Identification of a surface-exposed immunodominant epitope on outer membrane protein P1 of Haemophilus influenzae type b.

Eight murine monoclonal antibodies (MAbs) directed against outer membrane protein P1 of Haemophilus influenzae type b were generated and characterized. Seven of the eight MAbs reacted with recombinant P1 and purified P1 protein from H. influenzae type b strains MinnA and 1613; MAb P1.8 was specific for the latter strain. A panel of 32 nontypeable and 140 encapsulated Haemophilus strains recovered worldwide representing the major clonal families of serotypes a, b, and d was used to evaluate the distribution among Haemophilus strains of the epitopes identified by the P1-specific MAbs. The epitope reactive with the seven MAbs which recognized P1 from strains MinnA and 1613 was shared by 92% of the encapsulated Haemophilus isolates tested. The epitope is present in the H. influenzae type b strains from clonal families commonly recovered from cases of invasive disease in North America and Europe. A series of nested 5' and 3' deletions of the P1 gene were constructed and analyzed to localize the determinants on P1 recognized by the MAbs. MAbs P1.2, P1.4, P1.5, P1.6, and P1.7 recognized an epitope localized to the carboxy-terminal portion of P1. Murine MAbs P1.1 and P1.3 and two human MAbs, HiH-7 and HiH-10, recognized a complex epitope which was partially localized to the carboxy-terminal portion of the P1 protein. These data indicate that an immunodominant surface-exposed epitope is present on the carboxy-terminal portion of the P1 protein of type b Haemophilus isolates responsible for the majority of invasive disease in North America.     

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.     

Probing the idiotype/anti-idiotype antibody interaction with a set of synthetic peptide homologues.

Anti-idiotypic (anti-Id) antibodies were raised against two murine monoclonal antibodies (mAb 1/1 and mAb 2/1) which recognise two distinct and well-characterised epitopes on a 24-residue synthetic peptide representing part of the haemagglutinin (HA) of influenza virus. A monoclonal anti-Id antibody, specific for mAb 2/1, could bind to mAb 2/1 when the paratope of the latter was occupied with peptide, indicating that this anti-Id antibody is directed to a framework idiotope. In contrast, an anti-Id mAb derived from mAb 1/1-immunised mice was inhibited in its binding to Id by the parent peptide and also by the heptapeptide NVPEKQT which constitutes the epitope recognised by mAb 1/1. The small size of this synthetic peptide eliminates the possibility of significant steric inhibition in the system, and establishes that this mAb is a true paratope-directed anti-Id antibody. The interaction of this anti-Id mAb with the paratope of mAb 1/1 in the presence of a set of peptide homologues of the epitope was also examined. A peptide as short as 5 residues, which contains two of the three irreplaceable residues of the epitope, could inhibit binding between the two mAbs.     

Interstrain cross-reactive idiotypes on monoclonal antibodies to an encephalitogenic myelin basic protein peptide.

In order to assess the role of idiotype (Id) and the anti-Id network in murine experimental autoimmune encephalomyelitis (EAE), Id-bearing monoclonal antibodies (mAb) to human myelin basic protein (MBP) peptide acetyl 1-9, as well as mAb anti-Id, were developed in EAE-susceptible PL/J mice (H-2u). These mice recognize MBP residues acetyl 1-9 as an encephalitogenic determinant. Reactivities of PL/J Id-bearing mAbs to MBP and to MBP peptides were identical to those of mAbs generated against the same MBP peptide in EAE-resistant BALB/c mice (H-2d), even though isotypes of the mAbs differed. By using an inhibitory ELISA and immunoblotting, it was demonstrated that one PL/J mAb anti-Id recognized a public or framework Id, whereas another PL/J mAb-anti Id was directed to a private Id more restricted to the paratopic site. Two Id-bearing PL/J mAbs shared a cross-reactive Id (IdX) on the light chain, and an interstrain IdX was present on both the heavy and light chains of mAbs raised in PL/J and BALB/c mice to the same MBP peptide. The PL/J mAb anti-Id was capable of cross-regulating the production of Id-bearing mAbs by hybridomas across murine strains. These findings suggest that a restrictive family of germ-line genes encode for these Id-bearing antibodies to MBP peptide, irrespective of whether the MBP peptide is encephalitogenic in the murine strain immunized. Manipulation of the Id network may provide a means for modifying autoimmune demyelinating diseases of the central nervous system.     

Specificity of Monoclonal Anti-nucleosome Auto-antibodies Derived from Lupus Mice

Recently, anti-nucleosome antibodies, which do not bind to DNA or to individual histones, have been identified in longitudinal studies in lupus mice. These anti-nucleosome antibodies occur early in spontaneous SLE and are formed prior to other anti-nuclear specificities. However, nucleosomal epitopes are yet to be fully characterized. We selected a panel of six monoclonal anti-nucleosome antibodies (mAbs) (#2, #32, #34, PL2-6, LG8-1 and LG10-1) derived from lupus mice. These mAbs were tested in ELISA on subnucleosome structures and on a panel of 53 histone peptides, covering the entire sequence of the five histones. Two mAbs reacted with one of these peptides, but the reactivity hardly exceeded the background reactivity. Based on the nucleosome and subnucleosome ELISA we identified different recognition patterns. Three mAbs showed the highest reactivity towards the intact nucleosome. For two of them (#32 and LG8-1) the nucleosomal epitope was primarily located on H2A-H2B/DNA, whereas for mAb #34 this primary epitope was located on H3/H4/DNA. Two mAbs (#2 and PL2-6) showed the highest reactivity with H2A-H2B/DNA and one mAb (LG10-1) recognized H3-H4/DNA. In the subnucleosome ELISA all but one (mAb #32) recognized more than one epitope, including DNA complexed to a variety of cationic molecules. Comparing these reactivities we identified for all mAbs one specific nucleosomal epitope, whereas reactivity with other subnucleosomes was comparable to the reactivity towards DNA complexed with cationic molecules. In inhibition experiments both in ELISA and in immunofluorescence it was found that only one of the mAbs (i.e. PL2-6), recognizing an epitope on H2A-H2B/DNA as primary epitope, could be inhibited by H2A-H2B/DNA in fluid phase. The two mAbs recognizing an epitope on H3-H4/DNA as primary epitope could be inhibited by H3-H4/DNA in fluid phase. From these analyses, we conclude first that for these nucleosome specific mAbs linear histone peptides are not very important. Second, that these mAbs all recognize different epitopes on both H2A/H2B-DNA and H3/H4-DNA and third that some solid phase H2A/H2B-DNA epitopes are not expressed on fluid phase H2A/H2B-DNA. Our findings suggest that in SLE the nucleosome can act as auto-antigen and that there is no immunodominant β cell epitope within the nucleosome.     

Quantification of lupus anti-ribosome P antibodies using a recombinant P2 fusion protein and determination of the predicted amino acid sequence of the autoantigen in patients'' mononuclear cells.

The cDNA encoding the ribosomal protein P2 antigen was cloned from a human cDNA library constructed in the lambda gt11 expression vector. A beta-galactosidase-P2 fusion protein was purified to near homogeneity and used to develop an ELISA which was highly specific for anti-P antibodies produced in murine and human SLE. The median concentration of human IgG anti-P antibodies in serum was estimated to be 100 micrograms/ml (range 6-450 micrograms/ml). Pre-incubation of human anti-P sera with a synthetic peptide, corresponding to the C-terminal 22 amino acids of P2, completely inhibited reactivity with the fusion protein in the ELISA. These findings confirm that lupus anti-P sera show a striking restriction in epitope specificity and indicate that the P2 fusion protein is a useful alternative to the synthetic peptide antigen for detection and quantification of anti-P antibodies. To investigate the possibility that anti-P antibodies were induced by 'altered-self', cDNA encoding P2 were also cloned from lupus patients and control mononuclear cells. The predicted amino acid sequences of the patients' P2 were identical to that of the normal controls indicating that a primary structural abnormality of the P2 autoantigen was unlikely.     

Production and characterization of a human monoclonal anti-idiotype to anti-ribosomal P antibodies

Autoantibodies to ribosomal P protein (anti-P) are a specific hallmark of systemic lupus erythematous (SLE). Several authors found significant associations of anti-P antibodies with neuropsychiatric, hepatic, and renal disease. We now report the isolation by phage display of human anti-idiotype (Id) monoclonal antibody fragments as single-chain Fv fragment (scFv) against anti-P antibodies. The V gene repertoires were derived from the RNA obtained from the B cells of a SLE patient. Affinity-purified anti-P antibodies were used for the selection of bacterial clones producing anti-P-specific scFv antibody fragments and little reactivity with normal IgG and other IgG antibodies. The anti-Id antibody recognizes a public idiotope broadly cross-reactive with polyclonal anti-P antibodies and inhibited binding of anti-P to ribosomal P antigen in immunoassays and on Jurkat cells. The anti-Id scFv antibody fragment may have therapeutic implications in SLE. They may also be used as probes in the study of the structure of the idiotype.     

Structural and antigenic conservation of the P2 porin protein among strains of Haemophilus influenzae type b.

The P2 porin protein is the most abundant protein in the outer membrane of Haemophilus influenzae type b (Hib). Biochemical and immunochemical techniques were used to characterize the P2 proteins from a number of different Hib strains. P2 proteins from Hib outer membrane vesicles were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose for in situ tryptic digestion. Solid-phase tryptic digests of P2 from eight Hib strains were resolved by high-pressure liquid chromatography and shown to be similar if not identical. Radioimmunoprecipitation analysis involving Hib cells (containing intrinsically radiolabeled proteins or lipooligosaccharide) and Western blot (immunoblot) analysis were used to identify two P2-specific murine monoclonal antibodies (MAbs). These MAbs were shown to be reactive with 120 Hib strains tested in a colony blot radioimmunoassay. One of these MAbs bound to a surface-exposed P2 epitope that was antibody accessible on all Hib strains tested; the other MAb was directed against a P2 epitope that either was not exposed on the cell surface or was otherwise inaccessible to antibody.     

Generation of monoclonal antibodies of desired specificity using chimeric polyomavirus-derived virus-like particles

Foreign protein sequences presented on hamster polyomavirus (HaPyV) major capsid protein VP1-derived virus-like particles (VLPs) have been demonstrated to be highly immunogenic. The current study was aimed to evaluate VP1-derived chimeric VLPs as tools for hybridoma technology to generate monoclonal antibodies (mAbs) of desired specificity. Chimeric VLPs containing inserts of different size and origin were used as immunogens. Chimeric VLPs carrying a 9 amino acid (aa)-long cytotoxic T-cell epitope (STAPPVHNV) of human mucin 1 (MUC1) elicited a strong epitope-specific humoral immune response in mice and promoted the production of MUC1-specific mAbs. From a total of seven mAbs of IgG isotype generated against the chimeric VLPs, two mAbs were directed against the MUC1 epitope and five mAbs against the VP1-carrier. Two out of five anti-VP1 mAbs recognized epitopes located at the previously defined insertion site #2 (aa 223/224), which confirms its surface-exposed localization. Chimeric VLPs carrying a 120-aa long sequence of Puumala hantavirus (PUUV) nucleocapsid protein (NP) promoted the generation of five mAbs of IgG isotype specific to PUUV NP. All mAbs recognized the full-length NP of different PUUV strains. In contrast, no VP1-specific mAbs were obtained. The ability of chimeric VLPs to activate antigen-presenting cells was evaluated by studying the uptake of chimeric VLPs by murine spleen cell-derived dendritic cells (DCs). Efficient uptake of VLPs and activation of murine DCs were demonstrated, which may represent the basis of the strong immunogenicity of chimeric VLPs. In conclusion, chimeric VLPs effectively stimulated the production of IgG antibodies specific for foreign epitopes presented at surface-exposed regions. Thus, chimeric HaPyV VP1-derived VLPs represent efficient immunogens for hybridoma technology and provide a promising alternative to chemical coupling of synthetic peptides to carrier proteins.     

Outer membrane protein binding sites of complement component 3 during opsonization of Haemophilus influenzae.

Complement component 3 (C3) binding to Haemophilus influenzae type b (Hib) is an important step in host defense against invasive disease, but the details of this process remain poorly understood. We have shown that the P1 and P2 outer membrane proteins (OMPs) serve as binding sites for C3 on serum-opsonized Hib. Whole-cell lysates of opsonized Hib were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved proteins were transferred to nitrocellulose. Immunoblot analysis with monoclonal antibodies (MAbs) to the 49-kDa P1 and 39-kDa P2 OMPs demonstrated high-molecular-weight bands that were not present when the bacteria were opsonized with heat-inactivated or methylamine-treated serum. Immunoblot analysis with MAbs to the 98- or 16-kDa (P6) OMPs did not reveal additional bands. An unencapsulated Hib mutant still lacked C3 bound to the 98-kDa or P6 OMP, indicating that the absence of C3 binding to these proteins was not the result of epitope masking by the capsule. Studies with MAbs to C3 fragments confirmed that the anti-P1- and anti-P2-reactive bands were C3 fragments bound to these OMPs. The molecular weights of proteins reactive to anti-OMP and anti-C3 antibodies indicated that multiple C3 fragments may be bound to P1 or that C3 may be bound to P2 multimers. Finally, the presence of other anti-C3-reactive proteins indicated that several other proteins serve as C3 targets during the opsonization of Hib.     

Comparative analysis of the structures of the outer membrane protein P1 genes from major clones of Haemophilus influenzae type b.

P1 outer membrane proteins from Haemophilus influenzae type b are heterogeneous antigenically and with respect to apparent molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For determination of the molecular basis for the differences in the P1 proteins, the genes for the P1 proteins from strain 1613, representative of outer membrane protein subtype 3L, and strain 8358, representative of outer membrane protein subtype 6U, were cloned, sequenced, and compared with the previously reported gene for the P1 protein from strain MinnA, a strain with the outer membrane protein subtype 1H. These prototype strains are representatives of the three major clonal families of H. influenzae type b responsible for invasive disease in diverse areas of the world. The nucleotide sequences of the P1 genes from strains 1613 and 8358 were 94 and 90% identical to the MinnA sequence, respectively. The derived amino acid sequences were 91 and 86% identical, respectively. Heterogeneity between the MinnA and 1613 proteins was largely localized to two short variable regions; the protein from strain 8538 contained a third variable region not observed in the other P1 proteins. Thus, the outer membrane protein P1 genes are highly conserved; the variable regions may code for the previously demonstrated strain-specific antigenic determinants.     

Antigenic characterization and analysis of the human immune response to outer membrane protein E of Branhamella catarrhalis.

Outer membrane protein E (OMP E) is a 50-kDa major OMP of Branhamella catarrhalis. Polyclonal antisera and four monoclonal antibodies (MAbs) to OMP E were generated to study its antigenic structure. All antibodies recognized epitopes in all 19 B. catarrhalis strains tested by immunoblot assays. By flow cytometry, it was determined that MAbs 1B3 and 9G10d recognized epitopes which are expressed on the surface of the intact bacterium, while MAbs IC11 and 7C10 recognized epitopes which were buried within the outer membrane. A competitive enzyme-linked immunosorbent assay showed that MAbs 1B3 and 9G10d recognize the same or closely related epitopes. Proteinase K treatment of whole bacterial cells revealed that MAbs 1B3 and 9G10d recognize a surface-exposed epitope located in the 17-kDa region towards the amino terminus of OMP E. The human serum and mucosal antibody responses to OMP E in adults with chronic bronchitis were studied. A majority of these patients had immunoglobulin A to OMP E in sputum supernatants. None of ten adults who experienced lower respiratory tract infections due to B. catarrhalis demonstrated a clear-cut rise in antibody titer to OMP E in serum or sputum supernatant. This study has demonstrated that OMP E has at least one surface-exposed epitope which is highly conserved among strains of B. catarrhalis and which is located in the amino-terminal 184 amino acids of the molecule.     

Human and murine B-cells recognize the HBeAg/beta (or HBe2) epitope as a linear determinant.

The complete amino acid (aa) sequence of the hepatitis B virus (HBV) core protein (HBcAg), deduced from the genome of the HBV ayw subtype, was synthesized as decapeptides with five overlapping aas. The peptides were tested for reactivity with monoclonal antibodies (mAbs) to the beta (or HBe2) epitope of hepatitis B e antigen (HBe/b mAbs; 57/8, 78/3, 141/158 and 141/207). Cross-competition between the mAbs with a mAb to the HBe/alpha epitope (or HBe1) and an anti-HBc mAb showed that all the HBe/b mAbs specifically inhibited human anti-HBe/b binding. Screening the HBc/e peptides showed that all anti-HBe/b mAbs recognized a peptide covering the residues 126-135. Three of the mAbs, 78/3, 141/152 and 141/207, had a less restricted reactivity than the other two, suggesting the recognition of the HBe/b as a discontinuous determinant. Fine mapping of the region aa 126-135 was performed by synthesizing decapeptides with nine overlapping aas, covering residues aa 121-140. All mAbs, except 78/3, reacted with the linear sequence TPPAYR, at residues 128-133. An additional set of peptides was synthesized, where the six aas within the epitope 128-133 were substituted in turn by the other 19 possible aas. By this approach, the essential aas for mAb 57/8 were found to be the sequence of PPA at residues 129-131, and for mAb 141/158 the sequence PP-Y, at residues 129, 130 and 132, respectively. Human recognition of the linear HBe/b epitope was investigated by using a peptide covering residues 121-140 (p 33). Thirty-one sera from chronic carriers of HBsAg, of which seven were positive for HBeAg and the remaining 24 for anti-HBe, were investigated. Of the sera with HBeAg, two had low levels of anti/-HBe/b in the p 33 assay. Out of the sera with anti-HBe, eight were positive in the p 33 EIA. Thus, murine monoclonals and human sera may recognize the HBe/b epitope as a linear determinant residing around aa 130.     

Characterization of a heat-modifiable outer membrane protein of Haemophilus somnus.

In immunoblot analysis, a murine monoclonal antibody (MAb), 27-1, which was produced to an outer membrane protein (OMP) of Haemophilus somnus, showed that a major OMP is heat modifiable, having a molecular mass of 28 kDa when the N-lauroylsarcosine-insoluble OMP preparation was solubilized at 60 degrees C and a mass of 37 kDa when the OMP preparation was solubilized at 100 degrees C. The heat-modifiable OMP reacted intensely with convalescent sera obtained from calves with experimental H. somnus pneumonia in immunoblot analysis. Immunoelectron microscopic and antibody absorption studies revealed that the MAb 27-1 epitope was not surface exposed on the intact bacterium. However, a decrease in antibody reactivity to the heat-modifiable OMP in immunoblot analysis after absorption of convalescent serum with intact bacterial cells of H. somnus suggests that a surface-exposed portion of the heat-modifiable OMP is expressed on the intact bacterium. MAb 27-1 reacted with 45 of 45 strains of H. somnus tested in immunoblot analysis. The apparent molecular mass of the antigen varied among strains, and five reactivity patterns demonstrated by MAb 27-1 were observed. MAb 27-1 also reacted with six species in the family Pasteurellaceae, Escherichia coli, and Salmonella dublin, but not with the other eight species of gram-negative bacteria. The heat-modifiable OMP of H. somnus showed immunological cross-reactivity with the OmpA protein of E. coli K-12 and significant N-terminal amino acid sequence homology with the OmpA proteins of gram-negative bacteria. We conclude that a major, 37-kDa heat-modifiable OMP of H. somnus, which elicits an antibody response in H. somnus-infected animals, is a common antigen among H. somnus strains tested and is structurally related to the OmpA protein of E. coli.     

Identification and characterization of a protective immunodominant B cell epitope of pertactin (P.69) from Bordetella pertussis.

Epitopes defined by monoclonal antibodies (mAb) specific for the Bordetella pertussis outer membrane protein P.69 (pertactin) were mapped using a series of amino- and carboxy-terminal deletion mutants expressed in Escherichia coli. mAb were found to bind predominantly to a region of pertactin spanning a (Pro-Gln-Pro)5 repeat motif and one mAb was found to bind to another region spanning a (Gly-Gly-Xaa-Xaa-Pro)5 repeat motif. To localize further the mAb-binding sites, a panel of synthetic peptides, a series of 94 overlapping hexameric peptides, and a P.69 30-amino acid fusion to a hepatitis B core protein (HBcAg-69), were synthesized. This combined approach has identified the binding site for the mAb BBO5: Pro-Gly-Pro-Gln-Pro-Pro; mAb BBO7, E4A8 and E4D7: Ala-Pro-Gln-Pro-Pro-Ala-Gly-Arg; and mAb BPE3: Thr-Leu-Trp-Tyr-Ala-Glu-Ser-Asn-Ala-Leu-Ser-Lys-Arg. We have used a non-lethal murine respiratory model of B. pertussis infection to investigate the ability of a peptide containing the epitope of the mAb BBO5 to elicit protective immunity. Immunization of mice with the HBcAg-69 protein prevented growth of B. pertussis in the lungs compared to mice receiving HBcAg alone, and protection correlated with high titers of anti-P.69 antibodies.     

Comparison of Properties of Murine Monoclonal Anti-idiotypic Antibodies Generated with Idiotype-Bearing Monoclonal Antibodies to Myelin Basic Protein Peptides or Their Complementary Peptides

The present study was undertaken to compare the features of monoclonal antibody (mAb) anti-idiotope (Id) induced by complementary peptides, synthesized on the basis of inverted hydropathy for a myelin basic protein (MBP) peptide, or by conventional methodology using Id-bearing antibodies to the same MBP peptide as immunogen. The six reagents studied consisted of mAbs reactive with MBP peptide acetyl 1-9 and MBP peptide 80-89 and anti-Id reagents against these two mAbs prepared by either the complementary peptide or the conventional approach. ELISA, immunoblotting, immunoinhibition of hybridoma cell production of Idbearing mAb to MBP, and FACS indicated that the anti-Ids generated by either technique were similar although existing in a range reflecting biologic phenomena. mAbs anti-Id prepared by either method continued to show an IgM isotype preference, possibly related to technical considerations, and continued to recognize a cross-reactive Id on the κ light chain of the mAbs to MBP peptides acetyl 1-9 and 80-89. There was no indication that the anti-Ids prepared by the complementary peptide approach were restrictive or selective in a manner different from those made by the conventional approach.     

Major Trypanosoma cruzi antigenic determinant in Chagas'' heart disease shares homology with the systemic lupus erythematosus ribosomal P protein epitope.

A Trypanosoma cruzi lambda gt11 cDNA clone, JL5, expressed a recombinant protein which was found to react predominantly with chronic Chagas' heart disease sera. The cloned 35-residue-long peptide was identified as the carboxyl-terminal portion of a T. cruzi ribosomal P protein. The JL5 13 carboxyl-terminal residues shared a high degree of homology with the systemic lupus erythematosus (SLE) ribosomal P protein epitope. Synthetic peptides comprising the 13 (R-13), 10 (R-10), and 7 (R-7) carboxyl-terminal residues of the JL5 protein were used to study, by enzyme-linked immunosorbent assay, the specificity of the Chagas' disease anti-JL5 and SLE anti-P antibodies. The R-13 peptide defined a linear antigenic determinant of the JL5 recombinant protein. As was proved for JL5, R-13 defined antibody specificities which were significantly increased in chronic Chagas' heart disease patients. Only SLE anti-P positive sera were found to react with JL5 and R-13. Fine epitope mapping showed that Chagas' disease anti-JL5 and SLE anti-P antibodies define similar epitopes within the R-13 peptide. The binding of the SLE sera to JL5 was completely blocked by the R-13 peptide, indicating that the shared specificity between anti-JL5 and anti-P autoantibodies was exclusively limited to the conserved linear epitope(s) within the R-13 peptide. The prevalence of high anti-R-13 antibody titers in Chagas' heart disease patients supports the hypothesis that postulates the existence of autoimmune disorders in Chagas' heart disease.     

Antigenic structure of outer membrane protein E of Moraxella catarrhalis and construction and characterization of mutants

Outer membrane protein E (OMP E) is a 50-kDa protein of Moraxella catarrhalis which possesses several characteristics indicating that the protein will be an effective vaccine antigen. To study the antigenic structure of OMP E, eight monoclonal antibodies were developed and characterized. Three of the antibodies recognized epitopes which are present on the bacterial surface. Fusion peptides corresponding to overlapping regions of OMP E were constructed, and immunoblot assays were performed to localize the areas of the molecule bound by the monoclonal antibodies. These studies identified a surface-exposed epitope in the region of amino acids 80 through 180. To further study the protein, two mutants which lack OMP E were constructed. In bactericidal assays, the mutants were more readily killed by normal human serum compared to the isogenic parent strains. These results indicate that OMP E is involved in the expression of serum resistance of M. catarrhalis.     

Multiple alignment and sorting of peptides derived from phage-displayed random peptide libraries with polyclonal sera allows discrimination of relevant phagotopes.

Biopanning of phage-displayed random peptide libraries is a powerful technique for identifying peptides that mimic epitopes (mimotopes) for monoclonal antibodies (mAbs). However, peptides derived using polyclonal antisera may represent epitopes for a diverse range of antibodies. Hence following screening of phage libraries with polyclonal antisera, including autoimmune disease sera, a procedure is required to distinguish relevant from irrelevant phagotopes. We therefore applied the multiple sequence alignment algorithm PILEUP together with a matrix for scoring amino acid substitutions based on physicochemical properties to generate guide trees depicting relatedness of selected peptides. A random heptapeptide library was biopanned nine times using no selecting antibodies, immunoglobulin G (IgG) from sera of subjects with autoimmune diseases (primary biliary cirrhosis (PBC) and type 1 diabetes) and three murine ascites fluids that contained mAbs to overlapping epitope(s) on the Ross River Virus envelope protein 2. Peptides randomly sampled from the library were distributed throughout the guide tree of the total set of peptides whilst many of the peptides derived in the absence of selecting antibody aligned to a single cluster. Moreover peptides selected by different sources of IgG aligned to separate clusters, each with a different amino acid motif. These alignments were validated by testing all of the 53 phagotopes derived using IgG from PBC sera for reactivity by capture ELISA with antibodies affinity purified on the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the major autoantigen in PBC: only those phagotopes that aligned to PBC-associated clusters were reactive. Hence the multiple sequence alignment procedure discriminates relevant from irrelevant phagotopes and thus a major difficulty with biopanning phage-displayed random peptide libraries with polyclonal antibodies is surmounted.     

Mapping of B-cell epitopes on the outer membrane P2 porin protein of Haemophilus influenzae by using recombinant proteins and synthetic peptides.

The P2 protein of Haemophilus influenzae type b has a porin activity and is the most abundant protein in the outer membrane. We have employed fusion protein constructs and synthetic peptides along with monoclonal antibodies to map B-cell epitopes in this protein. A linear, surface-exposed epitope was identified between residues 158 and 174. A second surface-exposed epitope was identified near the carboxy-terminal end of the protein (residues 319 to 341). Two additional B-cell epitopes were identified. One was localized between residues 28 and 55, whereas the other was located between residues 148 and 174. These epitopes were not present on the surface of intact H. influenzae cells. Thus, four distinct immunogenic and antigenic regions on the P2 protein have been identified.