogida en su laboratorio, dándome la gran oportunidad de formar parte
de su grupo de investigación. También quisiera agradecerles las fructuosas discusiones y el constante
apoyo siempre que me sentía perdida entre “biacores” y péptidos...
A Jaume y Chary, de los SCT, porque siempre han traído un rayo de Sol a mis días de autismo cuando
tocaba hacer biacore (al final, la “unidad” paula se puede desacoplar del BIACORE 1000!)
Estoy igualmente en deuda con Wendy Fernández, Dr. Núria Verdaguer y Dr. Ignacio Fita por
presentarme el guapísimo mundo de la cristalografía de proteínas y por toda la paciencia que tuvieron
guiando mis pasitos infantiles en este campo de investigación (eh, Wendy? A que tendrás un
descansito, ahora que no estaré todo el rato preguntándote “y ahora qué se hace?”...muchas gracias,
de verdad! Eres un sol!).
Bueno, y ahora...cómo agradecer a todos vosotros que sois el alma del inolvidable (y enorme!) “grup
10”? Vuestra alegría y buena voluntad inagotables, las palabras de consuelo siempre que la ciencia se
resistía a colaborar (lo que suele ser la regla, no la excepción...), las tardes de cine (lo siento, sigo
preferiendo las V.O.S.!!! ...bueno, creo que solo tú, Mac, me comprendes en este tema...), las noches de
“vicio” por Poble Nou (no diré nombres para no daros mala fama, pero sabéis quienes sois), las
excursiones “torpedillas” por la montaña (demasiado “sanas” para mí, pero siempre podía gritar
“Javiiii, no me dejeeees! Me da miedo pasar por aquíiiii!!!!”), las clases de RMN y de ornitología del
grande (en altura y como persona) Doctor Millet, las carcajadas siempre que Maria José (la “femme
formidable”) decía alguna palabrota en gallego... La incansable Super-Molina y la mítica Cristina
Carreño (la mejor guía práctica de laboratorio que he conocido nunca)... el equipo “carmanyola”, que
también tenía compañeros “del otro pasillo”, y que animaba la hora de comer con temas de actualidad
tan diversos como la receta de codornices de Cris Chiva o la película de anoche, todo esto
acompañado por unas negociatas sospechosas relacionadas con las mafias de los puntos Danone...
DelFresni, qué tal si nos montamos un restaurante? (tu pones el arroz negro y yo el pulpo, vale?)
Tampoco puedo olvidar el super buen rollo del “lab 3”...empezando por Eva, mi compañera “aftosa” y
siguiendo por Alberto, con quien disfruté buenos momentos musicales “after nine”, cuando ya no
quedaba nadie (ní tu, Paul...) y podíamos poner la radio a “toda leche” (aunque los jóvenes no nos
entendieran cuando nos animábamos con algun “greatest hit” de los 80... debe ser el “generation
gap”...), y, como no, mi primera jefecilla, Mary-Light (ahora Señora Dueña), toda ella un encanto de
persona... también las nuevas generaciones han sido clave para trabajar en el lab 3 con placer:
Judit[h], aunque te estés haciendo mayorcita (cómo va Peter Pan?), seguirás siendo “mi
monstruito”...Ferrán, todo él un personaje, tan carismático como su “ForFi”...y el último fichage,
Miquel (buen chaval), con carita de ángel y comentarios de diablete...
Luego la inolvidable “Granada Connection”: la sonriente y altruísta Melena (buena contadora de
leyendas nazaríes), Miriam Royo, mi compañera en foto-adicción (podremos vender nuestras fotos a la
National Geographic, no?), y la dulce Lorena, de calidades humanas inigualables (aunque con ciertos
problemillas relacionados con calcetines blancos...).
Para cerrar el “sector en castellano”, debo expresar mi gratitud a Txell (alias, Cielitos, alias MaryHeaven) por su amistad y su valor, sea peleándose con retro-inversos ó con 400 millones de péptidos
en forma de ocho, sea aprendiendo el portugués (creo que Lia está de acuerdo en darte “matrícula”),
sea pateando todo Porto (con sus infinitas subidas y bajadas) arrastrada (literalmente) por mí...
Antes de mais devo agradecer à Prof. Doutora Maria Joaquina Amaral Trigo, não só por me apoiar e
acreditar em mim profissionalmente, mas também pela sua valiosa amizade.
Agradeço à Fundação Calouste Gulbenkian (Lisboa, Portugal) a bolsa de doutoramento que me foi
concedida, o apoio financeiro para assistir a congressos internacionais e a gentileza com que sempre
fui tratada.
À Lia, catalã por motivos geográfico-sentimentais, portuguesa pelo idioma (si beim qui teim um cerrto
sôtaqui dá cidádji dji São Paulo, né?) e com a bondade e a doçura de brasileira que é. És daquelas
jóias raras com quem se pode sempre contar.
Aos meus amigos de sempre, Helena, Carla e Alfredo, por continuarmos a ser uma espécie de
D’Artagnan e os, ou melhor, as Três Mosqueteiras...”voltar à terrinha” não seria o mesmo sem vocês e
nunca me cansarei de agradecer a vossa amizade!
Aos meus pais, Tina e Quim, e ao Richard, por
tudo o que são para mim, dedico a presente Tese.
General index
Abstract
Resumen
Abbreviations
Amino acids
Amino acid protecting groups
Resins, handles and coupling reagents
iii
iv
v
vii
ix
x
0. Introduction
Surface plasmon resonance biosensors
3
5
7
11
15
22
25
27
29
33
37
0.1 Surface plasmon resonance
0.2 Real-time biospecific interaction analysis
0.3 Measuring kinetics of biospecific interactions
References
Foot-and-mouth disease virus
0.4 Foot-and-mouth disease
0.5 Foot-and-mouth disease virus
0.6 The development of anti-FMDV vaccines
References
Objectives
45
1. SPR screening of synthetic peptides from the GH loop of FMDV
49
51
52
56
60
64
64
67
68
1.0 Introduction
1.1 Optimisation of the experimental set-up
1.2 Application to the systematic screening of FMDV peptides
1.3 Use of other site A – directed monoclonal antibodies
1.4 Probing subtle differences in peptide and mAb behaviour by SPR
1.5 Validity of the experimental kinetic constants
1.6 Relevance of the SPR data for FMDV studies
References
2. Antigenic determinants in the GH loop of FMDV C1-Barcelona (or C-S30)
2.0 Introduction
2.1 Peptides mimicking the GH loop of FMDV C1-Barcelona and the
corresponding partial mutants
2.2 SPR study of the C-S30 peptides
2.3 Competition ELISA analysis of the C-S30 pentadecapeptides
2.4 Size effects in the antigenicity of C-S30 peptides
2.5 Input from parallel X-ray diffraction studies
2.6 Effect of conformation in the antigenicity of C-S30 peptides
2.7 Antigenic evaluation of C-S30 peptides through solution affinity SPR analysis
2.8 Two-dimensional proton nuclear magnetic resonance studies of C-S30 peptides
2.9 Recapitulation
References
3. Antigenic peptides with non-natural replacements within the GH loop of FMDV
3.0 Introduction
3.1 Peptides that combine antigenicity-enhancing replacements in the GH loop
3.2 Direct kinetic SPR analysis
3.3 Indirect SPR kinetic analysis using a high molecular weight competitor antigen
3.4 Solution affinity SPR analysis of the peptide antigens
3.5 Two-dimensional 1H-NMR analysis of peptide A15(FPS)
3.6 X-ray diffraction crystallography analysis of a peptide-antibody complex
3.7 Recapitulation
References
69
71
71
75
83
85
89
90
95
101
107
109
111
113
113
116
119
127
131
134
144
145
i
Conclusions
147
4. Materials & Methods
151
153
153
156
157
157
158
159
159
162
164
165
165
174
175
176
176
177
178
4.1 General procedures
4.1.1 Solvents and chemicals
4.1.2 Instrumentation
4.1.3 Analytical methods
4.1.4 Chromatographic methods
References
4.2 Solid-phase peptide synthesis
4.2.1 Solid-phase peptide synthesis protocols
4.2.2 Synthesis of peptides from the GH loop of FMDV
References
4.3 Antigenic evaluation of the FMDV peptides
4.3.1 SPR analysis of peptide-antibody interactions
4.3.2 Enzyme-linked immunosorbent assays
References
4.4 Structural studies of the FMDV peptides
4.4.1 Two-dimensional proton nuclear magnetic resonance
4.4.2 Protein X-ray diffraction crystallography
References
ii
Abstract
A fast and direct surface plasmon resonance (SPR) method for the kinetic analysis of the interactions between
peptide antigens and immobilised monoclonal antibodies (mAb) has been established. Protocols have been
developed to overcome the problems posed by the small size of the analytes (< 1600 Da). The interactions
were well described by a simple 1:1 bimolecular interaction and the rate constants were self-consistent and
reproducible. The key features for the accuracy of the kinetic constants measured were high buffer flow rates,
medium antibody surface densities and high peptide concentrations. The method was applied to an extensive
analysis of over 40 peptide analogues towards two distinct anti-FMDV antibodies, providing data in total
agreement with previous competition ELISA experiments.
Eleven linear 15-residue synthetic peptides, reproducing all possible combinations of the four replacements
found in foot-and-mouth disease virus (FMDV) field isolate C-S30, were evaluated. The direct kinetic SPR
analysis of the interactions between these peptides and three anti-site A mAbs suggested additivity in all
combinations of the four relevant mutations, which was confirmed by parallel ELISA analysis. The four-point
mutant peptide (A15S30) reproducing site A from the C-S30 strain was the least antigenic of the set, in
disagreement with previously reported studies with the virus isolate. Increasing peptide size from 15 to 21
residues did not significantly improve antigenicity. Overnight incubation of A15S30 with mAb 4C4 in solution
showed a marked increase in peptide antigenicity not observed for other peptide analogues, suggesting that
conformational rearrangement could lead to a stable peptide-antibody complex. In fact, peptide cyclization
clearly improved antigenicity, confirming an antigenic reversion in a multiply substituted peptide. Solution NMR
studies of both linear and cyclic versions of the antigenic loop of FMDV C-S30 showed that structural features
previously correlated with antigenicity were more pronounced in the cyclic peptide.
Twenty-six synthetic peptides, corresponding to all possible combinations of five single-point antigenicityenhancing replacements in the GH loop of FMDV C-S8c1, were also studied. SPR kinetic screening of these
peptides was not possible due to problems mainly related to the high mAb affinities displayed by these synthetic
antigens. Solution affinity SPR analysis was employed and affinities displayed were generally comparable to or
even higher than those corresponding to the C-S8c1 reference peptide A15. The NMR characterisation of one
of these multiple mutants in solution showed that it had a conformational behaviour quite similar to that of the
native sequence A15 and the X-ray diffraction crystallographic analysis of the peptide – mAb 4C4 complex
showed paratope – epitope interactions identical to all FMDV peptide – mAb complexes studied so far. Key
residues for these interactions are those directly involved in epitope – paratope contacts (141Arg,
143
Asp,
146
His)
as well as residues able to stabilise a particular peptide global folding. A quasi-cyclic conformation is held up by
a hydrophobic cavity defined by residues 138, 144 and 147 and by other key intrapeptide hydrogen bonds,
delineating an open turn at positions 141, 142 and 143 (corresponding to the Arg-Gly-Asp motif).
iii
Resumen
Se diseñó un método rápido y sencillo para el análisis cinético por resonancia de plasmón superficial (RPS) de
las interacciones entre antígenos peptídicos de bajo peso molecular (< 1600 Da) y anticuerpos monoclonales
(AM) inmovilizados en la superficie de un chip sensor. Dichas interacciones se ajustaron a un modelo de
interacción bimolecular 1:1 y las constantes cinéticas obtenidas resultaron fiables y reproducibles. Los
parámetros clave para la calidad de las constantes cinéticas medidas fueron un flujo de tampón elevado, una
densidad superficial de AM intermedia y una elevada concentración de péptido. El método se extendió a más
de 40 análogos peptídicos frente a dos AM contra el virus de la fiebre aftosa (VFA), obteniéndose total
correlación con datos anteriores de ELISA competitivo.
Se sintetizaron once pentadecapéptidos con todas las combinaciones posibles de las cuatro mutaciones que
caracterizan el bucle GH del aislado C-S30 del VFA respecto a la secuencia de referencia C-S8c1. Los
resultados del análisis cinético directo, por RPS, de la antigenicidad de estos péptidos frente a tres AM
sugirieron que dichas combinaciones eran aditivas, observación que fué confirmada por ELISA competitivo.
Así, el tetramutante (A15S30) que mimetiza el bucle GH de C-S30 resultó ser el peor antígeno de la serie, en
contraste con resultados anteriores con este aislado. Aumentando el tamaño del tetramutante de 15 a 21
aminoácidos no afectó significativamente su antigenicidad. En cambio, una incubación prolongada con el AM
llevó a un aumento de reactividad no observado para otros análogos. Posiblemente una reordenación
conformacional del péptido pudo conllevar a la formación de un complejo estable con el anticuerpo.
Experimentos de RPS con un análogo cíclico del péptido A15S30 confirmaron una reversión en la
antigenicidad del tetramutante inducible a través de restricciones conformacionales. Estudios de ambos
péptidos, lineal y cíclico, por resonancia magnética nuclear (RMN) mostraron que características estructurales
anteriormente correlacionadas con la antigenicidad eran más pronunciadas en el análogo cíclico.
Se prepararon veintiseis péptidos con todas las posibles combinaciones de cinco sustituciones específicas en el
bucle GH del VFA C-S8c1. Dichas sustituciones individuales habían sido objeto de estudios anteriores,
obteniéndose una elevada antigenicidad para los correspondientes péptidos mutantes frente a AM anti-VFA.
No se pudo sistematizar el análisis cinético por RPS de los nuevos mutantes multiples, debido a problemas
tanto en la determinación de las constantes cinéticas de disociación, como en la regeneración de las superficies
de AM. Se utilizó así la RPS para la determinación de la afinidad péptido – AM en solución, obteniéndose
antigenicidades comparables o incluso superiores a las del péptido nativo A15 (VFA C-S8c1). Se estudió uno
de los mutantes multiples (A15FPS) por RMN, observándose una conformación identica a la del péptido
nativo. El estudio del complejo cristalino entre el péptido A15FPS y el AM 4C4 por difracción de RX mostró
que las interacciones parátopo – epítopo eran similares a las observadas con el péptido nativo. Se concluyió
que los residuos clave para el reconocimiento son tanto aquellos involucrados en contactos directos (141Arg,
143
Asp,
146
His) como aquellos que estabilizan el plegamiento adecuado del péptido. Así, una conformación casi
cíclica es soportada por una cavidad hidrofóbica definida por los residuos 138, 144 y 147 y por puentes de
hidrógeno intra-peptídicos clave, diseñándose un bucle abierto centrado en las posiciones 141, 142 and 143
(triplete Arg-Gly-Asp).
iv
Abbreviations
AA
Amino acid
AAA
Amino acid analysis
AcOH
Acetic acid
AM
2-[4-aminomethyl-(2,4-dimethoxyphenyl)phenoxy]acetic acid
APS
Ammonium persulphate
ATR
Attenuated total reflection
BSA
Bovine serum albumin
CDR
Complementarity determining region
Da
Dalton
DCM
Dichloromethane
DIEA
Diisopropylethylamine
DIP
Diisopropylcarbodiimide
DMF
dimethylformamide
EDC
N-ethyl-N’-(dimethylaminopropyl)carbodiimide
EDTA
Ethylenediaminotetraacetic acid
ELISA
Enzyme-linked immunosorbent assay
eq
equivalent
ESI
Electro-spray ionisation
Fab
Fragment, antigen-binding
Fc
Fragment, crystallisable
FMD
Foot-and-mouth disease
FMDV
Foot-and-mouth disease virus
FT-IR
Fourier-transform infrared spectroscopy
HBcAg
Hepatitis B core antigen
HCA
Human carbonic anhydrase
HEPES
4-(2-hydroxyethyl)piperazine-1-ethanesulphonic acid
HOBt
1-Hydroxybenzotriazole
HPLC
High performance liquid chromatography
HRV
Human rhino virus
HS
Heparan sulphate
IC50
Antigen concentration giving 50% inhibition
IFC
Integrated fluidic cartridge
Ig
Immunoglobulin
ka
Association rate constant / M-1s-1
KA
Affinity constant (association) / M-1
kd
Dissociation rate constant / s-1
KD
Affinity constant (dissociation) / M
KLH
Keyhole limpet hemocyanin
ks
Apparent/global rate constant / M-1s-1
LED
Light-emitting diode
v
mAb
Monoclonal antibody
MALDI-TOF Matrix-assisted laser desorption ionisation – time-of-flight
MAP
Multiple antigenic peptide
MBHA
p-methylbenzhydrylamine resin
MBS
m-maleimidobenzoyl-N-hydroxysuccinimide
MeCN
acetonitrile
MeOH
methanol
MPLC
Medium-pressure liquid chromatography
MS
Mass spectrometry
MW
Molecular weight
NHS
N-hydroxysuccinimide
NMM
N-methylmorpholine
NMP
N-methylpyrrolidone
NMR
Nuclear magnetic resonance
NOE
Nuclear Overhauser effect
NOESY
Nuclear Overhauser effect spectroscopy
OD
Optical density
PBS
Phosphate buffer saline
PEG
Polyethylene glycol
PS
Polystyrene
PVC
Polyvinyl chloride
R
Response
Req
Response at equilibrium
RI
Refractive index
Rmax
Maximal response
RNA
Ribonucleic acid
Rtot
Total response
RU
Resonance unit
SD
Standard deviation
SDS
Sodium dodecylsulphate
SDS-PAGE
Sodium dodecylsulphate – polyacrylamide gel electrophoresis
SPPS
Solid-phase peptide synthesis
SPR
Surface plasmon resonance
SPW
Surface plasmon wave
VP
Viral protein
TBTU
N-[(1H-benzotriazol-1-yl)dimethylaminomethylene]-N-methylmethaneaminium N-oxide
tetrafluoroborate
TEMED
N,N,N’,N’-tetramethylethylenediamine
TFA
Trifluoroacetic acid
TFE
2,2,2-trifluoroethanol
TIR
Total internal reflection
TOCSY
Total correlation spectroscopy
UV - Vis
Ultraviolet – visible spectroscopy
vi
Amino acids
Threeletter
One-letter
code
code
Ala
A
Name
Formula
Alanine
CH3
NH CH
Arg
Asn
R
N
Arginine
Asparagine
H2C
(CH2)2
NH CH
CO
CO
NH C NH2
NH
H2C CONH2
NH CH
Asp
D
Aspartic acid
CO
H2C COOH
NH CH
Cys
C
Cysteine
CO
H2C SH
NH CH
Gln
Q
Glutamine
H2C CH2
NH CH
Glu
E
Glutamic acid
G
Glycine
CONH2
CO
H2C CH2
NH CH
Gly
CO
COOH
CO
NH CH2 CO
N
His
H
Histidine
H2C
NH
HN CH CO
Ile
I
Isoleucine
CH(CH3)CH2CH3
NH CH
Leu
L
Leucine
CH2CH(CH3)2
NH CH
Lys
K
Lysine
M
Methionine
F
Phenylalanine
CO
CH2CH2SCH3
NH CH
Phe
CO
CH2(CH2)3NH2
NH CH
Met
CO
CO
H2C
HN CH CO
vii
Threeletter
One-letter
code
code
Pro
P
Name
Formula
Proline
N CH
Ser
S
Serine
H2C OH
NH CH
Thr
Trp
T
W
CO
Threonine
CO
CH(OH)CH3
NH CH
CO
H2C
NH
Triptophan
HN CH CO
Tyr
Y
CH3
Tyrosine
H2C
HN CH CO
Val
V
Valine
CH(CH3)2
NH CH
Ahx
*
6-aminohexanoic
acid
NH
(CH2)5
CO
CO
Table I Abbreviations used for amino acid residues according to the
Biochemistry Nomenclature Committee of the IUPAC-IUB [specified in Eur. J.
Biochem. 138, 9-37 (1984) and J. Biol. Chem. 264, 633-673 (1989)]. α carbon
side chains are presented in the non-ionic form for the twenty coded amino acids;
All amino-acid residues employed corresponded to the natural L-configuration.
* Ahx is a non-coded amino acid residue used in this work.
viii
Amino acid protecting groups
Abbreviation
Name
Stability
Formula
Boc
t-butyloxycarbonyl
Stable to bases,
labile to TFA
CH3
O
H3C C O
CH3
O
Fmoc
9-fluorenylmethyloxycarbonyl
Npys
3-nitro-2-pyridylsulphenyl
Stable to acids and
labile to bases
O
Stable to acids and
bases, labile to
nucleophiles
S
NO2
N
H3C
Pmc
2,2,5,7,8pentamethylchromane-6sulphonyl
Stable to bases,
labile to TFA
CH3
H3C
CH3
H3C
O S O
t
Bu
Trt
t-butyl
Triphenylmethyl (trityl)
Stable to bases,
labile to TFA
Stable to bases,
labile to 1% TFA
CH3
H3C C
CH3
C
Table II Amino acid protecting groups employed in this work.
ix
Resins, handles and coupling reagents
Abbreviation
Structure
OCH3 NH2
AM
H3CO
OH
O
O
MBHA
H2N
Polystyrene
PEG-PS
H2N
O
H
N
O
DIP
O
N
H
(
O)
n
H3C
H3C
H
N
N
H
O
CH N C N CH
O
N
H
CH3
CH3
CH3 CH3
H3C N
TBTU
N CH3
+
-
BF4
N
N
N+
O
-
Table III Resins, handles and coupling reagents used in this work.
x
Polystyrene
“En el campo hubo de todo: sequía, caracol, fiebre aftosa.”
Isabel Allende
in La casa de los espíritus
0. Introduction
Surface plasmon resonance biosensors
SPR as a tool in the functional analysis of an immunodominant site in FMDV
6
Surface plasmon resonance biosensors
0.1
Surface Plasmon Resonance
0.1.1
The physical phenomenon1-6
When a beam of light propagating through a first medium of higher refractive index, n1 (e.g. a glass
or quartz prism) meets an interface with a second medium of lower refractive index, n2 (e.g. an
aqueous solution), then it will be totally internally reflected for all incident angles greater than a
critical angle θc:
θc=sin-1(n2/n1)
(0.1)
where θ is the angle between the incident
beam and the axis normal to the plane of
the interface. This phenomenon is known
as total internal reflection (TIR)4. Despite
being totally reflected, the incident beam
establishes an electromagnetic field that
penetrates a small distance into the
second medium, where it propagates
parallel to the plane of the interface (Fig.
Figure 0. 1 Schematic view of the total internal reflection
(TIR) phenomenon3.
0.1). This electromagnetic field is called the evanescent wave. The intensity of the evanescent
electric field, I(z), decays exponentially with perpendicular distance z from the interface:
I(z)=Ioe-z/d
(0.2)
where d is the penetration depth for angles of incidence θ<θc and light of wavelength λo:
d=(λo/4π)(n12sin2θ-n22)
(0.3)
d is independent of incident light polarisation but depends on its wavelength. The evanescent
electric field intensity at z=0, Io, depends both on θ and the incident beam polarisation. When the
beam is polarised parallel to the plane of the interface, Io is given by Io//:
Io//=I//[4cos2θ(2sin2θ-n2)]/(n4cosθ+sin2θ-n2)
(0.4)
When the beam is polarised perpendicular to the plane of the interface, the field intensity at z=0 is
equal to:
7
SPR as a tool in the functional analysis of an immunodominant site in FMDV
Io⊥=I⊥(4cos2θ)/(1-n2)
(0.5)
I// and I⊥ are the intensities of the incident light beam polarised parallel or perpendicular to the
interface, respectively, and n=(n2/n1)<1. Therefore, two major characteristics of the evanescent
wave are worth to notice:
i)
the depth of the evanescent wave is typically less than a wavelength, thus extending a few
hundred nm into the dielectric (liquid phase of refractive index n2);
ii)
the evanescent field intensity, Io, for angles a few degrees above the critical angle θc is
several times the incident intensity, I.
When a thin metal film is inserted at the prism/dielectric interface, a new phenomenon called surface
plasmon resonance (SPR) can occur5. Surface plasmons are waves of oscillating surface charge
density (conducting electrons) propagating along the metal surface, at a metal (e.g. silver or
gold)/dielectric (e.g. aqueous solution) interface. The field amplitude of the surface plasmon is
maximal at the interface and decays evanescently, i.e., perpendicularly to it, with a penetration into
the dielectric of about 100-200 nm. Due to high loss in the metal, the surface plasmon wave
propagates with high attenuation in the visible and the near-infrared spectral regions. The
distribution of the electromagnetic field of a surface plasmon wave is highly asymmetric and the
majority of the field is concentrated in the dielectric (Table 0.1).
Table 0.1 Major characteristics of surface plasmon waves (SPW) at the metal-water interface6.
Metal layer supporting SPW
Wavelength (nm)
Propagation length(µm)
Penetration depth into metal (nm)
Penetration depth into dielectric (nm)
Concentration of field in dielectric (%)
Silver
630
19
24
219
90
Gold
630
3
29
162
85
850
57
23
443
95
850
24
25
400
94
Surface plasmons cannot be directly excited
(resonate) by light, since the frequency and
wave
vector
requirements
cannot
be
simultaneously matched. Nevertheless, indirect
excitation can be achieved by an evanescent
wave created by the internal reflection of a pFigure 0. 2 Schematic view of the surface plasmon
resonance (SPR) phenomenon3.
polarised incident beam at the metal-coated
surface of the prism.
This excitation, or resonance, occurs only at a well-defined angle of incidence, θsp, given by:
θsp =sin-1(ksp/ngko)
8
(0.6)
Surface plasmon resonance biosensors
where ng is the refractive index of the prism, λ is the wavelength of the incident light in the vacuum,
ksp is the wave vector of the surface plasmon and ko is the wave vector of the light in the vacuum
ko=2π/λ. When the resonance condition is fulfilled, energy from the incident light is transferred to
the non-radiative surface plasmon and converted to heat. This energy loss is recognised by a sharp
minimum (≤1o at half-width) in the angle-dependent reflectance (Fig. 0.2).
0.1.2
SPR optical sensors and their applications1-31
The phase matching, i.e., the resonance angle θsp, is very sensitive to changes in wavelength, metal
thickness and refractive indices of the prism (n1) and of the dielectric (n2). However, if the first three
factors are all kept constant, θsp will depend only on the refractive index (i.e., on the dielectric
constant) of the dielectric (n2). A change in the dielectric refractive index very close to the metal
surface will originate a change in the resonance angle θsp, which is the principle of SPR sensing.
Considering the unique characteristics of SPR detection, it is possible to design SPR sensors that,
under the proper geometries, allow the sensing of physical, chemical or biochemical phenomena
which give rise to changes in the optical properties of the solution (dielectric) very close to the metal
surface.
Generally, an SPR optical sensor is composed of an optical system, a transducing medium interrelating the optical and the reagent domains, and an electronic system controlling the optoelectronic
devices and allowing data processing. The transducing unit transforms changes in the refractive
index, determined by continuously monitoring the SPR angle, into changes in the quantity of
interest. Measurement of the angular dependence of reflectance from an SPR sensor surface requires
a monochromatic light source, such as a
small gas laser (HeNe at 633 nm) or a solid
state diode in the far red. The optical
pathway
can
include
elements
for
polarisation in the incident plane (ppolarisation), attenuation, spatial filtering
and shaping the beam to a convergent
wedge focused at the SPR surface of a
hemicylindrical glass prism (Fig. 0.3).
Figure 0. 3 Optical apparatus for the measurement of
the angular dependence of resonance1.
SPR sensing has a wide variety of applications. Measurement of physical properties such as
displacement and angular position using SPR sensors has been described7,8. Exploitation of physical
phenomena occurring in optical transducing materials allowed the development of specific SPR
sensors, such as humidity detectors using humidity-induced refractive index changes in porous thin
layers and polymers9. The thermooptic effect in hydrogenated amorphous silicon has also been used
to create an SPR-based temperature sensor10.
9
SPR as a tool in the functional analysis of an immunodominant site in FMDV
Chemical SPR sensors are based on the measurement of SPR variations due to adsorption or
chemical reaction of an analyte within a transducing medium which causes changes in its optical
properties. Examples of chemical applications of SPR due to analyte adsorption include: monitoring
of hydrocarbons, aldehydes and alcohols adsorbing on polyethyleneglycol films11; monitoring of
chlorinated
hydrocarbons
adsorbing
on
polyfluoroalkylsiloxane12;
detection
of
aromatic
hydrocarbons adsorbing on Teflon film13. SPR devices using palladium are effective in the detection
of molecular hydrogen14; also, chemisorption of NO2 on gold has been used for NO2 detection15.
Copper and nickel phtalocyanine films have been used for SPR detection of toluene16, while bromocresol purple films have been employed for the detection of NH3 vapours17. SPR detection of Cu
and Pb ions was also made possible by combination with anodic stripping voltammetry18.
Affinity SPR biosensors are the most
widely employed, where SPR, as a
surface-oriented method, allows real-time
analysis
of
biospecific
interactions
without the use of labelled biomolecules.
The SPR biosensor technology has been
Figure 0. 4 SPR detection caused by biospecific binding of
ligand in solution to an immobilised receptor22.
commercialised
central
tool
quantifying
and
for
has
become
characterising
biomolecular
a
and
interactions.
19
Since the first demonstration, in 1983, of the viability of SPR biosensing , SPR detection of
biospecific interactions was developed until the appearance, in 1994, of the first analysis methods
for surveying biomolecular interactions in real-time3. These methods have been improved for the
study of kinetic and thermodynamic constants of those interactions. Generally, SPR biosensing relies
on the immobilisation of the biological receptor at the chemically modified gold surface, which is in
contact with a buffer solution20. Upon addition of a specific ligand to the solution, binding occurs
close to the gold surface, allowing for SPR detection due to mass increase, and consequent change
in the refractive index in this region (Fig. 0.4)21. The shift in the resonance angle acts as a mass
detector and the continuous angular interrogation in the SPR-sensing device allows for the real-time
monitoring of binding, providing kinetic data on the biospecific interaction. Prism-based SPR
biosensors using angular interrogation have been employed in studies of antigen-antibody23-26,
protein-protein27,28, protein-DNA interactions29and epitope mapping30,31. Many other biomolecular
studies are presently among the applications of SPR biosensing, which has become a part of modern
analytical methods.
10
Surface plasmon resonance biosensors
0.2
Real-Time Biospecific Interaction Analysis
The use of optical biosensors for interaction analysis has made it possible to obtain affinity and
kinetic data for a large number of protein-protein, protein-peptide and protein-DNA systems.
Biosensor AB (Uppsala, Sweden) is undoubtedly the biosensor market leader, since it launched, in
late 1990, the first commercial SPR-based instrument, BIAcore32-36.
0.2.1
The BIAcore technology3,20-22,37,38
BIAcore uses SPR to investigate biospecific
interactions at the surface of a sensor chip. One
of the components in the interaction is
immobilised on the sensor chip surface and the
other flows over the surface free in solution. As
the interaction proceeds, the concentration
(mass) of analyte in the surface layer changes,
giving an SPR response which can be followed
in real-time in the form of a sensorgram (Fig.
0.5). The instrument consists of a processing
unit,
reagents
for
ligand
Figure 0. 5 Sensorgram: monitoring the SPR
response in terms of binding to receptor22.
immobilisation,
exchangeable sensor chips and a personal
computer for control and evaluation. The
processing unit contains the SPR detector and
an
integrated
microfluidic
cartridge
that,
together with an autosampler, controls the
delivery of sample plugs into a transport buffer
which flows continuously over the sensor chip
surface (Fig. 0.6). All the injection and
detection
systems
are
thermostatically
Figure 0. 6 Scheme of the BIAcore instrument21.
controlled so that BIAcore measurements are carried out at constant temperature. The light source
in BIAcore is a near-infrared light-emitting diode (LED) and light is focused on the gold film as a
wedge-shaped beam giving a fixed range of incident angles. The SPR response is monitored by a
fixed array of light-sensitive diodes covering the whole wedge of reflected light. Reagents, buffers
and samples are delivered to the sensor chip surface through a liquid handling system composed by
three main parts: the pumps, the sample injector and the integrated fluidic cartridge (IFC). One of
the pumps is used to maintain the continuous flow over the surface, while the other is used for
injection of samples and reagents via the autosampler (sample injector). This autosampler is
programmed to take defined volumes of liquid from specified sample positions to either other
sample positions or to the IFC injection port.
11
SPR as a tool in the functional analysis of an immunodominant site in FMDV
0.2.2
Ligand immobilisation3,20-22,37,39,40
Sensor chip architecture
The sensor chip is a glass slide onto which a 50-nm thick gold
film has been deposited. Immobilisation by physical adsorption
on gold has disadvantages, namely ligand denaturation, nonspecific binding and steric hindrance. Therefore, the gold surface
has been chemically modified to allow ligand covalent
immobilisation39 in order to obtain a stable ligand surface, with
the possibility of repeated analyses and maximum exposure of
the ligand to the solution containing the biospecific partner (Fig.
Figure 0. 7 Sensor chip CM521,22.
0.7).
Immobilisation chemistry
Proteins are, by far, the most widely employed ligands in biospecific interaction analysis. Therefore,
the development of chemistries for ligand immobilisation was based on protein chemistry, namely
on the reaction between protein primary amino groups and the carboxyl groups from the
carboxymethyldextran matrix to form amide bonds. Immobilisation starts by activation of the matrix
COOH groups as N-hydroxysuccinimide active esters, upon reaction with N-hydroxysuccinimide
(NHS) in the presence of N-ethyl-N’-(dimethylaminopropyl)carbodiimide (EDC), in water. Next, a
protein solution at low ionic strength and pH below the isoelectric point is passed over the surface
and protein-matrix amide bonds are formed (Fig. 0.8). The efficiency of the immobilisation step
relies simultaneously on two factors:
i)
Electrostatic pre-concentration of positively charged protein in the negatively charged
carboxymethyldextran matrix, and
ii)
Reaction between the protein primary amines and the matrix active esters.
1
2
3
4
Figure 0. 8 Steps in the standard ligand immobilisation on CM5 sensor chips: 1. COOH activation with
EDC/NHS; 2. Ligand coupling; 3. Blocking of remaining reactive NHS-ester groups with ethanolamine; 4.
Final ligand surface20.
12
Surface plasmon resonance biosensors
Remaining active esters after protein immobilisation are
converted into inactive amides via reaction with
ethanolamine. The SPR detector continuously monitors
the immobilisation steps (Fig. 0.8) and the amount of
immobilised protein can be controlled either by protein
concentration, reaction time or other factors such as ionic
strength or pH20. NHS-esters are also reactive with other
nucleophilic groups from the ligand, such as thiol or
aldehyde groups (Fig. 0.9). Other chemical modifications
Figure 0. 9 Ligand immobilisation
based on NHS-ester activation22.
based on NHS-active esters were proposed40:
i)
Formation of an amine derivative by reaction of the NHS-esters with ethylenediamine;
ii)
Similar preparation of a hydrazide derivative upon reaction of hydrazine with the NHSactive esters;
iii)
Obtention
of
a
maleimide
derivative
adding
sulfo-m-maleimidobenzoyl-N-
hydroxysuccinimide ester (sulfo-MBS) to the amine surface prepared according to i).
Tailor-made sensor chips
The high versatility of the carboxymethyldextran matrix in sensor chip CM5 is accompanied by a
very high binding capacity and low non-specific binding suitable for the majority of biospecific
analyses, particularly those involving kinetic studies on low-molecular weight analytes or
concentration analysis. However, the size and charge density of the matrix can be detrimental for
specific studies, such as those involving high-molecular weight molecules or complex culture media.
Presently, a set of sensor chips in which the dextran matrix has been tailored to suit various
experimental studies is available, ranging from chips with absent (C1) or shortened (F1) dextran
polymers to chips with reduced charge density (B1) in the dextran matrix. Other specific sensor
chips are also available, such as a plain gold surface (J1) suitable to create new surface chemistries,
a streptavidin (SA) surface to capture biotinylated ligands, a nitrilotriacetic acid (NTA) surface with
capture via nickel chelation and a flat hydrophobic (HPA) surface for membrane biochemistry.
0.2.3
General methodology3,21-31,37,41-54
Binding strategies
Methods for real-time biospecific interaction analysis include single- or multi-step binding to the
sensor chip surface and direct or indirect measurement of analyte. In single-step methods binding of
one component to the immobilised ligand is measured, while in multi-step methods sequential
binding of two or more components is monitored. When the interaction of the analyte itself with the
modified sensor surface is monitored, the method is direct and in such case the response increases
with increasing amount of analyte. Indirect methods rely upon measurement of a component which
either:
13
SPR as a tool in the functional analysis of an immunodominant site in FMDV
i)
interacts with analyte in solution and the remaining free concentration in solution is
measured (solution affinity), or
ii)
competes with the analyte of interest for the same ligand binding site (surface competition).
In indirect methods, the response is inversely related to the amount of analyte.
Direct single-step methods (Fig. 0.10) are the simplest
way to study biospecific interactions and are commonly
used
for
kinetic
studies
and
for
concentration
measurement of macromolecules at relatively high
quantities (medium to large analytes above ca. 1
Figure 0. 10 Direct single-step detection of
analyte on the sensor surface22.
µg/ml)23,27,28,41-45.
Direct multi-step methods are assays in which each
stage in the series of binding steps is recorded in the
sensorgram. A common use of these methods consists
in the immobilisation of a capturing molecule (e.g.
streptavidin, anti-immunoglobulin) that specifically
binds
the
ligand
(biotinylated
molecule,
immunoglobulin), which is the receptor of the target
analyte (Fig. 0.11)29,46-48. This affinity capture allows
Figure 0. 11 Direct multi-step detection of
analyte on the sensor surface22.
the use of non-pure samples of ligand (e.g. from cell culture media) and also the oriented noncovalent immobilisation of ligand. These methods are often employed in binding site analysis such
as epitope mapping30,31,49. Another application of multi-step methods is the use of a secondary
molecule to enhance analyte response in sandwich assays where an analyte binds an immobilised
ligand and a second macromolecule is then injected to bind the bound analyte.
Indirect methods are most widely employed for small
analytes (molecular weight<1000 Da) in solution. Direct
Sensor surface
detection of such analytes is often difficult and these
usually lack multiple independent binding sites necessary
for response enhancement with sandwich techniques. In
solution affinity experiments the analyte and a specific
Target
analyte
Analyte receptor
(measured molecule)
receptor interact in solution and, once equilibrium is
reached, the remaining free receptor is determined by
SPR using a sensor chip where another ligand (e.g. the
Figure 0. 12 Solution affinity studies –
the target analyte is pre-equilibrated with
its biospecific receptor in solution and
remaining free receptor is measured on the
surface.
14
analyte itself) is immobilised (Fig. 0.12)24,25,50,51. Thus,
the solution affinity between analyte and receptor can be
determined.
Surface plasmon resonance biosensors
In surface competition assays a high molecular
Sensor surface
weight analyte is usually employed to compete with
the low molecular weight target analyte for the
same ligand binding site. Since response due to
Target analyte
(small competitor)
small analyte binding is unappreciable, only the
response from the large analyte is monitored.
Large analyte
(measured molecule)
Figure 0. 13 Surface competition between the
small target analyte and the SPR-detected
macromolecule.
Therefore,
the
effects
on
the
kinetics
of
macromolecule binding due to additions of small
competing analyte can be measured and the
kinetics of small analyte binding can be indirectly
determined (Fig. 0.13)52.
Surface regeneration
Regeneration of the ligand surface allows for the re-utilisation of the same biospecific surface for
series of measurements, obviating the need to replicate identical surfaces. The most general
regeneration methods rely on pH reduction below 2.5 using strong inorganic acids such as 10-100
mM HCl or H3PO4 or weaker acids such as glycine buffers. Nevertheless, ligand tolerance to acids is
variable and, on the other hand, many ligand-analyte complexes may not be disrupted under acidic
conditions. Therefore, regeneration procedures must be optimised and many regenerating agents
other than acids (bases such as 10 mM NaOH, high ionic strength solutions such as 1M NaCl, etc.)
may be found to be more effective. A systematic regeneration optimisation protocol has been
recently described and successfully applied to antibody surfaces53,54. Stock solutions are mixtures of
similar components (e.g. all acids, all bases, all salts, all detergents, etc.) and regeneration cocktails
are different combinations of such stock solutions. Fine-tuning of regeneration cocktails may provide
the answer to problems not overcome with standard regeneration methods and allow for the use of
molecules that, otherwise, would not be suitable as easy-to-regenerate ligands.
0.3
Measuring kinetics of biospecific interactions
Characterisation of the affinities and rates of biospecific interactions is fundamental in many areas of
biochemical research. Methods that measure changes in optical parameters, such as fluorescence or
absorbance, can be employed for direct kinetic analysis. However, these methods require that one
of the reactants is often labelled with a radioactive or fluorescent probe and thus no longer in native
form. SPR detection is more general than these methods, since it is sensitive to changes in mass and
no labelling is required. When analyte is injected across a ligand surface, the resulting sensorgram
displays three essential phases, namely, association of analyte with ligand during sample injection,
equilibrium (if reached) during sample injection, where the rate of analyte binding is balanced with
complex dissociation, and dissociation of analyte-ligand complex due to buffer flow immediately
15
SPR as a tool in the functional analysis of an immunodominant site in FMDV
after the end of analyte injection. With suitable analysis of binding data, reliable affinity and kinetic
data can be obtained from SPR experiments. However, for the majority of experimental purposes,
semi-quantitative ranking of rates and/or affinities is sufficient.
0.3.1
Basic theory3,31,37,55,56
A 1:1 interaction between the analyte (A) continuously flowing in solution over the ligand (B)
surface may be described by:
A+B
ka
AB
kd
Considering that, in the association phase, the sensor surface is continuously replenished with free
analyte solution and the amount of bound analyte is negligible with respect to the total analyte
concentration (C), pseudo-first order kinetics can be assumed. Thus, the rate of complex formation
is given by the equation:
d[AB]/dt=ka[A][B]-kd[AB]
(0.7)
which, in terms of SPR response, can be expressed as:
dR/dt=kaC(Rmax-R)-kdR= kaCRmax-(kaC+kd)R
(0.8)
where:
!
R is the SPR response (in resonance units, RU) at time t;
!
Rmax is the maximum analyte binding capacity (in RU), which reflects the number of ligand
binding sites, i.e., total ligand concentration;
!
ka is the association rate constant;
!
kd is the dissociation rate constant.
Therefore, a plot of dR/dt against R will be a straight line of slope -(kaC+kd) or -ks, where ks is the
apparent binding rate, and a plot of ks against analyte concentration will give a straight line with
slope ka and intercept on the ordinate kd.
When equilibrium is reached, the total binding rate (dR/dt) is zero and, from equation 0.8:
kaC(Rmax-Req)=kdReq
(0.9)
Where Req is the total response at equilibrium. Considering that the affinity association constant (KA)
is given by ka/kd, binding affinity can be determined from equilibrium measurements, as it can be
inferred from substituting and rearranging equation 0.9:
16
Surface plasmon resonance biosensors
Req/C=KARmax-KAReq
(0.10)
Thus, by plotting Req/C against Req, a straight line is obtained and KA and Rmax can be calculated
from the slope and the intercept on the ordinate, respectively.
During the dissociation phase, analyte solution is replaced by a continuous flow of running buffer
solution and analyte concentration drops to zero. For the pseudo-first order kinetics model, complex
dissociation can be described by:
dR/dt=-kdR
(0.11)
which, in the logarithmic form, can be given by:
ln(R0/Rt)=kd(t-t0)
(0.12)
where R0 is the response at an arbitrary start dissociation time t0. Consequently, a plot of ln(Rt/R0)
will give a straight line with slope -kd.
This basic theoretical model only applies when the interaction is homogeneous and when the
pseudo-first order kinetics is actually observed.
0.3.2
Fitting and evaluating biosensor data55-58
Curve fitting methods
In early kinetic studies based on SPR biospecific interaction analysis, data evaluation relied upon
linearisation of the binding data, according to the equations described in the previous section.
Nevertheless, linear transformations also transform the parameter-associated errors, which decreases
the quality of primary data.
On the other hand, it requires data from many analyte
concentrations. Therefore, non-linear least squares analysis
has been introduced for fitting and evaluating biosensor
data57.
Non-linear
least
squares
methods
optimise
parameter values by minimising the sum of the squared
residuals (S), being the latter the difference between the
fitted (rf) and the experimental (rx) curves at each point
Schematic
Figure
0.
14
representation of non-linear least
squares fitting by minimising squared
residuals55.
(residuals are squared in order to equal the weight of
deviations above and below the experimental curve, Fig.
0.14, Eq. 0.13).
2
S = ∑ (r f − rx )
n
(0.13)
1
17
SPR as a tool in the functional analysis of an immunodominant site in FMDV
Non-linear least squares analysis has been applied to curve fitting based on the integrated rate
equations (Table 0.2, page 21). This analytical integration is the simplest tool for systems with rate
equations that can be readily integrated. However, many interactions studied on biosensors do not
fit simple kinetic models, which can be seen by curved plots when linearisation is applied or by poor
fits when using the integrated rate equations. The software currently employed for biosensor data
evaluation includes several kinetic fitting models (Table 0.2, page 21). Those models corresponding
to binding that can be described by well-known rate equations use analytical integration while more
complex models, such as interactions with mass transfer limitations or conformational changes, rely
upon curve fitting with numerical integration58. Numerical integration is more computationallyintensive but allows evaluation when the rate equations cannot be integrated analytically. In
numerical integration methods, each species is assigned an initial concentration and the reaction is
stepped through in discrete time intervals. At the end of each interval the concentration of each
species is calculated considering its rate of formation or disappearance according to the rate
equations. Numerical integration can be used to model any kinetic mechanism and also to analyse
biosensor data by curve fitting as is done with analytical integration. However, with numerical
methods data is usually analysed globally by fitting both the association and dissociation phases for
several concentrations simultaneously. Global curve fitting is advantageous, because it minimises the
possibility of having a good fit with a wrong kinetic model and it lowers the variance in the estimates
of the rate constants.
Evaluating fitted data
The fitting algorithms are purely mathematical tools without any biochemical “knowledge”.
Therefore, it is always important to examine the results of fitted data to check for “reasonableness”
of the parameters found. This must be kept in mind at the time of choosing the “best fit”. This best
fit depends on the ability of the fitting algorithm to converge for the true minimum in the sum of
squared residuals and on the number of parameters that can be varied in the model, i.e., the
complexity of the model. Increasing model complexity also increases the probability to fall in local
minima and obtain misleading fits. Wrong fits are usually evident from markedly poor curve fits or
unreasonable results and are often due to bad data quality or inadequate choice of the fitting model.
Increasing the complexity of a model will also increase the ability of fitting the experimental curves
to the equation, since there is a wider range for varying parameters in order to obtain a closer fit.
Therefore, it is important to accept the simplest model that fits the sensorgrams when evaluating
kinetic data and judge whether a slightly better fit with a more complex model is experimentally
significant. The quality of the fit is described by the chi squared statistical parameter, defined as:
χ2=S/(n-p)
(0.14)
where n is the number of data points, p is the number of fitted parameters and
S is the sum of the
residuals (Eq. 0.13). Since n>>p, χ2 reflects the average squared residual per data point and, when
18
Surface plasmon resonance biosensors
the model fully fits the experimental data, χ2 represents the mean square of the signal noise. In
practice it is useful to check for the shape of the residual plot, since non-random distribution of
residuals is often a symptom of an incorrect fit.
0.3.3
Deviations from the langmuirian behaviour2,3,37,51,55,58-72
a) Mass-transport limitations2,3,51,55,59-66
Transport of mobile analyte to the sensor surface (Fig.
0.15) may be a serious problem when the interaction is
fast. Insufficient transport rate will not allow to obtain
meaningful kinetics (the rate-limiting step will be the
diffusion into the dextran matrix and not the interaction
itself)
and
the
assumption
that
analyte
bulk
concentration is constant and equal to the injected
Figure 0. 15 Scheme of the different
factors influencing transport of mobile
analyte to the sensor surface with
immobilised ligand2.
concentration is no longer valid. Consequently, the rate
equations corresponding to pseudo-first-order kinetics
are not applicable to systems under diffusion-controlled kinetics. Diffusion effects can be minimised
using high flow rates (> 30 µl/min), low density ligand surfaces and high analyte concentrations.
Nevertheless, systems with very high interaction rates will be always diffusion-controlled, which
implies an upper limit to the range of association rate constants amenable to study by SPR. Another
effect related to mass-transport limitations is analyte rebinding during the dissociation phase. If
analyte depletion from the surface is not fast enough, analyte molecules will rebind to the ligand and
response no longer follows a single exponential decay.
b) Ligand heterogeneity2,3,55,58-61,67,68
Random immobilisation chemistries and high surface density
lead to heterogeinety of ligand sites, which therefore are no
longer equivalent neither independent (Fig. 0.16). This effect
is more pronounced with high analyte concentrations, i.e.,
with decreasing number of free “readily accessible⇔higher
Figure 0. 16 Illustration of
heterogeneous binding of analyte to
ligand molecules immobilised in
exposed and buried sites59.
affinity” ligand sites. Oriented attachment of ligand to the
dextran
layer,
low
analyte
concentrations
and
low
immobilisation levels are the best measures to avoid
heterogeneity effects.
c) Analyte heterogeneity
3,55,59
Although biospecific analysis allows for the utilisation of non-purified samples (concentration
measurements of bioactive molecules in biological samples, ligand fishing, etc.), it must be ensured
that samples for kinetic analysis do not contain molecules, other than the analyte, that can interact
with the ligand. Otherwise, the SPR response will reflect the sum of different binding events and
cannot be described by simple kinetics.
19
SPR as a tool in the functional analysis of an immunodominant site in FMDV
d) Steric hindrance2,59,60
The formation of a complex between a large analyte and
immobilised ligand can mask additional binding sites. Although it
could be argued that such steric hindrance would not affect
binding kinetics (it would only decrease Rmax), this is not strictly
true, since the flexibility/fluidity of the dextran matrix allows
temporarily masked sites to become accessible during analyte
Figure 0. 17 Illustration of the
parking problem: masking of
ligand
binding
sites
by
attachment of large analyte
molecules59.
injection, adding complexity to the kinetics of the interaction.
This problem, also named the parking problem, assumes greater
proportions for large macromolecular analytes, higher analyte
concentrations and high density ligand surfaces (Fig. 0.17).
e) Analyte multivalency and ligand co-operativity51,55,59,69
Poor fits with pseudo-first-order kinetics should be expected whenever analyte is multivalent (e.g.
antibody bivalency), since 1:1 stoichiometry is no longer observed. Also, it is difficult to ensure that
both analyte binding sites (in the case of bivalency) are equivalent and independent, as well as to
know to which extent has the interaction 1:1 or 1:2 stoichiometry. Another situation where binding
sites may not be independent occurs when there is ligand co-operativity. Although equivalent, the
ligand binding sites may not interact independently from each other and negative or positive cooperative interactions will prevent the system from following simple pseudo-first-order kinetics.
f) Conformational changes58-60
It has been suggested that non-conformity of sensorgrams with the langmuirian model could be due
to additional steps involving isomerisation of the AB complex. Such two-state-reactions, where there
is conformational change upon binding, are not well described by pseudo-first-order kinetics and
other models must be employed to fit the data.
0.3.4
Experimental design in kinetic SPR analysis2,3,37,51,55,61,68-72
Deviations to pseudo-first-order kinetics predicted for 1:1 interactions could be interpreted as due to
more complex interaction mechanisms describing the interactions. However, they are often
produced by artefacts which can be minimised by careful experimental design. Low ligand
immobilisation levels are advisable to avoid mass-transport limitations, ligand heterogeneity or steric
hindrance. Analyte concentration must be high enough to avoid diffusion-controlled kinetics and low
enough not to saturate the surface (between 0.1KD and 10 KD). Buffer flow must be kept at high rate
to minimise diffusion-controlled binding and, whenever possible, soluble ligand must be added to
buffer in the dissociation phase to avoid rebinding effects. Oriented immobilisation chemistries
should be used when random amine coupling is seen to be a significative source of surface
heterogeneities. Instrumental drifts or non-specific binding to the carboxymethyldextran matrix are
often eliminated by subtraction of a blank run, using either an inactive analyte or a suitable
reference cell with inactive ligand. If problems with non-specific binding are persistent, the choice of
another kind of surface (other model of sensor chip) may be the solution.
20
Surface plasmon resonance biosensors
Table 0.2 Rate equations used in the pre-defined fitting models included in the BIAevaluation 3.0 software55.
Simultaneous ka/kd fit
Differential equations
Total response
(a) 1:1 langmuirian
d[B]/dt=-(ka[A][B]-kd[AB])
binding
d[AB]/dt=ka[A][B]-kd[AB]
[AB]+RI
Reaction scheme
A+B⇔AB
[A]=C, [B]0=Rmax,
[AB]0=0
(b) 1:1 binding with
the same as in (a)
drifting baseline
the same as in (a)
ton)+RI
the same as in (a) plus
(c) 1:1 binding with
mass transfer
[AB]+drift(t-
d[A]/dt=kt(C-[A])- (ka[A][B]-kd[AB])
[AB]+RI
Abulk⇔A+B⇔AB
[A] bulk=C, [B]0=Rmax,
[AB]0=0
(d) Heterogeneous
d[B1]/dt=-(ka1[A][B1]-kd1[AB1])
ligand (2 different
d[AB1]/dt=ka1[A][B1]-kd1[AB1]
binding sites)
d[B2]/dt=-(ka2[A][B2]-kd2[AB2])
A+B1⇔AB1
[AB1]+[AB2]+RI
d[AB2]/dt=ka2[A][B2]-kd2[AB2]
A+B2⇔AB2
[A]=C, [B1]0=Rmax1,
[B2]0=Rmax2
[AB1]0=[AB2]0=0
(e) Heterogeneous
d[B]/dt=-(ka1[A1]mw1[B]-
analyte (competition
kd1[A1B])/mw1n1-(ka2[A2]mw2[B]-
between two different
kd2[A2B])/mw2n2
analytes)
A1+B⇔A1B
[A1B]+[A2B]+RI
d[A1B]/dt=ka1[A1]mw1[B]-kd1[A1B]
[A1B]0=[A2B]0=0
d[B]/dt=-(ka1[A][B]-kd1[AB])-
A+B⇔AB
(ka2[AB][B]-kd2[AB2])
d[AB]/dt=(ka1[A][B]-kd1[AB])-
[AB]+[AB2]+RI
(ka2[AB][B]-kd2[AB2])]
d[B]/dt=-(ka1[A][B]-kd1[AB])
change (two-state
d[AB]/dt=(ka1[A][B]-kd1[AB])-
reaction)
(ka2[AB]-kd2[AB*])
[AB]+[AB*]+RI
(h) 1:1 langmuirian
binding
A+B⇔AB⇔AB*
[A]=C, [B]0=Rmax,
[AB]0=[AB*]0=0
d[AB*]/dt=ka2[AB]-kd2[AB*]
Separate ka/kd fit
AB+B⇔AB2
[A]=C, [B]0=Rmax,
[AB]0=[AB2]0=0
d[AB2]/dt=ka2[AB][B]-kd2[AB2]
(g) Conformational
[A1]=C1, [A2]=C2,
[B]0=Rmax/mw1,
d[A2B]/dt=ka2[A2]mw2[B]-kd2[A2B]
(f) Bivalent analyte
A2+B⇔A2B
Integrated rate equations
R=
[
((Ck a + k d )t )
Ck a Rmax 1 − e −
Ck a + k d
R = R0e
-kdt
+ offset
] + RI
[AB]+RI
the same as in (a)
[AB]+offset
21
SPR as a tool in the functional analysis of an immunodominant site in FMDV
References
1 Garland, P. B. (1996) Optical evanescent wave methods for the study of biomolecular interactions,
Quart. Rev. Biophys. 2, 91-117.
2 Schuck, P. (1997) Use of surface plasmon resonance to probe the equilibrium and dynamic aspects of
interactions between biological macromolecules, Annu. Rev. Biophys. Biomol. Struct. 26, 541-566.
3 “BIAcore Instrument Handbook”, (Pharmacia Biosensor AB, 1994) Uppsala, Sweden.
4 Hirshcfield, T. (1967), Physics of Thin Films 9, 145.
5 Kretschmann, E. and Raether, H. (1968) Radiative decay of non-radiative surface plasmons excited
by light, Z. Natuforsch. 23A, 2135-2136.
6 Homola, J., Yee, S. S. and Gauglitz, G. (1999) Surface plasmon resonance sensors: review, Sens. &
Actuat. B 54, 3-15.
7 Margheri, G., Mannoni, A. and Quercioli F. (1996) A new high-resolution displacement sensor based
on surface plasmon resonance, Proc. SPIE 2783, 211-220.
8 Schaller, J. K., Czepluch, R. and Stojanoff, C. G. (1997) Plasmon spectroscopy for high resolution
angular measurements, Proc. SPIE 3098, 476-486.
9 Weiss, M. N., Srivastava, R. and Groger, H. (1996) Experimental investigation of a surface plasmonbased integrated-optic humidity sensor, Electron. Lett. 32, 842-843.
10 Chadwick, B. and Gal, M. (1993) An optical temperature sensor using surface plasmons, Japn. J.
Appl. Phys. 32, 2716-2717.
11 Miwa, S. and Arakawa, T. (1996) Selective gas detection by means of surface plasmon resonance
sensors, Thin Solid Films 281-282, 466-468.
12 Abdelghani, A., Chovelon, J. M., Jaffrezic-Renault, N., Ronot-Trioli C., Veillas, C. and Gagnaire, H.
(1997) Surface plasmon resonance fiber-optic sensor for gas detection, Sens. & Actuat. B 38-39, 407410.
13 Podgorsek, R. P., Sterdenburgh, T. Wolters, J. Ehrenreich, T., Nischwitz, S. and Franke, H. (1997)
Optical gas sensing by evaluating ATR leaky mode spectra, Sens. & Actuat. B 39, 349-352.
14 Chadwick, B. and Gal, M. (1994) A hydrogen sensor based on the optical generation of surface
plasmons in a palladium alloy, Sens. & Actuat. B 17, 215-220.
15 Ashwell, G. J. and Roberts, M. P. S. (1996) Highly selective surface plasmon resonance sensor for
NO2, Electron. Lett. 32, 2089-2091.
16 Granito, C., Wilde, J. N., Petty, M. C., Houston, S. and Iradale, P.J. (1996) Toluene vapor sensing
using copper and nickel phthalocyanine Langmuir-Blodgett films, Thin Solid Films 284-285, 98-101.
17 Van Gent, J., Lambeck, P. V., Bakker, R. J., Popma, T. J., Sudholter, E.J.R. and Reinhoudt, D.N.
(1991) Design and realization of a surface plasmon resonance-based chemo-optical sensor, Sens. &
Actuat. A 26, 449-452.
18 Chinowsky, T. M., Saban, S. B. and Yee, S. S. (1996) Experimental data from a trace metal sensor
combining surface plasmon resonance with anodic stripping voltammetry, Sens. & Actuat. B 35, 3743.
19 Liedberg, B., Nylander, C. and Lundström, K. (1983) Surface plasmons resonance for gas detection
and biosensing, Sens. & Actuat. 4, 299-304.
20 Johnsson, B., Löfås, S. and Lindquist, G. (1991) Immobilization of proteins to a
carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon
resonance sensors, Anal. Biochem. 198, 268-277.
21 Fägerstam, L. G., Frostel-Karlsson, Å., Karlsson, R., Persson, B. and Rönnberg, I. (1992) Biospecific
interaction analysis using surface plasmon resonance detection applied to kinetic, binding site and
concentration analysis, J. Chrom. 597, 397-410.
22 http://www.biacore.com/
23 Altschuh, D., Dubs, M. C., Weiss, E., Zeder-Lutz, G. and Van Regenmortel, M. H. V. (1992)
Determination of kinetic constants for the interaction between monoclonal antibody and peptides
using surface plasmon resonance, Biochemistry 31, 6298-6304.
24 Lasonder, E., Bloemhoff, W. and Welling, G. W. (1994) Interaction of lysozyme with synthetic antilysozyme D1.3 antibody fragments studied by affinity chromatography and surface plasmon
resonance, J. Chrom. A 676, 91-98.
25 Lasonder, E., Schellekens, G. A., Koedijk, D. G. A. M., Damhof, R. A., Welling-Wester S., Feijlbrrief,
M., Scheffer, A. J. and Welling, G. W. (1996) Kinetic analysis of synthetic analogues of linear-epitope
peptides of glycoprotein D of herpes simplex virus type I by surface plasmon resonance, Eur. J.
Biochem. 240, 209-214.
26 Houshmand, H., Fröman, G. and Magnusson, G. (1999) Use of bacteriophage T7 displayed peptides
for determination of monoclonal antibody specificity and biosensor analysis of the binding reaction,
Anal. Biochem. 268, 363-370.
27 Wu, Z., Johnson, K., Choi, Y. and Ciardelli, T. L. (1995) Ligand binding analysis of soluble interleukin
2-receptor complexes by surface plasmon resonance, J. Biol. Chem. 270, 16045-16051.
22
Surface plasmon resonance biosensors
28 Lessard, I. A. D., Fuller, C. and Perham, R. N. (1996) Competitive interaction of component enzymes
with the peripheral subunit-binding domain of the pyruvate-dehydrogenase multienzyme complex of
Baccilus stearothermophilus: kinetic analysis using surface plasmon resonance detection, Biochemistry
35, 16863-16870.
29 Cheskis, B. and Freedman, L. P. (1996) Modulation of nuclear receptor interactions by ligands: kinetic
analysis using surface plasmon resonance, Biochemistry 35, 3309-3318.
30 Dubs, M. C., Altschuh, D. and Van Regenmortel, M. H. V. (1992) Mapping of viral epitopes with
conformationally specific monoclonal antibodies using biosensor technology, J. Chrom. 597, 391396.
31 Saunal, H. and Van Regenmortel, M. H. V. (1995) Mapping of viral conformational epitopes using
biosensor measurements, J. Immunol. Meth. 183, 33-41.
32 Hodgson, J. (1994) Light, Angles, Action: Instruments for label-free, real-time monitoring of
intermolecular interactions, Biotechnology 12, 31-35.
33 Malmqvist, M. and Karlsson, R. (1997) Biomolecular interaction analysis: affinity biosensor
technologies for functional analysis of proteins, Curr. Op. Chem. Biol. 1, 378-383.
34 Pathak, S. and Savelkoul, H. F. J. (1997) Biosensors in immunology: the story so far, Immunol.
Today 18, 464-467.
35 Fivash, M., Towler, E. M. and Fisher, R. J. (1998) BIAcore for macromolecular interaction, Curr. Op.
Biotechnol. 9, 97-101.
36 Lakey, J. H. and Raggett, E. M. (1998) Measuring protein-protein interactions, Curr. Op. Struct. Biol.
8, 119-123.
37 “BIAapplications Handbook”, (Pharmacia Biosensor AB, 1994) Uppsala, Sweden.
38 Sjölander, S. and Urbaniczky, C. (1991) Integrated fluid handling system for biomolecular interaction
analysis, Anal. Chem. 63, 2338-2345.
39 Löfås, S. and Johnsson, B. (1990) A novel hydrogel matrix on gold surfaces in surface plasmon
resonance sensors for fast and efficient covalent immobilization of ligands, J. Chem. Soc., Chem.
Commun., 1526-1528.
40 O’Shannessy, D. J., Brigham-Burke, M. and Peck, K. (1992) Immobilization chemistries suitable for
use in the BIAcore surface plasmon resonance detector, Anal. Biochem. 205, 132-136.
41 Brigham-Burke, M., Edwards, J. R. and O’Shannessy, D. J. (1992) Detection of receptor-ligand
interactions using surface plasmon resonance: model studies employing the HIV-1 gp120/CD4
interaction, Anal. Biochem. 205, 125-131.
42 Lemmon. M. A., Ladbury, J. E., Mandiyan, V., Zhou, M. and Schlessinger, J. (1994) Independent
binding of peptide ligands to the SH2 and SH3 domains of Grb2, J. Biol. Chem. 269, 31653-31658.
43 Tamamura, H., Otaka, A., Murakami, T., Ishihara, T., Ibuka, T., Waki, M., Matsumoto, A.,
Yamamoto, N. and Fujii, N. (1996) Interaction of an anti-HIV peptide, T22, with gp120 and CD4,
Biochem. Biophys. Res. Comm. 219, 555-559.
44 Chao, H. Houston, M. E., Grothe, S., Kay, C. M., O’Connor-McCourt, M., Irvin, R. T. and Hodges, R.
S. (1996) Kinetic study on the formation of a de novo designed heterodimeric coiled-coil: use of
surface plasmon resonance to monitor the association and dissociation of polypeptide chains,
Biochemistry 35, 12175-12185.
45 England, P., Brégére, F. and Bedouelle, H. (1997) Energetic and kinetic contributions of contact
residues of antibody D1.3 in the interaction with lysozyme, Biochemistry 36, 164-172.
46 Huyer, G., Li, Z. M., Adam, M., Huckle, W. R. and Ramachandran, C. (1995) Direct determination of
the sequence recognition requirements of the SH2 domains of SH-PTP2, Biochemistry 34, 10401049.
47 Shen, B. J., Hage, T. and Sebald, W. (1996) Global and local determinants for the kinetics of
interleukin-4/interleukin 4 receptor α chain interaction: a biosensor study employing recombinant
interleukin-4 binding protein, Eur. J. Biochem. 240, 252-261.
48 Lookene, A., Chevreuil, O., Østergaard, P. and Olivecrona, G. (1996) Interaction of lipoprotein lipase
with heparin fragments and with heparan sulfate: stoichiometry, stabilization and kinetics,
Biochemistry 35, 12155-12163.
49 Van Regenmortel, M. H. V., Altschuh, D., Pellequer, J. L., Richalet-Sécordel, P., Saunal, H., Wiley, J.
A. and Zeder-Lutz, G. (1994) Analysis of viral antigens using biosensor technology, Methods: A
Comp. Meth. Enzymol. 6, 177-197.
50 Zeder-Lutz, G., Rauffer, N., Altschuh, D. and Van Regenmortel, M. H. V. (1995) Analysis of
cyclosporin interactions with antibodies and cyclophilin using BIAcore, J. Immunol. Meth. 183, 131140.
51 Nieba, L., Krebber, A. and Plükthun, A. (1996) Competition BIAcore for measuring true affinities:
large differences from values determined from binding kinetics, Anal. Biochem. 234, 155-165.
52 Karlsson, R. (1994) Real-time competitive kinetic analysis of interactions between low-molecularweight ligands in solution and surface-immobilized receptors, Anal. Biochem. 221, 142-151.
53 Andersson, K., Hamalainen, M. and Malmqvist, M. (1999) Identification and optimization of
regeneration conditions for affinity-based biosensor assays. A multivariate cocktail approach, Anal.
Chem. 71, 2475-2481.
23
SPR as a tool in the functional analysis of an immunodominant site in FMDV
54 Andersson, K., Areskoug, D. and Hardenborg, E. (1999) Exploring buffer space for molecular
interactions, J. Molec. Recogn. 12,
55 “BIAevaluation Software Handbook: version 3.0”, (Biosensor AB, 1997) Uppsala, Sweden.
56 Atkins P. W., “Physical Chemistry, 5th ed., Oxford University Press, Oxford, U. K. 1994.
57 O’Shannessy, D. J., Brigham-Burke, M., Soneson, K. K., Hensley, P. and Brooks, I. (1993)
Determination of rate and equilibrium binding constants for macromolecular interactions using surface
plasmon resonance: use of nonlinear least squares analysis methods, Anal. Biochem. 212, 457-468.
58 Morton, T. A., Myszka, D. and Chaiken, I. (1995) Interpreting complex binding kinetics from optical
biosensors: a comparison of analysis by linearization, the integrated rate equation and numerical
integration, Anal. Biochem. 227, 176-185.
59 O’Shannessy, D. J. and Winzor, D. J. (1996) Interpretation of deviations from pseudo-first-order
kinetic behavior in the characterization of ligand binding by biosensor technology, Anal. Biochem.
236, 275-283.
60 Bowles, M. R., Hall, D. R., Pond, S. M. and Winzor, D. J. (1997) Studies of protein interactions by
biosensor technology: an alternative approach to the analysis of sensorgrams deviating from pseudofirst-order kinetic behavior, Anal. Biochem. 244, 133-143.
61 Schuck, P. (1997) Reliable determination of binding affinity and kinetics using surface plasmon
resonance biosensors, Curr. Op. Biotech. 8, 498-502.
62 Glaser, R. W. (1993) Antigen-antibody binding and mass transport by convection and diffusion to a
surface: a two-dimensional computer model of binding and dissociation kinetics, Anal. Biochem. 213,
152-161.
63 Myszka, D., Arulanatham, P. R., Sana, T., Wu, Z., Morton, T. A. and Ciardelli, T. L. (1996) Kinetic
analysis of ligand binding to interleukin-2 receptor complexes created on an optical biosensor surface,
Prot. Sci. 5, 2468-2478.
64 Hall, D. R., Cann, J. R. and Winzor, D. J. (1996) Demonstration of an upper limit to the range of
association rate constants amenable to study by biosensor technology based on surface plasmon
resonance, Anal. Biochem. 235, 175-184.
65 Myszka, D., Morton, T. A., Doyle, M. L. and Chaiken, I. M. (1997) Kinetic analysis of a protein
antigen-antibody interaction limited by mass transport on an optical biosensor, Biophys. Chem. 64,
127-137.
66 Witz, J. (1999) Kinetic analysis of analyte binding by optical biosensors: hydrodynamic penetration of
the analyte flow into the polymer matrix reduces the influence of mass transport, Anal. Biochem. 270,
201-206.
67 Oddie, G. W., Gruen, L. C., Odgers, G. A., King, L. G. and Kortt, A. A. (1997) Identification and
minimization of nonideal binding effects in BIAcore analysis: ferritin/anti-ferritin Fab’ interaction as a
model system, Anal. Biochem. 244, 301-311.
68 Kortt, A. A., Oddie, G. W., Iliades, P., Gruen, L. C. and Hudson, P. J. (1997) Nonspecific amine
immobilization of ligand can be a potential source of error in BIAcore binding experiments and may
reduce binding affinities, Anal. Biochem. 253, 103-111.
69 Kalinin, N. L., Ward, L. D. and Winzor, D. J. (1995) Effects of solute multivalence on the evaluation
of binding constants by biosensor technology: studies with concanavalin A and interleukin-6 as
partitioning proteins, Anal. Biochem. 228, 238-244.
70 Catimel, B., Nerrie, M., Lee, F.T., Scott, A. M., Ritter, G., Welt, S., Old, L. J., Burgess, A. W. and
Nice, E. C. (1997) Kinetic analysis of the interaction between the monoclonal antibody A33 and its
colonic epithelial antigen by the use of an optical biosensor: a comparison of immobilisation strategies,
J. Chrom. A 776, 15-30.
71 Karlsson, R. and Fält, A. (1997) Experimental design for kinetic analysis of protein-protein interactions
with surface plasmon resonance biosensors, J. Immunol. Meth. 200, 121-133.
72 Ober, R. J. and Ward, E. S. (1999) The choice of the reference cell in the analysis of kinetic data using
BIAcore, Anal. Biochem. 271, 70-80.
24
Foot-and-mouth disease virus
SPR as a tool in the functional analysis of an immunodominant site in FMDV
26
Foot-and-mouth disease virus
0.4
Foot-and-mouth disease
0.4.1
General features1-5
Foot-and-mouth disease (FMD) is an acute systemic infection affecting even-toed ungulates, both
domesticated and wild, including cattle, swine, sheep and goats. FMD generally involves mortality
rates below 5%, but even so it is considered the most important disease of farm animals since it
causes important decreases in livestock productivity and trade. The main route of infection of
ruminants is the inhalation of airborne virus, but infection via the alimentary tract or skin lesions is
also possible, although requiring higher doses of virus. After primary replication in the pharynx, the
virus enters the bloodstream and, following a 3 to 5 days period of febrile viræmia, it spreads
throughout the organs and tissues where new sites for secondary infection are established. Some
clinical symptoms of FMD are fever, anorexia, weight loss, lameness, salivation and vesicular lesions
(mouth and skin). Although FMD only rarely causes death in adult animals, the virus can cause
severe lesions in the myocardium of young animals, leading in this case to high mortality rates1-3.
An asymptomatic persistent infection can be established in ruminants for periods of a few weeks to
several years as a consequence either of the acute infection or of vaccination with live-attenuated
virus. Animals affected by this long-term persistent infection are known as carrier animals and are an
important reservoir of the FMD virus in nature. Also, it has been suggested that carrier cattle are a
possible source of FMD outbreaks by virus transmission to susceptible animals. The impossibility to
cure carrier animals by vaccination, together with the extraordinary genetic and antigenic complexity
of the FMD virus, are major drawbacks for the control of the disease1-5.
0.4.2
Natural distribution of FMD
The earliest reports on FMD were descriptions of outbreaks in Northern Italy in 1514 and in
Southern Africa in 1780, written by Fracastorri6 and Le Vailant7, respectively. Seven
immunologically different serotypes of the FMD virus are known, namely A, O, C, Asia-1, SouthAfrican Territories (SAT) -1, -2 and -3, which comprise more than 65 subtypes. The global
distribution of the disease in 1997 was as represented in Fig. 0.18, with no significant changes over
the last 30 years. FMD is endemic in South America, sub-Saharan Africa, India and Middle/Far
East8. Countries such as Chile, French Guyana, Guyana and Surinam have been FMD free for the
last decade, while the members of the Mercasur (Argentina, Uruguay, Paraguay and Brazil) have
greatly improved the control of the disease through vaccination programmes8. In sub-Saharan
Africa, the control of FMD has been motivated by the exportation of beef to Europe. However, there
is occasional spread of the disease from the African buffalo, which is usually restricted to game
parks. On the other hand, poor surveillance and diagnostic facilities as well as deterioration of some
control programmes are causative of FMD spread to domestic cattle, including North-African
countries like Tunisia, Morocco and Algeria8.
27
SPR as a tool in the functional analysis of an immunodominant site in FMDV
Figure 0. 18 Estimated world distribution of FMD in 1997. Dark zones represent regions where the disease is
endemic, striped zones regions where the disease is controlled and under vaccination programmes and light
zones are FMD free (adapted from reference 8).
The control of the disease in India and other countries in the Far East is very difficult due to the
extremely large number of sheep, goats and cattle and to the poverty of many of the farmers. New
outbreaks occurred in Asian countries where the disease had been controlled (Malaysia, Philippines,
Japan) and FMD was recently introduced in Taiwan8. The uncontrollable movement of livestock
between countries of the Middle East has made it impossible to effectively control the disease in this
region of the world. Partial control of FMD was achieved only in Israel, upon immunisation with
vaccines produced in Europe. Of concern to Europe has been the situation in Turkey, since this is
the traditional route by which FMD enters the Balkans8. Sanitary measures such as movement
restrictions and quarantine, total slaughter of affected and in-contact animals (“stamping out”) and
extensive vaccination employing inactivated whole-virus have been successful in the control and
eradication of the disease in Europe (Fig. 0.19), which led to the decision of the European Union to
cease vaccination in 1991. This decision was followed, for the sake of trading agreements, by the
remaining European countries, and control of imports, quarantine and “stamping out” replaced
vaccination as the measures to exclude the disease. Nevertheless, several outbreaks have been
reported in Europe since 1991, namely in Bulgaria
35000
O
u
t
b
r
e
a
k
s
(1991, 1993, 1996), Italy (1993), Greece (1994, 1996),
30000
Russia (1995), Albania (1996), Macedonia (1996),
25000
Kosovo (1996) and the Turkish Thrace (1995, 1996)8.
20000
In the particular context of the Iberian Peninsula, the last
15000
10000
recorded outbreak occurred in Spain during 1986.
5000
Although the peninsula has been FMD-free since then,
0
1980
1975
1970
1965
1960
Year
Figure 0. 19 Estimated FMD outbreaks
in Europe from 1960 to 1982 (reproduced
from reference 1).
28
the large (and insufficiently controlled) flow in persons
and goods from and into Northern African territories is a
cause of grave concern for the animal health authorities,
even if not explicitly acknowledged.
Foot-and-mouth disease virus
0.5
Foot-and-mouth disease virus
0.5.1
The virus particle
Foot-and-mouth disease virus (FMDV) was the first recognized viral pathogen (by Loeffler and
Frosch in 18989) and is the sole member of the genus Aphthovirus belonging to the Picornaviridæ
family. The viral particle, or virion, contains a single-stranded RNA of positive polarity,
approximately 8500 nucleotides long. The RNA is covalently linked to a small protein, VPg, at its 5’
terminus and translation of the RNA yields a single polypeptide (L-P1-P2-P3) which is then cleaved
into the structural (from the P1 region) and non-structural (such as the viral-specific protease 3C and
the viral-specific RNA polymerase 3D) proteins. The virus capsid is non-enveloped and has
icosahedral symmetry with a diameter of approximately 300 Å, consisting of 60 copies of each of
the structural proteins VP1, VP2, VP3 and VP4 (Fig. 0.20). While the first three structural proteins
(MW≈24 kDa) have surface components, the fourth (MW≈8.5 kDa) is internal. The virion is also
usually composed by one or two units of VP0, the precursor of VP2 and VP410,11.
Figure 0. 20 Illustration of the structure of three picornaviruses (FMDV, Mengo and
HRV14) and their capsid proteins VP1-4 (reproduced from reference 11).
29
SPR as a tool in the functional analysis of an immunodominant site in FMDV
0.5.2
Architecture of the FMD virion
The structure of the FMDV particle was first resolved
for serotype O1 BFS 1860 by X-ray diffraction
analysis11. Since then, other serotypes of FMDV
have been crystallised and analysed, allowing some
of the phenotypic (e.g., high buoyant density in
CsCl, acid lability) and serological (immunological
and antigenic) properties of the virus to be
explained from a structural point of view12-15. The
Figure 0. 21 Structure of the viral capsid for
FMDV C-S8c1 (only α C are represented); VP1
– 4 are represented in blue, green, red and
yellow, respectively (reproduced from reference
overall
shape
of
the
outer
virus
surface
is
approximately spherical and relatively smooth. The
virus has icosahedral symmetry (Fig. 0.21); each
asymmetric unit (1/12 of the particle) is a pentamer11-15 formed upon assembly of five copies of the
biological protomer of FMDV16. The arrangement of VP1, VP2 and VP3 in the biological protomer
is as represented in Fig. 0.22, where the internal VP4 is not displayed.
The viral proteins VP1 – 3 of FMDV are quite similar in size, position, orientation and tertiary
structure to those of other picornaviruses, with VP1 showing the most significant rearrangements.
VP4 is the most variable protein among picornaviruses, the one belonging to FMDV being the larger
(Fig. 0.20).
0.5.3
Antigenic structure of FMDV
It has long been known that the main cell
attachment site and the immunodominant region
of FMDV are both located on a solvent exposed
region at the surface of the virion, namely in
trypsin-sensitive
areas
of
VP117,18.
Earlier
serological studies showed that different serotypes
of FMDV shared a highly variable region of VP1,
comprising residues 135 to 155 (Fig. 0.23)19, as
one of the major antigenic sites of the virus.
Several overlapping B-cell epitopes are located
within this region and are able to induce both
neutralising
and
non-neutralising
antibody
responses19-23. The high sequence variability
found in this region accounts for the low crossreactivity observed among different serotypes21-23.
30
Figure 0. 22 Ribbon protein diagram of the
FMDV C-S8c1 protomer composed of proteins
VP1 – blue, VP2 – green and VP3 – yellow
(reproduced from reference 16).
Foot-and-mouth disease virus
This immunodominant region was seen to correspond to the
loop which connects β-sheets G and H of the VP1 β-barrel,
named the GH loop11-15. Since the first evidences pointing to
the relevance of the GH loop in both the infectivity and
immune response in FMD, an enormous volume of research
has been focused on this region14,19-55. Unfortunately, the first
crystal structure of FMDV (strain O1 BFS) showed this region
to have very low electron density11, indicating high mobility
and thus lack of a defined structure. Based on the
assumption
that
such
disordered
conformation
was
dependent on a native disulphide bond linking Cys134 of
VP1, at the base of the loop, and Cys130 of VP2, the crystal
structure of FMDV O1 BFS was analysed under reducing
conditions and the conformation of the loop was thus
resolved12 (shown in yellow in Fig. 0.23). Other important
Figure 0. 23 Localisation of the GH
loop within the FMDV protomer
(above) and detailed illustration of the
conformation of this loop (below) –
yellow for isolate O1 BFS; magenta for
isolate C-S8c1. The RGD motif is
shown in detail (reproduced from
reference 51).
and 4 of FMDV O)
antigenic and immunogenic sites have been identified in
several FMDV serotypes; for instance, the C-terminal stretch
of VP1 (which, together with the GH loop, defines the main
antigenic site 1 in serotype O), or sites involving different
loops from the three accessible viral proteins (e.g. sites 2, 3
28,56-59
. The absence of cross-reactivity between the different types of FMDV,
together with the lack of steric hindrance between serotype-specific mAbs in competition
experiments, clearly show that antigenic sites in these serotypes are topologically independent from
each other. Resolution of the crystal structures of other FMDV variants, such as FMDV C-S8c115, or
peptide/virus – antibody complexes33,36,42,51,52, provided further evidence of such topological
differences, as shown in Fig. 0.23.
0.5.4
FMDV cell attachment sites: the Arg-Gly-Asp motif
Studies on surface topology, sequence conservation and inhibition of cell attachment of different
picornaviruses have shown that the majority of these pathogens share a common strategy for hiding
their cell attachment sites from the immune system. Such sites are usually placed inside canyons or
pits, out of reach from antibody footprints60. The absence of any such canyons or pits in the smooth
FMDV surface11-15, as well as the existence of a highly conserved Arg-Gly-Asp (RGD) motif within
the hypervariable GH loop of VP111-59, led to suspect that this motif could have a key role in
infectivity, since RGD is known to promote cell attachment in several different systems61.
Immunochemical and structural studies have shown that the RGD motif is, in fact, critically involved
in FMDV infectivity, upon cell attachment via the integrin αvβ3, the vitronectin receptor18,27,36,41,42,62-70.
Being placed in a highly exposed region of FMDV, the RGD motif has been surprisingly conserved
31
SPR as a tool in the functional analysis of an immunodominant site in FMDV
among the different serotypes, in spite of the high immune pressure exerted on this region. The
strategy of FMDV to elude antibody recognition is based on surrounding RGD with hypervariable
residues within a disordered loop. Thus, a mechanism for escape from antibody neutralisation
would involve subtle structural modifications which preserve the integrin-recognisable open-turn
conformation of the RGD triplet (Fig. 0.23)41,42,44,62-69.
Despite its obvious relevance, the RGD motif is not the only possible route for FMDV to be
internalised by the host cells70-77. Increasing evidence that FMDV clones lacking the RGD triplet can
infect host cells has made the essentiality of this motif questionable. In fact, it is now known that
there are at least three different mechanisms for cell recognition by FMDV. Apart from the RGDintegrin mechanism, there are isolates of FMDV which use heparan sulphate (HS) as the
predominant cell surface ligand72-75 (e.g., certain strains of FMDV O1, cell culture-adapted FMD
viruses) and even others which can establish RGD- and HS-independent infections76.
It has also been reported that FMDV can cause infection via the antibody-dependent enhancement
pathway, in which FMDV bound to virus-specific antibodies could enter cells via the Fc receptor,
thus bypassing the RGD mechanism70,71.
0.5.5
Antigenic and genetic variability of FMDV
RNA viruses are characterised by an error-prone RNA replication, which gives them great potential
for variation1,10. In FMDV genomes, the sequence homology between different serotypes can be as
low as 25-40% while homologies between subtypes of a same serotype are usually above 60-70%23.
Natural populations of FMDV from a single disease outbreak have been shown to be heterogeneous
and, moreover, “individual” isolates have been reported to include two different nucleotide
sequences. The high variability of FMDV (Table 0.3) led to the proposal that FMDV natural
populations are quasispecies, i. e., pools of variant genomes statistically defined but individually
indeterminate1,10,78. High mutation rates during replication allow FMD viruses to continuously evolve
and adapt to new environments. Although most mutations will be detrimental and eliminated by
natural selection (negative selection), others can be of value under the particular conditions where
the virus is replicating and are therefore selected (positive selection)1,10,75,78-87. Despite the high
heterogeneity of FMDV populations, there is a potential for long-term conservation of sequences
due to the continuous selection of a same consensus sequence in a situation of equilibrium79.
Whenever this equilibrium is ruptured, rapid evolution and selection of new master sequences take
place1,10,79.
One of the most troubling consequences of genetic variability is antigenic diversity. Immunochemical
studies have shown that isolates of the same geographical and chronological origin as well as viral
clones derived from single isolates may be antigenically distinct10,12,30,57,70,79-95. Antigenic variants
have been isolated under variable conditions, such as in partially immune animals, persistently
infected cattle4 and in cell culture96-98, in the latter case both in the presence or the absence of
immune pressure99,100. Therefore, antigenic variants result from the high mutation rates during RNA
32
Foot-and-mouth disease virus
replication and from the negative selection of most of the mutant phenotypes. This would mean that
substitutions at antigenic sites such as the FMDV GH loop are very likely to occur, since these are
disordered, flexible, and therefore, permissive sites, not subject to intensive negative selection.
This antigenic diversity has serious implications in vaccine design since synthetic vaccines should
include multiple independent epitopes in order to decrease the probability of selection of FMD
viruses resistant to the immune response.
Table 0.3 Variability of FMDV (reproduced from reference 12 and based on data from references therein).
Genetic heterogeneity
During a disease outbreak
Among consensus sequences of different isolates
Among consensus sequences of contemporary isolates
Among individual genomes of one isolate
Of clonal populations in cell culture
Among consensus sequences of independently passaged
plaque-purified viruses
Among individual genomes of clonal, passaged population
Frequency of mAb-resistant mutants
In viruses from lesions of infected animals
In viruses from cell culture fluid
Substitutions/genome
60 – 70
2 – 20
0.6 – 2
14 – 57
2–8
Evolution
2×
×10-6 – 2×
×10-5
-5
4×
×10
Substitutions/nucleotide/year
Rate of fixation of mutations
Acute disease
Persistent infection
<4×
×10-4 – 4.5×
×10-2
-3
9×
×10 – 7.4×
×10-2
0.6
The development of anti-FMDV vaccines
0.6.1
Conventional vaccines
Vaccination has been one of the most powerful tools for efficient control of infectious diseases such
as poliomyelitis, measles, yellow fever and smallpox, the latter having been totally eradicated worldwide. Conventional vaccines are whole-virus vaccines where attenuated variants or inactivated
viruses are employed. The possibility to control viral RNA quasispecies with classical vaccines relies
on two important factors: i. attempts of the virus to escape immune response upon mutation lead to
non-viable phenotypes which cannot adapt to the environment, and ii. the constant actualisation of
vaccine strains to include field variants from new outbreaks provides a broad coverage of the genetic
and antigenic heterogeneity found in the field. Nevertheless, high mutational rates are still an
obstacle to the efficacy of RNA viral vaccines. Also, on a more practical level, not all viruses are
easily grown in cell culture, a fact that can often prevent the production of classical vaccines, such as
hepatitis A101.
Most vaccines against FMDV are prepared by growing the virus in surviving bovine tongue epithelial
fragments, pig or calf kidney cell monolayers, or in baby hamster kidney cell culture and subsequent
inactivation with ethyleneimine (aziridine). The inactivated virus is then adsorbed onto aluminium
33
SPR as a tool in the functional analysis of an immunodominant site in FMDV
hydroxide and mixed with saponin prior to inoculation; vaccine delivery can also rely on emulsions
with an oil adjuvant. These classical anti-FMDV vaccines, given as a single dose, have been effective
in the control of the disease in Western Europe3,8,101-105.
The need of cold chains to keep viral vaccines at low temperatures in order to preserve their
immunogenicity is one of the reasons for the unsuccessful vaccination programmes in countries with
difficult terrain and climate conditions (e.g., tropical countries). Other disadvantages of whole-virus
vaccines arise from occasional deficient inactivation of the infective particle and consequent escape
to the field, causing new FMD outbreaks and eventually establishing persistent infections in cattle,
which act as important reservoirs and factories of new variants. But, clearly, the most important
problem of anti-FMDV classical vaccines comes from the high antigenic diversity of this virus, since
convalescent animals recovering from infection with a particular serotype are not protected against
other serotypes. Furthermore, each serotype consists of a wide spectrum of variant isolates and often
the virus strain used to prepare vaccines against a certain serotype does not offer the same degree of
protection against other isolates of the same serotype. Moreover, adaptation of an outbreak virus to
growth in cell culture can lead to the selection of variants that are antigenically different from their
parental virus1,2,10,12,30,70,79-98.
0.6.2
Synthetic vaccines
In view of the difficulties posed by conventional vaccines, the development of synthetic, molecularly
engineered vaccines has become a priority for the control of viral diseases. In particular, the use of
peptide-based synthetic vaccines offers significant advantages over classical procedures in terms of
stability, availability, safety, purity and cost101,106. These benefits are not easily achieved, however.
Thus, in order to design effective candidate vaccines, the antigenic and immunogenic determinants
of the pathogen must be adequately understood.
Intensive research has been focused on FMDV B-cell epitopes with the hope that they could be
mimicked by short linear peptides capable of eliciting protective virus-specific immune
responses20,25,26,39,43,45,107-115. As mentioned before, early studies allowed the recognition of major
antigenic sites located within protein VP1, namely the GH loop (antigenic site A) and the C-terminal
region (antigenic site C). Peptide vaccines based on site A or on constructs including both A and C
sites (the so-called “DiMarchi” peptides, Fig. 0.24) induced significant levels of anti-FMDV
neutralising antibodies and protected either guinea-pigs or natural hosts (pigs and cattle)116-118.
CysCys
VP1 C-terminus
(residues 200-213)
ProProSer
VP1 G-H loop
(residues 141-158)
ProCysGly
Figure 0. 24 The “DiMarchi” peptide antigen: the VP1 C-terminal and GH loop regions from FMDV O1
Kaufbeuren were brought together in a linear construct; a ProProSer spacer was used to induce a turn and
Cys residues were placed at each terminus to allow oligomerisation and bypass the use of a carrier protein.
34
Foot-and-mouth disease virus
The immunogenicity of synthetic FMDV antigens was shown to be
generally lower than that of classical vaccines in natural hosts. Also
differences between anti-virus and anti-peptide immune responses were
detected, namely, the good correlation between neutralising activity of anti-
Carrier protein
(BSA or KLH)
virus sera and host protection was not always well established in peptide-
+
immunised animals119-123. Attempts to increase the immunogenicity of small
FMDV peptides include the design of constructs containing tandem repeats
of the linear peptide, attachment of peptide to carrier proteins such as
bovine serum albumin (BSA) and keyhole limpet hæmocyanin (KLH)107-115
(Fig. 0.25) or insertion in scaffolds such as multiple antigenic peptide
(MAP) systems124 (Fig. 0.26), recombinant proteins (e.g., β-galactosidase
from Escherichia coli35,40,125,126) and hepatitis B virus core (HBc) protein
Carrier-peptide
conjugate
which self-assembles into a spherical virus-like particle, the hepatitis B core
Figure
0.
25
Representation of a
peptide-carrier protein
conjugate
(peptide
represented as a black
“loop”).
antigen (HBcAg)67,101 (Fig. 0.27). Recombinant technology is also
important in protection against FMDV, as shown by recent results using
recombinant viruses or transgenic plants where VP1 or the precursor
polypeptide P1 of FMDV capsid proteins have been inserted127-130.
NH2
NH2
NH2
NH
NH2
NH
NH
NH
NH
NH
+
NH2
NH2
NH2
NH2
NH
NH
Figure 0. 26 Multiple antigenic peptide (MAP) system: a poly-lysine scaffold
is used to present in a single chimera several copies (8, in the present case) of
the synthetic antigen, represented as a black “loop” (adapted from reference
101).
In terms of antigenicity and immunogenicity, comparison between different peptide vaccine
candidates clearly shows that peptide presentation and orientation are important25,35,40,101,125,126,131.
Thus, insertion of a peptide reproducing site A of FMDV C-S8c1 on different solvent-exposed loops
of the homotetrameric enzyme β-galactosidase yielded different antigenicity levels of the resulting
chimeras, some of them more antigenic than the corresponding KLH conjugate35,40,125,126. These
results prove the sensitivity of anti-FMDV antibody responses to peptide conformation, regardless of
the localisation of antigenic site A on a linear and flexible loop. Another evidence of such
dependence on orientation was reported by Schaaper et al., who observed anti-peptide immune
responses dependent on the peptide-carrier coupling method131.
35
SPR as a tool in the functional analysis of an immunodominant site in FMDV
+
HBc
protein
HBc
protein-peptide
HBcAg-peptide
assembly
Figure 0. 27 Antigenic peptide (black “loop”) insertion into hepatitis B virus
core protein and self-assembly of the latter into hepatitis B core antigen
(HbcAg) particle (adapted from reference 101).
Despite these differences, short linear and cyclic peptides have been shown to reproduce rather
faithfully the features of antigenic site A from different isolates, including C-S strains
29,31,34,36-
38,41,44,49,50
. This opened the possibility of analysing in detail the effects of amino acid replacements
found in natural isolates and the repercussions of antigenic variation in the field132,133. Moreover, it
allowed an extensive screening of the effects of single-point replacements of amino acids spanning
the entire GH loop41 (Fig. 0.28) which, together with the recently resolved structures of some
peptide-antibody complexes36,42,44, provided further insight into the mechanisms of interaction
between the GH loop and anti-FMDV neutralising antibodies. The knowledge of such mechanisms
at the molecular level can provide the basis for the design of FMDV peptides with strong antigenic
character, suitable to be inserted in constructs including T-cell epitopes and other immunogenic
components to produce efficient synthetic anti-FMDV vaccines. Recent advances with retro-inverso
FMDV peptides135-138 (increasing peptide resistance to host proteases) and with synthetic models of
important discontinuous antigenic sites (site D from FMDV C-S8c1 isolate)59 are also encouraging
regarding the future of fully synthetic anti-FMDV vaccines.
Y
T
A
S
A
. R
G
D
L
A
H
L
T
T
T
SD6
4C4
6D11
7JD1
7CA11
5A2
7FC12
infectivity
Figure 0. 28 Sequence of the GH loop from C-S8c1 clone of FMDV (adapted from reference 134).
Above the sequence: (●) variable residues found in 50 field isolates of serotype C FMDV; (▲) residues found replaced in 97
laboratory FMDV mutants (89 of them derived from C-S8c1) selected by antibodies; (▼) replaced residues found after 25
independent passages of FMDV in cell culture, in the absence of immune pressure. Below the sequence: Average effects of
single-point replacements within site A on antigenicity towards 7 anti-FMDV monoclonal antibodies, where a black box
stands for IC50>100, a vertically striped box for 30<IC50<100 and a white box to IC50<5. The last row represents the
average effects of single-point mutations within the GH loop on inhibition of infectivity (FMDV C-S8c1), where a black box
stands for IC50>300, a vertically striped box for 30<IC50<300 and a horizontally striped box for 5<IC50<30.
36
Foot-and-mouth disease virus
References
1 Domingo, E., Mateu, M. G., Martínez, M. A., Dopazo, J., Moya, A. and Sobrino, F. Genetic variability
and antigenic diversity of foot-and-mouth disease virus, in: Kurstak, E., Marusk, R. G., Murphy, S.A .
and Van Regenmortel, M. H. V., eds. “Applied Virology Research” – vol. 2, Plenum Publishing Co.,
New York: 1990; pp. 233-266.
2 Woodbury, R. L. (1995) A review of the possible mechanisms for the persistence of foot-and-mouth
disease virus, Epidemiol. & Infect. 114, 1-13.
3 Doel, T. R. (1996) Natural and vaccine-induced immunity to foot-and-mouth disease: the prospects
for improved vaccines, Ver. Sci. Tech. Off. Int. Epiz. 15, 883-911.
4 Gebauer, F., de la Torre, J. C., Gomes, I., Mateu, M. G., Barahona, H., Tiraboschi, B., Bergmann, I.,
Augé de Mello, P. and Domingo, E. (1988) Rapid selection of genetic and antigenic variants of footand-mouth disease virus during persistence in cattle, J. Virol. 62, 2041-2049.
5 Hernández, A. M. M., Carrilo, E. C., Sevilla, N. and Domingo, E. (1994) Rapid cell variation can
determine the establishment of a persistent viral infection, Proc. Natl. Acad. Sci. USA 91, 3705-3709.
6 Fracastorri, H. (translation) in: Wright, W. C., eds. “Contagion, Contagious diseases and their
treatment.”, London, Putnam: 1545
7 Le Vailant, “Travels into the interior parts of Africa 1780-1785” – vol. 2: 1795.
8 Kitching, R. P. (1998) A recent history of foot-and-mouth disease, J. Comp. Path. 118, 89-108.
9 Lœffler, F., Frosch, P. (1898) Berichte der Kommission zur Erforschung der Maul- und Klauenseuche
bei dem Institut für Infectrionskrankheiten in Berlin, Zbl. Bakter Abt. I. Orig. 23, 371-391.
10 Domingo, E., Escarmís, C., Martínez, M. A., Martínez-Salas, E. and Mateu, M. G. (1992) Foot-andmouth disease virus populations are quasispecies, Curr. Topics Microbiol. Immunol. 176, 33-47.
11 Acharya, R., Fry, E., Stuart, D., Fox, G., Rowlands, D. and Brown, F. (1989) The three-dimensional
structure of foot-and-mouth disease virus at 2.9 Å resolution, Nature 337, 709-716.
12 Parry, N., Fox, G., Rowlands, D., Brown, F., Fry, E., Acharya, R., Logan, D. and Stuart, D. (1990)
Structural and serological evidence for a novel mechanism of antigenic variation in foot-and-mouth
disease virus, Nature 347, 569-572.
13 Curry, S., Abu-Ghazaleh, R., Blakemore, W., Fry, E., Jackson, T., King, A., Lea, S., Logan, D.,
Newman, J. and Stuart, D. (1992) Crystallization and preliminary X-ray analysis of three serotypes of
foot-and-mouth disease virus, J. Mol. Biol. 228, 1263-1268.
14 Logan, D., Abu-Ghazaleh, R., Blakemore, W., Curry, S., Jackson, T., King, A., Lea, S., Lewis, R.,
Newman, J., Parry, N., Rowlands, D. J., Stuart, D. and Fry, E. (1993) Structure of a major
immunogenic site on foot-and-mouth disease virus, Nature 362, 566-568.
15 Lea, S., Hernández, J., Blakemore, W., Brocchi, E., Curry, S., Domingo, E., Fry, E., Abu-Ghazaleh,
R., King, A., Newman, J., Stuart, D. and Mateu, M. G. (1994) The structure and antigenicity of a type
C foot-and-mouth disease virus, Structure 2, 123-139.
16 http://www.rscb.org/pdb/ (structure code 1FMD)
17 Laporte, J. and Lenoir, G. (1973) Structural proteins of foot-and-mouth disease virus, J. Gen. Virol.
20, 161-168.
18 Cavanagh, D., Sangar, D. V., Rowlands, D. J. and Brown, F. (1977) Immunogenic and cell
attachment sites of foot-and-mouth disease virus: further evidence for their location in a single capsid
polypeptide, J. Gen. Virol. 35, 149-158.
19 Strohmaier, K., Franze, R. and Adam, K. H. (1982) Location and characterization of the antigenic
portion of the foot-and-mouth disease virus immunizing protein, J. Gen. Virol. 59, 295-306.
20 Oulridge, E. J., Barnett, P. V., Parry, N. R., Syred, A., Head, M. and Rweyemamu, M. M. (1984)
Demonstration of neutralizing and non-neutralizing epitopes on the trypsin-sensitive site of foot-andmouth disease virus, J. Gen. Virol. 65, 203-207.
21 Cheung, A., DeLamarter, J., Weiss, S. and Küpper, H. (1983) Comparison of the major antigenic
determinants of different serotypes of foot-and-mouth disease virus, J. Virol. 48, 451-459.
22 Beck, E., Feil, G. and Strohmaier, K. (1983) The molecular basis of the antigenic variation of footand-mouth disease virus, EMBO J. 2, 555-559.
23 Grubman, M. J., Zellner, M. and Wagner, J. (1987) Antigenic comparison of the polypeptides of footand-mouth disease virus serotypes and other picornaviruses, Virology 158, 133-140.
24 Robertson, B. H., Moore, D. M., Grubman, M. J. and Kleid, D. (1983) Identification of an exposed
region of the immunogenic capsid polypeptide VP1 on foot-and-mouth disease virus, J. Virol. 46,
311-316.
25 Parry, N. R., Syred, A., Rowlands, D. J. and Brown, F. (1988) A high proportion of anti-peptide
antibodies recognize foot-and-mouth disease virus particles, Immunology 64, 567-572.
26 Pfaff, E., Thiel, H. J., Strohmaier, K. and Scaller, H. (1988) Analysis of neutralizing epitopes on footand-mouth disease virus, J. Virol. 62, 2033-2040.
37
SPR as a tool in the functional analysis of an immunodominant site in FMDV
27 Fox, G., Parry, N. R., Barnett, P. V., McGinn, B., Rowlands, D. J. and Brown, F. (1989) The cell
attachment site on foot-and-mouth disease virus includes the amino acid sequence RGD (ArginineGlycine-Aspartic Acid), J. Gen. Virol. 70, 625-637.
28 McCahon, D., Crowther, J. R., Belsham, G. J., Kitson, J. D. A., Duchesne, M., Have, P., Meloen, R.
H., Morgan, D. O. and de Simone, F. (1989) Evidence for at least four antigenic sites on type O footand-mouth disease virus involved in neutralization; Identification by single and multiple-site
monoclonal antibody-resistant mutants, J. Gen. Virol. 70, 638-644.
29 Camarero, J. A., Andreu, D., Cairó, J. J., Mateu, M. G., Domingo, E. and Giralt, E. (1993) Cyclic
disulfide model of the major antigenic site of serotype C foot-and-mouth disease virus (synthetical,
conformational and immunochemical studies), FEBS Lett. 328, 159-164.
30 Krebs, O., Ahl, R., Strauß, O., C. and Marquardt, O. (1993) Amino acid changes outside the G-H
loop of capsid protein VP1 of type O foot-and-mouth disease virus confer resistance to neutralization
by anti-peptide G-H serum, Vaccine 11, 359-362.
31 Roig, X., Novella, I. S., Giralt, E. and Andreu, D. (1994) Examining the relationship between
secondary structure and antibody recognition in immunopeptides from foot-and-mouth disease virus,
Lett. Pept. Sci. 1, 39-49.
32 France, L. L., Piatti, P. G., Newman, J. F. E., Toth, I., Gibbons, W. A. and Brown, F. (1994) Circular
dichroism, molecular modeling and serology indicate that the structural basis of antigenic variation in
foot-and-mouth disease virus is α-helix formation, Proc. Natl. Acad. Sci. USA 91, 8442-8446.
33 Verdaguer, N., Mateu, M. G., Bravo, J., Tormo, J., Giralt, E., Andreu, D., Domingo, E. and Fita, I.
(1994) Crystallization and preliminary X-ray diffraction studies of a monoclonal Fab fragment against
foot-and-mouth disease virus and of its complex with the main antigenic site peptide, Proteins 18,
201-203.
34 Valero, M. L., Camarero, J. A., Adeva, A., Verdaguer, N., Fita, I., Mateu, M. G., Domingo, E., Giralt,
E. and Andreu, D. (1995) Cyclic peptides as conformationally restricted models of viral antigens:
application to foot-and-mouth disease virus, Biomed. Peptides, Prot. & Nucl. Acids 1, 133-140.
35 Benito, A., Mateu, M. G. and Villaverde, A. (1995) Improved mimicry of a foot-and-mouth disease
virus antigenic site by a viral peptide displayed on β-galactosidase surface, Biotechnology 13, 801804.
36 Verdaguer, N., Mateu, M. G., Andreu, D., Giralt, E., Domingo, E. and Fita, I (1995) Structure of the
major antigenic loop of foot-and-mouth disease virus complexed with a neutralizing antibody: direct
involvment of the Arg-Gly-Asp motif in the interaction, EMBO J. 14, 1690-1696.
37 Mateu, M. G., Camarero, J. A., Giralt, E., Andreu, D. and Domingo, E. (1995) Direct evaluation of
the immunodominance of a major antigenic site of foot-and-mouth disease virus in a natural host,
Virology 206, 298-306.
38 Mateu, M. G., Andreu, D. and Domingo, E. (1995) Antibodies raised in a natural host and
monoclonal antibodies recognize similar antigenic features of foot-and-mouth disease virus, Virology
210, 120-127.
39 Zamorano, P., Wigdorovitz, A., Pérez-Filguera, M., Carrillo, C., Escribano, J. M., Sadir, A. M. and
Borca, M. V. (1995) A 10-amino acid linear sequence of VP1 of foot-and-mouth disease virus
containing B- and T-cell epitopes induces protection in mice, Virology 212, 614-621.
40 Carbonell, X., Benito, A. and Villaverde, A. (1996) Converging antigenic structure of a recombinant
viral peptide displayed on different frameworks of carrier proteins, FEBS Lett. 397, 169-172.
41 Mateu, M. G., Valero, M. L., Andreu, D. and Domingo, E. (1996) Systematic replacement of amino
acid residues within an Arg-Gly-Asp containing loop of foot-and-mouth disease virus and effect on cell
recognition, J. Biol. Chem. 271, 12814-12819.
42 Verdaguer, N., Mateu, M. G., Bravo, J., Domingo, E. and Fita, I. (1996) Induced pocket to
accomodate the cell attachment site Arg-Gly-Asp motif in a neutralizing antibody against foot-andmouth disease virus, J. Mol. Biol. 256, 364-376.
43 Pegna, M., Molinari, H., Zetta, L., Melacini, G., Gibbons, W. A., Brown, F., Rowlands, D., Chan, E.
and Mascagni, R. (1996) the solution conformational features of two highly homologous antigenic
peptides of foot-and-mouth disease virus serotype A, variants A and USA, correlate with their
serological properties, J. Pep. Sci. 2, 91-105.
44 Verdaguer, N., Sevilla, N., Valero, M. L., Stuart, D., Brocchi, E., Andreu, D., Giralt, E., Domingo, E.,
Mateu, M. G. and Fita, I. (1998) A similar pattern of interaction for different antibodies with a major
antigenic site of foot-and-mouth disease virus: implications for intratypic antigenic variation, J. Virol.
72, 739-748.
45 Volpina, O. M., Yarov, A. V., Zhmak, M. N., Kuprianova, M. A., Chepurkin, A. V., Toloknov, A. S.
and Ivanov, V. T. (1996) Synthetic vaccine against foot-and-mouth disease based on a palmitoyl
derivative of the VP1 protein 135-159 fragment of the A22 virus strain, Vaccine 14, 1375-1380.
46 De Prat-Gay, G. (1997) Conformational preferences of a peptide corresponding to the major antigenic
determinant of foot-and-mouth disease virus: implications for peptide-vaccine approaches, Arch.
Biochem. Biophys. 341, 360-369.
38
Foot-and-mouth disease virus
47 Zamorano, P., Wigdorovitz, A., Pérez-Filguera, M., Escribano, J. M., Sadir, A. M. and Borca, M. V.
(1998) Induction of anti-foot-and-mouth disease virus T and B cell responses in cattle immunized with
a peptide representing ten amino acids of VP1, Vaccine 16, 558-563.
48 Valero, M. L., Giralt, E. and Andreu, D. (1999) A comparative study of cyclization strategies applied to
the synthesis of head-to-tail cyclic analogs of a viral epitope, J. Peptide Res. 53, 56-67.
49 Haack, T., Camarero, J. A., Roig, X., Mateu, M. G., Domingo, E., Andreu, D. and Giralt, E. (1997) A
cyclic disulfide peptide reproduces in solution the main structural features of a native antigenic site of
foot-and-mouth disease virus, Int. J. Biol. Macromol. 20, 209-219.
50 Valero, M. L., Camarero, J. A., Haack, T., Mateu, M. G., Domingo, E., Giralt, E. and Andreu, D.
(2000) Native-like cyclic peptide models of a viral antigenic site: finding a balance between rigidity
and flexibility, J. Mol. Recognit. 13, 5-13.
51 Hewat, E. A., Verdaguer, N., Fita, I., Blakemore, W., Brookes, S., King, A., Newman, J., Domingo, E.,
Mateu, M. G. and Stuart, D. I. (1997) Structure of the complex of an Fab fragment of a neutralizing
antibody with foot-and-mouth disease virus: positioning of a highly mobile antigenic loop, EMBO J.
16, 1492-1500.
52 Verdaguer, N., Schoehn, G., Ochoa, W. F., Fita, I., Brookes, S., King, A., Domingo, E., Mateu, M. G.,
Stuart, D. and Hewat, E. A. (1999) Flexibility of the major antigenic loop of foot-and-mouth disease
virus bound to a Fab fragment of a neutralizing antibody: structure and neutralization, Virology 255,
260-268.
53 Domingo, E., Verdaguer, N., Ochoa, W. F., Ruiz-Jarabo, C. M., Sevilla, N., Baranowski, E., Mateu,
M. G. and Fita, I. (1999) Biochemical and structural studies with neutralizing antibodies raised against
foot-and-mouth disease virus, Virus Res. 62, 169-175.
54 Feliu, J. X., Benito, A., Oliva, B., Avilés, F. X. and Villaverde, A. (1998) Conformational flexibility in a
highly mobile protein loop of foot-and-mouth disease virus: distinct structural requirements for integrin
and antibody binding, J. Mol. Biol. 283, 331-338.
55 Benito, A. and Van Regenmortel, M. H. V. (1998) Biosensor characterization of antigenic site A of
foot-and-mouth disease virus presented in different vector systems, FEMS Immunol. & Med.
Microbiol. 21, 101-115.
56 Barnett, P. V., Samuel, A. R., Pullen, L., Ansell, D., Butcher, R. N. and Parkhouse, R. M. E. (1998)
Monoclonal antibodies, against O1 serotype foot-and-mouth disease virus, from a natural bovine host,
recognize similar antigenic features to those defined by the mouse, J. Gen. Virol. 79, 1687-1697.
57 Mateu, M. G., Hernández, J., Martínez, M. A., Feigelstock, D., Lea, S., Pérez, J. J., Giralt, E., Stuart,
D., Palma, E. L. and Domingo, E. (1994) Antigenic heterogeneity of a foot-and-mouth disease virus
serotype in the field is mediated by very limited sequence variation at several antigenic sites, J. Virol.
68, 1407-1417.
58 Mateu, M. G., Escarmís, C. and Domingo, E. (1998) Mutational analysis of discontinuous epitopes of
foot-and-mouth disease virus using an unprocessed capsid protomer precursor, Virus Res. 53, 27-37.
59 Borràs, E., Giralt, E. and Andreu, D. (1999) A rationally designed synthetic peptide mimic of a
discontinuous viral antigenic site elicits neutralizing antibodies, J. Am. Chem. Soc. 121, 1193211933.
60 Chapman, M. S. and Rossmann, M. G. (1993) Comparison of surface properties of picornaviruses:
strategies for hiding the receptor site from immune surveillance, Virology 195, 745-756.
61 Hynes, R. O. (1992) Integrins: versatility, modulation and signaling in cell adhesion, Cell 69, 11-25.
62 Novella, I. S., Borrego, B., Mateu, M. G., Domingo, E., Giralt, E. and Andreu, D. (1993) Use of
substituted and tandem-repeated peptides to probe the relevance of the highly conserved RGD
tripeptide in the immune response against foot-and-mouth disease virus, FEBS Lett. 330, 253-259.
63 Berinstein, A., Roivainen, M., Hovi, T., Mason, P. W. and Baxt, B. (1995) Antibodies to the
vitronectin receptor (integrin αvβ3) inhibit binding and infection of foot-and-mouth disease virus to
cultured cells, J. Virol. 69, 2664-2666.
64 Villaverde, A., Feliu, J. X., Harbottle, R. P., Benito, A. and Coutelle, C. (1996) A recombinant,
arginine-glycine-aspartic acid (RGD) motif from foot-and-mouth disease virus binds mammalian cells
through vitronectin and, to a lower extent, fibronectin receptors, Gene 180, 101-106.
65 J. Hernández, Valero, M. L., Andreu, D., Domingo, E. and Mateu, M. G. (1996) Antibody and host
cell recognition of foot-and-mouth disease virus (serotype C) cleaved at the Arg-Gly-Asp (RGD) motif:
a structural interpretation, J. Gen. Virol. 77, 257-264.
66 Jackson, T., Sharma, A., Abu-Ghazaleh, R., Blakemore, W. E., Ellard, F. M., Simmons, D. L.,
Newman, J. W. I., Stuart, D. I. and King, A. M. Q. (1997) Arginine-glycine-aspartic acid-specific
binding by foot-and-mouth disease viruses to the purified integrin αvβ3 in vitro, J. Virol. 71, 83578361.
67 Sharma, A., Rao, Z., Fry, E., Booth, T., Jones, E. Y., Rowlands, D. J., Simmons, D. L. and Stuart, D.
I. (1997) Specific interactions between human integrin αvβ3 and chimeric hepatitis B virus core
particles bearing the receptor-binding epitope of foot-and-mouth disease virus, Virology 239, 150157.
39
SPR as a tool in the functional analysis of an immunodominant site in FMDV
68 Villaverde, A., Feliu, J. X., Arís, A., Harbottle, R.. P., Benito, A. and Coutelle, C. (1998) A cell
adhesion peptide from foot-and-mouth disease virus can direct cell targeted delivery of a functional
enzyme, Biotechnol. Bioeng. 59, 295-301.
69 Neff, S., Sá-Carvalho, D., Rieder, E., Mason, P. W., Blystone, S. D., Brown, E. J. and Baxt, B. (1998)
Foot-and-mouth disease virus virulent for cattle utilizes the integrin αvβ3 as its receptor, J. Virol. 72,
3587-3594.
70 Ruiz-Jarabo, C. M., Sevilla, N., Dávila, M., Gómez-Mariano, G., Baranowski, E. and Domingo, E.
(1999) Antigenic properties and population stability of a foot-and-mouth disease virus with an altered
Arg-Gly-Asp receptor recognition motif, J. Gen. Virol. 80, 1899-1909.
71 Mason, P. W., Baxt, B., Brown, F., Harber, J., Murdin, A. and Wimmer, E. (1993) Antibodycomplexed foot-and-mouth disease virus, but not poliovirus, can infect normally insusceptible cells via
the Fc receptor, Virology 192, 568-577.
72 Mason, P. W., Rieder, E. and Baxt, B. (1994) RGD sequence of foot-and-mouth disease virus is
essential for infecting cells via the natural receptor but can be bypassed by an antibody-dependent
enhancement pathway, Proc. Natl. Acad. Sci. USA 91, 1932-1936.
73 Jackson, T., Ellard, F. M, Abu-Ghazaleh, R., Brookes, S. M., Blakemore, W. E., Corteyn, A. H.,
Stuart, D. I., Newman, J. W. I. and King, A. M. Q. (1996) Efficient infection of cells in culture by type
O foot-and-mouth disease virus requires binding to cell surface heparan sulfate, J. Virol. 70, 52825287.
74 Sá-Carvalho, D., Rieder, E., Baxt, B., Rodarte, R., Tanuri, A. and Mason, P. W. (1997) Tissue culture
adaptation of foot-and-mouth disease virus selects viruses that bind to heparin and are attenuated in
cattle, J. Virol. 71, 5115-5123.
75 Fry, E. E., Lea, S. M., Jackson, T., Newman, J. I., Ellard, F. M., Blakemore, W. E., Abu-Ghazaleh, R.,
Samuel, A., King, A. M. Q. and Stuart, D. I. (1999) The structure and function of a foot-and-mouth
disease virus-oligosaccharide receptor complex, EMBO J. 18, 543-554.
76 Baranowski, E., Ruiz-Jarabo, C., Sevilla, N., Andreu, D., Beck, E. and Domingo, E. (2000) Cell
recognition by foot-and-mouth disease virus that lacks the RGD integrin-binding motif: flexibility in
Aphthovirus receptor usage, J. Virol. 74, 1641-1647.
77 Brown, F., Benkirane, N., Limal, D., Halimi, H., Newman, J. F. I., Van Regenmortel, M. H. V.,
Briand, J. P. and Müller, S. (2000) Delineation of a neutralizing subregion within the
immunodominant epitope (GH loop) of foot-and-mouth disease virus VP1 which does not contain the
RGD motif, Vaccine 18, 50-56.
78 Novella, I. S., Domingo, E. and Holland, J. J. (1996) Rapid viral quasispecies evolution: implications
for vaccine and drug strategies, Molec. Med. Today 53, 248-253.
79 Piccone, M. E., Kaplan, G., Giavedoni, L., Domingo, E. and Palma, E. L. (1988) VP1 of serotype C
foot-and-mouth disease virus: long-term conservation of sequences, J. Virol. 62, 1469-1473.
80 Sáiz, J. C. and Domingo, E. (1996) Virulence as a positive trait in viral persistence, J. Virol. 70, 64106413.
81 Sevilla, N. and Domingo, E. (1996) Evolution of a persistent Aphthovirus in cytolytic infections: partial
reversion of phenotypic traits accompanied by genetic diversification, J. Virol. 70, 6617-6624.
82 Domingo, E., Mateu, M. G., Escarmís, C., Martínez-Salas, E., Andreu, D., Giralt, E., Verdaguer, N.
and Fita, I. (1996) Molecular evolution of aphthoviruses, Virus Genes 11, 197-207.
83 Charpentier, N., Dávila, M., Domingo, E. and Escarmís, C. (1996) Long-term, large-population
passage of Aphthovirus can generate and amplify defective noninterfering particles deleted in the
leader protease gene, Virology 223, 10-18.
84 Martínez, M. A., Verdaguer, N., Mateu, M. G. and Domingo, E. (1997) Evolution subverting
essentiality: dispensability of the cell attachment Arg-Gly-Asp motif in multiply passaged foot-andmouth disease virus, Proc. Natl. Acad. Sci. 94, 6798-6802.
85 Carrillo, C., Borca, M., Moore, D. M., Morgan, D. O. and Sobrino, F. (1998) In vivo analysis of the
stability and fitness of variants recovered from foot-and-mouth disease virus quasispecies, J. Gen.
Virol. 79, 1699-1706.
86 Sevilla, N., Ruiz-Jarabo, C. M., Gómez-Mariano, G., Baranowski, E. and Domingo, E. (1998) An RNA
virus can adapt to the multiplicity of infection, J. Gen. Virol. 79, 2971-2980.
87 Baranowski, E., Sevilla, N., Verdaguer, N., Ruiz-Jarabo, C. M., Beck, E. and Domingo, E. (1998)
Multiple virulence determinants of foot-and-mouth disease virus in cell culture, J. Virol. 72, 63626372.
88 Mateu, M. G., Rocha, E., Vicente, O., Vayreda, F., Navalpotro, C., Andreu, D., Pedroso, E., Giralt, E.,
Enjuanes, L. and Domingo, E. (1987) Reactivity with monoclonal antibodies of viruses from an
episode of foot-and-mouth disease, Virus Res. 8, 261-274.
89 Mateu, M. G., da Silva, J. L., Rocha, E., de Brum, D. L., Alonso, A., Enjuanes, L., Domingo, E. and
Barahona, H. (1988) Extensive antigenic heterogeneity of foot-and-mouth disease virus serotype C,
Virology 167, 113-124.
40
Foot-and-mouth disease virus
90 Mateu, M. G., Martínez, M. A., Andreu, D., Parejo, J., Giralt, E., Sobrino, F. and Domingo, E. (1989)
Implications of a quasispecies genome structure: effect of frequent, naturally occurring, amino acid
substitutions on the antigenicity of foot-and-mouth disease virus, Proc. Natl. Acad. Sci. USA 86,
5883-5887.
91 Mateu, M. G., Martínez, M. A., Cappucci, L., Andreu, D., Giralt, E., Sobrino, F., Brocchi, E. and
Domingo, E. (1990) A single amino acid substitution affects multiple overlapping epitopes in the
major antigenic site of foot-and-mouth disease virus of serotype C, J. Gen. Virol. 71, 629-637.
92 Martínez, M. A., Hernández, J., Piccone, M. E., Palma, E. L., Domingo, E., Knowles, N. and Mateu,
M. G. (1991) Two mechanisms of antigenic diversification of foot-and-mouth disease virus, Virology
184, 695-706.
93 Feigelstock, D., Mateu, M. G., Piccone, M. E., de Simone, F., Brocchi, E., Domingo, E. and Palma, E.
L. (1992) Extensive antigenic diversification of foot-and-mouth disease virus by amino acid
substitutions outside the major antigenic site, J. Gen. Virol. 73, 3307-3311.
94 Feigelstock, D. A., Mateu, M. G., Valero, M. L., Andreu, D., Domingo, E. and Palma, E. L. (1996)
Emerging foot-and-mouth disease virus variants with antigenically critical amino acid substitutions
predicted by model studies using reference viruses, Vaccine 14, 97-102.
95 Sevilla, N., Verdaguer, N. and Domingo, E. (1996) Antigenically profound amino acid substitutions
occur during large population passages of foot-and-mouth disease virus, Virology 225, 400-405.
96 Díez, J., Dávila, M., Escarmís, C., Mateu, M. G., Domínguez, J., Pérez, J. J., Giralt, E., Melero, J. A.
and Domingo, E. (1990) Unique amino acid substitutions in the capsid protein of foot-and-mouth
disease virus from a persistent infection in cell culture, J. Virol. 64, 5519-5528.
97 Meyer, R. F. and Brown, F. (1995) Sequence identification of antigenic variants in plaque isolates of
foot-and-mouth disease virus, J. Virol. Meth. 55, 281-283.
98 Piatti, P., Hassard, S., Newman, J. F. E. and Brown, F. (1995) Antigenic variants in plaque-isolate of
foot-and-mouth disease virus: implications for vaccine production, Vaccine 13, 781-784.
99 Borrego, B., Novella, I. S., Giralt, E., Andreu, D. and Domingo, E. (1993) Distinct repertoire of
antigenic variants of foot-and-mouth disease virus in the presence or absence of immune selection, J.
Virol. 67, 6071-6079.
100 Holguín, A., Hernández, J., Martínez, M. A., Mateu, M. G. and Domingo, E. (1997) Differential
restrictions on antigenic variation among antigenic sites of foot-and-mouth disease virus in the
absence of antibody selection, J. Gen. Virol. 78, 601-609.
101 Brown, F. (1990) Synthetic peptides as potential vaccines against foot-and-mouth disease, Endeavour
14, 87-94.
102 Twomey, T., Newman, J., Burrage, T., Piatti, P., Lubroth, J. and Brown, F. (1995) Structure and
immunogeneicity of experimental foot-and-mouth disease and poliomielytis vaccines, Vaccine 13,
1603-1610.
103 Brown, F. (1999) Foot-and-mouth disease and beyond: vaccine design, past, present and future,
Arch. Virol. [Suppl.] 15, 179-188.
104 Mason, P. W., Piccone, M. E., McKenna, T. St.-C., Chinsangaram, J. and Grubman, M. J. (1997)
Evaluation of a live-attenuated foot-and-mouth disease virus as a vaccine candidate, Virology 227,
96-102.
105 Doel, T. R. (1999) Optimization of the immune response to foot-and-mouth disease vaccines, Vaccine
17, 1767-1771.
106 Sela, M. and Arnon, R. (1984) Synthetic antigens and vaccines, Interdisc. Sci. Rev. 9, 271-282.
107 Pfaff, E., Mussgay, M., Böhm, H. O., Schulz, G. E. and Schaller, H. (1982) Antibodies against a preselected peptide recognize and neutralize foot-and-mouth disease virus, EMBO J. 1, 869-874.
108 Geysen, H. M., Meloen, R. H. and Barteling, S. J. (1984) Use of peptide synthesis to probe viral
antigens for epitopes to a resolution of a single amino acid, Proc. Natl. Acad. Sci. USA 81, 39984002.
109 Francis, M. J., Fry, C. M., Rowlands, D. J., Brown, F., Bittle, J. L., Houghten, R. A. and Lerner, R. A.
(1985) Immunological priming with synthetic peptides of foot-and-mouth disease virus, J. Gen. Virol.
66, 2347-2354.
110 Geysen, H. M., Meloen, R. H. and Barteling, S. J. (1985) Small peptides induce antibodies with a
sequence and structural requirement for binding antigen comparable to antibodies raised against the
native protein, Proc. Natl. Acad. Sci. USA 82, 178-182.
111 Geysen, H. M., Rodda, S. J. and Mason, T. J. (1986) A priori delineation of a peptide which mimics a
discontinuous antigenic determinant, Molec. Immunol. 23, 709-715.
112 Meloen, R. H. and Barteling, S. J. (1986) Epitope mapping of the outer structural protein VP1 of three
different serotypes of foot-and-mouth disease virus, Virology 149, 55-63.
113 Meloen, R. H., Puyk, W. C., Meijer, D. J. A., Lankhof, H., Posthumus, W. P. A. and Shaaper, W. M.
M. (1987) Antigenicity and immunogenicity of synthetic peptides of foot-and-mouth disease virus, J.
Gen. Virol. 68, 305-312.
114 Francis, M. J., Hastings, G. Z., Clarke, B. E., Brown, A. L., Bedell, C. R., Rowlands, D. J. and Brown,
F. (1990) Neutralizing antibodies to all seven serotypes of foot-and-mouth disease virus elicited by
synthetic peptides, Immunology 69, 171-176.
41
SPR as a tool in the functional analysis of an immunodominant site in FMDV
115 Siligardi, G., Drake, A. F., Mascagni, P., Rowlands, D. J., Brown, F. and Gibbons, W. A. (1991) A CD
strategy for the study of polypeptide folding/unfolding: a synthetic foot-and-mouth disease virus
immunogenic peptide, Int. J. Peptide Protein Res. 38, 519-527.
116 DiMarchi, R., Brooke, G., Gale, C., Cracknell, V., Doel, T. and Mowat, N. (1986) Protection of cattle
against foot-and-mouth disease by a synthetic peptide, Science 232, 639-641.
117 Doel, T. R., Gale, C., Brooke, G. and DiMarchi, R. (1988) Immunization against foot-and-mouth
disease with synthetic peptides representing the C-terminal region of VP1, J. Gen. Virol. 69, 24032406.
118 Steward, M. W., Stanley, C. M., DiMarchi, R., Mulcahy, G. and Doel, T. R. (1991) High-affinity
antibody induced by immunization with a synthetic peptide is associated with protection of cattle
against foot-and-mouth disease, Immunology 72, 99-103.
119 Murdin, A. D. and Doel, T. R. (1987) Synthetic peptide vaccines against foot-and-mouth disease. I.
Duration of the immune response and priming in guinea pigs, rabbits and mice, J. Biol. Standardiz.
15, 39-51.
120 Murdin, A. D. and Doel, T. R. (1987) Synthetic peptide vaccines against foot-and-mouth disease. II.
Comparison of the response of guinea pigs, rabbits and mice to various formulations, J. Biol.
Standardiz. 15, 58-65.
121 Francis, M. J., Fry, C. M., Rowlands, D. J. and Brown, F. (1988) Qualitative and quantitative
differences in the immune response to foot-and-mouth disease virus antigens and synthetic peptides,
J. Gen. Virol. 69, 2483-2491.
122 Mulcahy, G., Gale, C., Robertson, P., Iysan, S. DiMarchi, R. and Doel, T. R. (1990) Isotype responses
of infected, virus-vaccinated and peptide-vaccinated cattle to foot-and-mouth disease virus, Vaccine 8,
249-256.
123 Taboga, O., Tami, C., Carrillo, E., Núñez, J. I., Rodríguez, A., Sáiz, J. C., Blanco, E., Valero, M. L.,
Roig, X., Camarero, J. A., Andreu, D., Mateu, M. G., Giralt, E., Domingo, E., Sobrino, F. and Palma,
E. L. (1997) A large-scale evaluation of peptide vaccines against foot-and-mouth disease: lack of solid
protection in cattle and isolation of escape mutants.
124 Francis, M. J., Hastings, G. Z., Brown, F., McDermed, J., Lu, Y. A. and Tam, J. P. (1991)
Immunological evaluation of the multiple antigen peptide (MAP) system using the major immunogenic
site of foot-and-mouth disease virus, Immunology 73, 249-254.
125 Feliu, J. X. and Villaverde, A. (1998) Engineering of solvent-exposed loops in Escherichia coli βgalactosidase, FEBS Lett. 434, 23-27.
126 Carbonell, X., Feliu, J. X., Benito, A. and Villaverde, A. (1998) Display-induced antigenic variation in
recombinant peptides, Biochem. Biophys. Res. Comm. 248, 773-777.
127 Sanz-Parra, A., Vázquez, B., Sobrino, F., Cox, S. J., Ley, V. and Salt, J. S. (1999) Evidence of partial
protection against foot-and-mouth disease in cattle immunized with a recombinant adenovirus vector
expressing the percursor polypeptide (P1) of foot-and-mouth disease virus capsid proteins, J. Gen.
Virol. 80, 671-679.
128 Wigdorovitz, A., Carrillo, C., dos Santos, M. J., Trono, K., Peralta, A., Gómez, M. C., Ríos, R. D.,
Franzone, P. M., Sadir, A. M., Escribano, J. M. and Borca, M. V. (1999) Induction of a protective
antibody response to foot-and-mouth disease virus in mice following oral or parenteral immunization
with alfalfa transgenic plants expressing the viral structural protein VP1, Virology 255, 347-353.
129 Sanz-Parra, A., Jímenez-Clavero, M. A., García-Briones, M. M., Blanco, E., Sobrino, F. and Ley, V.
(1999) Recombinant viruses expressing the foot-and-mouth disease virus capsid precursor polypeptide
(P1) induce cellular but not humoral antiviral immunity and partial protection in pigs, Virology 259,
129-134.
130 Wigdorovitz, A., Pérez-Filguera, D. M., Robertson, N., Carrillo, C., Sadir, A. M., Morris, T. J. and
Borca, M. V. (1999) Protection of mice against challenge with foot-and-mouth disease virus (FMDV)
by immunization with foliar extracts from plants infected with recombinant tobacco mosaic virus
expressing the FMDV structural protein VP1, Virology 264, 85-91.
131 Schaaper, W. M. M., Lankhof, H., Puijk, W. C. and Meloen, R. H. (1989) Manipulation of antipeptide immune response by varying the coupling of the peptide with the carrier protein, Molec.
Immunol. 26, 81-85.
132 Mateu, M. G., Andreu, D., Carreño, C., Roig, X., Cairó, J. J., Camarero, J. A., Giralt, E. and
Domingo, E. (1992) Non-additive effects of multiple amino acid substitutions on antigen-antibody
recognition, Eur. J. Immunol. 22, 1385-1389.
133 Carreño, C., Roig, X., Camarero, J. A., Cairó, J. J., Mateu, M. G., Domingo, E., Giralt, E. and
Andreu, D. (1992) Studies on antigenic variability of C-strains of foot-and-mouth disease virus by
means of synthetic peptides and monoclonal antibodies, Int. J. Peptide Protein Res. 39, 41-47.
134 Valero, M. L. “Mimetización estructural e inmunogénica del sitio antigénico principal del virus de la
fiebre aftosa” (Ph. D. Thesis), Department of Organic Chemistry – University of Barcelona: 1997.
135 Briand, J. P., Benkirane, N., Guichard, G., Newman, J. F. E., Van Regenmortel, M. H. V., Brown, F.
and Müller, S. (1997) A retro-inverso peptide corresponding to the GH loop of foot-and-mouth
disease virus elicits high levels of long-lasting protective neutralizing antibodies, Proc. Natl. Acad. Sci.
USA 94, 12545-12550.
42
Foot-and-mouth disease virus
136 Müller, S., Benkirane, N., Guichard, G., Van Regenmortel, M. H. V. and Brown, F. (1998) The
potential of retro-inverso peptides as synthetic vaccines, Exp. Opin. Invest. Drugs 7, 1429-1438.
137 Petit. M. C., Benkirane, N., Guichard, G., Du, A. P. C., Marraud, M., Cung, M. T., Briand, J. P. and
Müller, S. (1999) Solution structure of a retro-inverso peptide analogue mimicking the foot-and-mouth
disease virus major antigenic site, J. Biol. Chem. 274, 3686-3692.
138 Nargi, F., Kramer, E., Mezencio, J., Zamparo, J., Whetstone, C., Van Regenmortel, M. H. V., Briand,
J. P., Müller, S. and Brown, F. (1999) Protection of swine from foot-and-mouth disease with one dose
of an all-D retro peptide, Vaccine 17, 2888-2893.
43
SPR as a tool in the functional analysis of an immunodominant site in FMDV
44
1. SPR screening of synthetic
peptides from the GH loop of FMDV
SPR as a tool in the functional analysis of an immunodominant site in FMDV
50
SPR screening of synthetic peptides from the GH loop of FMDV
1.0
Introduction
The first objective of the present work was the study of the applicability of SPR biosensors1 to
kinetically characterise the interactions between peptides related to viral antigenic sites and relevant
monoclonal antibodies2. In particular, the research was focused on the interactions between antiFMDV mAbs and synthetic peptides reproducing an immunodominant region of FMDV (antigenic
site A, residues 136-150 of envelope protein VP1, isolate C-S8c1)3-5 to examine the main structural
features involved in the recognition of this site by neutralising antibodies. Synthetic peptides
reproducing different mutations at this site are particularly useful in identifying residues involved in
recognition or escape events6,7. Given the large number of such peptides and the relatively small
number of relevant mAbs, the most productive approach would seem to be mAb immobilisation and
analysis of the peptides as soluble analytes. However, a limitation of the SPR technique is that
interactions between low molecular weight (<5 kDaA) analytes and their immobilised binding
partners cannot, in principle, be studied directly since the increase in mass on the sensor chip is too
small to provide reliable data8. Not only small responses are a problem, but also bulk refractive
index effects together with non-specific
binding and mass-transport limitations can
affect true binding kinetics, particularly in
antigen
antigen
the direct detection of small analytes. A
possible
way
to
circumvent
detection
problems associated with small analytes is
to use a competitive kinetic analysis with a
high molecular weight analyte for the same
ligand
binding
site9.
However,
this
approach was not initially feasible, since a
high molecular weight representative of
kd
ka
ka
Fab
fragment
kd
Fab
fragment
Fc fragment
Figure 1. 1 Representation of an antigen – antibody
interaction; Fab stands for antigen-binding fragment and
Fc for crystallisable fragment.
antigenic site A, e. g., capsid protein VP1 of
FMDV, was unavailable. In view of this, it was decided to work with immobilised mAb and address
the difficulties associated with the small size (≈1.6 kDa) of the peptide analytes.
A 1:1 bimolecular interaction kinetics is to be expected for peptide-antibody interaction, if both
antigen-binding fragments are considered independent and equivalent (Fig. 1.1)10.
A
The experimental work described in the present thesis has been carried out using a BIACORE 1000 instrument. Since this
work was completed, improved versions of the BIAcore instrumentation with higher sensitivity (BIACORE 2000, 3000, X...)
have been commercialised.
51
SPR as a tool in the functional analysis of an immunodominant site in FMDV
1.1
Optimisation of the experimental set-up
Antigenic site A of FMDV C-S8c1 contains several distinct, overlapping, B-cell epitopes and is
located in the GH loop (residues 136 to 150) of the envelope protein VP12-7,11. It can be reproduced
by peptide A15B, corresponding to the sequence:
136
YTASARGDLAHLTT150T
A few sets of experiments, using A15 as analyte, were run on mAb SD6C surfaces with different
densities (8, 1.7 and 0.8 ng/mm2), using peptide concentrations between 1 and 2440 nM and two
different buffer flow rates (5 and 60 µl/min). mAb immobilisation and peptide injection procedures
are described in section 4.3.1.1.
1.1.1
High mAb density
In a first approach, a very high mAb surface density (8 ng/mm2) and high A15 concentrations were
employed in an attempt to overcome the low responses that were to be expected from the small size
of the analyte. Peptide injections were carried out at 5 µl/min, using the kinject mode to avoid
sample dispersion at injection plugs, and association and dissociation times were of 7 and 6
minutes, respectively. The surface was regenerated, at the end of each cycle, by a 3-min pulse of
100 mM HCl. The sensorgrams generated under these conditions (Fig. 1.2 A) could not be fitted to
the expected 1:1 bimolecular interaction kinetics, as inferred from the high and non-random
residuals observed in the dissociation phase (Fig. 1.2 B), and from the concentration-dependence of
the association rate constant, ka (Fig. 1.2 C).
B
Peptides used for optimisation and validation of the SPR experimental set-up were kindly given by Dr. Mari-Luz Valero
(Dept. Q.O. - U. B., Barcelona).
C
Monoclonal antibody SD6 is a site-A directed neutralising mAb raised against FMDV C-S8c1; it was kindly supplied by Dr.
Nuria Verdaguer and Wendy F. Ochoa (IBMB/CSIC – Barcelona).
52
SPR screening of synthetic peptides from the GH loop of FMDV
s
1.60E+06
C
1.40E+06
ka/M-1s-1
1.20E+06
1.00E+06
8.00E+05
6.00E+05
4.00E+05
2.00E+05
0.00E+00
0
200
400
600
800
1000
1200
Peptide concentration/nM
Figure 1. 2 First approach to the SPR kinetic analysis of the interaction between immobilised mAb SD6 and
peptide analyte A15: A. Experimental sensorgrams; B. Distribution of residual data points for the dissociation
phase corresponding to [A15]=310 nM (detailed view of the experimental and modelled curves); C. Variation
of ka with peptide concentration.
The shape of the dissociation curves suggested that some analyte rebinding to the surface was
occurring, affecting true kinetics. At the same time, the apparent association rate constant decreased
with increasing analyte concentration, i. e., the more peptide was injected, the more difficult became
its binding to mAb molecules. So, it appeared that antibody molecules were heterogeneously
distributed in the dextran matrix, with different accessibility levels12. Upon analyte injection, the first
peptide molecules would occupy the most accessible mAb receptors and the following ones would
have increasing difficulty in reaching free mAb binding sites, such effect becoming larger with higher
peptide concentrations. This seemed to be confirmed by the better fit obtained when a
heterogeneous ligand kinetic model was employed to fit the experimental data (data not shown).
This model, however, considers only two different types of ligand, which is most probably far from
reality. Since lowering peptide concentration (1 to 50 nM) did not improve the results (data not
shown), new conditions were searched in order to obtain experimental data consistent with a
langmuirian kinetic behaviour (1:1 bimolecular interaction).
53
SPR as a tool in the functional analysis of an immunodominant site in FMDV
1.1.2
Medium mAb density
The poor results obtained in the previous section were symptomatic of significant heterogeneity in
ligand accessibility and orientation. Also, diffusion-controlled delivery of analyte to the most
hindered SD6 molecules would be a further cause for the observed deviations. Therefore, a second
SD6 surface was prepared with much lower density (1.7 ng/mm2) and another set of injections was
run at the same flow rate, spanning peptide concentrations from 1 to 2440 nM. In this case, peptide
concentrations below 70 nM were too low for a clear response to be observed, since sensorgrams
were hardly distinguished from mere bulk refractive index effects. Higher peptide concentrations led
to results better than those described in section 1.1.1, but still presenting some degree of data
inconsistency (not shown).
1.1.3
Low mAb density
Further lowering of mAb surface density (to 0.8 ng/mm2), in an attempt to eliminate the non-ideal
effects observed so far, did not work either. In fact, this density was seen to be too low for the
detection of the FMDV peptides injected, even at analyte concentrations as high as 2.44 µM (not
shown).
1.1.4
High buffer flow rate
Since the previous results, all of them obtained at 5 µl/min flow, persistently deviated from the
expected behaviour at different mAb surface densities and peptide concentrations, the flow rate
seemed an important parameter to manipulate in order to optimise the SPR analysis. Low buffer
flow rate could be favouring diffusion-controlled kinetics, affecting true binding constants12-14.
Therefore, a fourth set of SPR experiments was run, this time using the medium density SD6 surface
(1.7 ng/mm2) and high A15 concentrations (152 to 2440 nM), and raising the buffer flow rate to 60
µl/min. Both association and dissociation times were decreased (90 and 240 seconds, respectively)
to diminish sample and buffer consumption. Under these experimental conditions, consistent and
apparently reliable data were obtained. Experimental and modelled curves were virtually
superimposable (Fig. 1.3 A) with a random distribution of residuals within an interval of ca. ±0.4 RU
(Fig. 1.3 B).
Linearity of ks versus peptide concentration over the 32-fold concentration range was observed, as
required for a concentration-independent ka (Fig.1.3 C). The chi-squared (χ2) value was smaller than
0.1 and data self-consistency15 was further confirmed by the total agreement between the values for
the equilibrium association constant, KA, obtained from either the ka/kd ratio or from the plot of Req
versus peptide concentration (Fig. 1.3 D)D.
D
The theoretical basis for SPR kinetic analysis is exposed in section 0.3.
54
SPR screening of synthetic peptides from the GH loop of FMDV
Data analysis produced good quality fits, reproducing the same rate and affinity constants
independently from fitting curves globally, locally or with separate association/dissociation phases to
the langmuirian kinetics model (Table 1.1).
40
1
30
Response / RU
25
152 nM
305 nM
610 nM
1220 nM
2440 nM
152 nM, calc
305 nM, calc
610 nM, calc
1220 nM, calc
2440 nM, calc
20
15
10
0.6
0.4
0.2
0
0
50
100
150
200
250
-0.2
5
-0.4
0
-0.6
-5
-0.8
-10
152 nM
305 nM
610 nM
1220 nM
2440 nM
B
0.8
Residuals / RU
A
35
-1
-10
40
90
140
190
Time / s
0.16
D
r2 = 0.9992
0.12
ks / s-1
0.1
0.08
0.06
0.04
ks (1/s) vs. Conc of analyte
19
Response at
equilibrium (Req) / RU
0.14
20
C
18
17
16
15
0.02
0
0.00E+00
Linear (ks (1/s) vs. Conc of
analyte)
5.00E-07
1.00E-06
1.50E-06
2.00E-06
Peptide concentration / M
2.50E-06
14
0.00E+00
Req (RU) vs. Conc of
analyte
5.00E-07
1.00E-06
1.50E-06
2.00E-06
2.50E-06
Peptide concentration / M
Figure 1. 3 Results obtained in the SPR kinetic analysis of the interaction between immobilised mAb
SD6 and soluble peptide A15: A. Sensorgrams (experimental and modelled); B. Distribution of residual
data points; C. Plot of locally fitted ks (apparent rate constant, see section 0.3) versus peptide
concentration; D. Plot of Req (response at equilibrium) vs. peptide concentration (see section 0.3).
55
SPR as a tool in the functional analysis of an immunodominant site in FMDV
Table 1.1 Quantitative data on the 1:1 langmuirian interaction between immobilised mAb SD6 and
soluble peptide A15.
Curve fitting
[peptide]/ nM
ka/M-1s-1
kd/s-1
KA/M-1
Global
-
6.2×104
2.6×10-3
2.3×107
Local, simultaneous ka/kd
152
305
610
1220
2440
6.0×104
5.8×104
5.9×104
6.1×104
6.2×104
2.4×10-3
2.6×10-3
2.6×10-3
2.7×10-3
2.9×10-3
2.5×107
2.3×107
2.3×107
2.3×107
2.1×107
Local, separate ka/kd
152
305
610
1220
2440
6.7×104
6.2×104
5.5×104
5.8×104
5.9×104
2.4×10-3
2.6×10-3
2.6×10-3
2.8×10-3
2.7×10-3
2.8×107
2.4×107
2.1×107
2.1×107
2.2×107
1.2
Application to the systematic screening of FMDV peptides
1.2.1
Screening of 44 FMDV peptides as antigens towards mAb SD6
Having found suitable experimental conditions for the kinetic analysis of the A15/SD6 interaction, a
similar protocol was applied to the systematic screening of 43 other A15 analogues. The antigenicity
of these peptides had been previously characterised by competition ELISA6, which made them
excellent models to evaluate the reliability of our SPR optimised analysis conditions.
An additional peptide, A15scr, with the same constitutive amino acids as A15 but randomly ordered
(RAGTATTLADLHYST), was used as a negative control. The scrambled sequence A15scr had no
apparent specific binding, but gave rise to a substantial bulk refraction index response (Fig. 1.4 A),
as observed for all other peptides analysed. Therefore, the curves for each site A peptide were
corrected by subtraction of the corresponding A15scr sensorgrams (Fig. 1.4 B and C). The
consistency and accuracy of the fitted kinetic data for the whole set of A15 analogues were in every
aspect similar to those described for A15 under the same conditions (Fig. 1.4 D, E and F). The
stability of the SD6 surface to the repeated strong acid regeneration cycles allowed the screening of
the entire set over the same surface without any detectable loss in mAb activity, thus providing
reliable comparison among the different peptides. This is a clear advantage of the present SPR
configuration, since in the alternative immobilisation of peptides one cannot control the similarity of
the different peptide surfaces. The constants obtained for the interaction between mAb SD6 and the
44 peptides screened are shown in Table 1.2.
56
SPR screening of synthetic peptides from the GH loop of FMDV
2
60
A
50
1.5
1
Residuals/RU
40
Response/RU
D
157 nM
314 nM
627 nM
1254 nM
2509 nM
30
20
10
0.5
0
0
50
100
150
200
250
-0.5
-1
0
-1.5
-10
-10
40
90
140
190
-2
240
Time/s
Time/s
80
0.1
B
70
163 nM
326 nM
652 nM
1305 nM
2610 nM
50
E
0.09
0.08
0.07
0.06
-1
40
ks/s
Response/RU
60
30
0.05
0.04
20
0.03
10
0.02
0
0.01
-10
-10
40
90
140
190
r2 = 0.9991
0
0.00E+00
240
5.00E-07
Time/s
2.00E-06
2.50E-06
0.000002
0.0000025
19.5
C
F
19
15
18.5
10
18
5
Req/RU
Response/RU
1.50E-06
Peptide concentration/M
25
20
1.00E-06
0
-5
163 nM
326 nM
652 nM
1305 nM
2610 nM
-10
-15
-20
17.5
17
16.5
16
15.5
15
-10
40
90
140
Time/s
190
240
0
0.0000005
0.000001
0.0000015
Peptide concentration/M
Figure 1. 4 A. Sensorgrams for the A15scr/SD6 interaction; B. Sensorgrams for the interaction between
SD6 and an FMDV peptide: A15 (140P); C. Sensorgrams of the same interaction as in B, after correction
upon subtraction of sensorgrams shown in A; D. Residual distribution after fitting sensorgrams C to the 1:1
bimolecular interaction kinetics model; E. Linear plot of locally fitted ks versus peptide concentration; F. Plot
of locally fitted Req versus peptide concentration (for comparison between the association equilibrium
constants as obtained from this plot or from the ka/kd ratio).
57
SPR as a tool in the functional analysis of an immunodominant site in FMDV
Table 1.2 Kinetic dataa of the interactions between mAb SD6 and 44 site A peptidesb.
PEPTIDE
ka/M-1s-1
kd/s-1
KA/M-1
ELISA
PEPTIDE
ka/M-1s-1
kd/s-1
KA/M-1
A15
A15(137P)
7.3×104
5.8×104
1.4×10-3
1.9×10-3
5.4×107
3.1×107
A15(148S)
9.4×104
ni
6.7×10-3
ni
1.4×107
ni
A15(138D)
A15(138P)
ni
ni
ni
A15(138E)
ni
ni
ni
A15(139P)
ni
ni
ni
A15(138F)
8.6×104
3.9×10-3
2.2×107
A15(140P)
7.4×104
2.1×10-3
3.5×107
A15(138K)
ni
ni
ni
A15(141P)
ni
ni
ni
A15(138R)
ni
A15(142P)
ni
ni
ni
A15(143P)
ni
ni
ni
A15(138V)
A15(138Y)
A15(144P)
ni
ni
ni
A15(145P)
ni
ni
A15(146P)
ni
A15(147P)
ni
A15(148P)
6.6×10
A15(137S)
1.2×105
A15(138S)
1.8×107
2.3×105
8.8×10-3
2.6×107
A15(145D)
ni
ni
ni
ni
A15(145E)
5.3×10
ni
ni
A15(145F)
ni
ni
ni
ni
A15(145I)
ni
ni
7
A15(145K)
4.5×10
3.5×10-3
3.5×107
A15(145R)
1.1×105
1.2×10-2
8.8×106
A15(140S)
1.5×105
8.6×10-4
A15(141S)
4.5×10
2.6×10
A15(142S)
6.1×104
A15(143S)
ni
A15(144S)
ni
A15(145S)
4.0×10
A15(147S)
1.3×104
4
6.2×10
ni
ni
1.4×104
9.7×10-3
1.4×106
A15(147A)
9.2×104
2.1×10-3
4.4×107
1.8×108
A15(147D)
ni
ni
ni
1.8×10
7
A15(147E)
6.7×10
5.6×10-3
1.1×107
A15(147G)
ni
ni
-3
5.1×10
1.1×10-2
-3
2.2×107
3.2×105
6.1×10-3
3.7×107
A15(147K)
6.6×104
3.4×10-3
2.0×107
4
3.1×10
A15(147N)
4.6×104
5.0×10-3
9.2×106
6
A15(147R)
7.9×10
3.5×10
-3
2.3×107
1.2×106
A15(147V)
9.2×104
6.6×10-3
1.4×107
ni
-3
4
4.1×10
1.3×107
1.1×107
ni
4
4.1×10
-3
-3
-2
1.1×10
4
7.1×10
ni
-3
4
1.3×10
ni
5
7.8×10
4
ELISA
corrected for non-specific binding; b qualitative relative antigenicities from ELISA competition assays are represented, with a black box corresponding to IC50>100, a dark grey box to
IC50 = 30 to 100, a light grey box to IC50 = 5 to 30 and a white box to IC50<5. “ni” - no measurable interaction.
58
a
SPR screening of synthetic peptides from the GH loop of FMDV
Table 1.2 also includes previous data from enzyme-linked immunosorbent assays (ELISA)6. These
data had been expressed as IC50 values (competitor peptide concentration giving a 50 % drop in
maximal absorbance), normalised to the IC50 of peptide A15 (see section 4.3.2). A general
agreement between both SPR and ELISA techniques was observed, thus supporting the reliability of
the functional characterisation of small antigenic FMDV peptides using SPR.
1.2.2
Reproducibility in the SPR constants measured on the SD6 surface
Although systematic repetition of assays for every analyte was not possible, given the large number
of peptides, the reproducibility of our SPR analysis was nevertheless assessed by repeated injection
of a representative sub-set of peptides. Six A15 analogues were independently analysed six times
under similar conditions, with the results shown in Table 1.3. Standard deviations of the measured
kinetic parameters oscillate between 2 and 11% of the mean value, which is quite good considering
the small size of the analytes. A sole exception was seen with kd for the SD6/A15(137I) complex
(SD=20%), which is not surprising given the very low dissociation rate observed for this complex,
making it more prone to be affected by experimental error.
Table 1.3 Reproducibility in kinetic SPR analyses of SD6/peptide interactions (six assays per peptide).
Peptide
ka/M-1s-1
5
A15 (137I)
mean±SD
A15 (138K)
mean±SD
-4
ka/M-1s-1
4
kd/s-1
6.5×10
2.88×10 (*)
3.74×10-3 (*)
9.7×104
4.7×10-4
4.69×104
4.13×10-3
9.1×104
4.3×10-4
5.76×104
3.63×10-3
4
-4
4
A15 (145E)
9.6×10
5.1×10
4.82×10
4.23×10-3
8.7×104 (*)
8.0×10-5 (*)
6.23×104
4.28×10-3
9.3×105 (*)
2.7×10-4 (*)
5.21×104
4.15×10-3
±0.4)×
×104
(9.6±
±1)×
×10-4
(5±
±0.6)×
×104
(5.3±
±0.3)×
×10-3
(4.1±
n.i.
n.i.
2.87×104 (*)
4.67×10-3 (*)
n.i.
n.i.
n.i.
n.i.
n.i.
mean±SD
4
4.15×10
4.55×10-3
4.86×104
4.00×10-3
n.i.
4.40×104
3.90×10-3
n.i.
n.i.
4
4.52×10
4.07×10-3
n.i.
n.i.
2.45×104
3.99×10-3
-
-
±0.3)×
×104
(4.5±
±0.3)×
×10-3
(4.1±
2.09×104 (*)
9.65×10-3 (*)
4
A15 (145K)
mean±SD
1.90×10-3
-3
3.05×10 (*)
5.40×10-3 (*)
1.44×104
9.01×10-3
2.44×10-3
1.38×104
9.46×10-3
1.00×105
1.88×10-3
1.37×104
1.03×10-3
5
-3
4
1.37×10
1.01×10-3
±0.03)×
×104
(1.39±
±0.6)×
×10-3
(9.7±
9.33×10 (*)
1.98×10 (*)
1.19×105
2.43×10-3
7.24×104
1.13×10
mean±SD
Peptide
1.0×10
1.22×105
A15(148I)
kd/s-1
±0.1)×
×105
(1.1±
4
A15(145R)
2.17×10
±0.2)×
×10-3
(2.1±
mean±SD
(*) data was not considered for calculating mean and standard deviation values.
59
SPR as a tool in the functional analysis of an immunodominant site in FMDV
1.3
Use of other site A-directed monoclonal antibodies
A desirable general applicability of our direct kinetic SPR antigenic analysis of small site A peptides
would obviously require not only the ability to distinguish between different analytes (antigens) but
also between different receptors (antibodies). Therefore, it was decided to adapt the procedure to a
new mAb, 4C4E, as the immobilised receptor.
1.3.1
Adaptation of the experimental set-up to a new mAb
In order to obtain good quality data with mAb 4C4, slight changes had to be introduced in the
protocols of SPR analysis previously described for mAb SD6. MAb 4C4 coupled more efficiently to
the dextran matrix under the same conditions employed for the immobilisation of SD6:
immobilisation levels had to be therefore adjusted by dilution of the mAb solution, to achieve a final
surface densities of ca. 1600 RU (1.6 ng/mm2).
Also, it was observed that 4C4 surfaces were not suitably regenerated with hydrochloric acid. A clear
symptom for this problem was that sensorgrams from the same 4C4 surface showed an increase in
baseline response and a concomitant decrease in signal for identical A15 concentrations over
repetitive cycles (not shown). Alternative regeneration procedures, using other acids (phosphoric or
formic) or bases (10 mM glycine, pH 12 or 10 mM sodium hydroxide) were tested and sodium
hydroxide was found to be the most efficient regenerating agent.
Further, while for SD6 the optimal analyte concentration range was generally between ca. 75 and
1250 nM, for 4C4 saturation was already reached at concentrations above 600 nM (not shown).
This observation suggested that mAb 4C4 possessed higher affinity than SD6 towards the site A
peptides, which was later confirmed upon peptide analysis on 4C4 surfaces (see following section).
1.3.2
Screening of 44 FMDV peptides as antigens towards mAb 4C4
A systematic screening similar to that described in section 1.2.1 was performed on a 4C4 surface
(Fig. 1.5). Surface mAb density and injection parameters were quite the same, with the only
difference being the peptide concentrations used (from 35 to 1250 nM). The kinetic data were fitted
as before, generally displaying identical accuracy and consistency levels. Once more, the global
agreement between SPR-derived affinities and previous ELISA data was remarkable and further
validated the experimental SPR set-up (Table 1.4).
E
mAb 4C4 is a site-A directed, anti-FMDV neutralising antibody, raised against strain C1-Brescia; it was kindly supplied by
Dr. Nuria Verdaguer and Ms. Wendy F. Ochoa (IBMB/CSIC – Barcelona, Spain).
60
SPR screening of synthetic peptides from the GH loop of FMDV
65
A
39 nM
78 nM
157 nM
314 nM
627 nM
1254 nM
Response/RU
45
B
115
35 nM
70 nM
140 nM
95
Response/RU
55
35
25
280 nM
560 nM
75
1120 nM
55
15
35
5
15
-5
-5
-10
40
90
140
190
-10
240
40
90
190
240
65
65
35 nM
70 nM
140 nM
280 nM
560 nM
1120 nM
55
45
35
25
D
55
45
Response/RU
C
Response/RU
140
Time/s
Time/s
35
70 nM (sim)
140 nM (sim)
280 nM (sim)
560 nM (sim)
1120 nM (sim)
35 nM
70 nM
140 nM
280 nM
560 nM
1120 nM
25
15
15
5
5
-5
35 nM (sim)
-5
-10
40
90
140
Time/s
190
240
-10
40
90
140
190
240
Time/s
Figure 1. 5 A. Binding curves for non-specific peptide A15scr on a mAb 4C4 surface. The remaining plots are sensorgrams for
binding of peptide A15(142S) to mAb 4C4: B. Raw data; C. After correction for non-specific binding; D. Overlay plot of
experimental (corrected) and simulated (sim) sensorgrams. [Note: higher total (Rtot) responses correspond to higher peptide concentrations,
except for 280 nM peptide injection (second smaller response) which presented a lower bulk RI jump].
61
SPR as a tool in the functional analysis of an immunodominant site in FMDV
Table 1.4 Kinetic dataa of the interactions between mAb 4C4 and 44 site A peptidesb.
PEPTIDE
A15
ka/M-1s-1
kd/s-1
KA/M-1
PEPTIDE
A15(148S)
ka/M-1s-1
A15(137P)
3.8×10
1.2×105
-3
1.9×10
6.1×10-4
1.9×10
2.0×108
A15(138D)
3.0×10
n.i.
A15(138P)
4.1×10
n.i.
5
5.1×10
n.i.
-2
8.0×10
n.i.
6
A15(138E)
n.i.
1.9×10
n.i.
-3
1.0×10
n.i.
8
A15(141P)
1.9×10
n.i.
5
A15(142P)
n.i.
n.i.
A15(143P)
n.i.
A15(144P)
A15(139P)
8
ELISA
5
A15(138F)
5
kd/s-1
7.9×10
n.i.
KA/M-1
-3
n.i.
3.8×10
n.i.
n.i.
5.5×10
5
A15(138K)
3.5×10
5
A15(138R)
1.4×105
1.9×10-2
7.4×106
n.i.
A15(138V)
2.7×105
1.4×10-3
2.0×108
n.i.
n.i.
A15(138Y)
n.i.
n.i.
n.i.
A15(145D)
4.0×105
n.i.
1.3×10-3
n.i.
3.0×108
n.i.
A15(145P)
n.i.
n.i.
n.i.
A15(145E)
1.5×105
2.2×10-3
6.9×107
A15(146P)
n.i.
n.i.
n.i.
A15(145F)
1.4×105
5.1×10-3
2.7×107
A15(147P)
n.i.
A15(140P)
n.i.
5.7×10
-3
9.8×107
2.3×10
-2
1.5×107
A15(145I)
1.7×105
6.4×10-3
2.7×107
7
A15(145K)
2.5×10
5
-3
1.1×108
5.9×10-3
n.i.
1.1×107
n.i.
n.i.
A15(148P)
1.6×10
A15(137S)
1.7×105
3.0×10-3
5.6×107
A15(145R)
A15(138S)
2.5×105
2.2×10-3
1.1×108
A15(147A)
6.6×104
n.i.
A15(140S)
2.5×105
2.4×10-3
1.1×108
A15(147D)
n.i.
n.i.
n.i.
A15(141S)
1.2×10
5
3.5×10
-3
3.2×10
7
A15(147E)
n.i.
n.i.
n.i.
3.8×10
n.i.
-3
1.6×10
n.i.
7
A15(147G)
n.i.
n.i.
n.i.
A15(143S)
6.3×10
n.i.
4
A15(147K)
n.i.
n.i.
n.i.
A15(144S)
n.i.
A15(147N)
n.i.
n.i.
n.i.
A15(147R)
n.i.
A15(142S)
A15(145S)
A15(147S)
3.8×10
n.i.
5
1.4×10
-2
n.i.
5
7.6×10
n.i.
1.2×10
n.i.
-3
5.1×10
n.i.
7
A15(147V)
1.4×10
2.3×10
n.i.
5
5.6×10
ELISA
7
n.i.
-2
2.5×106
corrected for non-specific binding; b qualitative relative antigenicities from ELISA competition assays are represented, with a black box corresponding to IC50>100, a dark grey box to IC50
= 30 to 100, a light grey box to IC50 = 5 to 30 and a white box to IC50<5. “ni” - no measurable interaction.
62
a
SPR screening of synthetic peptides from the GH loop of FMDV
1.3.3
Reproducibility in the constants measured on the 4C4 surface
As already described in section 1.2.1, reproducibility of the SPR-measured constants was evaluated
through repetitive analyses of a small set of site A peptides. Again, six peptides were independently
analysed six times on a mAb 4C4 surface. Results for mAb 4C4 are presented in Table 1.5 and
show very good reproducibility, with standard deviations less than 9% of the mean values.
Table 1.5 Reproducibility in kinetic SPR analysis of 4C4/peptide interactions (six assays per peptide).
Peptide
A15(138D)
mean±SD
ka/M-1s-1
kd/s-1
n.i.
1.06×105 (*)
2.16×10-2 (*)
n.i.
n.i.
5
1.46×10
1.94×10-2
n.i.
n.i.
1.45×105
1.88×10-2
n.i.
n.i.
1.51×105
2.08×10-2
n.i.
n.i.
5
1.42×10
1.94×10-2
n.i.
n.i.
1.39×105
1.88×10-2
_
_
±0.05)×105
(1.45±
±0.07)×10-2
(1.94±
A15(138K)
mean±SD
A15(138R)
mean±SD
5.54×105
5.06×10-3
2.56×105
6.59×10-4
6.00×10
6.23×10
5
2.90×10
1.34×10-3
4.70×105
5.44×10-3
2.49×105
1.15×10-3
5.68×105
5.55×10-3
2.67×105
1.34×10-3
5.70×105
6.04×10-3
2.72×105
1.60×10-3
4.90×10 (*)
1.16×10 (*)
2.83×10
1.62×10-3
±0.5)×105
(5.5±
±0.5)×10-3
(5.7±
3.05×105
2.35×10-2
4.46×105 (*)
1.41×10-3 (*)
3.62×105
2.33×10-2
3.99×105
1.32×10-3
3.74×10
2.33×10
5
4.05×10
1.12×10-3
3.78×105
2.40×10-2
3.82×105
1.41×10-3
3.38×105
2.25×10-2
3.80×105
1.48×10-3
-3
5
mean±SD
kd/s-1
n.i.
5
A15(138F)
ka/M-1s-1
5
A15(138V)
-3
5
mean±SD
A15(138Y)
-2
4.65×105 (*)
2.25×10-2 (*)
±0.3)×10
(3.5±
±0.06)×10
(2.32±
5
±0.2)
(2.7±
×105
3.82×105
-2
mean±SD
±0.2)×10-3
(1.4±
1.34×10-3
±0.3)×10
(4.0±
5
±0.1)×10-3
(1.3±
(*) data was not considered for calculating mean and standard deviation values.
63
SPR as a tool in the functional analysis of an immunodominant site in FMDV
1.4
Probing subtle differences in peptide and mAb behaviour by SPR
Comparison of the results in sections 1.2 and 1.3 leads to the immediate conclusion that not only
the different features of the peptides analysed can be distinguished through SPR, but also distinct
mAb “personalities” can be appreciated.
Peptides screened on the same mAb surface are mainly distinguished by their dissociation rate
constants. A closer look into Tables 1.2 or 1.4 shows that ka varies over a 10-fold range, while kd
varies over a 100-fold range. This observation has already been reported16,17 and it has been
proposed that the biologically relevant SPR-derived parameter is, in fact, kd, since it is a measure of
the life-time of the ligand-receptor complex. Correlations between dissociation rate constants and
neutralisation have also been found18.
On the other hand, comparison of data from the two mAb surfaces seems to suggest that each
antibody has its own “avidity range”, i. e., its own range of association rate constants, which provide
a measure of the accessibility of a particular paratope towards similar antigens. Thus, while average
ka for SD6 is 7.9×104 M-1s-1, for mAb 4C4 it is 2.4×105 M-1s-1, a three-fold increase.
The validity of the SPR approach for the screening of antigenic site A peptides is better illustrated in
Fig. 1.6, which shows the good correlation between antigenicity data measured with SPR and
previous results from competition ELISA. Also, the distinct recognition requirements imposed by
different antibodies is clearly demonstrated upon comparison of Figs. 1.6 A and 1.6 B, particularly
in what concerns recognition of A15 analogues displaying mutations at position 147 (corresponding
to a leucine in the native sequence). This provides further proof of the suitability of SPR to the
functional study of antigenic determinants in viral epitopes using synthetic peptides.
1.5
Validity of the experimental kinetic constants
Evaluation of mass-transport influence on kinetic data is often advisable. Tests should include
analysis over a concentration range from 0.1 to 10 KD, variation of the buffer flow rate and also
variation of the binding capacity using different surface densities13-15. In this work, consistent results
were observed for peptides analysed over a 30-fold concentration range, from as high as 10 KD
down to ca. 40 nM. The 0.1 KD condition was possible only for peptides with KD values at or above
the µM level, since response could not be accurately measured at lower peptide concentrations.
Mass-transport effects were evaluated on peptide A15 at two different buffer flow rates (2 and 60
µl/min) and three different surface capacities (ca. 0.5, 1.6 and 2.5 ng/mm2) as shown in Table 1.6.
Binding was not measurable at the lowest density surfaces, as expected from both previous results
(section 1.1) and small size of the analytes.
64
SPR screening of synthetic peptides from the GH loop of FMDV
120
A
ELISA
100
SPR
80
60
40
20
7V
7N
14
7D
7G
14
14
14
5I
5R
14
5E
14
8Y
14
8F
8R
13
13
8D
7S
4S
2S
0S
7S
7P
5P
3P
1P
9P
13
13
14
14
14
14
13
14
14
14
14
13
13
7P
0
120
B
ELISA
SPR
100
80
60
40
20
7V
7N
14
7G
14
14
7D
14
5I
5R
14
14
5E
8Y
14
8F
8R
13
13
8D
7S
4S
2S
0S
7S
7P
5P
3P
1P
9P
13
13
14
14
14
14
13
14
14
14
14
13
13
7P
0
Figure 1. 6 Comparison of SPR [relative KD =KD (peptide X)/KD (peptide A15)] and ELISA
[relative IC50=IC50 (peptide X)/IC50 (peptide A15)] affinity data for the 43 variants of A15
towards: A. mAb SD6; B. mAb 4C4. Peptides displaying IC50 or KD values too high to be
accurately measured are represented by bars truncated at 100. In the horizontal axis are
represented the A15 analogues screened, with the number corresponding to the A15 position
replaced and the letter corresponding to the capital case code of the amino acid residue
introduced at that position (only half of the peptide labels are shown for simplicity).
65
SPR as a tool in the functional analysis of an immunodominant site in FMDV
Higher densities did not show important differences in kinetic rate constants, with all data sets giving
best fits to the 1:1 langmuirian interaction model. Despite deviations observed when the smaller
buffer flow rate was employed, these hardly affected the magnitude of the kinetic rate constants or
the quality of the fitted data.
Table 1.6 Kinetic data for the mAb SD6/peptide A15 and mAb 4C4/peptide A15 binding interactions under
different buffer flow rate and surface density conditions.
Buffer
SD6/ng.mm-2
4C4/ ng.mm-2
flow rate
µL/min)
(µ
2
60
0.5
*
*
1.6
2.5
ka=5.9×104M-1s-1
ka=9.0×104M-1s-1
kd=1.3×10-3s-1
kd=1.2×10-3s-1
χ2=0.3
0.4
1.7
2.7
ka=2.1×105M-1s-1
ka=2.6×105M-1s-1
kd=1.6×10-3s-1
kd=8.4×10-4s-1
χ2=1.1
χ2=2
χ2=0.4
ka=7.3×104M-1s-1
ka=1.1×105M-1s-1
ka=3.8×105M-1s-1
ka=5.0×105M-1s-1
kd=1.4×10-3s-1
kd=1.8×10-3s-1
kd=1.9×10-3s-1
kd=2.0×10-3s-1
χ2=0.2
χ2=2.2
χ2=1.0
χ2=0.5
*
*
* no reliable measurements at this surface density.
Mass-transport limitations are not usually dramatic for small analytes and can be avoided with
careful experimental set-ups, where high buffer flow rates and low surface densities are key features.
However, even when careful experimental design is applied and apparently consistent data is
obtained, one cannot rule out the possibility of diffusion-controlled kinetics. Hence, the SPR-derived
kinetic rate constants cannot be considered as absolutely “true” values. Further, one cannot fully
compare the events taking place at the biosensor surface, where the biological receptor is
immobilised, with those occurring in solution or in physiologic media. Although agreement with
ELISA experiments provides a valuable check for the reliability of biosensor data, one cannot write
off the possibility that mass-transport affects actual ka and kd values by a similar factor, thus
providing thermodynamic constants apparently consistent with equilibrium experiments.
Nevertheless, the real usefulness of the SPR technology lies in the comparative analysis of the kinetic
behaviour of analogous analytes screened under the same experimental conditions and this was the
purpose of the present work.
66
SPR screening of synthetic peptides from the GH loop of FMDV
1.6
Relevance of the SPR data for FMDV studies
Antigenic site A is a key component of the immune response against FMDV, and some of its
constituent amino acid residues play a decisive role in the mechanisms of FMDV escape under
immune pressure19. The involvement of the RGD tripeptide motif in both antibody and host cell
recognition, as well as the importance of key adjacent residues such as Leu 144 and Leu 147 were
well-established in previous studies, where site A variant peptides proved very useful in probing the
antigenic structure of this site5,6,11,20. Since only equilibrium data had been reported so far, the
dynamic aspects of site A peptide-antibody interactions remained unexplored and real-time
biospecific SPR analysis seemed the right tool to perform such an exploration. Forty-three analogues
of the site A reference peptide A15 (from the C-Sc8c1 FMDV clone) were chosen to show how
structural variation within site A can be correlated with and adequately explained by kinetic SPR
data.
The choice of peptides focused on several structural features of antigenic site A. A proline scan was
first performed from residues 137 to 148 of the GH loop. The well-known structure-disrupting effect
of Pro was reflected in a complete absence of measurable binding when Pro was replacing residues
within the RGD triplet or the following short helical stretch at Asp 142 – Leu 1475,6. Replacement at
the N-terminal region did not affect binding in positions 137 and 140, but produced a slight
decrease and a significant loss in antigenicity for positions 138 and 139, respectively. This agrees
with reported observations that Ser 139 participates in important polar interactions6 in which Pro is
unable to engage. Next, a serine scan was performed, given the striking preservation of antigenicity
in site A variants having Ser at critical positions6. Ser replacements at the 137 – 142 region are in
general well-tolerated, including positions 141 and 142, corresponding to Arg and Gly of the RGD
motif. On the other hand, changes at either Asp 143, Leu 144 and Leu 147 were clearly detrimental
to recognition, a result which can be explained by (i) the role of the Asp residue of the RGD motif in
antibody recognition and (ii) the fact that both Leu 144 and Leu 147 are involved in hydrophobic
interactions in all available three-dimensional structures of peptide A15 – antibody complexes5,6.
A third group of variant peptides included replacements at positions 138, 145 and 147 to illustrate
the subtle effects that structural variation can bring about in antibody recognition. For instance,
replacements with charged basic (Arg, Lys) residues at Ala 138 are better tolerated by mAb 4C4
than by SD6. This is in agreement with the higher percentage of residue contact observed for the
latter mAb in the crystal structure of A15 – antibody complexes6. Non-polar aliphatic or aromatic
amino acids seem to be acceptable by both mAbs at this position. Changes at position 145 (Ala) are
similarly interesting. While SD6 does not recognise peptides with non-polar replacements, 4C4
easily binds the same mutated peptides. Even more striking is the reactivity of both mAbs with the
Glu-replaced peptide, whereas the Asp mutation is not recognised. Finally, position 147 provides
the more critical differentiation between both mAbs assayed: while SD6 is quite tolerant to
mutations (except Asp), 4C4 is extremely sensitive to changes at this position.
67
SPR as a tool in the functional analysis of an immunodominant site in FMDV
References
1 Fägerstam, L. G., Frostell-Karlsson, A., Karlsson, R., Persson, B. and Rönnberg, I. (1992) Biospecific
interaction analysis using surface plasmon resonance detection applied to kinetic, binding site and
concentration analysis, J. Chromatogr. 597, 397.
2 Van Regenmortel, M. H. V., Altschuh, D., Pellequer, J. L., Richalet-Sécordel, P., Saunal, H., Wiley, J.
A., Zeder-Lutz, G. (1994) Analysis of viral antigens using biosensor technology. Methods: A Comp.
Meth. Enzymol. 6, 177.
3 Carreño, C., Roig, X., Camarero, J., Mateu, M. G., Domingo, E., Giralt, E., Andreu, D. (1992) Studies
on antigenic variability of C strains of foot-and-mouth disease virus by means of synthetic peptides
and monoclonal antibodies. Int. J. Peptide Protein Res. 39, 41.
4 Feigelstock, D. A., Mateu, M. G., Valero, M. L., Andreu, D., Domingo, E., Palma, E. L. (1996)
Emerging foot-and-mouth disease virus variants with antigenically critical amino acid substitutions
predicted by model studies using reference viruses. Vaccine 14, 97.
5 Verdaguer, N., Mateu, M. G., Andreu, D., Giralt, E., Domingo, E., Fita, I. (1995) Structure of the
major antigenic loop of foot-and-mouth disease virus complexed with a neutralizing antibody: direct
involvement of the Arg-Gly-Asp motif in the interaction. EMBO J. 14, 1690.
6 Verdaguer, N., Sevilla, N., Valero, M. L., Stuart, D., Brocchi, E., Andreu, D., Giralt, E., Domingo, E.,
Mateu, M. G., Fita, I. (1998) A similar pattern of interaction for different antibodies with a major
antigenic site of foot-and-mouth disease virus: implications for intratypic antigenic variation. J. Virol.
72, 739.
7 Mateu, M.G., Andreu, D., Carreño, C., Roig, X., Cairó, J. J., Camarero, J. A., Giralt, E. and
Domingo, E. (1992) Non-additive effects of multiple amino acid substitutions on antigen-antibody
recognition. Eur. J. Immunol. 22, 1385.
8 Karlsson, R., Ståhlberg R. (1995) Surface plasmon resonance detection and multispot sensing for
direct monitoring of interactions involving low-molecular-weight analytes and for determination of low
affinities. Anal. Biochem. 228, 274.
9 Karlsson, R. (1994) Real-time competitive kinetic analysis of interactions between low-molecularweight ligands in solution and surface-immobilized receptors. Anal. Biochem. 221, 142.
10 Abbas, A. K., Lichtman, A. H. and Pober, J. S. “Cellular and molecular immunology”, 3rd edition; W.
B. Saunders Co., New York (1997).
11 Mateu, M. G., Valero, M. L., Andreu, D. and Domingo, E. (1996) Systematic replacement of amino
acid residues within an Arg-Gly-Asp-containing loop of foot-and-mouth disease virus and effect on cell
recognition. J. Biol. Chem. 271, 12814.
12 O’Shannessy, D. J., Winzor, D. J. (1996) Interpretation of deviations to pseudo-first-order kinetic
behavior in the characterization of ligand binding by biosensor technology. Anal. Biochem. 236, 275.
13 Schuck, P. (1997) Use of surface plasmon resonance to probe the equilibrium and dynamic aspects of
interactions between biological macromolecules. Ann. Rev. Biophys. Biomol. Struct. 26, 541.
14 Schuck, P. (1997) Reliable determination of binding affinity and kinetics using surface plasmon
resonance biosensors. Curr. Op. Biotechnol. 8, 498.
15 Shuck, P. and Minton, A. P. (1996) Kinetic analysis of biosensor data: elementary tests for autoconsistency. Trends Biochem. Sci. 21, 458-460.
16 Altschuh, D., Dubs, M. C., Weiss, E., Zeder-Lutz, G., Van Regenmortel, M. H. V. (1992)
Determination of kinetic constants for the interactions between a monoclonal antibody and peptides
using surface plasmon resonance. Biochemistry 31, 6298.
17 England, P., Brégére, F. and Bedouelle, H. (1997) Energetic and kinetic contributions of contact
residues of antibody D1.3 in the interaction with lysozyme. Biochemistry 36, 164.
18 VanCott, T. C., Bethke, F. R., Polonis, V. R., Gorny, M. K., Zolla-Pazner, S., Redfield, R. R. and Birx,
D. L. (1994) Dissociation rate of antibody-gp120 binding interactions is predictive of V3-mediated
neutralization of HIV-1. J. Immunol. 153, 449.
19 Martínez, M. A., Hernández, J., Piccone, M. E., Palma, E. L., Domingo, E., Knowles, E. and Mateu,
M. G. (1991) Two mechanisms of antigenic diversification of foot-and-mouth disease virus. Virology
184, 695.
20 Hernández, J., Valero, M. L., Andreu, D., Domingo, E. and Mateu, M. G. (1996) Antibody and host
cell recognition of foot-and-mouth disease virus (serotype C) cleaved at the Arg-Gly-Asp (RGD) motif:
a structural interpretation. J. Gen. Virol. 77, 257.
68
2. Antigenic determinants
in the GH loop of FMDV C1-Barcelona
SPR as a tool in the functional analysis of an immunodominant site in FMDV
70
Antigenic determinants in the GH loop of FMDV C1-Barcelona
2.0
Introduction
On previous genomic studies of FMDV field isolates, a natural variant, C1-Barcelona (or C-S30), was
characterised as containing four mutations in the GH loop (Ala138→Thr, Ala140→Thr,
Leu147→Val and Thr149→Ala) relative to the reference strain C-S8c1 (Table 2.1). Analyses of this
four-point mutant by immuno-enzymatic assays showed that it was fully recognised by site
A-directed mAbs such as 4C4. Further, this behaviour was confirmed in antigenicity studies of
peptide – keyhole limpet hemocyanin (KLH) conjugates reproducing the four relevant mutations1-7.
The fact that one of the mutations, Leu147→Val, was found to be detrimental for antibody and cell
recognition of site A peptides, makes the GH loop of FMDV C-S30 an interesting example to learn
more about antigen-antibody recognition mechanisms in FMDV.
The second objective of the present work was, therefore, the synthesis and analysis of peptides
mimicking not only the GH loop of the C-S30 strain but also all possible partial mutants of this
natural isolate.
2.1
Peptides mimicking the GH loop of FMDV C1-Barcelona and the corresponding
partial mutants
A set of fifteen pentadecapeptides was synthesised, corresponding to all possible combinations of
the four mutations found in antigenic site A of C1-Barcelona (C-S30), taking peptide A15 (GH loop
of FMDV C-S8c1) as the reference sequence (Table 2.1).
These peptides were synthesised by machine-assisted parallel solid-phase peptide synthesis, using
standard Fmoc/tBu protocols
8-10
as shown in Fig. 2.1 and described in more detail in section 4.2
(Materials & Methods). Crude peptides were obtained following cleavage from the resin and
submitted to further purification (Fig. 2.2) by medium-pressure liquid chromatography (MPLC).
Purified products were all satisfactorily identified (MALDI-TOF MS, AAA) as the target peptides, with
global yields of ca. 50% (Table 2.2). Peptides were lyophilised and stored at – 20 oC prior to their
utilisation in subsequent studies.
71
SPR as a tool in the functional analysis of an immunodominant site in FMDV
Table 2.1 Pentadecapeptides reproducing all possible combinations of the mutations found in the GH
loop of FMDV C1-Barcelona (C-S30).
Name
A15
A15(138T)
A15(140T)
A15(147V)
A15(149A)
A15(138T,140T)
A15(138T,147V)
A15(138T,149A)
A15(140T,147V)
A15(140T,149A)a
A15(147V,149A)
A15(138T,140T,147V)
A15(138T,140T,149A)
A15(138T,147V,149A)
A15(140T,147V,149A)
A15(138T,140T,147V,149A)b
Sequence
Mutants
YTASARGDLAHLTTT
--T---------------T--------------------V---------------A--T-T-----------T--------V----T----------A----T------V------T--------A-----------V-A--T-T------V----T-T--------A--T--------V-A----T------V-A--T-T------V-A-
GH loop of FMDV C-S8c1
One-point
Two-point
GH loop of FMDV C1-Brescia
Two-point
Three-point
GH loop of FMDV C-S30
a
termed A15Brescia further on.
b
termed A15S30 further on.
MPLC
0
30 min
5% B
95% B
0
30 min
5% B
35% B
Figure 2. 2 Typical HPLC profiles obtained in the synthesis (left) and
purification (right) of the FMDV C-S30 peptides.
72
Antigenic determinants in the GH loop of FMDV C1-Barcelona
PG
PG
TFA
Boc
+ Fmoc-linker-OH
+ coupling agent
piperidine
+ Fmoc-OH
+ coupling mixture
Fmoc
Fmoc
PG
+ Fmoc-OH
+ coupling mixture
PG
Fmoc
resin bead (MBHA, PEG-PS, ...)
PG
amino acid residue
PG
protecting groups (tBu, OtBu, Trt, Pmc, ...)
PG
PG
PG
PG
bi-functional spacer (handle)
bond scission
PG
PG
PG
TFA (+ scavengers)
Figure 2. 1 Schematic representation of the general protocol in Fmoc/tBu solid-phase peptide synthesis (described under Materials & Methods, section 4.2)8-10.
73
SPR as a tool in the functional analysis of an immunodominant site in FMDV
Table 2.2 Yield, purity (HPLC) and characterisation (MALDI-TOF MS and AAA) of the C-S30 series.
Peptide
Purity
(% HPLC)
90
81
MW
found
1607.2
1607.1
MW
expected
1607
1607
Amino acid analysis
(AAA)
A15(138T)
A15(140T)
Global yield
(%)
52
49
A15(147V)
43
87
1562.9
1563
Thr, 4.06 (4); Ser, 0.97 (1); Gly, 0.98 (1); Ala 3.06 (3); His, 0.86 (1); Arg, 0.89 (1)
A15(149A)
51
80
1547.1
1547
Asp, 0.92 (1); Ser, 0.80 (1); Gly, 1.03 (1); Ala 4.10 (4); His, 1.06 (1); Arg, 0.99 (1)
Asp, 1.12 (1); Ser, 0.94 (1); Gly, 0.98 (1); Ala 2.03 (2); His, 1.08 (1); Arg, 0.85 (1)
Asp, 0.97 (1); Ser, 0.85 (1); Gly, 1.05 (1); Ala 2.06 (2); His, 1.09 (1); Arg, 0.88 (1)
A15(138T,140T)
46
85
1636.9
1637
Asp, 1.10 (1); Ser, 0.88 (1); Gly, 1.07 (1); Ala 1.06 (1); Leu, 2.01 (2); His, 0.87 (1)
A15(138T,147V)*
10
76
1592.8
1593
Asp, 0.96 (1); Ser, 1.13 (1); Val, 0.95 (1); Leu, 1.15 (1); His, 0.95 (1); Arg 1.01 (1)
A15(138T,149A)
50
96
1576.9
1577
Asp, 0.94 (1); Ser, 0.91 (1); Gly, 1.06 (1); Leu, 2.04 (2); His, 0.97 (1); Arg, 0.91 (1)
A15(140T,147V)
41
94
1592.7
1593
Asp, 0.90 (1); Ser, 0.87 (1); Gly, 1.07 (1); Ala 2.09 (2); Leu, 1.10 (1); Arg, 0.84 (1)
A15Brescia
36
91
1576.9
1577
Asp, 0.95 (1); Ser, 0.87 (1); Gly, 1.06 (1); Ala 3.06 (3); Leu, 1.99 (2); His, 0.94 (1)
A15(147V,149A)*
21
79
1533.1
1533
Asp, 0.71 (1); Gly, 1.07 (1); Ala 4.04 (4); Val, 0.68 (1); Leu, 1.03 (1); Arg, 0.86 (1)
A15(138T,140T,147V)
48
93
1623.6
1623
Asp, 1.03 (1); Ser, 0.88 (1); Gly, 1.09 (1); Ala 1.08 (1); Leu, 1.08 (1); Arg, 0.84 (1)
A15(138T,140T,149A)
41
91
1607.6
1607
Asp, 0.92 (1); Ser, 0.81 (1); Gly, 1.06 (1); Ala 2.14 (2); Leu, 1.96 (2); His, 0.92 (1)
A15(138T,147V,149A)
52
98
1562.9
1563
Asp, 0.98 (1); Ser, 0.90 (1); Gly, 1.01 (1); Ala 3.06 (3); Leu, 1.04 (1); Arg, 1.01 (1)
98
1562.9
1563
Asp, 1.02 (1); Ser, 0.86 (1); Gly, 1.03 (1); Ala 3.04 (1); Leu, 1.05 (1); Arg, 0.82 (1)
99
1592.7
1593
Asp, 1.10 (1); Ser, 0.88 (1); Gly, 1.07 (1); Ala 1.06 (1); Leu, 2.01 (2); His, 0.87 (1)
A15(140T,147V,149A)
A15S30
59
74
* These syntheses were carried out under sub-optimal conditions due to instrumental malfunction.
Relative amino acid ratios found by AAA are followed by the expected value in parenthesis.
Antigenic determinants in the GH loop of FMDV C1-Barcelona
2.2
SPR study of the C-S30 peptides
Having found suitable experimental conditions for the SPR kinetic study of interactions between
FMDV peptides in solution and immobilised anti-FMDV antibodies, as described in chapter 1, SPR
was again chosen for the characterisation of the C-S30 peptides. This would allow the detailed study
of the effects caused by the stepwise introduction of the four mutations found in the GH loop of
FMDV C-S30 and provide a possible explanation for the peculiar behaviour of this virus isolate.
The C-S30 pentadecapeptides were, therefore, screened by SPR against three anti-site A
monoclonal antibodies, SD6, 4C4 and 3E5A.
The three mAbs were immobilised on CM5 sensor chips following standard protocols, with final
immobilisation densities of about 1600 RU. Sensorgrams were obtained and analysed as previously
described (chapter 1) and all measurable interactions fitted to the 1:1 langmuirian interaction kinetic
model (often considering baseline drift). Whenever interactions could not be reliably measured,
sensorgrams had a square-wave like shape, either due to bulk refractive index response or to
extremely fast on/off rates. Although the monitored on/off rates were generally high, and
consequently liable to be under mass-transport effects, the quantitative data obtained appeared to
be self-consistent and were thus considered reliable. Discussion of the results is presented in the
following sections.
2.2.1
One-point mutants
As observed in previous studies by competition ELISA, SPR analysis showed that substitutions
Ala140→Thr and Thr149→Ala were well tolerated by the three mAbs. This was to be expected,
since both replacements are present in field isolate C1-Brescia, previously shown to be recognised by
these mAbs4.
Mutations Ala138→Thr and Leu147→Val affected antibody recognition to different extents: mAb
SD6 was more sensitive to Ala138→Thr than to Leu147→Val, quite the opposite to mAb 4C4,
which tolerated the first replacement much better than the second one. Similar effects were observed
for mAb 3E5. These results are consistent with recent crystallographic studies, where it was found
that Ala138 has a higher percentage of residue contact with mAb SD6 than with mAb 4C414.
Peptide affinities to each mAb are mainly reflected in the different dissociation rate constants
observed for the corresponding peptide-mAb complexes (Table 2.3), as further illustrated in Fig. 2.3.
A
this anti-site A mAb was raised against FMDV strain C1-Brescia. Ascitic fluid of mAb 3E5 was kindly supplied by Dr.
Emiliana Brocchi (IZSLE – Brescia, Italy).
75
SPR as a tool in the functional analysis of an immunodominant site in FMDV
Table 2.3 Kinetic SPR analysis of the interactions between FMDV C-S30 peptides and mAbs SD6, 4C4 and 3E5.
mAb
SD6
4C4
3E5
Peptide
ka/M-1s-1
kd/s-1
KA/M-1
ka/M-1s-1
kd/s-1
KA/M-1
ka/M-1s-1
kd/s-1
KA/M-1
A15
7.3×104
1.4×10-3
5.4×
×107
3.8×105
1.9×10-3
1.9×
×108
1.6×105
1.6×10-3
9.4×
×107
A15(138T)
1.0×105
1.5×10-2
6.5×
×106
2.5×105
5.9×10-3
4.2×
×107
1.1×105
8.5×10-3
1.3×
×107
A15(140T)
1.4×105
3.0×10-3
4.7×
×107
6.0×105
2.6×10-3
2.3×
×108
2.6×105
1.5×10-3
1.8×
×108
A15(147V)
1.1×105
1.0×10-2
1.0×
×107
9.5×104
4.4×10-2
2.2×
×106
3.3×105
5.0×10-2
6.6×
×106
A15(149A)
1.2×105
2.2×10-3
5.5×
×107
6.4×105
3.1×10-3
2.1×
×108
4.8×105
1.5×10-3
3.2×
×108
A15(138T,140T)
1.6×105
1.5×10-2
1.1×
×107
2.6×105
8.2×10-3
3.1×
×107
2.4×105
8.4×10-3
2.9×
×107
A15(138T,147V)
3.8×104
4.2×10-2
9.1×
×105
2.4×105
4.2×10-2
5.7×
×106
2.6×105
4.2×10-2
6.3×
×106
A15(138T,149A)
1.2×105
1.7×10-2
7.0×
×106
2.5×105
3.2×10-3
7.6×
×107
3.2×105
8.3×10-3
3.9×
×107
A15(140T,147V)
7.8×104
1.3×10-2
6.1×
×106
2.4×105
6.5×10-2
3.7×
×106
3.8×105
4.8×10-2
8.0×
×106
A15Brescia
9.0×104
8.0×10-3
1.2×
×107
2.6×105
1.6×10-3
1.6×
×108
4.4×105
4.2×10-3
1.0×
×108
A15(147V,149A)
1.0×105
1.7×10-2
6.0×
×106
2.1×105
4.4×10-2
4.7×
×106
4.8×105
3.7×10-2
1.3×
×107
3.4×105
1.6×10-1
2.1×
×106
5
-3
A15(138T,140T,147V)
A15(138T,140T,149A)
ni
2.2×10
5
A15(138T,147V,149A)
2.2×10
-2
9.7×
×10
6
ni
2.1×10
9.6×10
2.2×
×10
7
ni
3.4×10
5
1.4×10-2
2.5×
×107
3.1×105
5.2×10-2*
6.0×
×106
3.6×105
4.1×10-2
8.9×
×106
A15(140T,147V,149A)
1.7×105
1.8×10-2
9.1×
×106
4.4×105
7.2×10-2*
6.1×
×106
5.9×105
5.2×10-2*
1.1×
×107
A15S30
3.8×104
8.8×10-2*
4.3×
×105
2.2×105
1.2×10-1*
2.0×
×106
3.2×105
7.2×10-2*
4.5×
×106
76
* Although resulting from apparently reliable data fits, kd values equal or higher than 5×10-2 should be regarded with some caution, since this value is considered the limit of
reliable SPR measurement of rate constants; “ni” denotes interactions which could not be reliably measured.
Antigenic determinants in the GH loop of FMDV C1-Barcelona
90
115
A
A15138T 39 nM
A15138T 78 nM
A15138T 156 nM
A15138T 312 nM
A15138T 625 nM
A15138T 1250 nM
70
Response/RU
60
50
A15140T 39 nM
A15140T 78 nM
A15140T 156 nM
A15140T 312 nM
A15140T 625 nM
A15140T 1250 nM
B
95
Response/RU
80
40
30
20
10
75
55
35
15
0
-10
-5
-10
40
90
140
190
240
290
-10
40
90
Time/s
35
190
240
290
135
A15147V 39 nM
A15147V 78 nM
A15147V 156 nM
A15147V 312 nM
A15147V 625 nM
A15147V 1250 nM
25
15
5
-5
-15
A15149A 39 nM
A15149A 78 nM
A15149A 156 nM
A15149A 312 nM
A15149A 625 nM
A15149A 1250 nM
D
115
95
Response/RU
C
Response/RU
140
Time/s
75
55
35
-25
15
-35
-45
-5
-10
40
90
140
190
Time/s
240
290
340
-10
40
90
140
190
240
290
Time/s
Figure 2. 3 Sensorgrams obtained in the SPR kinetic analysis of the interactions between immobilised mAb 4C4 and: A. peptide A15(138T); B. peptide
A15(140T); C. peptide A15(147V) and D. peptide A15(149A). This figure illustrates the differences observed in the dissociation rate constants for each
mAb-peptide interaction.
77
SPR as a tool in the functional analysis of an immunodominant site in FMDV
2.2.2
Two- and three-point mutants
Analysis of data in Table 2.3 immediately suggests that two- and three-point combinations of the
amino acid replacements are additive. Indeed, antigenicities of the two- and three-point mutant
peptides towards the three mAbs employed reflect the combined effects of the single-point mutations
present in each particular sequence. This effect is further confirmed when comparing the
experimental relative affinities [KArel=KA(peptide)/KA(A15)] with the calculated relative affinities
assuming additive effects in the combination of single-point mutations [expected KArel =KArel(singlepoint mutant 1) × KArel(single-point mutant 2) × ... × KA(peptide A15)], as illustrated in Fig. 2.4.
Peptide A15Brescia (with replacements A140→T and T149→A) was the most antigenic within the
group of multiple-point mutants (Table 2.4). Interestingly, for this peptide the correlation between
experimental and calculated relative affinities was poorer than for all the other mutants, suggesting a
compensatory effect (i.e., lack of additivity) between both replacements. On the other hand,
peptides containing the L147→V substitution were the poorest antigens, particularly when the
A138→T replacement was also present. Further, affinities were again almost exclusively determined
by the dissociation rates of peptide – mAb complexes (compare data within each mAb set in Table
2.3).
2.2.3
Four-point mutant: peptide A15S30
The most striking result obtained in this SPR screening of FMDV peptides was the low affinity
observed for peptide A15S30 (reproducing the GH loop of FMDV C-S30) towards all three mAbs
assayed. Although such low antigenicity was totally in agreement with the additive effects observed
when combining the different individual substitutions in the partial mutants (Fig. 2.4), previous
studies with FMDV C-S30 had shown that this natural isolate was neutralised by mAb 4C415 and,
further, that the KLH conjugate of a 21-mer peptide reproducing the C-S30 loop had been fully
recognised by the same mAb in immuno-enzymatic assays6,7.
Despite the fact that SPR data was apparently self-consistent and reliable, we decided to perform a
qualitative comparison between the SPR affinities of peptides A15, A15Brescia and A15S30 using a
reverse SPR configuration: peptide immobilisation and mAb as soluble analyte, as described under
Materials & Methods (section 4.3.1). Even though this configuration was not optimised (to avoid
artefacts such as diffusion controlled kinetics or ligand heterogeneity11), the performance of all assays
under identical conditions and the high molecular weight of the mAb analytes would provide both
high SPR responses and a reliable qualitative comparison between the three peptide antigens. As
shown in Table 2.4 and Fig. 2.5, this assay further confirmed the above SPR data: although rate
constants for peptide – mAb interaction depended on the analysis format (consistently lower in the
format with mAb as analyte), thermodynamic affinities were virtually the same and the antigenicity
ranking was identical to the one derived from the first SPR analysis of these peptides. Since these
78
Antigenic determinants in the GH loop of FMDV C1-Barcelona
SPR results did not agree with the neutralisation and immuno-enzymatic data discussed above, a
competition ELISA screening of all C-S30 peptides synthesised towards the three mAbs was
performed, as described in section 2.3.
0.90
0.80
A
experimental
calculated
Relative affinity
0.70
0.60
0.50
0.40
0.30
0.20
Peptide
A15S30
A15(140T,147V,149A)
A15(138T,140T,149A)
A15(147V,149A)
A15(140T,149A)
A15(140T,147V)
A15(138T,149A)
A15(138T,147V)
0.00
A15(138T,140T)
0.10
1.35
B
experimental
1.15
calculated
Relative affinity
0.95
0.75
0.55
0.35
5.95
C
A15S30
experimental
calculated
4.95
Relative affinity
A15(140T,147V,149A)
A15(138T,147V,149A)
Peptide
A15(138T,140T,149A)
A15(138T,140T,147V)
A15(147V,149A)
A15(140T,149A)
A15(140T,147V)
A15(138T,149A)
A15(138T,147V)
-0.05
A15(138T,140T)
0.15
3.95
2.95
1.95
A15S30
A15(140T,147V,149A)
A15(138T,147V,149A)
A15(138T,140T,149A)
Peptide
A15(147V,149A)
A15(140T,149A)
A15(140T,147V)
A15(138T,149A)
A15(138T,147V)
-0.05
A15(138T,140T)
0.95
Figure 2. 4 Experimental and calculated affinities of A15 mutant
peptides towards mAbs SD6 (A), 4C4 (B) and 3E5 (C). Calculated values
have been determined assuming additive effects of the amino acid
replacements (see text).
79
SPR as a tool in the functional analysis of an immunodominant site in FMDV
45
35
25
20
15
A15S30, 39 nM
A15S30, 78 nM
A15S30, 156 nM
A15S30, 312 nM
A15S30, 625 nM
B
75
Response/RU
30
Response/RU
95
A15, 38 nM
A15, 76 nM
A15, 152 nM
A15, 305 nM
A15, 610 nM
A
40
55
35
10
fast dissociation
5
15
Peptide
bound
0
slow dissociation
Peptide
bound
-5
-5
-10
40
90
140
190
240
290
340
-10
40
90
140
Time/s
190
240
290
Time/s
135
115
Response/RU
95
75
55
135
95
31 nM
62 nM
125 nM
250 nM
500 nM
75
55
35
35
15
15
-5
4C4,
4C4,
4C4,
4C4,
4C4,
D
115
Response/RU
4C4, 16 nM
4C4, 31 nM
4C4, 62 nM
4C4, 125 nM
4C4, 250 nM
4C4, 500 nM
C
-5
-10
40
90
140
190
Time/s
240
290
340
-10
40
90
140
190
240
290
340
Time/s
80
Figure 2. 5 Sensorgrams from the SPR direct analysis of the interactions between peptides A15 and A15S30 with mAb 4C4: A. immobilised mAb vs.
A15; B. immobilised mAb vs. A15S30; C. immobilised A15 vs. 4C4; D. immobilised A15S30 vs. 4C4.
Antigenic determinants in the GH loop of FMDV C1-Barcelona
Table 2.4 Kinetic analyses using peptides immobilised on the chip.
mAb
Peptide
A15
SD6
4C4
3E5
ka/M-1s-1
4
kd/s-1
KA/M-1
A15Brescia*
1.2×10
1.8×104
2.5×10
-
-4
5.0×107
-
A15S30
2.9×103
1.8×10-2
1.6×105
A15
2.3×104
A15Brescia*
1.8×104
2.2×10-4
-
1.1×108
-
A15S30
2.5×104
1.1×10-2
2.3×106
A15
1.2×105
A15Brescia*
1.7×104
7.3×10-4
-
1.7×108
-
A15S30
3.5×104
1.1×10-2
3.2×106
* kd too small to be reliably measured; ka determined from the linear
dependence of ks (global rate constant=ka×C+kd) on analyte concentration.
2.2.4
A possible significance for kinetic rate constants in antigen-antibody interactions
As already mentioned, a consistent observation in the present study was that peptide – antibody
affinities seem mainly determined by the dissociation rate constant, kd (Table 2.3). Hence, for a
given family of peptide analogues, a higher or lower antigenicity towards a given mAb would
exclusively depend on the half-life (t1/2) of the complex. Thus, all analogues would be similarly
capable of approaching the mAb paratope but, once there, their different ability to establish
complex-stabilising interactions would dictate the life-time of the complex and, consequently,
peptide – antibody affinity (KD). If this interpretation was true, the association rate constant (ka)
would be sequence-independent and only determined by the global fitness of the antibody paratope
(KD ∝ kd ⇒ ka = constant) to a series of analogue antigens. Indeed, a closer look at the kinetic data
of the SPR screening of 15-mer peptides (Table 2. 3) suggests that this might be the case. Plotting
affinities (KD) against dissociation rate constants (kd) gives for each mAb a set of points for which a
correlation line with a satisfactory r2 coefficient (>0.9) can be derived (Fig. 2.6). The slopes of these
three lines correlate well with the reciprocal of the average association rates of the interactions
among each mAb and the entire set of peptide antigens. Thus, slope of SD6 correlation line is
1×10-5 vs. 1/average ka = 9.0×10-6. Similarly, for 4C4, slope = 4×10-6 vs. 1/average ka = 3.1×10-6;
and for 3E5, slope = 3×10-6 vs. 1/average ka = 3.4×10-6.
Of course, these correlations would only apply to relatively similar sets of analogue antigens; more
drastic changes in antigen size, folding or amino acid composition would certainly be expected to
lead to substantial changes in both dissociation and association rate constants.
81
SPR as a tool in the functional analysis of an immunodominant site in FMDV
7.00E-07
6.00E-07
mAb SD6
mAb 4C4
mAb 3E5
slope = 1 x 10
r2 = 0.94
-5
slope = 4 x 10-6
2
r = 0.99
KD/M
5.00E-07
4.00E-07
3.00E-07
2.00E-07
slope = 3 x 10
r2 = 0.92
-6
1.00E-07
0.00E+00
0.00E+00
2.00E-02
4.00E-02
6.00E-02
8.00E-02
1.00E-01
1.20E-01
-1
kd/s
Figure 2. 6 Correlation between thermodynamic affinity (KD) and kinetic dissociation constant (kd) for the
SPR-measured interactions between C-S30 peptides and three anti-FMDV mAbs SD6, 4C4 and 3E5.
82
Antigenic determinants in the GH loop of FMDV C1-Barcelona
2.3
Competition ELISA analysis of the C-S30 pentadecapeptides
As already mentioned, in view of the surprisingly low affinity shown by the A15S30-4C4 complex
on SPR, a competition ELISA (Fig. 2.7) screening of all the C-S30 pentadecapeptides was
performed16,17. For this purpose, 96-well plates were coated with an FMDV C-S8c1 reference
antigen, the KLH conjugate of peptide A21 (corresponding to a 6-residue extension at the Cterminus of peptide A15):
YTASARGDLAHLTTTHARHLP
A21
Equilibrium mixtures of the competitor at different concentrations with constant concentrations of
the mAb were incubated and the amount of mAb which bound to antigen was measured. Plotting
the variation of plate-bound mAb with competitor peptide concentration gave inhibition curves such
as those in Fig. 2.8, thus providing an evaluation of competitor peptide antigenicity. This
antigenicity was expressed in terms of relative IC50 (concentration of competitor producing 50%
inhibition), where relative means normalised with respect to the IC50 of peptide A15. As can be seen
from Figs. 2.8 and 2.9, data derived from competition ELISA were in good agreement with previous
SPR results, in the sense that the poorest antigens in SPR were also the worse competitors in ELISA.
These results demonstrate that the low A15S30-4C4 affinity previously obtained did not come from
eventual artefacts in SPR analysis.
Plate-bound antigen (competed)
Competitor antigen in solution
Specific monoclonal antibody
Anti-Fc antibody (conjugated to a carrier
that provides a means for detection)
Figure 2. 7 Scheme of the central steps
in competition ELISA analysis: preequilibrated peptide competitor – mAb
mixtures are incubated with plate-bound
antigen; the amount of mAb that
preferably binds the immobilised antigen
is detected after incubation of a tagged
(e.g. peroxidase) anti-Fc antibody.
83
SPR as a tool in the functional analysis of an immunodominant site in FMDV
120
A15S30/SD6
A15/SD6
A15S30/4C4
A15/4C4
A15S30/3E5
A15/3E5
100
% absorbance
80
60
40
20
0
0.1
1
10
100
1000
peptide concentration (pmol/100 µl)
Figure 2. 8 Competition between plate-bound A21 – KLH and pentadecapeptides A15
and A15S30 for anti-site A mAbs.
% Percentage of the maximal absorbance measured for mAb incubated with plate-bound antigen in
the absence of peptide competitor; all absorbances were corrected by subtraction of mean absorbance
obtained for negative controls.
60
mAb SD6
mAb 4C4
mAb 3E5
relative IC50
50
40
30
20
10
Figure 2. 9 Screening of the C-S30 pentadecapeptides by competition ELISA.
IC50 values were normalised to IC50 of peptide A15; IC50 higher than the maximum
competitor concentration are truncated at 60.
84
A15S30
A15(140T,147V,149A)
A15(138T,147V,149A)
A15(138T,140T,149A)
A15(147V,149A)
A15(138T,140T,147V)
Peptide
A15Brescia
A15(140T,147V)
A15(138T,149A)
A15(138T,147V)
A15(138T,140T)
A15(149A)
A15(147V)
A15(140T)
A15(138T)
0
Antigenic determinants in the GH loop of FMDV C1-Barcelona
2.4
Size effects in the antigenicity of C-S30 peptides
The observation of a low antigenicity for peptide A15S30 in solution, and the confirmation that it
was not artefactual (e.g. peptide instability or aggregation) prompted us to investigate if the
discrepancies between our measurements and previous immunoenzymatic results1-7 could be due to
the small size of our peptides. Therefore, two additional 21-mer versions of antigenic site A in FMDV
strains C-S8c1 (peptide A21) and C-S30 (peptide A21S30) were synthesised and analysed by SPR
and competition ELISA.
2.4.1
YTASARGDLAHLTTTHARHLP
A21
YTTSTRGDLAHVTATHARHLP
A21S30
Synthesis of 21-mer peptides reproducing antigenic site A from FMDV strains C-S8c1 and
C-S30
Synthetic procedures and results of the synthesis and purification of these 21-mer peptides were
identical to those described in section 2.1, employing either manual or machine-assisted Fmoc/tBu
solid-phase peptide synthesis (see Materials & Methods, section 4.2). The results are summarised in
Table 2.5.
Table 2.5 Synthesis data of A21 and A21S30.
Peptide
A21
Global
yield (%)
Purity
(% HPLC)
MW
found
MW
expected
AAA*
37
94
2302.6
2303
Asp, 0.97 (1); Gly, 1.06 (1); Ala, 3.89 (4);
Leu, 3.19 (3); His, 2.87 (3); Pro, 1.12 (1)
Asp, 0.93 (1); Gly, 0.91 (1); Ala, 3.12 (3);
Leu, 2.17 (2); His, 3.02 (3); Pro, 1.08 (1)
* Relative amino acid ratios given by AAA are followed by the theoretical values in parenthesis.
A21S30
31
98
2287.8
2288
85
SPR as a tool in the functional analysis of an immunodominant site in FMDV
2.4.2
Antigenic analyses of A21 and A21S30 by SPR and competition ELISA
Peptides A21 and A21S30 were studied by SPR using peptide as either analyte or immobilised
ligand. As already explained in section 2.2.3, SPR analyses using immobilised peptide were not fully
optimised for artefacts such as ligand heterogeneity or mass-transport limitations. This is reflected in
the lower kinetic constants measured when using mAb as analyte. Nevertheless, one of our major
goals in the present work has been the application of SPR analysis to screen small antigenic peptides
as analytes and all experiments in the reverse format should be regarded as merely comparative.
SPR data of the interactions between peptide A21S30 and the anti-GH loop mAbs are displayed in
Table 2.6. Parallel analyses of peptide A21 were also performed but could not be accurately
quantitated due to either insufficient surface regeneration (using mAb immobilised on the chip) or
extremely small off-rates (using peptide immobilised on the chip). Only the interaction between
immobilised SD6 and A21 could be measured: ka=1.3×105 M-1s-1, kd=6.1×10-3 s-1, KA=2.1×107 M-1.
Although this was a serious drawback for an accurate evaluation of the antigenicity of A21, it
provided further evidence of the high affinity of the C-S8c1 peptides towards anti-GH loop mAbs. In
turn, peptide A21S30 displayed high dissociation rate constants (kd) as previously observed with the
shorter 15-mer peptide A15S30 (Fig. 2.10). A slight increase in affinity could be observed upon
addition of further 6 amino amino acid residues to the sequence of the C-S30 GH loop, but such
increase was also qualitatively observed for the C-S8c1 sequence, thus maintaining the antigenicity
ranking already observed and discussed in previous sections.
Table 2.6 Interactions of anti-site A mAbs with A21S30.
mAb
SD6
4C4
3E5
86
Peptide
A21S30
as analyte
immobilised
on the chip
ka/M-1s-1
8.6×104
5.4×103
kd/s-1
2.9×10-2
4.3×10-3
KA/M-1
3.0×
×106
1.2×106
ka/M-1s-1
2.4×105
2.5×104
kd/s-1
4.3×10-2
3.7×10-3
KA/M-1
5.6×
×106
6.8×
×106
ka/M-1s-1
2.8×105
5.0×104
kd/s-1
5.0×10-2
4.8×10-3
KA/M-1
5.6×
×106
1.0×
×107
Antigenic determinants in the GH loop of FMDV C1-Barcelona
12780
95
A
A21S30, 39 nM
A21S30, 78 nM
A21S30, 155 nM
A21S30, 310 nM
A21S30, 621 nM
A21S30, 1242 nM
75
12760
65
12750
12740
12730
A21, 40 nM
A21, 80 nM
A21, 159 nM
A21, 318 nM
A21, 636 nM
A21, 1278 nM
12720
12710
12700
Response/RU
Response/RU
B
85
12770
55
45
35
25
15
5
-5
12690
-10
40
90
140
190
240
-10
290
40
90
3495
190
240
290
90
4C4, 15 nM
4C4, 31 nM
4C4, 62 nM
4C4, 125 nM
4C4, 250 nM
4C4, 500 nM
2995
2495
1995
1495
80
D
4C4, 15 nM
4C4, 31 nM
4C4, 62 nM
4C4, 125 nM
4C4, 250 nM
4C4, 500 nM
70
60
Response/RU
C
Response/RU
140
Time/s
Time/s
50
40
30
20
995
10
495
0
-5
-10
-10
40
90
140
190
Time/s
240
290
340
-10
40
90
140
190
240
290
Time/s
Figure 2. 10 Interactions between mAb 4C4 and peptides A21 and A21S30: A. peptide A21 vs. immobilised mAb (note the increasing baseline response
due to incomplete surface regeneration); B. peptide A21S30 vs. immobilised mAb (note the high dissociation rate); C. mAb 4C4 vs. immobilised A21
(note the extremely low dissociation rate); D. mAb 4C4 vs. immobilised A21S30 (high dissociation rate).
87
SPR as a tool in the functional analysis of an immunodominant site in FMDV
Competition ELISA analysis of the two 21-mer peptides was also performed as described in section
2.3. Once again, peptide A21S30 was seen to be less antigenic than peptide A21 towards the three
mAbs assayed (Fig. 2.11).
120
% absorbance
100
80
60
A21S30_SD6
A21S30_4C4
A21S30_3E5
A21_SD6
A21_4C4
A21_3E5
40
20
0
0.1
1
10
100
1000
Peptide concentration (pmol/100 µl)
Figure 2. 11 Screening of peptides A21 and A21S30 by competition ELISA.
In summary, results from the antigenic characterisation of these 21-mer peptides showed that
peptide size did not account for the low antigenicity observed for peptide A15S30 in solution. So the
question of why the C-S30 peptide sequences were less antigenic than expected from previous
immunological studies1-7 remained open.
The total agreement between SPR and ELISA experiments proved that the discrepancies between
our data and previous immunoenzymatic results could not come from technical or experimental
artefacts.
At this point, we could only envisage a last-resource explanation, namely that in this particular case
peptide conformation was responsible for the different behaviour of peptide A15S30 (or A21S30)
when analysed by either ELISA/SPR or by immunodot. This remote possibility would run contrary
to the general observation that the continuous antigenic site A is perfectly mimicked by linear
peptides under all circumstances.
88
Antigenic determinants in the GH loop of FMDV C1-Barcelona
2.5
Input from parallel X-ray diffraction studies
At this point of our work we had access to X-ray diffraction studies performed by Wendy F. Ochoa
and co-workers (IBMB, CSIC - Barcelona) showing that peptide A15S30 could form a complex with
the Fab fragment of mAb 4C418. The peptide adopted a nearly cyclic conformation in the complex,
almost identical to the one observed for a similar complex with peptide A15. In the A15S30-4C4
complex, the more critical positions, residues 138 and 147, showed very few direct contacts with the
antibody. Therefore, these residues influence antigenicity by altering the conformation of the peptide
as a whole, rather than by local interactions. In the A15S30-4C4 complex, an additional water
molecule (which could not occupy the same position in the A15-4C4 complex due to steric
hindrance) was seen to bridge the side chain hydroxyl group of
atoms of
144
Leu and
138
Thr and the main chain oxygen
147
Val (Fig. 2.12). Thus, this water molecule seemed to be a key feature in
holding the compact fold of the peptide, which has been further confirmed by molecular dynamics
simulations performed by the same researchers18.
Figure 2. 12 Conformation of peptide A15S30 complexed with the
Fab fragment of mAb 4C4; interactions between peptide (dark blue)
and antibody (light blue) or water molecules (red spheres) are marked
with dashed lines; the RGD motif is located in an open turn
conformation, closely related to the one previously observed for the
viral loop as part of the virion; the water molecule that bridges
residues 138 (Thr side chain hydroxyl), 144 and 147 (main chain
oxygen atoms) is the one further on the right side of the image.
(Figure was provided by Ms. Wendy F. Ochoa).
89
SPR as a tool in the functional analysis of an immunodominant site in FMDV
2.6
Effect of conformation in the antigenicity of C-S30 peptides
When earlier FMDV C-S30 studies, recent X-ray diffraction data and our own results on antigenicity
of C-S30 peptides are all brought together, it seems plausible that the apparent discrepancies
between the former and the latter data are due to factors such as distinct conformations of the
antigenic site under the different assay systems employed. Therefore, we decided to analyse the
effect of introducing conformational constraints in the C-S30 peptides. For that purpose, two
additional cyclic peptides (cyc16S30 and cyc16147Val) were synthesised.
2.6.1
Synthesis of peptides cyc16S30 and cyc16147Val
The design and synthesis of these cyclic peptides was based on previous research in our group,
involving the same type of constructs based on the GH loop sequence belonging to the C-S8c1 viral
strain. Cyclic versions of the viral antigenic site were formed by intra-molecular disulphide formation
between N- and C-terminal cysteine residues added to the sequences. An extra 6-aminohexanoic
acid (Ahx) residue was also included to give some flexibility to the constructs, so that the known
disordered structure of the viral loop could be better mimicked19. Production of these cyclic peptides
relied on the synthesis and purification of the corresponding linear bis-thiol precursors (A15S30 and
A16147Val) using methods similar to those previously described in section 2.1, followed by air
oxidation of the thiol groups at pH 8 and high peptide dilution (Fig. 2.13)20. Cyclization was
monitored by HPLC and the qualitative Ellman21 assay (see Materials & Methods), reaching
completion within one hour (Fig. 2.14). Cyclic peptides were then lyophilised without further
purification. General data on the synthesis and cyclization of these peptides are presented in Table
2.7.
CysThrXaaSerXaaArgGlyAspLeuAlaHisValThrYaaAhxCys
SH
SH
[O], pH 8
CysThrXaaSerXaaArgGlyAspLeuAlaHisValThrYaaAhxCys
Figure 2. 13 Oxidation of bis-thiol precursors to cyclic peptides
cyc16S30 (Xaa=Thr, Yaa=Ala) and cyc16147Val (Xaa=Ala,
Yaa=Thr); air oxidation was performed at pH 8 and high dilution
(50 µM) to favour intra-molecular cyclization.
90
Antigenic determinants in the GH loop of FMDV C1-Barcelona
B
A
C
D
A16S30
cyc16S30
0
30 min
5%B
0
95%B
30 min
5%B
0
30 min
5%B
95%B
95%B
0
30 min
5%B
95%B
Figure 2. 14 HPLC profiles showing both linear and cyclic forms of the different steps in the synthesis of
cyc16S30; A. crude A16S30 (bis-thiol form), B. MPLC-purified A16S30, C. air oxidation at 30 min, D.
oxidation at 1 hour.
Table 2.7 Synthesis of the C-S30 cyclic peptides and their bis-thiol precursors.
MW
found
MW
expected
97
1647.2
1647
95
1645.4
1645
Peptide
Global
yield
(%)
Purity
(%HPLC)
A16S30
37
cyc16S30
82
AAA*
Asp, 1.02 (1); Ser, 1.02 (1); Gly, 0.99 (1);
Ala, 1.99 (2); Leu, 0.98 (1)
Asp, 0.99 (1); Ser, 1.07 (1); Gly, 0.97 (1);
Ala, 1.99 (2); Leu, 0.99 (1)
Asp, 1.05 (1); Ser, 1.01 (1); Gly, 0.99 (1);
Ala, 2.76 (3); Leu, 0.97 (1)
Asp, 1.07 (1); Ser, 1.04 (1); Gly, 0.93 (1);
cyc16147Val
85
93
1615.9
1616
Ala, 2.78 (3); Leu, 0.96 (1)
*Relative amino acid proportions given by AAA are followed by the expected value in parenthesis.
A16147Val
34
94
1617.9
1618
91
SPR as a tool in the functional analysis of an immunodominant site in FMDV
2.6.2
Antigenic evaluation of cyclic C-S30 peptides using SPR
The affinities of the cyclic peptides towards the three anti-site A mAbs were measured by SPR
analysis, both with peptide as analyte and as immobilised ligand (Table 2.8 and Fig. 2.15).
Table 2.9 Interactions between peptides cyc16S30 and cyc16147Val and anti-site A mAbs.
Peptide
4C4
3E5
immobilised on the chip
cyc16S30
cyc16 Val
cyc16S30
cyc16147Val
ka/M-1s-1
7.2x104
1.8x105
2.0x104
1.5x104
kd/s-1
1.8x10-2
4.5x10-3
4.1x10-3
1.1x10-3
KA/M-1
4.0x106
4.1x107
4.8x106
1.3x107
ka/M-1s-1
5.0x105
1.7x105
9.3x103
1.5x104
kd/s-1
3.5x10-3
5.0x10-3
8.9x10-5
2.6x10-4
KA/M-1
1.4x108
3.3x107
1.1x108
5.0x107
ka/M-1s-1
4.5x105
1.6x105
1.0x104
1.0x104
kd/s-1
5.4x10-3
2.9x10-3
2.0x10-4
4.1x10-4
KA/M-1
8.4x107
5.5x107
5.0x107
2.5x107
mAb
SD6
as analyte
147
Before discussing the SPR data of the cyclic peptides, it must be re-emphasised that analyses using
mAb as analyte were not subject to optimisation and, therefore, data from such analyses should be
regarded as purely comparative. In fact, kinetic constants in Table 2.9 show that mass-transport
limitations are probably occurring when mAb is used as analyte, since rate constants measured
under this analysis configuration are lower than when the small peptides are the analytes11-13.
However, since both rate constants seem to be affected by mass-transport artefacts to a similar
extent, the affinities displayed are within the same order of magnitude as those measured in the
reverse configuration and, furthermore, the antigenicity ranking of the peptides is maintained in
both analysis formats.
92
Antigenic determinants in the GH loop of FMDV C1-Barcelona
30
25
A
15
10
Response/RU
10
Response/RU
B
20
20
0
-10
cyc16S30, 39 nM
cyc16S30, 78 nM
cyc16S30, 156 nM
cyc16S30, 312 nM
cyc16S30, 625 nM
cyc16S30, 1250 nM
-20
-30
5
0
-5
-10
cyc16Val, 39 nM
cyc16Val, 156 nM
cyc16Val, 312 nM
cyc16Val, 625 nM
cyc16Val, 1250 nM
-15
-20
-25
-30
-40
-10
40
90
140
190
240
-10
290
40
90
Time/s
695
190
240
290
115
595
495
395
295
4C4, 15 nM
4C4, 31 nM
4C4, 62 nM
4C4, 125 nM
4C4, 250 nM
4C4, 500 nM
D
95
Response/RU
4C4, 15 nM
4C4, 31 nM
4C4, 62 nM
4C4, 125 nM
4C4, 250 nM
4C4, 500 nM
C
Response/RU
140
Time/s
75
55
35
195
15
95
-5
-5
-10
40
90
140
190
Time/s
240
290
340
-10
40
90
140
190
240
290
Time/s
Figure 2. 15 Sensorgrams of the SPR kinetic analysis of the interactions between mAb 4C4 and: peptide cyc16S30 as analyte (A) and as immobilised
ligand (C); peptide cyc16147Val as analyte (B) and as immobilised ligand (D).
93
SPR as a tool in the functional analysis of an immunodominant site in FMDV
Data in Table 2.9 show that cyclic versions of peptides A15S30 (i.e., peptide cyc16S30) and
A15(147Val) (i.e., peptide cyc16147Val) are clearly more antigenic than their linear counterparts
against all three mAbs. Further, the increase in affinity is reflected in both association and,
especially, dissociation rate constants, indicating that cyclic peptides bind more readily to the mAbs
and that the resulting complexes are stabilised to a greater extent. A similar result had been
observed by M. L. Valero and co-workers in the analysis of the interactions between mAb SD6 and
both peptide A15 and its corresponding cyclic disulphide analogue (AhxA16SS)19. In this previous
study, an increase of about one order of magnitude in mAb affinity was observed upon peptide
cyclization (KA from 1.9×107 to 1.2×108 M-1), but almost exclusively due to an increase in association
rate constant (ka from 3.7×103 to 2.6×104 M-1s-1). This indicated that, despite the easier entry of the
cyclic peptide into the mAb paratope, the final complex had the same half-life as the one formed
with the linear analogue (kd was 2.2×10-4 and 2.0×10-4 s-1 for the cyclic and linear peptides,
respectively).
The most striking observation made with our cyclic peptides corresponding to FMDV C-S30 and to
a hypothetical Leu147→Val mutant (a field isolate with such single-point replacement has not yet
been isolated) was the fact that, while the C-S30 sequence was less antigenic than the
147
Val mutant
towards mAb SD6, this ranking was inverted when the other two mAbs were considered (the results
with mAb 4C4 being the most significant). This was precisely what had been observed in earlier
immuno-enzymatic studies of field isolate C-S30 as well as of KLH conjugates of site A peptides
displaying either the corresponding four replacements or the single Leu147→Val substitution.
Further, this was the first evidence, using small peptides, of the reversion observed in 4C4 – antigen
affinity when both Ala138→Thr and Leu147→Val replacements were brought together.
Additionally, these results confirmed what had been postulated by W. F. Ochoa and co-workers,
concerning the fact that residues
138
Thr and
147
Val in the A15S30-4C4 complex have only minor
contacts with the antibody paratope and, thus, differences in binding affinities observed for peptides
with replacements at these positions would be due to reduced stability of such peptides in the “mAbrecognisable” conformation18.
A question still remained, however: peptide A15S30 can be crystallised in complex with antibody
4C4, so both molecules can undergo a considerable induced fit to form a stable complex. Therefore,
would prolonged (e.g., overnight) incubation of peptide with antibody, both species in solution,
result in higher affinities than those measured in kinetic SPR or competition ELISA assays?
94
Antigenic determinants in the GH loop of FMDV C1-Barcelona
2.7
Antigenic evaluation of C-S30 peptides through solution affinity SPR analysis
2.7.1
Basic concepts22,23
Measurement of affinity in solution with SPR biosensors is based on the determination of the free
concentration of one of the interactants in equilibrium mixtures. Measurements are made on known
concentrations of the free interactant for a standard curve to be built and also on the equilibrium
mixtures for determination of affinity. If binding in solution is written as:
A+B
AB
the experiment is designed so that a constant concentration of B is incubated with a series of known
concentrations of A and, then, SPR is used to measure the remaining free concentration of B in
solution (Fig. 2.16). Such measurement is performed on a sensor chip surface where a specific
ligand for B (A’) had been previously immobilised and on which a calibration curve using known
concentrations of B had been built. The affinity constant can then be calculated with the
BIAEvaluation software24, where the variation of free B with the concentration of A is fitted to the
equation:
[B] − [A] − K D
2
+
([A] + [B] + K D )2 − [A]× [B]
4
(2.1)
In the particular case of antigen –
antibody interactions (taking, for
Chip-bound antigen
instance, B for antibody and A for
Antigen in solution
antigen), one should ensure that
Fab fragment of specific mAb
reactions take place at a 1:1
stoichiometry. Thus, antibody Fab
fragments instead of the whole
immunoglobulin
must
be
employed. Fab fragments of the
relevant antibodies were produced
by
digestion
described
with
under
papain,
as
Materials
&
Figure 2. 16 Main steps in
solution affinity SPR analysis:
pre-incubation
of
both
interactants
in
solution
(above) and injection of the
equilibrium mixtures over a
sensor chip surface with preimmobilised specific antigen
(below).
MethodsB (section 4.3.1.3).
B
Purified Fab of SD6 was kindly supplied by Dr. Esteban Domingo and Ms. Mercedes Dávila (CBMSO – UAM, Madrid); Fab
4C4 was a kind gift of Dr. Nuria Verdaguer and Ms. Wendy F. Ochoa (IBMB – CSIC, Barcelona); Fab from mAb 3E5 was
prepared by digestion of mAb isolated from ascitic fluid kindly supplied by Dr. Emiliana Brocchi (IZSLE, Brescia – Italy).
95
SPR as a tool in the functional analysis of an immunodominant site in FMDV
The main steps in antibody digestion and Fab purification were monitored by SDS-PAGE (Fig.
2.17).
Figure 2. 17 SDS-PAGE monitoring (at 12%
acrylamide) of the digestion and purification of the
Fab fragment of mAb 3E5; lanes A and B: purified
mAb 4C4 and its corresponding Fab fragment,
used instead of MW protein standards; lane C:
papain digestion of mAb 3E5 at 3 h of reaction;
starting mAb, plus Fab2 and Fab products can be
distinguished; lane D: Fab fragment of mAb 3E5
after a two-step (affinity and gel filtration
chromatography) purification.
A
B
C
D
Solution affinity SPR analysis relies on concentration measurements. In the SPR biosensor,
concentration measurements are based either on binding level (avoiding bulk refractive index
contributions) or on binding rate determinations. Under conditions of limiting mass transfer of
analyte to the surface, the initial binding rate is independent of ligand density and interaction
kinetics, being exclusively determined by analyte concentration and diffusion characteristics. In the
present study, we were able to see that SPR analyses with immobilised peptide and antibody as
analyte were influenced by mass-transport artefacts even at flow rates as high as 60 µl/min (sections
2.2.3, 2.4.2 and 2.6.2). Thus, free Fab concentrations in the present study had to be derived from
initial binding rate measurements at 5 µl/min on sensor chips with peptide A15 (0.3 ng/mm2) preimmobilised by standard procedures (see Materials & Methods, section 4.3.1.3). Binding rates were
taken from curve slopes at a given injection time, chosen as the earliest possible where the influence
of bulk refractive index or other artefacts at injection plugs would be negligible.
It could be argued that there was no evidence that the Fab – A15 interactions were 100% diffusioncontrolled. Nevertheless, all measurements were performed on the same A15 surface and the
analyte (Fab) was always the same for each data set, so antigenicity ranking of the peptide
analogues screened under these conditions is totally meaningful.
The dependence of the measured free Fab concentration on antigen concentration gives an
inhibition curve that can be fitted to Eq. 2.1 (BIAEvaluation general fit→solution affinity model) so
that affinity is calculated (as either KA or its reciprocal KD). However, the influence of immobilised
antigen (A15) on free Fab concentration measurements is not taken into account when fitting data
as described; in fact, the immobilised peptide is competing with the soluble analogue for the same
Fab molecules as in competition ELISA. The Cheng and Prusoff’s formula (Eq. 2.2)25 can be used to
obviate this problem:
Ki = 1 +
96
K ' A [B ]
IC50
(2.2)
Antigenic determinants in the GH loop of FMDV C1-Barcelona
where Ki is the “real” affinity for the interaction of A and B in solution, K’A is the affinity for the
interaction of B with immobilised A’ and IC50 is the concentration of A causing a 50% drop in the
total concentration of B.
2.7.2
Results
Injection of known Fab (SD6, 4C4 and 3E5) standards on the A15 surface allowed the building of
calibration curves (Fig. 2.18), which were subsequently employed in the determination of Fab
molecules that remained free after overnight incubation with peptide antigens in solution.
3.5
3
data points (SD6)
data points (4C4)
data points (3E5)
fitted curve (SD6)
fitted curve (4C4)
fitted curve (3E5)
Slope/RU.s
-1
2.5
2
1.5
1
0.5
0
0.00E+00
5.00E-08
1.00E-07
1.50E-07
2.00E-07
2.50E-07
3.00E-07
3.50E-07
Fab concentration/M
Figure 2. 18 Plots of initial binding rate/RU.s-1 vs. Fab concentration (M) for the three antibodies
employed in the present study. Measurements were made at a 5 µl/min flow rate on a 0.3 ng/mm2 A15
surface; data points were fitted to a four-parameter equation using BIAEvaluation software in order to
build the corresponding calibration curves.
The quantification of remaining free Fab in solution for each incubated mixture (where Fab total
concentration is constant and peptide antigen concentrations varied) allowed to build inhibition
curves (Fig. 2.19) from which the peptide – antibody solution affinities were calculated, either
through direct fitting to the BIAEvaluation solution affinity model (Eq. 2.1) or using the Cheng &
Prusoff’s formula (Eq. 2.2). Results are summarised in Table 2.9.
97
SPR as a tool in the functional analysis of an immunodominant site in FMDV
1.00E-07
A15
A15Brescia
A15(147V)
A15S30
A21S30
cyc16val
cyc16S30
A15Scr
9.00E-08
Fab concentration/M
8.00E-08
7.00E-08
6.00E-08
5.00E-08
4.00E-08
3.00E-08
2.00E-08
1.00E-08
0.00E+00
0.00E+00
1.00E-07
2.00E-07
3.00E-07
4.00E-07
5.00E-07
6.00E-07
7.00E-07
Peptide concentration/M
Figure 2. 19 Inhibition curves obtained in the SPR analysis of the interactions between Fab 4C4 and
peptides A15, A15Brescia, A15(147Val), A15S30, A21S30, cyc16S30, cyc16147Val and A15Scr in solution;
a constant total Fab concentration was used and competitor peptide concentrations were varied from 0 to
625 nM.
Table 2.9 Affinity data of interactions between peptides and mAbs in solution.
Fab
Peptide
A15
A15(147V)
A15Brescia
SD6
A15S30
A21S30
cyc16S30
cyc16147Val
A15
A15(147V)
A15Brescia
4C4
A15S30
A21S30
cyc16S30
cyc16147Val
A15
A15(147V)
A15Brescia
3E5
A15S30
A21S30
cyc16S30
cyc16147Val
a
KA (solution
affinity fit)a/M-1
Ki (Cheng &
Prusoff’s)b/M-1
KA (kinetic
analysis)c/M-1
4.3×107
3.6×106
4.3×107
6.0×105
1.6×106
2.8×106
5.5×107
8.2×107
3.8×106
5.5×107
2.3×106
5.2×106
1.4×108
2.7×107
1.6×108
4.5×106
6.5×107
2.1×106
2.9×106
4.4×107
4.5×107
6.3×
×107
4.5×
×106
6.5×
×107
ND
ND
7.5×
×106
5.0×
×107
2.0×
×108
2.8×
×107
1.5×
×108
1.1×
×107
3.8×
×107
1.8×
×108
8.2×
×107
2.0×
×108
2.8×
×107
1.1×
×108
8.2×
×106
1.1×
×107
1.1×
×108
8.5×
×107
5.4×107
1.0×107
1.2×107
4.3×105
3.0×106
4.0×106
4.1×107
1.9×108
2.2×106
1.6×108
2.0×106
5.6×106
1.4×108
3.3×107
9.4×107
6.6×106
1.0×108
4.5×106
5.6×106
8.4×107
5.5×107
Direct curve fitting with the BIAEvaluation software.
Application of the Cheng & Prusoff’s formula.
c
Data from previous SPR kinetic assays.
ND, not determined.
b
98
Antigenic determinants in the GH loop of FMDV C1-Barcelona
2.7.3
Discussion
SPR measurement of affinities of C-S8c1, C1-Brescia and C-S30 peptides for anti-site A mAbs SD6,
4C4 and 3E5 provided a conclusive confirmation of the data discussed in the present chapter.
Indeed, as can be seen in Fig. 2.20 and Table 2.9, antigenicity rankings observed for these
interactions were in good agreement with those previously obtained by competition ELISA and
kinetic SPR analysis. Peptide A15S30 was less antigenic than its counterparts from strains C-S8c1
(peptide A15) or C1-Brescia (A15Brescia). At the same time, important differences were observed
and are discussed in the following paragraphs.
i) Solution affinities obtained by curve fitting or by the Cheng & Prusoff’s formula
Comparing the affinities in Table 2.9 as obtained by solution affinity fit or by the Cheng & Prusoff’s
formula, the influence of immobilised antigen A15 becomes quite evident. Thus, Eq. 2.1 describes
phenomena such as those occurring in competition ELISA. In this case, peptides with lower
antigenicity will be the most affected by the immobilised antigen competitor and fitted affinities will
be lower than affinities determined in solution. In contrast, the Cheng & Prusoff’s formula (Eq. 2.2)
allows to obtain data independent from the immobilised antigen and major differences between
both methods of affinity calculation can be observed for the least antigenic peptides A15(147V),
A15S30 and A21S30 towards mAbs 4C4 and 3E5. Differences of about one order of magnitude can
be found in these cases, relative to affinities calculated by direct fit of the inhibition curves.
ii) Solution affinity versus kinetic SPR data
Comparing affinity data calculated from the inhibition curves using Eq. 2.2 with previous data
obtained by kinetic SPR analysis, an excellent agreement is observed with three important
exceptions: peptides A15(147V), A15S30 and A21S30 (towards mAbs 4C4 and 3E5). Even though
relative ranking of all antigens is maintained, an increase in affinity of about one order of magnitude
is measured in solution equilibrium experiments involving these peptides. The fact that such
observation is more pronounced for these three particular peptide mutants appears to be quite
significant. Indeed, it seems that when antigen and antibody are allowed to interact overnight both
free in solution, they can rearrange so that more stabilised complexes are formed. Thus, the lower
affinities measured in kinetic SPR analysis would possibly be due to interaction times (1.5 min) too
small for such conformational changes to be detected. This ability of the C-S30 GH loop to
rearrange into a mAb-recognisable structure, leading to stable antibody-antigen complexes, could be
the basis for the recognition and neutralisation of FMDV C-S30 by mAb 4C4.
99
SPR as a tool in the functional analysis of an immunodominant site in FMDV
iii) A role for peptide conformation
The above observations are further supported by the modulation of peptide antigenicity upon
cyclization. Both four-point and one-point (147Leu→Val) replaced sequences produced important
increases in mAb affinities when presented as cyclic peptides. Particularly in the case of mAb 4C4, it
was observed that the cyclic C-S30 sequence was about one order of magnitude more antigenic
than the cyclic one-point mutant
147
Leu→Val, which confirms previous studies suggesting a positive
reversion in the antigenicity of the four-point mutant1-7. These observations have important
implications vis à vis the simplistic view of continuous antigenic sites as conformation-independent.
If this was in fact the case, peptide cyclization would have only minor effects on antigenicity. Further,
the loss of antigenicity due to amino acid replacements in positions that are not in direct contact with
the antibody paratope18 (e.g.,
138
Ala→Thr and
147
Leu→Val in linear peptides) cannot be easily
explained by factors other than peptide conformation.
100
Antigenic determinants in the GH loop of FMDV C1-Barcelona
2.8
Two-dimensional proton nuclear magnetic resonance studies of C-S30 peptides26
Most linear and many cyclic peptides possess a high conformational flexibility, meaning that they
can adopt a fairly large set of interconvertible conformations in solution. In a few favourable cases,
the population of one conformer is large enough to be distinctly detected by spectroscopy. Among
the techniques commonly employed for the detection and identification of peptide conformation
such as circular dichroism, Raman spectroscopy, FT-IR and NMR, the latter has a clear advantage in
that it allows not only the global detection of the preferred structure, but also the characterisation of
the individual amino acid residues defining such structure. Thus, it provides a picture of peptide
folding in aqueous solution, which is desirable when searching for structure – activity relationships.
Due to their natural abundance, gyromagnetic constant and localisation in peptides, protons are the
best probes for peptide conformational NMR studies. Although a simple one-dimensional NMR
spectrum of the peptide should always be obtained in order to check for peptide purity,
concentration or aggregation, the level of complexity is usually too high for a complete assignment.
So, two-dimensional NMR experiments are needed for a full and unequivocal proton assignment in
peptide studies.
Peptides A15S30 and cyc16S30 were both studied by 2D – 1H NMR in solution, in an attempt to
define secondary structure elements that could explain the antigenic features of both peptides. NMR
experiments (TOCSY27, NOESY28 and ROESY29) were carried out both in aqueous solution and in
the presence of the structuring agent trifluoroethanol, as described under Materials & Methods
(section 4.4.1).
2.8.1
Basic concepts
2D – 1H NMR spectra of peptides are interpreted according to the sequential assignment method
developed by Wüthrich for proteins27. The first step in the procedure relies on the total correlation
scalar experiment (TOCSY), in which peaks are detected for protons that can correlate with each
other by means of a magnetisation transfer sequence involving, at each step, H – H couplings. This
allows the identification of each amino acid residue independently from all the others in the
sequence, since magnetisation cannot be transferred through the amide bond from one residue to
the following one. Thus, each amide proton (HN) will be correlated with all other protons from the
same spin system; the number and chemical shift of such protons provide an identification of the
amino acid residue in question. If the peptide includes an amino acid residue that is unique in the
whole sequence, the assignment is immediate and unequivocal. However, if a certain amino acid
residue is repeatedly present along the sequence, it will be necessary to carry out another kind of
NMR experiment where protons from different amino acid residues are correlated. Such correlation
is based on nuclear Overhauser effect (NOE) experiments (NOESY28, ROESY29). The NOE arises
from the dipolar relaxation that occurs between two nuclei that are spatially close to each other,
101
SPR as a tool in the functional analysis of an immunodominant site in FMDV
regardless of their belonging or not to the same spin system. An NOE between a pair of protons is,
therefore, observed when there is a population of peptide structures where both nuclei are within
4.5 Å from each other. Thus, when all residues are attributed a set of signals in the TOCSY
spectrum, the second step will be the use of NOE experiments to establish connectivities among
them. This will be possible if sequential distances such as dαNi,i+1, dNni,i+1, dβNi,i+1, etc., can be
observed, i. e., correlations between proton Hα of residue i with HN of residue i+1, or HN of residue i
with HN of residue i+1, or Hα of residue i with HN of residue i+1, respectively. Since the α proton of
a given residue is usually close in space to the HN of the following residue, sequential dαNi,i+1 NOEs
are useful to assign the amino acid sequence.
Given the slow time-scale of NMR spectroscopy relative to optical spectroscopy, one must keep in
mind that NMR spectral parameters are all averaged. Thus, all conformational information provided
by NMR corresponds to the average of all structures adopted by the peptide in solution. Of the
several parameters that can be used in NMR peptide structural studies, only conformational chemical
shifts and NOEs have been considered in the present work.
Chemical shifts are the most easily measured NMR parameters and are quite susceptible to subtle
changes in the chemical environment of the proton. The large number of protein structures assigned
by NMR made possible a statistical study correlating differences in chemical shifts with peptide
secondary structure. The most useful chemical shift differences have been found to be those
between the Hα of folded and random coil structures, the latter ones derived from model
oligopeptides. Thus, Hα conformational chemical shift differences (defined as ∆δHα=δexp – δ random coil)
are found to be negative for helices (average, - 0.39 ppm) and β turns, and positive for β sheets
(average, + 0.37 ppm). NOEs usually provide the most unambiguous information about peptide
structure. The sole observation of a NOE between two protons implies that there are conformational
populations in which these two protons are spatially close (d ≤ 4.5 Å), independently of their being
or not close in the primary sequence. Energy studies on the conformational space of proteins allow
to establish NOE patterns that correlate with peptide secondary structure. A general guide for
structural interpretation of NOEs is presented in Table 2.10.
Table 2.10 Useful NOEs for the identification of peptide secondary structure elements.
NOE
dαNi,i+1
dNNi,i+1
dNNi,i+2
dαNi,i+2
dαNi,i+3
dαNi,i+4
dαβi,i+3
Helix
Structure
β sheet
β turn
α
✔✔
310
✔✔
✔✔✔
✔✔✔
✔
✔
✔
✖
✔✔✔ (2)
✔✔
✔✔✔ (1)
✔
✔✔✔
I
✔✔
II
✔✔✔
✔
✔✔
✖
✔✔
✔✔
✔✔
✔✔
✖
✔
✔
✔
✖
✖
✖
✖
✔
✔
✖
✖
✖
✔ - expected NOE (the number of ticks corresponds to the expected intensities); ✖ - not observed.
102
Antigenic determinants in the GH loop of FMDV C1-Barcelona
2.8.2
Results
Sequence assignment based on TOCSY and NOESY/ROESY spectra is shown in Fig. 2.20 for both
A15S30 and cyc16S30 peptides in water. Chemical shifts observed for both peptides, in water as
well as in 30% TFE, are presented in Table 2.11.
150T
145A
147V
144L
140T
143D
148T
139S
138T
141R
146H
142G
149A
137T
150Ahx
147V
145A
146H
144L
149A
143D
138T
141R
139S
142G
148T
137T
140T
151C
Figure 2. 20 Expansion of the TOCSY experiments (70 ms) of peptides A15S30 (above) and
cyc16S30 (below) performed at 25 oC in water; the different spin systems are indicated and were
assigned upon analysis of TOCSY, NOESY and ROESY spectra.
103
SPR as a tool in the functional analysis of an immunodominant site in FMDV
Table 2.11 Chemical shifts (ppm) measured in the 2D – 1H NMR study of peptides A15S30 and cyc16S30 in water and in 30% TFE at 25 oC.
A15S30
Residue
H
136
Tyr/Cys
cyc16S30
H 2O
N
H
α
nd
4.32
8.49
4.47
30% TFE
β
N
α
H
Other
H
3.15
3.19
4.15
nd
nd
4.32
1.20
8.40
4.52
H
H 2O
β
N
α
H
Other
H
3.13
3.20
4.20
nd
nd
4.48
1.24
8.34
4.42
H
30% TFE
β
Hα
Hβ
Other
nd
4.48
ne
8.03
4.22
3.40
3.26
4.18
1.28
H
Other
H
3.34
3.23
4.25
ne
1.20
N
137
Thr
138
Thr
8.36
4.32
4.25
1.24
8.25
4.41
4.32
1.28
8.29
4.38
4.28
1.20
8.25
4.45
4.31
1.25
Ser
8.43
4.55
ne
8.33
4.60
8.43
4.57
3.88
ne
8.33
4.60
4.37
1.20
8.18
4.40
1.25
8.36
4.40
4.24
1.20
8..18
4.43
3.96
3.90
4.33
ne
8.27
3.91
3.98
4.31
ne
Thr
3.86
3.92
4.27
1.24
Arg
8.35
4.32
4.30
8.44
3.94
3.22
1.64
ne
4.36
3.95
1.89
1.76
ne
8.28
8.32
3.20
1.70
ne
4.34
3.93
1.90
1.80
ne
8.36
8.40
3.30
1.66
ne
8.28
Gly
1.89
1.77
ne
8.34
3.97
1.93
1.80
ne
3.22
1.70
ne
Asp
8.32
4.66
ne
8.21
4.65
2.86
ne
8.25
4.61
2.73
ne
8.15
4.67
2.83
ne
8.19
4.29
2.85
2.80
1.61
0.92
0.85
ne
8.08
4.26
1.71
0.95
0.90
ne
8.16
4.24
1.66
0.92
0.84
ne
8.14
4.24
1.69
0.95
0.90
ne
139
140
141
142
143
144
Leu
145
Ala
8.12
4.23
1.32
8.02
4.22
1.38
8.10
4.18
1.28
8.03
4.20
1.34
146
His
8.36
4.71
nd
8.09
4.67
nd
8.16
4.70
8.05
4.69
8.05
4.12
1.00
8.09
4.17
3.30
3.14
2.10
nd
0.92
3.37
3.23
2.20
0.94
8.04
4.18
3.43
3.20
2.19
1.00
1.21
8.07
4.37
4.29
1.25
8.26
4.32
4.21
1.20
8.07
4.36
4.29
1.24
7.91
4.32
1.41
1.30
1.50
2.33
7.66
3.22
1.65
8.25
4.76
3.32
3.04
147
Val
8.19
4.18
3.15
3.26
2.07
148
Thr
8.33
4.33
4.19
Ala
8.44
4.41
1.41
8.12
4.40
1.47
8.18
4.25
1.36
Thr/Ahx
8.11
4.29
4.24
1.20
7.87
4.33
nd
1.23
7.84
3.15
1.63
-/Cys
-
-
-
-
-
-
-
-
8.44
4.68
3.30
3.00
149
150
151
nd
1.35
1.54
2.35
104
ne, non existing; nd, not determined.
Antigenic determinants in the GH loop of FMDV C1-Barcelona
Conformational chemical shifts
The first structural information derived from the NMR experiments was based on the conformational
chemical shifts plotted in Fig. 2.21. As can be seen, neither peptide shows any marked tendency to
adopt a predominant canonical (helix or β-sheet) conformation in solution. The small absolute
values of the conformational chemical shifts suggest that both peptides exist mainly in aperiodic
(random coil) form in solution. This lack of structuration could not be modified by environmental
changes, as shown by the similarity between conformational chemical shifts observed in water and
in 30% TFE.
0.6
0.4
∆δHα
α/ppm
∆δ
0.2
0
-0.2
-0.4
cyc16S30, 30% TFE
cyc16S30, water
A15S30, 30% TFE
A15S30, water
-0.6
-0.8
-1
X1
T
T
S
T
R
G
D
L
A
H
V
T
A
X2
X3
residue
Figure 2. 21 Conformational chemical shifts (∆δHα) observed at 25 oC for peptides A15S30 (Xaa=Tyr,
Yaa=Thr, Zaa= - ) and cyc16S30 (Xaa=Cys, Yaa=Ahx, Zaa=Cys) both in water and in 30% TFE.
Nevertheless, a slight tendency to structuration in the central region of the peptides can be
distinguished. As previously observed by T. Haack and co-workers for the linear (A15) and cyclic
disulphide (AhxA16SS) versions of FMDV C-S8c119,30, the conformational chemical shifts in the
region that includes the RGD tripeptide are compatible with a tendency for an open turn
conformation. Further, the cluster of negative conformational chemical shifts from
143
Asp to
146
His
could be suggestive of an incipient short helix in this region, as previously observed for the C-S8c1
peptides by the same authors. A significant difference between the peptides of both strains is,
however, the fact that this short helix extends, in the C-S8c1 peptides, to the
147
Leu residue. In the
case of the C-S30 peptides, the replacement of leucine by valine at this position seems to shorten
this pre-helical stretch, and this could be related to the lower antigenicities observed in peptides
including this replacement. This seems further supported by the fact that absolute values of
conformational chemical shifts in this region are higher for cyclic peptide cyc16S30, which is the
best of the two C-S30 antigens under study.
105
SPR as a tool in the functional analysis of an immunodominant site in FMDV
Structural information from NOEs
Data from NOE experiments were not particularly conclusive. A continuous series of αNi,i+1 and
βNi,i+1 NOEs was observed for both peptides either in water or in 30% TFE (Fig. 2.22). These NOEs
are compatible with practically all conformations and therefore not very informative. Some weak
NNi,i+1 NOEs – compatible with α helix – were observed for peptide A15S30 in 30% TFE, in the
same region where a tentative assignment of incipient helix had been made (see previous section).
In turn, peptide cyc16S30 displayed weak NNi,i+1 NOEs both in water and 30% TFE (Fig. 2.23).
Peak assignment was often ambiguous due to identical chemical shifts for different HN and peak
overlap possibly prevented the detection of other informative HN – HN connectivities.
A15S30
Y
Η2Ο
NN(i,i+1)
αN(i,i+1)
βN(i,i+1)
30% TFE
NN(i,i+1)
αN(i,i+1)
βN(i,i+1)
cyc16S30
C
T
T
T
T
S
S
T
T
R
R
G
G
D
D
L
L
A
A
H
H
V
V
T
T
A
A
T
X
C
NN(i,i+1)
αN(i,i+1)
NN(i,i+2)
βN(i,i+1)
Η2Ο
NN(i,i+1)
αN(i,i+1)
NN(i,i+2)
βN(i,i+1)
30% TFE
Figure 2. 22 Summary of the NOEs observed for peptides A15S30 and cyc16S30 in water and 30% TFE;
relative NOE intensities are represented by bar thickness; dotted lines stand for overlapping or ambiguous
NOEs.
145
A - 146H
149
V - 148T ?
A - 150T
147
A - 146H
145
143D
140T
-
144L
A - 150Ahx
149
- 141R
C - 137T ?
151
142
G - 143D
T - 139S ?
138
G - 143D
142
Figure 2. 23 Expansion of the ROESY experiments (200 ms) performed at 25 oC: peptide A15S30 in 30%
TFE (left) and peptide cyc16S30 in water (right). Diagonal peaks (along the dashed lines) were omitted for
simplicity.
106
Antigenic determinants in the GH loop of FMDV C1-Barcelona
2.9
Recapitulation
The work described in the present chapter involved a major goal:
Finding out why FMDV C-S30 was recognised and neutralised by anti-site A antibodies such as 4C4,
even though it possesses, within this site, mutations known to be detrimental for mAb recognition
(e.g., 147L→V).
To accomplish such purpose, a total of sixteen peptides were synthesised and studied by SPR.
ELISA and NMR analyses were also performed on some of these peptides, to further complement
the study of the GH loop from FMDV C-S30. Extensive research previously reported on 15-mer
peptide mimics of FMDV antigenic site A provided a solid basis for the adequacy of such peptides as
models of this antigenic site.
The first set of eleven peptides corresponded to linear pentadecapeptides reproducing all possible
combinations of the four mutations (138A→T,
140
A→T,
147
L→V,
149
T→A) present in FMDV C-S30
antigenic site A (taking as reference sequence the antigenic site A of FMDV C- S8c1).
A direct kinetic SPR analysis of the mAb – peptide interactions was performed according to a
protocol previously optimised and validated with similar peptide FMDV antigens (chapter 1). Results
pointed to additive effects in all combinations of the four relevant mutations. For each mAb,
association rate constants were virtually equal, while dissociation rate constants varied in a relatively
broad range, increasing with decreasing peptide antigenicity.
The four-point mutant linear 15-mer peptide from the C-S30 GH loop (A15S30) was shown to be
the least antigenic of the set.
The surprisingly low antigenicity of peptide A15S30 led to a competition ELISA screening of all 15mer peptides, in order to confirm the SPR data.
Peptide A15S30 was again shown to be a poor competitor in this format of analysis, thus
confirming its low antigenicity relative to C-S8c1 or C1-Brescia peptides.
Having confirmed that the unexpected results obtained for A15S30 were not due to any technical
artefact from the SPR biosensor, our attention was then focused on the peptide itself: was the
peptide too short?
Two 21-mer linear peptide models of the C-S8c1 and C-S30 GH loops were then studied by kinetic
SPR analysis and competition ELISA.
Peptide C-S30 was, once again, clearly less antigenic than peptide C-S8c1.
107
SPR as a tool in the functional analysis of an immunodominant site in FMDV
At this point of the investigation, parallel studies performed by Wendy F. Ochoa and co-workers
showed that the 15-mer peptide A15S30 could be crystallised in complex with the Fab fragment of
mAb 4C4. The peptide adopted a nearly cyclic conformation similar to the one previously described
for the C-S8c1 peptide A15 in a similar complex. Further, it was observed that the two more critical
mutations (138A→T and
147
L→V), although not in direct interaction with the antibody, were both
involved in keeping the peptide in a pseudo-cyclic conformation, through hydrogen bonding
involving the Thr side chain hydroxyl, one water molecule and the main chain oxygen atoms of
144
Leu and 147Val.
So, a new question was immediately raised: Were the linear C-S30 peptides too flexible in solution?
Two cyclic disulphide peptides were thus studied by SPR, one of them reproducing the C-S30
sequence (cyc16S30) and the other containing the one-point mutation
147
L→V (cyc16147Val) in
order to analyse the effects of conformation modulation on peptide antigenicity.
An increase in peptide affinity was observed upon cyclization. While the single-point was still
more antigenic than the four-point mutant towards mAb SD6, a reversion in this ranking was
observed with mAb 4C4.
The higher antigenicity of C-S30 cyclic peptides and the results obtained by Wendy F. Ochoa with
the linear C-S30 peptide led to another question: Would the flexible peptide A15S30 be able to
rearrange in order to form a stable complex with anti-site A mAb 4C4 in solution, after prolonged
incubation?
To answer this question, a solution affinity SPR experiment was performed using peptides
A15(147V), A15S30, cyc16S30 and cyc16147Val as target analytes and peptides A15 and
A15Brescia as reference analytes. Despite confirming that linear C-S30 peptides were again less
antigenic than their C-S8c1 or C1-Brescia counterparts after overnight incubation with mAb in
solution, a significant increase (about one order of magnitude) in 4C4 affinity towards peptide
A15S30 was observed. This suggests that, indeed, incubation of 4C4 with A15S30 can lead the
peptide to rearrange and form a stable complex with antibody.
Solution conformation NMR studies of both A15S30 and cyc16S30 peptides, though not totally
conclusive, were quite suggestive. Both peptides were seen to be very flexible in solution, even in
the presence of conformation-inducing solvents. Nevertheless, both displayed a tendency for an
open turn in the RGD region, followed by an incipient short helical path, as previously observed
for C-S8c1 linear and cyclic peptides. Remarkably, while in the C-S8c1 peptides this helical
stretch extends up to the
147
147
Leu, in the C-S30 peptides it stops at the
146
His, suggesting that a
Leu→Val helix-disruptive mutation could be the basis for the lower antigenicites observed in
peptides including this mutation. Further, this short helix is more pronounced in the cyclic model
of the C-S30 GH loop, which can be a reason for the higher antigenicities observed for this
peptide.
108
Antigenic determinants in the GH loop of FMDV C1-Barcelona
References
1 Mateu, M. G., Rocha, E., Vicente, O., Vayreda, F., Navalpotro, C., Andreu, D., Pedroso, E., Giralt, E.,
Enjuanes, L. and Domingo, E. (1987) Reactivity with monoclonal antibodies of viruses from an
episode of foot-and-mouth disease, Virus Res. 8, 261-274.
2 Mateu, M. G., da Silva, J. L., Rocha, E., de Brum, D. L., Alonso, A., Enjuanes, L., Domingo, E. and
Barahona, H. (1988) Extensive antigenic heterogeneity of foot-and-mouth disease virus serotype C,
Virology 167, 113-124.
3 Mateu, M. G., Martínez, M. A., Andreu, D., Parejo, J., Giralt, E., Sobrino, F. and Domingo, E. (1989)
Implications of a quasispecies genome structure: effect of frequent, naturally occurring, amino acid
substitutions on the antigenicity of foot-and-mouth disease virus, Proc. Natl. Acad. Sci. USA 86,
5883-5887.
4 Mateu, M. G., Martínez, M. A., Cappucci, L., Andreu, D., Giralt, E., Sobrino, F., Brocchi, E. and
Domingo, E. (1990) A single amino acid substitution affects multiple overlapping epitopes in the
major antigenic site of foot-and-mouth disease virus of serotype C, J. Gen. Virol. 71, 629-637.
5 Martínez, M. A., Hernández, J., Piccone, M. E., Palma, E. L., Domingo, E., Knowles, N. and Mateu,
M. G. (1991) Two mechanisms of antigenic diversification of foot-and-mouth disease virus, Virology
184, 695-706.
6 Mateu, M. G., Andreu, D., Carreño, C., Roig, X., Cairó, J. J., Camarero, J. A., Giralt, E. and
Domingo, E. (1992) Non-additive effects of multiple amino acid substitutions on antigen-antibody
recognition, Eur. J. Immunol. 22, 1385-1389.
7 Carreño, C., Roig, X., Camarero, J. A., Cairó, J. J., Mateu, M. G., Domingo, E., Giralt, E. and
Andreu, D. (1992) Studies on antigenic variability of C-strains of foot-and-mouth disease virus by
means of synthetic peptides and monoclonal antibodies, Int. J. Peptide Protein Res. 39, 41-47.
8 Fields, G. B. and Noble, R. L. (1990) Solid-phase peptide synthesis utilizing 9fluorenylmethoxycarbonyl amino acids, Int. J. Peptide Protein Res. 53, 161-214.
9 Knorr, R., Trzeciak, A., Bannwarth, W. and Gillesen, D. (1989) New coupling reagents in peptide
chemistry, Tetrahedron Lett. 30, 1927-1930.
10 Bernatowicz, M. C., Daniels, S. B. and Köster, H. (1989) A comparison of acid labile linkage agents
for the synthesis of peptide C-terminal amides, Tetrahedron Lett. 30, 4645-4648.
11 O’Shannessy, D. J. and Winzor, D. J. (1996) Interpretation of deviations from pseudo-first-order
kinetic behavior in the characterization of ligand binding by biosensor technology, Anal. Biochem.
236, 275-283.
12 Schuck, P. (1997) Reliable determination of binding affinity and kinetics using surface plasmon
resonance biosensors, Curr. Op. Biotech. 8, 498-502.
13 Hall, D. R., Cann, J. R. and Winzor, D. J. (1996) Demonstration of an upper limit to the range of
association rate constants amenable to study by biosensor technology based on surface plasmon
resonance, Anal. Biochem. 235, 175-184.
14 Verdaguer, N., Sevilla, N., Valero, M. L., Stuart, D., Brocchi, E., Andreu, D., Giralt, E., Domingo, E.,
Mateu, M. G. and Fita, I. (1998) A similar pattern of interaction for different antibodies with a major
antigenic site of foot-and-mouth disease virus: implications for intratypic antigenic variation, J. Virol.
72, 739-748.
15 Mateu, M. G., personal communication
16 Abbas, A. K., Lichtman, A. H. and Pober, J. S. “Cellular and molecular immunology”, 3rd ed., W. B.
Saunders Co., United States of America (1997).
17 Mateu, M. G., Andreu, D. and Domingo, E. (1995) Antibodies raised in a natural host and
monoclonal antibodies recognize similar antigenic features of foot-and-mouth disease virus, Virology
210, 120-127.
18 Ochoa, W. F., Kalko, S., Mateu, M., Gomes, P., Andreu, D., Domingo, E., Fita, I. and Verdaguer, N.
(2000) A multiply substituted GH loop from foot-and-mouth disease virus in complex with a
neutralizing antibody: a role for water molecules, J. Gen. Virol. 81, 1495-1505.
19 Valero, M. L., Camarero, J. A., Haack, T., Mateu, M. G., Domingo, E., Giralt, E. and Andreu, D.
(2000) Native-like cyclic peptide models of a viral antigenic site: finding a balance between rigidity
and flexibility, J. Mol. Recognit. 13, 5-13.
20 Andreu, D., Albericio, F., Solé, N. A., Munson, M. C., Ferrer, M. and Barany, G. Formation of
disulfide bonds in synthetic peptides and proteins in “Methods in molecular biology, vol. 35: Peptide
synthesis protocols”, Pennington, M. W. and Dunn, B. M. (Eds.), Humana Press Inc., Totowa, New
Jersey (1994), pp 91-169.
21 Ellman, G. L. (1958) A colorimetric method for determining low concentrations of mercaptans, Arch.
Biochem. Biophys. 74, 443-450.
22 “BIAapplications Handbook”, (Pharmacia Biosensor AB, 1994) Uppsala, Sweden.
23 Nieba, L., Krebber, A. and Plükthun, A. (1996) Competition BIAcore for measuring true affinities:
large differences from values determined from binding kinetics, Anal. Biochem. 234, 155-165.
109
SPR as a tool in the functional analysis of an immunodominant site in FMDV
24 “BIAevaluation Software Handbook: version 3.0”, (Biosensor AB, 1997) Uppsala, Sweden.
25 Lazareno, S. and Birdsall, N. J. (1993) Estimation of competitive antagonist affinity from functional
inhibition curves using the Gaddum, Schild and Cheng-Prusoff equations, British J. Pharmacol. 109,
1110-1119.
26 Wüthrich, K. “NMR of proteins and nucleic acids”, Wiley, New York (1986).
27 Braunschweiler, L. and Ernst, R. R. (1983), J. Magn. Reson. 53, 521.
28 Kumar, A., Ernst, R. R. and Wüthrich, K. (1980), Biochem. Biophys. Chem. Comm. 95, 1.
29 Bothner-By, A. A., Stephens, R. L., Lee, J., Warren, C. D. and Jeanloz, R. W. (1984) Structure
determination of a tetrasaccharide: transient nuclear overhauser effects in the rotating frame, J. Am.
Chem. Soc. 106, 811-813.
30 Haack, T., Camarero, J. A., Roig, X., Mateu, M. G., Domingo, E., Andreu, D. and Giralt, E. (1997) A
cyclic disulfide peptide reproduces in solution the main structural features of a native antigenic site of
foot-and-mouth disease virus, Int. J. Biol. Macromol. 20, 209-219.
110
3. Antigenic peptides with non-natural
replacements within the GH loop of FMDV
SPR as a tool in the functional analysis of an immunodominant site in FMDV
112
Antigenic peptides with non-natural replacements within the GH loop of FMDV
3.0
Introduction
In an attempt to analyse the contribution of each amino acid residue to the antigenicity of site A of
FMDV C-S8c1, M. L. Valero and co-workers evaluated a set of 250 peptides corresponding to the
systematic replacement of all residues within the sequence of peptide A151-3. Peptide antigenicity
was quantitated by competition ELISA, using a panel of seven anti-site A mAbs: SD6, 4C4, 5A2,
6D11, 7DJ1, 7FC12 and 7CA11. In this systematic screening, five singly replaced peptides were
found to be antigenic for at least three mAbs, being comparable to or even better than the native
A15 sequence. These peptides corresponded to the substitutions Thr137→Ile, Ala138→Phe,
Ala140→Pro, Gly142→Ser and Thr148→Ile. Although the first two and the last residue
replacements correspond to hyper-variable regions of the GH loop, the contrast in size between the
Phe and the Ala residues, the structural “personality” of Pro and, even more, the Gly→Ser mutation
within the highly conserved RGD motif, altogether raise many questions about what would be the
contribution of these residues in antibody recognition.
In the present chapter, further studies on the above mentioned amino acid replacements are
presented. Both the one-point mutants (Table 3.1), and a set of peptides reproducing all possible
combinations of the five mutations were synthesised and antigenically characterised by SPR. Some
NMR and X-ray diffraction studies were also performed, as a structural complement to the functional
SPR characterisation of the peptide antigens.
3.1
Peptides that combine antigenicity-enhancing replacements in the GH loop
Thirty-one peptides, corresponding to the combinations of the five mutations (Table 3.1), were
synthesised (Fig. 3.1, Table 3.2) by methods similar to those mentioned in chapter 2 and further
described in section 4.2 (Materials & Methods).
MPLC
Figure 3. 1 HPLC of crude
(left) and purified (right)
peptide A15(138F,140P,142S),
representative of the thirty-one
15-mers of this chapter.
0
30 min
0
30 min
113
SPR as a tool in the functional analysis of an immunodominant site in FMDV
Table 3.1 Pentadecapeptide library reproducing all possible combinations of the substitutions Thr137→Ile,
Ala138→Phe, Ala140→Pro, Gly142→Ser and Thr148→Ile.
Name
Sequence
Mutants
A15
YTASARGDLAHLTTT
-I--------------F---------------P---------------S-------------------I--IF------------I--P----------I----S--------I----------I---F-P-----------F---S---------F---------I-----P-S-----------P-------I-------S-----I--IF-P----------IF---S--------IF---------I--I--P-S--------I--P-------I--I----S-----I---F-P-S---------F-P-------I---F---S-----I-----P-S-----I--IF-P-S--------IF-P-------I--IF---S-----I--I--P-S-----I---F-P-S-----I--IF-P-S-----I--
GH loop of FMDV C-S8c1
A15(137I)
A15(138F)
A15(140P)
A15(142S)
A15(148I)
A15(137I,138F)
A15(137I,140P)
A15(137I,142S)
A15(137I,148I)
A15(138F,140P)
A15(138F,142S)
A15(138F,148I)
A15(140P,142S)
A15(140P,148I)
A15(142S,148I)
A15(137I,138F,140P)
A15(137I,138F,142S)
A15(137I,138F,148I)
A15(137I,140P,142S)
A15(137I,140P,148I)
A15(137I,142S,148I)
A15(138F,140P,142S)
A15(138F,140P,148I)
A15(138F,142S,148I)
A15(140P,142S,148I)
A15(137I,138F,140P,142S)
A15(137I,138F,140P,148I)
A15(137I,138F,142S,148I)
A15(137I,140P,142S,148I)
A15(138F,140P,142S,148I)
A15(137I,138F,140P,142S,148I)
114
One-point
Two
Point
Three
Point
Four-point
“
“
Five-point
Antigenic peptides with non-natural replacements within the GH loop of FMDV
Table 3.2 General data (yield and product characterisation by HPLC, ES or MALDI-TOF MS and AAA) of the syntheses of the 15-mer peptides.
Peptide
Global
yield (%)
Purity
(% HPLC)
MW
found
MW
expected
AAA
Asp, 1.07 (1); Ser, 1.04 (1); Gly, 1.08 (1); Ala, 3.10 (3); Leu, 1.96 (2); His, 0.92 (1)
A15(137I)
89
99
1589.2
1589
Asp, 0.96 (1); Ser, 0.96 (1); Gly, 1.03 (1); Ala, 2.05 (2); Leu, 2.05 (2); His, 0.99 (1)
A15(138F)
65
97
1653.3
1653
Asp, 1.04 (1); Ser, 0.97 (1); Gly, 1.05 (1); Ala, 2.07 (2); Leu, 1.96 (2); Arg, 1.01 (1)
A15(140P)
84
98
1603.1
1603
Asp, 1.05 (1); Ser, 1.99 (2); Ala, 3.08 (3); Leu, 2.00 (2); His, 0.92 (1); Arg, 0.95 (1)
A15(142S)
79
99
1607.2
1607
Asp, 1.05 (1); Ser, 1.01 (1); Gly, 1.04 (1); Ala, 3.03 (3); Leu, 1.82 (2); Arg, 0.94 (1)
A15(148I)
87
98
1589.1
1589
Asp, 1.00 (1); Ser, 0.98 (1); Gly, 1.03 (1); Ala, 1.98 (2); Leu, 1.98 (2); His, 1.02 (1)
A15(IF)*
15
93
1665.1
1665
Asp, 1.06 (1); Ser, 1.00 (1); Gly, 1.10 (1); Ala, 1.93 (2); Pro, 0.99 (1); Arg, 1.02 (1)
A15(IP)
55
91
1615.6
1615
Asp, 0.99 (1); Ser, 2.07 (2); Ala, 3.08 (3); Leu, 2.03 (2); His, 0.88 (1); Arg, 0.95 (1)
A15(IS)
79
90
1619.2
1619
Asp, 1.00 (1); Ser, 0.99 (1); Gly, 1.01 (1); Ala, 3.04 (3); His, 0.92 (1); Arg, 0.99 (1)
A15(II)
78
97
1601.3
1601
Asp, 1.06 (1); Pro, 1.01 (1); Gly, 1.09 (1); Ala, 1.05 (1); Leu, 1.89 (2); Arg, 1.07 (1)
A15(FP)
72
95
1679.6
1679
Asp, 1.05 (1); Ser, 2.10 (2); Ala, 2.05 (2); Leu, 1.88 (2); His, 0.92 (1); Arg, 1.00 (1)
A15(FS)
86
89
1684.1
1684
Asp, 1.03 (1); Ser, 0.95 (1); Gly, 1.03 (1); Ala, 2.00 (2); His, 0.90 (1); Arg, 1.09 (1)
A15(FI)
86
91
1665.4
1665
Asp, 1.02 (1); Pro, 1.03 (1); Ala, 2.04 (2); Leu, 1.93 (2); His, 0.92 (1); Arg, 1.11 (1)
A15(PS)
81
87
1632.4
1633
Asp, 1.03 (1); Ser, 0.95 (1); Gly, 1.00 (1); Ala, 2.01 (2); His, 0.95 (1); Arg, 1.07 (1)
A15(PI)
79
89
1615.6
1615
Asp, 1.05 (1); Ser, 1.96 (2); Ala, 3.10 (3); Tyr, 0.87 (1); His, 0.92 (1); Arg, 1.14 (1)
A15(SI)
79
86
1619.6
1619
Asp, 1.03 (1); Ser, 0.96 (1); Gly, 1.05 (1); Ala, 1.01 (1); Pro, 0.98 (1); Arg, 1.05 (1)
A15(IFP)
78
94
1691.6
1691
Asp, 1.10 (1); Ser, 1.90 (2); Tyr, 0.91 (1); Phe, 1.01 (1); His, 0.97 (1); Arg, 1.08 (1)
A15(IFS)
95
86
1694.5
1695
Asp, 1.01 (1); Ser, 0.91 (1); Gly, 1.01 (1); Ala, 1.97 (2); His, 1.07 (1); Arg, 1.04 (1)
A15(IFI)
83
95
1677.4
1677
Asp, 1.03 (1); Pro, 1.04 (1); Ala, 2.01 (2); Leu, 1.90 (2); His, 0.91 (1); Arg, 1.12 (1)
A15(IPS)
82
94
1644.3
1645
Asp, 1.05 (1); Ser, 0.99 (1); Pro, 1.00 (1); Gly, 1.08 (1); Ala, 1.93 (2); Arg, 1.03 (1)
A15(IPI)
71
92
1626.6
1627
Asp, 1.05 (1); Ser, 2.07 (2); Ala, 2.97 (3); Leu, 1.84 (2); His, 0.96 (1); Arg, 0.95 (1)
A15(ISI)
80
92
1631.1
1631
Asp, 0.97 (1); Ser, 1.97 (2); Ala, 1.04 (1); Leu, 2.08 (2); Phe, 1.03 (1); Arg, 1.02 (1)
A15(FPS)
73
97
1708.3
1709
Asp, 1.02 (1); Pro, 1.00 (1); Gly, 1.06 (1); Ala, 1.02 (1); Phe, 0.86 (1); His, 0.90 (1)
A15(FPI)
87
90
1690.1
1691
Asp, 1.07 (1); Ala, 2.09 (2); Leu, 1.85 (2); Tyr, 0.87 (1); Phe, 0.91 (1); His, 1.07 (1)
A15(FSI)
88
91
1695.0
1695
Asp, 1.00 (1); Pro, 1.02 (1); Ala, 2.02 (2); Tyr, 0.93 (1); His, 0.91 (1); Arg, 1.11 (1)
A15(PSI)
89
92
1645.0
1645
Asp, 0.98 (1); Ser, 1.91 (2); Pro, 0.98 (1); Ala, 0.99 (1); His, 1.07 (1); Arg, 1.07 (1)
A15(IFPS)
77
95
1720.9
1721
Asp, 0.99 (1); Ser, 0.94 (1); Pro, 0.96 (1); Gly, 1.03 (1); Ala, 0.96 (1); Arg, 1.01 (1)
A15(IFPI)
74
94
1703.1
1703
Asp, 1.07 (1); Ser, 2.06 (2); Ala, 2.04 (2); Leu, 1.91 (2); His, 0.93 (1); Arg, 0.89 (1)
A15(IFSI)
84
98
1706.7
1707
Asp, 1.02 (1); Ser, 1.91 (2); Pro, 1.02 (1); Ala, 1.98 (2); His, 0.96 (1); Arg, 1.11 (1)
A15(IPSI)
80
91
1656.0
1657
Asp, 1.04 (1); Ser, 2.01 (2); Pro, 1.04 (1); Ala, 1.01 (1); Phe, 0.91 (1); Arg, 1.08 (1)
A15(FPSI)
56
92
1721.2
1721
Asp, 1.09 (1); Ser, 2.03 (2); Pro, 1.02 (1); Ala, 1.05 (1); Leu, 1.99 (2); Arg, 0.95 (1)
A15(IFPSI)
80
94
1732.1
1733
Note: relative amino acid proportions given by AAA are followed by the expected value in parenthesis (for simplicity, mutant peptides are designed only with the capital case letter
code for the replaced amino acids without the corresponding position number).* synthesis performed under sub-optimal conditions due to instrumental malfunction.
115
SPR as a tool in the functional analysis of an immunodominant site in FMDV
3.2
Analysis of the mutated peptides by direct kinetic SPR
Kinetic SPR screening of the thirty-one peptide antigens was performed as previously described in
chapters 1 and 2. In this particular case, it was observed that most interactions could not be
quantitated, either due to high association rates, extremely slow dissociation rates or even
insufficient surface regeneration (Fig. 3.2). This was particularly frequent with mAbs 4C4 and 3E5.
Also, surface saturation was observed for peptide concentrations higher than ca. 600 nM.
Interaction data that could be reasonably fitted as a 1:1 bimolecular interaction (Table 3.3)
presented, in most cases, rate constants in the limit of reliable kinetic information4-6. Non-ideal
effects, such as ligand rebinding in the dissociation phase, seemed to be affecting binding kinetics
(Fig. 3.3).
15
25
B
A
13
20
11
Response/RU
Response/RU
9
7
5
Negligible
descent
3
1
-1
15
Steep
ascent
10
5
0
-3
A15(138F,148I), 41 nM
A15(137I,138F)
-5
-5
-10
40
90
140
190
240
290
-10
40
90
Time/s
140
190
240
Time/s
11590
C
A15(IFI), 36 nM
A15(IFI), 73 nM
A15(IFI), 146 nM
A15(IFI), 292 nM
A15(IFI), 584 nM
A15(IFI), 1167 nM
11580
Response/RU
11570
11560
11550
11540
11530
Increasing
baseline level
11520
Decreasing response
(for increasing concentrations)
11510
-10
40
90
140
190
240
290
Time/s
Figure 3. 2 Sensorgrams illustrating problems often observed in the kinetic SPR analysis of the
interactions between anti-site A mAbs and peptides combining mutations Thr137→Ile, Ala138→Phe,
Ala140→Pro, Gly142→Ser and Thr148→Ile; A. extremely slow dissociation (as can be observed, slope
is, in fact, positive, possibly due to the sum of a negligible dissociation slope and a positive slope from
instrumental drift); B. extremely fast association (a steep ascent can be observed, giving the sensorgram
a square wave-like shape in the association phase); C. Insufficient surface regeneration: the baseline
level increases and the response level decreases from cycle to cycle.
116
290
Antigenic peptides with non-natural replacements within the GH loop of FMDV
mAb
Peptide
A15
A15(137I)
A15(138F)
A15(140P)
A15(142S)
A15(148I)
A15(IF)
A15(IP)
A15(IS)
A15(II)
A15(FP)
A15(FS)
A15(FI)
A15(PS)
A15(PI)
A15(SI)
A15(IFP)
A15(IFS)
A15(IFI)
A15(IPS)
A15(IPI)
A15(ISI)
A15(FPS)
A15(FPI)
A15(FSI)
A15(PSI)
A15(IFPS)
A15(IFPI)
A15(IFSI)
A15(IPSI)
A15(FPSI)
A15(IFPSI)
ka/M-1s-1
SD6
kd/s-1
7.3×104
9.6×104
8.6×104
7.4×104
6.1×104
1.1×105
2.9×105
1.6×105
7.3×104
2.3×105
1.1×105
1.1×105
2.6×105
1.4×10-3
5.0×10-4
3.9×10-3
2.1×10-3
5.6×10-3
2.1×10-3
5.1×10-3
2.6×10-3
4.1×10-3
3.0×10-3
8.5×10-3
1.1×10-2
8.4×10-4
5.4×
×107
1.9×
×108
2.2×
×107
3.5×
×107
1.1×
×107
5.3×
×107
5.6×
×107
5.9×
×107
1.8×
×107
7.6×
×107
1.3×
×107
1.0×
×107
3.0×
×108
5
-3
KA/M-1
✖
1.5×10
3.7×104
3.5×10
6.0×10-3
4.0×
×10
6.1×
×106
3.5×104
1.2×105
9.3×104
3.5×10-3
4.2×10-3
5.4×10-4
1.0×
×107
2.9×
×107
1.7×
×108
4.5×104
8.5×104
1.4×105
5.0×104
5.9×104
1.5×105
2.5×105
1.3×105
1.7×105
8.6×104
2.1×105
1.5×10-3
8.8×10-3
4.3×10-3
9.4×10-3
7.1×10-4
4.2×10-3
5.5×10-4
1.7×10-3
2.5×10-3
6.1×10-3
2.2×10-4
3.1×
×107
9.7×
×106
3.3×
×107
5.4×
×106
8.4×
×107
3.6×
×107
4.5×
×108
7.7×
×107
6.7×
×107
1.4×
×107
9.8×
×107
✖
✖
7
ka/M-1s-1
4C4
kd/s-1
3.8×105
2.9×105
5.5×105
1.9×105
6.3×104
7.0×105
6.4×105
1.9×10-3
2.0×10-3
5.7×10-3
1.9×10-3
3.8×10-3
2.3×10-3
1.7×10-3
1.9×
×108
1.4×
×108
9.8×
×107
1.0×
×108
1.6×
×107
3.0×
×108
3.8×
×108
2.8×105
1.0×10-3
2.8×
×108
3.6×105
5.9×10-4
6.0×
×108
2.0×105
7.9×10-4
2.5×
×108
2.0×105
7.0×10-4
2.9×
×108
7.6×104
1.8×10-3
4.3×
×107
2.0×105
4.2×10-4
4.8×
×108
4.6×105
4.0×105
1.6×10-3
2.9×10-4
1.6×
×108
1.4×
×109
4.7×105
8.5×10-4
5.5×
×108
✖
✖
✖
✖
✖
✖
✖
✖
✖
✖
✖
✖
✖
✖
✖
✖
KA/M-1
ka/M-1s-1
3E5
kd/s-1
1.6×105
3.5×105
6.0×105
2.0×105
1.8×105
5.9×105
1.6×10-3
1.0×10-3
1.4×10-3
1.5×10-3
7.0×10-3
1.1×10-3
✖
✖
✖
✖
✖
✖
✖
✖
✖
✖
✖
✖
✖
✖
✖
✖
✖
✖
✖
✖
✖
✖
✖
✖
✖
✖
KA/M-1
9.4×
×107
3.4×
×108
4.0×
×108
1.4×
×108
2.5×
×107
6.0×
×108
Table 3.3 Kinetic SPR
analysis of the peptides
towards mAbs SD6, 4C4 and
3E5.
Notes:
Although
resulting
from
apparently reliable data fits,
kinetic parameters should be
regarded with some caution,
since most ka values are in the
limit
for
reliable
kinetic
measurements not affected by
mass-transport limitations.
✖ non-measurable interactions.
117
SPR as a tool in the functional analysis of an immunodominant site in FMDV
20
A
A15(140P,142S), 20 nM
A15(140P,142S), 39 nM
A15(140P,142S), 78 nM
A15(140P,142S), 156 nM
A15(140P,142S) 312 nM
A15(140P,142S), 625 nM
Response/RU
15
10
5
0
-5
-10
40
90
140
190
240
290
Time/s
0.12
B
r2=0.984
0.10
r2=0.999
0.06
ks/s
-1
0.08
0.04
0.02
Peptide concentration/M
0.00
0.00E+00 1.00E-07 2.00E-07 3.00E-07 4.00E-07 5.00E-07 6.00E-07 7.00E-07
Figure 3. 3 Sensorgrams of the interaction between peptide A15(140P,142S) and mAb 4C4 (A); despite the
good quality of data when using global curve fitting, slight deviations were observed in the dissociation rate
constants (increasing with peptide concentration) when local curve fitting was employed; this fact together with
the slight curvature observed when plotting ks against peptide concentration (B) indicate that non-ideal effects
were affecting binding kinetics.
118
Antigenic peptides with non-natural replacements within the GH loop of FMDV
3.3
Indirect SPR kinetic analysis using a high molecular weight competitor antigen7
As explained in section 0.2.3, there are indirect SPR methods for the kinetic characterisation of
biospecific interactions between an immobilised receptor and small ligands in solution. One of them
is a kind of surface competition assay, where a high molecular weight ligand, specific to the
immobilised receptor, is injected and detected on the sensor chip surface. The kinetics of the
macromolecular ligand – receptor interaction is characterised and, then, mixtures of this
macromolecule with varying concentrations of the small analyte of interest are injected (Fig. 3.4).
The effects observed on the macromolecule – receptor interaction upon addition of the small
competitor analyte provide an indirect means to measure the kinetics of the small analyte – receptor
interaction.
Figure 3. 4 Main steps in the surface competition SPR kinetic analysis: left – the kinetics of the interaction
between surface – immobilised receptor (brown) and the macromolecular analyte (yellow) is measured; right –
effects due to addition of a small competitor (target analyte, green) on the macromolecule – receptor interaction
are evaluated and the kinetics of the small analyte – receptor interaction is thus determined.
Due to problems often observed in the direct kinetic SPR described in the previous section, it was
thought that perhaps an indirect approach would be more appropriate. Indeed, peptide – antibody
interactions seemed to be affected by diffusion-controlled kinetics, due to the apparently high
association rates involved. Further, symptoms of both analyte rebinding and surface saturation at
higher analyte concentrations were equally observed. Thus, it would seem advisable to decrease
mAb surface density and analyte concentrations as much as possible, in order to minimise such nonideal effects4-6. However, this would imply severe losses in response levels, given the small size of the
target analytes. Therefore, an indirect approach based on the detection of a macromolecular analyte
would be a possible solution for these problems7.
In the absence of a natural macromolecular FMDV antigen available for such surface competition
SPR assays, alternative high molecular weight antigens were chosen, as described in the following
sections.
119
SPR as a tool in the functional analysis of an immunodominant site in FMDV
3.3.1
Peptide – protein (1:1) conjugate: the A15 – human carbonic anhydrase I (HCA I) construct
To ensure unambiguous mechanisms for peptide – macromolecule competitive binding to the
antibody paratope, a macromolecular antigen with only one specific epitope per molecule was
needed. This implied the conjugation of a site A representative peptide such as A15 to a protein
carrier in a 1:1 stoichiometry. A safe way to achieve such stoichiometry would be to link through a
heterodisulphide bond the A15 sequence (with an additional Cys residue) to a carrier protein
bearing a single Cys.
Search in the Protein Data Bank for a protein suitable for such purposes (i.e., with a single cysteine
residue, weighing over ca. 10 kDa, commercially available and affordable) led to human carbonic
anhydrase I (HCA I – E. C. 4.2.1.1).
3.3.1.1 Synthesis of peptide (Npys)Cys – A15 and heterodimerisation with protein HCA I
The A15 sequence was assembled as described in previous sections, then Boc–Cys(Npys)–OH was
coupled as N-terminal residue by similar protocols and cleavage/side chain deprotection with TFA
was then performed as usual (see Materials & Methods, section 4.3), leading to peptide (Npys)Cys–
A15 (Table 3.4). The 3-nitro-2-pyridylsulphenyl (Npys) group is stable to TFA and therefore not
removed in the cleavage. This thiol protecting group was chosen for its well-known applicability to
direct peptide – protein conjugation through cysteine residues. Such property of the Npys group is
due both to the fact that it is stable to the standard acidolytic cleavage conditions (even to hydrogen
fluoride acidolysis in Boc/Bzl chemistry) and also to its thiol-activating character, which allows
regiospecific peptide – protein coupling through cysteine residues (Fig. 3.5)8.
Table 3.4 General data concerning the synthesis of peptide (Npys)Cys – A15.
Global
yield (%)
Purity
(% HPLC)
MW
found
MW
expected
AAA
67
81
1833.6
1833
Asp, 1.04 (1); Ser, 0.93 (1); Gly, 1.07 (1);
Ala, 3.10 (3); Leu, 2.00 (2); His, 0.97 (1)
S
S
pH 4.2
SH
S
N
S
S
+
HN
NO2
NO2
Figure 3. 5 Regiospecific formation of a disulphide heterodimer via the Npys thiol activation; Npys not only
serves as thiol-protection during peptide synthesis, but also activates the Cys sulphur atom toward other
nucleophiles, such as other Cys thiol groups8.
120
Antigenic peptides with non-natural replacements within the GH loop of FMDV
The heterodimerisation reaction was carried out overnight under acidic, denaturating conditions (6
M guanidine hydrochloride in water, pH 4.2) using excess peptide, and was monitored by HPLC
(Fig. 3.6). The final reaction mixture was dialysed (MW cut-off = 15 to 20 kDa) against decreasing
concentrations of guanidine hydrochloride in water for 48 hours. The major product in the final
dialysed solution was characterised by MALDI-TOF MS (Fig. 3.7) as the target HCA I – CysA15 1:1
conjugate.
1
A
Figure 3. 6 HPLC monitoring of the
heterodimerisation reaction: A. crude
(Npys)Cys – A15 peptide; B. HCA I;
C, D and E progress of the reaction at
2, 8 and 24 h, respectively.
B
15 - 65%B, 30 min
C
2
2
15 - 65%B, 30 min
E
D
3
2
1
1
3
3
1
2
15 - 65%B, 30 min
15 - 65%B, 30 min
15 - 65%B, 30 min
Figure 3. 7 MALDI-TOF MS spectrum of the final
heterodimerisation product, which contains unreacted HCA I
protein (the MALDI-TOF MS spectrum of the commercial HCA I
is shown in the upper left corner).
121
SPR as a tool in the functional analysis of an immunodominant site in FMDV
3.3.1.2 Antigenic evaluation of the HCA I – CysA15 conjugate
The adequacy of the HCA I – CysA15 construct as an FMDV antigen was evaluated by direct SPR
detection on SD6, 4C4 and 3E5 surfaces (with mAb densities of approximately 600 RU, i. e, 0.6
ng/mm2). Unfortunately, absence of specific response was repeatedly observed, showing that the
construct was not antigenic towards these mAbs (Fig. 3.8). Some assays at pH values (5.8 and 8)
different from the usually employed (7.3) did not improve the results (only non-specific binding
between protonated protein – negatively-charged dextran layer was observed at the lowest pH).
These results were confirmed by competition ELISA experiments using A21 – KLH conjugate as the
plate-bound antigen (Fig. 3.9). Inadequate conformational presentation or inaccessibility of peptide
A15 were considered as probable causes for such lack of antigenicity.
34800
B
31 nM
62 nM
125 nM
250 nM
500 nM
29800
A
31 nM
62 nM
125 nM
250 nM
500 nM
35
19800
14800
9800
4800
-200
-10
25
40
90
140
190
240
290
340
Time/s
24800
C
15
31 nM
62 nM
125 nM
250 nM
500 nM
19800
Response/RU
Response/RU
45
Response/RU
24800
55
5
-5
-10
40
90
140
190
240
290
Time/s
340
14800
9800
4800
-200
-10
40
90
140
190
240
290
340
Time/s
Figure 3. 8 SPR analysis of conjugate HCA I – CysA15: A. injection of conjugate samples over a mAb 4C4
surface (600 RU), employing the conditions used for FMDV peptides; B. and C. injection of protein samples at
pH 5.8 over the same 4C4 surface and over an non-derivatised sensor chip surface, respectively.
140
120
% absorbance
100
Figure 3. 9 Inhibition curves from
competition ELISA analysis of the
HCA I – CysA15 conjugate (curves
for reference peptide A15 were
omitted).
80
60
40
HCA-A15 vs. 3E5
HCA-A15 vs. SD6
HCA-A15 vs. 4C4
20
0
0.1
1
10
µL)
conjugate concentration (pmol/100µ
122
100
1000
Antigenic peptides with non-natural replacements within the GH loop of FMDV
3.3.2
Recombinant engineered proteins bearing the FMDV GH loop peptide: protein JX249A
3.3.2.1 Preliminary assays
The extensive research work on recombinant proteins bearing the FMDV C-S8c1 GH loop carried
out by A. Villaverde and co-workers9-12 opened the possibility to use one of these engineered
proteins as such macromolecular antigen.
A recombinant β-galactosidase from Escherichia coli, protein JX249A, has solvent exposed-loops
which have been engineered for the insertion of a peptide from the GH loop of FMDV C-S8c1
(TT136YTASARGDLAHLTT150THARHLP). JX249A is a homotetramer with four GH loops per
protein, having a total molecular weight of 472 kDa. Given its high antigenicity, JX249A was tested
in preliminary SPR assaysA, where each protein molecule would be regarded as four independent
antigenic monomers to simplify data processing. The first assays confirmed the antigenicity of
JX249A towards mAbs SD6, 4C4 and 3E5, and preliminary analyses also indicated that peptide
A15 competed with JX249A in binding to surface-immobilised mAb (surface densities of about 600
RU), while non-specific peptide A15Scr did not (Fig. 3.10). Nevertheless, important problems due to
insufficient surface regeneration and consequent protein accumulation on the surface led to short
surface life-times and prevented a systematic screening of the peptide antigens.
110
A
JX249A, 10 nM
JX249A, 20 nM
JX249A, 40 nM
JX249A, 80 nM
JX249A, 160 nM
90
Response/RU
70
Figure 3. 10 SPR assays using JX249A for
indirect SPR kinetic analysis of peptide-antibody
interactions: A. JX249A – mAb 4C4 interaction;
B. competition between JX249A with peptide
A15; C. competition between JX249A and peptide
A15Scr; JX249A was used at a constant 80 nM
concentration in the competition assays.
50
30
10
-10
-10
40
90
140
190
240
290
Time/s
70
90
decreasing slope
with increasing
peptide
concentration
B
60
80
C
A15Scr, 0 nM
A15Scr, 20 nM
A15Scr, 40 nM
A15Scr, 80 nM
A15, 160 nM
slope independent
of peptide
concentration
70
60
Response/RU
Response/RU
50
A15, 0 nM
A15, 19 nM
A15, 38 nM
A15, 76 nM
A15, 152 nM
40
30
20
50
40
30
20
10
10
faster protein dissociation and lower bound protein
level with increasing peptide concentration
0
dissociation rate and amount of protein bound
independent of peptide concentration
0
-10
-10
-10
40
90
140
190
Time/s
240
290
340
-10
40
90
140
190
240
290
340
Time/s
A
Protein engineering, production and antigenic evaluation by ELISA were performed by Dr. A. Villaverde and co-workers (U.
A. B., Bellaterra – Barcelona), who kindly offered protein samples to the author.
123
SPR as a tool in the functional analysis of an immunodominant site in FMDV
3.3.2.2 Screening of alternative regeneration conditions
Insufficient surface regeneration was, once again, preventing the study of the kinetics of peptide –
mAb interactions by SPR. Therefore, a study of regeneration conditions, as described by Andersson
and co-workers13, was carried out. This study was based on the screening of several regeneration
cocktails, consisting of mixtures of the stock solutions presented in Table 3.5. This multi-cocktail
approach is based on the principle that what one kind of chemical property (acidic, basic, saline,
organic, denaturating) cannot disrupt, perhaps another one can or, even better, combination of
several distinct chemical properties will act synergistically and solve the regeneration problem.
Table 3.5 Stock solutions13used for the multi-cocktail surface regeneration approach in SPR analysis.
Cocktail
A
B
I
U
D
C
Main chemical properties Composition
Equal volumes of 0.15 M phosphoric, formic and
Acid
malonic acids, adjusted to pH 5 with 4 M NaOH
Equal volumes of 0.20 M ethanolamine, sodium
Basic
phosphate, piperazine and glycine, adjusted to pH 9
with 2 M HCl
Potassium thiocianate (0.46 M), magnesium chloride
Ionic/denaturating
(1.83 M), urea (0.92 M) and guanidine hydrochloride
(1.83 M)
Equal volumes of dimethylsulfoxide, formamide,
Non-polar/organic
acetonitrile, ethanol and 1-butanol
0.3% (w/w) CHAPS, 0.3% (w/w) zwittergent 3-12,
Detergent
0.3% (v/v) Tween 80, 0.3% (v/v) Tween 20 and
0.3% (v/v) Triton X-100
Chelating
20 mM EDTA aqueous solution
The general protocol consists of a screening and an optimisation step. In the first step, diluted
solutions or simple binary combinations of the above described cocktails are tested (Table 3.6). The
evaluation of the screening cocktails is carried out in the biosensor and, afterwards, an optimisation
step is performed upon combinatorial mixing of the stock cocktails rated as the best.
Table 3.6 Cocktails used in the screening step of the multi-cocktail regeneration approach13.
Composition of the screening cocktails
Bww Iww Dww Uww Cww BDw BCw Aiw Adw Auw Acw Idw Icw Duw DCw Ucw ABw
* equivalent amounts (v/v) of each cocktail represented by the corresponding letter (see Table 3.5); w stands for water.
124
Antigenic peptides with non-natural replacements within the GH loop of FMDV
Having an immunoglobulin as the surface-immobilised receptor, the use of cocktails I or D, as
defined in Table 3.5, could be harmful. Therefore, cocktails A, B, C, U and a modified ionic cocktail
I’ (differing from I in that denaturating chemicals such as urea or guanidine hydrochloride were not
added) were screened as described. The screening was performed on high density mAb surfaces (ca.
3 ng/mm2) to ensure high responses for an unequivocal evaluation of the cocktail regeneration
efficacy. In neither case was a significant improvement observed (Fig. 3.11).
1980
Cocktail U
Cocktail I’
Cocktail A
Cocktail C
Cocktail B
Response/RU
1480
980
480
650 RU
640 RU
638 RU
630 RU
627 RU
1152 RU
-20
-20
180
380
580
780
980
1180
1380
1580
1780
Time/s
Figure 3. 11 Successive injections of stock regeneration solutions Bww, Cww, Aww, I’ww and Uww (see Table
3.6) on a mAb 4C4 surface (3 ng/mm2), after binding of protein JX249A (300 nM).
Results in Fig. 3.11 show the inefficacy of the stock regeneration solutions tested for recovering the
initial baseline level. Further tests, either with triplicate injections of each regeneration cocktail or
with binary combinations of the stock solutions (AI’ and CI’ at different proportions), did not
improve the results. The increase in response level observed when injecting cocktail Uww was
attributed to compression of the hydrophilic dextran matrix due to the organic solvents present in
this cocktail.
The cocktail approach was also tested on some problematic peptide – mAb interactions (mentioned
in 3.2), but results were none the better.
125
SPR as a tool in the functional analysis of an immunodominant site in FMDV
3.3.2.3 Capping of JX249A free cysteine thiol groups
We hypothesised that the free thiol groups from the JX249A cysteine residues might be the source
for the irreversible binding of the protein to the mAb surfaces, upon disulphide bridge cross-linking.
Usually, experiments with bacterial β-galactosidases at room temperature require the addition of βmercaptoethanol, in order to maintain the cysteine thiol groups in their native free form and to
avoid protein aggregation. In the above SPR experiments, β-mercaptoethanol was not added, since
protein solutions would be put in contact with an antibody surface and native folding of the
immunoglobulin had to be preserved. Thus, capping of the JX249A cysteine thiol groups seemed to
be a wise precaution to avoid both protein aggregation and, possibly, disulphide cross-linking to the
mAb surfaces.
Capping of the free thiol groups was carried out by nucleophilic substitution with iodoacetic acid
(Fig. 3.12) as described under Materials & Methods (section 4.3); this converted cysteine residues
into carboxymethylcysteine14. Protein integrity after carboxymethylation was checked by SDS-PAGE
(Fig. 3.13) and capping yield (75%) was assessed by AAA, using carboxymethylcysteine standards.
SH
COOH
I
COOH
OH , ∆
S
+
HI
Figure 3. 12 Nucleophilic attack of a thiol sulphur atom on the methylene group of iodoacetic acid14.
29 kDa
45 kDa
JX249A
66 kDa
JX249A after
carboxymethylation
97 kDa
116 kDa
Figure 3. 13 SDS-PAGE (10% acrylamide) analysis of
protein JX249A before (lane A) and after (lane C)
carboxymethylation of the cysteine side-chain thiol
groups; the following protein standards (lane B) were
employed: from higher to lower MW – myosin, βgalactosidase, phosphorylase B, bovine albumin,
ovalbumin and carbonic anhydrase.
205 kDa
A
B
C
Use of the capped JX249A in SPR assays as those described in the previous section did not,
unfortunately, solve the regeneration problems already observed (not shown). This indicated that
such problems were due either to intrinsic features of the protein – surface interactions or to the
presence of some free JX249A mixed with the carboxymethylated protein.
126
Antigenic peptides with non-natural replacements within the GH loop of FMDV
3.4
Solution affinity SPR analysis of the peptide antigens
Several features of anti-FMDV mAb interactions with either the peptide antigens or protein JX249A
prevented their dynamic characterisation by means of SPR kinetic analysis. Therefore, the
characterisation of the synthetic peptides under study was alternatively carried out by solution
affinity SPR analysis15,16, as previously described for the FMDV C-S30 peptides (previous chapter,
section 2.7). The same experimental set-up was employed under similar conditions (described in
Materials & Methods, section 4.3). A confirmative competition ELISA screening of the peptides was
carried out in parallel, as described in section 2.3.
Inhibition curves such as those exemplified in Fig. 3.14 were observed and affinity constants were
determined by the Cheng & Prusoff’s formula17, as previously exposed in section 2.7. These
constants are presented in Table 3.7, where the corresponding results from kinetic SPR analysis and
competition ELISA are included, for comparison purposes.
9.0E-08
Free Fab Concentration/M
8.0E-08
7.0E-08
A15scr
6.0E-08
A15(138F)
5.0E-08
A15(140P)
A15(142S)
4.0E-08
A15(FPS)
3.0E-08
A15
2.0E-08
1.0E-08
0.0E+00
0.0E+00
1.0E-07
2.0E-07
3.0E-07
4.0E-07
5.0E-07
6.0E-07
Peptide Concentration/M
Figure 3. 14 Inhibition curves in the SPR analysis of the interactions between Fab 4C4 and
peptide antigens in solution; a constant 80 nM total concentration of Fab was employed; peptide
A15Scr was included as a negative control.
Analysis of quantitative data in Table 3.7 shows that all the peptides are highly antigenic, taking the
native A15 antigen as reference. Differences observed between SPR data in kinetic or in solution
equilibrium experiments were not generally significant. This is a further evidence of the reliability of
the kinetic SPR methodology employed along the present study of FMDV peptides. More
pronounced differences were due either to mass transport-influenced kinetic data or to the fact that
immobilised mAb – free peptide and free mAb – free peptide interactions are intrinsically different
and therefore only peptide ranking should be compared.
127
SPR as a tool in the functional analysis of an immunodominant site in FMDV
Table 3.7 Affinity constants for the interactions between mAbs (Fab) SD6, 4C4 and 3E5 with the peptide antigens bearing combinations of the replacements Thr137→Ile,
Ala138→Phe, Ala140→Pro, Gly142→Ser and Thr148→Ile (columns labelled KA-sol.aff.).
3E5
Fab
4C4
SD6
Peptide
E
KA-sol.aff. /M-1
KA-kin /M-1
KA-sol.aff. /M-1
KA-kin /M-1
E
KA-sol.aff. /M-1
KA-kin /M-1
E
7
8
7
8
8
A15
++
++
++
5.4×10
1.9×10
9.4×107
6.3×
×10
2.0×
×10
2.0×
×10
A15(137I)
++
++
++
1.9×108
1.4×108
3.4×108
2.0×
×108
1.4×
×108
8.5×
×107
7
7
7
8
8
A15(138F)
++
++
++
2.2×10
9.8×10
4.0×108
3.6×
×10
2.1×
×10
2.1×
×10
A15(140P)
++
++
++
3.5×107
1.0×108
1.4×108
7.1×
×107
1.8×
×108
1.6×
×108
A15(142S)
++
++
++
1.1×107
1.6×107
2.5×107
7.3×
×107
6.2×
×107
2.7×
×107
A15(148I)
++
++
++
5.3×107
3.0×108
6.0×108
6.7×
×107
2.0×
×108
2.0×
×108
✖
A15(IF)
++
++
++
5.6×107
3.8×108
5.1×
×107
2.2×
×108
1.6×
×108
✖
✖
A15(IP)
++
++
++
5.9×107
2.1×
×108
1.3×
×108
6.1×
×107
7
8
7
8
✖
A15(IS)
++
++
++
1.8×10
2.8×10
5.2×
×10
1.9×
×10
1.3×
×108
✖
✖
A15(II)
++
++
++
7.6×107
9.1×
×107
2.3×
×108
2.0×
×108
✖
✖
A15(FP)
++
++
++
1.3×107
2.2×
×108
2.0×
×108
2.8×
×107
✖
A15(FS)
++
++
++
1.0×107
6.0×108
7.1×
×106
2.0×
×108
1.6×
×108
✖
✖
A15(FI)
++
++
++
3.0×108
2.1×
×107
2.1×
×108
1.9×
×108
✖
✖
A15(PS)
++
++
++
2.5×108
2.0×
×108
6.2×
×107
3.6×
×107
✖
✖
A15(PI)
++
++
++
4.0×107
6.0×
×107
1.8×
×108
1.9×
×108
✖
A15(SI)
++
++
++
6.1×106
2.9×108
1.6×
×107
1.9×
×108
1.2×
×108
✖
✖
✖
A15(IFP)
++
++
++
2.2×
×108
1.9×
×108
7.4×
×107
✖
A15(IFS)
++
++
++
1.0×107
4.3×107
2.0×
×107
8.5×
×107
5.9×
×107
✖
✖
A15(IFI)
++
++
++
2.9×107
5.6×
×107
2.4×
×108
1.8×
×108
✖
✖
A15(IPS)
++
++
++
1.7×108
1.4×
×108
2.2×
×108
7.4×
×107
✖
✖
✖
A15(IPI)
++
++
++
7.9×
×107
2.2×
×108
2.0×
×108
✖
✖
A15(ISI)
++
++
++
3.1×107
4.5×
×107
2.2×
×108
1.7×
×108
✖
A15(FPS)
++
++
++
9.7×106
4.8×108
2.1×
×108
1.3×
×108
6.5×
×106
✖
✖
A15(FPI)
++
++
++
3.3×107
5.0×
×107
1.3×
×108
1.8×
×108
✖
A15(FSI)
++
++
++
5.4×106
1.6×108
1.3×
×107
2.1×
×108
1.5×
×108
✖
A15(PSI)
++
++
++
8.4×107
1.4×109
2.1×
×108
1.5×
×108
2.5×
×107
✖
✖
A15(IFPS)
++
++
++
3.6×107
4.7×
×107
2.2×
×108
1.3×
×108
✖
✖
A15(IFPI)
++
++
++
4.5×108
7.1×
×107
2.0×
×108
1.8×
×108
✖
A15(IFSI)
++
++
++
7.7×107
5.5×108
2.1×
×108
1.2×
×108
1.9×
×107
✖
✖
A15(IPSI)
++
++
++
6.7×107
5.0×
×107
1.5×
×108
1.3×
×108
✖
✖
A15(FPSI)
++
++
++
1.4×107
1.7×
×107
2.2×
×108
1.3×
×108
✖
✖
A15(IFPSI)
++
++
++
9.8×107
1.7×
×108
1.9×
×108
3.8×
×107
128
Note: values measured by kinetic SPR are included in columns labelled KA-kin; total Fab concentrations were kept constant for each peptide dilution series and peptide concentrations varied
between 0 and 625nM; results from a competition ELISA screening of the peptides are also included (column E), where signs – , + and ++ stand for low (IC50 rel>30), medium (30<IC50rel<10)
and high antigenicity (IC50 rel<10), respectively.
Antigenic peptides with non-natural replacements within the GH loop of FMDV
The one-point mutants are closely equivalent regarding antigenicity. In spite of this, mAb SD6
slightly “resents” mutations A138→F and G142→S, while the other two mAbs only disfavour the
mutation within the RGD motif. The higher involvement of residue 138 in peptide-SD6 complexes
and the important role of the RGD triplet are both in agreement with these observations1,18-21. All
mAbs are “indifferent” to mutations T137→I and T148→I, which is consistent with the almost
absent participation of these residues in the mAb-peptide interactions21.
Generally, the multiply substituted peptides displayed similar affinities, close to those expected from
additive effects in the combination of the one-point mutations (Fig. 3.15). However, for mAbs 4C4
and 3E5, affinities of multiple mutants containing the G142→S replacement were systematically
superior to those expected from the “additivity rule”. Unless there was an undetected error in the
affinities of all peptides containing this mutation towards both 4C4 and 3E5, these differences
suggest a small positive synergistic effect in these multiple mutants. So it seems that mutations
outside the RGD triplet compensate the slight decrease in affinity provoked by replacing glycine by
serine in this important motif. Possibly, such compensation comes from peptide conformational
features, which are not as favourable in the A15(142S) single-point mutant as when the other
replacements are combined with the RSD motif.
Interestingly, peptides including multiple mutations within the GH loop of C-S8c1 FMDV still display
antigenicities as high as those of the native sequence. This is even more relevant when one of these
mutations is located in the RGD triplet and involves the substitution of a glycine by a serine residue.
For these reasons, the multiply substituted peptide A15(FPS) was submitted to further structural
studies, both by two-dimensional 1H-NMR of free peptide in solution and by X-ray diffraction
crystallography of its complex with antibody 4C4. Peptide A15(FPS) was chosen since it combines
the three more relevant mutations. Also, W. F. Ochoa and co-workers have observed very low
electron densities for residues placed at both ends of FMDV pentadecapeptides (residues≤137 and
≥148) preventing the unequivocal location of such residues in the structure of peptide-4C4
complexes. This has been interpreted as due to the lack of strong interactions between the terminal
residues and the antibody paratope21.
129
SPR as a tool in the functional analysis of an immunodominant site in FMDV
1.0E+08
9.0E+07
2.5E+08
A
-1
2.0E+08
Affinity constant/M
7.0E+07
6.0E+07
5.0E+07
4.0E+07
3.0E+07
1.5E+08
1.0E+08
5.0E+07
2.0E+07
FPSI
peptide
peptide
C
measured
expected
1.5E+08
1.0E+08
5.0E+07
FPSI
IFPSI
IPSI
IFSI
IFPI
IFPS
PSI
FSI
FPI
FPS
ISI
IPI
IPS
IFI
IFS
peptide
130
IFP
SI
PI
PS
FI
FS
FP
II
IS
IF
0.0E+00
IP
Figure 3. 15 Comparison between
measured and expected (for additive
combination of partial mutations) solution
affinity constants.
Affinity constant/M
-1
2.0E+08
IFPSI
IFSI
IPSI
IFPI
IFPS
FSI
PSI
FPI
ISI
FPS
IPI
IFI
IPS
IFS
SI
IFP
PI
FI
PS
FS
II
FP
IS
IF
IP
FPSI
0.0E+00
IFPSI
IFSI
IPSI
IFPI
IFPS
FSI
PSI
FPI
ISI
FPS
IPI
IFI
IPS
IFS
SI
IFP
PI
FI
PS
FS
II
FP
IF
0.0E+00
IS
1.0E+07
IP
-1
8.0E+07
Affinity constant/M
measured
expected
B
measured
expected
Antigenic peptides with non-natural replacements within the GH loop of FMDV
3.5
Two-dimensional 1H-NMR analysis of peptide A15(FPS)
The structural features of peptide A15(FPS) in solution were analysed by 2D – 1H NMR22, under
conditions identical to those described in section 2.8 for C-S30 peptides. In the present case, peptide
A15(FPS) was studied both in water and in 30% TFE, through TOCSY and NOESY/ROESY
experiments (Fig. 3.16)23-25. The chemical shifts measured are presented in Table 3.8.
144L
145A
150T
139S
149T
146H
142S
138F
147L
148T
137T
141R
143D
149T -150T
145A -146H
138F -139S
142S -143D
142S -144L
Figure 3. 16 Expansions of the TOCSY (above) and ROESY at 200 ms (below) spectra of
peptide A15(FPS) in water at 25 oC.
131
SPR as a tool in the functional analysis of an immunodominant site in FMDV
Table 3.8 Chemical shifts measured in the 2D 1H-NMR analysis of of peptide A15(FPS) at 25 oC.
A15(FPS)
Residue
H 2O
H
H
α
30% TFE
β
4.76
3.84
3.78
2.30
1.99
3.73
3.57
4.68
3.75
4.36
2.30
1.95
8.35
4.30
1.86
1.75
Ser
8.29
4.40
Asp
8.49
4.68
8.07
4.26
3.90
3.85
2.85
2.80
1.60
142
144
Leu
145
Ala
8.08
4.25
1.30
146
His
8.29
4.69
Leu
8.25
4.42
3.27
3.15
1.60
1.90
3.67
3.60
1.65
3.20
7.31
4.38
8.03
4.28
1.90
1.77
8.06
4.37
8.34
4.66
3.97
3.89
2.85
0.90
0.85
8.01
4.23
7.96
4.17
8.04
4.63
8.07
4.36
7.98
7.93
148
Thr
8.28
4.45
4.25
7.60
7.18
0.90
0.85
1.26
149
Thr
8.23
4.46
4.30
1.20
150
Thr
8.12
4.33
4.28
1.20
147
7.30
8.04
8.20
Arg
3.09
4.34
7.29
141
4.03
7.04
6.84
1.16
4.64
4.22
3.07
Pro
3.05
8.25
4.28
4.58
140
Other
4.23
8.24
8.34
8.39
Ser
Hβ
3.03
Thr
139
Hα
Other
Phe
137
138
4.23
Tyr
H
N
H
7.14
6.80
1.12
136
143
N
1.68
3.22
7.36
1.70
1.62
1.38
0.93
0.88
4.42
3.38
3.24
1.78
1.66
4.34
8.06
7.33
0.93
0.90
1.26
4.47
4.28
1.25
ambiguous due to peak overlap
0.3
0.2
∆δHα
α/ppm
∆δ
0.1
0
Y
T
F
S
P
R
S
D
L
A
H
L
T
T
T
-0.1
-0.2
-0.3
Water
30% TFE
-0.4
-0.5
residue
Figure 3. 17 Conformational chemical shifts observed for peptide A15(FPS) in water and
30% TFE, at 25 oC.
132
Antigenic peptides with non-natural replacements within the GH loop of FMDV
Conformational chemical shifts
Fig. 3.17 shows absolute conformational chemical shifts slightly higher for peptide A15(FPS) than
those previously observed for the C-S30 peptides (chapter 2). The global shape of the plots
resembles those observed for peptides C-S30 or C-S8c1 under identical conditions. However, the
region containing the open turn at the Arg-Gly-Asp triplet and the following short helix usually
observed in FMDV peptides26,27 is longer for peptide A15(FPS). This region extends in a continuous
manner from position 140 (in which an alanine is replaced by a proline) to the
146
His-147Leu
positions, including the altered Arg-Ser-Asp motif. The almost identical plots obtained either in
water or in 30% TFE suggest that peptide A15(FPS) is not particularly sensitive to structure-inducing
solvents, and thus conformationally stable.
NOEs observed
The above observations are further supported by the NOEs observed for this peptide (Fig. 3.16):
despite its globally disordered structure, peptide A15(FPS) presented some interesting NOEs
corresponding to NNi,i+1 and NNi,i+2 connectivities, located precisely in the short helical path
mentioned above (Fig. 3.18). These NOEs reinforce that helical character is indeed present in the
142
S – 146H stretch.
Compared to what was described in chapter 2 for C-S30 peptides, peptide A15(FPS) is more prone
to adopt a defined structure in solution. In fact, linear peptide A15S30 did not exhibit any clearly
observable NOEs in water. Only in the presence of a structuring solvent (TFE) did the C-S30
peptide exhibit NOEs similar to those observed in the case of A15(FPS). As for the more antigenic
version cyc16S30, NOEs coincident to those described for A15(FPS) were observed both in water
and 30% TFE. These observations further support a significant relationship between a stable peptide
conformation in solution and antigenicity.
Solvent
H2O
30% TFE
NOE
αNi,i+1
NNi,i+1
NNi,i+2
βNi,i+1
Y T F S P R S D L A H L T T T
αNi,i+1
NNi,i+1
NNi,i+2
βNi,i+1
Figure 3. 18 Distribution of NOEs observed for peptide A15(FPS) in water and 30% TFE; NOE
relative intensity is represented by the thickness of the bars and dotted lines correspond to possibly
overlapping NOEs.
133
SPR as a tool in the functional analysis of an immunodominant site in FMDV
3.6
X-ray diffraction crystallography analysis of a peptide – antibody complex
3.6.0
Introduction
X-ray diffraction crystallography was employed in the structural study of the complex formed by
peptide A15(138F,140P,142S) and the Fab fragment of mAb 4C4. This study was performed in
collaboration with W. F. Ochoa, Dr. N. Verdaguer and Dr. I. Fita, who had been working on similar
peptide – antibody complexes1,19-21, including those between Fab 4C4 and FMDV peptides A15,
A15(138F), A15(140P) and A15(142S). The present structural study had the purpose of analysing
in detail eventual structural changes in the paratope – epitope interaction region, caused by the
simultaneous introduction of three important mutations in the FMDV GH loop. Also, we were
interested in observing how both antibody and peptide molecules managed to fit together forming a
stable complex, in the presence of such amino acid replacements.
3.6.1
Determination of protein structure by X-ray crystallography28
Solving the three-dimensional structure of proteins by X-ray crystallography requires well-ordered
and strongly X-ray diffracting crystals. However, well-ordered protein crystals are difficult to grow,
due to the large size and irregular surface of protein molecules. These pack in a crystal forming large
channels that occupy more than half the volume of the crystal and are filled with solvent molecules.
Different protein molecules within a crystal seldom are in direct contact with each other and
interactions are indirect, through several solvent layers. This feature is the reason why structures of
proteins determined by X-ray crystallography are considered as the same as those for the
biologically active proteins in solution. The high solvent content makes protein crystals much less
resistant than their inorganic counterparts and protein crystallisation is difficult to achieve, being
critically dependent on factors such as pH, temperature, protein concentration and purity, solvent,
protein
solution
glass
cover
ionic strength and precipitant. Crystals
are formed upon slow precipitation from
supersaturated
solutions.
The
most
widely employed technique for protein
crystallisation is the hanging-drop vapour
seal
diffusion method (Fig. 3.19), in which a
precipitant
solution
Figure 3. 19 Scheme of the hanging-drop method used in
protein crystallisation (adapted from reference 30).
droplet of protein solution (plus adequate
additives) is placed on a glass cover,
facing down a larger reservoir of a similar
solution
(with
higher
precipitant
concentration and without protein). The
droplet looses water gradually by vapour
134
Antigenic peptides with non-natural replacements within the GH loop of FMDV
diffusion to the reservoir and precipitation occurs.
Crystals are submitted to X-ray diffraction analysis. X-rays are short wavelength electromagnetic
radiation, resulting from electronic transitions from excited to low energy levels. Conventional X-ray
sources are high-voltage tubes with a metal plate (anode) that is bombarded with accelerating
electrons, X-rays of specific wavelength being emitted. Rotating anode X-ray generators are the most
commonly found in X-ray crystallography laboratories. Much more powerful X-ray generators are
synchrotron storage rings, in which electrons or positrons travel close to the speed of light. Strong
radiation is emitted at all wavelengths, covering the X-ray spectrum. After passing through a
collimator, monochromatic X-ray radiation is produced with an intensity several orders of
magnitude higher than that produced by conventional X-ray sources. This allows very short
exposure times in diffraction experiments and useful data can be collected from small and more
sensitive crystals.
The primary beam must strike the crystal from several different directions so that all possible
diffraction spots are produced. The crystal is therefore rotated during the experiment and the
diffraction spots are recorded either on film or by an electronic detector. Electronic area detectors,
such as the imaging plate detector, are a kind of electronic film where a plate covered with a
photosensitive material is used to store the diffraction spots. The image thus produced is then
digitised into a computer.
When a crystal is put in the path of an X-ray primary beam, some of the X-rays interact with the
electrons on each crystal atom, causing them to oscillate. The oscillating electrons, in turn, emit new
X-rays in all directions, a phenomenon known as scattering. Due to the regular three-dimensional
arrangement of atoms in a crystal, the radiations emitted by the different electrons interfere with
each other and, in most cases, cancel each other out. Some of them interfere positively, giving
beams that are recorded as diffraction spots (Fig. 3.20).
A
diffracted
beams
primary beam
X-ray
source
crystal
detector
Figure 3. 20 Representation
of a diffraction experiment: A.
diffracted X-ray beams after
the primary beam hits the
crystal; B. a diffraction pattern
from a protein crystal (adapted
from reference 30).
B
135
SPR as a tool in the functional analysis of an immunodominant site in FMDV
Thus, each spot is originated by interference of all X-rays emerging from all crystal atoms with
identical diffraction angle. According to Bragg’s law, diffraction is regarded as reflection of the
primary beam by a set of parallel planes through the unit cells of the crystal. X-rays reflected from
adjacent planes travel different distances and diffraction only occurs when this difference equals the
wavelength of the beam. The position of the diffraction data on the detector film relates each spot to
a specific set of planes through the crystal, from which the size of the unit cell can be determined.
Each recorded spot is related to a diffracted beam characterised by its amplitude, wavelength and
phase. These three parameters are needed to determine the spatial arrangement of the atoms.
However, the phase is lost in X-ray diffraction experiments and this is the major problem in X-ray
crystallography. The classical way to circumvent this problem in protein X-ray crystallography is
based on the preparation of heavy atom protein derivatives. X-ray diffraction data from the protein
alone, from the heavy atom alone and from the heavy atom protein derivative are then used to
attribute initial phases to the protein atoms.
A simpler method to determine initial phases to work with is the molecular replacement method,
which requires a known protein structure similar to the molecule under study. The phases belonging
to the search model are assigned as the initial phases of the new protein structure and an electron
density map is calculated.
Then, with the aid of computer graphics, a trial-and-error process is started in order to build up a
model: the polypeptide main chain and side chains are matched with the electron densities and
computer-aided crystallographic refinement of the model is performed. In this refinement, the model
is slightly changed to minimise the differences between the experimental diffraction data and the
calculated model. This difference can be given in terms of the R factor, a residual disagreement that
is zero for total agreement and around 0.59 for total disagreement. The R factor lies between 0.15
and 0.20 for high quality data.
Non-zero R factors are seldom due to errors in the protein model. Rather, they derive from
imperfections in the experimental data, such as variations in protein conformation, inaccurate
solvent corrections or orientation of the micro-crystals. Therefore, the final model is an average of
molecules that differ slightly both in conformation and orientation, not corresponding exactly to the
real crystal.
136
Antigenic peptides with non-natural replacements within the GH loop of FMDV
General structure of immunoglobulins28
3.6.2
The basic structure of all immunoglobulins (Ig) involves two identical heavy chains and two identical
light chains, linked through disulphide bonds (Fig. 3.21). The major type of immunoglobulin in
human serum is the class G Ig (IgG), which is a monomer of the basic structural unit. The IgG
polypeptide chain is divided into domains of 110 amino acid residues each; the light chains contain
two of such domains and the heavy chains contain four. A light chain is composed of a variable
amino-terminal domain (VL) and a constant carboxy-terminal (CL), whereas a heavy chain is built up
from an amino-terminal variable domain (VH) followed by three constant domains (CH1, CH2 and
CH3). The variable VL and VH domains coincide with the antigen binding sites of the IgG and are not
uniformly variable along their lengths: three sub-domains, called hypervariable or complementarity
determining regions (CDR1, CDR2 and
antigen binding sites
CDR3), show much higher variability,
both in sequence and size. The CDRs
VH
VL
hinge
region
CH1
are the regions that determine the
CDR1
CDR2
CDR3
specificity of the antigen – antibody
interactions. Complete IgG molecules
CL
are difficult to crystallise, but their
light
chain
CH2
CH3
+
papain
enzymatic digestion with papain or
+
pepsin cleaves the Ig by the hinge
region, with one Fc and two identical
heavy
chain
Fab
Fab
Fc
Figure 3. 21 Basic structure of an IgG and its fragments,
produced by enzymatic digestion with papain (partially
adapted from reference 30).
Fab fragments being obtained (Fig.
3.21). High resolution X-ray structural
information on these fragments has
shown all domains to have a similar
structure, either in light or heavy chains, either in variable or constant regions. This structure is the
so-called immunoglobulin fold, where a constant domain is formed by seven anti-parallel strands,
four of which form one β sheet and the remaining three form another.
Both β sheets are closely packed together in a barrel-like arrangement (Fig. 3.22 A). The loops
connecting the strands are short and thus the majority of the framework invariant residues are in the
β sheets. These structural features are similar for both heavy and light chains. Variable domains are
structurally similar to constant domains, but contain nine instead of seven β strands. The two
additional strands are placed in the important loop region that contains the hypervariable CDR2
(Fig. 3.22 B). These extra strands provide the scaffold that renders CDR2 closer to the other two
hypervariable loops CDR1 and CDR3. CDR2 and CDR3 are hairpin loops linking different strands
in the five-strand β sheet, while CDR1 is a cross-over between one strand from the five-strand sheet
and another from the four-strand sheet.
137
SPR as a tool in the functional analysis of an immunodominant site in FMDV
CDR1
N
CDR3
N
CDR2
C
The
Figure
3.
22
immunoglobulin fold: A.
general structure of a
constant IgG domain; B.
general structure of a
variable
IgG
domain
(adapted from reference
30).
C
A
B
The Fab fragment is the “arm” of the IgG molecule
antigen
that contains an intact antigen binding site. In this
fragment, the heavy and light chains are tightly and
extensively associated, in such a way that CL
associates with CH1 and VL with VH. Thus, a Fab
fragment consists of two globular regions, one with
the two constant domains and the other with the two
CDRs
VL
VH
CL
CH1
variable domains (Fig. 3.23). While the constant
domains associate by close interactions between the
almost perpendicular four-strand sheets from CH and
CL, the variable domains associate in a very different
manner. In this case, the interaction area is formed
by the five-strand β sheets, almost parallel to each
other, and defining a barrel structure of eight (four
from each five-strand sheet) antiparallel β strands.
This allows the CDR loops from both variable
domains to be located at the same end of the barrel,
forming the complete antigen binding site.
138
Figure 3. 23 Schematic representation of
a Fab fragment: VL/VH and CL/CH1 domains
associate in such a way that CDRs can
“grab” the antigen.
Antigenic peptides with non-natural replacements within the GH loop of FMDV
3.6.3
Molecular structure of the A15(FPS) – 4C4 complex in the crystal state
The protocols previously described for the crystallisation of similar FMDV peptide – Fab 4C4
complexes20,21 proved to be readily applicable to the present case (see Materials & Methods, section
4.4.2) and crystals as those shown in Fig. 3.24 were formed. Despite the difficulties found in growing
clean and perfect crystals, good diffraction data were acquired using synchrotron radiation at the
European Synchrotron Radiation Facility at Grenoble, France.
Figure 3. 24 Crystals of the A15(FPS) – 4C4
complex.
After evaluating and internally scaling the diffraction data29 (Table 3.9), initial phases were assigned
taking the known structure of Fab 4C4 as the search model20,30. The model was initially subjected to
rigid body refinement
and then treating each constant and variable domains as independent
structural units. The computed electron density maps clearly showed extra density at the antigen
binding site, corresponding to the peptide ligand. The peptide was then added to the model
structure, which was improved by cycles of manual rebuilding with program O31 and refinement with
the CNS package32. The final model had an R factor of 0.22 at a 2.3 Å resolution (Table 3.9) and is
represented in Fig. 3.25. The epitope – paratope contacts through hydrogen bonds are listed in
Table 3.10 and matched those previously observed with the native peptide A15 in complex with the
same mAb1.
A better illustration of the present structural study requires a global appreciation of the complex, as
well as of those formed between the same mAb and the three relevant single-point mutants,
previously solved by W. F. Ochoa and co-workers21. All four peptides were shown to interact with
Fab 4C4. As can be seen in Fig. 3.26, the three single-point mutants and the triple mutant all adopt
a similar quasi-cyclic conformation, also shared by the native sequence A151,21. This conformation
seems therefore to be a key feature in the antibody – FMDV peptide recognition process. A
stereoview of the A15(FPS) peptide fold in complex with Fab 4C4 is shown in Fig. 3.27.
139
SPR as a tool in the functional analysis of an immunodominant site in FMDV
Table 3.9 Crystallisation and diffraction data of
the A15(FPS) – 4C4 complex.
Crystalisation and data collection
Space group
P212121
Cell parameters (Å)
48.417
68.792
145.404
25 – 2.2
Resolution (Å)
Overall Completeness (%)
99.7
Rsymm (%)
7.2
10.2
Average I/σ
Total # of residues
Fab
429
13
Peptide
Total
#
of
solvent
molecules
269
Volume solvent (%)
48.81
Diffraction agreement
Resolution (Å)
15 – 2.3
# of reflections
22153
Rfree
0.266
Rfactor
0.225
Figure 3. 25 Structure of the A15(FPS) – 4C4
complex (only Fab variable domains are
shown); peptide side chains and some peptide
– antibody hydrogen bonds are shown in more
detail (structures built with program SETOR33).
rms deviations from ideal distance
Bond length (Å)
0.0179
Bond angle (º)
2.2193
Average thermal factor (Å)
Fab
24.8
Peptide
24.3
Stereochemistry of main chain
Omega angle std. dev.
2.0
Bad contacts/100 res.
0.8
Zeta angle std. dev.
2.0
Stereochemistry of side chain
Chi-1 pooled std. dev.
140
11.5
Figure 3. 26 Superposition of the structures
adopted by peptides A15(138F) – red,
A15(140P) – dark blue; A15(142S) – orange
and A15(FPS) – light blue, when complexed
with Fab 4C4 (structures built using SETOR33).
Antigenic peptides with non-natural replacements within the GH loop of FMDV
Table 3.10 Hydrogen bonds between antigenic peptide
A15(FPS) and the Fab fragment of mAb 4C4.
Bond interaction site in
Peptide
Fab
Location
Tyr136 O
Asn34 Nδ2
L1
Distance
(Å)
2.6
Thr137 O
Asp104 N
H3
2.8
Thr137 Oγ1
Ser103 Oγ
H3
3.0
Ser139 Oγ
Asn96 Oδ1
L3
2.5
Arg141 O
Asp98 N
L3
2.9
Arg141 Nη1
Asn96 O
L3
3.3
Arg141 Nη2
Asn96 Nδ2
L3
3.1
Asp143 O
Tyr59 Oη
H2
3.8
Asp143 Oδ1
Thr50 Oγ1
H2
2.7
Asp143 Oδ1
Arg99 Nε
H3
2.7
Asp143 Oδ2
Arg99 Nη2
H3
2.7
His146 Nδ1
Tyr59 Oη
H2
2.9
His146 Nδ2
Thr33 Oγ1
H1
3.0
Note: letters L and H in the location column stand for light and
heavy chain, respectively, and are followed by numbers indicating
the CDR where the antibody residue is located.
Figure 3. 27 Stereoview of the Fo-Fc omit map of peptide A15(FPS) at 2.3 Å resolution; the peptide final
model, including water molecules, was also shown for clarity; residues 149 and 150 were not considered in the
model (image generated with program SETOR33).
141
SPR as a tool in the functional analysis of an immunodominant site in FMDV
The similarity found in both peptide folding and epitope – paratope interactions for all FMDV
peptide – antibody complexes studied so far is remarkable. In view of this, we can devise the
following requirements for the recognition of FMDV peptides by anti-site A mAbs:
Fab-peptide interactions
Important interactions between peptide residues and the Fab cannot be bypassed, which is
confirmed by the absence of Fab-recognition of peptide mutants where key residues had been
replaced1,34,35. These interactions mainly involve peptide residues 141, 143 and 146 (Table 3.10).
They are similar to several other resolved structures1,20,21 and strong hydrogen bonds are seen to
stabilise the complex scaffold. Other observed interactions, particular to each complex, can explain
affinity differences between the different complexes, but do not seem to be absolutely necessary.
Peptide conformation
The hydrogen bonds between peptide residues and Fab do not seem to be sufficient to ensure a
strong interaction, since a precise peptide conformation seems to be an important requirement for
peptide-Fab union. Such conformation involves two important features:
Hydrophobic cavity
In all peptides studied a hydrophobic cavity was observed1,18-21, mainly formed by
residues 138, 144 and 147. These residues engage in strong hydrophobic interactions
through their side chains, stabilising peptide conformation. Mutations at these
positions would imply the loss of such cavity and, therefore, a decrease in the stability
of the peptide-Fab complex. However, that does not occur with the
138
Ala→Phe
replacement; in this case, the Phe side chain is oriented into the cavity, and not only
does not disrupt hydrophobic interactions, but in fact stabilises the cavity itself (Fig.
3.26, 3.27).
Intrapeptide interactions
There is a set of interactions between the different peptide residues which contribute to
peptide folding, such as hydrogen bonds between the different nitrogen and oxygen
atoms of the main chain and also the presence of several water molecules bridgebonding peptide residues. These are key interactions that restrict mutations to those
amino acids able to preserve this type of structural arrangement. A remarkable
example is the
142
Gly→Ser mutation, in which the side chain hydroxyl group of Ser
replaces a water molecule present in the structures where
142
Gly is conserved (Fig.
3.28), maintaining the turn characteristic of the RXD motif (X=Gly or Ser). Pro at
position 140 also helps in the stabilisation of such a turn.
142
Antigenic peptides with non-natural replacements within the GH loop of FMDV
Figure 3. 28 Detailed view of the RXD turn, with X=Gly and X=Ser for the upper
[peptide A15(138F)] and lower [peptide A15(142S)] structures, respectively; the
corresponding Fo-Fc omit maps are also shown. As it can be seen, the RGD turn is
held up by hydrogen bridging between Ser139 O – H2O – Leu144 N (above), while
the RSD turn is stabilised by similar hydrogen bridging between Ser139 O – Ser142
Oγ – Leu144 N (below). Structures were built with SETOR33.
143
SPR as a tool in the functional analysis of an immunodominant site in FMDV
3.7
Recapitulation
This chapter was focused on the study of 15-residue peptides from the GH loop of FMDV. The
peptide sequences were based on the reference FMDV strain C-S8c1, bearing combinations of the
replacements
137
T→I,
138
A→F,
140
A→P,
142
G→S and
148
T→I. These amino acid replacements were
interesting in the sense that the corresponding single-point mutants had been previously found to be
significantly antigenic towards several anti-GH loop neutralising mAbs1. Therefore, the question was
raised whether multiple combination of these replacements could lead to positive synergistic effects,
yielding promising peptide antigens. Also, three such replacements deserved further attention:
position 138 was known to play a role in intrapeptide interactions1,20 and the much larger size of the
Phe side chain seemed unlikely to be “ignored” by antibodies; the introduction of a Pro in the loop,
with its peculiar structural behaviour, deserved to be analysed; and the effect of replacing Gly by Ser
in the highly conserved, key RGD motif, also captured our attention.
These peptides were fully characterised as FMDV antigens by means of SPR studies, towards three
anti-site A mAbs. It was immediately observed that this peptide family was quite different from the
one discussed in chapter 2. In fact, all these peptides displayed high antigenicities, which prevented
the kinetic study of their interactions with the mAbs due to mass-transport limitations. High
association rate constants, very slow dissociations and/or incomplete surface regeneration were
persistently observed and alternative solution affinity SPR analyses were performed. These
confirmed the high peptide – antibody affinities expected, generally comparable to or even higher
than those displayed by the native sequence represented by peptide A15.
The fact that these multiple mutants were fully recognised by anti-site A neutralising mAbs was quite
interesting and led to other questions, such as how did the mutations affect peptide conformation
and how did the antibody paratope adapt to these mutations. We approached the first question
through a two-dimensional 1H-NMR study of peptide A15(FPS) in solution and the second one by
means of an X-ray diffraction crystallography study of the complex formed between mAb 4C4 and
the same peptide. The NMR characterisation of peptide A15(FPS) in solution showed that this
peptide had a conformational behaviour quite similar to that previously observed for the native
sequence A1526,27. Data suggested an open turn in the RSD region followed by an incipient short αhelix up to residue 147, features which had been previously recognised in peptide A15 and
regarded as antigenically relevant26,27,34.
144
Antigenic peptides with non-natural replacements within the GH loop of FMDV
The diffraction study of the A15(FPS) – 4C4 complex showed that the pattern of antibody – antigen
interactions is identical for all FMDV peptides studied so far1,18-21. Thus, a stable mAb – peptide
complex can be formed as long as some key requisites are fulfilled. These involve specific residues
committed in direct epitope – paratope contacts (141Arg,
143
Asp,
146
His) and residues able to stabilise
a particular peptide conformation. This conformation corresponds to a quasi-cyclic folding around a
hydrophobic cavity defined by residues 138, 144 and 147 and to other important intrapeptide
hydrogen bonds defining the central open turn involving positions 141, 142 and 143. Amino acid
replacements that not only do not disrupt, but even help to promote these essential requirements for
mAb recognition can yield peptides with significant reactivity towards anti-FMDV neutralising mAbs
and thus useful as FMDV antigens.
References
1 Verdaguer, N., Sevilla, N., Valero, M. L., Stuart, D., Brocchi, E., Andreu, D., Giralt, E., Domingo, E.,
Mateu, M. G. and Fita, I. (1998) A similar pattern of interaction for different antibodies with a major
antigenic site of foot-and-mouth disease virus: implications for intratypic antigenic variation, J. Virol.
72, 739-748.
2 Valero, M. L. “Mimetización estructural e inmunogénica del sitio antigénico principal del virus de la
fiebre aftosa” (Ph. D. Thesis), Department of Organic Chemistry – University of Barcelona: 1997.
3 Mateu, M. G., Valero, M. L., Andreu, D. and Domingo, E. (1996) Systematic replacement of amino
acid residues within an Arg-Gly-Asp containing loop of foot-and-mouth disease virus and effect on cell
recognition, J. Biol. Chem. 271, 12814-12819.
4 O’Shannessy, D. J. and Winzor, D. J. (1996) Interpretation of deviations from pseudo-first-order
kinetic behavior in the characterization of ligand binding by biosensor technology, Anal. Biochem.
236, 275-283.
5 Schuck, P. (1997) Reliable determination of binding affinity and kinetics using surface plasmon
resonance biosensors, Curr. Op. Biotech. 8, 498-502.
6 Hall, D. R., Cann, J. R. and Winzor, D. J. (1996) Demonstration of an upper limit to the range of
association rate constants amenable to study by biosensor technology based on surface plasmon
resonance, Anal. Biochem. 235, 175-184.
7 Karlsson, R. (1994) Real-time competitive kinetic analysis of interactions between low-molecularweight ligands in solution and surface-immobilized receptors, Anal. Biochem. 221, 142-151.
8 Albericio, F., Andreu, D., Giralt, E., Navalpotro, C., Pedroso, E., Ponsati, B. and Ruiz-Gayo, M.
(1989) Use of the Npys thiol protection in solid phase peptide synthesis, Int. J. Peptide Protein Res.
34, 124-128.
9 Benito, A., Mateu, M. G. and Villaverde, A. (1995) Improved mimicry of a foot-and-mouth disease
virus antigenic site by a viral peptide displayed on β-galactosidase surface, Biotechnology 13, 801804.
10 Carbonell, X., Benito, A. and Villaverde, A. (1996) Converging antigenic structure of a recombinant
viral peptide displayed on different frameworks of carrier proteins, FEBS Lett. 397, 169-172.
145
SPR as a tool in the functional analysis of an immunodominant site in FMDV
11 Feliu, J. X., Benito, A., Oliva, B., Avilés, F. X. and Villaverde, A. (1998) Conformational flexibility in a
highly mobile protein loop of foot-and-mouth disease virus: distinct structural requirements for integrin
and antibody binding, J. Mol. Biol. 283, 331-338.
12 Feliu, J. X. and Villaverde, A. (1998) Engineering of solvent-exposed loops in Escherichia coli βgalactosidase, FEBS Lett. 434, 23-27.
13 Andersson, K., Hamalainen, M. and Malmqvist, M. (1999) Identification and optimization of
regeneration conditions for affinity-based biosensor assays. A multivariate cocktail approach, Anal.
Chem. 71, 2475-2481.
14 Lapko, V. N., Jiang, X. Y. and Smith, D. L. (1998) Surface topography of phytocrome A deduced
from specific chemical modification with iodoacetamide, Biochemistry 37, 12526-12535.
15 Nieba, L., Krebber, A. and Plükthun, A. (1996) Competition BIAcore for measuring true affinities:
large differences from values determined from binding kinetics, Anal. Biochem. 234, 155-165.
16 “BIAapplications Handbook”, (Pharmacia Biosensor AB, 1994) Uppsala, Sweden.
17 Lazareno, S. and Birdsall, N. J. (1993) Estimation of competitive antagonist affinity from functional
inhibition curves using the Gaddum, Schild and Cheng-Prusoff equations, British J. Pharmacol. 109,
1110-1119.
18 Verdaguer, N., Mateu, M. G., Andreu, D., Giralt, E., Domingo, E. and Fita, I. (1995) Structure of the
major antigenic loop of foot-and-mouth disease virus complexed with a neutralizing antibody: direct
involvment of the Arg-Gly-Asp motif in the interaction, EMBO J. 14, 1690-1696.
19 Verdaguer, N., Mateu, M. G., Bravo, J., Domingo, E. and Fita, I. (1996) Induced pocket to
accomodate the cell attachment site Arg-Gly-Asp motif in a neutralizing antibody against foot-andmouth disease virus, J. Mol. Biol. 256, 364-376.
20 Ochoa, W. F., Kalko, S., Mateu, M., Gomes, P., Andreu, D., Domingo, E., Fita, I. and Verdaguer, N.
(2000) A multiply substituted GH loop from foot-and-mouth disease virus in complex with a
neutralizing antibody: a role for water molecules, J. Gen. Virol. 81, 1495-1505.
21 Ochoa, W. F. et al., manuscript in preparation.
22 Wüthrich, K. “NMR of proteins and nucleic acids”, Wiley, New York (1986).
23 Braunschweiler, L. and Ernst, R. R. (1983), J. Magn. Reson. 53, 521.
24 Kumar, A., Ernst, R. R. and Wüthrich, K. (1980), Biochem. Biophys. Chem. Comm. 95, 1.
25 Bothner-By, A. A., Stephens, R. L., Lee, J., Warren, C. D. and Jeanloz, R. W. (1984) Structure
determination of a tetrasaccharide: transient nuclear overhauser effects in the rotating frame, J. Am.
Chem. Soc. 106, 811-813.
26 Haack, T., Camarero, J. A., Roig, X., Mateu, M. G., Domingo, E., Andreu, D. and Giralt, E. (1997) A
cyclic disulfide peptide reproduces in solution the main structural features of a native antigenic site of
foot-and-mouth disease virus, Int. J. Biol. Macromol. 20, 209-219.
27 Valero, M. L., Camarero, J. A., Haack, T., Mateu, M. G., Domingo, E., Giralt, E. and Andreu, D.
(2000) Native-like cyclic peptide models of a viral antigenic site: finding a balance between rigidity
and flexibility, J. Mol. Recognit. 13, 5-13.
28 Branden, C. and Tooze, J., “Introduction to protein structure”, Garland Publishing Inc., New York
(1991).
29 Otwinowsky, Z. and Minor, W. (1997) Processing of X-ray diffraction data collected in oscillation
mode, Methods Enzymol. 276, 307-326.
30 Rossman, M. G. (Ed.) “The molecular replacement method”, Gordon & Breach, New York (1972).
31 Jones, T. A., Zou, J. Y., Cowan, S. W. and Kjeldgaard, M. (1991) Improved methods for building
protein models in electron density maps and the location of errors in these models, Acta Crystallogr. A
47, 110-119.
32 Brünger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang,
J.-S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T. and Warren, G.
L. (1998) Acta Crystallogr. D 54, 905-921.
33 Evans, S. V. (1993) SETOR: hardware-lighted three-dimensional solid model representations of
macromolecules, J. Molec. Graphics 11, 134-138.
34 Mateu, M. G. (1995) Antibody recognition of picornaviruses and escape from neutralisation: a
structural view, Virus Res. 38, 1-24.
35 Domingo, E., Verdaguer, N., Ochoa, W. F., Ruiz-Jarabo, C. M., Sevilla, N., Baranowski, E., Mateu,
M. G. and Fita, I. (1999) Biochemical and structural studies with neutralising antibodies raised against
foot-and-mouth disease virus, Virus Res. 62, 169-175.
146
Conclusions
SPR as a tool in the functional analysis of an immunodominant site in FMDV
148
Conclusions
1
A reliable SPR method for the kinetic analysis of binding between peptide antigens and
immobilised antibodies has been established, despite the small size of the analytes. The
interactions were well described by a simple 1:1 bimolecular interaction model and data
were self-consistent, reproducible and in total agreement with previous competition ELISA
screenings.
SPR kinetic analysis was, therefore, proven to be adequate for the functional
characterisation of small FMDV peptide antigens.
2
Different combinations, reproduced by linear 15-residue peptides, of the four amino acid
replacements found in the GH loop of FMDV isolate C-S30 were seen to be additive in
ELISA and kinetic SPR assays. Whereas increasing the size of the C-S30 peptide did not
cause any marked effect, overnight incubation with mAb in solution led to an antigenic
reversion of peptide A15S30 towards mAbs 4C4 and 3E5, but not SD6. A similar effect was
observed upon peptide cyclization. Solution NMR studies of both linear and cyclic C-S30
peptides showed that structural features formerly associated with peptide antigenicity were
more pronounced in the cyclic peptide.
Although the FMDV GH loop is a continuous (i.e., linear) antigenic region usually well
mimicked by linear peptides, conformation seems to have subtle, but important, effects
in the reproduction of recognition events involving peptides derived from field isolate
C-S30.
3
Antigenic FMDV peptides, comparable to or even better antigens than the wild type
sequence, can be obtained by combination of adequate amino acid replacements. The
peptide mutants display conformational and antibody – binding behaviour similar to those
characterising the native peptide.
A stable mAb – peptide complex can be formed as long as key requisites are fulfilled,
involving both residues committed in direct mAb – peptide contacts (141Arg,
146
143
Asp,
His), and residues able to promote/stabilise a quasi-cyclic folding held up by a
hydrophobic cavity (defined by positions 138, 144 and 147) and by intra-peptide
hydrogen bonds delineating an open turn at the central region (positions 141, 142 and
143).
149
SPR as a tool in the functional analysis of an immunodominant site in FMDV
150
4. Materials & Methods
SPR as a tool in the functional analysis of an immunodominant site in FMDV
152
Materials & Methods
4.1 General procedures
4.1.1
Solvents and chemicals
Supplier
Amino acids and resins for SPPS
Advanced Chemtech, Propeptide,
Bachem Feinchemikalien AG and
Calbiochem-Novabiochem
Coupling reagents for SPPS
DIP, HOBt
TBTU
Fluka
Propeptide/Neosystem
Solvents and other reagents for SPPS
DCM, Normasolv p. a.
DMF, peptide synthesis
NMP, peptide synthesis
NMM, peptide synthesis
DIEA, p. s.
Piperidine, p. a.
TFA, p. s.
Tert-butylmethylether* >99%
Water
MeOH, MeCN, hplc grade
Glacial AcOH, p. a.
Anisole, p. s.
1,2-ethanedithiol, 90%+
Thioanisole, >99%
Triethylsilane, > 99%
Hydrochloric acid 37%, p. a.
Phenol, p. s.
Ninhydrin, p. a.
Scharlau
Scharlau/Panreac
Applied Biosystems
Merck
Merck, Acros
Aldrich
KalieChemie
Fluka
De-ionised and filtered with a MilliQ Plus
system (Millipore) to a resistivity superior
to 18 MΩ cm-1
Merck, Panreac, Scharlau
Merck
Merck
Aldrich
Fluka
Aldrich
Merck
Aldrich
Merck
*Stabilised with BTH and stored with sodium
153
SPR as a tool in the functional analysis of an immunodominant site in FMDV
Materials and reagents for ELISA
PBS, tablets for buffer preparation
BSA, fraction V
Goat-anti-mouse IgG antibody - peroxidase
conjugate, blotting grade
orto-phenylenediamine
Hydrogen peroxide 35%, stabilised p. a.
Tween 20, Plusone
PVC 96-well plates, highly activated
Sigma
Boehringer Manheim
Bio Rad
Sigma
Acros
Pharmacia Biotech
Titertek, 77-172-05
Materials and reagents for SPR analysis
EDC (amine coupling kit), BIAcertified
NHS (amine coupling kit), BIAcertified
Ethanolamine hydrochloride (amine coupling
kit), BIAcertified
HBS buffer, BIAcertified
Sensor chip CM5, BIAcertified
Oxalic acid, p. s.
orto-phosphoric acid, p. s.
Formic acid, p. a.
Malonic acid, p. a.
Sodium phosphate, p. a.
Ethanolamine, p. a.
Piperazine, p. a.
Glycine, p. a.
Potassium thiocianate, p. s.
Magnesium chloride, p. a.
154
Biosensor AB
Merck
Scharlau
Merck
Merck
Merck
Aldrich
Aldrich
Fluka
Merck
Merck
Materials & Methods
General reagents
biochemistry
and
materials
for
KLH, 18 mg/ml in 65% aqueous
ammonium sulphate
Glutaraldehyde, p. a.
HCA I (E. C. 4.2.1.1)
Guanidine hydrochloride, purum >98%
Citric acid, p. a.
Sodium citrate, p. a.
E.D.T.A. (Titriplex III), p. a.
Sodium chloride, p. a.
Tris, p. a.
Urea, p. a.
Iodoacetic acid, p. a.
Cysteine, for molecular biology
β-mercaptoethanol, purum >99%
Ammonium sulphate, for biochemistry
Sodium azide, purum p. a. >99%
Papain (E. C. 3.4.22.2)
Dialysis membrane (MW cut-off=15-20 kDa),
∅=16 mm
Centriprep-3 concentrators
Calbiochem
Sigma
Sigma, C-4396
Fluka
Merck
Merck
Merck
Merck
Merck
Merck
Merck
Sigma
Fluka
Merck
Fluka
Sigma, P-4762
Servapor, 4415
Amicon
Reagents for SDS-PAGE
Amresco
Serva
Merck
Sigma
Boehringer
Sigma
Bio Rad
Bio Rad
Acrylamide/Bis 37.5:1, ultrapure grade
APS, p. a.
TEMED, p. a.
Glycine, for molecular biology
SDS
Glycerol, p. a.
Bromophenol blue
Coomassie brilliant blue R-250
Solvents
and
spectroscopy
materials
for
Deuterium oxide, 99.95% Uvasol
2,2,2-Trifluoroethanol - d3
1,4 – dioxane, for spectroscopy Uvasol
NMR quartz tubes, 5 mm OD (high
magnetic field)
NMR
Merck
SDS
Merck
SDS
SDS
155
SPR as a tool in the functional analysis of an immunodominant site in FMDV
4.1.2
Instrumentation
Amino acid analysis*
Beckman System 6300
- elution with sodium salts
- 250 × 4 mm column containing a
polysulphonate resin for cationic exchange
- post-column detection by the ninhydrin
reaction
Mass spectrometry**
- ES-MS
- MALDI-TOF (matrices: ACH, SA)
Fisons Instruments VG Quatro
Finnigan MAT Lasermat 2000, Bruker II Biflex
UV-Vis spectrometry
Perkin-Elmer Lambda 5
pH-meter
Crison MicropH 2002
Centrifuge
Beckman GS-15R
Lyophiliser
Virtis Freezemobile 12EL
ELISA spectrometer
Labsystem Multiskan MS
SPR instrument
BIAcore 1000, IFC4 with recovery
NMR spectrometry***
Varian VXR500
X-ray diffraction****
MarResearch image plate detector (180 × 0.10
mm, 1800 pixels); Rigaku RU-200B rotating
anode
SDS-PAGE
Bio Rad Mini-PROTEAN II electrophoresis cell;
Bio Rad gel dryer, model 583.
* amino acid analyses were performed by Dr. C. Carreño, Dr. M. L.Valero and Ms. M. E. Méndez, at the Servei de
Sintesi de Pèptids de la Universitat de Barcelona.
** mass spectra were acquired by Drs. I. Fernández, M. Vilaseca, M. L. Valero and E. de Oliveira, at the Servei de
Espectrometria de Masses de la Divisió de Ciències de la UB.
*** NMR spectra were recorded by Dr. M. A. Molins at the Servei de RMN, SCT, Universitat de Barcelona.
**** X-ray diffraction data were acquired by Ms. W. F. Ochoa at the European Synchrotron Facility in Grenoble.
156
Materials & Methods
4.1.3
Analytical methods
4.1.3.1. Qualitative ninhydrin assay
This assay serves to detect free amine groups on polymeric supports (resins) for SPPS and is
performed as described by Kaiser et al.1
4.1.3.2 Qualitative Ellman assay
This assay allows the detection of free thiol groups either in solution or on polymeric supports
compatible with aqueous media, according to Ellman et al.2
4.1.3.3. Amino acid analysis
The content and proportion of amino acid residues present in a free or resin-bound peptide are
determined by amino acid analysis (AAA), following a previous hydrolysis step. To hydrolyse a
peptide-resin3, 1 – 10 mg of dried resin are placed into a Pyrex glass tube and 250 µl of a 1:1 (v/v)
mixture of 12 M hydrochloric and propionic acids are added. The tube is sealed and hydrolysis is
carried out at 155 oC for 90 minutes. The procedure for a free peptide4 is similar, hydrolysing with 6
M HCl for 45 minutes. The hydrolysed mixture is then evaporated to dryness and the residue
dissolved in a known volume of a 0.06 M citrate buffer, pH 2.0. After filtration through a nylon filter
(∅pore=0.45 µm), the sample is ready for AAA.
4.1.4
Chromatographic methods
4.1.4.1 High performance liquid chromatography
Analytical HPLC is performed in either of the following systems:
Waters – composed by a controller and a quaternary pump 600E with a low pressure
mixer, an automatic injection system Waters 712, a variable wavelength UV-Vis detector
490E and a integrator/recorder either D-2000 (Merck-Hitachi) or Chromatopac C-R5A.
Shimadzu – composed by two LC-6A pumps with a high pressure mixer, an SCL-6B
controller, an SIL-6B auto-injection system, a variable wavelength UV-Vis detector SPD-6A
and a integrator/recorder Chromatopac C-R6A.
The HPLC column is a 250 × 4 mm Nucleosil C18, with a reverse solid phase of octadecylsyloxane
(∅beads=5 µm; ∅pore=120 Å). The mobile phases are gradients of H2O (0.045% v/v TFA) and MeCN
(0.036% v/v TFA) at a 1 ml/min flow.
157
SPR as a tool in the functional analysis of an immunodominant site in FMDV
4.1.4.2 Medium pressure preparative liquid chromatography
Peptides are purified by MPLC in systems composed by LCD/Milton or Duramat ProMinent piston
pumps, variable wavelength Applied Biosystems or single wavelength Uvicord 2158 SD (LKB)
detectors at 220 nm, Ultrorac 2070II (LKB) or Gilson FC205 fraction collectors and Servoscribe
(Phillips) or Pharmacia-LKB REC101 recorders. Glass columns (!=200-300 mm, ∅internal=25 mm)
with Vydac C18 reverse phase (∅beads=15-20µm, ∅pore=300 Å) are used and the mobile phase
consisted on a binary linear gradient of H2O and MeCN with 0.05% TFA at a constant flow of 120150 ml/h.
References
1 Kaiser, E., Colescott, R. L., Bossinger, C. D. and Cook, P. I. (1970) Color test for detection of free
terminal amino groups in solid-phase synthesis of peptides, Anal. Biochem. 34, 595-598.
2 Ellman, G. L. (1958) A colorimetric method for determining low concentrations of mercaptans, Arch.
Biochem. Biophys. 74, 443-450.
3 Scotchler, J., Lozier, R. and Robinson, A. B. (1970) Cleavage of single amino acid residues from
Merrifield resin with hydrogen chloride and hydrogen fluoride. J. Org. Chem. 35, 3151-3152.
4 Steward, J. M. and Young, J. D. in “Solid Phase Peptide Synthesis” , 2nd ed., Pierce Chemical Co.,
Rockford, Illinois (1984).
158
Materials & Methods
4.2 Solid-phase peptide synthesis
4.2.1
Solid-phase peptide synthesis protocols
4.2.1.1 Preparation of resins for peptide synthesis
The dry resin (MBHA1 or PEG-PS2) is placed in a polypropylene syringe containing a polyethylene
filter. The resin is allowed to swell in 40% TFA in DCM (1 × 1 min + 1 × 20 min), filtered and
neutralised with 5% DIEA in DCM (3 × 1 min), then washed with DCM (5 × 30 s) and DMF (3 × 1
min).
A mixture of the chosen two-functional spacer (or handle) and HOBt (3 eq each) is dissolved in a
minimum volume of DMF and added to the resin in the syringe. The coupling agent DIP (3 eq) is
then added and coupling is allowed to proceed overnight or until a negative ninhydrin test is
obtained. If the ninhydrin test remains positive after 18 hours of reaction, the resin is washed and a
re-coupling step (1 eq of all reactants in similar conditions) is performed. When coupling is
complete, the resin is filtered, thoroughly rinsed with DMF and dried.
4.2.1.2 Fmoc/ tBu chemistry
4.2.1.2.1
Manual synthesis3
Manual syntheses are performed in polypropylene syringes with a polyethylene porous filter. The
volumes of solvents and reagent solutions added should cover the entire resin to allow optimal
solvating and swelling of the beads. Stirring is done with a teflon rod and, at the end of each cycle,
excess reagents, by-products and solvents are eliminated by filtration and washing with DMF and
DCM.
Peptide chain elongation is performed according to the following steps:
159
SPR as a tool in the functional analysis of an immunodominant site in FMDV
Step
1
2
3
4
5
6
7
8
Reagenta
DMF
20 % piperidine in DMF
20 % piperidine in DMF
DMF
Fmoc-AA-OH/coupling agent/DIEAb
DMF
DMF
Ac2O/DIEA 1:1 in DMF
9
DCM
a
b
Treatment
Wash
Pre-equilibrate
Deblock (Fmoc removal)
Wash
Coupling
Wash
Wash
Acetylation (block non-reacted
amino groups)
Wash
Time/min
3×1
1
10
3×1
45 – 60
3×1
3×1
15
3×1
volumetric reagent/solvent proportions
coupling agents used: DIP, TBTU; Base (DIEA) is required with TBTU only (2 eq DIEA/ 1 eq TBTU)
The coupling agents employed in this work were DIP and TBTU4, the latter requiring the addition of
base (DIEA) in a 2:1 molar proportion between base and reagent. The Fmoc-AA-OH were dissolved
in the minimum volume of DMF and, once coupling time was over and washing steps performed
(step 6), a ninhydrin assay was done. When the assay was negative, chain elongation proceeded to
the incorporation of the following amino acid residue, starting with the removal of the Fmoc group
(step 2 and the following). When the ninhydrin test was positive, a recoupling cycle was performed
(step 5 and the following). If the addition of recoupling steps could not improve coupling efficiency,
then acetylation (steps 7 – 9) could be used to block the non-reacted amino groups.
4.2.1.2.2
Machine-assisted synthesis
Peptides can also be synthesised in a MilliGen 9050 Plus PepSynthesiser, which dissolves amino
acids and coupling reagents and works with a continuous flow Fmoc/tBu chemistry (instead of
filtration steps after each cycle). The inlet/outlet detectors allow a constant monitoring of the
synthesis at each step and it is possible to choose synthesis scale and coupling reagents.
The general protocol consists on Fmoc-AA-OH/coupling agent dissolution in DMF, followed by
addition of 0.6 M DIEA in DMF. The coupling mixture is activated through a 5 min bubbling step
with nitrogen and subsequent transfer of the solution to the column reactor, which contains the
previously de-blocked resin. Chain elongation proceeds as follows:
Step
1
2
3
4
5
6
7
a
b
Reagenta
DMF
20 % piperidine in DMF
20 % piperidine in DMF
DMF
Activated Fmoc-AA-OHb
Activated Fmoc-AA-OH
DMF
Treatment
Wash
Pre-equilibrate
Deblock (Fmoc removal)
Wash
Coupling
Coupling
Wash
Flow /ml.min-1
3
3
3
3
3
3
3
Time
15 s
1 min
5 min
7 min
5s
60 min
4 min
volumetric reagent/solvent proportions
coupling agents used: TBTU
Once the synthesis is completed, the peptide-resin is transferred to a polypropylene syringe to be
washed and dried as described in 4.2.1.2.1.
160
Materials & Methods
4.2.1.2.3
Machine-assisted parallel synthesis
Multiple peptide synthesis can be performed on an Abimed MAS 422 synthesiser, which allows the
simultaneous synthesis of up to 48 peptide-resins, using Fmoc/tBu chemistry with in situ activation.
The synthesis programmes are quite flexible in what concerns synthesis scale, number of coupling
steps per amino acid residue and duration of each step. This synthesiser operates as follows: the
Fmoc-AA-OH (0.6 M in DMF, except for Fmoc-His(Trt)-OH and Fmoc-Phe-OH, which are
dissolved in NMP), the coupling reagent (0.5 M TBTU) and the base (4 M NMM), which are
previously dissolved and placed in appropriate racks and sealed with septa, are added to 2 ml
syringes containing previously deblocked and washed resin, and fitted to a 48-port manifold system.
The addition is done in a pre-defined sequence, according to the reactivity of each amino acid
residue which is added to each syringe. Chain elongation proceeds according to steps 1-6:
Step Reagenta
Treatment
1
2
3
4
5
6
7d
8
9
10
11
12
Deblock
Wash
Wash
Coupling
Wash
Wash
Wash
Wash
Deblock
Wash
Wash
Wash
20 % piperidine in DMF
DMF
DMF
Fmoc-AA-OH/TBTU/NMM
DMF
DMF
DMF
DMF
20 % piperidine in DMF
DMF
DMF
DCM
Number of
repeats
2 × 1 ml
2 × 1 ml
3 × 0.3 ml
1
12 × 1 ml
2 × 0.3 ml
2 × 1 ml
3 × 0.3 ml
2 × 1 ml
2 × 1 ml
3 × 0.3 ml
3 × 0.3 ml
Time/min
5b
0.5
0.5
30c
0.5
0.5
0.5
0.5
5b
0.5
0.5
0.5
a
volumetric reagent/solvent proportions;
reaction time is increased along chain elongation;
c
100 µl of DCM are added at 80% of the coupling total time ;
d
steps 7-12 correspond to the final cycles for resin deblocking, washing and drying.
b
4.2.1.2.4
Peptide cleavage from the resin and removal of side-chain protecting groups
Up to 500 mg of dry resin (Nα - Fmoc previously removed) are placed in a Falcon centrifuge tube.
The cleavage reagent (cocktail R5) is prepared: 90% TFA, 2% anisole, 5% thioanisole and 3% 1,2 –
ethanedithiol, and added to the resin at the proportion of 1 ml cocktail : 100 mg resin. The reaction
is carried out at room temperature for 2 hours with constant shaking. Anhydrous tertbutylmethylether (40 ml) is then added and the mixture cooled at -78 oC for peptide precipitation.
The suspension is stirred, then centrifuged at 4 oC and 4000 r. p. m. for 15 minutes, after which the
supernatant is decanted. The procedure is repeated 5 times from the ether addition step. The final
peptide precipitate is dried with nitrogen, resuspended in AcOH 10% and filtered through a
polypropylene syringe containing a polyethylene filter. The peptide solution is then lyophilised.
161
SPR as a tool in the functional analysis of an immunodominant site in FMDV
4.2.2
Synthesis of peptides from the GH loop of FMDV
4.2.2.1 Peptides for SPR and ELISA
Peptide sequences and their characterisation are compiled in chapters 2 and 3.
4.2.2.1.1
Linear 15-mer peptides from the FMDV strain C1-Barcelona (C-S30)
These peptides were prepared by machine-assisted parallel synthesis at a 25 µmol scale (section
4.2.1.2.3) on an Fmoc-AM-MBHA resin (0.51 mmol/g), where the handle AM6 was incorporated as
described in 4.2.1.1. The usual side-chain protecting groups in Fmoc/tBu synthesis were employed:
Asp(OtBu), Arg(Pmc), His(Trt), Ser(tBu), Thr(tBu) and Tyr (tBu).
The peptides, which were obtained as C-terminal carboxamides, were cleaved and deprotected as
described in 4.2.1.2.4, with some modifications: a polystyrene pipette tip was adapted to each resincontaining syringe and the latter was introduced in a 10 ml Sarsted polypropylene tube, previously
containing the cleavage cocktail (1 ml). The cocktail was then sucked up into the syringe and, after
air bubbles were carefully expelled, the reaction was carried out as previously described. Once the
first 2-hour period was over, the peptide crudes were expelled from the syringes to the Sarsted tubes
and additional 0.5 ml of fresh cleavage cocktail were sucked up into the syringes and reaction
proceeded for further 30 minutes. The second filtrates were mixed with the first ones and processed
as described in chapter 4.2.1.2.4.
Crude products were analysed by HPLC (5→95% B and 10→45% B), AAA and ES MS(+) or
MALDI-TOF MS (section 4.1.4.1). Peptides with more than 15% of byproducts were purified by
reverse phase MPLC (5→25% B, section 4.1.4.2).
4.2.2.1.2
Larger versions of the FMDV GH loop: peptides A21 and A21S30
The 21-residue peptides A21 and A21S30 were synthesised either by manual Fmoc/ tBu chemistry
(section 4.2.1.2.1) or by machine-assisted synthesis on a MilliGen 9050 Plus PepSynthesiser (section
4.2.1.2.2) at a 50 µmol scale. In either case, an Fmoc-AM-PEG-PS resin (0.20 mmol/g) was
employed and procedures were as already described in previous chapters. Peptide cleavage from
the deblocked resin was done as described in 4.2.1.2.4. Both peptides were purified by reverse
phase MPLC (10→30% B) and characterised as usual.
4.2.2.1.3
Cyclic versions of the FMDV GH loop: peptides cyc16S30 and cyc16147Val
The cyclic peptides, cyc16S30 and cyc16147Val, were synthesised by intra-molecular disulphide
bridge formation7 of the corresponding linear bis-thiol precursors (see chapter 2). The linear bis-thiol
162
Materials & Methods
peptides were synthesised by similar methods as those described in 4.2.2.1.2, using Fmoc-Cys(Trt)OH and Fmoc-Ahx-OH in addition to the other protected amino acids usually employed (section
4.2.2.1.1). Peptide cleavage, characterisation and purification by MPLC (10→25% B) were
performed as already described, having the extra care that peptides were always kept under acidic
conditionsA to avoid intermolecular disulphide bridge cross-linking. Once the linear bis-thiol
precursors were purified and lyophilised, cyclization proceeded by air oxidation at high peptide
dilution and pH 8. The peptide was added stepwise to 100 mM ammonium bicarbonate buffer, pH
8, to a final concentration of 50 µM, under vigorous stirring, and left to react at open air.
The extent of cyclization was monitored by HPLC (10→45% B) and by the Ellman qualitative assay,
and usually reached completion within 1 hour. The reaction was stopped upon dropwise addition of
glacial acetic acid until pH 3. Cyclic peptides were characterised by HPLC, AAA and MALDI-TOF
MS and repeatedly lyophilised from water to eliminate the ammonium salts.
4.2.2.1.4
Linear 15-mer peptides bearing the mutations
and
T→I,
137
A→F,
138
140
A→P,
142
G→ S
T→ I
148
These peptides were prepared by machine-assisted parallel synthesis at a 25 µmol scale on an
Fmoc-AM-MBHA resin (0.51 mmol/g), by similar methods as those described in section 4.2.2.1.1.
Peptide cleavage was performed according to the same section and, again, crude peptides having
more than 15% of impurities were purified by reverse phase MPLC (15→45% B). Peptide
characterisation and quantification was as previously described.
4.2.2.2 Single syntheses of peptides for structural studies
The set of FMDV peptides for NMR and X-ray diffraction studies were prepared in individual
syntheses and exhaustively purified to meet the purity requirements of both techniques. Syntheses
were performed at a 100 µmol scale on an Fmoc-AM-MBHA resin (0.20 mmol/g) in the MilliGen
9050 Plus PepSynthesiser by methods similar to those described. Peptide purification by MPLC was
carried out as usual, regarding that at least 5 mg of 99% pure peptide should be obtained.
A
MPLC peptide purification fractions were collected on tubes containing 100 µl of 0.1 M AcOH.
163
SPR as a tool in the functional analysis of an immunodominant site in FMDV
References
1 Barany, G. and Merrifield, R. B. in “The peptides”, vol. 2, 1st ed., Gross, E. and Meinhofer, R. B.
(Eds.), Academic Press, New York (1980).
2 Barany, G. and Albericio, F., Mild orthogonal solid-phase peptide synthesis, in “Peptides 1990:
Proceedings of the 21st European Peptide Symposium” , Giralt, E. and Andreu, D. (Eds.), ESCOM,
Leiden (1991), pp 139.
3 Fields, G. B. and Noble, R. L. (1990) Solid-phase peptide synthesis utilizing 9fluorenylmethoxycarbonyl amino acids, Int. J. Peptide Protein Res. 53, 161-214.
4 Knorr, R., Trzeciak, A., Bannwarth, W. and Gillesen, D. (1989) New coupling reagents in peptide
chemistry, Tetrahedron Lett. 30, 1927-1930.
5 Albericio, F., Kneib-Cordonier, N., Biancalana, S., Gera, L., Masada, R. I., Hudson, D. and Barany,
G. (1990) Preparation and application of the 5-(4-(9-fluorenylmethyloxycarbonyl)-aminomethyl-3,5dimethoxyphenoxy)valeric acid (PAL) handle for the solid-phase peptide synthesis of C-terminal
peptide amides under mild conditions, J. Org. Chem. 55, 3730-3743.
6 Bernatowicz, M. C., Daniels, S. B. and Köster, H. (1989) A comparison of acid labile linkage agents
for the synthesis of peptide C-terminal amides, Tetrahedron Lett. 30, 4645-4648.
7 Andreu, D., Albericio, F., Solé, N. A., Munson, M. C., Ferrer, M. and Barany, G. Formation of
disulfide bonds in synthetic peptides and proteins in “Methods in molecular biology, vol. 35: Peptide
synthesis protocols”, Pennington, M. W. and Dunn, B. M. (Eds.), Humana Press Inc., Totowa, New
Jersey (1994), pp 91-169.
164
Materials & Methods
4.3 Antigenic evaluation of the FMDV peptides
4.3.1
SPR analysis of peptide-antibody interactions
The technical and scientific bases for real-time surface plasmon resonance biospecific interaction
analysis are exposed in sections 0.1 to 0.3 of the present work. A BIAcore SPR biosensor was used,
and standard amide immobilisation chemistry on a CM5 sensor chip were employed. Both
immobilisation chemistry and sensor chip features are described in section 0.2. Standard
procedures, following manufacturer’ s instructions, were employed as far as possible. Equipments
and reagents for biosensor analysis are specified in section 4.1.
4.3.1.1 Peptide and mAb solutions for biosensor analysis
Peptide stock solutions ca. 2.5 mM in 0.1 M acetic acid were prepared and quantitated by AAA.
Solutions for BIAcore analysis were obtained by 1000-fold and subsequent serial dilutions in HBS.
Stock solutions of mAbs SD6 and 4C4 (in PBS with 0.02% sodium azide, pH 7.3) were desalted
and buffer-exchanged on an NAP-5 Sephadex G-25 column (Pharmacia Biotech) and final mAb
concentrations were determined by measurement of optical density at 280 nm, considering that 1
OD280 ≈ 0.75 mg (protein)/ml.
4.3.1.2 Optimisation of the direct kinetic analysis of immobilised mAb – peptide interactions
SD6 solutions (100 and 50 µg/ml, in either 10 mM sodium acetate, pH 5.5, or 5 mM sodium
maleate, pH 6.5) were separately injected (30 µl) at 5 µl/min over a non-activated sensor surface, to
determine which gave the most efficient mAb pre-concentration into the dextran matrix.
Three SD6 surfaces were prepared using the standard amine coupling procedure as described by the
manufacturer1: each carboxymethyl surface was activated with a 35 µl injection (at 5 µl/min) of a
solution containing 0.2 M EDC and 0.05 M NHS, and SD6 was then coupled at three different
densities by injecting over each surface 35 µl of 50, 5 and 3 µg/ml SD6 in 10 mM sodium acetate
165
SPR as a tool in the functional analysis of an immunodominant site in FMDV
buffer, pH 5.5, respectively. Non-reacted activated groups were then blocked by a 30 µl injection of
ethanolamine hydrochloride and remaining non-covalently bound material was washed off in a
regeneration step with a 3-min pulse of 100 mM HCl. Surface densities obtained were of 8000,
1700 and 800 RU, respectively, where 1 RU (resonance unit) corresponds to 1 ng (protein)/mm2
(surface).
A few sets of experiments, using A15 as analyte, were run on the three SD6 surfaces at different
peptide concentrations (ranging from 1 to 2500 nM) and flow rates (5 and 60 µl/min). All
experiments were done with HBS as running buffer at 25
o
C, using the kinjection mode.
Sensorgrams were generated with 7-min peptide injections in the HBS flow, followed by 6-min
dissociation in running buffer and then by a 2-min regeneration step with 100 mM HCl.
Biosensor data were prepared, modelled and fitted by means of the BIAEvaluation 3.0.2 software2
(Biosensor AB, 1994-97, run on Windows ’ 95). The quality of the fits was assessed by visual
comparison between experimental and modelled sensorgrams, as well as by statistical parameters
such as χ2 and standard errors associated to the calculated constants, or by further inspection of
residual distribution.
4.3.1.3 Systematic screening of FMDV peptide antigens: validation of the SPR methodology
Immobilisation of mAbs SD6 and 4C4 was performed as described in the previous section.
Biospecific surfaces were obtained by injecting 35 and 16 µl of the 5 µg/ml SD6 and 4C4 solutions
in 10 mM acetate buffer pH 5.5, respectively. Following the capping step with ethanolamine
hydrochloride, remaining non-covalently bound molecules were washed off with a 3-min pulse of
100 mM HCl or 10 mM NaOH for SD6 or 4C4 surfaces, respectively. The final immobilisation
responses were of about 1600 RU.
All kinetic SPR analyses were run at a 60 µl/min HBS flow and each peptide was analysed at 6
different concentrations, ranging from ca. 80 to 2500 nM for SD6 and ca. 40 to 1250 nM for 4C4.
Sensorgrams were generated by kinjections of peptide solutions with 90 s association steps followed
by 240 s dissociation in running buffer. A 90 s pulse of 100 mM HCl or 10 mM NaOH (SD6 and
4C4 surfaces, respectively) was applied to regenerate the surfaces at the end of each cycle and wash
steps (needle, IFC, system flush) were added to avoid carry-over. The pentadecapeptide A15scr,
containing the constituent amino acids of A15 in scrambled form, was injected under the same
conditions as a control for non-specific binding to the sensor chip surfaces.
After subtracting the response of peptide A15scr to the responses of the relevant peptides, data were
prepared, modelled and fitted by means of BIAevaluation software as already described.
166
Materials & Methods
4.3.1.4 Antigenic evaluation of FMDV C-S30 peptides by direct SPR kinetic analysis
Peptides from the FMDV C-S8c1 and C-S30 GH loops (syntheses described in section 4.2.2) were
screened by SPR as described in section 4.3.1.3. This screening included an additional anti-FMDV
monoclonal antibody, mAb 3E5. This mAb was purified from ascitic fluid as follows.
4.3.1.5 Purification of mAb 3E5 from ascitic fluid
Ascitic fluid was unfrozen and divided into 1 ml aliquots, which were then centrifuged for 5 min at
10000 r.p.m. (4 oC). The supernatants were pooled and an equivalent volume of buffer A was
added (buffer A: 112.4 g/l glycine, 175.4.g/l NaCl, pH 8.9). This mixture was again divided into 1
ml aliquots and centrifuged. The aqueous fraction was separated from lipids and pellets.
A HiTrap protein A – Sepharose affinity column (Pharmacia Biotech), coupled to a 2132
Microperpex (LKB) peristaltic pump, was prepared by extensive rinsing, with 100 mM sodium citrate
buffer, pH 3, then with buffer A, at a constant flow of 20 ml/h.
The sample was applied to the column and eluted with buffer A until OD280≤0.01; elution then
proceeded with 100 mM sodium citrate buffer, pH 5.0, and fractions were collected on glass tubes
containing 100 µl of 100 mM Tris-HCl buffer, pH 8.5. Fractions were monitored at 280 nm and,
once an absorbance peak was observed, the elution buffer was changed to 100 mM sodium citrate,
pH 3, until a second smaller peak was observed. Fractions collected at pH 5 with OD280≥0.5 were
pooled and dialysed against PBS overnight (PBS, phosphate buffered saline: 137 mM NaCl, 2.7
mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.3; 3 × 1 l) and final concentration was
determined by optical density at 280 nm. For the present purpose, further steps for mAb
concentration (upon precipitation with ammonium sulphate and subsequent dialysis against PBS
and gel filtration on Sephadex G-100) were not required.
4.3.1.6 SPR interaction analysis of free FMDV C-S30 peptides with immobilised mAbs SD6, 4C4
and 3E5
Immobilisation of mAbs SD6 and 4C4 were as described in section 4.3.1.3. MAb 3E5 was
immobilised similarly to the described for mAb 4C4. Kinetic analyses were run as described in
section 4.3.1.3 and peptide concentrations injected over the 3E5 surface ranged from ca. 35 to ca.
625 nM. Data evaluation procedures were as already described, using the 1:1 langmuirian binding
(either with or without baseline drift) kinetic model2.
167
SPR as a tool in the functional analysis of an immunodominant site in FMDV
4.3.1.7 SPR interaction analysis of immobilised FMDV C-S30 peptides with free mAbs SD6, 4C4
and 3E5
Peptides A15, A15S30, A21, A21S30, A15Brescia, cyc16S30, cyc16147Val and A15scr were
immobilised by methods identical to those described for mAb immobilisation. Following a surfaceactivation step similar to that previously described, 25 µl of the peptide solutions (200 µg/ml in 10
mM acetate buffer, pH 5.0) were injected over the surface and final immobilisation responses of ca.
170 RU were obtained. Surfaces were regenerated by 2-min pulses of 100 mM HCl.
mAb solutions, 25 to 800 nM in HBS, were injected as previously described for peptides and
sensorgrams were run, modelled and fitted as before.
4.3.1.8 Antigenic evaluation of multiply substituted FMDV peptides by direct SPR kinetic analysis
Direct SPR kinetic analysis of peptides reproducing combinations of the substitutions
138
A→F,
140
A→P,
142
G→S and
137
T→I,
148
T→I could not be accomplished due to experimental problems
related either to extremely slow complex dissociation or to insufficient surface regeneration (see
chapter 3).
4.3.1.9 Alternative strategies for surface regeneration
A screening of alternative regeneration conditions, based on the multi-cocktail approach of
Andersson and co-workers5, was performed (see chapter 3, Tables 3.5 and 3.6).
The evaluation of the screening cocktails is performed by a 10-min analyte injection at a flow rate of
2 µl/min, followed by 30 s injections (at 20 µl/min) of a given cocktail until analyte level decreases to
30% or less of the original value. At this point, a new analyte injection is applied, followed by
injections of another regeneration cocktail. After a regeneration effectiveness is attributed to each
cocktail, a more extensive optimisation can be performed, relying on combinatorial mixing of the
different stock cocktails.
Cocktails including stock solutions A, B, C, U or I’ (see chapter 3) were screened as described, but
none led to satisfactory results.
4.3.1.10 Indirect SPR kinetic analysis by competition assays using an FMDV peptide – carrier
protein conjugate
The preparation of a 1:1 peptide – protein conjugate made use of the chemistry of the Npys thiol
protecting group7. An Fmoc-peptide A15-AM-MBHA resin was prepared by solid phase peptide
Fmoc/tBu chemistry (scale 50 µmol) on an Fmoc-AM-MBHA resin (0.20 mmol/g) in the MilliGen
9050 Plus PepSynthesiser. After removal of the Nα - Fmoc protecting group and subsequent
168
Materials & Methods
washing cycles, Boc-Cys(Npys)-OH was manually coupled (using 4 eq. of DIP in DCM) to the
peptide-resin. Once coupling was completed and resin conveniently washed and dried, peptide
cleavage and side-chain deprotection (except for the Npys group, stable to strong acids) was
performed with a 90% TFA, 5% H2O and 5% Et3Si cleavage mixture The Cys(Npys)A15 peptide
was characterised by AAA, HPLC and MALDI-TOF MS and lyophilised prior to heterodimerisation
with protein HCA I (human carboxy anhydrase I, a 28 kDa monomer with a single Cys residue)8.
The reaction of disulphide heterodimerisation between Cys(Npys)A15 and HCA I was performed
under denaturating acidic conditions. HCA I (5 mg, 0.17 µmol) was dissolved in the minimum
amount of 6 M guanidine hydrochloride in water (pH 4.2) and left under magnetic stirring for 30
min, until a positive Ellman test was obtained. Peptide Cys(Npys)A15 (3.13 mg, 1.7 µmol) was
added to the protein solution and reaction was allowed to proceed overnight, monitored by HPLC
(15→65% B) and by the qualitative Ellman assay. When reaction reached equilibrium, the mixture
was diluted to 2 ml with 6 M guanidine hydrochloride and dialysed during 48 h against decreasing
concentrations of guanidine hydrochloride (4 M, 1 × 1 l → 2 M, 1 × 1 l → 1 M, 1 × 1 l → water, 3
× 1 l). The dialysed solution was characterised by HPLC, MALDI-TOF MS and AAA, and the total
protein content was determined by optical density at 280 nm. After adding sodium azide (0.03%),
the protein solution was divided into 500 µl aliquots that were stored at –20 oC prior to SPR assays.
The protein heterodimer HCA I – CysA15 was injected on sensor chip flow cells where mAbs SD6,
4C4 and 3E5 had previously been immobilised by standard procedures (ca. 600 RU of each mAb
were immobilised). A total protein concentration of 300 nM was injected over the three mAb
surfaces and also over a fourth mock surface (EDC/NHS activation plus ethanolamine hydrochloride
capping, without protein injection) for non-specific binding evaluation. Injections were performed as
already described for peptide injection. Protein response was studied at three different pH values,
upon protein dilution in either HBS (pH 7.3), 10 mM Tris – HCl (pH 8.5) or 10 mM sodium acetate
(pH 5.5) buffers. In neither case was a specific response observed. A non-specific response was
observed at pH 5.5, due to the electrostatic attraction between protonated protein – pI ≈ 6 – and the
negatively charged carboxymethyl dextran matrix.
4.3.1.11 Indirect SPR kinetic analysis by competition assays using an engineered recombinant
protein expressing the GH loop of FMDV C-S8c1
Protein JX249A is a recombinant β-galactosidase from Escherichia coli9, with solvent exposed loops
where
a
24-residue
136
(TT YTASARGDLAHLTT
peptide
150
from
10
THARHLP)
the
GH
loop
of
FMDV
C-S8c1
has been inserted. The protein is a 472 kDa
homotetramer with one GH loop per monomer and is highly antigenic towards a panel of anti-GH
loop antibodies.
Protein samples (305 µg/ml in buffer Z: 0.06 M sodium hydrogen phosphate, 0.04 M sodium dihydrogen phosphate, 0.01 M potassium chloride and 1 mM magnesium sulphate) were diluted in
HBS to a total FMDV peptide concentration of 624 nM (and subsequent serial dilutions) and tested
169
SPR as a tool in the functional analysis of an immunodominant site in FMDV
for SPR analysis. Protein was injected over SD6, 4C4 and 3E5 surfaces (ca. 600 RU of mAb
immobilisation level), as previously described. Insufficient mAb surface regeneration was observed,
which could not be overcome by alternative regeneration procedures based on the multi-cocktail
approach described in section 4.3.1.9. The regeneration strategy that yielded better results consisted
on three successive 1-min injections of 40 mM NaOH, but even so surface life-time was significantly
reduced due to the inefficient removal of bound protein and consequent decreasing availability of
mAb binding sites.
In spite of the surface regeneration problems observed, a set of preliminary tests for SPR surface
competition analysis was performed using peptides A15 and A15scr as competitors. A constant
amount of protein JX249A (total final concentration in FMDV peptide = 160 nM) was added to six
peptide solutions with concentrations ranging from 0 to 300 nM. The peptide – protein mixtures
were then injected as previously described for peptide injections and three 1-min pulses of 40 mM
NaOH for partial surface regeneration were added at the end of each injection. Data was processed
with the BIAEvaluation 3.0.2 software, using the heterogeneous analyte kinetic model2 (competition
between two different analytes, section 0.3).
4.3.1.12 Indirect SPR kinetic analysis by competition assays using cysteine-capped protein JX249A
Since one of the possible causes for JX249A irreversible binding to mAb surfaces could be the fact
that all cysteine thiol groups in native bacterial β-galactosidases are reduced, capping of the thiol
groups was performed using iodoacetic acid11. A denaturating solution (2 ml, 7.5 M urea, 4 mM
EDTA, 0.25 Tris-HCl, pH 8.5) was added to a 170 µg/ml JX240A solution in buffer Z (2 ml),
corresponding to 46 nmol of total cysteine. β-mercaptoethanol was added (2 µl, 26 µmol) to cleave
any disulphide bonds in the protein and the mixture was left to stand at 60 oC for 1 h in the dark,
followed by another hour at room temperature. Then, iodoacetic acid (5 µl, 39 mM in 0.1 M NaOH)
was added to the mixture and reaction was allowed to proceed for further 30 min in the dark at
room temperature. Reaction was quenched by excess β-mercaptoethanol (100 µl) and the mixture
was then dialysed for 48 hours against decreasing concentrations of urea (5 M, 1 × 1 l → 2 M, 1 ×
1 l → 1 M, 1 × 1 l → water, 3 × 1 l).
The dialysed solution was analysed by AAA to determine the degree of cysteine carboxymethylation
(sample and two carboxymethylcysteine standards were submitted to the same AAA protocol) and
by SDS-PAGE to check for protein integrity.
SDS-PAGE analysis12 of the protein JX249A before and after cysteine carboxymethylation was
performed on a BIO RAD Mini-PROTEAN II electrophoretic cell. An 8% acrylamide gel (7 × 8 cm)
was prepared by mixing a 40% acrylamide solution (2 ml of an acrylamide/bis-acrylamide mixture,
37.5:1) with H2O (5.5 ml), “ lower” buffer (2.5 ml, 1.5 M Tris-HCl and 0.4% SDS, pH < 8.7), 15 %
APS (40 µl) and TEMED (5 µl); the mixture was poured in the aligned clamp assembly, covered
with a water layer and left to polimerise at room temperature for 40 min. The upper gel layer for
sample loading was prepared by mixing the 40% acrylamide solution (150 µl) with H2O (1.3 ml),
170
Materials & Methods
“upper” buffer (500 µl, 0.5 M Tris-HCl and 0.4% SDS, pH < 8.7), 15% APS (20 µl) and TEMED (2
µl); this mixture was poured over the lower solidified gel and left to polimerise at room temperature
for 50 min (a teflon comb was used to mould the sample loading wells). Protein samples (JX249A
and carboxymethylated JX249A, 200 µg/ml) were diluted in “ sample” buffer (1:1 v/v dilution in
20% glycerol, 4% SDS, 0.125 M Tris-HCl, 0.04% bromophenol blue, pH 6.8) and, after adding βmercaptoethanol (2 µl), were heated at 110 oC for 2 min. A mixture of protein molecular weight
standards including carbonic anhydrase (28 kDa), ovalbumin (45 kDa), bovine albumin (66 kDa),
phosphorylase B (97 kDa), β-galactosidase (116 kDa) and myosin (205 kDa) was prepared by
similar methods. The gel assembly was introduced in the inner cooling core and completely covered
with “ running” buffer (500 ml, 1.92 M glycine, 0.25 M Tris-HCl, 1 % SDS, pH<8.7). Both samples
and standards were loaded (20 µl) in the corresponding wells. The gel was then run at a constant
voltage of 150 V for approximately 1 hour. After cutting off the upper layer, the gel was submerged
into a Coomassie blue staining bath (0.1% Coomassie blue R-250 in fixative medium: 40%
MeOH/10% AcOH) and left under mechanic shaking for 30 min. Destaining of background colour
was done with several changes of 40% MeOH/10% AcOH (3 changes, overnight). Colourdeveloped gel was dried under vacuum and heat (2 h) on a BIO RAD 583 gel dryer, using a slowly
increasing temperature gradient, followed by constant heating at 80 oC and a final fast cooling step.
The carboxymethylated JX249A fraction was analysed by SPR under conditions identical to those
described for the original protein. Similar results were obtained.
4.3.1.13 Indirect SPR affinity analysis by solution competition experiments
A solution competition SPR approach was employed for the determination of peptide – antibody
affinities13 (section 0.2). In this approach, similar to a competition ELISA experiment, a known
constant Fab concentration is incubated with known increasing competitor antigen (peptide)
concentrations. When equilibrium is reached, the peptide – antibody mixtures are put in contact
with a surface covered with specific antigen (for instance, the C-S8c1 GH loop peptide A15) and the
relationship between free Fab in competitor concentration provides a measure for competitor
peptide – antibody affinity.
Fab fragments of both SD6 and 4C4 monoclonal antibodies were kindly supplied by Ms. Wendy F.
Ochoa and Dr. Nuria Verdaguer (IIQAB – CSIC, Barcelona). Isolation and purification of Fab 3E5
were performed at 4 oC (except where mentioned otherwise) as follows: mAb was purified from
ascitic fluid as described in section 4.3.1.5 and then concentrated by precipitation with 45%
ammonium sulphate. The suspension was centrifuged (10000 r.p.m.) for 20 min and pellet was
resuspended in the minimum volume of PBS buffer. This suspension was then dialysed against PBS
overnight (3 × 1 l). To a Falcon centrifuge tube containing the antibody solution (3 mg in 2 ml of
PBS) were added the following reagents: 24 µl of 0.1 M EDTA, 126 µl of 100 mM cysteine and 30
µg of papain. The volume was completed to 3 ml with PBS buffer and the tube was sealed and left
171
SPR as a tool in the functional analysis of an immunodominant site in FMDV
at 37 oC for 5 hours. The digestion was then quenched by addition of iodoacetamide (180 µl). The
digestion mixture was analysed by SDS-PAGE on a 12% acrylamide gel as described in section
4.3.1.12, except for pre-treatment of samples, which were not submitted to heating neither to βmercaptoethanol addition prior to loading in the gel. mAb and Fab 4C4 samples were used as
standards. Proteins in the digestion mixture were precipitated with 85% ammonium sulphate and
the suspension was centrifuged (10000 r.p.m.) for 20 min. Pellet was resuspended in the minimum
volume of 1:1 PBS/buffer A and the suspension dialysed overnight against buffer A (3 × 1 l). After
centrifuging at 12000 r.p.m. to remove remaining solid particles, the protein solution was eluted in a
protein A – Sepharose column as previously described for mAb purification. Fractions with
OD280≥0.5 (first elution peak) were pooled and concentrated to a final volume of 2 ml, using a
Centriprep-3 concentrator* at 2000 r. p. m.
Fab was purified by gel filtration on a Sephadex G-100 support previously conditioned and
equilibrated (overnight) at a constant PBS flow of 20 ml/h. Sample elution was performed at the
same buffer flow and monitored at 280 nm. Three peaks were collected and their composition was
analysed by SDS-PAGE as previously described in this section. Fab-containing fractions were
pooled, concentrated with a Centriprep-3 concentrator and quantitated by optical density at 280
nm.
A biospecific surface was prepared upon peptide A15 immobilisation on a CM5 sensor chip as
previously described (section 4.3.1.7), injecting 50 µl of the peptide solution (200 µg/ml in 10 mM
acetate buffer, pH 5.5) in order to obtain high surface peptide density (ca. 300 RU) and therefore
favour mass transport limitations (see chapter 3).
Fab SD6, 4C4 and 3E5 stock solutions were diluted in HBS to a final concentration of 320 nM (and
subsequent serial dilutions). Series of 7 different Fab concentrations (ranging from 0 to 320 nM)
were injected over the sensor chip surface with immobilised A15: 5-min injections at 5 µl/min were
applied, and 1-min pulses of 100 mM HCl were used for regeneration. Under mass transport
limitations, initial binding rate is related to analyte concentration14, therefore a calibration curve for
initial rate = ƒ (Fab concentration) could be built from the dependence of curve initial slope
(measured at the 100th second of injection time with a 10-second time window) on Fab
concentration (see chapter 3).
*
this system is used for concentration and desalting of 5 – 15 ml samples, having a 3 kDa molecular weight cut-off. Sample is
placed in a container where a filtrate collector is immersed and twist-locked. Immersion creates a slight hydrostatic pressure
differential which is increased upon centrifugation of the assembly. Therefore, solvent and materials below the molecular
weight cut-off are forced through the membrane into the filtrate collector until equilibrium is reached (hydrostatic pressure
differential = 0). Upon removal of filtrate solution, the differential is restablished and successive concentration cycles can be
carried out.
172
Materials & Methods
Peptide solutions (5 to 1250 nM in HBS) were incubated with Fab (80 nM) overnight at 4 oC. The
solutions were then allowed to stand at room temperature for 1 hour for re-equilibration prior to
injection in the SPR system. Each peptide – Fab mixture was then injected over the sensor chip
surface (5-min injections at 5 µl/min) and 100 mM HCl 1-min pulses were used to regenerate the
peptide surface after each injection. Remaining free Fab in each injected mixture was measured
from curve initial slope and subsequent intrapolation in the corresponding calibration curve. The
dependence of remaining free Fab on competitor peptide concentration was plotted and processed
by the following two methods:
Data points from the titration series where free Fab concentration was measured from the binding
rate were fitted to the equation (Fab total concentration is constant, peptide concentration is the
independent variable and KD is the fitted parameter):
[Fab] − [ peptide] − K D
2
+
([peptide] + [Fab] + K D )2 − [ peptide]× [Fab]
4
that is included in the BIAEvaluation solution affinity model2 (chapter 3). This evaluation of KD
(KD=1/KA) does not take into account the effects of the immobilised peptide antigen.
Another method, that takes into account the influence of the immobilised peptide, is based on the
Cheng and Prusoff’ s formula15 (chapter 3):
Ki = 1 +
K A [Fab]
IC50
where KA is the immobilised peptide – Fab affinity (determined independently, for instance, by SPR
kinetic analysis), [Fab] is the Fab total concentration and IC50 is the 50% inhibitory concentration for
the competitor peptide in solution (determined from the free Fab = ƒ (peptide concentration) plot).
173
SPR as a tool in the functional analysis of an immunodominant site in FMDV
4.3.2
Enzyme-linked immunosorbent assays – ELISA
The antigenicity of the FMDV synthetic peptides towards mAbs SD6, 4C4 and 3E5 was also
determined by immuno-enzymatic assays16, namely, competition ELISA17. Procedures were as
follows:
Peptide A21 conjugated to KLH# (5 pmol of peptide in 100 µl PBS per well) was incubated
overnight at 4 oC as coating antigen in micro-titer ELISA 96-well plates. The latter were saturated for
3 h with 5% BSA in PBS and then liquid was removed upon suction under reduced pressure with a
Pasteur pipette. This and all subsequent steps were carried out at room temperature. Then, 100 µl of
a solution containing a non-saturating, constant amount of mAb – pre-incubated for 1.5 h with
different concentrations of the competitor peptide antigens (serial dilutions from 243 to 0.1
pmol/100 µl) in 1% BSA in PBS – was added to the wells and further incubated for 1 h. After
washing with 0.1% BSA, 0.1% Tween 20 in PBS, 100 µl of peroxidase-conjugated goat anti-mouse
IgG (1:3000 dilution in PBS) were added to each well. Incubation was for 1 h, followed by thorough
rinsing with 0.1% BSA, 0.1% Tween 20 in PBS. Bound antibody was detected using H2O2 and ortophenylenediamine as substrate. Colour was allowed to develop in the dark for 10 minutes and
reaction was quenched upon addition of 2 M H2SO4 (100 µl/well). The absorbance at 492 nm was
immediately read.
The assay included a series of positive and negative controls: a positive control A21-KLH + mAb
(without competitor peptide) in triplicate and five negative controls, respectively, A21-KLH + PBS
(× 2), PBS + mAb (× 2) and PBS + PBS (× 1). Absorbances were corrected upon subtraction of
the average absorbance measured for negative controls and expressed as percentages of the
maximum absorbance (average of positive controls). Competitor peptide antigenicity was expressed
as IC50, that is, 50% of inhibitory concentration (competitor concentration leading to a 50%
decrease in maximum absorbance) and normalised to the IC50 obtained for the standard peptide
A15 (IC50 rel = IC50 competitor/IC50 A15, sections 2 and 3).
#
this conjugate had been already prepared by Dr. M. L. Valero and M. E. Méndez.
174
Materials & Methods
References
1 Johnsson, B., Löfås, S. and Lindquist, G. (1991) Immobilization of proteins to a
carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon
resonance sensors, Anal. Biochem. 198, 268-277.
2 “BIAevaluation Software Handbook: version 3.0”, (Biosensor AB, 1997) Uppsala, Sweden.
3 Morton, T. A., Myszka, D. and Chaiken, I. (1995) Interpreting complex binding kinetics from optical
biosensors: a comparison of analysis by linearization, the integrated rate equation and numerical
integration, Anal. Biochem. 227, 176-185.
4 O’ Shannessy, D. J., Brigham-Burke, M., Soneson, K. K., Hensley, P. and Brooks, I. (1993)
Determination of rate and equilibrium binding constants for macromolecular interactions using surface
plasmon resonance: use of nonlinear least squares analysis methods, Anal. Biochem. 212, 457-468.
5 Andersson, K., Hamalainen, M. and Malmqvist, M. (1999) Identification and optimization of
regeneration conditions for affinity-based biosensor assays. A multivariate cocktail approach, Anal.
Chem. 71, 2475-2481.
6 Karlsson, R. (1994) Real-time competitive kinetic analysis of interactions between low-molecularweight ligands in solution and surface-immobilized receptors, Anal. Biochem. 221, 142-151.
7 Albericio, F., Andreu, D., Giralt, E., Navalpotro, C., Pedroso, E., Ponsati, B. and Ruiz-Gayo, M.
(1989) Use of the Npys thiol protection in solid phase peptide synthesis, Int. J. Peptide Protein Res.
34, 124-128.
8 http://www.rscb.org/pdb/ (structure code 1ca1)
9 Jacobson, R. H., Zhang, X. J., DuBose, R. F. and Matthews, B. W. (1994) Three-dimensional
structure of β-galactosidase from E. coli, Nature 369, 761-766.
10 Carbonell, X., Feliu, J. X., Benito, A. and Villaverde, A. (1998) Display-induced antigenic variation in
recombinant peptides, Biochem. Biophys. Res. Comm. 248, 773-777.
11 Lapko, V. N., Jiang, X. Y. and Smith, D. L. (1998) Surface topography of phytocrome A deduced
from specific chemical modification with iodoacetamide, Biochemistry 37, 12526-12535.
12 Hames, B. D. and Rickwood, D. “Gel electrophoresis of proteins – a practical approach”, 2nd ed., IRL
Press, Oxford (1990).
13 Nieba, L., Krebber, A. and Plükthun, A. (1996) Competition BIAcore for measuring true affinities:
large differences from values determined from binding kinetics, Anal. Biochem. 234, 155-165.
14 “BIAapplications Handbook”, (Pharmacia Biosensor AB, 1994) Uppsala, Sweden.
15 Lazareno, S. and Birdsall, N. J. (1993) Estimation of competitive antagonist affinity from functional
inhibition curves using the Gaddum, Schild and Cheng-Prusoff equations, British J. Pharmacol. 109,
1110-1119.
16 Abbas, A. K., Lichtman, A. H. and Pober, J. S. “Cellular and molecular immunology” , 3rd ed., W. B.
Saunders Co., United States of America (1997).
17 Mateu, M. G., Andreu, D. and Domingo, E. (1995) Antibodies raised in a natural host and
monoclonal antibodies recognize similar antigenic features of foot-and-mouth disease virus, Virology
210, 120-127.
175
SPR as a tool in the functional analysis of an immunodominant site in FMDV
4.4 Structural studies of the FMDV peptides
4.4.1
Two-dimensional proton nuclear magnetic resonance1
NMR spectra were acquired both in aqueous solution (85% H2O + 15% D2O) and in the presence
of the structuring agent TFE (30% TFE + 60% H2O + 10% D2O) at a peptide concentration of 2
mM. Experiments were run at 25 oC and 1,4-dioxane was added to all samples as an internal
reference standard.
All experiments were carried out with a Varian VXR-500 S NMR spectrometer and further processed
with the VNMR3 software programs. The 2D 1H-NMR experiments performed were:
!
TOCSY3, with 70 millisecond mixing time;
!
NOESY4, with mixing time of either 200 or 400 milliseconds;
!
ROESY5, with 200 millisecond mixing time.
Water signal elimination was carried out either upon pre-saturation or using the WATERGATE6
method. Prior to the Fourier transform, both FIDs and interferograms were multiplied by an
exponential function.
176
Materials & Methods
4.4.2
Protein X-ray diffraction crystallography7,8
4.4.2.1 Protein crystallisation
Crystals of the complex between the Fab of 4C4 and peptide A15(138F,140P,142S) were obtained
by the hanging drop vapour diffusion9 technique and subsequent micro and macro-seeding steps.
Fab (40 µl, 18 mg/ml in PBS) and peptide (8 µl, 10 mg/ml in H2O) were incubated at 4 oC for 2
hours. A simple search for crystallisation conditions was performed in the vicinity of the conditions
found by W. F. Ochoa for the crystallisation of similar Fab 4C4 – FMDV peptide complexes at 20
o
C: 1 µl droplets of the peptide-Fab mixture were mixed with equivalent volumes of the precipitating
agents; these agents were based on different dilutions of PEG 4K in water, 100 mM Tris-HCl buffer
at variable pH and 400 mM LiCl. Each precipitating solution (1 ml) was poured on a well of 24-well
cell culture plates, which acted as solution reservoirs. Protein droplets were put on pre-treated& glass
covers that were then inverted and stuck, using silicone grease, to the top of the corresponding
solution reservoir.
Small twined needles were formed at 18% polyethyleneglycol (PEG) 4K, pH 8.5 and then used for
micro-seeding: a cat whisker was soaked in a needle-containing drop and then in a fresh protein
drop that was equilibrated against a solution reservoir as previously described. This micro-seeding
produced larger needles at 16% PEG 4K, which were harvested (upon suction with a capillary
quartz tube, ∅=0.2 mm) and washed in crystallising solution. These needles were used for macroseeding in 2 µl droplets containing 7 mg/ml of Fab, 1.8 mg/ml of peptide, 6.5% PEG 4K, 0.2 M LiCl
with 50 mM Tris HCl (pH=8.5), equilibrated against a reservoir containing 13% PEG 4K equally
buffered at room temperature. Small needle-shaped crystals (0.6 × 0.05 × 0.03 mm) were
reproducibly formed under these conditions and, occasionally, unstable hexagonal crystals were also
observed.
4.4.2.2 Data collection
Crystals for cryogenic data collection were soaked in harvesting solutions with 20% of glycerol and
flash-frozen under a stream of boiled-off nitrogen at 100 K. X-ray data sets were collected by W. F.
Ochoa on a MarResearch image plate detector (180 × 0.10 mm, 1800 pixels) system using a Rigaku
RU-200B rotating anode, on the European Synchrotron Radiation Facility at Grenoble. A 2.2 Å
resolution data set was collected with 1o rotations (a total of 91 rotations) at a crystal-detector
distance of 180 mm. Crystals were orthorhombic, space group P212121, and unit cell parameters as
presented in chapter 3, containing one molecule of the complex per asymmetric unit. Diffraction
data were auto-indexed and integrated and merged using programs DENZO and SCALEPACK10.
&
glass covers were treated as follows: 30 min in a dichloromethylsilane bath (hood) → 30 min in a water bath → 30 min in a
fresh water bath → 30 min in an ethanol bath; the covers were then allowed to dry prior to their utilisation.
177
SPR as a tool in the functional analysis of an immunodominant site in FMDV
4.4.2.3 Structure solution and refinement
Crystals of the complex seemed related to crystals formed with the same Fab and the wild-type
peptide A15, whose structure had been previously solved. However, the unit cell parameters
differed and the structure was newly determined by molecular replacement11 using the AmoRe
package12, employing the 4C4 Fab coordinates as searching model. The initial solutions were then
optimised by allowing to move as four separated rigid bodies the variable heavy, variable light,
constant heavy and constant light domains. Examination of the electron density maps, calculated at
this stage, clearly showed extra densities corresponding to peptide occupying the antigen binding
site. The final model for the structure of the complex was obtained by iterative cycles of manual
modelling of water molecules and rebuilding of protein/peptide chains using the program O13,
alternating with positional refinement using standard protocols in the CNS package14. Bulk solvent
correction was applied, allowing the use of all reflections in the resolution shell 15.0 – 2.3 Å. The
refined models converged to satisfactory crystallographic agreement factors, as presented in chapter
3. Structural refinement analysis was done with PROCHECK15 and graphic representation of the
structure was processed with program SETOR16.
References
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
178
Wüthrich, K. “NMR of proteins and nucleic acids”, Wiley, New York (1986).
Braunschweiler, L. and Ernst, R. R. (1983), J. Magn. Reson. 53, 521.
http://www.varianinc.com/nmr/
Kumar, A., Ernst, R. R. and Wüthrich, K. (1980), Biochem. Biophys. Chem. Comm. 95, 1.
Bothner-By, A. A., Stephens, R. L., Lee, J., Warren, C. D. and Jeanloz, R. W. (1984) Structure
determination of a tetrasaccharide: transient nuclear overhauser effects in the rotating frame, J. Am.
Chem. Soc. 106, 811-813.
Piotto, M., Saudek, V. and Sklenár, V. (1992), J. Biomol. NMR 2, 661-665.
Drenth, J., “Principles of protein X-ray crystallography” , Cantor, C. R. (Ed.), Springer-Verlag, New
York (1987).
Ducruix, A. and Giegé, R., “Crystallization of nucleic acids and proteins – a practical approach.”,
Oxford University Press, Oxford (1992).
McPherson, A., “ Preparation and analysis of protein crystals”, Wiley, New York (1982).
Otwinowsky, Z. and Minor, W. (1997) Processing of X-ray diffraction data collected in oscillation
mode. Methods Enzymol. 276, 307-326.
Rossman, M. G. (Ed.) “The molecular replacement method”, Gordon & Breach, New York (1972).
Navaza, J. (1994) AmoRe: an automated package for molecular replacement, Acta Crystallogr. A 50,
157-163.
Jones, T. A., Zou, J. Y., Cowan, S. W. and Kjeldgaard, M. (1991) Improved methods for building
protein models in electron density maps and the location of errors in these models, Acta Crystallogr. A
47, 110-119.
Brünger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang,
J.-S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T. and Warren, G.
L. (1998) Acta Crystallogr. D 54, 905-921.
Laskowski, R. A., MacArthur, M. W., Smith, D. K., Jones, D. T., Hutchinson, E. G., Morris, A. L.,
Naylor, B., Moss, D. and Thornton, J. M. “PROCHECK Manual, version 3.0”, Oxford Molecular Ltd.,
Oxford, UK (1994).
Evans, S. V. (1993) SETOR: hardware-lighted three-dimensional solid model representations of
macromolecules, J. Molec. Graphics 11, 134-138.