Unique intermolecular bactericidal epitope involving the homosialopolysaccharide capsule on the cell surface of group B Neisseria meningitidis and Escherichia coli K1

The N-propionylated group B meningococcal polysaccharide mimics a unique bactericidal epitope on the surface of group B meningococci and Escherichia coli K1. This was confirmed when both the above organisms were able to absorb the bactericidal antibodies from a mouse-anti-N-propionylated group B meningococcal polysaccharide-tetanus toxoid conjugate serum. By using affinity columns it was possible to divide the conjugate antiserum into three distinct populations of both group B polysaccharide cross-reactive and non-cross-reactive antibodies, one of which contained most of the bactericidal activity. The cross-reactive (IgG1) antibodies were absorbed by an affinity column in which the group B polysaccharide was linked to the solid support by a long spacer arm, thereby isolating a population of non-cross-reactive (IgG1) antibodies. Surprisingly the above column also retained another population of non-cross-reactive (IgG2a) and (IgG2b) antibodies which contained most of the bactericidal activity. These latter antibodies were not absorbed by a similar group B polysaccharide-affinity column in which a short spacer arm was employed. Thus the above experiments not only effected a separation of highly bactericidal antibodies but also provided evidence that the long spacer arm is functional in the binding of the bactericidal antibodies to the affinity column. This indicates that the bactericidal epitope is mimicked by the group B polysaccharide in the presence of the long spacer arm, which supports the hypothesis that the epitope is polysaccharide-associated and is probably intermolecular in nature.     

Mapping of monoclonal antibodies specific to P64k: a common antigen of several isolates of Neisseria meningitidis

P64k is a minor outer membrane protein from Neisseria meningitidis. This protein has been produced at high levels in Escherichia coli. We generated a group of monoclonal antibodies (mAbs) against recombinant P64k, which recognise four non-overlapping epitopes, as shown using competition assays with biotinylated mAbs. The P64k sequences involved in mAbs binding were mapped with synthetic overlapping peptides derived from the P64k protein, and located in the previously determined three-dimensional structure of the protein. These antibodies were also characterised by whole-cell ELISA and bactericidal tests against N. meningitidis. Only two of the recognised epitopes were exposed on the bacterial surface, and none of the mAbs showed bactericidal activity. The relationship between these results and the structural data on the epitopes bound by the mAbs is discussed.     

Gonococcal outer-membrane protein PIB: comparative sequence analysis and localization of epitopes which are recognized by type-specific and cross-reacting monoclonal antibodies.

Comparison of the inferred amino acid sequence of outer-membrane protein PIB from gonococcal strain P9 with those from other serovars reveals that sequence variations occur in two discrete regions of the molecule centred on residues 196 (Var1) and 237 (Var2). A series of peptides spanning the amino acid sequence of the protein were synthesized on solid-phase supports and reacted with a panel of monoclonal antibodies (mAbs) which recognize either type-specific or conserved antigenic determinants on PIB. Four type-specific mAbs reacted with overlapping peptides in Var1 between residues 192-198. Analysis of the effect of amino acid substitutions revealed that the mAb specificity is generated by differences in the effect of single amino acid changes on mAb binding, so that antigenic differences between strains are revealed by different patterns of reactivity within a panel of antibodies. The variable epitopes in Var1 recognized by the type-specific mAbs lie in a hydrophilic region of the protein exposed on the gonococcal surface, and are accessible to complement-mediated bactericidal lysis. In contrast, the epitope recognized by mAb SM198 is highly conserved but is not exposed in the native protein and the antibody is non-bactericidal. However, the conserved epitope recognized by mAb SM24 is centred on residues 198-199, close to Var1 , and is exposed for bactericidal killing.     

Monoclonal antibodies to meningococcal factor H binding protein with overlapping epitopes and discordant functional activity

Background

Meningococcal factor H binding protein (fHbp) is a promising vaccine candidate. Anti-fHbp antibodies can bind to meningococci and elicit complement-mediated bactericidal activity directly. The antibodies also can block binding of the human complement down-regulator, factor H (fH). Without bound fH, the organism would be expected to have increased susceptibility to bacteriolysis. Here we describe bactericidal activity of two anti-fHbp mAbs with overlapping epitopes in relation to their different effects on fH binding and bactericidal activity.

Methods and Principal Findings

Both mAbs recognized prevalent fHbp sequence variants in variant group 1. Using yeast display and site-specific mutagenesis, binding of one of the mAbs (JAR 1, IgG3) to fHbp was eliminated by a single amino acid substitution, R204A, and was decreased by K143A but not by R204H or D142A. The JAR 1 epitope overlapped that of previously described mAb (mAb502, IgG2a) whose binding to fHbp was eliminated by R204A or R204H substitutions, and was decreased by D142A but not by K143A. Although JAR 1 and mAb502 appeared to have overlapping epitopes, only JAR 1 inhibited binding of fH to fHbp and had human complement-mediated bactericidal activity. mAb502 enhanced fH binding and lacked human complement-mediated bactericidal activity. To control for confounding effects of different mouse IgG subclasses on complement activation, we created chimeric mAbs in which the mouse mAb502 or JAR 1 paratopes were paired with human IgG1 constant regions. While both chimeric mAbs showed similar binding to fHbp, only JAR 1, which inhibited fH binding, had human complement-mediated bactericidal activity.

Conclusions

The lack of human complement-mediated bactericidal activity by anti-fHbp mAb502 appeared to result from an inability to inhibit binding of fH. These results underscore the importance of inhibition of fH binding for anti-fHbp mAb bactericidal activity.     

Protective activity of monoclonal antibodies to genome-derived neisserial antigen 1870, a Neisseria meningitidis candidate vaccine

Genome-derived neisserial Ag (GNA) 1870 is a meningococcal vaccine candidate that can be subdivided into three variants based on amino acid sequence variability. Variant group 1 accounts for approximately 60% of disease-producing group B isolates. The Ag went unrecognized until its discovery by genome mining because it is expressed in low copy number by most strains. To investigate the relationship between Ab binding to GNA1870 and complement-mediated protective functions, we prepared a panel of four murine IgG mAbs against rGNA1870 (variant 1) and evaluated their activity against nine genetically diverse encapsulated Neisseria meningitidis strains expressing subvariants of variant 1 GNA1870. Based on flow cytometry with live encapsulated bacteria, surface accessibility of the epitopes recognized by the mAbs appeared to be low in most strains. Yet mAb concentrations <1 to 5 micro g/ml were sufficient to elicit bactericidal activity with human complement and/or activate C3b deposition on the bacterial surface. Certain combinations of mAbs were highly bactericidal against strains that were resistant to bactericidal activity of the respective individual mAbs. The mAbs conferred passive protection against bacteremia in infant rats challenged by strains resistant to bacteriolysis, and the protective activity paralleled the ability of the mAb to activate C3b deposition. Thus, despite low GNA1870 surface exposure, anti-GNA1870 variant 1 Abs are bactericidal and/or elicit C3b deposition and confer protection against bacteremia caused by encapsulated N. meningitidis strains expressing GNA1870 subvariant 1 proteins. The data support GNA1870 as a promising vaccine candidate for prevention of meningococcal group B disease caused by GNA1870 variant 1 strains.     

Mouse antibodies to the insulin receptor developing spontaneously as anti-idiotypes. I. Characterization of the antibodies

Mice immunized to ungulate insulins were found to develop antibodies of two specificities: insulin antibodies that were mostly IgG1 and IgG2 antibodies that acted both as anti-idiotypes to specific mouse insulin antibodies and as antibodies to the insulin receptor. There was a negative association between the presence of anti-idiotypic receptor antibodies and insulin antibodies bearing the specific idiotype; the specific idiotypic antibodies were confined to the early phase of the primary response while the anti-idiotypic receptor antibodies were detected only after the idiotypic antibodies had disappeared. To map the insulin epitope that triggered the specific idiotypic response, we chemically altered the insulin molecule so as to inhibit its interaction with the insulin receptor. The altered insulins triggered high titers of antibodies binding to antigenic determinants on native insulin, but no anti-idiotypic receptor antibodies. Thus, the epitope responsible for the specific idiotypic-anti-idiotypic network was probably the part of the insulin molecule whose conformation is recognized by the insulin receptor.     

Characterization and comparative bactericidal activity of monoclonal antibodies to Bordetella pertussis lipo-oligosaccharide A.

Spleen cells from mice immunized with a Bordetella pertussis N-lauroyl sarcosine membrane extract (SME) were used to generate hybridoma cells lines producing monoclonal antibodies (mAbs). Seven mAbs were shown to be specific to B. pertussis lipo-oligosaccharide (LOS) by immunoblotting of SME or purified LOS following SDS-PAGE. All mAbs reacted with the B. pertussis Tohama I strain of the LOS AB phenotype, and did not react with the atypical variant strain 134 of the LOS B phenotype. The immune reactivity of the mAbs was retained after treatment of SME with proteinase K and was lost after sodium periodate treatment. No cross-reactivity was observed with the mAbs when tested against B. parapertussis and other Gram-negative bacteria. However, all mAbs reacted with B. bronchiseptica. Binding assays with live B. pertussis cells demonstrated that mAbs strongly reacted with cell surface exposed antigenic determinants. High bacterial cell lytic capability was observed for five of these mAbs. Concentrations between 0.22 and 2.2 micrograms mAb ml-1 (0.1 and 1 microgram per 450 microliter assay) purified by protein A were required to kill at least 50% of the bacteria. Competition immunoassays with biotinylated antibodies showed that the bacteriolytic and non-bacteriolytic mAbs were directed to different epitopes of the B. pertussis LOS A.     

Common epitopes of protein antigens in Neisseria and Moraxella

Cross reactions between N. meningitidis and M. catarrhalis proteins were studied with the use of a panel of monoclonal antibodies to M. catarrhalis protein antigens. All antigenic preparations under study were shown to give cross reactions between N. meningitidis serotype porin of 39 kD (strain B125) and M. catarrhalis proteins of 40-41 kD. These M. catarrhalis proteins belonged to main proteins of class F and had the function of porins in the cell. In addition, the epitope of 41-kD antigen, detected by monoclonal antibodies 3E10, is common for both N. meningitidis porin and N. meningitidis iron-regulated proteins of 70 and 50 kD. The epitope of M. catarrhalis protein of 67 kD, detected by monoclonal antibodies 1G6, is common for N. meningitidis porin and N. meningitidis iron-regulated proteins of 50 and 55 kD.     

The cellular location of a foreign B cell epitope expressed by recombinant bacteria determines its T cell-independent or T cell-dependent characteristics.

We have targeted two foreign B cell antigenic determinants to different locations in the Escherichia coli cell to examine what effect this had on antibody responses elicited by the recombinant bacteria. The two epitopes were the 132-145 peptide from the PreS2 region of hepatitis B virus and the C3 neutralization epitope of poliovirus type 1. They were each expressed in two forms either on the surface, as part of the outer-membrane protein LamB, or soluble in the periplasm, as part of the periplasmic protein MalE. When live bacteria expressing the foreign epitope at the cell surface were used for immunization of mice, they induced T cell-independent antibody responses characterized by a rapid induction of IgM and IgG antibodies. In contrast, when the same foreign epitope was inserted into the MalE protein, the antibody response was only detectable after 3 wk, belonged only to the IgG class and was strictly T cell dependent. This study has therefore identified two major pathways by which epitopes expressed by bacterial cells can stimulate specific antibody responses. The first pathway is mediated by direct activation of B cells by bacterial cell-surface Ag and does not require T cell help. The second pathway is T cell dependent and concerns Ag that can be released from the bacteria in a soluble form. We have also studied the effect of the exact position of the B cell antigenic determinant within the LamB protein and with respect to the outer membrane by comparing the immunogenicity of the PreS epitope inserted at three different permissive sites of LamB. The data indicated that to obtain an antibody response with intact bacteria, the epitope must be protruding sufficiently from the outside of the outer membrane. In contrast, when semipurified hybrid proteins were used as immunogen, the exact position of the B cell antigenic determinant within solubilized LamB protein does not influence its immunogenicity.     

Monoclonal antibodies against O and K antigens of uropathogenic Escherichia coli O4 : K12 : H− as opsonins

Abstract Monoclonal antibodies of subclasses IgG1 and IgG2b and specific for the O4 antigen of Escherichia coli 20025 (O4 : K12 : H⁻) and the capsular K12 polysaccharide of the same strain (IgM) were obtained with the hybridoma technique using spleen cells from Balb/c mice, immunized with a crude bacterial extract, and Sp2/O-Ag8 myeloma cells. The anti-O4 antibodies reacted exclusively with the O4 lipopolysaccharide and not with those from serologically O-cross reactive E. coli . The anti-K12 antibodies recognized as epitope (part of) the KDO moiety of the capsular K12 polysaccharide. Not only anti-K12, but also anti-O4 antibodies effectively phagoopsonized encapsulated E. coli 20025. The opsonized bacteria were killed in subsequent in vitro phagocytosis by human leokocytes in the presence of human serum complement.     

Serum bactericidal activity in a secondary school population following an outbreak of meningococcal disease: effects of carriage and secretor status

Abstract Sera obtained from 106 children following an outbreak of Neisseria meningitidis (B:4:P1.15) were screened for bactericidal antibodies against isolates of meningococci and Neisseria lactamica . Most had high titres of antibodies to N. lactamica and N. meningitidis NG:4:- but not to capsulate isolates: B:4:P1.15; B:15:P1.16; B:4:-; C:4:-. Bactericidal activity was higher for both carriers and secretors but the differences were not significant. Bactericidal activity was not associated with total or specific IgA or IgM. Carriers had significantly higher levels of IgG to N. lactamica but not to NG:4:- in sera with bactericidal activity for each of the capsulate strains. Among non-carriers, higher levels of IgG to N. lactamica were associated with killing of B:4:P1.15 and B:4:-. Secretors' sera with bactericidal activity had significantly higher levels of IgG to N. lactamica compared with sera that were not bactericidal. This was not observed among non-secretors. Antibodies to the outbreak strain were adsorbed by all Neisseria isolates tested and absorption of sera with N. lactamica alone completely removed the bactericidal activity against the outbreak strain.     

Production of rabbit precipitating antisera to subclasses of human IgG]

Precipitating antisera to human subclasses IgG were obtained by immunization of rabbits by whole molecules IgG2, IgG3, IgG4 and gamma 1-chains derived from IgG1H (Pr). Analysis of the antisera obtained demonstrated that rabbits produced specific antibodies to the antigenic subclass determinants IgG3 well, to IgG2, IgG4--much worse, and failed to produce specific antibodies to subclass IgG1 (in immunization with whole molecules of this protein). Antisera contained antibodies to the antigenic determinants common of IgG, and antibodies to light chains which were removed by immunosorption, for which purpose a sorbent on the basis of BrCN sepharose conjugated with IgG of the three other subclasses and Fab-fragment was used.     

Monoclonal antibodies against human beta-glucocerebrosidase

Monoclonal antibodies were obtained against the membrane-bound lysosomal enzyme beta-glucocerebrosidase (acid beta-glucosidase), which is deficient in Gaucher's disease. BALB/c mice were immunized with homogeneous enzyme protein extracted from a sodium dodecyl sulphate/polyacrylamide gel. The mice were subsequently hyperimmunized with partially purified enzyme prior to fusion of spleen cells with myeloma cells. After fusion, 32 primary hybrid cell populations were obtained which continued to produce antibodies against beta-glucocerebrosidase after prolonged time of culture. All antibodies reacted with both native and denatured enzyme. Four primary cell populations were subcloned and the antibodies produced were characterized. The antibodies were all four of the IgG1 subclass. Three of these antibodies bind to protein A whereas one does not. The results of binding assays indicated that three of the antibodies react with the same antigenic domain (epitope 1), but the fourth with a different one (epitope 2). Probably two antigenic determinants are present in epitope 1 since one of the antibodies with specificity for epitope 1 is inactivated after iodination by the chloramine-T procedure whereas a second one is not.     

Study of various presentation forms for a peptide mimetic of Neisseria meningitidis serogroup B capsular polysaccharide

The formulation of a broadly protective vaccine to prevent the serogroup B Neisseria meningitidis (MenB) disease is still an unmet medical need. We have previously reported the induction of bactericidal and protective antibodies against MenB after immunization of mice with a phage-displayed peptide named 4 L-5. This peptide mimics a capsular polysaccharide (CPS) epitope in MenB. With the aim of developing vaccine formulations that could be used in humans, we evaluate in this study various forms of presentation to the immune system of the 4 L-5 sequence, based on synthetic peptides. We synthesized the following: (i) a linear 4 L-5 peptide, (ii) a multiple antigen peptide containing four copies of the 4 L-5 sequence (named MAP), which was then dimerized, and the product named dimeric MAP, and (iii) a second multiple antigen peptide, in this case with two copies of the 4 L-5 sequence and a copy of a T-helper cell epitope of tetanus toxoid, which was then dimerized and the product named MAP-TT. The linear peptide, the MAP, and the dimeric MAP were conjugated to the carrier protein P64K by different conjugation methods. Plain antigens and antigens coupled to P64K were used to immunize BALB/c mice. Of those variants that gave immunogenic results, MAP-TT rendered the highest levels of specific antipeptide IgG antibodies and serum bactericidal activity. These results can find application in the development of meningococcal vaccine candidates and in peptide-based vaccines strategies.     

Antibodies to meningococcal H.8 (Lip) antigen fail to show bactericidal activity

Purified H.8 (Lip) antigen was coupled to tresyl-activated Sepharose 4B and used in affinity columns to purify anti-Lip antibodies from convalescent patient sera and from immune rabbit sera. Affinity-purified anti-Lip antibodies isolated from two convalescent patient sera contained 1000 and 1280 ELISA units of antibody and included antibodies of IgG, IgA, and IgM isotypes. An anti-Lip mouse monoclonal ascites (2-1-CA2) had 28,400 ELISA units of antibody. Bactericidal assays were performed using three different case strains of Neisseria meningitidis group B, namely 44/76, 8532, and 8047. Neither preparation of purified human anti-Lip antibodies had detectable bactericidal activity against strains 44/76 and 8532, but one of the two had a titer of 1:4 against strain 8047. Anti-Lip antibodies that were purified from immune rabbit serum and contained 1600 ELISA units of anti-Lip antibodies also failed to show detectable bactericidal activity. The rabbits were immunized with purified Lip antigen and showed specific antibody levels of 2000-2200 units by ELISA, but even the unfractionated sera had little or no bactericidal activity against the test strains. The high titer mouse monoclonal ascites had no bactericidal activity against the test strains. The poor bactericidal activity associated with monoclonal and polyclonal antibodies to the Lip antigen suggest that in spite of other attractive properties it may not be useful as a meningococcal vaccine.     

Immunological detection of heterogeneous O-antigen containing lipopolysaccharides in Escherichia coli

The components of the cell envelopes of Escherichia coli O1:K1, O7:K1, O18:K1 and O83:K1 strains were separated on SDS-polyacrylamide gels. Longitudinal slices (50 microns thick) of the gel were incubated with typing sera for E. coli O1, O7, O18 and O83, followed by detection of the bound antibodies with 125I-labelled protein A and autoradiography. The antisera reacted with many cell envelope components of strains both with the homologous O-serotype and heterologous O-serotypes. With O-typing sera cross-reactions with heterologous cells and cells boiled for 2 h were found. Up to 40 serotype-specific bands at regular positions with molecular weights between 12000 and 100000 were demonstrated. Since these bands were also observed when purified lipopolysaccharide and unabsorbed homologous O-typing sera were used, it was concluded that these bands represented lipopolysaccharide molecules with increasing molecular weight, all of which contained O-antigen specific immunodeterminants. The band patterns were not influenced by the growth conditions of the cells or the various isolation procedures for the cell envelopes. Comparison of various strains serotyped as O18 revealed strain differences with respect to their lipopolysaccharide band patterns. In the case of O21- and O83-serotyped strains lipopolysaccharide cross-reactions, which were detected by agglutination, were analysed in detail using the gel immunoradioassay method. These cross-reactions appeared to be caused by the presence of common determinants on their lipopolysaccharides and polysaccharide-like material. The cross-reacting antibodies could be removed by cross-absorption. It is concluded that the immunological detection of lipopolysaccharides and other components of E. coli in gels is an important tool in (1) the control of the specificity of typing antisera, (2) the study of the nature of cross-reacting antigens and (3) the study of the nature and uniformity of the various O- and K-serotypes.     

The application of an improved solid phase synthetic technique to the delineation of an antigenic site of apolipoprotein A-II

Previous studies have shown that the antigenic sites of human plasma high-density apolipoprotein A-II (apoA-II) are separate from their lipid-binding determinants in human high density lipoproteins (HDL). A specific radioimmunoassay has shown that three distinct antigenic sites are located in residues 4-23, 31-46, and 56-77; these studies suggested that an antigenic site might be restricted to residues 60-77 in the 56-77 fragment. To further delineate this site, we have developed a solid phase radioimmunoassay technique using an improved solid support on which selected sequences of peptides were synthesized, deprotected with HF, and the resulting peptidyl-resins tested for their capability of binding purified 125I-anti-apoA-II antibodies. Amino acid analyses and solid phase sequence analyses were performed to verify the sequence of the synthetic peptide on the solid support. Using this technique, 125I-anti-apoA-II antibodies had achieved 50% of maximal binding when residues 61-77 were attached to the solid support. The maximal binding was achieved by the addition of one more residue, Leu60, thus confirming our suggestion that a major antigenic site is located in residues 60-77. The binding to the peptidyl-resin was inhibited by a synthetic fragment corresponding to residues 60-77 indicating that the antibodies were specifically bound to the resin.     

Subtractive immunization yields monoclonal antibodies that specifically inhibit metastasis

Subtractive immunization allowed the isolation and characterization of monoclonal antibodies that specifically inhibit metastasis but not proliferation of highly metastatic human tumor cells. The tolerizing agent cyclophosphamide was used to suppress the immune system in mice to dominant immunodeterminants present on a non-metastatic variant (M-) of the human epidermoid carcinoma cell line (HEp3). Mice were then inoculated with a highly metastatic variant (M+) of HEp3 to enhance an immune response to antigenic determinants present on metastatic cells. Hybridomas were generated and screened by ELISA for differential reactivity to M+ HEp3 over M- HEp3 cells. This experimental approach, termed subtractive immunization (S.I.), was compared to a control immunization protocol, which eliminated the cyclophosphamide treatment. The S.I. protocol resulted in an eight-fold increase in the proportion of mAbs that react with molecules enriched on the surface of the M+ HEp3 cells. Two of the mAbs derived from the S.I. protocol, designated DM12-4 and 1A5, were purified and examined for their effect in a metastasis model system in which chick embryos are transplanted with primary HEp3 tumors. Purified mAbs DM12-4 and 1A5, inoculated i.v. into the embryos, inhibited spontaneous metastasis of HEp3 cells by 86 and 90%, respectively. The mAbs are specifically anti-metastatic in that they have no effect on the growth of HEp3 cells in vitro nor did they inhibit primary tumor growth in vivo. The mAbs recognize M+ HEp3 cell surface molecules of 55 kD and 29 kD, respectively. These data demonstrate that the S.I. protocol can be used for the development of unique mAbs that are reactive with antigenic determinants whose expression is elevated on metastatic human tumor cells and which function mechanistically in the metastatic cascade.     

Properties of three monoclonal antibodies that recognize an 80-kDa phytohemagglutinin-binding glycoprotein from porcine lymphocytes

Monoclonal antibodies (mAbs) directed against porcine splenocyte phytohemagglutinin receptor glycoproteins were produced in BALB/c mice. Three antibody-producing, stable hybridomas were cloned and expanded in the peritoneal cavity of BALB/c mice. The mAbs (A7, B1, and H3) were purified and belong to the IgG2 subclass of immunoglobulins (kappa light chain). Each 125I-labeled mAb bound to purified porcine splenocytes with an (apparent) affinity KA congruent to 10(8) M-1 (Scatchard analysis). The number of (apparent) binding sites was 5 x 10(4) sites/cell in the case of B1 and H3, and approximately 15 x 10(4) sites/cell for A7. Immunoprecipitation experiments showed that the three mAbs recognized a single antigenic protein of Mr 80 kilodaltons (gp80). In addition, each mAb recognized a different epitope of gp80, as observed by Western blot analyses. Assessment of the relative ability of anti-gp80 mAbs to stimulate porcine splenocytes as determined by [3H]thymidine incorporation showed weak (A7 and B1) or no (H3) mitogenic activity. Cross-linked anti-gp80 mAbs were not mitogenic, except in the case of B1. In contrast, each anti-gp80 mAb (cross-linked or untreated) showed synergistic mitogenic properties when used in combination with a suboptimal concentration of phytohemagglutinin. The mechanism involved in this synergistic effect is discussed.     

The potential protective effect of immunization with outer-membrane protein I from Neisseria gonorrhoeae

Immunization of rabbits with outer membranes (OM) of Neisseria gonorrhoeae produced antibodies directed against outer-membrane proteins PI and PIII. The antibodies directed against PIII reacted equally well on Western blots with all strains tested, but antibodies directed against PI reacted only with the homologous strain. When purified PIB was used for immunization the immune response was quite different: the sera obtained reacted with both homologous and heterologous PIB types and also reacted with strains expressing PIA. Western blotting of peptides produced by sequential cleavage of PIB revealed that the antigenic determinants recognized by anti-OM sera were predominantly located in the central surface-exposed region of PIB, as is the epitope recognized by the protective anti-PIB monoclonal antibody SM24. In contrast antibodies produced by immunization with purified PI reacted with antigenic determinants in the N-terminal portion of PIB. Nevertheless these determinants are accessible to immune attack on the native protein since the anti-PI sera were opsonic and were strongly bactericidal for both PIA- and PIB-expressing strains.