Developing subunit immunogens using B and T cell epitopes and their constructs derived from the F1 antigen of Yersinia pestis using novel delivery vehicles

Yersinia pestis is the etiological agent of pneumonic and bubonic plague. As the currently licensed vaccines for plague have their own limitations, there is a need for a rational and more effective form of a subunit vaccine to combat both forms of the disease. Newer methods of antigen delivery coupled with adjuvant offer an alternative approach toward a plague vaccine. In order to develop a new generation vaccine against plague, we chose an immunodominant, outer membrane capsular protein, F1 of Y. pestis. The immunogenicity of the peptide sequences, predicted to possess B (three sequences, B1, B2 and B3) and T (two sequences, T1 and T2) cell determinants, was studied in a murine model with different genetic backgrounds, using alhydrogel and liposomes as delivery vehicles. All the peptide sequences are immunogenic in all mouse strains and showed primary and secondary immune response. B2 peptide was found to be most immunogenic, followed by B1 and B3 peptides. Chimeras made between B and T structures proved highly immunogenic and the antibody levels are comparable with native F1 antigen, thereby proving that T1 and T2 are helper sequences. Interestingly, the liposome mode of immunization was found to be more immunogenic and generated higher affinity antibodies than the alum-based preparation. Immunization using a mixture of all the peptides further proved B2 to be immunodominant. The IgG isotype profile showed predominance of IgG1, IgG2b followed by IgG2a for all the formulations irrespective of mode of antigen delivery. Lymphocyte proliferation of spleen cells primed in vivo with peptides, B-T conjugates and F1 antigen followed by in vitro stimulation with these antigens in soluble (medium) and particulate (liposome) form, showed dose-dependent stimulation of T cells, while B-T constructs showed a higher stimulation index, comparable to F1 antigen. The liposome mode of antigen presentation showed higher lymphoproliferation of spleen cells. Of all the peptides tested, T1 and T2 sequences showed the highest stimulation indices. The pattern of cytokine levels was in the following order: interferon-gamma>interleukin-2>interleukin-4. In vivo protective studies of the B-T conjugates revealed that B1T1 and a mixture of conjugates showed a survival rate of 10 days. Thus, the study highlights the importance of B and T cell epitopes as peptide-based immunogens, being a serious alternative for plague vaccine.     

The fimA gene encoding the type-1 fimbrial subunit of Escherichia coli. Nucleotide sequence and primary structure of the protein

The nucleotide sequence was determined of a region of 1450 base pairs encompassing the fimA gene for the subunit of type 1 fimbriae of Escherichia coli as well as flanking regions containing potential regulator sequences. The 'translated' protein contains a 23-residue signal peptide; the processed fimbrial subunit consists of 158 amino acid residues yielding a relative molecular mass of 15706. The elucidated sequence shows significant homology with those of other E. coli fimbrial proteins.     

鼠疫耶尔森菌F1抗原的性质研究

鼠疫菌F1抗原是鼠疫亚单位新疫苗最重要的候选抗原,对其性质的充分认识,将有助于抗原制造工艺和新疫苗的开发。F1抗原的性质研究包括:微观结构,一级核苷酸、氨基酸序列,二级结构,高分子聚集形态,以及F1抗原的理化性质。     

Nucleotide sequence and structural organization of Yersinia pestis insertion sequence IS100

Abstract The L1 major protein of human papillomavirus type 16 was expressed in Sf-21 insect cells with a recombinant baculovirus vector. Virus-like particles similar in appearance to empty verions were identified by electron microscoy at densities of 1.29–1.30. Purified particles reacted with monoclonal anti-HPV-16-L1 antibody in Western blot and immuno dot blot suggesting that conformational epitopes are present in the recombinant particles. Immunodot blot assays using human sera correlated with the detection of HPV-16 DNA by the polymerase chain reaction. The results suggest that HPV-16-L1 virions produced by the baculovirus system might be useful for developing serologic tests to measure antibodies to conformational epitopes and may offer potential for vaccine development.     

Identification of immunodominant epitope of F1 antigen of Yersinia pestis

The F1 antigen of Yersinia pestis has been identified as one of the major protective antigens of this bacterium. The present study aims to delineate major and minor antigenic sites of F1 antigen. Using algorithmic predictions, five peptide sequences (P1, P2, P3, P4 and P5) spanning the C-terminal region were identified and synthesized. Antibodies were generated in mice against the peptides, native F1 protein and polymerized F1 antigen using liposomes as mode of immunization. Cross-reactivity between F1 antigen and peptides was tested using both solid and solution phase assays. Similar assays were done with rabbit anti-F1 sera. Competitive inhibition assays using a different combination of antisera and competing antigen identified P2 peptide FFVRSIGSKGGKLAAGKYTDAVTV (142-165) as the immunodominant sequence. The results indicate that this sequence appears to be exposed on the surface of F1 molecule. In a solid phase binding assay, P2 peptide was recognized even at high F1 antisera dilution. However, when antisera raised to different peptides were tested for binding to F1 antigen, antisera to P4 peptide showed maximal immunoreactivity. This implies more accessibility of this region during immobilization on solid surface. There was consistency in the results obtained for different strains of mice as well as for the rabbit antisera. Such a sequence of F1 antigen, which is recognized widely in animals of different genetic background, would be useful for diagnosis and subunit vaccine.     

Nucleotide sequence of the gene encoding respiratory syncytial virus matrix protein.

The amino acid sequence of the matrix protein of the human respiratory syncytial virus (RS virus) was deduced from the sequence of a cDNA insert in a recombinant plasmid harboring an almost full-length copy of this gene. It specifically hybridized to a single 1,050-base mRNA from infected cells. The recombinant containing 944 base pairs of RS viral matrix protein gene sequence lacked five nucleotides corresponding to the 5' end of the mRNA. The nucleotide sequence of the 5' end of the mRNA was determined by the dideoxy sequencing method and found to be 5' NGGGC, wherein the C residue is one nucleotide upstream of the cloned viral sequence. The initiator ATG codon for the matrix protein is embedded in an AATATGG sequence similar to the canonical PXXATGG sequence present around functional eucaryotic translation initiation codons. There is no conserved sequence upstream of the polyadenylate tail, unlike vesicular stomatitis virus and Sendai virus, in which four nucleotides upstream of the polyadenylate tail are conserved in all genes. There is no equivalent of the eucaryotic polyadenylation signal AAUAAA upstream of the polyadenylate tail. The matrix protein of 28,717 daltons has 256 amino acids. It is relatively basic and moderately hydrophobic. There are two clusters of hydrophobic amino acid residues in the C-terminal third of the protein that could potentially interact with the membrane components of the infected cell. The matrix protein has no homology with the matrix proteins of other negative-strand RNA viruses, implying that RS virus has undergone extensive evolutionary divergence. A second open reading frame potentially encoding a protein of 75 amino acids and partially overlapping the C terminus of the matrix protein was also identified.     

Nucleotide sequence of the gene encoding mouse transition protein 2

The gene encoding the testis-specific basic chromosomal protein, mouse transition protein 2, is split by a single small intron that falls between the first and second nucleotides of a codon. Since the genes encoding protamines 1 and 2 and transition protein 1 in mammals contain a single intron in the same position, protamines and transition proteins appear to be evolutionarily related.     

Nucleotide sequence of the gene encoding adenovirus type 2 DNA binding protein.

We have determined the nucleotide sequence of the gene encoding adenovirus type 2 (Ad2) DNA binding protein (DBP). From the nucleotide sequence the complete amino acid sequence of Ad2 DBP has been deduced. A comparison of the amino acid sequences of Ad2 and Ad5 DBP, both 529 residues long, reveals that the C-terminal 354 residues of both sequences are identical. Within the N-terminal 175 amino acid residues Ad2 and Ad5 show nine differences. The site of mutation in Ad2 ND1ts23, a mutant with a temperature-sensitive DNA replication, was mapped at the nucleotide level. A single nucleotide alteration in the DBP gene, resulting in a leucine leads to phenylalanine substitution at position 282 in the amino acid sequence is responsible for the temperature-sensitive character of this mutant. Previously, we localized the mutation of another DBP mutant with a temperature-sensitive DNA replication (H5ts125) at position 413 in the amino acid sequence of the DBP molecule (Nucleic Acids Res. 9 (1981) 4439-4457). These mapping data are discussed in relation to the structure and function of the DBP molecule.     

重组鼠疫菌F1抗原在大肠杆菌中的表达及免疫原性分析

利用基因重组技术,用pET42(b+)质粒在大肠杆菌DE3中表达鼠疫菌F1抗原。经分析rF1抗原基因序列与天然F1抗原结构基因序列完全一致,电泳扫描测其表达量为25%:W estern B lot结果表明,rF1抗原可与F1特异性抗体相互作用,具有天然F1抗原的活性。用镍离子亲和层析纯化rF1抗原免疫BALB/c小鼠,在其血清中可检测到高滴度的抗F1抗体。     

Nucleotide sequence of the gene encoding the matrix protein of Newcastle disease virus.

The nucleotide sequence of the gene encoding the matrix (M) protein of the Beaudette C strain of Newcastle disease virus (NDV) has been determined from overlapping cDNA clones. Control sequences typical of paramyxovirus mRNA start and polyadenylation signals have been identified. Assuming that the M gene starts and finishes at these sequences, the M gene is 1241 nucleotides long and encodes one long open reading frame of 364 amino acids, corresponding to a polypeptide of molecular weight 39605, in good agreement with estimates from SDS gels. The M protein has an amino acid sequence that is both hydrophobic and highly basic. The NDV M protein has sequence homologies to the M proteins of Sendai, measles, canine distemper and respiratory syncytial viruses.     

Nucleotide sequence of the gene encoding the nitrogenase iron protein of Thiobacillus ferrooxidans.

The DNA sequence was determined for the cloned Thiobacillus ferrooxidans nifH and part of the nifD genes. A putative T. ferrooxidans nifH promoter was identified whose sequences showed perfect consensus with those of the Klebsiella pneumoniae nif promoter. Two putative consensus upstream activator sequences were also identified. The amino acid sequence was deduced from the DNA sequence. In a comparison of nifH DNA sequences from T. ferrooxidans and eight other nitrogen-fixing microbes, a Rhizobium sp. isolated from Parasponia andersonii showed the greatest homology (74%) and Clostridium pasteurianum (nifH 1) showed the least homology (54%). In a comparison of the amino acid sequences of the Fe proteins, the Rhizobium sp. and Rhizobium japonicum showed the greatest homology (both 86%) and C. pasteurianum (nifH 1 gene product) demonstrated the least homology (56%) to the T. ferrooxidans Fe protein.     

Nucleotide sequence of gene PBII encoding salivary proline-rich protein P-B

The nucleotide sequence of gene PBII encoding salivary proline-rich protein P-B was determined. PBII is 7.1 kb long and contains 3 exons. PBII exhibits considerable nucleotide sequence homology not only in exons but also in introns with PBI (accession number D89501), the gene whose nucleotide sequence was determined previously [Isemura and Saitoh (1997) J. Biochem. 121, 1025-1030]. PBI and II constitute a gene family distinct from that to which the majority of salivary proline-rich protein ones belong. The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession number AB031740.