Conserved lipoprotein H.8 of pathogenic Neisseria consists entirely of pentapeptide repeats

The pathogenic Neisseria, N. gonorrhoeae and N. meningitidis, possess an outer membrane protein (OMP), designated H.8, with a conserved monoclonal antibody (MAb)-binding epitope. We determined the DNA sequence of a gonococcal H.8 gene, and confirmed the relationship between the cloned gene and the H.8 OMP by constructing a gonococcal mutant lacking H.8. The predicted H.8 OMP is a lipoprotein 71 amino acids in length, composed of 13 repeats of a consensus sequence AAEAP with perfect 5-residue periodicity. The AAEAP units form a repeating epitope that comprises the entire predicted sequence of the protein.     

The H8 antigen of pathogenic Neisseriae

We cloned and sequenced the H8 gene from N. meningitidis FAM18. The predicted amino acid sequence included a consensus lipoprotein signal sequence processing site, consistent with lipid modification that could account for the unusual electrophoretic and solubilization properties of H8. The amino acid sequence was rich in alanine and proline, especially in an imperfectly periodic region near the amino terminus, which encompassed the epitope recognized by available monoclonal antibodies. In a panel of neisserial strains, the presence of DNA homologous to the H8 gene correlated with the expression of an H8 protein. We cloned a gene from N. meningitidis JB515 that was distinct from the H8 gene but encoded a protein also recognized by an anti-H8 monoclonal antibody. Mice were not protected from meningococcemia by passive immunization with such an antibody.     

Identification of epitopes recognized by monoclonal antibodies SM1 and SM2 which react with all pili of Neisseria gonorrhoeae but which differentiate between two structural classes of pili expressed by Neisseria meningitidis and the distribution of their encoding sequences in the genomes of Neisseria spp

The pili expressed by all isolates of Neisseria gonorrhoeae react with two monoclonal antibodies, SM1 and SM2. In contrast, although many isolates of Neisseria meningitidis also express pili (class I) which react with antibodies SM1 and SM2, a proportion express pili (class II) which fail to react. In order to define the epitopes recognized by these antibodies, a series of overlapping peptides corresponding to the amino acid sequence of conserved regions of gonococcal pili have been synthesized. The minimum epitope recognized by antibody SM1 was found to comprise a linear peptide EYYLN, corresponding to residues 49-53 of mature pilin. In contrast, antibody SM2 reacted with a number of peptides from around the cysteine residue (Cys 1) at position 120, suggesting that an extended region may contribute to a conformational epitope recognized by this antibody in the native protein. The identification of the two epitopes defines structural differences between the classes of pili expressed by meningococci. In order to determine the distribution of pilin gene sequences in Neisseria we used as hybridization probes an oligonucleotide (PS1) with the sequence 5'-GAGTATTACCTGAATCA-3' which spans the coding region for the SM1 epitope, and a fragment of the 3' end of the gonococcal pilE gene which contains conserved sequences flanking the two Cys codons and encodes the SM2 epitope. All strains of N. gonorrhoeae and N. meningitidis tested, regardless of piliation phenotype, harboured DNA sequences homologous to those encoding the carboxy-terminus of meningococcal class I pilin. Furthermore, all gonococci and all meningococci producing class I pili hybridized with oligonucleotide probe PS1. Non-reverting non-piliated derivatives of previously class I pilus-producing strains showed reduced hybridization signals with this probe, but nevertheless retained sequences homologous to the coding sequence for the SM1 epitope. However, meningococci producing class II pili could be divided into two groups on the basis of their reaction with the PS1 probe: half the strains tested failed to react, which is consistent with our previous analysis of silent class I pilin sequences; the remainder reacted (relatively weakly) with the probe, suggesting that the silent pil sequences in these strains extend further towards the 5' end of the pilin gene than in strains studied previously. Some strains of Neisseria lactamica reacted weakly with both types of probe but failed to produce SM1-reactive pili. In contrast, isolates of Neisseria flava, Neisseria pharyngis, Neisseria sicca and a series of unrelated bacteria failed to react with both SM1 antibody and the DNA probes. This confirms that possession of 'gonococcal' pilin sequences is limited to the pathogenic neisseriae.     

Characterization of the neisserial lipid-modified azurin bearing the H.8 epitope

The pathogenic Neisseria have multiple genes encoding proteins that bind monoclonal antibody (MAb) H.8. We previously reported the cloning and sequencing of a meningococcal gene (laz) encoding an H.8 MAb-binding protein with a consensus lipoprotein processing site, an N-terminal domain containing the epitope for H.8 MAb binding, and a C-terminal domain with extensive similarity to the sequences of azurins from other organisms. In the current study, we showed that the product of the cloned gene could be labelled with palmitic acid, that it was subject to globomycin-sensitive processing, and that it was immunologically cross-reactive with azurin from Pseudomonas aeruginosa. All neisserial species tested, both pathogens and commensals, produced a protein recognized by anti-azurin serum. Southern blots with oligonucleotide probes specific for the azurin domain of the gene showed that it was present in a single copy in the chromosome; it was highly conserved in gonococci and meningococci, and less conserved in commensal Neisseria species.     

Phosphorylcholine decoration of lipopolysaccharide differentiates commensal Neisseriae from pathogenic strains: identification of licA-type genes in commensal Neisseriae

Phosphorylcholine (ChoP) is a potential candidate for a plurispecific vaccine, because it is present on surface components of many mucosal organisms, including Haemophilus influenzae, Streptococcus pneumoniae and Pseudomonas aeruginosa. In addition, ChoP has been detected on pili of Neisseria meningitidis and Neisseria gonorrhoeae. In this study, we demonstrate the presence of the phosphorylcholine epitope on the lipopolysaccharides (LPSs) of several species of commensal Neisseriae (Cn), a property that differentiates commensal from the pathogenic strains of Neisseriae. In an extended survey of 78 strains, we confirmed the exclusive expression of the ChoP epitope on pili of pathogenic Neisseriae. Despite the presence of pili on Cn, which are homologous to Class II pili of N. meningitidis, they did not react with anti-ChoP antibody. This observation was further supported by the fact that 14C-labelled choline was incorporated only in the LPSs of Cn. Analysis of the LPS of N. lactamica strain NL4 revealed two distinct and interconvertible molecular species of LPS with high and low levels of reactivity with anti-ChoP antibody. In addition, on/off phase variation gave rise to frequent modulation in the levels of antibody reactivity. A concurrent modulation was also observed in the binding of C-reactive protein, CRP, a ChoP-binding reactant that is implicated in bacterial clearance. Genetic analysis showed the presence of a gene in several Cn spp. with significant sequence identity to H. influenzae licA. This gene encodes choline kinase and is also involved in phase variation of the LPS-associated ChoP in H. influenzae. In contrast, licA-like genes were not identified in the pathogenic Neisseria strains tested. They are absent from N. meningitidis strain Z2491 genome database. These data suggest that the genetic basis for ChoP incorporation in Cn LPS resembles that in H. influenzae spp. and may be distinct from that generating the ChoP epitope on pili of pathogenic Neisseriae. Further, the modulation of ChoP expression on Cn LPS, and corresponding modulation of CRP binding, has the potential to confer the property of immune avoidance and thus of persistence on mucosa.     

Structural features of an antigen required for cellular interactions and for T cell activation in a MHC-restricted response

The protein Ag, tobacco mosaic virus protein, (TMVP) and its tryptic peptide number 8 (residues 93-112 of the protein) exhibit cross-reactivity on the T cell level in some strains of mice (e.g., C3H.SW, C57BL/10); these strains are termed cross-reactive (CR). In other strains such as A/J or B10.BR, no cross-reactivity is exhibited; these strains are termed non-cross-reactive (NCR). Genetic experiments indicated that the cross-reactivity is dominant and that it is mapped to the I-A or I-E region of the MHC, with cross-reactivity exhibited by the I-Ab haplotype but not by I-Ak or I-Ek. Cell reconstitution experiments have indicated that the non-cross-reactivity is associated with the inability of the NCR APC to present Ag. Analysis of the area(s) on peptide 8 which serve(s) as epitope revealed that both strains recognize an overlapping area consisting of 11 amino acid residues in the middle of peptide 8 (residues 97-107), which by itself is nonstimulatory to TMVP- or peptide 8-immune T cells of the CR or the NCR strains. However, the addition of a few amino acid residues of the sequence of peptide 8 to this area converts it to a complete stimulatory epitope. Additivity experiments revealed that the CR strain contains two major T cell populations each recognizing this middle region of peptide 8 when elongated by a few amino acids N-terminally and C-terminally, respectively. In contrast, the NCR strain contains one major T cell population recognizing elongation only N-terminally. Because TMVP (but not peptide 8) requires processing before presentation to T cells, it is postulated that, during processing of TMVP, there occur alterations in the area of the proximal three or four N-terminal amino acids of the region consisting of peptide 8, destroying the only region containing the T cell epitope recognized by the NCR strain, hence TMVP and peptide 8 do not exhibit cross-reactivity in this strain. The same alterations of TMVP still leave intact an epitope consisting of amino acid residues C-terminal to the altered area which is recognized by the CR strain, hence the cross-reactivity exhibited by this strain. The results suggest that the difference in cross-reactivity on the T cell level between TMVP and peptide 8 exhibited by the strains may be due to differences in the orientation of presentation and the subsequent cell recognition of an epitope contained within peptide 8.(ABSTRACT TRUNCATED AT 400 WORDS)     

Epitopes on the major capsid protein of simian virus 40

Thirteen monoclonal antibodies which react with the major capsid protein (VP1) of simian virus 40 (SV40) have been isolated. Of these, five neutralized viral infectivity when added in sufficient concentration. Seven of the antibodies reacted with denatured VP1 and also recognized fragments generated by protease or cyanogen bromide cleavage. The region of VP1 recognized by all seven antibodies was mapped within a nine-amino-acid segment located in the carboxyl portion of the protein (from amino acid positions 312 to 321). This region is likely to protrude from the surface of the protein as judged by high hydrophilicity and low hydropathy predicted from the amino acid sequence and lack of secondary structure by contrast with the rest of the protein for which predominantly beta-sheet structure is predicted. Competition between these antibodies and synthetic peptides for binding to virus particles confirmed that the continuous epitope is contained within the nine-amino-acid sequence. Competition between the different monoclonal antibodies suggested that the continuous epitope was also part of more complex discontinuous epitopes recognized by some of the other antibodies. These results support a model in which a segment of the carboxyl-terminal portion of VP1 protrudes from the surface of the virus to form an antigenic structure.     

Sequence analysis of recent H7 avian influenza viruses associated with three different outbreaks in commercial poultry in the United States

The hemagglutinin (HA) and neuraminidase (NA) genes of H7 avian influenza virus (AIV) isolated between 1994 and 2002 from live-bird markets (LBMs) in the northeastern United States and from three outbreaks in commercial poultry have been characterized. Phylogenetic analysis of the HA and NA genes demonstrates that the isolates from commercial poultry were closely related to the viruses circulating in the LBMs. Also, since 1994, two distinguishing genetic features have appeared in this AIV lineage: a deletion of 17 amino acids in the NA protein stalk region and a deletion of 8 amino acids in the HA1 protein which is putatively in part of the receptor binding site. Furthermore, analysis of the HA cleavage site amino acid sequence, a marker for pathogenicity in chickens and turkeys, shows a progression toward a cleavage site sequence that fulfills the molecular criteria for highly pathogenic AIV.     

Epitope mapping of monoclonal antibodies for the Deinococcus radiodurans bacteriophytochome

Bacteriophytochromes (BphP) are phytochrome‐like light sensing proteins in bacteria, which use biliverdin as a chromophore. In order to study the biochemical properties of the DrBphP protein, five (2B8, 2C11, 3B2, 3D2, and 3H7) anti‐DrBphP monoclonal antibodies were produced through the immunization of mice with purified full‐length DrBphP and DrBphN (1–321 amino acid) proteins, and epitope mapping was then carried out. Among the five antibodies, 2B8 and 2C11 preferentially recognized the N‐terminal region of BphP whereas 3B2, 3D2, and 3H7 showed preference for the C‐terminal region. We performed further epitope mapping using recombinant truncated BphP proteins to narrow down their target sequences. The results demonstrated that each of the five monoclonal antibodies recognized different regions on the DrBphP protein. Additionally, epitopes of 2B8 and 3H7 antibodies were discovered to be shorter than 10 amino acids (2B8: RDPLPFFPP, 3H7: PGEIEEA). These two antibodies with such specific recognition epitopes could be especially valuable for developing new peptide tags for protein detection and purification.     

Molecular cloning and analysis of genes for sialic acid synthesis in Neisseria meningitidis group B and purification of the meningococcal CMP-NeuNAc synthetase enzyme.

The gene encoding for the CMP-NeuNAc synthetase enzyme of Neisseria meningitidis group B was cloned by complementation of a mutant of Escherichia coli defective for this enzyme. The gene (neuA) was isolated on a 4.1-kb fragment of meningococcal chromosomal DNA. Determination of the nucleotide sequence of this fragment revealed the presence of three genes, termed neuA, neuB, and neuC, organized in a single operon. The presence of a truncated ctrA gene at one end of the cloned DNA and a truncated gene encoding for the meningococcal sialyltransferase at the other confirmed that the cloned DNA corresponded to region A and part of region C of the meningococcal capsule gene cluster. The predicted amino acid sequence of the meningococcal NeuA protein was 57% homologous to that of NeuA, the CMP-NeuNAc synthetase encoded by E. coli K1. The predicted molecular mass of meningococcal NeuA protein was 24.8 kDa, which was 6 kDa larger than that formerly predicted (U. Edwards and M. Frosch, FEMS Microbiol. Lett. 96:161-166, 1992). Purification of the recombinant meningococcal NeuA protein together with determination of the N-terminal amino acid sequence confirmed that this 24.8-kDa protein was indeed the meningococcal CMP-NeuNAc synthetase. The predicted amino acid sequences of the two other encoded proteins were homologous to those of the NeuC and NeuB proteins of E. coli K1, two proteins involved in the synthesis of NeuNAc. These results indicate that common steps exist in the biosynthesis of NeuNAc in these two microorganisms.     

Evolution of an Autotransporter: Domain Shuffling and Lateral Transfer from Pathogenic Haemophilus to Neisseria

The genomes of pathogenic Haemophilus influenzae strains are larger than that of Rd KW20 (Rd), the nonpathogenic laboratory strain whose genome has been sequenced. To identify potential virulence genes, we examined genes possessed by Int1, an invasive nonencapsulated isolate from a meningitis patient, but absent from Rd. Int1 was found to have a novel gene termed lav, predicted to encode a member of the AIDA-I/VirG/PerT family of virulence-associated autotransporters (ATs). Associated with lav are multiple repeats of the tetranucleotide GCAA, implicated in translational phase variation of surface molecules. Laterally acquired by H. influenzae, lav is restricted in distribution to a few pathogenic strains, including H. influenzae biotype aegyptius and Brazilian purpuric fever isolates. The DNA sequence of lav is surprisingly similar to that of a gene previously described for Neisseria meningitidis. Sequence comparisons suggest that lav was transferred relatively recently from Haemophilus to Neisseria, shortly before the divergence of N. meningitidis and Neisseria gonorrhoeae. Segments of lav predicted to encode passenger and beta-domains differ sharply in G+C base content, supporting the idea that AT genes have evolved by fusing domains which originated in different genomes. Homology and base sequence comparisons suggest that a novel biotype aegyptius AT arose by swapping an unrelated sequence for the passenger domain of lav. The unusually mobile lav locus joins a growing list of genes transferred from H. influenzae to Neisseria. Frequent gene exchange suggests a common pool of hypervariable contingency genes and may help to explain the origin of invasiveness in certain respiratory pathogens.     

Identification of the region including the epitope for a monoclonal antibody which can neutralize human parvovirus B19.

In this study, we identified a region in the human parvovirus structural protein which involves the neutralization of the virus by a monoclonal antibody and site-specific synthetic peptides. A newly established monoclonal antibody reacted with both viral capsid proteins VP1 and VP2. The epitope was found in six strains of independently isolated human parvovirus B19. The monoclonal antibody could protect colony-forming unit erythroid in human bone marrow cell culture from injury by the virus. The monoclonal antibody reacted with only 1 of 12 peptides that were synthesized according to a predicted amino acid sequence based on nucleotide sequences of the coding region for the structural protein of B19 virus. The sequence recognized by the antibody was a site corresponding to amino acids 328 to 344 from the amino-terminal portion of VP2. This evidence suggests that the epitope of the viral capsid protein is located on the surface of the virus and may be recognized by virus-neutralizing antibodies.     

Complete amino acid sequence of rat kidney ornithine aminotransferase: identity with liver ornithine aminotransferase

The complete amino acid sequence of rat kidney ornithine aminotransferase [EC 2.6.1.13] is presented. The 404-residue sequence was determined by analysis of peptides generated by digestion of the S-carboxyamidomethylated protein with CNBr, Achromobacter protease I, arginylendopeptidase, or Staphylococcus aureus V8 protease. Mueckler and Pitot have reported the amino acid sequence of the rat liver enzyme (440 residues) as predicted from the nucleotide sequence of the cDNA [Mueckler, M.M. & Pitot, H.C. (1985) J. Biol. Chem. 260, 12993-12997]. The amino acid sequence of the rat kidney enzyme presented herein coincides with residue 36 (Gly) through 440 (Phe) of the predicted precursor protein, indicating that the liver and kidney enzymes are identical, and that the enzyme is processed at the amino-terminal region after translation.     

应用噬菌体随机肽库技术筛选戊型肝炎病毒中和表位模拟肽

以识别戊型肝炎病毒（HEV）构象依赖性中和表位的单克隆抗体8C11、8H3作为固相筛选分子,对噬菌体随机7肽库进行4轮筛选后,随机挑取单克隆噬菌体进行测序。合成优势7肽序列基因,将其插入HBcAg-AA78-83位置之中,进行原核表达,所获重组蛋白经蛋白印迹实验证实可与相应单抗结合,电镜下可见重组蛋白能形成与HBcAg相似的类病毒颗粒。化学合成单抗8H3筛选出的优势7肽,所获7肽经生物传感器结合实验证实与单抗8H3结合。这些结果提示用噬菌体7肽库可以筛选出部分模拟构象性表位的短肽,为亚单位疫苗的研制提供了新的思路。     

Mapping of neutralizing epitopes on Renibacterium salmoninarum p57 by use of transposon mutagenesis and synthetic peptides

Renibacterium salmoninarum is a gram-positive bacterium that causes bacterial kidney disease in salmonid fish. The virulence mechanisms of R. salmoninarum are not well understood. Production of a 57-kDa protein (p57) has been associated with isolate virulence and is a diagnostic marker for R. salmoninarum infection. Biological activities of p57 include binding to eukaryotic cells and immunosuppression. We previously isolated three monoclonal antibodies (4D3, 4C11, and 4H8) that neutralize p57 activity. These monoclonal antibodies (MAbs) bind to the amino-terminal region of p57 between amino acids 32 though 243; however, the precise locations of the neutralizing epitopes were not determined. Here, we use transposon mutagenesis to map the 4D3, 4C11, and 4H8 epitopes. Forty-five transposon mutants were generated and overexpressed in Escherichia coli BL21(DE3). The ability of MAbs 4D3, 4H8, and 4C11 to bind each mutant protein was assessed by immunoblotting. Transposons inserting between amino acids 51 and 112 disrupted the 4H8 epitope. Insertions between residues 78 and 210 disrupted the 4C11 epitope, while insertions between amino acids 158 and 234 disrupted the 4D3 epitope. The three MAbs failed to bind overlapping, 15-mer peptides spanning these regions, suggesting that the epitopes are discontinuous in conformation. We conclude that recognition of secondary structure on the amino terminus of p57 is important for neutralization. The epitope mapping studies suggest directions for improvement of MAb-based immunoassays for detection of R. salmoninarum-infected fish.     

Mapping and Characterization of a Sequential Epitope on the Rabies Virus Glycoprotein Which Is Recognized by a Neutralizing Monoclonal Antibody,RG719

We have established a murine hybridoma cell line RG719 which produces a rabies virus-neutralizing IgM-type monoclonal antibody (referred to as MAb RG719). Immunoblot analysis indicated that the antibody recognized a sequential epitope of G protein. Among four rabies virus strains tested, the antigenicity to MAb RG719 was absent from the Nishigahara strain, while the other three strains (HEP, ERA and CVS) reacted to the MAb. Studies with deletion mutants of the G protein indicated that the epitope was located in a middle region of the primary structure of G protein, ranging from position 242 to 300. By comparing the estimated amino acid sequence of the four strains, we found in this region two amino acids (at positions 263 and 291) which are common to three of those strains but are not shared by the Nishigahara strain. The site-directed point mutagenesis revealed that replacement of phenylalanine-263 by leucine destroyed the epitope of the HEP G protein, while the epitope was generated on the Nishigahara G protein whose leucine-263 was replaced by phenylalanine. These observations suggest that phenylalanine-263 is essential for constructing the epitope for MAb RG719. The synthetic 20-mer peptide produced by mimicking the amino acid sequence (ranging from amino acid positions 249 to 268) of the presumed epitope region was shown to bind specifically to MAb RG719 and also to raise the virus-neutralizing antibodies in rabbits. Vaccination with the HEP vaccine produced in Japan induced in humans and rabbits production of significant amounts of the antibodies which reacted with the 20-mer peptide.     

Tandem copies of a human rotavirus VP8 epitope can induce specific neutralizing antibodies in BALB/c mice

The VP8 subunit protein of human rotavirus (HRV) plays an important role in viral infectivity and neutralization. Recombinant peptide antigens displaying the amino acid sequence M(1)ASLIYRQLL(10), a linear neutralization epitope on the VP8 protein, were constructed and examined for their ability to generate anti-peptide antibodies and HRV-neutralizing antibodies in BALB/c mice. Peptide antigen constructs were expressed in E. coli as fusion proteins with thioredoxin and a universal tetanus toxin T-cell epitope (P2), in order to enhance the anti-peptide immune response. The peptide antigen containing three tandem copies of the VP8 epitope induced significantly higher levels of anti-peptide antibody than only a single copy of the epitope, or the peptide co-administered with the carrier protein and T-cell epitope. Furthermore, the peptide antigen containing three copies of the peptide produced significantly higher virus-neutralization titres, higher than VP8, indicating that a peptide antigen displaying repeating copies of the amino acid region 1-10 of VP8 is a more potent inducer of HRV-neutralizing antibodies than VP8 alone, and may be useful for the production of specific neutralizing antibodies for passive immunotherapy of HRV infection.     

A single-amino-acid variant of the H60 CD8 epitope generates specific immunity with diverse TCR recruitment

TCR of CD8 T cells recognizes peptides of 8–9 amino acids in length (epitope) complexed with MHC class I. Peptide ligands differing from an epitope by one or two amino acids are thought to modulate the immune response specific to that epitope. H60 is a minor histocompatibility antigen for which the specific CD8 T-cell response dominates during alloresponse after MHC-matched allogeneic transplantation. In the present study, we developed a transgenic mouse (designated H60H Tg) expressing a variant of H60, designated H60H, in which the arginine residue at position 4 of the H60 epitope sequence (LTFNYRNL) is replaced by a histidine residue (LTFHYRNL). Immunization of female C57BL/6 mice with splenocytes from male H60H Tg induced a CD8 T cell primary response and memory response after re-challenge. The response was CD4 help-dependent, demonstrating the potency of H60H as a cellular antigen. The response induced by the H60H cellular antigen was comparable to that induced by H60 in its peak magnitude and overall immune kinetics. H60H challenge recruited broadly diverse TCRs to the specific response, shaping a TCR repertoire different from that of the natural H60 epitope. However, some of the TCRs did overlap between the H60H- and H60-specific CD8 T cells, suggesting that H60H might modulate the H60-specific response. These results may provide a basis for the modulation of the H60-specific CD8 T-cell response.     

The class 1 outer membrane protein of Neisseria meningitidis: gene sequence and structural and immunological similarities to gonococcal porins

The class 1 protein is a major protein of the outer membrane of Neisseria meningitidis, and an important immunodeterminant in humans. The complete nucleotide sequence for the structural gene of a class 1 protein has been determined. The sequence predicts a protein of 374 amino acids, preceded by a typical signal peptide of 19 residues. The hydropathy profile of the predicted protein sequence resembles that of the Escherichia coli and gonococcal porins. The predicted protein sequence of the class 1 protein exhibits considerable structural similarity to the gonococcal porins PIA and PIB. Western blot studies also reveal immunologically conserved domains between the class 1 protein, PIA and PIB. A restriction fragment from the class 1 gene hybridizes to gonococcal genomic fragments in Southern blots. In addition to the class 1 gene coding region there is a large open reading frame on the opposite strand.