Immunogenicity of overlapping synthetic peptides covering the entire sequence of Haemophilus influenzae type b outer membrane protein P2.

Haemophilus influenzae type b is a major cause of bacterial meningitis in young children. Antibodies against the outer membrane protein P2 are protective in the infant rat model of bacteremia. To identify conserved, surface-exposed, and protective epitopes of P2, 17 overlapping peptides covering the entire sequence of the protein were synthesized. Antisera from mice, guinea pigs, and rabbits raised against chromatographically purified P2 were tested for their reactivities to the peptides by enzyme-linked immunosorbent assays (ELISA). Three major linear immunodominant B-cell epitopes were mapped to residues 53 to 81, 241 to 265, and 314 to 341 of mature P2. Human convalescent-phase antisera also reacted strongly with these three epitopes. Rabbit antisera against all peptide-keyhole limpet hemocyanin conjugates except two peptides containing residues 8 to 19 and 302 to 319 recognized the corresponding peptides in ELISA and reacted with P2 on immunoblots. Immunization with all unconjugated peptides, except the 19 N-terminal residues, induced very strong peptide-specific antibody responses, and these antisera reacted with P2 on immunoblots. Rabbit antisera raised against peptides corresponding to residues 1 to 14, 125 to 150, 193 to 219, and 241 to 319 also recognized P2 purified from H. influenzae nontypeable isolates. Identification of these immunodominant B-cell epitopes and conserved regions is a first step toward the rational design of a universal H. influenzae vaccine.     

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.     

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.     

Strain-specific and immunodominant surface epitopes of the P2 porin protein of nontypeable Haemophilus influenzae.

The P2 porin protein is the major outer membrane protein of nontypeable Haemophilus influenzae. Five monoclonal antibodies to P2 of four strains of nontypeable H. influenzae were developed by immunizing mice with whole bacterial cells. All five antibodies recognized epitopes on P2 in immunoblot assays of whole organism lysates, purified outer membrane, and purified P2. Competitive enzyme-linked immunosorbent assays and immunoblot assays of cyanogen bromide-digested P2 showed that two antibodies to the P2 protein of strain 1479 recognized different epitopes on the molecule. Immunofluorescence and immunoelectron microscopy demonstrated that each of the five antibodies recognized epitopes that were abundantly expressed on the bacterial surface. Analysis of 120 H. influenzae strains indicated that three of the five antibodies were reactive exclusively with the homologous strain. The remaining two antibodies were reactive with less than 3% of the strains. These studies indicate that the P2 protein expresses a highly strain-specific and immunodominant epitope on the bacterial surface. The expression of strain-specific and immunodominant epitopes on the bacterial surface may represent a mechanism by which the bacterium induces antibodies that will protect against recurrent infection by the homologous strain but will not protect against infection by heterologous strains.     

Detection of the major epitopes of human protamine P1 recognized by rabbit and mouse antibodies

The characterization of the major antigenic determinants present in human protamine P1 has been carried out by the use of specific rabbit polyclonal and mouse monoclonal antisera raised against protamine P1. This basic protein, the full amino acid sequence of which has been determined here, has been cleaved by cyanogen bromide and/or by pepsin to generate a discrete number of peptides. These have been purified, characterized by partial amino acid sequencing and used for the determination of their antigenic reactivities with antisera to native protamine P1. Both rabbit polyclonal and mouse monoclonal antibodies were able to recognize the NH2-terminal CNBr peptide encompassing residues 1-36 to the same extent as the intact protamine. A minor epitope present on the COOH-terminal peptide 37-50 could be detected only with the polyclonal rabbit antisera. Attempts to further cleave the P1 molecule in order to isolate peptides shorter than fragments 1-36 whilst retaining full antigenic reactivities, were unsuccessful. This suggests that the epitopes in P1 are conformation-dependent and located for the most part on the amino-terminal half of the molecule, which comprises the characteristic central arginine cluster. The implication of these findings for the studies of the specificities of autoantibodies in sera from infertile and vasectomized individuals is discussed.     

Immunogenicity of peptides simulating a neutralization epitope of transmissible gastroenteritis virus.

Previously, an epitope recognized by a set of neutralizing monoclonal antibodies directed against the S protein of transmissible gastroenteritis has been identified. This neutralization epitope can be simulated by a single peptide combining residues 380 to 387 and 1176 to 1184 of the S protein; this combination peptide (SFFSYGEI-QLAKDKVNE) was more antigenic than it single constituents. Here we describe the immunogenicity of this combination peptide, in comparison with monomer and tandem peptides of both constituents, and with a cyclic peptide consisting of residues 373 to 398. All antisera, raised in rabbits, bound to the peptide used as immunogen. Only sera that recognized the residues 380 to 387 bound to whole virus. Three of the four antisera with the highest binding titers to whole virus also had neutralization activity. Analysis of the fine-specificity of the antisera with PEPSCAN peptides indicated that the spectrum of antibodies induced by the 380 to 387 sequence depended on the presentation of this sequence in a peptide to the immune system. The nonbinding and nonneutralizing anti-(380 to 387)-sera appeared to contain a limited spectrum of antipeptide antibodies. Furthermore, the lack of neutralization of the antiserum against the combination peptide could be explained by the immunodominance in rabbits of the 1176 to 1184 sequence over the 380 to 387 sequence. These findings demonstrate a few fundamental problems of simulating discontinuous epitopes by single synthetic peptides.     

Epitope analysis of an immunodominant domain on the P1 protein of Haemophilus influenzae type b using synthetic peptides and anti-idiotypic antibodies.

Synthetic peptides, anti-idiotypic antibodies (anti-Id) and human and murine monoclonal antibodies (mAbs) were used to further define a major antigenic domain on the outer membrane P1 protein (OMP) of Haemophilus influenzae type b (Hib). Synthetic peptides were elaborated from the known primary sequences of the P1 protein of prototype Hib strains MinnA (OMP subtype 1H) and 8358 (OMP subtype 6U). By peptide mapping, antibodies are categorized into three groups: A, B and C. A first epitope on the P1 from strain MinnA was identified by the reactivity of one set of murine anti-P1 mAbs with the two overlapping peptides 11H and 13H, corresponding to amino acid residues 384-412 and 400-437, respectively. On the basis of their reactivity with both peptides, these mAbs were designated as group A. Anti-Id obtained from mice immunized with two group A mAbs reacted specifically with all group A mAbs. A second epitope on the same P1 protein was identified by the reactivity of the peptide 13H with another distinct set of murine anti-P1 mAbs assigned to group B. This group of mAbs did not recognize the peptide 11H. Murine anti-Id which were prepared against one group B mAb inhibited the attachment of this mAb to outer membrane preparations, whereas the binding of the other group B mAbs was not affected, suggesting that these mAbs represent a heterologous group of mAbs. The epitope(s) recognized by two human anti-P1 mAbs was (were) distinct from the ones recognized by murine mAbs since no reactivity with the peptides was observed. Similarly, the binding of the two human mAbs to the P1 antigen was not inhibited by anti-Id raised against group A or B mAbs. Interestingly, an epitope on a different P1 protein recovered from strain 8358 was identified by the reactivity of group C murine mAbs with the peptide 13U, which occupies the same position on the P1 protein as 13H but differs from the latter by 10 amino acid residues. Our studies demonstrated the presence of several distinct surface-exposed B-cell epitopes within the antigenic domain which was defined previously on the P1 protein of Hib MinnA. Furthermore, we showed the immunodominance of this region on two different P1 proteins. None of the mAbs, however, had a bacteriolytic or protective activity against Hib strains. We suggest that the surface-exposed immunodominant region on the OMP P1 of Hib do not induce protective antibodies against Hib infection.     

Antibodies to synthetic peptides corresponding to variable-region first-framework segments of T cell receptors

Recent studies at the gene level have shown that T cells express rearranged genes for four types of T cell receptors that are strongly homologous to classical immunoglobulins in the joining region and in the framework 1 (Fr1) and 3 segments of the variable region. Based upon the homologies in gene sequence, it follows that the gene products would show similarities in amino acid sequence and in the folding of the proteins so that cross-reactivities in antigenic determinants would be expected between variable regions of the T cell receptors and classical immunoglobulins. We have synthesized peptides corresponding to predicted protein sequences of the Fr1 residues of T cell receptor alpha, beta- and gamma-chains and have produced antibodies in rabbits against these synthetic peptides. Use of antisera and affinity-purified antipeptide antibodies indicated that high-titer antibodies could be raised that were specific for individual Fr1 peptides. Cross-reactions among Fr1 peptides of T cell receptors and immunoglobulin light chains were observed. In addition, some rabbit antisera raised against classical polyclonal immunoglobulins or affinity-purified immunoglobulin-like T cell receptors were found to exhibit binding activity against Fr1 peptides of T cell receptor beta- and gamma-chains. The sequence homology, although real among the Fr1 of T cell receptors and immunoglobulin light chains, is moderate and the antigenic cross-reaction must reflect the configuration and types of amino acids present. The development of antipeptide antibodies holds promise for the characterization of T cell receptors of various T cell sources and also offers a new means for the identification of molecules related to rearranging immunoglobulins.     

Mapping epitopic regions of cholera toxin B-subunit protein.

Continuous overlapping synthetic hexapeptides representing the entire 103 amino acid sequence of the immunodominant B-subunit protein of cholera enterotoxin were used to examine reactivities of a variety of antisera in attempts to detect and define sequence-related (continuous) antigenic regions. The validity of the methods was established by the reactions of polyclonal antisera raised against longer synthetic peptides with appropriate synthetic hexapeptides. An unexpected cross-reaction is attributed to the presence of three identical amino acids (Gln16-Ile17-His18)--although in different order (Gln56-His57-Ile58)--in two parts of the B-subunit chain. Adsorption studies using polyclonal rabbit antisera revealed that, in many instances, denatured B-subunit protein more effectively removed reactivity with hexapeptides than did the native protein. Native holotoxin was more effective than native B-subunit. Sera from human cholera convalescents gave diffuse patterns of reactivity with synthetic hexapeptides--primarily against regions of reactive hexapeptides rather than with clearly defined continuous epitopes. Among many epitopic regions encountered, a strongly reactive tetramer, Ser-Gln-His-Ile (SQHI), was discovered in a highly conserved region, residues 55-58, of the B-subunit amino acid sequence. Adsorption studies revealed that this epitope is apparently exposed on the surface of the native protein. Amino acid substitution revealed the essentiality of Gln and His residues to this epitope. Gly54 was not part of the epitope but substitution of acidic residues Glu and Asp for Gly eliminated reactivity with antibody. The results suggest that continuous epitopes may contribute to the antigenicity of the native toxin protein and may be potentially useful for development of a peptide vaccine.     

Neutralization of HPV16, 18, 31, and 58 pseudovirions with antisera induced by immunizing rabbits with synthetic peptides representing segments of the HPV16 minor capsid protein L2 surface region

Neutralizing antibody against human papillomavirus (HPV) minor capsid protein L2 can cross-neutralize different HPV genotypes in vitro. To identify the segments containing the cross-neutralization epitopes of HPV16 L2, we characterized antisera obtained by immunizing two rabbits with each of the ten synthetic peptides of 14 to 20 amino acids (aa) long, which represents a part of the HPV16 L2 sequence from aa 14 to 144. The antisera against the peptides within the region from aa 18 to 144 efficiently bound to HPV16 L1/L2-capsids and neutralized HPV16 pseudovirions, indicating that the region is displayed on the surface of the capsids and contains several neutralization epitopes. Antiserum against the peptide from aa 18 to 38 (anti-P18/38) cross-neutralized HPV18. Anti-P56/75 cross-neutralized HPV18, 31, and 58. Anti-P61/75 and anti-P64/81 cross-neutralized HPV18 and 58. Anti-P96/115 and the antiserum induced by a mutant P96/115 (S and T at aa 101 and 112 were replaced with L and S, respectively) cross-neutralized HPV31 and 58. The mixture of equal volumes of three antisera, anti-P18/38, anti-P56/75, and anti-mutant P96/115, neutralized HPV16, 18, 31, and 58 more efficiently than anti-P56/75 alone, suggesting that there is a synergistic effect of antibodies on the cross-neutralization. The cross-neutralization appears to be correlated with conserved aa sequences among HPV types. The data in this study provide a basis for designing vaccine antigens effective against a broader spectrum of the high-risk HPVs.     

Antigenic cross-reactivity potential of synthetic peptides immobilized on polyethylene rods.

A number of continuous epitopes of tobacco mosaic virus protein (TMVP) have been defined by the pepscan technique using polyclonal and monoclonal antibodies to TMVP as well as antisera raised against synthetic peptides. In general, the location of continuous epitopes agreed with the results of earlier studies with peptides synthesized by classical methods although there were some notable exceptions. Results obtained with the different types of antibodies used in this study indicated that a homology of three residues was sufficient to give rise to antigenic cross-reactions. In the case of antibodies raised against a peptide conjugated to ovalbumin, some unexpected cross-reactivities could be explained by assuming that antibodies to the carrier molecule recognized homologous tripeptide sequences in TMVP and ovalbumin.     

Selection of glutamate-rich protein long synthetic peptides for vaccine development: antigenicity and relationship with clinical protection and immunogenicity

Antibodies against three long synthetic peptides (LSPs) derived from the glutamate-rich protein (GLURP) of Plasmodium falciparum were analyzed in three cohorts from Liberia, Ghana, and Senegal. Two overlapping LSPs, LR67 and LR68, are derived from the relatively conserved N-terminal nonrepeat region (R0), and the third, LR70, is derived from the R2 repeat region. A high prevalence of antibody responses to each LSP was observed in all three areas of endemic infection. Levels of cytophilic immunoglobulin G (IgG) antibodies against both GLURP regions were significantly correlated with protection from clinical P. falciparum malaria. Protected children from the Ghana cohort possessed predominantly IgG1 antibodies against the nonrepeat epitope and IgG3 antibodies against the repeat epitope. T-cell proliferation responses, studied in the cohort from Senegal, revealed that T-helper-cell epitopes were confined to the nonrepeat region. When used as immunogens, the LR67 and LR68 peptides elicited strong IgG responses in outbred mice and LR67 also induced antibodies in mice of different H-2 haplotypes, confirming the presence of T-helper-cell epitopes in these constructs. Mouse antipeptide antisera recognized parasite proteins as determined by immunofluorescence and immunoblotting. This indicates that synthetic peptides derived from relatively conserved epitopes of GLURP might serve as useful immunogens for vaccination against P. falciparum malaria.     

腺病毒六邻体蛋白型间线性化抗原位点的研究

目的 对人类腺病毒（adenovirus,AdV)六邻体型间抗原位点的特性进行研究。方法 通过计算机对腺病毒六邻体氨基酸序列进行比较分析，结合抗原性的预测结果和六邻体蛋白三维空间结构的肽段暴露状态，选定了保守性抗原位点进行多肽合成或通过构建重组质粒表达蛋白，将合成的六邻体肽段和表达纯化的蛋白抗原免疫动物后，用免疫印迹和间接免疫荧光方法检测抗血清的免疫特异性，结果 免疫印迹分析显示，抗多肽抗体和抗重组蛋白抗体均与腺病毒的六邻体蛋白特异性识别，间接免疫荧光显示，腺病毒感染HeLa细胞核内荧光着色，并且抗血清有较好的腺病毒型间反应性，合成多肽抗体与含有相同肽段的重组蛋白抗原产生特异性结合。结论 在腺病毒六邻体蛋白中存在有线性化的型间抗原位点，全盛的六邻体多肽和表达重组蛋白可用于诊断价值抗体的研制。     

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.     

Comparison of hemagglutinating pili of Haemophilus influenzae type b with similar structures of nontypeable H. influenzae.

Thirty-eight clinical isolates of nontypeable Haemophilus influenzae were tested for the presence of hemagglutinating pili similar to those of H. influenzae type b (Hib) that mediate buccal epithelial cell adherence. Four endogenously hemagglutinating (HA+) strains were identified, and eight additional HA+ variants were obtained from HA- strains by erythrocyte enrichment. All 12 HA+ nontypeable H. influenzae isolates bound antisera directed against denatured pilins of Hib, but none bound antisera against assembled native pili of Hib. In erythrocyte- and buccal-cell-binding assays, HA+ nontypeable H. influenzae binding was reduced compared with HA+ Hib binding and was not significantly different from HA- nontypeable H. influenzae binding. Both HA- and HA+ nontypeable H. influenzae binding was increased over binding of HA- Hib. HA+ nontypeable H. influenzae strains agglutinated adult erythrocytes that possess the Anton antigen, which is thought to be the receptor for Hib pili, and did not agglutinate cord or Lu(a-b-) dominant erythrocytes, which lack the Anton antigen. Electron microscopy of HA- and HA+ variants of three nontypeable H. influenzae strains showed few or no surface appendages on the HA- organisms, but piluslike structures were seen on many organisms from two HA+ nontypeable H. influenzae strains and on a few organisms from one strain. Thus, nontypeable H. influenzae appears to possess structures that are immunologically similar to the pilins that make up the hemagglutinating pili of Hib. However, nontypeable H. influenzae appears to also possess mechanisms for erythrocyte and buccal cell adherence that are not directly correlated with the presence of a hemagglutinating pilus.     

Epitope mapping of the outer membrane protein P5-homologous fimbrin adhesin of nontypeable Haemophilus influenzae

To identify potential immunodominant and/or adhesin binding domains of the outer membrane protein P5-homologous fimbrin adhesin of nontypeable Haemophilus influenzae (NTHI), three sets of synthetic peptides were synthesized and assayed in an adherence inhibition assay, by Western blotting, and in a biomolecular interaction analysis (BIA) system. The first series of 34 8- to 10-mer peptides represented the entire mature protein sequentially. The second set of four peptides (each 19 to 28 residues) represented the four predicted major surface-exposed regions (or loops) of this adhesin. The third series of seven peptides (each 27 to 34 residues) were specifically designed to map the third surface-exposed region. Data obtained by BIA indicated limited reactivity of a panel of high-titered immune chinchilla sera to the 8- to 10-mer peptides representing the mature protein, likely because these linear peptides did not represent continuous epitopes. However, several of these short peptides did inhibit adherence of multiple NTHI strains to a human respiratory epithelial cell. Overall, greatest relative reactivity in both BIA and adherence inhibition assays was demonstrated against, or shown by, peptides mapping to the third and fourth predicted surface-exposed regions of this adhesin, thereby indicating the presence of immunodominant and adhesin binding domains at these sites. Middle ear fluids sequentially recovered from a chinchilla with an ongoing NTHI-induced otitis media (OM) as well as sera from children with OM due to NTHI also reacted exclusively with peptides representing the third and fourth surface-exposed regions of the P5-fimbrin adhesin, indicating a similarity in immune recognition of this bacterial protein by these two hosts. Collectively, these data together with the previously demonstrated protective efficacy of immunogens derived from this adhesin in chinchilla models support the continued development of P5-fimbrin based vaccine components.     

Identification of antigenic epitopes in an alanine-rich repeating region of a surface protein antigen of Streptococcus mutants.

A surface protein antigen (PAc) of Streptococcus mutans with a molecular mass of 190 kDa is considered to play an important role in the initial attachment of this streptococcus to the tooth surface. Two internal repeating amino acid sequences are present in the PAc molecule. One repeating region located in the N-terminal region is rich in alanine (A-region), and the other, located in the central region, is rich in proline (P-region). To identify antigenic epitopes on the A-region of the PAc protein, 82 sequential overlapping synthetic decapeptides covering one of the repetitive units of the A-region were synthesized. In the epitope scanning analyses using murine antisera raised against recombinant PAc (rPAc), multiple antigenic epitopes were found in the repetitive unit of the A-region, and some of them reacted with antisera to rPAc from BALB/c, B10, B10.D2, and B10.BR mice. In particular, a peptide YEAALKQY (residues 366 to 373) was recognized by anti-rPAc sera from all four strains of mice. The reactivities of anti-rPAc sera in the epitope scanning were confirmed by using a purified synthetic peptide, NAKATYEAALKQYEADLAA (corresponding to residues 361 to 379). Furthermore, antisera against a surface protein antigen PAg (SpaA) of Streptococcus sobrinus from BALB/c mice reacted strongly to residues 330 to 337, 362 to 369, and 366 to 373 of the PAc protein by the epitope scanning analysis. An AKATYEAALKQY (residues 362 to 373 of the PAc protein)-like sequence, AKANYEAKLAQY, was found within the A-region of S. sobrinus PAg, suggesting that the amino acid sequences AKA-YEA and YEA-L-QY may be major cross-reactive epitopes of the S. mutans PAc protein and the S. sobrinus PAg protein.     

Mapping of linear B-cell epitopes of the S2 subunit of pertussis toxin.

The linear immunogenic and antigenic structure of the S2 subunit of pertussis toxin was investigated with synthetic peptides corresponding to regions of the protein sequence predicted to contain surface-exposed hydrophilic beta turns. Five peptides as peptide-bovine serum albumin conjugates were recognized by anti-pertussis toxin antiserum and were thus designated "immunogenic epitopes." Two prominent immunogenic epitopes were specified by peptides corresponding to sequences spanning R107-120 and R186-199, whereas peptides corresponding to residues R35-50 and R91-106 were only bound in low titer. Three peptides as thyroglobulin conjugates elicited antisera in rabbits that bound intact pertussis toxin by enzyme-linked immunosorbent assay and immunoblot. These peptides were designated "antigenic epitopes." The most prominent antigenic determinant was localized to the N-terminal end of the S2 sequence encompassing residue R1-7. Peptides R35-50 and R91-106 represented two minor antigenic epitopes. Antisera to two additional peptides corresponding to residues R134-149 and R186-199 recognized the S2 subunit only by Western blotting (immunoblotting). Only antiserum raised against peptide R91-106 also recognized the S3 subunit by Western blotting, indicating a marked antigenic and probably also structural difference between the two highly homologous subunits.     

Synthetic peptides representing epitopes of outer membrane protein F of Pseudomonas aeruginosa that elicit antibodies reactive with whole cells of heterologous immunotype strains of P. aeruginosa.

By using the published amino acid sequence for mature outer membrane protein F of Pseudomonas aeruginosa, a computer-assisted analysis was performed to identify sites with potential as surface-exposed, antigenic regions located throughout the length of the protein molecule. Synthetic peptides 13 to 15 amino acid residues in length were synthesized for 10 such regions. Mice were immunized with each of the 10 synthetic peptides conjugated to keyhole limpet hemocyanin. An enzyme-linked immunosorbent assay (ELISA) of the antisera was performed by using each of the synthetic peptides as the ELISA antigen to verify that immunoglobulin G (IgG) antibodies capable of reacting with the peptide used as immunogen were elicited by each peptide. Each of the antipeptide antisera was screened for the presence of IgG antibodies that could bind to the surface of intact cells of strains representing the seven heterologous Fisher-Devlin immunotypes of P. aeruginosa by use of an ELISA with whole cells of the various strains as the ELISA antigen. Three peptides elicited antibodies capable of reacting with whole cells of all seven immunotype strains. Peptide 10, corresponding to amino acid residues 305 to 318, elicited whole-cell-reactive antibodies at high titers. Peptide 9, corresponding to amino acid residues 261 to 274, elicited whole-cell-reactive antibodies at more intermediate titers. Peptide 7, corresponding to amino acid residues 219 to 232, elicited such antibodies only at low titers. The carboxy-terminal portion of the mature protein appears to be the immunodominant portion. In particular, peptides 10 (NATAEGRAINRRVE) and 9 (TDAYNQKLSERRAN) appear to have potential for use as immunogens in a synthetic vaccine for immunoprophylaxis against infections caused by P. aeruginosa. Antisera from mice immunized with either peptide 9 or 10 mediated opsonophagocytic uptake by human polymorphonuclear leukocytes of wild-type cells of P. aeruginosa but exhibited no opsonic activity against a protein F-deficient mutant of P. aeruginosa.     

Species-specific B-cell epitope on the C-terminal region of the α antigen fromMycobacterium scrofulaceum

In the amino acid (AA) sequences of α antigen from mycobacteria, C-terminal regions were variable among a variety of mycobacterial species though the N-terminal regions were relatively conserved. These regions may possess some species-specific antigenic determinants of the α antigen fromMycobacterium scrofulaceum(S-α). AAs288–300 of S-α fused to β-galactosidase was reactive with the antisera raised against S-α. The same fused peptide did not react with the antisera raised against the α antigen fromMycobacterium avium(A-α) andMycobacterium bovisBCG (B-α). B-cell epitope mapping then was performed focusing on the C-terminal region of S-α using the synthetic peptides. Their reactivities with antisera raised against the α antigens of three different mycobacterial species were assessed by ELISA. AAs279–286 were a cross-reactive common immunodominant region among three mycobacterial species. This region may be one of the cross-reactive common epitopes in mycobacterial species. And AAs291–300 were reactive only with the antisera raised against S-α. This region may possess a species-specific epitope.