Basic Study On Highly Pathogenic Avian Influenza H5N1 Virus Universal Vaccines

The highly pathogenic avian influenza (HPAI) H5N1 virus (refer to"H5N1 virus"thereinafter) has posed severe threat to public health worldwide, not only because of the sporadically occurred cases of infection and death, but also because of the transmission of the virus in human populations, leading to a potential new influenza pandemic. Therefore, it is especially necessary to develop effective vaccines against H5N1 virus. However, H5N1 viruses have currently evolved into many different clades due to rapid variation of the viral antigens. Consideration of the high variation of H5N1 virus and the limitation of current vaccine production, it is particularly important to vaccinate humans in advance with conserved antigen-based universal vaccines that may induce cross-protection against different clades of H5N1 virus. In this study, a comprehensive research was carried out to develop universal influenza vaccines based on the highly conserved M2e of H5N1 virus. As such, two candidate vaccines, respectively designated M2e4-MAP and M2e-ASP-1, were developed, with the aim to elucidate effective prophylactic tools against H5N1 virus and strengthen the abilities to prevent potential H5N1 influenza pandemic.1. Analysis of the conservation and serum cross-reactivity of H5N1 M2eTo analyze the conservation and determine the target antigen of M2e, the full-length sequences of viral M2 protein from various hosts and different periods of H5N1 isolates were collected from NCBI Influenza Virus Resource and used for sequencing alignment analysis, based on which the consensus sequences of M2e of H5N1 were identified. It was shown that there are some amino acid variations between M2e consensus sequence of H5N1 virus and that of human influenza virus. Further analysis of the serum cross-reactivity indicated that H5N1 M2e-specific antibody was detectable in sera of patients with H5N1 influenza, but undetectable in those from non-H5N1 influenza patients. It was also found that the titer of H5N1 M2e-specfic antibodies is less than 102 in the sera of mice specific to M2e of human influenza virus. The above results suggest that there is little cross-reactivity of M2e between H5N1 virus and human influenza virus. Therefore, the consensus sequence of M2e of H5N1 was selected as the target in this study to design universal vaccines against H5N1 viruses.2. Development and evaluation of HPAI H5N1 universal vaccines based on M2e4-MAPTo effectively evaluate cross-clade protection of M2e-based influenza vaccines against divergent H5N1 viruses, a BALB/c mouse model was initially established by challenge of the na?ve mice with two clades (clade1: A/Vietnam/1194/04; clade 2.3.4: A/Shenzhen/406H/06) of H5N1 virus. The mice showed the dramatic body weight loss and absolute death within 10 days after challenge with 10LD50 of the viruses, and the peak level of viral titers were found in mouse lungs at 5 days post-challenge, which was accompanied by pulmonary histopathological damages similar to clinical signs. Therefore, lung viral titers, pulmonary histopathogical damages, body weight changes and survival rates were defined as the four most important indicators for the evaluation of M2e-induced protection.To enhance the immunogenicity of M2e and mimic the homotetrameric structure of native M2e, a tetra-branched multiple antigenic peptide carrying 4 chains of consensus sequence of H5N1 M2e (M2e4-MAP) was synthesized, and its immunogenicity and cross-clade protection against divergent H5N1 viruses were evaluated in the established mouse model. After lethal challenge (10LD50) with clade 1 (A/Vietnam/1194/04) or clade 2.3.4 (A/Shenzhen/406H/06) of H5N1 virus, the M2e4-MAP-vaccinated mice demonstrated significant decrease of viral replications and obvious alleviation of histopathological damages in their lungs, as well as significant increase of survival rate (even reached to 70%～80%). The above results suggest that M2e4-MAP may significantly enhance the immunogenicity of M2e, being able to provide effective cross-clade protection against divergent H5N1 viruses.3. Development and evaluation of HPAI H5N1 universal vaccines based on M2e-ASP-1 fusion proteinThe study in this section was carried out by fusing the consensus sequence of M2e of H5N1 virus with a novel adjuvant protein, activation associated protein-1 (ASP-1). The strategies of increasing M2e tandem copies and linking M2e to adjuvant components were applied to construct recombinants fusing one copy of M2e to ASP-1 (M2e-ASP-1) or three tandem copies of M2e to ASP-1 (M2e3-ASP-1) and subsequent expression of the recombinant proteins in E.coli expression system. A recombinant protein fusing three M2e copies with trxA (a pET32a(+) vector-derived chaperone without adjuvanticity) was expressed simultaneously as the matched control. Mice were vaccinated with recombinant proteins, followed by challenged with two clades of H5N1 virus. The results demonstrated that M2e-ASP-1, particularly M2e3-ASP-1, were able to induce high titers of M2e-specific antibodies and effective cellular immune responses without the help of adjuvants. After 10LD50 of lethal challenge with two clades (clade 1:A/Vietnam/1194/04; clade2.3.4:A/Shenzhen/406H/06) of H5N1 virus, M2e3-ASP-1-vaccinated mice indicated dramatic decrease of lung viral replications and significant alleviation of histopathological damages, accompanied with high survival rate reaching 70%-80%. All mice in the M2e3-ASP-1-vaccination group could survive from 3LD50 of lethal virus challenge. These results imply that M2e3-ASP-1 containing three M2e copies induced better immune responses and protection than M2e-ASP-1 with one M2e copy, providing complete cross-clade protection against divergent H5N1 viruses.To further understand the efficacy of M2e-induced cross-protection in the case of inablility of hemagglutinin (HA) protein in inducing protective immunity due to virus variation, a recombinant protein encoding HA of A/Vietnam/1194/04(H5N1) (rHA) was expressed in baculovirus expression system, and subsequently conjugated with M2e3-ASP-1 to vaccinate mice. According to the regular rHA vaccination schedule, mice were vaccinated twice with the mixed protein at 4-week interval, followed by lethal challenge (10LD50) of H5N1 virus clade1 (A/Vietnam/1194/04) or clade2.3.4 (A/Shenzhen/406H/06). As a result, rHA-M2e3-ASP-1 vaccination induced high titers of antibodies specific to both HA and M2e, as well as potent neutralizing antibodies against HA of A/Vietnam/1194/04 that completely protected vaccinated mice against homologous H5N1 virus challenge. The results also revealed that although no neutralizing antibodies were detected against HA of heterologous stain (A/Shenzhen/406H/06) of H5N1 virus, 60% of the rHA-M2e3-ASP-1-vaccinated mice could survive from challenge with this heterologous isolate. In contrast, in the absence of M2e3-ASP-1, all rHA-vaccinated mice died from the same lethal challenge (10LD50) of heterologous (A/Shenzhen/406H/06) H5N1 virus. Therefore, these above results further illustrated the cross-protective ability of M2e3-ASP-1 protein in the absence of HA-induced protection.4. Evaluation of cross-protective immunity conferred by H5N1 M2e4-MAP against 2009 H1N1 pandemic influenzaA great concern has been raised for the potential influenza pandemic due to the reassortment of HAPI H5N1 virus with the 2009 swine-origin H1N1 influenza virus. Based on our analysis, there is only one amino acid difference between H5N1 M2e and H1N1 M2e consensus sequences. Thus, in order to further investigate the H5N1-M2e-induced cross-protection against 2009 pandemic H1N1 virus, mice were vaccinated with H5N1-M2e4-MAP, followed by lethal challenge with the H1N1 virus. It was shown that vaccination with H5N1-M2e4-MAP induced high titers of cross-reactive antibodies against H1N1-M2e, which provided complete protection against H1N1 virus challenge, with the survival rate reaching 100%.In summary, this study has demonstrated that the consensus sequence of H5N1-M2e plays valuable roles in the development of universal vaccines against H5N1 viruses, the results of which have proven the practicity of the designed vaccine strategies in improving the immunogenicity and cross-protection of M2e-based H5N1 universal vaccines. The study also revealed the necessity of these vaccine strategies in improving vaccine efficacy, further suggesting that the developed H5N1 universal vaccines based on the current research will play important roles in preventing and fighting against any future influenza epidemic and pandemic potentially caused by H5N1 virus.