Development of universal Influenza vaccine in chicken with insights on the extracellular domain of Matrix protein

Avian influenza (AI) is an infectious disease of avian species caused by type A influenza viruses with a significant economic impact on the poultry industry. In addition to affecting poultry, different subtypes of AI viruses can infect other species, thus complicating prevention and control. Vaccination is one of the main disease prevention strategies in many countries worldwide. Current influenza vaccines based on the highly variable hemagglutinin (HA) protein can provide effective protection against specific, antigenically matching virus, but little protection against more distant strains, even those belonging to the same subtype, and they do not provide heterosubtypic immunity. Therefore, to protect chickens against new strain of AI virus, as well as control and prevent virus spread among farms, new vaccines needed to be designed to overcome the limitations of conventional vaccines. One of the approaches for new vaccine design is targeting conserved regions of the influenza genome as possible universal vaccines to induce cross protective immunity against different strains and to eliminate constant vaccine updates based on circulating virus. The extracellular domain of ion channel M2 protein (M2e) is highly conserved among different AI strains suggesting that it would be a promising candidate for developing a universal influenza vaccine. Due to poor immunogenicity of the small M2e, various M2e conjugating carriers and adjuvant formulations have been used to enhance its immunogenicity and protective efficacy. In this study, our goal is to evaluate the potential use of M2e-based vaccines for the control of AI in chickens. In the first part of the study, a recombinant avian M2e protein expressed on the surface of the norovirus P particle (M2e-PP) was tested for its immunogenicity and protective efficacy against challenges with 3 different AI viruses in chickens. Two-weekold specific pathogen free chickens were vaccinated 3 times with M2e-PP either subcutaneously (SQ) with oil adjuvant or transmucosally (intranasal, IN; eye drop, ED; microspray, MS) without adjuvant. M2e-PP vaccination via the SQ route induced significant IgG antibody responses which were increased by each booster vaccination. Neither IgG nor IgA responses were detected from sera nor nasal wash of transmucosally immunized birds. Upon intranasal challenge, M2e-PP vaccination via the SQ route significantly reduced virus shedding from both the trachea and the cloaca for all three challenge viruses. Despite the absence of detectable IgG and IgA responses in birds vaccinated with the M2e-PP via the IN route, a similar level of reduction in virus shedding was observed in the IN group compared to the SQ group. In conclusion, M2e- PP vaccination in chicken has demonstrated its high immunogenicity and its ability to protect chickens against challenge with 3 different AI virus subtypes.;A combination of M2e-PP recombinant protein with inactivated influenza vaccine (IIV) was tested in chickens, as an approach to overcome the limited strain specific protection of the IIV. Co-immunization of birds with both vaccines did not affect production of M2e specific IgG antibody compared to the M2e-PP alone vaccinated group. However, the co-immunized birds showed significantly higher hemagglutination inhibition antibody titers against vaccine and challenge viruses as well as cross reactive antibody responses against the H5, H6, and H7 viruses compared to the IIV alone vaccinated group. Upon intranasal homologous and heterologous virus challenges, combined vaccine groups showed greater reduction in viral titers from tracheal swabs compared to those groups receiving IIV alone. Moreover, M2e-PP antisera from vaccinated birds were able to bind to their native M2e target expressed on whole virus and infected cells and to inhibit viral replication.;Overall, our results supports that the universal vaccine approach using an M2e based vaccine can provide cross-protection against challenge viruses among different HA subtypes. Additionally, supplementing IIV with M2e-PP can expand the vaccine protective efficacy. Further improvements of the vaccine immunogenicity and efficacy are required for this vaccine to be practical. A better understanding of the protective immune mechanism will also be critical for the optimization of an M2e-based vaccine in chickens.