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

Hemagglutinin (HA) contains a head domain with a high degree of variability and a relatively conserved stem region. HA is the major viral antigen on the surface of the influenza virus. To define the biologic activities of chimeric HA bearing different head domains and stem regions or their potential use, a HA chimeric gene containing the head domain of the H7 subtype virus and stem region of the H3 subtype virus was modified and expressed using a baculovirus expression vector. Then, the secreted protein was purified and its biologic activities characterized. Approximately 1.4 mg/mL cH7/3 HA could be obtained, and its molecular weight was ≈ 70 kD. The trimer form of cH7/3 protein had hemagglutination activity and could be recognized by specific antibodies. The method described here can be used for further studies on the screening of HA stem-reactive antibodies or the development of vaccines with conserved epitopes.
Antibodies, Viral ; immunology ; Baculoviridae ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Hemagglutination ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; immunology ; Humans ; Influenza Vaccines ; genetics ; immunology ; Influenza, Human ; prevention & control ; virology

OBJECTIVETo prepare the 4 candidate vaccine strains of H5N1 avian influenza virus isolated in China.

METHODSRecombinant viruses were rescued using reverse genetics. Neuraminidase (NA) and hemagglutinin (HA) segments of the A/Xinjiang/1/2006, A/Guangxi/1/2009, A/Hubei/1/2010, and A/Guangdong/1/2011 viruses were amplified by RT-PCR. Multibasic amino acid cleavage site of HA was removed and ligated into the pCIpolI vector for virus rescue. The recombinant viruses were evaluated by trypsin dependent assays. Their embryonate survival and antigenicity were compared with those of the respective wild-type viruses.

RESULTSThe 4 recombinant viruses showed similar antigenicity compared with wild-type viruses, chicken embryo survival and trypsin-dependent characteristics.

CONCLUSIONThe 4 recombinant viruses rescued using reverse genetics meet the criteria for classification of low pathogenic avian influenza strains, thus supporting the use of them for the development of seeds and production of pre-pandemic vaccines.

Animals ; Chick Embryo ; Chickens ; China ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; metabolism ; Influenza A Virus, H5N1 Subtype ; immunology ; Influenza Vaccines ; immunology ; Influenza in Birds ; prevention & control ; virology ; Neuraminidase ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Vaccines, Synthetic ; immunology

Novel reassortant H3N2 swine influenza viruses (SwIV) with the matrix gene from the 2009 H1N1 pandemic virus have been isolated in many countries as well as during outbreaks in multiple states in the United States, indicating that H3N2 SwIV might be a potential threat to public health. Since southern China is the world's largest producer of pigs, efficient vaccines should be developed to prevent pigs from acquiring H3N2 subtype SwIV infections, and thus limit the possibility of SwIV infection at agricultural fairs. In this study, a high-growth reassortant virus (GD/PR8) was generated by plasmid-based reverse genetics and tested as a candidate inactivated vaccine. The protective efficacy of this vaccine was evaluated in mice by challenging them with another H3N2 SwIV isolate [A/Swine/Heilongjiang/1/05 (H3N2) (HLJ/05)]. Prime and booster inoculation with GD/PR8 vaccine yielded high-titer serum hemagglutination inhibiting antibodies and IgG antibodies. Complete protection of mice against H3N2 SwIV was observed, with significantly reduced lung lesion and viral loads in vaccine-inoculated mice relative to mock-vaccinated controls. These results suggest that the GD/PR8 vaccine may serve as a promising candidate for rapid intervention of H3N2 SwIV outbreaks in China.
Animals ; Female ; Influenza A Virus, H3N2 Subtype/*genetics/immunology ; Influenza Vaccines/genetics/immunology/*therapeutic use ; Mice ; Mice, Inbred BALB C ; Orthomyxoviridae Infections/immunology/*prevention & control/virology ; Reassortant Viruses/genetics/immunology ; Reverse Genetics/methods/*veterinary ; Swine ; Swine Diseases/immunology/*prevention & control/virology ; Vaccines, Inactivated ; Virus Replication

Infectious bronchitis virus (IBV) poses a severe threat to the poultry industry and causes heavy economic losses worldwide. Vaccination is the most effective method of preventing infection and controlling the spread of IBV, but currently available inactivated and attenuated virus vaccines have some disadvantages. We developed a chimeric virus-like particle (VLP)-based candidate vaccine for IBV protection. The chimeric VLP was composed of matrix 1 protein from avian influenza H5N1 virus and a fusion protein neuraminidase (NA)/spike 1 (S1) that was generated by fusing IBV S1 protein to the cytoplasmic and transmembrane domains of NA protein of avian influenza H5N1 virus. The chimeric VLPs elicited significantly higher S1-specific antibody responses in intramuscularly immunized mice and chickens than inactivated IBV viruses. Furthermore, the chimeric VLPs induced significantly higher neutralization antibody levels than inactivated H120 virus in SPF chickens. Finally, the chimeric VLPs induced significantly higher IL-4 production in mice. These results demonstrate that chimeric VLPs have the potential for use in vaccines against IBV infection.
Animals ; Antibodies, Viral/blood ; *Chickens ; Chimera/genetics/immunology ; Coronavirus Infections/prevention & control/*veterinary/virology ; Female ; *Immunity, Innate ; Infectious bronchitis virus/genetics/*immunology ; Influenza A Virus, H5N1 Subtype/genetics/immunology ; Injections, Intramuscular/veterinary ; Mice ; Mice, Inbred BALB C ; Neuraminidase/genetics ; Poultry Diseases/*prevention & control/virology ; Recombinant Fusion Proteins/genetics/immunology ; Spike Glycoprotein, Coronavirus/genetics/*immunology ; Vaccines, Synthetic/administration & dosage/genetics/immunology ; Vaccines, Virus-Like Particle/administration & dosage/genetics/*immunology ; Viral Proteins/genetics

This study aims to investigate the characteristics of genomic variation of pandemic A/H1N1/2009 influenza virus isolated in Fujian Province, China. Complete genome sequence analysis was performed on 14 strains of pandemic A/H1N1/2009 influenza virus isolated from Fujian during 2009-2012. All virus strains were typical low-pathogenic influenza viruses, with resistance to amantadine and sensitivity to neuraminidase inhibitors. Eight genome fragments of all strains were closely related to those of A/California/07/2009 (H1N1) vaccine strain, with > or = 98.2% homology. Compared with the vaccine strain, the influenza strains from Fujian had relatively large variation, and variation was identified at 11 amino acid sites of the HA gene of A/Fujiangulou/SWL1155/2012 strain, including 4 sites (H138R, L161I, S185T, and S203T) involved inthree antigen determinants (Ca, Sa, and Sb). In conclusion, the influenza vaccine has a satisfactory protective effect on Fujian population, but the influenza strains from Fujian in 2012 has antigenic drift compared with the vaccine strain, more attention should therefore be paid to the surveillance of mutations of pandemic A/H1N1/2009 influenza virus.
Antiviral Agents ; pharmacology ; China ; epidemiology ; Drug Resistance, Viral ; genetics ; Genome, Viral ; genetics ; Genomics ; Humans ; Influenza A Virus, H1N1 Subtype ; drug effects ; genetics ; immunology ; physiology ; Influenza, Human ; epidemiology ; prevention & control ; Pandemics ; prevention & control ; Viral Vaccines ; immunology

The desired effect of vaccination is to elicit protective immune responses against infection with pathogenic agents. An inactivated influenza vaccine is able to induce the neutralizing antibodies directed primarily against two surface antigens, hemagglutinin and neuraminidase. These two antigens undergo frequent antigenic drift and hence necessitate the annual update of a new vaccine strain. Besides the antigenic drift, the unpredictable emergence of the pandemic influenza strain, as seen in the 2009 pandemic H1N1, underscores the development of a new influenza vaccine that elicits broadly protective immunity against the diverse influenza strains. Cold-adapted live attenuated influenza vaccines (CAIVs) are advocated as a more appropriate strategy for cross-protection than inactivated vaccines and extensive studies have been conducted to address the issues in animal models. Here, we briefly describe experimental and clinical evidence for cross-protection by the CAIVs against antigenically distant strains and discuss possible explanations for cross-protective immune responses afforded by CAIVs. Potential barriers to the achievement of a universal influenza vaccine are also discussed, which will provide useful guidelines for future research on designing an ideal influenza vaccine with broad protection without causing pathogenic effects such as autoimmunity or attrition of protective immunity against homologous infection.
Adaptive Immunity ; Antigens, Viral/immunology ; *Cross Protection ; Genome, Viral ; Humans ; Immunity, Innate ; Influenza Vaccines/*immunology/therapeutic use ; Influenza, Human/*prevention & control ; Orthomyxoviridae/genetics/immunology ; Vaccines, Attenuated

Since 2002, H7 subtype avian influenza viruses (AIVs) have caused more than 100 human infection cases in the Netherlands, Italy, Canada, the United States, and the United Kingdom, with clinical illness ranging from conjunctivitis to mild upper respiratory illness to pneumonia. On March 31st, three fatal cases caused by infection of a novel reassortant H7N9 subtype were reported in Shanghai City and Anhui Province in China. With the ability of H7 subtype to cause severe human disease and the increasing isolation of subtype H7 AIVs, we highlighted the need for continuous surveillance in both humans and animals and characterization of these viruses for the development of vaccines and anti-viral drugs.
Animals ; Chickens ; Ducks ; Humans ; Influenza A virus ; genetics ; isolation & purification ; pathogenicity ; physiology ; Influenza Vaccines ; genetics ; immunology ; Influenza in Birds ; immunology ; prevention & control ; virology ; Influenza, Human ; immunology ; prevention & control ; virology ; Poultry Diseases ; immunology ; prevention & control ; virology ; Turkeys

To construct a recombinant T7 phage expressing matrix protein 2 ectodomain (M2e) peptides of avian influenza A virus and test immunological and protective efficacy in the immunized SPF chickens. M2e gene sequence was obtained from Genbank and two copies of M2e gene were artificially synthesised, the M2e gene was then cloned into the T7 select 415-1b phage in the multiple cloning sites to construct the recombinant phage T7-M2e. The positive recombinant phage was identified by PCR and sequencing, and the expression of surface fusion protein was confirmed by SDS-PAGE and Western-blot. SPF chickens were subcutaneously injected with 1 X 10(10) pfu phage T7-M2e, sera samples were collected pre- and post-vaccination, and were tested for anti-M2e antibody by ELISA. The binding capacity of serum to virus was also examined by indirect immunofluorescence assay in virus- infected CEF. The immunized chickens were challenged with 200 EID50 of H9 type avian influenza virus and viral isolation rate was calculated to evaluate the immune protective efficacy. A recombinant T7 phage was obtained displaying M2e peptides of avian influenza A virus, and the fusion protein had favorable immunoreactivity. All chickens developed a certain amount of anti-M2e antibody which could specially bind to the viral particles. In addition, the protection efficacy of phage T7-M2e vaccine against H9 type avian influenza viruses was 4/5 (80%). These results indicate that the recombinant T7 phage displaying M2e peptides of avian influenza A virus has a great potential to be developed into a novel vaccine for the prevention of avian influenza infection.
Animals ; Antibodies, Viral ; blood ; Bacteriophage T7 ; genetics ; immunology ; metabolism ; Chickens ; Enzyme-Linked Immunosorbent Assay ; Gene Expression Regulation, Viral ; Immunization ; Influenza A virus ; genetics ; immunology ; Influenza Vaccines ; immunology ; Influenza in Birds ; immunology ; metabolism ; prevention & control ; Peptides ; genetics ; immunology ; metabolism ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; Specific Pathogen-Free Organisms ; Viral Matrix Proteins ; genetics ; immunology ; metabolism

Virus-like particles (VLPs) are composed of multiple copies of one or more expressed recombinant viral structural proteins which spontaneously assemble into particles upon expression. VLPs are non infectious because they assemble without incorporating genetic material. VLPs have structural characteristics and antigenicity similar to the parental virus because they mimick the wild-type virus structure. Hence, they are recognized readily by the immune system which induces strong anti-viral immune responses to stop virus infection. VLPs have therefore shown dramatic effectiveness as candidate vaccines and diagnostic reagent for virus. Here, in order to provide reference to the research of influenza VLPs, we reviewed the current research progress of influenza VLPs, and discussed the characteristics associated with producing VLPs for influenza virus.
Animals ; Humans ; Influenza Vaccines ; genetics ; immunology ; Influenza, Human ; immunology ; virology ; Orthomyxoviridae ; genetics ; immunology ; physiology ; Viral Proteins ; genetics ; immunology ; Virion ; genetics ; immunology ; physiology ; Virus Assembly