Construction, Optimization And Immunological Experiment Study Of Virus Like Particles Vaccine Of H5N1 Influenza

The highly pathogenic avian influenza (HPAI) H5N1 virus has become highly enzootic since 2003 and has dynamically evolved to undergo substantial evolution. Clades 2.3.2.1 and 2.3.4 have become the most dominant lineage in recent years. While the pandemic risk posed by HPAI H5N1 viruses is widely appreciated, confounding efforts to stockpile vaccines that may be needed if an avian-origin influenza pandemic were to occur. The current inactived influenza vaccine has been poorly immunogenic to heterologous virus challenge. Recombinant non-infectious VLPs produced using a baculovirus expression system represent a low-costly and biosafety system for the production of vaccines against influenza viruses. As they could induce cellular immune response against heterologous influenza viruses, and they may serve as vaccines against highly mutation influenza viruses. This study used a baculovirus expression system to generate H5N1 influenza VLPs, optimized through inclusion of additional proteins and investigated the immunogenicity and protective efficacy of heterologous virus challenge.First, virus-like particles (VLP) based on A/meerkat/Shanghai/SH-1/2012 (clade 2.3.2.1) and comprising hemagglutinin (HA), neuraminidase (NA), and matrix (M1) were produced using a baculovirus expression system. The identification result showed H5N1 VLPs contained NA, HA and Ml with functional activity, resembled influenza virions in morphology and size, and were structurally intact. Mice immunized with VLP demonstrated stronger humoral and cellular immune responses than mice immunized with whole influenza virus (WIV), with 20-fold higher IgG antibody titers against A/meerkat/Shanghai/SH-1/2012 after boost. Notably, the WIV vaccine group showed partial protection (80% survival) to homologous challenge and little protection (40% survival) to heterologous (A/duck/Jilin/JL-SIV/2013, clade 2.3.4) challenge, whereas all mice in the VLP group survived. These results provide insight for the development of effective prophylactic vaccines based on VLPs with cross-clade protection for the control of current H5 HPAI outbreaks.In order to improve the immunogenicity of H5N1 VLPs, we inclusion of additional proteins which can serve as immunological adjuvants in VLPs to enhance adaptive immune responses following vaccination. Here, we report the generation of chimeric VLPs composed of membrane-anchored forms of the Escherichia coli heat-labile enterotoxin B subunit protein (LTB) or the Toll-like receptor 5 ligand, flagellin (Flic) or Granulocyte-macrophage colony-stimulating factor (GM-CSF). The identification result showed H5N1-, LTB-, Flic-, and GM-CSF-VLPs contained the expected proteins, resembled influenza virions in morphology and size, and were structurally intact. Mice intramuscularly, intranasally or orally immunized with VLPs containing LTB, Flic or GM-CSF generated greater humoral and cellular immune responses when compared with H5N1 VLPs without LTB, Flic or GM-CSF. Intramuscular and intranasal immunization with VLPs protected mice from a lethal challenge with homologous or heterologous H5N1 viruses irrespective of whether VLPs additionally included LTB, Flic or GM-CSF. In contrast, oral immunization of mice with LTB-VLPs, Flic-VLPs or GM-CSF-VLPs conferred partial protection from lethal challenge with both homologous and heterologous H5N1 influenza viruses, whereas mice immunized orally with VLPs lacking LTB, Flic or GM-CSF universally succumbed to infection. Mice immunized orally with LTB-or Flic-VLPs showed 10-fold higher virus-specific IgG titers when compared to mice immunized with H5Nl-VLPs lacking LTB and Flic.Collectively, these results indicate that VLP based on baculovirus expression system may provide protection of homologous and heterologous virus challenge, and inclusion of immunostimulatory proteins such as LTB, Flic or GM-CSF in VLP-based vaccines may represent a promising new approach for the control of current H5N1 HPAI outbreaks by eliciting higher quality immune responses and conferring improved cross-clade protection.