A novel M2e-multiple antigenic peptide providing heterologous protection in mice.

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.

Efficacy of a high-growth reassortant H1N1 influenza virus vaccine against the classical swine H1N1 subtype influenza virus in mice and pigs.

Swine influenza (SI) is an acute, highly contagious respiratory disease caused by swine influenza A viruses (SwIVs), and it poses a potential global threat to human health. Classical H1N1 (cH1N1) SwIVs are still circulating and remain the predominant subtype in the swine population in China. In this study, a high-growth reassortant virus (GD/PR8) harboring the hemagglutinin (HA) and neuraminidase (NA) genes from a novel cH1N1 isolate in China, A/Swine/Guangdong/1/2011 (GD/11) and six internal genes from the high-growth A/Puerto Rico/8/34(PR8) virus was generated by plasmid-based reverse genetics and tested as a candidate seed virus for the preparation of an inactivated vaccine. The protective efficacy of this vaccine was evaluated in mice and pigs challenged with GD/11 virus. Prime and boost inoculation of GD/PR8 vaccine yielded high-titer serum hemagglutination inhibiting (HI) antibodies and IgG antibodies for GD/11 in both mice and pigs. Complete protection of mice and pigs against cH1N1 SIV challenge was observed, with significantly fewer lung lesions and reduced viral shedding in vaccine-inoculated animals compared with unvaccinated control animals. Our data demonstrated that the GD/PR8 may serve as the seed virus for a promising SwIVs vaccine to protect the swine population.

Protective efficacy of a high-growth reassortant swine H3N2 inactivated vaccine constructed by reverse genetic manipulation.

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.

An M2e-based synthetic peptide vaccine for influenza A virus confers heterosubtypic protection from lethal virus challenge.

BACKGROUND: Vaccination is considered as the most effective preventive method to control influenza. The hallmark of influenza virus is the remarkable variability of its major surface glycoproteins, HA and NA, which allows the virus to evade existing anti-influenza immunity in the target population. So it is necessary to develop a novel vaccine to control animal influenza virus. Also we know that the ectodomain of influenza matrix protein 2 (M2e) is highly conserved in animal influenza A viruses, so a vaccine based on the M2e could avoid several drawbacks of the traditional vaccines. In this study we designed a novel tetra-branched multiple antigenic peptide (MAP) based vaccine, which was constructed by fusing four copies of M2e to one copy of foreign T helper (Th) cell epitope, and then investigated its immune responses. RESULTS: Our results show that the M2e-MAP induced strong M2e-specific IgG antibody,which responses following 2 doses immunization in the presence of Freunds' adjuvant. M2e-MAP vaccination limited viral replication substantially. Also it could attenuate histopathological damage in the lungs of challenged mice and counteracted weight loss. M2e-MAP-based vaccine protected immunized mice against the lethal challenge with PR8 virus. CONCLUSIONS: Based on these findings, M2e-MAP-based vaccine seemed to provide useful information for the research of M2e-based influenza vaccine. Also it show huge potential to study vaccines for other similarly viruses.

Protective efficacy of a broadly cross-reactive swine influenza DNA vaccine encoding M2e, cytotoxic T lymphocyte epitope and consensus H3 hemagglutinin.

BACKGROUND: Pigs have been implicated as mixing reservoir for the generation of new pandemic influenza strains, control of swine influenza has both veterinary and public health significance. Unlike human influenza vaccines, strains used for commercially available swine influenza vaccines are not regularly replaced, making the vaccines provide limited protection against antigenically diverse viruses. It is therefore necessary to develop broadly protective swine influenza vaccines that are efficacious to both homologous and heterologous virus infections. In this study, two forms of DNA vaccines were constructed, one was made by fusing M2e to consensus H3HA (MHa), which represents the majority of the HA sequences of H3N2 swine influenza viruses. Another was made by fusing M2e and a conserved CTL epitope (NP147-155) to consensus H3HA (MNHa). Their protective efficacies against homologous and heterologous challenges were tested. RESULTS: BALB/c mice were immunized twice by particle-mediated epidermal delivery (gene gun) with the two DNA vaccines. It was shown that the two vaccines elicited substantial antibody responses, and MNHa induced more significant T cell-mediated immune response than MHa did. Then two H3N2 strains representative of different evolutional and antigenic clusters were used to challenge the vaccine-immunized mice (homosubtypic challenge). Results indicated that both of the DNA vaccines prevented homosubtypic virus infections completely. The vaccines' heterologous protective efficacies were further tested by challenging with a H1N1 swine influenza virus and a reassortant 2009 pandemic strain. It was found that MNHa reduced the lung viral titers significantly in both challenge groups, histopathological observation showed obvious reduction of lung pathogenesis as compared to MHa and control groups. CONCLUSIONS: The combined utility of the consensus HA and the conserved M2e and CTL epitope can confer complete and partial protection against homologous and heterologous challenges, respectively, in mouse model. This may provide a basis for the development of universal swine influenza vaccines.

Genetic diversity of H9N2 influenza viruses from pigs in China: a potential threat to human health?

Pandemic strains of influenza A virus might arise by genetic reassortment between viruses from different hosts. Pigs are susceptible to both human and avian influenza viruses and have been proposed to be intermediate hosts or mixing vessels, for the generation of pandemic influenza viruses through reassortment or adaptation to the mammalian host. In this study, we summarize and report for the first time the coexistence of 10 (A-J) genotypes in pigs in China by analyzing the eight genes of 28 swine H9N2 viruses isolated in China from 1998 to 2007. Swine H9N2 viruses in genotype A and B were completely derived from Y280-like and Shanghai/F/98-like viruses, respectively, which indicated avian-to-pig interspecies transmission of H9N2 viruses did exist in China. The other eight genotype (C-J) viruses might be double-reassortant viruses, in which six genotype (E-J) viruses possessed 1-4 H5-like gene segments indicating they were reassortants of H9 and H5 viruses. In conclusion, genetic diversity of H9N2 influenza viruses from pigs in China provides further evidence that avian to pig interspecies transmission of H9N2 viruses did occur and might result in the generation of new reassortant viruses by genetic reassortment with swine H1N1, H1N2 and H3N2 influenza viruses, therefore, these swine H9N2 influenza viruses might be a potential threat to human health and continuing to carry out swine influenza virus surveillance in China is of great significance.