PURPOSE: To identify new immunogenic HLA-A*33;03-restricted epitopes from the human papillomavirus (HPV) 16 E7 protein for immunotherapy against cervical cancer. MATERIALS AND METHODS: We synthesized fourteen overlapping 15-amino acid peptides and measured intracellular interferon-γ (IFN-γ) production in PBMC and CD8+ cytotoxic T lymphocytes (CTLs) after sensitization with these peptides using flow cytometry and ELISpot assay. The immunogenicity of epitopes was verified using a ⁵¹Cr release assay with SNU1299 cells. RESULTS: Among the fourteen 15-amino acid peptides, E7₄₉₋₆₃ (RAHYNIVTFCCKCDS) demonstrated the highest IFN-γ production from peripheral blood mononuclear cells (PBMCs), and CD8+ CTLs sensitized with E7₄₉₋₆₃ showed higher cytotoxic effect against SNU1299 cells than did CD8+ CTLs sensitized with other peptides or a negative control group. Thirteen 9- or 10-amino acid overlapping peptides spanning E7₄₉₋₆₃, E7₅₀₋₅₉ (AHYNIVTFCC), and E7₅₂₋₆₁ (YNIVTFCCKC) induced significantly higher IFN-γ production and cytotoxic effects against SNU1299 cells than the other peptides and negative controls, and the cytotoxicity of E7₅₀₋₅₉- and E7₅₂₋₆₁-sensitized PBMCs was induced via the cytolytic effect of CD8+ CTLs. CONCLUSION: We identified E7₅₀₋₅₉ and E7₅₂₋₆₁ as novel HPV 16 E7 epitopes for HLA-A*33;03. CD8+ CTL sensitized with these peptides result in an antitumor effect against cervical cancer cells. These epitopes could be useful for immune monitoring and immunotherapy for cervical cancer and HPV 16-related diseases including anal cancer and oropharyngeal cancer.
Amino Acid Sequence ; CD8-Positive T-Lymphocytes/immunology/metabolism ; Epitopes/*immunology/therapeutic use ; Female ; *HLA-A Antigens ; Human papillomavirus 16/*immunology ; Humans ; *Immunotherapy ; Interferon-gamma/analysis/*biosynthesis ; Leukocytes, Mononuclear/immunology/metabolism ; T-Lymphocytes, Cytotoxic/immunology/metabolism ; Uterine Cervical Neoplasms/*therapy

Swine influenza viruses (SwIVs) cause considerable morbidity and mortality in domestic pigs, resulting in a significant economic burden. Moreover, pigs have been considered to be a possible mixing vessel in which novel strains loom. Here, we developed and evaluated a novel M2e-multiple antigenic peptide (M2e-MAP) as a supplemental antigen for inactivated H3N2 vaccine to provide cross-protection against two main subtypes of SwIVs, H1N1 and H3N2. The novel tetra-branched MAP was constructed by fusing four copies of M2e to one copy of foreign T helper cell epitopes. A high-yield reassortant H3N2 virus was generated by plasmid based reverse genetics. The efficacy of the novel H3N2 inactivated vaccines with or without M2e-MAP supplementation was evaluated in a mouse model. M2e-MAP conjugated vaccine induced strong antibody responses in mice. Complete protection against the heterologous swine H1N1 virus was observed in mice vaccinated with M2e-MAP combined vaccine. Moreover, this novel peptide confers protection against lethal challenge of A/Puerto Rico/8/34 (H1N1). Taken together, our results suggest the combined immunization of reassortant inactivated H3N2 vaccine and the novel M2e-MAP provided cross-protection against swine and human viruses and may serve as a promising approach for influenza vaccine development.
Animals ; Antibodies, Viral/blood ; Antigens, Viral/genetics/*immunology ; Body Weight ; Cross Protection/*immunology ; Disease Models, Animal ; Epitopes, T-Lymphocyte/genetics/immunology ; Female ; Influenza A Virus, H3N2 Subtype/genetics/*immunology ; Influenza Vaccines/*immunology ; Mice ; Mice, Inbred BALB C ; Orthomyxoviridae Infections/*immunology/mortality/pathology/prevention & control ; Peptides/genetics/*immunology ; Random Allocation ; Survival Analysis ; Vaccines, Synthetic/immunology ; Virus Replication

To study the CTL antigen epitopes and drug resistance mutations of HIV-1 gag and pol genes through analyzing gag and pol gene sequences. The HIV-1 gag and pol gene fragments were amplified using nested polymerase chain reaction. A total of 23 PCR sequences, 449 cloned gag sequences and 402 cloned pol sequences were obtained. Sequence analyses showed the 23 samples were subtype B or B'. A total of 4 in 8 CTL antigen epitopes appeared 8 mutations in consensus sequence of subtype B and B'. There were no mutations found in the PCR sequences, whereas a few mutations were found in clone sequences (9.80%) in 5 antigen epitopes in p24 region. Eighteen PIs-related mutations and 24 RTIs-related mutations were found in PCR sequences and clone sequences in pol gene region, in which 17 (94.44%) PIs-related mutations and 15 (62.50%) RTIs-related mutations were found only in the clone sequences, respectively. The results showed that the prevalence of HIV-1 drug resistance strains in this study was at a higher level (17.39%), suggesting that some samples were resistant.to existing antiviral drugs.
Antigens, Viral ; immunology ; DNA Mutational Analysis ; Drug Resistance, Viral ; genetics ; Epitopes ; immunology ; HIV-1 ; classification ; drug effects ; genetics ; immunology ; Human Immunodeficiency Virus Proteins ; genetics ; Mutation ; Phylogeny ; T-Lymphocytes, Cytotoxic ; immunology ; gag Gene Products, Human Immunodeficiency Virus ; genetics ; pol Gene Products, Human Immunodeficiency Virus ; genetics

To evaluate the effectiveness of rabies vaccination, we developed the SPA-ELISA method to detect the antibodies against rabies virus (RV) using the main antigenic determinant of nucleoprotein (RV N1) as antigen. The complete Nucleoprotein (N) gene and the partial N1 gene (1 000-1 353 bp) of RV Flury LEP strain were amplified using RT-PCR and PCR approaches. The two fragments were inserted into pGEX-6P-1 respectively. Then we transformed the recombinant plasmids into Escherichia coli BL21(DE3) strain and expressed them by adding 1 mmol/L of IPTG (isopropyl-beta-D-thiogalactopyranoside). SDS-PAGE analysis showed that both of the two recombinant proteins were presented as inclusion bodies. Compared with the complete nucleoprotein, the partial protein (RV N1) was expressed at a much higher level in E. coli BL21(DE3). The antigenic specificity of the partial N1 protein was confirmed by Western blotting. By coating the plates with purified RV N1 as an antigen, an SPA-ELISA method for the detection of the antibodies against RV was established. By optimizing this method, the optimal concentration of RV N1 coating the ELISA plate was 2 mg/L. The optimal concentration of serum samples and SPA-HRP was 1:100 and 1:4 000 respectively. Compared with a commercially available ELISA kit coating RV as antigen, the coincidence rate of SPA-ELISA was 94.1%. Our results show that the developed SPA-ELISA based on the RV N1 was useful for the detection of the antibodies against RV in the sera of dogs.
Animals ; Antibodies, Viral ; analysis ; immunology ; Dogs ; Enzyme-Linked Immunosorbent Assay ; methods ; Epitopes ; immunology ; Escherichia coli ; genetics ; Genetic Vectors ; genetics ; Nucleocapsid Proteins ; immunology ; Rabies virus ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Staphylococcal Protein A

To understand the HA1 genetic variation characterization of influenza H3N2 virus isolates in Zhu-hai during 2008-2009, we selected 20 of H3N2 Influenza strains cultured in MDCK cell. Viral RNAs were extracted and amplified by using RT-PCR. The amplified products were purified after identified by gel electrophoresis and then the nucleotide sequences of the amplicons were determined. The results were analyzed by the software ClustalX and MEGA4. 1. When compared with the amino acid sequences of the epitopes of HA1 district of H3N2 influenza vaccine recommended by WHO in 2008, changes were found in those of H3N2 influenza strains in Zhuhai in 2008: K140I in all of H3N2 influenza strains, L157S in 08-0343 and 08-0677, K158R in 08-0466, 08-0620 and 08-0667, K173E in 08-0466 and 08-0620, K173N in 08-0667, and I192T in 08-0667. The epitopes of HA1 district of H3N2 influenza strains in Zhuhai in 2009 are different from that of H3N2 influenza vaccine during the same time: K173Q and P194L occur in all of H3N2 influenza strains, N144K, K158N, and N189K occur in the strains except the strain 09-0056. HA1 domain of H3N2 influenza strains in 2009 has lost a glycosylation site at amino acid position 144 while the glycosylation sites of HA1 domain of H3N2 influenza stains isolated in 2008 remained. This study suggested that H3N2 influenza virus in Zhuhai in 2008 was not evolved a novel variant and H3N2 influenza variant in 2009 was attributed to antigenic drift in HA1 district.
Animals ; Antigens, Viral ; immunology ; Cell Line ; China ; Dogs ; Epitopes ; immunology ; Glycosylation ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; immunology ; metabolism ; Humans ; Influenza A Virus, H3N2 Subtype ; classification ; genetics ; immunology ; isolation & purification ; Mutation ; Phylogeny ; Sequence Analysis, DNA

OBJECTIVETo prepare monoclonal antibody (mAb) against prM epitope.

METHODSThe gene encoding prM was isolated using RT-PCR from brain of JEV infected mouse and cloned into prokaryotic expression vector pET-32a. Recombinant plasmid was transformed into E.coli BL21/DE3/LysS, then the transformed cells were expressed with the induction of IPTG. The expression and purification of the prM protein was analyzed by SDS-PAGE. The BALB/c mice were immunized with purified prM protein. Hybridoma cell lines secreting monoclonal antibodies against prM were established after cell fusion of mouse splenic cell and P3-X63-Ag8.653 cells. The specificity of mAb was identified by ELISA, Western Blot and Immunohistochemistry assay.

RESULTSmAb against prM epitope of JEV was prepared successfully.

CONCLUSIONThe obtained prM specific mAb was valuable for the prevention and dignosis of Japanese encephalitis.

Animals ; Antibodies, Monoclonal ; analysis ; immunology ; isolation & purification ; Antibody Specificity ; BALB 3T3 Cells ; Cell Line ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Encephalitis Virus, Japanese ; genetics ; immunology ; Epitopes ; immunology ; Escherichia coli ; genetics ; Mice ; Plasmids ; genetics ; metabolism ; Prokaryotic Cells ; metabolism ; Sequence Analysis, DNA ; Viral Proteins ; biosynthesis ; genetics ; immunology ; isolation & purification

OBJECTIVETo study the characteristics of the virology background of HLA-A2 restricted HBcAg(18-27) epitope mutations in HBV infected patients in China.

METHOD30 HBV sequences with different genotypes from Genbank were analyzed by bioinformatics and the mismatched primers were designed for constructing a PCR-RFLP method to screen HBcAg(18-27)V/I in China. The distributions of HBcAg(18-27)V/I of 160 samples with HBV genotype B/C infection from 8 areas in China were screened and analyzed by PCR-RFLP and sequencing. The affinity of HBcAg(18-27)V/I to HLA-A0201 was analyzed through referencing the bioinformatics websites.

RESULTSWe successfully constructed a PCR-RFLP method for screening HBcAg(18-27)V/I from genotype B/C, and only 3 samples with HBcAg(18-27)V sequence were found in the 160 samples (3/160, 1.88%). The affinity of HBcAg(18-27)I to HLA-A 0201 was lower than the one of HBcAg(18-27)V through bioinformatic analysis (HLA ligand score was 123 vs 156, and the SYFPEITHI score was 22 vs 24).

CONCLUSIONThe last amino acid of most HBcAg(18-27) sequences of epidemic HBV strains in China is isoleucine, and not valine. Therefore HBcAg(18-27) sequence background in different HBV genotypes should be thoroughly considered when using it as a reference or control in immunological research about HBV.

Adult ; China ; epidemiology ; Computational Biology ; DNA, Viral ; genetics ; Epitopes, T-Lymphocyte ; immunology ; Female ; Genotype ; HLA-A Antigens ; immunology ; Hepatitis B Core Antigens ; genetics ; immunology ; Hepatitis B virus ; classification ; immunology ; Hepatitis B, Chronic ; epidemiology ; immunology ; virology ; Humans ; Male ; Mutation ; Sequence Analysis, DNA ; T-Lymphocytes, Cytotoxic ; immunology

OBJECTIVETo identify a naturally presented HLA-A2-restricted epitope of MAGE-A3 antigen, FLWGPRALV (MAGE-A3(271 - 279)), on the surface of a human hepatocellular carcinoma (HCC) cell line HLE.

METHODSSynthetic peptide FLWGPRALV, served as positive control target, was analyzed by HPLC and HPLC-ESI-TOF-MSMS, in order to determine its HPLC elution time, mass-spectrometric characteristics and the lowest detection limitation by the two approaches. 3 x 10(9) HLE cells were collected, peptides naturally presented by major histocompatibility complex (MHC) molecules on the cell surface were isolated by mild acid elution, and concentrated by lyophilization, then the mixtures of peptides were fractioned by HPLC. The ingredient ranged from 2 min before the elution time determined by the synthetic peptide to 2 min after that was collected, concentrated by lyophilization, and analyzed by HPLC-ESI-TOF-MSMS, to identify the existence of the MAGE-A3(271 - 279) peptide.

RESULTSThe HPLC-ESI-TOF-MSMS detection provided an evidence for the existence of a doubly charged ion of (m/z)(2) 529.9, which was further analyzed by collision induced dissociation. The doubly charged ion was ultimately identified as the MAGE-A3(271 - 279) peptide, its amino sequence was FLWGPRALV and its molecular weight was 1058.4 Da.

CONCLUSIONSMAGE-A3(271 - 279) epitope could be naturally presented by HLA-A2 molecules to the surface of HCC cell line and MAGE-A3(271 - 279) peptide may have potential immunotherapeutic value in HCC patients.

Amino Acid Sequence ; Antigen Presentation ; Antigens, Neoplasm ; analysis ; isolation & purification ; Carcinoma, Hepatocellular ; immunology ; pathology ; Cell Line, Tumor ; Chromatography, High Pressure Liquid ; Epitopes, T-Lymphocyte ; analysis ; isolation & purification ; HLA-A2 Antigen ; immunology ; Humans ; Liver Neoplasms ; immunology ; pathology ; Mass Spectrometry ; Neoplasm Proteins ; analysis ; isolation & purification

Amino Acid Sequence ; Amyloid beta-Peptides ; genetics ; immunology ; Enzyme-Linked Immunosorbent Assay ; Epitope Mapping ; Epitopes ; genetics ; immunology ; Humans ; Immunoglobulin Fragments ; genetics ; immunology ; Immunoglobulin Variable Region ; genetics ; immunology ; Molecular Sequence Data ; Peptide Library ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA

OBJECTIVETo screen the severe acute respiratory syndromes (SARS) mimotopes with random phage peptide library and to investigate their immunogenicity.

METHODSUsing SARS sera as selective molecule, a 12 mer phage peptide library was biopanned and positive clones containing the mimic epitopes were selected. The immuno-characteriation of the epitopes were then investigated.

RESULTS2 positive clones that having specific affinity to SARS sera were obtained. The DNA sequencing data showed no homology between the sequences of the deduced amino acid of the two mimic antigen peptides and the sequence of SARS.

CONCLUSIONSARS mimotopes were obtained by phage peptide library screening. This method might provide a new approach for SARS therapy and vaccine development.

Animals ; Antigens, Viral ; Base Sequence ; Biomimetic Materials ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Epitopes ; genetics ; immunology ; Humans ; Peptide Library ; Sequence Analysis, DNA ; Severe Acute Respiratory Syndrome ; immunology