Development of immune response against H9N2 avian influenza after vaccination

My project described in this thesis aimed to figure out why H9N2 vaccination failed in poultry and developed some new vaccines to improve vaccine efficacy to stop transmission of H9N2 AIV in poultry. In chapter 2, we figured out that H9N2-specific MDAs were one of reasons for H9N2 vaccination failure in poultry. I went to several poultry farms in China and did an epidemiological investigation for more than three months. We found that newly hatched broilers before vaccination had already got high antibodies against H9N2 AIV, which we interpreted as MDAs. Those newly hatched broilers with high titers of MDAs couldn’t get potent immune responses after vaccination. Due to natural degradation of MDAs, 21-day-old broilers contained few MDAs, and these broilers got high immune responses after vaccination. All ages of SPF chickens got high humoral immune responses after vaccination. Furthermore, we confirmed the results by using PTAs to mimic MDAs in SPF chickens in laboratory and found that vaccinated chickens with high titers of PTAs still shed virus after homologous challenge. Finally, we identified that high (HI = 9 Log2) and medium (HI = 6 Log2) titers of PTAs interfered with immune response, while low (HI = 3 log2) titers of PTAs didn’t. In chapter 3, we developed a new adjuvant for the H9N2 IWV vaccine to overcome MDAs interference. We cloned different types of CpG ODN and their combination into plasmids as the adjuvant of the H9N2 IWV vaccine. Results showed that T vector enriched the combination of CpG-A and CpG-B ODN (T-CpG-AB) could significantly up-regulate mRNA expression of interleukins in vitro and in vivo. Furthermore, T-CpG-AB plasmid (30 µg per chicken)-based vaccine could enhance both strong humoral and cellular immune responses in the presence of high titers of PTAs (HI = 9 Log2) in chickens, which indicated that T-CpG-AB plasmid could be an excellent adjuvant candidate for H9N2 IWV vaccine to overcome MDAs interference in chickens. In chapter 4, we developed a viral vector vaccine and evaluated its efficacy in stopping transmission of H9N2 AIV among chickens with high titers of PTAs (HI = 9 Log2). HVT was used as a vaccine vector to express H9 hemagglutinin (HA) protein. The recombinant viral vector vaccine (rHVT-H9) could constantly express HA protein in vitro and in vivo. The rHVT-H9 successfully induced high humoral and cellular immune responses in the presence of PTAs in chickens, reducing viral shedding period after homologous challenge. More importantly, rHVT-H9 decreased the reproduction ratio (R) value estimated by SIR stochastic model, which suggested that the recombinant viral vector vaccine rHVT-H9 could reduce transmission of H9N2 AIV in poultry. In chapter 5, we explored the mechanisms of MDAs interference. Antibodies from different species against different antigens were passively transferred into SPF chickens to mimic complete H9N2-specific MDAs (F(ab)2 + Fc), the antigen-binding portion of H9N2-specific MDAs (F(ab)2) and the Fc-binding portion of H9N2-specific MDAs (Fc). We found that only the complete MDAs hinder the immune response rather than F(ab)2 or Fc. Intravenously injecting chicken type I interleukins into chickens with high titers of PTAs (H9N2-specific antibodies, HI = 12 log2) elicited potent immune responses after vaccination. Furthermore, the flow cytometry analysis showed that the induction of total B+ cells was significantly affected by PTAs after vaccination, while CD4+ and CD8+ T cells were not significantly influenced. In the last chapter, chapter 6, we discussed immune responses after vaccination, some new measurements to assess the efficacy of vaccines in stopping transmission of H9N2 AIV and future perspectives of vaccines in poultry based on new measurements.