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

OBJECTIVETo further study the therapy of wasting muscle by myostatin as a new targets, the eucaryotic expression vector coupled the foreign T-helper epitope of tetanus toxin (TT) to the N terminus of myostatin was constructed, and the effects of the gene vaccine on forelimb grip were tested in immunized mice.

METHODSA DNA fragment encoding the TT epitope followed by the N terminus of mature myostatin (330bp) was synthesized. The eucaryotic expression vector of myostatin was constructed and the chinese hamster ovary (CHO) cells were infected with the recombinant plasmids pVAC-TT-Ms by liposome transfection according to routine laboratory procedure. The myostatin expression was tested by cell immunofluorescence technique in transfected CHO. The forelimbs grip were tested in immunized mice with myostatin gene vaccine.

RESULTSThe eucaryotic expression vector of myostatin coupled TT epitope was constructed successfully through the restriction analysis and sequencing. The recombinant plasmids pVAC-TT-Ms met quality criterion as gene vaccine by analysis OD260/280 and electrophoresis. The myostatin expression was detected obviously in transfected CHO. The forelimb grip in immunized mice had an obvious increase. The average value of forelimb grip of the mice immunized with pVAC-TT-Ms was about 29.88% greater than that of control mice.

CONCLUSIONThe construction of eucaryotic expression vector of myostatin coupled TT epitope is successful in expression for recombinant human mature peptide of myostatin. The gene vaccine of myostatin meet quality criterion. The immunized mice has an obvious increase in forelimb grip.

Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Epitopes, T-Lymphocyte ; Genetic Vectors ; Hand Strength ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Muscle, Skeletal ; physiology ; Myostatin ; genetics ; immunology ; Plasmids ; Transfection ; Vaccines, DNA ; genetics ; immunology

We investigated the enhanced immune response of a recombinant T cell immunogen as an effective cellular immune adjuvant. The T cell immunogen named TI contained several T cell epitopes from the VP1, VP4, 3A and 3D proteins of foot-and-mouth disease virus (FMDV) and two pan-T helper (T(H)) cell sites to broaden the immunogenicity of the protein. Meanwhile, another fusion protein named OA-VP1 was expressed in bacteria, which contained two VP1 proteins of O and Asia1 type FMDV. Mice were vaccinated with commercially inactivated vaccine or OA-VP1 protein with or without the TI immunogen. The results show that mice inoculated with inactivated vaccine or OA-VP1 protein supplemented with TI immunogen produced significantly higher level of neutralizing antibodies (P < 0.01 or P < 0.05) than the mice only inoculated with inactivated vaccine or OA-VP1 protein by microneutralization assay. An obvious increase in T cell number by flow cytometric analysis and significantly higher concentration of IFN-gamma secreted in culture media of spleen lymphocytes were observed in groups supplemented with TI immunogen (P < 0.01). TI immunogen was an effective stimulator for humoral and cellular immunity and could help improve the immunogenicity of inactivated vaccine or protein subunit vaccine.
Adjuvants, Immunologic ; pharmacology ; Animals ; Capsid Proteins ; genetics ; immunology ; Epitopes, T-Lymphocyte ; genetics ; immunology ; Foot-and-Mouth Disease ; immunology ; prevention & control ; virology ; Foot-and-Mouth Disease Virus ; immunology ; Immunization ; Mice ; Viral Vaccines ; genetics ; immunology ; pharmacology

The purpose is to screen and identify the specific H-2d restricted T-cell epitopes. These epitopes are used to investigate the cellular immune response of BALB/c (H-2d) mice immunized with a HIV-1 vaccine which expresses six antigens of gp160, gag, pol, rev, tat and nef of HIV subtype B'/C. A replicating DNA vaccine and a non-replicating recombinant vaccinia virus vector, both expressing the six antigens mentioned above, were used to immune BALB/c (H-2d) mice in a prime-boost regiment. The six peptide libraries of HIV B'/C corresponding respectively to the six complete antigens were pooled according to a designed matrix format and used to test for IFN-gamma production from splenocytes of immunized mice by an enzyme-linked immunospot (IFN-gamma ELISPOT) assay. The ELISPOT data indicated that two of seven Gag-specific T-cell epitope peptides were identified to be the novel epitopes. One of three Pol-specific T-cell epitope is unreported. One novel epitope was confirmed in two gp160-specific T-cell epitope peptides. One Nef-specific T-cell epitope was identified. Three Tat-specific T-cell epitope peptides were continuous sequences in Tat peptide library and all contained either complete or partial sequence reported. Rev-specific T-cell epitope was not be found. The specific T-cell epitopes (H-2d restricted) were identified by IFN-7 ELISPOT assay, which could be used to detect the cellular immune response of BALB/c mice immunized with the HIV-1 vaccine expressing six antigens of gp160, gag, pol, rev, tat and nef of HIV subtype B'/C.
Amino Acid Sequence ; Animals ; Enzyme-Linked Immunospot Assay ; methods ; Epitopes, T-Lymphocyte ; chemistry ; genetics ; immunology ; Female ; H-2 Antigens ; chemistry ; genetics ; immunology ; HIV Antigens ; chemistry ; genetics ; immunology ; HIV Infections ; immunology ; virology ; HIV-1 ; classification ; genetics ; immunology ; Histocompatibility Antigen H-2D ; Humans ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Peptide Mapping ; methods

Untreated human immunodeficiency virus (HIV) infections usually lead to death from AIDS, although the rate of the disease progression varies widely among individuals. The cytotoxic T lymphocyte (CTL) response, which is restricted by highly polymorphic MHC class I alleles, plays a central role in controlling HIV replication. It is now recognized that the antiviral efficacy of CTLs at the single cell level is dependent on their antigen specificity and is important in determining the quality of host response to viruses so that the individual will remain asymptomatic. However, because of the extreme mutational plasticity of HIV, HIV-specific CTL responses are continuously and dynamically changing. In order to rationally design an effective vaccine, the questions as to what constitutes an effective antiviral CTL response and what characterizes a potent antigenic peptide to induce such responses are becoming highlighted as needing to be answered.
Animals ; Antigens, Viral ; immunology ; metabolism ; Epitopes, T-Lymphocyte ; Evolution, Molecular ; Genetic Variation ; HIV Infections ; immunology ; virology ; HIV-1 ; genetics ; pathogenicity ; physiology ; Host-Pathogen Interactions ; Humans ; Immunodominant Epitopes ; T-Lymphocytes, Cytotoxic ; immunology ; metabolism ; virology ; Virus Replication

Amino Acid Sequence ; Epitopes, T-Lymphocyte ; immunology ; Forecasting ; HLA Antigens ; immunology ; Hepacivirus ; genetics ; immunology ; Hepatitis C ; immunology ; virology ; Hepatitis C Antigens ; immunology ; Humans ; T-Lymphocytes, Cytotoxic ; immunology ; virology ; Viral Hepatitis Vaccines ; immunology

The 23 open reading frame (ORF) 5 sequences of Korean type II porcine reproductive and respiratory syndrome virus (PRRSV) were collected from viremic sera from the (modified live vaccine) MLV-vaccinating and non-vaccinating farms from 2007 to 2008. The samples were phylogenetically analyzed with previous ORF5 sequences, including type I Korean PRRSV, and previously reported or collected sequences from 1997 to 2008. A MN184-like subgroup of type II Korean PRRSV was newly identified in the viremic sera collected from 2007 to 2008. And of the type I PRRSVs, one subgroup had 87.2~88.9% similarity with the Lelystad virus, showing a close relationship with the 27~2003 strain of Spain. The maximum parsimony tree of type II PRRSV from 1997 to 2008 showed that they had evolved to four lineages, subgroups 1, 2, 3 and 4. Most of the recently collected type II PRRSVs belonged to subgroup 4 (48%). The region of three B-cell epitopes and two T-cell epitopes of ORF5 amino acids sequences was considerably different from the MLV in subgroups 3 and 4. In conclusion, the existence of type I PRRSV, which was genetically different from Lelystad virus (Prototype of type I PRRSV), and heterologous type II PRRSVs of viremic pigs detected even in the MLV-vaccinating farms indicated the need for new vaccine approaches for the control of PRRSV in Korea.
Animals ; Epitopes, B-Lymphocyte/immunology ; Epitopes, T-Lymphocyte/immunology ; Evolution, Molecular ; Korea ; *Open Reading Frames ; Phylogeny ; Pilot Projects ; Porcine Reproductive and Respiratory Syndrome/blood/genetics/immunology/*virology ; Porcine respiratory and reproductive syndrome virus/*genetics/immunology ; RNA, Viral/chemistry/genetics ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Swine ; Viral Vaccines/immunology/standards ; Viremia/genetics/immunology/virology

OBJECTIVETo predict and screen the efficient antigenic epitopes in genus-specific envelope protein LipL41 of Leptospira interrogans and to determine the immunoreactive diversity of LipL41s from different genotypes.

METHODSBioinformatic methods were applied to predict the T/B combined epitope candidates in LipL41/1 and LipL41/2 molecules. The nucleotide fragments encoding epitopes were amplified by PCR. Phage display system with SDS-PAGE was performed to obtain the recombinant PIIIs containing different T/B combined epitopes. Western Blot assays were performed to determine the immunoreactivity of recombinant PIIIs to various antisera including antiserum against rLipL41/1, rLipL41/2 and whole cell of L.interrogans strain Lai, and serum from patients with leptospirosis.

RESULTBased on the predicting data, eight common or differential combined epitopes in LipL41s were selected. The nucleotide fragments encoding the epitopes were obtained by PCR. All the T/B combined epitope fragments were correctly inserted into the N end of phage PIII protein and then successfully expressed. All the antisera were able to recognize each of the epitopes but the hybridization signal intensity was different. Among these epitopes, the common T/B combined epitopes LipL41/1-30 and LipL41/1-233 showed a stronger and stable hybridization signals.

CONCLUSIONAll 8 selected T/B combined epitopes in the study are the efficient antigenic epitopes. The common T/B epitopes LipL41/1-30 and LipL41/1-233 can be first used in development of leptospiral MAP vaccine. The cross immunoreaction is between the differential T/B epitopes LipL41s-89,LipL41s-299 and the different antisera.

Amino Acid Sequence ; Antigens, Bacterial ; genetics ; immunology ; Bacterial Vaccines ; genetics ; immunology ; Cloning, Molecular ; Epitopes, B-Lymphocyte ; genetics ; immunology ; Epitopes, T-Lymphocyte ; genetics ; immunology ; Genotype ; Molecular Sequence Data ; Peptide Library

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

BACKGROUNDImmunoglobulin heavy chain variable region (IgHV) is a well-characterized tumor antigen for B-cell malignancies. It can function as a target for T cell-mediated immune response. Clinical trials of IgHV protein vaccines against lymphoma have demonstrated induction of tumor-specific cytotoxic T lymphocyte (CTL) responses. However, complementary determining regions-based individual vaccines have disadvantages for wide clinical application. Although a recent study demonstrated that immunogenic peptides are derived from framework regions (FR) shared among patients with B-cell lymphoma, how to choose the appropriate peptides for each patient is still unsolved. The aim of this study was to investigate whether immunoglobulin heavy chain FR-derived peptides shared in each IgHV family are potential CTL epitopes presented by B-cell acute lymphoblastic leukemia (B-ALL). Such CTL epitopes might be beneficial to shifting vaccination strategies against B-ALL from individual specificity to family specificity.

METHODSSeven IgHV gene families were amplified respectively by PCR and sequenced directly from 71 childhood B-ALL cases. Bioinformatics was applied in analyzing characteristics of sequences available and predicting HLA-A*0201-restricted CTL epitopes for each IgHV family. An antigen-specific T cell expansion system was used to generate peptide-specific CTLs. The cytotoxicity of CTLs against B-ALL cells was assessed in the lactate dehydrogenase release assay.

RESULTSComplete IgHV rearrangements were identified in all of the 71 B-ALL cases. All of 40 sequences available showed > or = 98% homology with the nearest germline IgHV genes, indicating IgHV genes in B-ALL of germline nature. Twelve nonapeptides of high HLA-A*0201-binding scores were obtained from 26 productive IgHV protein sequences. Ten (83%) of the peptides were located in FR1 and FR3 shared among the corresponding IgHV family. CTLs specific for the peptide QLVQSGAEV located in FR1 (3 - 11) shared among the IgHV1 family could be successfully generated from peripheral blood mononuclear cells of two HLA-A*0201 + healthy donors in vitro and were capable of killing HLA-matched B-ALL cell clones belonging to the IgHV1 family.

CONCLUSIONAnti-B-ALL CTLs against immunoglobulin heavy chain FR-derived peptides have family-specific cytotoxicity.

Amino Acid Sequence ; Burkitt Lymphoma ; genetics ; immunology ; Epitopes, T-Lymphocyte ; genetics ; immunology ; Genes, Immunoglobulin Heavy Chain ; genetics ; Humans ; Immunoglobulin Heavy Chains ; chemistry ; genetics ; immunology ; Immunoglobulin Variable Region ; chemistry ; genetics ; immunology ; Molecular Sequence Data ; Oligopeptides ; immunology ; Polymerase Chain Reaction ; Protein Binding ; T-Lymphocytes, Cytotoxic ; immunology