Development of Pre-pandemic Influenza Vaccines against Highly Pathogenic H5 Strains

Abstract

The emergence of highly pathogenic avian influenza A viruses (HPAI) and their potential to cause human infections poses substantial public health threats. Among HPAI, H5 viruses are of particular concern given their global spread in avian species, zoonotic infection to humans and the high mortality rate in humans. While HPAI H5N1 human infections have typically been reported in Asian countries, Canada reported a case of fatal human infection by the HPAI H5N1 virus in 2014. The genome of the causative virus A/Alberta/01/2014 (H5N1) (AB14 (H5N1)) has been reported; however, the isolate had not been evaluated for its pathogenicity in animal models. In the first part of my thesis study, I characterized the pathogenicity of AB14 (H5N1) in mice and found that AB14 (H5N1) is highly lethal in mice. The virus caused systemic viral infection and erratic proinflammatory cytokine gene expression in different organs, including lung, spleen, and brain. This study not only provided experimental evidence to complement the specific human case report but also established an animal model for HPAI H5N1 virus, which is valuable and essential for evaluating vaccine and antiviral candidates against the potential influenza pandemics. Vaccination is the most effective intervention to prevent possible pandemic outbreaks. The conventional egg-based vaccine production strategy takes up to six months for vaccine development, during which the antigenically distinct pandemic viruses are allowed to spread in the naïve population leading to a rapid disease progression. Traditional inactivated vaccines against zoonotic avian influenza viruses often suffer from low immunogenicity due to the intrinsic poor antigenicity of avian influenza viruses, or they are poor inducers of cellular immunity. In my second part of the study, I aimed to develop a new subunit H5 influenza vaccine to achieve better immune protection against HPAI H5N1 virus infection. I expressed and purified hemagglutinin (HA) derived from AB14 (H5N1) using both mammalian (m) and bacterial (b) expression systems. The purified recombinant proteins were formulated with a proprietary adjuvant (TriAdj); their efficacy as vaccine candidates was then evaluated in the mouse model I established in the first part of the study. Intramuscular vaccination of two doses of TriAdj-formulated mammalian-expressed HA (m-HA/TriAdj) provided full protection against a lethal challenge of AB14 (H5N1) in mice. In contrast, bacterially expressed HA with TriAdj (b-HA/TriAdj), b-HA without adjuvant or m-HA without adjuvant did not result in protection in immunized mice. Furthermore, I analyzed the immune responses and found that m-HA/TriAdj elicited significantly higher levels of balanced Th1 and Th2 responses and neutralizing antibody. All of the mice in this group survived a lethal AB14 (H5N1) challenge and showed no signs of disease or infection as demonstrated by no loss of body weight or detectable virus in the lungs. My results suggest that m-HA formulated with TriAdj has the potential to protect against pandemic H5N1 in the event of its cross over to the human host. Overall, the two parts of this thesis provide an animal model for the HPAI AB14 (H5N1) virus and a highly effective vaccine candidate against the HPAI H5N1 virus. Understanding the pathogenicity of HPAI H5N1 virus in an animal model and developing the anti-H5 vaccine candidate will all contribute to the preparedness for a potential influenza pandemic caused by HPAI H5N1 viruses.