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The Journal of Immunology, 2001, 167: 6487-6496.
Copyright © 2001 by The American Association of Immunologists

A Novel Mimetic Antigen Eliciting Protective Antibody to Neisseria meningitidis1

Dan M. Granoff2,*, Gregory R. Moe*, Marzia M. Giuliani{dagger}, Jeannette Adu-Bobie{dagger}, Laura Santini{dagger}, Brunella Brunelli{dagger}, Francesca Piccinetti{dagger}, Patricia Zuno-Mitchell*, Sharon S. Lee*, Paolo Neri{ddagger}, Luisa Bracci{ddagger}, Luisa Lozzi{ddagger} and Rino Rappuoli{dagger}

* Children’s Hospital Oakland Research Institute, Oakland, CA 94609; {dagger} Instituto Ricerche Immunobiologiche, Chiron, Siena, Italy; and {ddagger} Department of Molecular Biology, University of Siena, Siena, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Molecular mimetic Ags are of considerable interest as vaccine candidates. Yet there are few examples of mimetic Ags that elicit protective Ab against a pathogen, and the functional activity of anti-mimetic Abs has not been studied in detail. As part of the Neisseria meningitidis serogroup B genome sequencing project, a large number of novel proteins were identified. Herein, we provide evidence that genome-derived Ag 33 (GNA33), a lipoprotein with homology to Escherichia coli murein transglycosylase, elicits protective Ab to meningococci as a result of mimicking an epitope on loop 4 of porin A (PorA) in strains with serosubtype P1.2. Epitope mapping of a bactericidal anti-GNA33 mAb using overlapping peptides shows that the mAb recognizes peptides from GNA33 and PorA that share a QTP sequence that is necessary but not sufficient for binding. By flow cytometry, mouse antisera prepared against rGNA33 and the anti-GNA33 mAb bind as well as an anti-PorA P1.2 mAb to the surface of eight of nine N. meningitidis serogroup B strains tested with the P1.2 serosubtype. Anti-GNA33 Abs also are bactericidal for most P1.2 strains and, for susceptible strains, the activity of an anti-GNA33 mAb is similar to that of an anticapsular mAb but less active than an anti-P1.2 mAb. Anti-GNA Abs also confer passive protection against bacteremia in infant rats challenged with P1.2 strains. Thus, GNA33 represents one of the most effective immunogenic mimetics yet described. These results demonstrate that molecular mimetics have potential as meningococcal vaccine candidates.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
There is considerable interest in using molecular mimetic Ags to elicit protective immune responses to different pathogens (1, 2, 3, 4, 5, 6, 7, 8, 9). Mimetics also are being investigated for the treatment of cancer (10) and autoimmune diseases (11). For vaccines for prevention of infectious diseases, the mimetic approach has the greatest utility when the nominal Ag is toxic or difficult to purify, or when it is desirable to direct the immune response to a limited number of epitopes. Examples of experimental mimetic Ags include the lipooligosaccharide of Neisseria gonorrhoeae (3), the fusion protein of respiratory syncytial virus (1), and conserved epitopes on different strains of HIV-1 (7) or hepatitis C virus (5).

A large number of studies have investigated the vaccine potential of mimetic Ags. For example, anti-idiotype vaccines are being tested in humans for the treatment of melanoma and colon cancer (reviewed in Ref. 10). In contrast, the experimental data, to date, on the use of mimetic vaccines for the prevention of infectious diseases have not been sufficiently compelling to justify testing in humans.

As part of the recently completed meningococcal serogroup B genome sequencing project (12), a large number of novel proteins were discovered that were conserved in sequence, appeared to be surface-exposed on encapsulated Neisseria meningitidis serogroup B (NmB)3 strains, and elicited serum bactericidal Ab responses (13). Among these was a lipoprotein, designated genome-derived Ag 33 (GNA33). The predicted amino acid sequence of GNA33 is homologous to that of membrane-bound lytic murein transglycosylase from Escherichia coli and Synechocystis sp. (14), and is highly conserved across N. meningitidis (13). Mice immunized with rGNA33 developed high-serum bactericidal Ab titers. The magnitude of the Ab response was similar to that of control animals immunized with an outer membrane vesicle (OMV) vaccine prepared from strain 2996. Because OMV vaccines are known to elicit protective serum Ab responses in humans (15, 16, 17), GNA33 was considered a promising candidate for development of a meningococcal vaccine capable of eliciting broad-based protection.

In this report, we show that GNA33 elicits protective Ab to NmB by mimicking a surface-exposed epitope on loop 4 of porin A (PorA) of strains with the P1.2 serosubtype. This unexpected identification of an unique mimetic Ag of PorA allowed us to compare the functional activity of Ab raised to a mimetic Ag to that raised by PorA, using in vitro and in vivo functional assays that historically have been relevant for predicting protection of meningococcal disease in humans (18).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains

The 22 N. meningitidis strains (21 in serogroup B and one in serogroup C) included in this study are summarized in Table IGo. The strains were isolated from patients with meningococcal disease residing in different countries and were collected over a period of 36 years.


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  		Table I. Binding of anti-GNA33 Abs to the surface of live, encapsulated N. meningitidis strains as measured by flow cytometry in relation to serological classification and PorA VR designation

 
Mutants of strains MC58, BZ232, and NMB (MC58{Delta}GNA33, BZ232{Delta}GNA33, and NMB{Delta}GNA33, respectively) in which the gna33 gene was deleted and replaced by allelic exchange with an antibiotic cassette were prepared by transforming the parent strain with the plasmid pBSUD33ERM. This plasmid contains the upstream and downstream flanking gene regions for allelic exchange and the ermC gene (erythromycin resistance). Briefly, the upstream flanking region (including the start codon) from -867 to +75 and the downstream flanking region (including the stop codon) from +1268 to +1744 were amplified from MC58 using the following primers: U33 forward, 5'-GCTCTAGAGATGAGTCGAACACAATGAACAATGTCCTGA; U33 reverse, 5'-TCCCCCGGGCTCTTGCTTTGGCAGGCGGCGA-3'; D33 forward, 5'-TCCCCCGGGCACGGGATATGTGTGGC-3'; D33 reverse, 5'-CCCGCTCGAGAGTAGGGACAACCGG-3'.

The fragments were cloned into pBluescript (Stratagene, Milan, Italy) and transformed into E. coli DH5{alpha} using standard techniques (19). Once all subcloning was complete, naturally competent NmB strains MC58, BZ232, and NMB were transformed by selecting a few colonies grown overnight on chocolate agar plates and mixing them with 20 µl of 10 mM Tris·HCl (pH 6.5) containing 1 µg of plasmid DNA. The mixture was spotted onto a chocolate agar plate, incubated for 6 h at 37°C, 5% CO2, then diluted in PBS and spread on chocolate agar plates containing 7 µg/ml erythromycin. The absence of the gna33 gene in the genome of erythromycin-resistant colonies for each of the three strains was confirmed by PCR using the primers (F33, GCTCTAGAGGGCGACGACAGGCGG-3'; and R33, 5'-CCCGCTCGAGTTACGGGCGGTATTCGG-3') that correspond to the 5'-sense and 3'-antisense strands, respectively, of the gna33 gene. Lack of GNA33 expression in the three strain isolates was confirmed by Western blot analysis, performed as described below.

mAb reagents

Abs used for flow cytometry, bactericidal, and in vivo protection experiments included the following: an anti-meningococcal PorA P1.2-specific subtyping mAb (MN16C13F4, subclass IgG2a) obtained from Rijksinstituut Voor Volksgezondheid en Mileu (Bilthoven, The Netherlands) or from W. Zollinger (Walter Reed Army Institute of Research, Washington, DC); and anti-polysaccharide mAbs specific for encapsulated serogroup B (20) (SEAM 12 (subclass IgG2a) and SEAM 3 (subclass 2b)) and serogroup C (mAb 181.1 (21), subclass IgG3). mAb 181.1 was provided by K. Stein (U.S. Food and Drug Administration, Bethesda, MD). The negative control consisted of a mouse IgG mAb (VIG10) of irrelevant specificity.

Expression and purification of GNA33

The gna33 open reading frame was amplified by PCR on chromosomal DNA from strain 2996 (22) with synthetic oligonucleotides used as primers. The amplified DNA fragment was cloned into pET-21b+ vector (Novagen, Madison, Wisconsin) to express the protein as His-tagged GNA33 or as a soluble protein without the signal and lipid modification sequences (rGNA33). The expression of recombinant protein was evaluated by SDS-PAGE, performed as described below. The His-tagged fusion protein was purified by affinity chromatography on Ni2+-conjugated chelating fast-flow Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden) and the untagged form was purified by FPLC using a mono S ion-exchange resin (Amersham Pharmacia Biotech).

Preparation of polyclonal anti-GNA33 antisera

To prepare antisera against GNA33, 20 µg of purified His-tagged GNA33 or untagged rGNA33 was used to immunize 6-wk-old CD1 female mice (4–10 mice per group). The mice were obtained from Charles River Breeding Laboratories (Italia, Calco, Italy, or Hollister, CA). The recombinant protein was given i.p., together with CFA for the first dose and IFA for the second (day 21) and third (day 35) booster doses. Blood samples were taken on days 34 and 49. As a negative control, mouse polyclonal antiserum was prepared against E. coli proteins from the strain used to express rGNA33.

Preparation of mAbs

Four- to six-week-old female CD1 mice were immunized as described above, except that the third dose was given without adjuvant. Three days later, mice were sacrificed and their spleen cells were fused with myeloma cells P3 x 63-Ag8.653 at a ratio of five spleen cells to one myeloma cell. After a 2-wk incubation in hypoxanthine/aminopterin/thymidine selective medium, hybridoma supernatants were screened for Ab binding activity by ELISA performed on microtiter plates coated with the noncapsulated N. meningitidis strain, M7 (23), which had been inactivated by treatment with 0.025% paraformaldehyde. Hybridomas secreting GNA33-specific Ab were cloned twice by limiting dilution and then expanded and frozen for subsequent use in tissue culture, or for ascites production in BALB/c mice.

The subclasses of the mAbs were determined using a mouse mAb isotyping kit (Amersham Pharmacia Biotech). Among the selected mAbs, one IgG2a anti-GNA33 mAb, designated mAb 25, was used in all of the binding and functional studies described below. This mAb was purified from mouse ascites by Hi-Trap affinity columns (Amersham Pharmacia Biotech) and, after exhaustive dialysis in PBS buffer, the concentration of the purified mAb was determined using a modified Lowry method with BSA as a standard (Bio-Rad DC Protein Assay; Bio-Rad, München, Germany). Specificity of mAb 25 binding was determined by Western blot using membrane proteins prepared from NmB strains MC58, BZ232, and NMB, and their respective GNA33 knockouts (MC58{Delta}GNA33, BZ232{Delta}GNA33, and NMB{Delta}GNA33; see Results).

Binding of antisera to the surface of live encapsulated meningococci

The ability of monoclonal or polyclonal antisera elicited by the recombinant gna33 protein to bind to the surface of live pathogenic strains of NmB was determined using a flow cytometric detection of indirect fluorescence assay, performed as described previously (24). In brief, bacterial cells were grown to mid-log phase in Mueller-Hinton broth, harvested by centrifugation, and resuspended in blocking buffer (PBS containing 1% (w/v) BSA and 0.2% (w/v) sodium azide) at a density of ~108 cells per milliliter. Dilutions of test or control antisera or mAbs were then added and allowed to bind to the cells, which were maintained on ice for 2 h. Following two washes with blocking buffer, the cells were incubated with FITC-conjugated F(ab')2 goat anti-mouse IgG (H and L chains) (Jackson ImmunoResearch Laboratories, West Grove, PA) and fixed with 0.25% formaldehyde in PBS buffer, and the bacterial cells were analyzed by flow cytometry.

Positive control Abs included meningococcal-specific serotyping or subtyping mAbs (MN2C3B, MN16C13F4; Rijksinstituut Voor Volksgezondheid en Mileu) and SEAM 12, an anti-polysaccharide mAb that is specific for encapsulated group B strains. The negative control consisted of a mouse IgG mAb (VIG10) of irrelevant specificity, or mouse polyclonal antiserum prepared against E. coli proteins from the strain used to express rGNA33.

Complement-dependent bactericidal Ab activity

Bactericidal activity was measured as previously described (24). Except where noted, the complement source was human serum from a healthy adult (25) with no detectable anticapsular Ab to serogroup B or C polysaccharide as tested by ELISA, and no detectable intrinsic bactericidal activity against the target strains when tested at a final serum concentration of 20% or 40%. In the present studies, this human complement source together with different test sera gave similar bactericidal titers to those measured when human serum from a patient with agammaglobulinemia was used as the complement source (our unpublished data). In a few experiments described in Results, bactericidal activity also was measured using serum from a patient with untreated agammaglobulinemia (26), infant rabbit serum, or adult rat serum as complement sources.

Animal protection

The ability of anti-GNA33 Abs to confer passive protection against NmB bacteremia was tested in infant rats challenged i.p. The assay was performed as previously described (27). In each experiment, 5- to 8-day-old pups from litters of outbred Wistar rats (Charles River Breeding Laboratories, Raleigh, NC) were randomly redistributed to the nursing mothers. A total of four experiments were performed using three different NmB strains (M986, BZ232, and 8047). Each strain had been serially passaged three times in infant rats and stored frozen at -80°C in skim milk. On the day before challenge, freshly thawed bacteria were inoculated onto chocolate agar and grown overnight at 37°C in 5% CO2. On the morning of the challenge, colonies were picked, inoculated into a broth culture, and grown and prepared as described above for the bactericidal assay. In experiment one, to enhance sensitivity of the assay, the bacteria from strain 8047 or M986 were suspended in different concentrations of anti-GNA33 or control antisera immediately before the challenge. A total of 100 µl of the suspensions was then administered i.p. Heparinized blood specimens were obtained by cardiac puncture 18 h after the bacterial challenge. Aliquots of 1, 10, and 100 µl of blood were plated onto chocolate agar. The CFU per milliliter of blood were determined after overnight incubation of the plates at 37°C in 5% CO2. In experiments two, three, and four, animals were treated i.p. with anti-GNA33 mAb or control mAbs at time 0. Two hours later the animals were challenged i.p. with bacteria from strain M986 (experiment two) or BZ232 (experiments three and four). Blood samples were obtained 18 h later and quantitation of bacteremia was performed as described for experiment one.

SDS-PAGE and Western blots

Total cell extracts of meningococcal bacteria were prepared as follows. Single colonies were grown in 7 ml of Mueller-Hinton broth (Difco, Detroit, MI) supplemented with 0.25% glucose to an A620 nm of 0.5–0.7. The bacteria were collected by centrifugation at 5000 x g for 15 min and resuspended in PBS. After freeze-thawing, the bacterial suspension was mixed with sample buffer (0.06 M Tris-HCl (pH 6.8), 10% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) 2-ME) and boiled for 10 min. Purified proteins (0.5 µg/lane) or total cell extracts (25 µg) derived from meningococcal strains were loaded onto 12.5% SDS-polyacrylamide gels (28) and transferred to a nitrocellulose membrane (29). The bound proteins were detected with anti-GNA33 mAb 25 at a final concentration of 6 µg/ml, or a 1/100 dilution of an anti-PorA P1.2 mAb followed by a 1/2000 dilution of HRP-labeled anti-mouse Ig (DAKO, Glostrup, Denmark).

Peptide spot synthesis

Peptide spot synthesis was performed on an amino-polyethylene glycol-cellulose membranes (Abimed Analyes-Technik, Langenfeld, Germany) using a model ASP 222 automated spot synthesizer (Abimed Analyes-Technik) and diisopropylcarbodiimide/N-hydroxybenzotriazole activation (30). After the final cycle, all the peptides were N-terminally acetylated with 2% acetic anhydride. At the end of the synthesis the side chain protecting groups were removed using a mixture of trifluoroacetic acid/triisobutylsilane/water/dichloromethane (50/3/2/45).

Peptide binding assay

Cellulose-bound peptides were soaked in ethanol to prevent hydrophobic interactions between the peptides. Nonspecific binding was blocked by incubating cellulose sheets overnight at 4°C with 10 ml of 2% casein in TBS (50 mM Tris-HCl, 137 mM NaCl, and 27 mM KCl (pH 7.0)), containing 0.05% Tween 20. The sheets were incubated for 2 h at 37°C with the anti-GNA33 mAb25 (6 µg/ml) or an anti-PorA 1.2 mAb, followed by a 1/3000 dilution of alkaline phosphatase-conjugated goat anti-mouse IgG (Bio-Rad). Sheets were developed with bromo-4-chloro-3-indolyl-phosphate (Sigma-Aldrich, Steinheim, Germany) and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl-tetrazolium bromide (Sigma-Aldrich) substrates in buffer (100 mM Tris (pH 8.9), 100 mM NaCl, and 2 mM MgCl2). Quantitative evaluation of the signal was obtained using a Umax Speedy II 2200 optical scanner (Umax Technologies, Fremont, CA).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Binding of anti-GNA33 Abs to the bacterial cell surface as determined by indirect fluorescence flow cytometry

Polyclonal Abs obtained from CD1 mice immunized with rGNA33 (encoded by the gene from strain 2996) were tested for their ability to bind to live bacterial cells of various NmB strains by a flow cytometry binding assay. Fig. 1GoA shows binding of polyclonal anti-rGNA33 antisera to four representative NmB strains, the parent strain, 2996 (P1.5,2), and three other strains, M3735 (P1.5,2), M4207 (P1.5), and MC58 (P1.7,16). The anti-GNA33 polyclonal antiserum reacted only with strains 2996 and M3735. The anticapsular positive control mAb bound to all four strains, whereas the negative control antiserum prepared from animals immunized with E. coli proteins showed only background binding. Fig. 1GoB shows the results of similar experiments measuring binding of the anti-GNA33 mAb 25 to the bacterial cell surface of three strains (M3735 (P1.5,2), M4207 (P1.5), and MC58 (P1.7,16)). The mAb bound only to strain M3735 (P1.5,2).



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  		FIGURE 1. A, Binding of polyclonal anti-GNA33 antisera and control mAbs to live encapsulated NmB strains 2996, M3735, M4207, and MC58 as determined by indirect fluorescence flow cytometry. The control mAbs and antisera include an anti-serogroup B capsular-specific murine mAb (SEAM 12), an N. meningitidis serosubtype mAb anti-PorA P1.2, and polyclonal antisera from mice immunized with E. coli OMVs. B, Binding of anti-GNA33 mAb 25 and control mAbs to NmB strains M3735, M4207, and MC58. The murine control mAbs include a mAb having an irrelevant specificity (VIG10), and the same anticapsular and anti-PorA P1.2 mAbs described in A.

 
Table IGo summarizes the results of flow cytometry experiments measuring the ability of anti-GNA33 antisera or mAb 25 to bind to the surface of live bacteria from 22 genetically diverse encapsulated meningococcal strains (21 in serogroup B and one in serogroup C). The anti-GNA33 Ab bound only to strains with the P1.5,2 or P1.2 serosubtypes (nine of nine vs zero of 13 strains with other PorA serosubtypes; p < 0.001). One of the nine positive strains, M986, showed lower binding than the other eight strains (vide infra). There was no binding to three strains (M4207, 1000, and BZ83) that express the P1.5 epitope (present on loop 1 of PorA; see Ref. 31) but not the P1.2 epitope (loop 4). Also, there was no binding to strain M136, which does not express PorA (i.e., P1-). These data indicate that binding of anti-GNA33 Ab to the bacterial surface correlates with expression of the PorA serosubtype P1.2.

Western blot of total membrane fractions prepared from different N. meningitidis group B strains

The apparent association between binding of anti-GNA33 Ab to the bacterial surface and expression of the P1.2 serosubtype was investigated further by SDS-PAGE Western blot of total membranes prepared from representative strains. Results from four serogroup B strains, two that were negative for anti-GNA33 surface binding by flow cytometry (NG3/88 (P1.7,1) and MC58 (P1.17,16)) and two that were positive (BZ232 and NMB (both P1.5,2)), are shown in Fig. 2Go. Data also are shown for total membrane preparations from three strains (MC58, BZ232, and NMB) in which the genes encoding GNA33 have been inactivated. In Fig. 2GoA, a single band with an apparent mass of ~48 kDa was detected by the anti-GNA33 mAb 25 in membrane preparations from the two non-P1.2 strains, NG3/88 (Fig. 2GoA, lane 3) and MC58 (Fig. 2GoA, lane 4). The band has an apparent molecular mass expected for rGNA33 (Fig. 2GoA, lane 1) and was absent in total protein prepared from the control E. coli strain (Fig. 2GoA, lane 2) or from the GNA33 knockout in strain MC58 (Fig. 2GoA, lane 5). In Fig. 2GoA, lanes 6 and 8 contain total membrane proteins prepared from strains BZ232 and NMB, respectively. Both of these strains have the PorA serosubtype P1.5,2. In each of the lanes there are two anti-GNA33-reactive bands. The higher 48-kDa band is absent from the GNA33 knockouts derived from BZ232 and NMB (Fig. 2GoA, lanes 7 and 9, respectively), a result confirming that this protein is GNA33.



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  		FIGURE 2. Western blots of rGNA33 and total proteins prepared from E. coli or N. meningitidis strains using anti-GNA33 mAb25 (A) or anti-PorA P1.2 (B) as the primary Ab. Lane 1, Recombinant GNA33; lane 2, total proteins prepared from E. coli strain used to express rGNA33; lane 3, NmB strains NG3/88; lane 4, MC58; lane 5, MC58{Delta}GNA33; lane 6, BZ232; lane 7, BZ232{Delta}GNA33; lane 8, NMB; and lane 9, NMB{Delta}GNA33.

 
Fig. 2GoB shows a Western blot of the same protein samples as described for Fig. 2GoA, but using the anti-PorA P1.2 mAb as the primary detecting Ab. As expected, there was no reactivity of the anti-PorA mAb with rGNA33 (Fig. 2GoB, lane 1), with the negative control E. coli proteins (Fig. 2GoB, lane 2), or with total membranes prepared from NmB strains that do not express PorA P1.2 (Fig. 2GoB, lanes 3–5). However, a protein having an apparent mass expected for PorA was detected in total membrane preparations from strains BZ232 (Fig. 2GoB, lane 6), BZ232{Delta}GNA33 (Fig. 2GoB, lane 7), NMB (Fig. 2GoB, lane 8), and NMB{Delta}GNA33 (Fig. 2GoB, lane 9) that express PorA P1.2. The results suggest that the protein with an apparent mass of 41 kDa reacting with the anti-GNA33 mAb in Fig. 2GoA was PorA.

Peptide mapping of the PorA and GNA33 epitopes recognized by the anti-GNA33 mAb 25

To identify the specific amino acid sequence recognized by the anti-GNA33 mAb 25, we prepared overlapping linear decapeptides spanning the entire amino acid sequences of GNA33 and PorA loop 4 from strain 2996 (GenBank accession number X57180). The peptides that were positive (>=8 dye units) with mAb 25 are summarized in Table IIGo. All eight of the positive GNA33 peptides share a tripeptide, QTP. The QTP sequence is also present in all five positive PorA P1.2 peptides that reacted with mAb 25. However, the QTP sequence is not sufficient for anti-GNA33 binding, as there was no mAb binding to three loop 4 peptides that contained QTP but lacked the preceding FVQ sequence.


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  		Table II. Epitope mapping of anti-GNA33 mAb 25 against overlapping peptides prepared from GNA33 and loop 4 of PorA P1.2 (strain 2996)

 
To define the minimal peptide sequence from each protein that is sufficient for anti-GNA mAb 25 binding, we prepared progressively smaller GNA33 peptides beginning with AQAFQTPVHS (see Fig. 3GoA) and PorA P1.2 peptides beginning with TPAHFVQQTP (see Fig. 3GoB). The mAb bound strongly with GNA33 peptides containing FQTPV and PorA P1.2 peptides containing FVQQTP, but not with any of the smaller peptides. The same minimal epitopes for each protein were identified by systematic alanine or glycine substitutions of amino acids contained within the relevant peptides of loop 4 of PorA and the GNA33 (data not shown).



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  		FIGURE 3. Binding of anti-GNA33 mAb 25 to progressively smaller peptides corresponding to segments from GNA33 (A) and PorA P1.2 (strain 2996) (B). The respective peptides shown were identified from mapping studies with overlapping 10-mer peptides prepared from each protein and shown to contain an epitope recognized by mAb 25 (see Table IIGo).

 
These data suggest that the Abs elicited by rGNA33 have bactericidal activity against Nm strains expressing the P1.2 epitope as the result of cross-reactivity with the P1.2 epitope of PorA that contains the sequence QTP.

Comparative binding of anti-GNA33 and anti-PorA P1.2 Abs to P1.2 NmB strains

The unexpected finding that anti-rGNA33 Abs cross-react with the PorA P1.2 epitope provided an opportunity to compare the activity of Ab raised to rGNA33 with that elicited by PorA serosubtype P1.2. With one exception, the concentration-dependent binding of the anti-GNA33 mAb determined by flow cytometry was similar to that of a control anti-PorA P1.2 mAb for the nine P1.2 strains tested (see representative data for strains 8047 and BZ232 in Fig. 4GoA). The exception, strain M986, showed relatively weaker anti-GNA33 Ab binding when compared with binding to the other P1.2 strains (see Fig. 4GoB). In contrast, binding by the anti-PorA P1.2 mAb was similar for all P1.2 strains, including M986.



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  		FIGURE 4. Binding of murine mAbs to live encapsulated NmB strains as determined by indirect fluorescence flow cytometry. The mAbs tested are described in Fig. 1GoB. A, Concentration-dependent binding of anti-GNA33 mAb 25 to strains 8047 (BC50 = 15 µg/ml with human complement) and BZ232 (BC50 > 150 µg/ml with human complement). Both strains were susceptible to bacteriolysis when tested with rabbit complement (see Table IIIGo). B, Concentration-dependent anti-GNA33 binding to strains M986 (PorA VR2 type P1.2) and M5682 (PorA VR2 type P1.2), as compared with strain 8047 (PorA VR2 type P1.2-2). M986 was resistant to anti-GNA33 bacteriolysis (human or rabbit), M5682 was susceptible (rabbit only), and strain 8047 was susceptible (human or rabbit).

 
The VR2 sequence type of strain M986 is reported to be P1.2 (GenBank accession number U92912), which is defined by a loop 4 sequence that includes the segment FVQQTPK, as opposed to FVQQTPQ for strains 8047 and BZ232 (VR2 type P1.2-2; see Table IGo). The VR2 type is based on the amino acid sequence of the particular P1.2 epitope (see Ref. 31 or http://mlst.zoo.ox.ac.uk for recently revised PorA VR designation conventions). Two other strains reported to have VR2 sequence type P1.2 (strains M3735 and M5682) showed strong anti-GNA33 Ab binding which, in each strain, was comparable to the respective binding of the control anti-PorA P1.2 mAb (see, for example, binding data with strain M3735 in Fig. 1GoA and strain M5682 in Fig. 4GoB). The VR2 sequence type of PorA loop 4 in strains M986, M3735, and M5682 was confirmed to be P1.2 by a repeated independent nucleotide sequencing experiment (M986 in our laboratory; M3735 and M5682 by C. Sacchi, personal communication). Therefore, the sequence difference (K->Q) does not appear to be sufficient to explain the decreased anti-GNA33 binding activity with strain M986.

Comparative bactericidal activity of anti-GNA33 and anti-PorA P1.2 Abs against P1.2 Nm strains

We compared the complement-dependent bactericidal activity of murine mAbs to PorA P1.2, rGNA33 (mAb 25), and serogroup B (SEAM 12) and C (mAb 181.1) polysaccharide capsules. With the exception of the serogroup C anticapsular mAb (subclass IgG3) that was used to test NmC strain M5954, the subclass of all of the other mAbs was IgG2a. The BC50 of the anti-PorA P1.2 mAb in the presence of human complement was <=0.5 µg/ml for all nine strains. The corresponding BC50 values of the serogroup B anticapsular mAb were higher, ranging from 5 to 12 µg/ml, and for the serogroup C mAb (strain M5954), <1 µg/ml.

As summarized in Table IIIGo, the bactericidal activity of the anti-GNA33 mAb was variable and was dependent on the complement source used. For three of the strains (8047, NMB, and M3735), BC50 values of the anti-GNA33 mAb in the presence of human complement ranged from 7 to 15 µg/ml. The respective values for these strains were similar to the bactericidal titers of the anticapsular Ab measured with this human complement source (serum from a healthy adult, without intrinsic bactericidal activity (25)). For the remaining six strains (2996, BZ232, M5545, M5682, M5954, and M986) there was no killing observed with the anti-GNA33 mAb in the presence of this human complement source (BC50 > 60 µg/ml; Table IIIGo), or with a second human complement source (serum from a patient with agammaglobulinemia; BC50 > 30 µg/ml). When infant rabbit serum was used as the complement source, all but one of the six strains that were resistant to killing with human complement were susceptible to anti-GNA33-induced lysis with the rabbit complement. The BC50 values of the susceptible strains when tested with rabbit complement ranged from <=1 µg/ml to 8 µg/ml (see Table IIIGo). The exception was strain M986, where no killing was observed with the anti-GNA33 mAb when tested with human or rabbit complement (BC50 values, >150 and >30 µg/ml, respectively). Lack of bacteriolysis for this strain may be related to the lower surface binding of the mAb as measured by flow cytometry (see Fig. 4GoB). We also tested the bactericidal activity of the polyclonal anti-rGNA33 antiserum against five of the strains using human complement (MAS). The respective bactericidal titers corresponded to the results measured with anti-GNA33 mAb 25 (see Table IIIGo).


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  		Table III. Bactericidal activity of anti-GNA33 Abs against different Nm strains

 
Passive protection by anti-GNA33 Abs in infant rats

We investigated the ability of mouse anti-GNA33 Ab to confer passive protection against NmB bacteremia in an infant rat model. The NmB strains used included a strain susceptible to anti-GNA33 bacteriolysis in the presence of human or rabbit complement 8047, a strain resistant to anti-GNA33 bacteriolysis with human complement but susceptible with rabbit complement (BZ232), and a strain resistant to anti-GNA33 bacteriolysis in the presence of human or rabbit complement (M986; see Table IIIGo). Bactericidal activity of the anti-GNA33 mAb against the latter two strains also was tested using adult rat serum as a complement source. BZ232 was susceptible (BC50 value < 1 µg/ml), whereas strain M986 was resistant (BC50 > 30 µg/ml). The results of the passive protection experiments with the different strains are summarized in Table IVGo.


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  		Table IV. Anti-GNA33 Ab passive protection in infant rats challenged with N. meningitidis serogroup B strains 8047, M986, or BZ232

 
Experiment one was performed with polyclonal anti-GNA33 antiserum that had been used in our initial flow experiments (Fig. 1GoA) and that had also been tested for bactericidal activity (Table IIIGo). A total of 100 µl of a 1/5 or 1/25 dilution of the polyclonal mouse anti-rGNA33 antiserum mixed with 5.8 x 103 CFU of strain 8047 and given i.p. completely protected rats against bacteremia measured 18 h after the challenge. In the same experiment, all animals treated with 100 µl of a 1/5 or 1/25 dilution of the anti-GNA33 antiserum and challenged with 6.5 x 103 CFU of strain M986, a strain resistant to anti-GNA33 bacteriolysis, developed bacteremia. Despite lack of bactericidal activity, the geometric mean CFU per milliliter of blood of the animals treated with the anti-GNA33 antiserum and challenged with strain M986 was 10- to 20-fold lower than that of control animals treated with a negative control antiserum prepared against E. coli proteins (p = 0.02). Similar results were obtained in experiment two with the anti-GNA33 mAb 25. All six rats pretreated with 20 µg of mAb 25 i.p. at time 0 and challenged 2 h later with 3.5 x 103 CFU of strain M986 had bacteria present in blood samples obtained 18 h after challenge. However, the geometric mean CFU per milliliter was <0.3% of that of control animals pretreated with an irrelevant mAb (p < 0.02). In the same experiment, 20 µg per rat of the anti-PorA P1.2 mAb was completely protective against strain M986, and 2 µg per rat was partially protective (only one of six treated animals developed bacteremia).

In experiments three and four, rats were challenged with strain BZ232 (resistant to anti-GNA33 bacteriolysis with human complement but susceptible with rabbit or rat complement). The protective activity of the anti-GNA33 mAb against this strain was similar to or higher than that of the control anticapsular Ab and only slightly less than that of the anti-PorA P1.2 mAb.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown that mouse Abs produced as the result of immunization with rGNA33 are able to mediate bacteriolysis of N. meningitidis strains in the presence of complement because of cross-reactivity of anti-GNA33 Abs with the P1.2 epitope of the porin protein, PorA. This result was unexpected because GNA33 and PorA have no significant sequence homology, are structurally and functionally unrelated, and are physically located in different bacterial substructures. Hence, GNA33 can be described as an immunologic mimic of PorA.

Molecular mimicry can be defined as the functional similarity of two unrelated molecules. With respect to Ab-Ag interactions, molecular mimetics may exhibit antigenic mimicry by binding to the same paratope as the nominal Ag. In some cases, antigenic mimetics exhibit immunogenic mimicry by eliciting a protective Ab response against a pathogenic organism (representative references include Refs. 1, 2, 4, 6, 9, 32 , and 33). The majority of immunogenic mimetics reported have been either peptide or anti-idiotype Abs for the treatment of cancer (reviewed in Ref. 10) or prevention of infectious diseases (selected examples include Refs. 1, 2, 4, 9 , and 32). Although there are numerous publications in the field of mimetic vaccines for prevention of infectious diseases, few of the reports have been confirmed by independent laboratories. Also, there have been few studies comparing the binding and functional activity of Abs elicited by the mimetic with those of Abs raised to the nominal Ag. In this context, the molecular mimicry exhibited by GNA33 is exceptional. First, GNA33 is a non-Ig protein that, as described above, is unrelated to PorA. Second, rGNA33 elicits an Ab response that, in many respects, is similar in functional activity to that elicited by native PorA in OMV preparations. Third, the polyclonal mouse anti-rGNA33 antisera described in this work were prepared in two independent laboratories (Chiron, Siena, Italy, and Children’s Hospital Oakland Research Institute, Oakland, CA), and the bactericidal data from the polyclonal and monoclonal anti-GNA33 Abs were independently replicated in the two laboratories.

In previous studies, immunization with peptides corresponding to loop 4 of PorA P1.2 failed to elicit Abs that bound to the native protein or mediated bacteriolysis in the presence of complement (34). Presumably, the smaller peptide fragments were unable to adopt stable conformations present in the native porin. Similarly, immunization with rPorA expressed in E. coli or Bacillus subtilus failed to elicit bactericidal Ab unless the conformation of the surface-accessible PorA epitopes in the recombinant protein were reconstituted using liposomes or detergents (35, 36). These results suggest that the epitopes on PorA responsible for eliciting bactericidal Ab are conformational. In contrast, immunization with the rGNA33 mimetic elicited bactericidal Ab that cross-reacted with the P1.2 epitope of PorA loop 4. Unlike rPorA, this occurred when the rGNA33 protein used as the immunogen was simply mixed with Freund’s adjuvant, without the need for renaturation of the recombinant molecule.

Abs generated to rGNA33 bound to the surface of most serosubtype P1.2 NmB strains as well as an anti-PorA mAb that defines the P1.2 epitope. For some strains, the bactericidal activity of the anti-mimetic Abs was similar to that of a mAb that binds to the polysaccharide capsule. However, for reasons that are unknown, the anti-mimetic Abs were less active than the corresponding anti-P1.2 mAb in activating complement-mediated bacteriolysis in vitro. This difference was particularly striking when human complement was used in the assay. With human complement only one-third of the P1.2 strains tested were susceptible to anti-rGNA33 bacteriolysis, and these strains required on average 10- to 20-fold higher Ab concentrations for bacteriolysis than the corresponding anti-PorA P1.2 mAb (see Table IIIGo).

The clinical importance of the lack of bactericidal activity of anti-GNA33 Abs against some P1.2 strains when tested with human complement is unclear. With the exception of strain M986, all of the strains that were resistant to bacteriolysis when tested with human complement were highly susceptible when tested with rabbit (see Table IIIGo) or rat complement (data not shown). Anti-GNA33 Abs also were protective in infant rats challenged with different P1.2 strains (see Table IVGo). For some strains, such as BZ232, the relative protective activity observed with the anti-GNA33 mAb25 was similar to that observed with the anti-PorA P1.2 mAb. Furthermore, both polyclonal and monoclonal anti-GNA33 Abs conferred protection against strain M986, even though that strain was highly resistant to anti-GNA33 bacteriolysis in the presence of human, rabbit, or rat complement in vitro. In the absence of bacteriolysis, the most likely mechanism for the observed protection against strain M986 is opsonization. The lower functional activity of the anti-mimetic Ab with human complement, as compared with that of the anti-PorA Ab, may result from subtle differences in binding avidity, or to subtle differences in the surface location of the respective epitopes recognized by anti-GNA33 mAb 25 and the anti-PorA P1.2 mAb.

Considerable data indicate that immunization of humans with OMV vaccines elicits serum bactericidal Ab responses and confers protection against developing meningococcal disease (reviewed in Ref. 37). In human infants immunized with OMV vaccines, the bactericidal Ab response is directed primarily against surface-exposed epitopes on PorA (38), primarily loops 1 and 4, which contain the most surface-exposed PorA epitopes on encapsulated meningococcal strains. In immunized adults, the protective Ab responses also appear to be directed against the Opc protein (39). The discovery that GNA33 exhibits immunologic mimicry of the PorA P1.2 epitope suggests that GNA33 may be a vaccine candidate for the prevention of disease caused by P1.2 strains, which represent ~8% of serogroup B isolates in the U.S. (40). Further, by substituting other PorA loops into GNA33 or into subdomains of GNA33, it may be possible to generate immunogenic mimetics of other serosubtype PorA epitopes that could be used as Ags in a multivalent meningococcal vaccine. Such a vaccine would have many advantages over vaccines based on using rPorA. For example, the preparation of such a rGNA33 vaccine would be greatly simplified as rGNA33 can be conveniently expressed in large amounts in noninfectious E. coli, without the need for detergent extractions, refolding, or reconstitution in lipid vesicles. Also, the epitope-containing segments of PorA variants from newly emergent NmB strains causing disease could be substituted into GNA33 as needed.


   Acknowledgments
 
We thank Katherine Alter for performing flow cytometry assays and Claudio Sacchi (Centers for Disease Control, Atlanta, GA) for kindly resequencing the porA genes from several strains he provided for this study. We are grateful to Alexander Lucas for reviewing the manuscript and providing helpful suggestions.


   Footnotes
 
1 This work was supported by Grants RO1 AI45642 and AI46464 from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health. The studies also were supported in part by a grant from the Chiron Corporation. J. A.-B. is a Marie Curie Fellow. Back

2 Address correspondence and reprint requests to Dr. Dan M. Granoff, Children’s Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609. E-mail address: dgranoff{at}chori.org Back

3 Abbreviations used in this paper: NmB, Neisseria meningitidis serogroup B; PorA, porin A; GNA33, genome-derived Ag 33; OMV, outer membrane vesicle. Back

Received for publication August 6, 2001. Accepted for publication September 25, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Chargelegue, D., O. E. Obeid, S. C. Hsu, M. D. Shaw, A. N. Denbury, G. Taylor, M. W. Steward. 1998. A peptide mimic of a protective epitope of respiratory syncytial virus selected from a combinatorial library induces virus-neutralizing antibodies and reduces viral load in vivo. J. Virol. 72:2040.[Abstract/Free Full Text]
  2. Fleuridor, R., A. Lees, L. Pirofski. 2001. A cryptococcal capsular polysaccharide mimotope prolongs the survival of mice with Cryptococcus neoformans infection. J. Immunol. 166:1087.[Abstract/Free Full Text]
  3. Gulati, S., D. P. McQuillen, J. Sharon, P. A. Rice. 1996. Experimental immunization with a monoclonal anti-idiotope antibody that mimics the Neisseria gonorrhoeae lipooligosaccharide epitope 2C7. J. Infect. Dis. 174:1238.[Medline]
  4. Pincus, S. H., M. J. Smith, H. J. Jennings, J. B. Burritt, P. M. Glee. 1998. Peptides that mimic the group B streptococcal type III capsular polysaccharide antigen. J. Immunol. 160:293.[Abstract/Free Full Text]
  5. Puntoriero, G., A. Meola, A. Lahm, S. Zucchelli, B. B. Ercole, R. Tafi, M. Pezzanera, M. U. Mondelli, R. Cortese, A. Tramontano, et al 1998. Towards a solution for hepatitis C virus hypervariability: mimotopes of the hypervariable region 1 can induce antibodies cross-reacting with a large number of viral variants. EMBO J. 17:3521.[Medline]
  6. Schreiber, J. R., K. L. Nixon, M. F. Tosi, G. B. Pier, M. B. Patawaran. 1991. Anti-idiotype-induced, lipopolysaccharide-specific antibody response to Pseudomonas aeruginosa. II. Isotype and functional activity of the anti-idiotype-induced antibodies. J. Immunol. 146:188.[Abstract]
  7. Scala, G., X. Chen, W. Liu, J. N. Telles, O. J. Cohen, M. Vaccarezza, T. Igarashi, A. S. Fauci. 1999. Selection of HIV-specific immunogenic epitopes by screening random peptide libraries with HIV-1-positive sera. J. Immunol. 162:6155.[Abstract/Free Full Text]
  8. Steward, M. W., C. M. Stanley, O. E. Obeid. 1995. A mimotope from a solid-phase peptide library induces a measles virus-neutralizing and protective antibody response. J. Virol. 69:7668.[Abstract]
  9. Westerink, M. A., P. C. Giardina, M. A. Apicella, T. Kieber-Emmons. 1995. Peptide mimicry of the meningococcal group C capsular polysaccharide. Proc. Natl. Acad. Sci. USA 92:4021.[Abstract/Free Full Text]
  10. Bhattacharya-Chatterjee, M., S. K. Chatterjee, K. A. Foon. 2000. Anti-idiotype vaccine against cancer. Immunol. Lett. 74:51.[Medline]
  11. Falcioni, F., K. Ito, D. Vidovic, C. Belunis, R. Campbell, S. J. Berthel, D. R. Bolin, P. B. Gillespie, N. Huby, G. L. Olson, et al 1999. Peptidomimetic compounds that inhibit antigen presentation by autoimmune disease-associated class II major histocompatibility molecules. Nat. Biotechnol. 17:562.[Medline]
  12. Tettelin, H., N. J. Saunders, J. Heidelberg, A. C. Jeffries, K. E. Nelson, J. A. Eisen, K. A. Ketchum, D. W. Hood, J. F. Peden, R. J. Dodson, et al 2000. Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science 287:1809.[Abstract/Free Full Text]
  13. Pizza, M., V. Scarlato, V. Masignani, M. M. Giuliani, B. Arico, M. Comanducci, G. T. Jennings, L. Baldi, E. Bartolini, B. Capecchi, et al 2000. Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing. Science 287:1816.[Abstract/Free Full Text]
  14. Lommatzsch, J., M. F. Templin, A. R. Kraft, W. Vollmer, J. V. Holtje. 1997. Outer membrane localization of murein hydrolases: MltA, a third lipoprotein lytic transglycosylase in Escherichia coli. J. Bacteriol. 179:5465.[Abstract/Free Full Text]
  15. Bjune, G., J. K. Gronnesby, E. A. Hoiby, O. Closs, H. Nokleby. 1991. Results of an efficacy trial with an outer membrane vesicle vaccine against systemic serogroup B meningococcal disease in Norway. NIPH Ann. 14:125.[Medline]
  16. Cartwright, K., R. Morris, H. Rumke, A. Fox, R. Borrow, N. Begg, P. Richmond, J. Poolman. 1999. Immunogenicity and reactogenicity in UK infants of a novel meningococcal vesicle vaccine containing multiple class 1 (PorA) outer membrane proteins. Vaccine 17:2612.[Medline]
  17. Zollinger, W. D., J. Boslego, L. O. Froholm, J. S. Ray, E. E. Moran, B. L. Brandt. 1987. Human bactericidal antibody response to meningococcal outer membrane protein vaccines. Antonie Van Leeuwenhoek 53:403.[Medline]
  18. Goldschneider, I., E. C. Gotschlich, M. S. Artenstein. 1969. Human immunity to the meningococcus. I. The role of humoral antibodies. J. Exp. Med. 129:1307.[Abstract]
  19. Sambrook, J., D. W. Russell. 2001. Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
  20. Granoff, D. M., A. Bartoloni, S. Ricci, E. Gallo, D. Rosa, N. Ravenscroft, V. Guarnieri, R. C. Seid, A. Shan, W. R. Usinger, et al 1998. Bactericidal monoclonal antibodies that define unique meningococcal B polysaccharide epitopes that do not cross-react with human polysialic acid. J. Immunol. 160:5028.[Abstract/Free Full Text]
  21. Garcia-Ojeda, P. A., M. E. Monser, L. J. Rubinstein, H. J. Jennings, K. E. Stein. 2000. Murine immune response to Neisseria meningitidis group C capsular polysaccharide: analysis of monoclonal antibodies generated in response to a thymus-independent antigen and a thymus-dependent toxoid conjugate vaccine. Infect. Immun. 68:239.[Abstract/Free Full Text]
  22. Van Der Ley, P., J. T. Poolman. 1992. Construction of a multivalent meningococcal vaccine strain based on the class 1 outer membrane protein. Infect. Immun. 60:3156.[Abstract/Free Full Text]
  23. Stephens, D. S., J. S. Swartley, S. Kathariou, S. A. Morse. 1991. Insertion of Tn916 in Neisseria meningitidis resulting in loss of group B capsular polysaccharide. Infect. Immun. 59:4097.[Abstract/Free Full Text]
  24. Moe, G. R., P. Zuno-Mitchell, S. S. Lee, A. H. Lucas, D. M. Granoff. 2001. Functional activity of anti-neisserial surface protein A monoclonal antibodies against strains of Neisseria meningitidis serogroup B. Infect. Immun. 69:3762.[Abstract/Free Full Text]
  25. Santos, G. F., R. R. Deck, J. Donnelly, W. Blackwelder, D. M. Granoff. 2001. Importance of complement source in measuring meningococcal bactericidal titers. Clin. Diagn. Lab. Immunol. 8:616.[Abstract/Free Full Text]
  26. Steele, N. P., Jr R. S. Munson, D. M. Granoff, J. E. Cummins, R. P. Levine. 1984. Antibody-dependent alternative pathway killing of Haemophilus influenzae type b. Infect. Immun. 44:452.[Abstract/Free Full Text]
  27. Moe, G. R., S. Tan, D. M. Granoff. 1999. Differences in surface expression of NspA among Neisseria meningitidis group B strains. Infect. Immun. 67:5664.[Abstract/Free Full Text]
  28. Laemmli, U. K.. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680.[Medline]
  29. Towbin, H., T. Staehelin, J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350.[Abstract/Free Full Text]
  30. Frank, R., H. Overwin. 1996. SPOT synthesis: epitope analysis with arrays of synthetic peptides prepared on cellulose membranes. Methods Mol. Biol. 66:149.[Medline]
  31. Sacchi, C. T., A. M. Whitney, T. Popovic, D. S. Beall, M. W. Reeves, B. D. Plikaytis, N. E. Rosenstein, B. A. Perkins, M. L. Tondella, L. W. Mayer. 2000. Diversity and prevalence of PorA types in Neisseria meningitidis serogroup B in the United States, 1992–1998. J. Infect. Dis. 182:1169.[Medline]
  32. Chen, Y. C., K. Delbrook, C. Dealwis, L. Mimms, I. K. Mushahwar, W. Mandecki. 1996. Discontinuous epitopes of hepatitis B surface antigen derived from a filamentous phage peptide library. Proc. Natl. Acad. Sci. USA 93:1997.[Abstract/Free Full Text]
  33. Magliani, W., L. Polonelli, S. Conti, A. Salati, P. F. Rocca, V. Cusumano, G. Mancuso, G. Teti. 1998. Neonatal mouse immunity against group B streptococcal infection by maternal vaccination with recombinant anti-idiotypes. Nat. Med. 4:705.[Medline]
  34. McGuinness, B., A. K. Barlow, I. N. Clarke, J. E. Farley, A. Anilionis, J. T. Poolman, J. E. Heckels. 1990. Deduced amino acid sequences of class 1 protein (PorA) from three strains of Neisseria meningitidis: synthetic peptides define the epitopes responsible for serosubtype specificity. J. Exp. Med. 171:1871.[Abstract/Free Full Text]
  35. Christodoulides, M., J. L. Brooks, E. Rattue, J. E. Heckels. 1998. Immunization with recombinant class 1 outer-membrane protein from Neisseria meningitidis: influence of liposomes and adjuvants on antibody avidity, recognition of native protein and the induction of a bactericidal immune response against meningococci. Microbiology 144:3027.[Abstract]
  36. Idänpään-Heikkila, I., S. Muttilainen, E. Wahlström, L. Saarinen, M. Leinonen, M. Sarvas, P. H. Mäkelä. 1995. The antibody response to a prototype liposome vaccine containing Neisseria meningitidis outer membrane protein P1 produced in Bacillus subtilis. Vaccine 13:1501.[Medline]
  37. Frasch, C. E.. 1995. Meningococcal vaccines: past, present and future. K. Cartwright, ed. Meningococcal Disease 245. Wiley, New York.
  38. Tappero, J. W., R. Lagos, A. M. Ballesteros, B. Plikaytis, D. Williams, J. Dykes, L. L. Gheesling, G. M. Carlone, E. A. Hoiby, J. Holst, et al 1999. Immunogenicity of 2 serogroup B outer-membrane protein meningococcal vaccines: a randomized controlled trial in Chile. JAMA 281:1520.[Abstract/Free Full Text]
  39. Rosenqvist, E., E. A. Hoiby, E. Wedege, K. Bryn, J. Kolberg, A. Klem, E. Ronnild, G. Bjune, H. Nokleby. 1995. Human antibody responses to meningococcal outer membrane antigens after three doses of the Norwegian group B meningococcal vaccine. Infect. Immun. 63:4642.[Abstract]
  40. Tondella, M. L., T. Popovic, N. E. Rosenstein, D. B. Lake, G. M. Carlone, L. W. Mayer, B. A. Perkins. 2000. Distribution of Neisseria meningitidis serogroup B serosubtypes and serotypes circulating in the United States: the Active Bacterial Core Surveillance Team. J. Clin. Microbiol. 38:3323.[Abstract/Free Full Text]
  41. Sacchi, C. T., A. P. Lemos, M. E. Brandt, A. M. Whitney, C. E. Melles, C. A. Solari, C. E. Frasch, L. W. Mayer. 1998. Proposed standardization of Neisseria meningitidis PorA variable-region typing nomenclature. Clin. Diagn. Lab. Immunol. 5:845.[Abstract/Free Full Text]
  42. Nurminen, M., S. Butcher, I. Idänpään-Heikkila, E. Wahlström, S. Muttilainen, K. Runeberg-Nyman, M. Sarvas, P. H. Mäkelä. 1992. The class 1 outer membrane protein of Neisseria meningitidis produced in Bacillus subtilis can give rise to protective immunity. Mol. Microbiol. 6:2499.[Medline]
  43. McGuinness, B. T., I. N. Clarke, P. R. Lambden, A. K. Barlow, J. T. Poolman, D. M. Jones, J. E. Heckels. 1991. Point mutation in meningococcal por A gene associated with increased endemic disease. Lancet 337:514.[Medline]



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