J. gen. Virol. (1988), 69, 3005-3013.
Printed in Great Britain
3005
Key words: vaccinia virus/VEE virus~recombinant virus
Recombinant Vaccinia/Venezuelan Equine Encephalitis (VEE) Virus
Expresses VEE Structural Proteins
By R I C H A R D M. K I N N E Y , 1. J O S E P H J. E S P O S I T O , 2
B A R B A R A J. B. J O H N S O N , 1 J O H N T. R O E H R I G , 1
J A M E S H. M A T H E W S , 1 A L A N D. T. B A R R E T T 3 AND D E N N I S W. T R E N T 1
1Division of Vector-Borne Viral Diseases, Center for Infectious Diseases, Centers for Disease
Control Public Health Service, U.S. Department of Health and Human Services, P.O. Box 2087,
Fort Collins, Colorado 80522, 2Division of Viral Diseases, Center for Infectious Diseases, Centers
for Disease Control Public Health Service, U.S. Department of Health and Human Services,
1600 Clifton Road, Atlanta, Georgia 30333, U.S.A. and 3Department of Microbiology,
University of Surrey, Guildford GU2 5XH, Surrey, U.K.
(Accepted 18 August 1988)
SUMMARY
c D N A molecules encoding the structural proteins of the virulent Trinidad donkey
and the TC-83 vaccine strains of Venezuelan equine encephalitis (VEE) virus were
inserted under control of the vaccinia virus 7.5K promoter into the thymidine kinase
gene of vaccinia virus. Synthesis of the capsid protein and glycoproteins E2 and E1 of
VEE virus was demonstrated by immunoblotting of lysates of CV-1 cells infected with
recombinant vaccinia/VEE viruses. VEE glycoproteins were detected in recombinant
virus-infected cells by fluorescent antibody (FA) analysis performed with a panel of
VEE-specific monoclonal antibodies. Seven E2-specific epitopes and two of four Elspecific epitopes were demonstrated by FA.
INTRODUCTION
Venezuelan equine encephalitis (VEE) virus is a mosquito-borne alphavirus which has caused
numerous epidemics in equines and humans in the Americas. VEE virus infection often
produces a fatal encephalitis in equines, with mortality and fatality rates in various epidemics of
19 to 4 0 ~ and 38 to 83 ~o, respectively (Groot, 1972). Although the disease is generally milder in
humans, with fatality rates lower than 1~ , human morbidity can be significant. In particular,
during the 1962 to 1964 VEE outbreak in Venezuela, an estimated 23 000 human cases occurred,
with 960 cases of central nervous system infection and 156 deaths. In an epidemic in Ecuador in
1969, an estimated 31000 human cases occurred, with 310 deaths. Children under 15 years old
are the most seriously affected (Groot, 1972). Between 1969 and 1971, a VEE epizootic epidemic
spread from South America through Central America and Mexico and reached the U.S.A. in
1971, causing over 1400 equine deaths in Texas (Lord, 1974).
The live, attenuated TC-83 vaccine strain of VEE virus was derived from its virulent parent
VEE strain, Trinidad donkey (TRD), by serial passage of T R D virus in embryonic guinea-pig
heart cells (Berge et al., 1961). The TC-83 vaccine proved invaluable in controlling the 1969 to
1971 epizootic of VEE in Central America (Walton et al., 1972; Eddy et al., 1972) and Texas
(Calisher & Maness, 1975; Baker et al., 1978). Although TC-83 virus is an effective vaccine,
concerns remain about its possible reversion to virulence (Berge et al., 1961) and entry of the
revertant into a mosquito-vertebrate host cycle (McKinney, 1972). The reactogenicity of the
TC-83 vaccine virus in humans (Alevizatos et al., 1967 ; McKinney et al., 1963) and the possible
teratogenic potential of VEE virus (London et al., 1977) are also of concern. A formaldehydeinactivated preparation of TC-83 virus (C-84) has proven inferior to live, attenuated TC-83 virus
0000-8559
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R. M. KINNEY AND OTHERS
in protecting hamsters f r o m p e r i p h e r a l or aerosol challange w i t h virulent T R D virus (Jahrling &
Stephenson, 1984).
W e h a v e investigated expression o f V E E virus proteins via r e c o m b i n a n t v a c c i n i a / V E E virus
as a possible a l t e r n a t i v e V E E vaccine. T h e potential for success of this strategy was
d e m o n s t r a t e d by R i c e et al. (1985) w h o o b t a i n e d expression o f the structural proteins of a n o t h e r
alphavirus, Sindbis virus, in a r e c o m b i n a n t v a c c i n i a / S i n d b i s virus and by F r a n k e et al. (1985)
w h o showed that cattle i m m u n i z e d w i t h r e c o m b i n a n t v a c c i n i a / S i n d b i s virus d e v e l o p e d antiSindbis virus neutralizing antibody.
W e h a v e r e p o r t e d the cloning and s e q u e n c i n g of c D N A molecules c o n t a i n i n g the entire
structural p r o t e i n - c o d i n g region o f the R N A g e n o m e s of V E E T R D ( K i n n e y et al., 1986) and
TC-83 ( J o h n s o n et al., 1986) viruses. W e report here the c o n s t r u c t i o n of r e c o m b i n a n t viruses,
V A C C / T C - 8 3 and V A C C / T R D , t h a t express the structural proteins of these viruses.
METHODS
Cells and viruses. Seed stocks of plaque-purified VEE viruses were prepared as frozen ( - 70 °C) aliquots of foetal
bovine serum-enriched (12%) Eagle's MEM from virus-infected Vero cells. Vaccinia virus was the New York City
Board of Health strain three times plaque-purified from a vial of smallpox vaccine (Dryvax, Wyeth Laboratories).
Vaccinia virus and vaccinia/VEE recombinant virus stocks were grown in human 143B or CV-1 monkey cell
cultures. Vero cell monolayers grown in six-well plates were used for virus plaque titrations. Overlay medium
consisted of M-199 medium made with Earle's balanced salts solution, 2% (v/v) foetal bovine serum, 1% (w/v)
Noble agar, 100 units/ml penicillin G, 100 gg/ml streptomycin sulphate and 0.12% (w/v) neutral red.
Construction o f chimeric plasmid. Plasmid pGS-62, a derivative of the pGS-20 vector (Mackett & Smith, 1986;
Mackett et al., 1984) with a single EcoRl site downstream of the vaccinia 7.5K promoter (Esposito et al., 1987),
was used for insertion ofcDNA encoding the structural proteins of VEE virus (Fig. 1). Restriction enzyme Tthl 11I
cleavage of pTC-5 (Johnson et al., 1986), pTRD-1 (Kinney et al., 1986) or pTRD-20 (unpublished results)
recombinant plasmids released the cDNA so that the translational start codon for the complete structural genes of
VEE virus could be ligated into pGS-62 immediately downstream of the vaccinia virus 7.5K early-late promoter
and mRNA start site by using EcoRl linkers. The mRNA transcript should initiate translation at the start codon
and terminate at the UGA stop codon located at the end of the cDNA in glycoprotein El.
Insertion o f VEEgenes into vaccinia virus. Recombinant vaccinia viruses were produced essentially as detailed by
Mackett et al. (1982, 1984). Recombinant virus was selected by plaque titration in TK- 143B cell monolayers
grown in six-well plates under overlay medium containing 30 gg/ml bromodeoxyuridine. Individual virus plaques
were grown in 143B cell cultures in 24-well plates, and screened for the presence of VEE genes by DNA dot blot
hybridization using nick-translated, 32p-labelled VEE-specific cDNA as probe. Recombinants were screened for
expression by immune dot blotting. Recombinants were then plaque-purified three times in 143B cells in the
presence of bromodeoxyuridine, and once in CV-1 cells without bromodeoxyuridine. CV-1 cell monolayers grown
in Eagle's MEM supplemented with 10 % foetal bovine serum and antibiotics on 150 cm 2 flasks were infected with
plaque-purified or recombinant vaccinia virus at a multiplicity of 0-05 p.f.u./cell. When c.p.e, was observed in
90% of the cells, they were harvested using a rubber policeman and pelleted by centrifugation at 6000 r.p.m, for 20
min in a GSA rotor. The cells were enucleated, trypsinized, homogenized and sonicated (three 30 s bursts at 50 W),
and the virus was pelleted through a 40~ (w/w) sucrose cushion as described by Esposito et al. (1981). The virus
pellet was resuspended in TE buffer (10 mM-Tris-HCl pH 9.0, 1 mM-disodium EDTA) and stored at - 70 °C until
use.
Analysis o f recombinant vaccinia/VEE virus genome structure. Genomic DNA was isolated from vaccinia or
vaccinia/VEE recombinant virus as previously described (Esposito et al., 1981). Restricted DNA was
electrophoresed in 0.6% agarose in TBE buffer (1 M-Tris-HC1 pH 8.3, 0.91 M-boric acid, 20 mM-disodium EDTA)
for 12 to 24 h at 30 V. Southern blotting and hybridization were performed as described by Maniatis et al. (1982).
Preparation o f VEE virus antisera. Polyclonal hyperimmune ascitic fluid to VEE TC-83 virus was prepared in
NIH Swiss outbred mice as described by Hammon & Sather (1969). The VEE virus monoclonal antibodies used
in this study were prepared and characterized by Roehrig et al. (1982).
Analysis o f protein expression. VEE viral proteins were visualized by immunofluorescence or by immunoblotting
of lysates of infected CV-1 cells. CV-1 cell monolayers (25 cm 2) were infected with virus (20 p.f.u./cell). At 18 h
post-infection, lysates were prepared by washing cells with cold phosphate-buffered saline (PBS) and then adding
0.7 ml of RIPA buffer (1% w/v sodium deoxycholate, 1% v/v Triton X-100, 0-1% w/v SDS, 50 m•-Tris HC1 pH
7.4) containing 1% (v/v) aprotinin (Sigma) to each monolayer. Cell lysates were forced several times through a 25gauge needle to shear DNA and then stored at - 70 °C until use. For immunoblotting, aliquots of cell lysates were
electrophoresed in 10 % (w/v) polyacrylamide-N,N'diallyltartardiamide (Bio-Rad) gels as described by France et
al. (1979). Polypeptides were transferred to nitrocellulose (Towbin et al., 1979) at 60 V for 4 h in transfer buffer (50
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Protein expression by vaccinia/VEE virus
Tthl1
1
I
~
-
~
-UGA
~/~ "EcoRI
3007
EcoRI
Small~
BamHI~
pUC18
ut with
Tthl111
lenow + dNTPs
igate
linker
EcoRI
EcoRI
ut with
sotate VEE cDNA
from agarose gel
Ligate
Fig. 1. Construction of the pTC-5A chimeric plasmid. VEE cDNA (bold line) was removed from
pUC 18 and joined to the vaccinia 7-5K early late promoter in the pGS-62 plasmid. TK indicates DNA
segments from the vaccinia virus TK gene.
mM-Tris base, 380 mM-glycine, 20~ v/v methanol) using a Transphor electrophoresis unit (Hoeffer Scientific
Instruments, San Francisco, Ca., U.S.A.). The nitrocellulose filter was blocked with 3 ~ (w/v) bovine serum
albumin (Fraction V, Reheis Chemical Co., Phoenix, Ariz., U.S.A.) in PBS and then incubated with anti-TC-83
mouse hyperimmune ascitic fluid (1:200 dilution) in PBS containing 0.05 ~ (v/v) Tween 20 (Sigma) for 2 h at room
temperature. The filter was washed with several rinses of PBS/Tween 20 and incubated for 1 h with goat antimouse, alkaline phosphatase-conjugated antibody (Jackson Immunoresearch Laboratories, Avondale, Pa.,
U.S.A.). Polypeptide bands were visualized using an NBT/BCIP kit (Kirkegaard & Perry Laboratories,
Gaithersburg, Md., U.S.A.).
Indirect immunofluorescence tests for VEE proteins were done with CV-1 cells (four- or eight-chamber LabTek tissue culture slides, Miles Laboratories) infected with virus at a multiplicity of 10 or 20 p.f.u./cell. At 24 h
post-infection, cell monolayers were rinsed twice with PBS and air-dried with or without prior acetone fixation.
Expression of VEE virus epitopes was determined using acetone-fixed spot slides of virus-infected CV-1 cells that
were scraped from 25 cm 2 flasks. Binding of virus-specific antibodies was detected with fluorescein
isothiocyanate-conjugated goat anti-mouse IgG (Sigma).
RESULTS
Genome structure analysis of recombinant vaccinia/VEE viruses
Three recombinants were produced: VACC/TC-5A, expressing the genes encoding the
structural proteins of VEE TC-83 virus, and two vaccinia/TRD virus recombinants,
VACC/TRD-1A and VACC/TRD-20A, expressing the structural proteins of VEE TRD virus.
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3008
R . M. K I N N E Y
(a)
1
AND OTHERS
(b)
2
3
4
5
1
2
3
4
5
11930~
6728~,
4093~
1907~,
1232~
Fig. 2. Detection of VEE virus cDNA in recombinant vaccinia/VEE virus genomes. HindlII-digested
DNA was electrophoresed in an agarose gel and transferred to nitrocellulose. The HindlII J fragment
containing the TK gene of vaccinia virus is indicated by the lower arrow. This fragment was altered in
size by insertion of VEE genes in the vaccinia/VEE recombinants (top arrow). Molecular sizes (bp) of
marker bands containing VEE cDNA are shown (lanes 1). (a) Ethidium bromide-stained gel; (b)
hybridization using 32p-labelled VEE cDNA probe. Lanes 2, vaccinia virus; lanes 3, TC-5A; lanes 4,
TRD-IA; lane 5, TRD-20A.
To confirm insertion of VEE virus c D N A into the vaccinia virus thymidine kinase (TK) locus,
D N A purified from wild-type of recombinant vaccinia virus was digested with HindlII for
Southern blot hybridization in which VEE virus-specific c D N A probes were used (Fig. 2b). The
D N A gel patterns in Fig. 2 (a) show that the approximately 5 kbp HindlII J fragment of vaccinia
virus (lower arrow) was not present in recombinant virus D N A (TC-5A, TRD-1A, TRD-20A),
which instead showed the appearance of a new fragment of about 9 kbp (upper arrow), which is
the predicted size of the HindlII J fragment containing the respective VEE virus c D N A
fragment plus the 7.5K promoter sequence from the chimeric plasmid. Radiolabelled V E E virus
c D N A hybridized to the 9 kbp HindlII fragment of the recombinant vaccinia/VEE virus D N A s
(Fig. 2b).
Expression of VEE structural proteins
Immunoblotting of VACC/TC-5A, V A C C / T R D - 1 A and V A C C / T R D - 2 0 A cell lysates
clearly showed a polypeptide band that comigrated with the capsid protein in the VEE TC-83
and T R D cell lysates and in dissociated purified virions (Fig. 3). Lysates of cells infected with
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Protein expression by vaccinia/VEE virus
1
2
3
4
5
6
7
8
3009
9
~t E2
~EI
Fig. 3. Immunoblot of VEE structural polypeptides in lysates of CV-1 cells infected for 18 h with
recombinant VACC/TC-5A, VACC/TRD-1A or VACC/TRD-20A virus or with VEE TC-83 or TRD
virus. Lanes 1 to 7 represent equal volumes of cell lysates. Proteins of purified VEE TC-83 virus and
TRD virus are shown in lanes 8 and 9 respectively. VEE capsid (C), E2 and E1 polypeptides are
indicated. Anti-TC-83 mouse ascitic fluid was used to detect VEE-specific polypeptides. Cells were
uninfected (lane 1) or infected with vaccinia virus (lane 2), VACC/TC-5A (lane 3), VACC/TRD-1A
(lane 4), VACC/TRD-20A (lane 5), VEE TC-83 (lane 6) or VEE TRD (lane 7).
the three recombinant viruses also contained a polypeptide that comigrated with the E1
envelope glycoprotein band present in cells infected with wild-type VEE virus or virions. The
VEE E2 polypeptide in lysates of VACC/TRD-1 A-infected cells appeared to migrate somewhat
faster and to be expressed at a higher level than in cells infected with VACC/TC-5A or
VACC/TRD-20A virus. Since all three recombinant viruses were constructed using VEE TC-83
or TRD cDNA clones containing equivalent genomic regions, including the native VEE codons
for initiation and termination of translation, we are unable to explain these differences in the
expression of E2. There was no evidence of E 1, E2 or capsid polypeptide in lysates of uninfected
or vaccinia virus-infected CV-1 cells (Fig. 3). The polypeptide band intensities in Fig. 3
indicated that the level of VEE virus polypeptide expression in recombinant virus-infected
CV-1 cells was severalfold lower than that in VEE virus-infected cells. Immune precipitation
of radiolabeUed lysates showed the presence of pE2, the intracellular precursor to the E2
glycoprotein, in CV-1 cells infected with each of the recombinant viruses (data not shown).
Expression of VEE proteins in recombinant virus-infected cells was demonstrated further by
fluorescent antibody (FA) analysis using anti-TC-83 mouse hyperimmune ascitic fluid (1:200
dilution) (Fig. 4). VEE antigens were detected in acetone-fixed and unfixed VEE TC-83 (Fig. 4b
and e respectively) and recombinant VACC/TC-5A (Fig. 4c andfrespectively) virus-infected
CV-I cells. VEE antigens were not present in uninfected CV-I cells (Fig. 4a, d). Although the
level of expression of VEE structural proteins in recombinant virus-infected cells was lower than
that in cells infected with VEE viruses (Fig. 3), many recombinant virus-infected acetone-fixed
ceils (Fig. 4c) showed FA intensity equal to that of TC-83 virus-infected cells (Fig. 4b). Surface
expression of VEE antigens as indicated by FA analysis of unfixed cells was lower in
recombinant virus-infected cells (Fig. 4 f ) than in cells infected with TC-83 virus (Fig. 4e). Time
course experiments showed that VACC/TC-5A or VACC/TRD-1A virus-infected CV-1 cells
expressed FA-detectable levels of VEE antigen in fixed cells by 4 h post-infection. VEE T R D
and VACC/TRD-1A virus-infected cells fluoresced to the same degree as TC-83 and
VACC/TC-5A virus-infected cells, respectively (data not shown).
Expression of VEE glycoprotein epitopes
Antiserum prepared against vaccinia virus showed positive fluorescence only with vaccinia
and vaccinia/VEE virus-infected cells. Polyvalent anti-TC-83 ascitic fluid reacted with cells
infected with VEE virus or vaccinia/VEE recombinant virus (Table 1). The availability of
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R . M. K I N N E Y
AND
OTHERS
/u)
(d)
F i g . 4. D e t e c t i o n o f V E E p r o t e i n s , u s i n g a n t i - T C - 8 3 m o u s e h y p e r i m m u n e ascitic fluid, b y i n d i r e c t
i m m u n o f l u o r e s c e n c e o f a c e t o n e - f i x e d (a to c) o r u n f i x e d (d to f ) CV-1 cell m o n o l a y e r s 24 h a f t e r
i n f e c t i o n w i t h T C - 8 3 v i r u s (b, e) o r r e c o m b i n a n t V A C C / T C - 5 A v i r u s (c,f). U n i n f e c t e d cell c o n t r o l s a r e
s h o w n (a, d). All p h o t o g r a p h s a r e i d e n t i c a l e x p o s u r e s . B a r m a r k e r r e p r e s e n t s 10 p.m.
Table
1.
Indirect immunofluorescence of acetone-fixed CV-1 cells 24 h post-infection
c
Vaccinia
V i r u s i n f e c t i n g cells
~
VACC/TC-5A
VACC/TRD-1A
Antibody*
Specificity
Uninfected
cells
TC-83
TRD
Mouse serum
MHIAF~
Vaccinia virus
VEE TC-83
-
3t
-
3
3
3
3
4
4
5B4D-6
1A4A-1
1A6C-3
1A3A-5
1A4D-1
1A3A-9
1A3B-7
E2 a T C - 8 3
E2 ~ PTF-39
E2 d EVE
E2 e P676
E2 f TRD
E2g P T F - 3 9
E2 h PTF-39
-
-
3
3
2
3
3
3
1
3
2
3
3
1
4
4
2
4
4
4
4
1
4
2
2
4
4
4
3B2D-5
3B2A-9
5B6A-6
3A5B-1
E1 a T C - 8 3
E1 b T C - 8 3
E 1~ T C - 8 3
E1 d T C - 8 3
.
-
-
3
3
3
3
3
3
3
4
3
3
1
4
.
.
-
.
* A l p h a n u m e r i c d e s i g n a t i o n s a r e a n t i - V E E v i r u s m o n o c l o n a l a n t i b o d i e s ( m o u s e a s c i t i c fluids). E p i t o p e
specificity a n d v i r u s u s e d to elicit m o n o c l o n a l a n t i b o d y a r e s h o w n . All a n t i b o d y p r e p a r a t i o n s w e r e tested a t 1 : 300
dilution.
"~ R e l a t i v e f l u o r e s c e n c e as d e t e r m i n e d b y t w o o b s e r v e r s in a t least t w o e x p e r i m e n t s . - , N e g a t i v e ; 4, m a x i m u m .
:~ M H I A F , m o u s e h y p e r i m m u n e ascitic fluid.
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Protein expression by vaccinia/VEE virus
3011
well characterized monoclonal antibodies identifying epitopes in the E1 and E2 envelope
glycoproteins of VEE virus (Roehrig et al., 1982; Roehrig & Mathews, 1985) enabled us to
investigate expression of VEE virus-specific antigenic determinants in some detail (Table 1).
The antibody (2A4B-12) defining the E2 b epitope of VEE virus was unavailable for this study.
The anti-E2 monoclonal antibodies shown in Table 1 generally reacted similarly with cells
infected with VEE or vaccinia/VEE virus. In particular, the TC-83-specific antibody 5B4D-6
(epitope E2 a) (Roehrig et al., 1982; Roehrig & Mathews, 1985) also reacted more strongly with
recombinant VACC/TC-5A-infected cells than with VACC/TRD-1A-infected cells. Monoclonal antibodies 1A4A-1 (E2°), 1A4D-1 (E2 f) and 1A3A-9 (E2g) showed positive FA
reactivities with cells infected with VACC/TC-5A, VACC/TRD-1A, TC-83 or TRD virus.
Although antibody 1A3A-5 (E2 e) reacted poorly with recombinant virus-infected cells, a pattern
of TC-83 antigen specificity similar to that seen with VEE virus-infected cells was observed.
Antibody 1A6C-3 (E2d), which reacted poorly with the cells expressing TC-83, TRD or
VACC/TRD-1A virus antigens, failed to react with VACC/TC-5A virus-infected cells. The
1A3B-7 (E2 h) antibody failed to react well with VACC/TRD-1A recombinant-infected cells,
while reacting strongly with cells infected with VACC/TC-5A, TC-83 or TRD virus.
Of the four E1 glycoprotein epitopes identified for VEE virus (Roehrig et al., 1982), only the
E1 b and E1 d epitopes were detected in recombinant VACC/TC-5A or VACC/TRD-1A virusinfected cells (Table 1). Although the E1 a and E1 ~ epitopes were detected in VEE TC-83 and
TRD virus-infected, acetone-fixed cells, these two epitopes were not evident by FA in
recombinant VACC/TC-5A or VACC/TRD-1A virus-infected cells analysed under identical
conditions.
DISCUSSION
The structural proteins of alphaviruses are translated as a polyprotein from a subgenomic 26S
mRNA in the order 5'-capsid-E3-E2-6K-E 1-3' (Schlesinger & K/i/iri/iinen, 1980). This precursor
polyprotein is processed by proteolytic cleavage to produce capsid, E2 and E1 proteins which are
incorporated into the mature virion. The short protein segments (E3, which is cleaved from pE2
to yield the E2 glycoprotein, and 6K) are cleavage products that are apparently not incorporated
into mature VEE virions.
As shown for recombinant vaccinia virus expressing the structural proteins of Sindbis virus
(Rice et al., 1985), VEE mRNA was apparently transcribed in recombinant vaccinia/VEE
virus-infected cells and translated into a polyprotein precursor that was processed to yield VEE
structural proteins as cleavage products, thereby effectively mimicking translation of the 26S
subgenomic mRNA in cells infected with VEE virus.
Indirect immunofluorescence using monoclonal antibodies demonstrated authentic expression of E2 a, E2 c, E2 e, E2 f, E2~, E1 b and E1 d epitopes in cells infected with recombinant
V A C C / T C - 5 A or V A C C / T R D - 1 A virus. We are unable to explain the reduced expression of the
E2 d epitope in VACC/TC-5A virus-infected cells. The pTRD-1 eDNA clone, however, encodes
a Lys substitution in the E2 protein at amino acid position 209 (Glu in TRD genomic RNA)
(Kinney et al., 1986). It is possible that this cloning artefact may alter the binding efficiency of
anti-E2 h monoclonal antibody to the E2 polypeptide expressed in VACC/TRD-1A virusinfected cells. The E1 a and E1 e epitopes, which were evident by FA in cells infected with VEE
TC-83 or TRD virus, were not detected by FA (under identical conditions) in recombinant
virus-infected cells. The reason for the apparent lack of expression of these two E 1 epitopes in
recombinant virus-infected cells is as yet unknown.
Documentation of the expression of VEE surface glycoprotein epitopes in cells infected with
recombinant vaccinia/VEE viruses has positive implications for the use of these recombinant
viruses as alternative live, self-replicating VEE vaccines. Expression of the E2 c epitope is
especially important because it defines the critical neutralization site of VEE virus (Roehrig et
al., 1982; Roehrig & Mathews, 1985). Earlier reports demonstrated that VEE E2 glycoprotein
induces neutralizing and haemagglutination-inhibiting antibodies in immunized rabbits
(France et al., 1979; Trent et al., 1979). Those epitopes (E2°, E2 f, E2g and E2 h) which are defined
by VEE-neutralizing monoclonal antibodies with different neutralization capacities are located
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R . M. K I N N E Y A N D O T H E R S
in the same spatial domain on the glycoprotein spike (Roehrig et al., 1982; Roehrig & Mathews,
1985). Mice passively immunized with monoclonal antibodies 1A4A-1 (E2C), IA3A-9 (E2o) and
1A3B-7 (E2h), which efficiently block attachment of VEE virus to cells in vitro (Roehrig et al.,
1988), are protected from lethal peripheral challenge with several virulent VEE subtype viruses
(Roehrig & Mathews, 1985). The E2 c and E2 h epitopes are the most conserved epitopes of the E2
glycoprotein among viruses of the VEE antigenic complex, and antibody elicited against these
two epitopes should provide protection from heterologous VEE virus challenge.
Monoclonal antibody 3B2A-9 (El b) has a low level of neutralizing activity and protective
capability in mice passively immunized against VEE virus (Roehrig et al., 1982; Mathews &
Roehrig, 1982). Passive administration of non-neutralizing anti-E ld monoclonal antibodies can
prevent lethal encephalitis in mice (Mathews & Roehrig, 1982; Schmaljohn et al., 1982; Hunt &
Roehrig, 1985). These results indicate that expression of VEE epitopes E1 b and E1 d in addition
to E2 epitopes by a genetically engineered vaccine may be a more effective immunogen than
recombinant vaccines that express E2 antigenic determinants alone. Experiments are now in
progress to determine the protective efficacy of the recombinant VACC/TC-5A virus in
iinmunized mice.
This work was supported by contract n u m b e r DAMD-87-PP-7876 from the U.S. A r m y Research and
Development C o m m a n d .
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