Development of Virus-like Particle Plant-Based Vaccines against Avian H5 and H9 Influenza A Viruses

Simple Summary Avian influenza virus (AIV) is a highly infectious viral disease, some subtypes of which (e.g., H5 and H9) cause high mortality in poultry and can be transmitted to humans. AIV continues to cause severe losses despite the availability of commercial vaccines. Haemagglutinin (HA) is the main antigen of the virus and neutralising antibodies are elicited against it upon infection. Virus-like particles (VLPs) mimic the shape of the virus but are devoid of the inner genetic material, so they are non-infectious but provoke a strong immune response. The aim of this project was to produce cheap, safe and effective plant-based vaccines by producing H9 and H5 VLPs in Nicotiana benthamiana. A transmission electron microscope (TEM) was used to visualise VLPs derived from plant extracts. Mice were initially immunised with multiple doses of the H5 and H9 VLPs to confirm that they elicited robust antibody and T-cell immune responses. It was then demonstrated that a single dose of H5 VLPs given to chickens stimulated an antibody response that could neutralise virus infectivity. These results show that plant-based vaccines are a promising alternative to traditional vaccines, especially in developing countries, and further development is desirable.Abstract Avian influenza A virus (AIV) is a significant cause of mortality in poultry, causing substantial economic loss, particularly in developing countries, and has zoonotic potential. For example, highly pathogenic avian influenza (HPAI) viruses of the H5 subtype have been circulating in Egypt for around two decades. In the last decade, H5N1 viruses of clade 2.2.1 have been succeeded by the antigenically distinct H5N8 clade 2.3.4.4b viruses. Furthermore, H9N2 viruses co-circulate with the H5N8 viruses in Egyptian poultry. It is widely recognised that effective vaccination against IAV requires a close antigenic match between the vaccine and viruses circulating in the field. Therefore, approaches to develop cost-effective vaccines that can be rapidly adapted to local virus strains are required for developing countries such as Egypt. In this project, the haemagglutinin (HA) proteins of Egyptian H5 and H9 viruses were expressed by transient transfection of plants (Nicotiana benthamiana). The formation of virus-like particles (VLPs) was confirmed by transmission electron microscopy. Mice were immunised with four doses of either H5 or H9 VLPs with adjuvant. Antibody and cellular immune responses were measured against the corresponding recombinant protein using ELISA and enzyme-linked immunosorbent assay (ELISpot), respectively. Chickens were immunised with one dose of H5 VLPs, eliciting HA-specific antibodies measured by ELISA and a pseudotyped virus neutralisation test using a heterologous H5 HA. In conclusion, plant-based VLP vaccines have potential for producing an effective vaccine candidate within a short time at a relatively low cost.