FEMS Immunology and Medical Microbiology 27 (2000) 227^233
www.fems-microbiology.org
Immunogenicity of in vitro folded outer membrane protein PorA of
Neisseria meningitidis
Carmen Jansen a , Betsy Kuipers b , Jenny van der Biezen a , Hans de Cock a ,
Peter van der Ley b , Jan Tommassen a; *
a
b
Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
Laboratory of Vaccine Research, National Institute of Public Health and the Environment, Antonie van Leeuwenhoeklaan 9, 3720 BA Bilthoven,
The Netherlands
Received 26 July 1999; accepted 18 November 1999
Abstract
In vitro folded and the denatured form of PorA P1.6 from Neisseria meningitidis strain M990 were used for immunization studies in mice.
Previously, the antigen was isolated from cytoplasmic inclusion bodies, folded and purified. Its immunogenicity without adjuvant appeared
to be low. The addition of the adjuvant QuilA, but not of galE lipooligosaccharide, considerably enhanced the immunogenicity. Moreover,
when immunized with folded PorA P1.6 plus QuilA, a clear switch towards the IgG2a subclass of antibodies and concomitantly, the
appearance of serum bactericidal activity, which is believed to be important for protective immunity, was observed. Hence, a tool for
preparing vaccines against serogroup B meningococci devoid of endotoxin is available. ß 2000 Federation of European Microbiological
Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Meningococcal vaccine; PorA porin; Bactericidal antibody; LPS; QuilA ; Neisseria meningitidis
1. Introduction
Neisseria meningitidis is a human pathogen and one of
the major causes of bacterial meningitis [1]. Vaccines
based on capsular polysaccharide o¡er no protection
against serogroup B meningococci, which cause the majority of outbreaks in the western world. The group B polysaccharide is poorly immunogenic in humans, probably
because of immunotolerance resulting from cross-reactivity between this polysaccharide and host glycoproteins of
the neonatal brain tissue [2]. Other major surface components, such as the outer membrane porin PorA (class 1
protein), are nowadays considered as potential vaccine
candidates. Various porA genes have been cloned and sequenced, and a topology model has been proposed [3].
This model predicts a structure composed of 16 amphipathic L-strands, which traverse the outer membrane and
generate eight surface-exposed loops. The longest surface-
* Corresponding author. Tel. : +31 (30) 2532999;
Fax: +31 (30) 2513655; E-mail: [email protected]
exposed loops, 1 and 4, correspond to two variable regions
(VR1 and VR2, respectively) on which subtyping of the
meningococci is based [4].
Bactericidal monoclonal antibodies (mAbs) directed
against PorA have been raised and shown to give protection against infection in an infant rat model [5]. In Norway, an outer membrane vesicle (OMV) vaccine was
shown to provide 57% protection during the course of
the trial [6], but, more importantly, it turned out that
the presence of bactericidal antibodies in the vaccinated
humans correlated with the presence of antibodies directed
against the PorA porin [7]. Furthermore, a hexavalent
PorA OMV vaccine induced bactericidal immune responses in mice and humans [8,9]. The epitopes that contributed predominantly to the bactericidal activity were
present in loops 1 and 4 of the PorA protein [10]. PorA
is expressed by almost all meningococcal isolates [11], and
its heterogeneity (several subtypes) is limited [12], making
it even more suitable as a potential vaccine.
Vaccines based on OMV require special safety for preparation. The bacteria, which are used to prepare OMV,
have to be inactivated. Furthermore, the presence of lipooligosaccharide (LOS, endotoxin) in the vaccine can cause
toxicity. From these points of view, vaccines based on
0928-8244 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 8 - 8 2 4 4 ( 9 9 ) 0 0 1 8 9 - 3
FEMSIM 1169 9-2-00
228
C. Jansen et al. / FEMS Immunology and Medical Microbiology 27 (2000) 227^233
puri¢ed outer membrane proteins (OMPs) isolated from
an LPS-free environment in non-pathogenic bacteria, have
a preference. Previously, we obtained high yields of the
PorA protein of N. meningitidis strain M990 (subtype
P1.6) by expressing the part of the gene encoding only
the mature domain of the protein in Escherichia coli [13].
The resulting cytoplasmic inclusion bodies were solubilized
in 8 M urea and, consequently, the protein exhibited a
non-native structure. It is commonly accepted that a native conformation is a prerequisite for eliciting bactericidal
antibodies. For example, although recombinant PorA
solubilized in either SDS or guanidine hydrochloride was
strongly immunogenic, no bactericidal antibodies were
elicited [14]. Only when the recombinant PorA was folded
into a native-like conformation in the presence of LPS [14]
or phospholipid vesicles [15] and the concentration of the
denaturant was decreased, bactericidal antibodies were obtained. Hence, we have established conditions to fold the
PorA protein, produced as inclusion bodies in E. coli, in
vitro [13]. Biophysical and functional characterization con¢rmed the native-like structure of the in vitro folded protein [13]. In the present study, we performed immunization
experiments to investigate the immunogenic properties of
the folded porin.
2. Materials and methods
2.1. Bacterial strains and growth conditions
The N. meningitidis strains used in this study are M990
(B:6:P1.6) [16] and H44/76 (B:15:P1.7,16) [17]. They were
grown overnight at 37³C on GC medium (Difco) supplemented with IsoVitaleX Enrichment (Beckton Dickinson)
in a humid atmosphere containing 5% CO2 .
2.2. PorA preparation and adjuvants
PorA P1.6 was isolated from E. coli, folded and puri¢ed
as described [13]. For control experiments, the in vitro
folded PorA was denatured by heating for 30 min at
100³C in 10 mM Tris-HCl pH 7.0 and 3 mM n-dodecylN,N-dimethyl-1-ammonio-3-propanesulfonate (SB12, Fluka ; puri¢ed as described [18]). Quillaja saponin A (QuilA)
was obtained from Iscotec AB (Lulea«, Sweden). GalE LOS
of the galE derivative of strain H44/76 was isolated by the
hot-phenol extraction method [19].
2.3. Immunization of mice
Balb/c mice, ¢ve animals each group, were subcutaneously immunized on days 0, 14 and 28 with 10 Wg PorA
dissolved in 0.5 ml of 10 mM Tris-HCl pH 7.0, 3 mM
SB12. To stimulate the immune response, either 20 Wg
QuilA or 5 Wg LOS were included in the antigen preparations. Sera were collected at day 42 and stored at 4³C.
2.4. ELISA
Whole cell ELISAs were performed as described [20].
The titer is de¢ned as the dilution of the serum where
50% of the ODmax in the assay is reached. Isotypes were
determined using goat-anti-mouse (GAM) Ig isotype-speci¢c conjugates, labelled with horseradish peroxidase
(HRP) (Southern Biotechnology Associates; dilution
1:5000, except for K-IgG1-HRP, which was diluted
1:2500).
For peptide ELISAs, microtiter plates were coated overnight at room temperature with 100 Wl of 4 Wg ml31 avidin
(Sigma) in 0.01 M phosphate-bu¡ered saline (PBS) pH
7.2. After washing, the wells were ¢lled with 100 Wl of 5
Wg ml31 biotinylated peptide solution in PBS, 0.1% (v/v)
Tween-80 (Merck) and incubated for 2 h at 37³C. After
washing, the wells were incubated for 1.5 h at 37³C with
twofold serial dilutions of the individual mouse sera in
PBS, 0.1% Tween-80 (starting dilution 1: 250). The mAb
MN37C7.24, directed against loop 5 of the PorA protein
(P. van der Ley, unpublished results), was used as a control. After extensive washing, the IgG conjugate GAMHRP (Southern Biotechnology Associates; dilution
1:5000) in PBS, 0.1% Tween-80, 0.5% (w/v) protifar (Nutricia) was added to the wells and incubated for 1 h at
37³C. The color reaction was performed as described [20].
The titer was de¢ned as the reciprocal value of the highest
serum dilution were the OD450 W0.1.
2.5. Serum bactericidal assay
Serum bactericidal activity was determined as described
[21] with some modi¢cations. The meningococcal cells
were grown until the OD620 had reached a value between
0.24 and 0.26. This yielded approximately 109 colony
forming units (cfu) ml31 . The bacterial cells were diluted
to 105 cfu ml31 in assay bu¡er consisting of Gey's balanced salt solution (Sigma) with 0.5% (w/v) bovine serum
albumin (BSA, Sigma). The total volume in each well of
the plate was 24 Wl, i.e. 12 Wl of serially diluted serum in
assay bu¡er with a starting dilution of 1:4, 6 Wl of bacterial suspension and 6 Wl of baby rabbit complement (Pel
Freeze Clinical Systems, ¢nal concentration 20% v/v in
assay bu¡er). Sera from mice were heat-inactivated for
30 min at 56³C prior to use. Serum samples and bacteria
were incubated for 10 to 15 min at room temperature
prior to the addition of complement. The serum bactericidal titer is de¢ned as the reciprocal value of the highest
serum dilution that results in v90% killing.
The bactericidal mAb MN19D6.13 (P. van der Ley,
unpublished results), directed against PorA P1.6, was
used as a control.
For peptide inhibition bactericidal assays, heat-inactivated mice sera (6 Wl, dilution 1:8) were mixed with 6 Wl
of mixes of peptides (total concentration 4 mg ml31 ) corresponding to either loop 1, 4 or 5 and incubated for 2 h
FEMSIM 1169 9-2-00
C. Jansen et al. / FEMS Immunology and Medical Microbiology 27 (2000) 227^233
229
at 37³C. Subsequently, 6 Wl of bacterial suspension and 6 Wl
of complement were added, and the bactericidal serum
activity was determined.
2.6. Synthesis of peptides
Oligopeptides of 15 residues, corresponding to loops 1,
4 and 5, with ¢ve amino acid residues overlap between the
peptides within each loop, were synthesized on a 10-Wmol
scale by using an automated multiple peptide synthesizer,
equipped with a 48-column reaction block (AMS 422,
ABIMED Analysen-Technik GmbH, Langenfeld, Germany) as described earlier [22]. Double couplings were
performed with £uorenylmethoxycarbonyl (Fmoc)-amino
acids (50 Wmol), benzotriazolyloxy-Tris-[N-pyrrolidino]phosphonium hexa£uorophosphate (50 Wmol), and Nmethylmorpholine (100 Wmol) and the Fmoc group was
cleaved with piperidine/N,N-dimethylacetamide, 2:8 (v/v),
as described earlier [21,22]. Fmoc-Lys(biotinylcaproyl)OH was coupled at the N terminus, followed by Fmoccleavage and acetylation.
2.7. SDS-PAGE and Western blotting
SDS-polyacrylamide gels were prepared as described
[23], except that the stacking and running gels contained
no SDS. The gels were run at 20 mA in a temperaturecontrolled room at 4³C to prevent denaturation of various
folded forms of the PorA protein during electrophoresis.
Prior to electrophoresis, samples with folded or denatured
PorA were incubated for 10 min at room temperature or
100³C, respectively, in sample bu¡er [23] containing 0.05%
or 2% SDS, respectively.
Western blotting was performed as described [24]. The
mice sera were diluted 1:100.
3. Results
3.1. Immunogenicity of in vitro folded PorA P1.6
Groups of ¢ve mice each were immunized with in vitro
folded PorA P1.6 and, as a control, with the denatured
protein, both without any adjuvant. The sera of each
group were pooled and analyzed for the antibody response
in a whole cell ELISA using the homologous strain
M990 as the coating antigen. The immune response was
moderate with the mean titers of 3409 and 3859 for denatured and folded PorA, respectively. The antibodies elicited with the denatured PorA were mainly of isotype
IgG1, while the antibodies elicited with folded PorA
showed a more equal distribution of isotypes IgG1,
IgG2a and IgG2b (results not shown), which initially
seemed promising, since it has been demonstrated that
bactericidal antisera contain signi¢cant titers of the complement-binding IgG2a and IgG2b isotypes [21,25]. How-
Fig. 1. Total IgG responses measured in whole cell ELISAs with cells
of strain M990 as immobilized antigen. Mice were immunized with either denatured (d) or folded (f) PorA P1.6 together with QuilA or LOS
as indicated. The titers of the individual mice (1^5) can be discriminated
in the bars.
ever, no bactericidal antibodies were detected in either of
the antisera.
3.2. Immunogenicity of in vitro folded PorA P1.6 with
adjuvants
The poor immunogenicity of the puri¢ed, in vitro folded
PorA, as compared with PorA in OMVs, may be explained by the absence of LOS in these preparations,
which has been reported to have immune response stimulating properties [26]. Consistently, it has previously been
demonstrated that the immune response elicited by outer
membrane complexes (OMC) of an LOS-de¢cient meningococcal strain was poor, but could be restored by the
addition of meningococcal LOS [25]. Moreover, also less
toxic compounds, such as the adjuvant QuilA, turned out
to restore the immune response [25]. Hence, to improve
the immune response, we combined the PorA immunogen
with either meningococcal galE LOS or with QuilA as an
adjuvant. Groups of ¢ve mice each were immunized and
sera of individual mice were tested in whole cell ELISAs
with strain M990 as antigen (Fig. 1). The meningococcal
galE LOS did not drastically enhance the immunogenicity
of the PorA protein. In contrast, PorA supplemented with
QuilA as an adjuvant resulted in four to seven times higher antibody titers as compared with the results of immunization experiments with LOS as an adjuvant (Fig. 1).
Furthermore, a clear switch towards the IgG2a subclass
of antibodies was observed when folded PorA plus QuilA
(group c) was compared to denatured PorA plus QuilA
(group a) (Fig. 2C and A, respectively).
Sera elicited with folded PorA recognized the corresponding native as well as denatured PorA proteins in
Western blots (results not shown), demonstrating that at
least some of the antibodies raised recognized linear epitopes. Reversibly, sera elicited with denatured PorA recognized, besides the corresponding denatured protein, also
the native protein in Western blots (results not shown),
demonstrating that similar linear epitopes were accessible
in the denatured as well as in the native porin.
FEMSIM 1169 9-2-00
230
C. Jansen et al. / FEMS Immunology and Medical Microbiology 27 (2000) 227^233
Fig. 2. IgG isotype responses. The panels A^D correspond to the groups of mice a^d as indicated in Fig. 1. The titers of the individual mice (1^5) can
be discriminated in the bars. The coated antigens were whole cells of the homologous strain M990.
3.3. Bactericidal assays
Since bactericidal antibodies are believed to be important for protective immunity, we tested the sera of the
individual mice for their bactericidal activity. With one
exception, the sera of all mice immunized with folded
PorA P1.6 and QuilA contained bactericidal antibodies
(Table 1), in correspondence with the high titers of the
complement-binding IgG2a and 2b isotypes in these sera
(Fig. 2). Furthermore, bactericidal antibodies were detected in the sera of some mice immunized with denatured
PorA and QuilA or with folded PorA and LOS, but there
was a great variability among the individual mice within
these groups, with the majority of the sera being inactive
(Table 1). No bactericidal antibodies were detected in the
sera of mice immunized with denatured PorA and LOS.
The heterogeneity in antibody response among the members within a single group can not readily be explained.
Table 1
Bactericidal antibody titersa
Antigen
Denatured PorA P1.6
Folded PorA P1.6
Adjuvant
+QuilA
+LOS
+QuilA
+LOS
Mice group
group a
group b
group c
group d
Mouse
Mouse
Mouse
Mouse
Mouse
64
2048
128
64
64
64
64
64
64
64
64
128
512
1024
32
64
64
64
256
64
a
1
2
3
4
5
The bactericidal antibody titers were de¢ned as described in Section 2.
As a positive control for the assay, the bactericidal mAb MN19D6.13
was used, which resulted in a titer of 1600.
Since no complement-mediated killing was observed for
mouse 1 of group c, immunized with folded PorA and
QuilA (Table 1), we considered the possibility that the
serum contained blocking antibodies. To test this possibility, the serum of mouse c.1 was mixed with the bactericidal mAb MN19D6.13, directed against PorA P1.6, and
bactericidal assays were performed with strain M990 (results not shown). However, no blocking of the activity of
the bactericidal mAb was observed.
3.4. Peptide ELISA
To determine to which part of the protein the antibodies
in the sera with the highest antibody titers, i.e. those of
groups a and c, are mainly directed, peptide ELISAs were
performed. Overlapping 15-mer peptides, corresponding to
the variable loops 1, 4 and 5, were used. The antibodies of
the individual mice were mainly directed towards the predicted apex of loop 4, i.e. amino acid residues T172^V181
(Table 2). Subsequently, we performed peptide inhibition
bactericidal assays to determine against which loop the
bactericidal antibodies of the sera of group c mice were
directed. However, peptide mixes corresponding to either
loops 1, 4 or 5 could not inhibit the bactericidal activity in
the sera of the individual mice c.2 and c.4 completely
(Table 3). Hence, the bactericidal antibodies in these sera
appeared to be directed against linear epitopes in several
loops or against one or several conformational epitopes,
rather than against a linear epitope in one speci¢c loop. In
the case of the antiserum of mouse c.5, the bactericidal
activity was lost by incubation with the loop 5 peptide mix
(Table 3), suggesting that the bactericidal activity is directed against loop 5. However, considering the low bac-
FEMSIM 1169 9-2-00
C. Jansen et al. / FEMS Immunology and Medical Microbiology 27 (2000) 227^233
231
Table 2
Peptide ELISA titersa
a
The titers were de¢ned as described in Section 2. The mAb MN37C7.24, directed against loop 5 of strain M990, was used as a control. Titers of
16 000 and higher are shaded in light gray and gray, respectively.
b
The sera of the individual mice immunized with denatured PorA and QuilA (group a) and folded PorA and QuilA (group c) were tested.
tericidal titer of this antiserum, this result has to be considered preliminary.
4. Discussion
The immunogenicity of in vitro folded and puri¢ed
PorA P1.6 without adjuvant was moderate. Strikingly,
the galE LOS did not enhance the immunogenicity. A
possible explanation is that a direct physical interaction
of LOS with the immunogen is required to establish its
function as an adjuvant. Such an interaction might be
disturbed by the presence of the detergent SB12 in the
micelles that were used for immunization. In this respect,
it should be noted that LOS was able to enhance the
immune response only to OMCs derived from an LOSde¢cient meningococcal mutant but not to whole cells of
these bacteria [25]. In that case, it was argued that LOS is
able to interact with OMCs but probably not with the
whole cells. However, importantly, the non-toxic adjuvant
QuilA could enhance the immunogenicity of the in vitro
folded PorA P1.6. Moreover, the QuilA adjuvant induced
Table 3
Peptide inhibition bactericidal assaya
Serum
c.2
c.4
c.5
Peptide mix
^
L1
L4
L5
^
L1
L4
L5
^
L1
L4
L5
Titer
128
64
32
32
1024
1024
512
512
32
32
64
6 32
a
The peptide inhibition bactericidal assay was performed as described in Section 2. The serum of mouse c.1 did not contain bactericidal activity (Table
1) and was therefore excluded from this experiment. No serum was left from mouse c.3 to perform this assay. Mixes of the four peptides corresponding
to loop 1 (L1), loop 4 (L4) and loop 5 (L5) (see also Table 2) or bu¡er (^) was added to the sera of the mice to inhibit bactericidal activity, and the remaining bactericidal activity was measured.
FEMSIM 1169 9-2-00
232
C. Jansen et al. / FEMS Immunology and Medical Microbiology 27 (2000) 227^233
a switch towards the IgG2a subclass of antibodies and,
concomitantly, bactericidal activity was detected. Bactericidal antibodies were also observed in some of the sera
elicited with denatured PorA P1.6 and QuilA. It should
be noted that the denatured antigen was obtained by heating the folded protein for 30 min at 100³C. In the absence
of any denaturant, this appeared to result in denaturation
of v95%, but still, some folded protein remained detectable (results not shown). Furthermore, the combination of
3 mM SB12 and 40 Wg ml31 QuilA appeared to induce
folding to some extent (unpublished observation). Therefore, we can not exclude the possibility that some refolding
occurred after denaturation of the antigen. The small
amount of folded PorA possibly present in the preparation
may have elicited the bactericidal antibodies present in
these sera.
In conclusion, the PorA, devoid of LPS due to its isolation from cytoplasmic inclusion bodies, and folded and
puri¢ed as described [13] is able to elicit bactericidal antibodies, provided that QuilA is added as an adjuvant.
Although the majority of antibodies recognizing linear
epitopes were directed against loop 4, the bactericidal antibodies were not (only) directed against linear epitopes in
loop 4. Since high amounts of pure and native-like PorA
can easily be obtained by the procedures employed, a new
vaccine against serogroup B meningococci that is free of
endotoxin can be developed. Such a vaccine should preferentially be based on several subtypes of puri¢ed PorA.
The same principles can probably be applied to other outer membrane protein antigens.
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
References
[17]
[1] Peltola, H. (1984) Meningococcal disease: still with us. Rev. Infect.
Dis. 5, 71^91.
[2] Finne, J., Leinonen, M. and Ma«kela«, P.H. (1983) Antigenic similarities between brain components and bacteria causing meningitidis.
Implications for vaccine development and pathogenesis. Lancet ii,
355^357.
[3] van der Ley, P., Heckels, J.E., Virji, M., Hoogerhout, P. and Poolman, J.T. (1991) Topology of outer membrane porins in pathogenic
Neisseria spp. Infect. Immun. 59, 2963^2971.
[4] Frasch, C.E., Zollinger, W.D. and Poolman, J.T. (1985) Serotype
antigens of Neisseria meningitidis and a proposed scheme for designation of serotypes. Rev. Infect. Dis. 7, 504^510.
[5] Saukkonen, K., Leinonen, M., Abdillahi, H. and Poolman, J.T.
(1989) Comparative evaluation of potential components for group
B meningococcal vaccine by passive protection in the infant rat and
in vitro bactericidal assay. Vaccine 7, 325^328.
[6] Bjune, G., HÖiby, E.A., GrÖnnesby, J.K., Arnesen, Ò., Fredriksen,
J.H., Halstensen, H., Holten, E., Lindbak, A., NÖkleby, H., Rosenqvist, E., Solberg, L.K., Closs, O., Eng, J., FrÖholm, L.O., Lystad,
A., Bakketeig, L.S. and Hareide, B. (1991) E¡ect of outer membrane
vesicle vaccine against serogroup B meningococcal disease in Norway. Lancet 338, 1093^1096.
[7] Rosenqvist, E., HÖiby, E.A., Wedege, E., Bryn, K., Kolberg, J.,
Klem, A., RÖnnild, E., Bjune, G. and NÖkleby, H. (1995) Human
antibody responses to meningococcal outer membrane antigens after
[18]
[19]
[20]
[21]
[22]
[23]
three doses of the Norwegian group B meningococcal vaccine. Infect.
Immun. 63, 4642^4652.
Claassen, I., Meylis, J., van der Ley, P., Peeters, C., Brons, H.,
Robert, J., Borsboom, D., van der Ark, A., van Straaten, I., Roholl,
P., Kuipers, B. and Poolman, J.T. (1996) Production, characterization and control of a Neisseria meningitidis hexavalent class 1 outer
membrane protein containing vesicle vaccine. Vaccine 14, 1001^1008.
Peeters, C.C.A.M., Ru«mke, H.C., Sundermann, L.C., Rouppe van
der Voort, E.M., Meulenbelt, J., Schuller, M., Kuipers, A.J., van
der Ley, P. and Poolman, J.T. (1996) Phase I clinical trial with a
hexavalent PorA containing meningococcal outer membrane vesicle
vaccine. Vaccine 14, 1009^1015.
Rouppe van der Voort, E., van der Ley, P., van der Biezen, J.,
George, S., Tunnela, O., van Dijken, H., Kuipers, B. and Poolman,
J. (1996) Speci¢city of human bactericidal antibodies against PorA
P1.7.16 induced with a hexavalent meningococcal outer membrane
vesicle vaccine. Infect. Immun. 64, 2745^2751.
Tsai, C.M., Frasch, C.E. and Mocca, L.F. (1981) Five structural
classes of major outer-membrane proteins in Neisseria meningitidis.
J. Bacteriol. 146, 69^78.
Abdillahi, H. and Poolman, J.T. (1988) Neisseria meningitidis group
B serosubtyping using monoclonal antibodies in whole cell ELISA.
Microb. Pathog. 4, 27^32.
Jansen, C., Wiese, A., Reubsaet, L., Dekker, N., de Cock, H., Seydel,
U. and Tommassen, J. Biochemical and biophysical characterization
of in vitro folded outer membrane porin PorA of Neisseria meningitidis. Submitted for publication.
Nurminen, M., Butcher, S., Ida«npa«a«n-Heikkila«, I., Wahlstro«m, E.,
Muttilainen, S., Runeberg-Nyman, K., Sarvas, M. and Ma«kela«,
P.H. (1992) The class I outer membrane protein of Neisseria meningitidis produced in Bacillus subtilis can give rise to protective immunity. Mol. Microbiol. 6, 2499^2506.
Muttilainen, S., Ida«npa«a«n-Heikkila«, I., Wahlstro«m, E., Nurminen,
M., Ma«kela«, P.H. and Sarvas, M. (1994) The Neisseria meningitidis
outer membrane protein P1 produced in Bacillus subtilis and reconstituted into phospholipid vesicles elicits antibodies to native P1 epitopes. Microb. Pathogen. 18, 423^436.
Maiden, M.C.J., Suker, J., McKenna, A.J., Bygraves, J.A. and Feavers, I.M. (1991) Comparison of the class I outer membrane proteins
of eight serological reference strains of Neisseria meningitidis. Mol.
Microbiol. 5, 727^736.
Holten, E. (1979) Serotypes of Neisseria meningitidis isolated from
patients in Norway during the ¢rst six months of 1978. J. Clin.
Microbiol. 9, 186^188.
Dekker, N., Merck, K., Tommassen, J. and Verheij, H.M. (1995) In
vitro folding of Escherichia coli outer-membrane phospholipase A.
Eur. J. Biochem. 232, 214^219.
Westphal, O. and Jann, J.K. (1965) Bacterial lipopolysaccharide extraction with phenol-water and further application of the procedure.
Methods Carbohydr. Chem. 5, 83^91.
Abdillahi, H. and Poolman, J.T. (1987) Whole cell ELISA for typing
Neisseria meningitidis with monoclonal antibodies. FEMS Microbiol.
Lett. 48, 367^371.
Hoogerhout, P., Donders, E.M.L.M., van Gaans-van den Brink,
J.A.M., Kuipers, B., Brugghe, H.F., van Unen, L.M.A., Timmermans, H.A.M., ten Hove, G.J., de Jong, A.P.J.M., Peeters,
C.C.A.M., Wiertz, E.J.H.J. and Poolman, J.T. (1995) Conjugates of
synthetic cyclic peptides elicit bactericidal antibodies against a conformational epitope on a class 1 outer membrane protein of Neisseria
meningitidis. Infect. Immun. 63, 3473^3478.
Brugghe, H.F., Timmermans, J.A.M., van Unen, L.M.A., ten Hove,
G.J., van de Werken, G., Poolman, J.T. and Hoogerhout, P. (1994)
Simultaneous multiple synthesis and selective conjugation of cyclized
peptides derived from a surface-loop of a meningococcal class 1 outer
membrane protein. Int. J. Pept. Protein Res. 43, 166^172.
Lugtenberg, B., Meijers, J., Peters, R., van der Hoek, P. and van
Alphen, L. (1975) Electrophoretic resolution of the `major outer
FEMSIM 1169 9-2-00
C. Jansen et al. / FEMS Immunology and Medical Microbiology 27 (2000) 227^233
membrane protein' of Escherichia coli K-12 into four bands. FEBS
Lett. 58, 254^258.
[24] de Cock, H., Scha«fer, U., Potgeter, M., Demel, R., Mu«ller, M. and
Tommassen, J. (1999) A¤nity of the periplasmic chaperone Skp of
Escherichia coli for phospholipids, lipopolysaccharides and non-native outer membrane proteins. Role of Skp in the biogenesis of outer
membrane protein. Eur. J. Biochem. 259, 96^103.
233
[25] Steeghs, L., Kuipers, B., Hamstra, H.J., Kersten, G., van Alphen, L.
and van der Ley, P. (1999) Immunogenicity of outer membrane proteins in a lipopolysaccharide-de¢cient mutant of Neisseria meningitidis : In£uence of adjuvants on the immune response. Infect. Immun.
67, 4988^4993.
[26] Johson, A.G. (1994) Molecular adjuvants and immunomodulators:
new approaches to immunization. Clin. Microbiol. Rev. 7, 277^289.
FEMSIM 1169 9-2-00