Mapping of bactericidal epitopes on the P2 porin protein of nontypeable Haemophilus influenzae.

The P2 porin protein is the major outer membrane protein of nontypeable Haemophilus influenzae and is a potential target of a protective immune response. Nine monoclonal antibodies (MAbs) to P2 were developed by immunizing mice with nontypeable H. influenzae whole organisms. Each MAb reacted exclusively with the homologous strain in a whole-cell immunodot assay demonstrating exquisite strain specificity. All nine MAbs recognized abundantly expressed surface-exposed epitopes on the intact bacterium by immunofluorescence and immunoelectron microscopy. Each MAb was bactericidal to the homologous strain in an in vitro complement-mediated killing assay. Immunoblot assay of cyanogen bromide cleavage products of purified P2 indicated that MAb 5F2 recognized the 10-kDa fragment, and the other eight MAbs recognized the 32-kDa fragment. Competitive ELISAs confirmed that 5F2 recognized an epitope that is different from the other eight MAbs. To further localize epitopes, MAbs 5F2 and 6G3 were studied in protein footprinting by using reversed-phase high-performance liquid chromatography. Three potential epitope-containing peptides which were reactive in an enzyme-linked immunosorbent assay with both 5F2 and 6G3 were isolated. These peptides were identified by N-terminal amino acid sequence and localized to loops 5 and 8 of the proposed model for P2. Fusion proteins consisting of glutathione S-transferase fused with variable-length peptides from loops 5 and 8 were expressed in the pGEX-2T vector. Immunoblot assay of fusion peptides of loops 5 and 8 confirmed that 5F2 recognized an epitope within residues 338 to 354 of loop 8; 6G3 and the remaining MAbs recognized an epitope within residues 213 to 229 of loop 5. These studies indicate that nontypeable H. influenzae contains bactericidal epitopes which have been mapped to two different surface-exposed loops of the P2 molecule. These potentially protective epitopes are strain specific and abundantly expressed on the surface of the intact bacterium.     

Immune recognition of porin and lipopolysaccharide epitopes of Salmonella typhimurium in mice

We investigated the antigenic specificity of the humoral immune response to infection by Salmonella typhimurium, by competitive inhibition enzyme-linked immunosorbent assay and Western immunoblots. A panel of eight murine monoclonal antibodies, raised to OmpC and OmpD porins and lipopolysaccharide (LPS)-O antigens, was used to define the specificity of the polyclonal immune response in mice. The monoclonal antibody panel recognized five distinct epitopes; these were localized to surface-exposed loops of OmpC and OmpD porin, to the "eye-let" forming loop L3 of OmpC/OmpD, and to LPS-O4 and O5 factors. The immune mouse serum raised to infections with S. typhimurium LT-2 strain WB600 (wild-type) competitively inhibited the binding of biotin-labelled monoclonal antibodies to the epitopes that they recognize, indicating that all five epitopes were targets of the host immune response to natural infection. However, only two epitopes, one within a surface-exposed loop of OmpC porin, and the other in the LPS-O4 factor, were immunodominant. Furthermore, the bacterial LPS core and O-antigen structure influenced the immune response to the porins. Surface epitopes of porins were dominant in the rough strain SH5014 (rfa), whereas the immune recognition of LPS epitopes was predominant in mice infected with the smooth, wild-type strain (WB600). Finally, the immune response to LPS epitopes O4 and O5 was more pronounced in mice immunized with heat-killed cells than those infected with live S. typhimurium.     

Surface-exposed antigenic cleavage fragments of Neisseria gonorrhoeae proteins 1A and IB.

Whole bacteria, isolated outer membranes, and purified protein I (PI) from one transparent (O-) and two different opaque (O+) phenotype gonococcal strains (serogroups I, II, and III; PI serotypes 1, 5, and 9b) were each treated with tolylsulfonyl phenylalanyl chloromethyl ketone-trypsin, alpha-chymotrypsin, and proteinase K. Protein IA (PIA) of strain 7122 (O-, serotype 1, serogroup I) was resistant to proteolysis by tolysulfonyl phenylalanyl chloromethyl ketone-trypsin and alpha-chymotrypsin and only slightly affected by proteinase K, as long as it was associated with intact bacteria or isolated outer membranes. Purified PIA however was cleaved by these enzymes, resulting in two to five fragments. In contrast, all preparations of strains 5766 opaque phenotype (O+, serotype 7, serogroup II) and 1955 (O+, serotype 9b, serogroup III) were accessible to proteolysis, resulting in cleavage fragments of PIB compatible to those described previously by O. Barrera and J. Swanson (Infect. Immun. 44:565-568, 1984), M. S. Blake et al. (Infect. Immun. 33:212-222, 1981), and Blake (in G. K. Schoolnik, ed., The Pathogenic Neisseriae, 1985). Our data indicated that the purified PIB fraction was more accessible to proteases than the PIBs of whole bacteria or outer membranes. The fragmentation pattern of PIA cleavage products were quite different from PIB fragments, consistent with the different structure of these two groups of PI molecules. Time-dependent cleavage experiments with proteases, i.e., alpha-chymotrypsin, indicated that PIA was subsequently cleaved into smaller fragments. Highly reactive monoclonal antibodies, each specific for a surface-exposed epitope of PIA of strain 7122 or PIB of strains 5766 and 1955, as assessed by coagglutination, Western blot, and immunofluorescence, were reacted with PIA and PIB cleavage fragments in Western blot experiments. All cleavage fragments of the purified PIA and PIB preparations with molecular weights of greater than or equal to 14,200 showed immune reaction in Western blotting, whereas whole cell and outer membrane PIB fragments were less reactive with the specific monoclonal antibodies.     

Mapping of a strain-specific bactericidal epitope to the surface-exposed loop 5 on the P2 porin protein of non-typeable Haemophilus influenzae

The P2 protein is the major outer-membrane protein of non-typeable Haemophilus influenzae (NTHI) and shows extreme heterogeneity among strains. Based on the analysis of antigenic structure, the P2 protein consists of eight potentially surface-exposed loops. Previous studies of monoclonal antibodies (mABs) to a single strain of NTHI showed that P2 contains potentially immunodominant epitopes in loop 5 of the molecule. The goal of the current work is to test the hypothesis that strain-specific and potentially immunodominant epitopes are located in loop 5 of P2 in other strains of NTHI as well. Gene fragments which encode peptides of loop 5 of strains 2019 and 5657 were cloned into an expression vector and subjected to immunoassays with mABs which recognize surface-exposed, bactericidal, strain-specific epitopes. Each mAB recognized loop 5 of the P2 protein of the homologous strain. Analysis of mutant clones with minor amino acid changes showed a loss of reactivity with the mABs. These observations indicate that loop 5 of the P2 molecule contains strain-specific, abundantly expressed surface-exposed epitopes. This further supports the hypothesis that loop 5 is an immunodominant region of the P2 molecule.     

Fine mapping of outer membrane protein P2 antigenic sites which vary during persistent infection by Haemophilus influenzae.

Antigenic drift of the major outer membrane protein (MOMP) P2 of nonencapsulated Haemophilus influenzae as observed during persistent infections in patients with chronic bronchitis was mimicked in a rabbit model in which H. influenzae persisted in subcutaneous cages. The antigenic drift resulted from amino acid substitutions in potentially surface-exposed loops of MOMP P2. Since in a rabbit model the appearance of antigenic variants was associated with the presence of strain-specific bactericidal antibodies (L. Vogel, B. Duim, F. Geluk, P. Eijk, H. Jansen, J. Dankert, and L. van Alphen, Infect. Immun. 64:980-986, 1996), we determined the epitope specificities of these bactericidal antibodies. The eight loops of MOMP P2 of H. influenzae d1 were separately expressed as fusion proteins with glutathione S-transferase. Sera of rabbits persistently infected with H. influenzae reacted with the loop 5 and loop 6 fusion proteins in immunoblotting and enzyme-linked immunosorbent assay. For fine mapping of the epitopes with pepscan analysis, overlapping synthetic peptides consisting of 12 amino acids were made. Rabbit sera contained antibodies reacting with peptides derived from loop 5 and peptides containing amino acids of the side of loop 6. In addition, MOMP P2 variant-specific reactions with the amino acids located at the tip of loop 6 were detected. The rabbit sera showed variant-specific complement-dependent bactericidal activities, which were eliminated by affinity chromatography with fusion proteins of loop 6 but not of loop 5. We conclude that, during persistence of H. influenzae in rabbits, variant-specific bactericidal antibodies are elicited to the variable tip of MOMP P2 loop 6.     

Role of surface-exposed loops of Haemophilus influenzae protein P2 in the mitogen-activated protein kinase cascade

The outer membrane of gram-negative bacteria contains several proteins, and some of these proteins, the porins, have numerous biological functions in the interaction with the host; porins are involved in the activation of signal transduction pathways and, in particular, in the activation of the Raf/MEK1-MEK2/mitogen-activated protein kinase (MAPK) cascade. The P2 porin is the most abundant outer membrane protein of Haemophilus influenzae type b. A three-dimensional structural model for P2 was constructed based on the crystal structures of Klebsiella pneumoniae OmpK36 and Escherichia coli PhoE and OmpF. The protein was readily assembled into the beta-barrel fold characteristic of porins, despite the low sequence identity with the template proteins. The model provides information on the structural features of P2 and insights relevant for prediction of domains corresponding to surface-exposed loops, which could be involved in the activation of signal transduction pathways. To identify the role of surface-exposed loops, a set of synthetic peptides were synthesized according to the proposed model and were assayed for MEK1-MEK2/MAPK pathway activation. Our results show that synthetic peptides corresponding to surface loops of protein P2 are able to activate the MEK1-MEK2/MAPK pathways like the entire protein, while peptides modeled on internal beta strands are unable to induce significant phosphorylation of the MEK1-MEK2/MAPK pathways. In particular, the peptides corresponding to loops L5 (Lys206 to Gly219), L6B (Ser239 to Lys253), and L7 (Thr280 to Lys287) activate, as the whole protein, essentially JNK and p38.     

Antibodies to meningococcal class 1 outer membrane proteins in South African complement-deficient and complement-sufficient subjects.

Inhibition assays were used to investigate human serum antibodies to the meningococcal class 1 outer membrane proteins. We adapted the whole-cell enzyme-linked immunosorbent assay technique to determine the ability of sera to inhibit the binding of murine subtyping monoclonal antibodies. Serum samples from 33 South African subjects with a deficiency in the sixth component of complement as well as serum samples from various groups of complement-sufficient subjects were investigated. Subjects were subdivided according to whether they were (i) convalescent from Neisseria meningitidis infections, (ii) nonconvalescent, or (iii) controls. Preliminary subtyping investigations had shown that P1.2 was present on 36% of meningococcal clinical isolates from Cape Province, South Africa. Assays with the anti-P1.2 antibodies showed the presence of high antibody levels in many deficient sera and moderately elevated levels in some sera from the complement-sufficient convalescent patients. P1.2, P1.4, P1.15, and P1.16 are epitopes situated on loop 4 of the class 1 outer membrane proteins, whereas P1.7 is on loop 1. Inhibition assays showed that human sera that inhibited binding by P1.2 monoclonal antibodies tended to inhibit the other monoclonal antibodies directed to loop 4 epitopes. This suggests that the epitopes recognized by the human antibodies are not exactly the same as the epitopes recognized by the murine monoclonal antibodies and raises the possibility of the importance of other epitopes.     

Antibodies to loop 6 of the P2 porin protein of nontypeable Haemophilus influenzae are bactericidal against multiple strains

The P2 porin protein is the most abundant protein in the outer membrane of nontypeable Haemophilus influenzae (NTHI). Analysis of sequences of P2 from different strains reveals the presence of both heterogeneous and conserved surface-exposed loops of the P2 molecule among strains. The present study was undertaken to test the hypothesis that antibodies to a conserved surface-exposed loop are bactericidal for multiple strains of NTHI and could thus form the basis of vaccines to prevent infection due to NTHI. Polyclonal antiserum to a peptide corresponding to loop 6 was raised and was immunopurified over a loop 6 peptide column. Analysis of the antibodies to whole organisms and peptides corresponding to each of the eight loops of P2 by immunoassays revealed that the antibodies were highly specific for loop 6 of P2. The immunopurified antibodies bound to P2 of 14 of 15 strains in immunoblot assays. These antibodies to loop 6 demonstrated complement-mediated bactericidal killing of 8 of 15 strains. These results support the concept of using conserved regions of the P2 protein as a vaccine antigen.     

Mutations on the FG surface loop of human papillomavirus type 16 major capsid protein affect recognition by both type-specific neutralizing antibodies and cross-reactive antibodies

The aim of this study was to further characterize the conformational neutralizing epitopes present on the surface-exposed FG loop of human papillomavirus (HPV) type 16 L1 major capsid protein. We have generated previously two chimeric L1 proteins by insertion of a foreign peptide encoding an epitope of the hepatitis B core (HBc) antigen within the FG loop. In addition, three other chimeric L1 proteins were obtained by replacing three different FG loop sequences by the HBc motif and three others by point mutations. All these chimeric L1 proteins retained the ability to self-assemble into virus-like particles (VLPs), with the exception of the mutant with substitution of the L1 sequence 274-279 by the HBc motif. The eight chimeric VLPs were then analyzed for differential reactivity with a set of six HPV-16 and HPV-31 monoclonal antibodies that bound to conformational and linear epitopes. The binding patterns of these monoclonal antibodies confirmed that the FG loop contained or contributed to neutralizing conformational epitopes. The results obtained suggested that the H31.F7 antibody, an anti-HPV-31 cross-reacting and neutralizing antibody, recognized a conformational epitope situated before the 266-271 sequence. In addition, H16.E70 neutralizing antibody reactivity was reduced with L1 VLPs with an Asn to Ala point mutation at position 270, suggesting that Asn is a part of the epitope recognized by this antibody. This study contributes to the understanding of the antigenic structure of HPV-16 and -31 L1 proteins by confirming that the FG loop contributes to neutralizing epitopes and suggesting the existence of both type-specific and cross-reactive conformational epitopes within the FG loop.     

Sequence polymorphism, predicted secondary structures, and surface-exposed conformational epitopes of Campylobacter major outer membrane protein

The major outer membrane protein (MOMP), a putative porin and a multifunction surface protein of Campylobacter jejuni, may play an important role in the adaptation of the organism to various host environments. To begin to dissect the biological functions and antigenic features of this protein, the gene (designated cmp) encoding MOMP was identified and characterized from 22 strains of C. jejuni and one strain of C. coli. It was shown that the single-copy cmp locus encoded a protein with characteristics of bacterial outer membrane proteins. Prediction from deduced amino acid sequences suggested that each MOMP subunit consisted of 18 beta-strands connected by short periplasmic turns and long irregular external loops. Alignment of the amino acid sequences of MOMP from different strains indicated that there were seven localized variable regions dispersed among highly conserved sequences. The variable regions were located in the putative external loop structures, while the predicted beta-strands were formed by conserved sequences. The sequence homology of cmp appeared to reflect the phylogenetic proximity of C. jejuni strains, since strains with identical cmp sequences had indistinguishable or closely related macrorestriction fragment patterns. Using recombinant MOMP and antibodies recognizing linear or conformational epitopes of the protein, it was demonstrated that the surface-exposed epitopes of MOMP were predominantly conformational in nature. These findings are instrumental in the design of MOMP-based diagnostic tools and vaccines.     

Two monoclonal antibodies with defined epitopes of P44 major surface proteins neutralize Anaplasma phagocytophilum by distinct mechanisms

Anaplasma phagocytophilum is an obligatory intracellular bacterium that causes human granulocytic anaplasmosis. The polymorphic 44-kDa major outer membrane proteins of A. phagocytophilum are dominant antigens recognized by patients and infected animals. However, the ability of anti-P44 antibody to neutralize the infection has been unclear due to a mixture of P44 proteins with diverse hypervariable region amino acid sequences expressed by a given bacterial population and lack of epitope-defined antibodies. Monoclonal antibodies (MAbs) 5C11 and 3E65 are directed to different domains of P44 proteins, the N-terminal conserved region and P44-18 central hypervariable region, respectively. Passive immunization with either MAb 5C11 or 3E65 partially protects mice from infection with A. phagocytophilum. In the present study, we demonstrated that the two monoclonal antibodies recognize bacterial surface-exposed epitopes of naturally folded P44 proteins and mapped these epitopes to specific peptide sequences. The two MAbs almost completely blocked the infection of the A. phagocytophilum population that predominantly expressed P44-18 in HL-60 cells by distinct mechanisms: MAb 5C11 blocked the binding, but MAb 3E65 did not block binding or internalization. Instead, MAb 3E65 inhibited internalized A. phagocytophilum to develop into microcolonies called morulae. Some plasma from experimentally infected horses and mice reacted with these two epitopes. Taken together, these data indicate the presence of at least two distinct bacterial surface-exposed neutralization epitopes in P44 proteins. The results indicate that antibodies directed to certain epitopes of P44 proteins have a critical role in inhibiting A. phagocytophilum infection of host cells.     

Identification of surface-exposed B-cell epitopes on high molecular-weight adhesion proteins of nontypeable Haemophilus influenzae.

We previously reported that two surface-exposed high-molecular-weight proteins, HMW1 and HMW2, expressed by a prototypic strain of nontypeable Haemophilus influenzae (NTHI) mediate attachment to human epithelial cells. These proteins are members of a family of highly immunogenic proteins common to most nontypeable Haemophilus strains. We also reported that immunization with an HMW1-HMW2 mixture modified the course of disease in an animal model of otitis media, suggesting the potential usefulness of these proteins as NTHI vaccine components. Identification of surface-accessible B-cell epitopes could be important to efforts to develop recombinant or synthetic peptide vaccines based upon these high-molecular-weight proteins. Thus, the purpose of the present study was to identify surface-accessible epitopes on the HMW1 and HMW2 proteins by using monoclonal antibodies (MAbs) and to determine the prevalence of these epitopes among the high-molecular-weight proteins expressed by heterologous nontypeable Haemophilus strains. MAbs were generated by immunizing mice with high-molecular-weight proteins purified from prototype strains and were screened by immunoelectron microscopy (IEM) for the ability to recognize surface epitopes. Two MAbs, designated AD6 and 10C5, that recognized surface epitopes by IEM were recovered. In order to map the epitopes recognized by these two MAbs, we constructed a set of HMW1 and HMW2 recombinant fusion proteins using the pGEMEX vectors and examined the reactivity of the MAbs with these fusion proteins. MAb AD6 recognized an epitope in both HMW1 and HMW2 which mapped to the last 75 amino acids at the carboxy termini of the two proteins. When examined for reactivity with heterologous strains, MAb AD6 recognized high-molecular-weight proteins in 75% of 125 unrelated nontypeable Haemophilus strains and, in addition, reacted with three of three such strains when examined by IEM. MAb 10C5 recognized an epitope that mapped to a 155-amino-acid segment near the carboxy terminus of HMW1. This epitope was adjacent to but distinct from the AD6 epitope and was absent from HMW2. The 10C5 epitope was expressed by 40% of the AD6 reactive strains. Identification of shared surface-exposed epitopes on the high-molecular-weight adhesion proteins suggests the possibility of developing recombinant or synthetic peptide-based vaccines protective against disease caused by the majority of NTHI strains.     

Outer membrane proteins as surface display systems

Outer membrane proteins (OMPs) of gram-negative bacteria can be used as carrier proteins to present foreign peptide epitopes on the bacterial cell surface. They all have a common structural motif of a beta-barrel that is composed of a variable number of transmembrane beta-strands connected at the periplasmic side with short turns and at the outside with long surface-accessible loops. Outer membrane proteins occur as monomers like OmpA, or assemble into trimers like the porins. Foreign gene products have been fused to surface-accessible regions of several outer membrane proteins including the porins OmpC, PhoE and LamB, lipoproteins as well as the OmpA protein. Short epitopes that are inserted into outer membrane proteins induce epitope-specific antibody responses, and are thus appealing candidates for live recombinant vaccines. Also large insertions, of more than 100 amino acids, are in some cases tolerated and do not affect the overall conformation of the carrier protein. The possible applications for outer membrane display include recombinant vaccines, peptide library screening, development of biocatalysts or whole-cell adsorbents, and adhesin-receptor interaction studies. It is expected that in the near future, development of new display systems will still increase the utilization of this emerging exciting technology.     

The Amino Acid Sequence of Neisseria lactamica PorB Surface-Exposed Loops Influences Toll-Like Receptor 2-Dependent Cell Activation

Toll-like receptors (TLRs) play a major role in host mucosal and systemic defense mechanisms by recognizing a diverse array of conserved pathogen-associated molecular patterns (PAMPs). TLR2, with TLR1 and TLR6, recognizes structurally diverse bacterial products such as lipidated factors (lipoproteins and peptidoglycans) and nonlipidated proteins, i.e., bacterial porins. PorB is a pan-neisserial porin expressed regardless of organisms' pathogenicity. However, commensal Neisseria lactamica organisms and purified N. lactamica PorB (published elsewhere as Nlac PorB) induce TLR2-dependent proinflammatory responses of lower magnitude than N. meningitidis organisms and N. meningitidis PorB (published elsewhere as Nme PorB). Both PorB types bind to TLR2 in vitro but with different apparent specificities. The structural and molecular details of PorB-TLR2 interaction are only beginning to be unraveled and may be due to electrostatic attraction. PorB molecules have significant strain-specific sequence variability within surface-exposed regions (loops) putatively involved in TLR2 interaction. By constructing chimeric recombinant PorB loop mutants in which surface-exposed loop residues have been switched between N. lactamica PorB and N. meningitidis PorB, we identified residues in loop 5 and loop 7 that influence TLR2-dependent cell activation using HEK cells and BEAS-2B cells. These loops are not uniquely responsible for PorB interaction with TLR2, but NF-κB and MAP kinases signaling downstream of TLR2 recognition are likely influenced by a hypothetical "TLR2-binding signature" within the sequence of PorB surface-exposed loops. Consistent with the effect of purified PorB in vitro, a chimeric N. meningitidis strain expressing N. lactamica PorB induces lower levels of interleukin 8 (IL-8) secretion than wild-type N. meningitidis, suggesting a role for PorB in induction of host cell activation by whole bacteria.     

Mapping and topographic localization of epitopes of the Yersinia pseudotuberculosis invasin protein.

The Yersinia pseudotuberculosis invasin protein is a 986-amino-acid outer membrane protein that promotes bacterial penetration into mammalian cells by binding to beta 1-chain integrin receptors. We previously showed that the integrin binding domain is encoded by the carboxyl-terminal 192 amino acids. To further investigate the structure of this protein, we characterized a set of 32 monoclonal antibodies (MAbs) directed against invasin. Invasin deletion derivatives and fusion proteins carrying different segments of invasin were used to map the epitopes of this set of MAbs into 10 overlapping but distinct intervals. Indirect immunofluorescence of intact bacteria expressing invasin demonstrated that two large regions of invasin contain epitopes exposed on the bacterial surface. To assess the role of these surface-exposed regions in the binding and invasion of mammalian cells, each of the MAbs was tested for its ability to inhibit these processes. All of the MAbs that recognized bacterial surface-exposed epitopes in the cell binding domain of invasin inhibited both cell attachment and cell penetration, and no other MAbs inhibited either activity.     

Immunogenic B-cell epitopes of Babesia bovis rhoptry-associated protein 1 are distinct from sequences conserved between species.

Babesia bovis merozoite apical membrane polypeptide Bv60 was found to be rhoptry associated by immuno-electron microscopy and was redesignated rhoptry-associated protein 1 (RAP-1). The N-terminal 300 amino acids of RAP-1 have a high level of sequence similarity to the same N-terminal region of p58, its homolog from Babesia bigemina. However, the interspecies conserved sequences did not include RAP-1 surface-exposed B-cell epitopes as defined by monoclonal antibodies. Furthermore, neither heterologous B. bigemina immune nor monospecific anti-p58 bovine serum binds to whole RAP-1, indicating that the major B-cell epitopes recognized by these sera are also not encoded by the conserved sequences. Truncated RAP-1, expressed by a subclone encoding the N-terminal 235 amino acids, is weakly bound by antibodies in heterologous sera. A peptide representing the longest conserved amino acid sequence (amino acids 121 to 134) in this region is also weakly bound by antibodies in immune bovine sera, and rabbit antibodies induced by and strongly reactive with the peptide alone fail to bind native or denatured RAP-1. Thus, although the conserved region may contain one or more poorly immunogenic B-cell epitopes, these epitopes are inaccessible to antibody in whole RAP-1. The results indicate that the major immunogenic B-cell epitopes of RAP-1, including surface-accessible epitopes bound by monoclonal antibodies, are distinct from the conserved sequences representing putative functional domains.     

Immunoprotection by monoclonal antibodies to the porins and lipopolysaccharide ofSalmonella typhimurium

Monoclonal antibodies (MAbs) were raised against the outer membrane (OM) antigens ofSalmonella typhimurium.Enzyme-linked immunosorbent assays and Western immunoblots indicated that 10 MAbs in the panel were specific for surface epitopes, and 10 recognized buried epitopes of OmpC or OmpD porins; three MAbs reacted with smooth lipopolysaccharide (LPS), two bound rough LPS, and the remaining three MAbs apparently reacted with a porin–LPS complex. We screened these MAbs and immune polyclonal sera in CAF₁(Ity^r) mice for their relative immunoprotective potential against a challenge with 10 to 500 LD₅₀of the virulentS. typhimuriumLT-2 strain WB600, or against two LD₅₀of purified OM from this organism. Polyclonal sera that contained high titers of antibodies to porin monomers and trimers, and LPS, provided significant protection (33 to 100% survivors). Antiporin MAbs, when administered individually, did not protect or prolong the survival of mice. A mixture of MAbs with specificity for the surface, but not buried epitopes of porins, prolonged the survival of mice against endotoxemia, but none provided significant protection against mouse typhoid. MAbs specific for smooth (but not rough) LPS on the other hand, conferred significant protection against endotoxemia and mouse typhoid. Finally, MAbs that presumably recognized epitopes present in porin–LPS complexes, were also protective against endotoxemia and mouse typhoid. These results support the role of antibodies to LPS O-chains, porin–LPS complexes, and to a lesser degree, native porins in acquired resistance to infection byS. typhimurium.     

Cross-reactivity of surface-exposed epitopes of outer membrane antigens of Haemophilus influenzae type b.

The cross-reactivity of exposed surface epitopes of outer membrane proteins from a spectrum of Haemophilus influenzae type b isolates that varied in their evolutionary distance from each other and in their outer membrane protein composition was analyzed by using an immunoblot assay. The results for outer membrane proteins a, n, and b/c were as follows. (i) A total of 13 of 14 strains possessing a protein a with similar mobilities on gels (i.e., the same apparent molecular weight) as protein a of strain Eag absorbed antibodies to protein a of strain Eag. These strains represented a broad spectrum on a scale of evolutionary distance. (ii) In contrast, only one of seven strains possessing a protein a with different mobilities absorbed these antibodies. (iii) Of five isolates close to strain Eag on the evolutionary scale, the four with a protein n with the same mobility as protein n of strain Eag absorbed antibodies to protein n of strain Eag. (iv) In contrast, of five isolates distant from strain Eag on the evolutionary scale, none absorbed antibodies to protein n, including one strain that had a protein n of the same mobility as that of strain Eag. (v) All strains that absorbed antibodies to protein b/c also absorbed antibodies to lipopolysaccharide, and the reverse of this was also true. Evolutionary distance and mobility of protein b/c on gels were not factors. Control experiments indicated that this result was an artifact due to the strong association of lipopolysaccharide with protein b/c on the gel and subsequent blot. The important conclusions from these experiments, especially pertinent for consideration of these proteins in either whole or peptide vaccines, are that proteins with apparently identical molecular weights can possess different surface-exposed epitopes, that proteins with different molecular weights can possess cross-reactive surface-exposed epitopes, and that some surface-exposed epitopes have been conserved even though the bacterium has undergone evolutionary divergence. In addition, experiments were also performed to determine whether H. influenzae type b strains maintained their integrity during the absorption step, i.e., incubation in antiserum. Strain Eag, which was used as a prototype type b strain, released a small proportion of its membrane (0.13%), but this did not result in exposure of epitopes that were usually buried. In contrast, strain S2, an unencapsulated mutant of strain Eag, was quite unstable, releasing three times as much membrane and a large proportion of its periplasmic proteins.     

Identification of surface-exposed B-cell epitopes recognized by Haemophilus influenzae type b P1-specific monoclonal antibodies.

A panel of P1 synthetic peptides was synthesized to map the surface-exposed epitopes of Haemophilus influenzae type b outer membrane protein P1 recognized by three murine monoclonal antibodies (MAbs 7C8, 3E12, and 6B1). By using peptide-specific enzyme-linked immunosorbent assays, MAbs 6B1, 7C8, and 3E12 were shown to recognize distinct epitopes localized within residues 60 to 88, 165 to 193, and 400 to 437 of mature P1, respectively. Since MAb 7C8 was shown previously to be protective against certain H. influenzae type b subtypes in the infant rat model of bacteremia, its cognate epitope was further characterized by using truncated peptide analogs. Fine mapping of the 7C8 epitope by competitive inhibition studies revealed that it was localized within residues 184 and 193.     

Porin flexibility in Providencia stuartii: cell-surface-exposed loops L5 and L7 are markers of Providencia porin OmpPst1

Epidemiologically unrelated Providencia stuartii strains isolated in hospitals in the south of France were investigated for their porin sequences and profiles. Noticeable resistance to β-lactams was found to be associated with production of extended spectrum β-lactamases or AmpC overproduction, but not metallo-β-lactamases. At the same time, the expression level of outer membrane porins was unmodified in these isolates. The identity of the amino acid sequences of the major porin OmpPst1 was less than 90% in the tested clinical strains, whereas sequences of the second major porin OmpPst2 were found to be identical in all isolates. Sequence diversity identified in the OmpPst1 porins was mainly located in two cell-surface-exposed loops (L5 and L7): these loops were found to be responsible for 80% of the main movements of the protein. Parallel tempering MD simulations indicated possible coordinated movement of these loops that might affect the electrostatic interaction of the porin with membrane components (e.g. LPS) or with external molecules/surfaces. This suggests that such flexibility of surface-exposed domains of OmpPst1 may participate in bacterial adaptation to the environment.