Control of avian influenza (AI) has been continuously challenged by the extreme diversity and complicated ecology of the causative agent, avian lineage type A influenza virus. Poultry species serves unique roles in AI ecology as providing an environment for viral evolution and act as an intermediate host to spread novel viral strains to other animal species including humans. Thus, to minimize the socio-economic impact from the emergence of novel strains and zoonotic infection, development of a strong immunity in poultry is desirable as a preventive measure. But currently available influenza vaccines for poultry, inactivated vaccines (IV) or recombinant viral vector vaccines, provide limited protection range confined to a specific target antigen and they require periodic updates to keep pace with the frequent viral mutations. As an alternative, live attenuated influenza vaccine (LAIV) can supplement the current vaccines for providing a broader protective immunity against diverse strains.
The long-term aim of this study is to develop a broadly effective LAIV for the use in poultry. Among diverse strategies to produce LAIV candidates, we focused on influenza A virus mutants that encode C-terminally truncated nonstructural protein 1 (NS1-truncated mutants). Nonstructural protein 1 (NS1 protein) is a multifunctional protein which provides the virus a favorable environment, primarily by interfering with host type I interferon (IFN) induction and signaling system. In turn, the NS1-truncated mutants can be a promising live vaccine candidate for their highly attenuated phenotype as well as an induction of a boosted immune response following the unimpeded type I IFN response. Previously, four genetically stable plaque purified NS1 truncated mutants, pc1- to pc4-LAIV, were evaluated for their potential as LAIV candidates in chickens. All the four candidates showed limited replication in chickens, but interestingly, the protective efficacy was not equal among viruses; only the pc3-and pc4-LAIV induced a high level serum antibody response and provided better heterologous protection. In a later study, the in vitro type I IFN induction ability of pc3-, or pc4-LAIV was observed to be higher than pc1- or pc2-LAIV, suggesting a correlation between in vitro type I IFN induction ability and the in vivo protective efficacy among NS1 truncated mutants.
Extended from the previous findings, we hypothesized that the higher immunogenicity of pc4-LAIV than pc2- LAIV is due to enhanced induction of type I IFN responses. The first phase of this study was aimed to demonstrate a correlation between type I IFN response and efficacy of pc4-LAIV in vivo. We first demonstrated that pc4-LAIV could induce higher levels of type I IFN stimulated genes (ISGs), 2’,5’-OAS and Mx genes, in chicken trachea and spleen than pc2-LAIV. Also, we unexpectedly found that pc4-LAIV not only induce higher level of serum antibody response than pc2-LAIV, but also accelerated the serum antibody response which was noticeable as early as 7 days post vaccination. For further demonstration of the role of a type I IFN response in the efficacy of pc4-LAIV, we treated chickens with recombinant chicken type I IFN and assessed its effect on IFN/ ISGs transcription, serum antibody response, and heterologous protective efficacy. The type I IFN treatment induced an accelerated serum antibody response against IV and upregulation of ISGs as observed with pc4-LAIV. More interestingly, type I IFN treatment directly enhanced the protective efficacy of pc2-LAIV to a similar level as pc4-LAIV. The results showed that the higher immunogenicity of pc4-LAIV than pc2-LAIV is correlated with the type I IFN related responses. Also, in addition to the previously observed serum antibody response and protective efficacy, the pc4-LAIV was efficient in stimulating an innate type I IFN related signaling and accelerating the adaptive immune response.
Based on the findings from the first phase of our study, we expected that the pc4-LAIV is less likely to be affected by the maturity of the chicken immune system due to its innate immune stimulatory effect and the ability to accelerate an adaptive immune response. In the second phase of this study, we aimed to compare the efficacy of pc4-LAIV in immunologically immature 1-day-old chickens to IV, which is known to be less immunogenic in young birds. As a result, 1-day-old vaccination with pc4-LAIV induced higher innate signaling sensitization, mucosal IgA response and better clearance of a heterologous challenge virus than the IV. But a serum antibody response was observed to be higher in IV groups, and the lower antibody response by pc4-LAIV seems to be due to the lack of proper mucosal adjuvant that can be used with pc4-LAIV. These findings indicate that each vaccine provides a distinctive advantage; pc4-LAIV has a higher priming effect on 1-day-old chickens but a serum antibody response can be more effectively elicited by the IV.
Based on the findings from the second part of study, we hypothesized that the most ideal protective efficacy could be achieved by combining the advantageous traits of each vaccine. So in the third phase of study, we tested the prime-boost strategy of pc4-LAIV vaccination at one-day-old followed by a IV vaccination at 3-week-old. The priming effect of pc4-LAIV vaccination at 1-day of age synergistically enhanced the efficacy of the boost vaccination at 3-week of age in that the prime boost regimen showed the highest serum antibody production. Also as a combined effect, the prime boost regimen produced early serum antibody response, broadened the cross reactivity of the antibody and induced a mucosal IgA response.
In summary, our study demonstrated that pc4-LAIV is efficient in eliciting an in vivo innate signaling sensitization and accelerating the adaptive immune response which correlated with heightened protection in both immunologically mature and immature birds. Also, pc4-LAIV priming was successfully incorporated into the current IV application without hindrance of each other’s efficacy and the effect of two vaccines could be synergistic. Our findings serve as a basis for the development of a broad spectrum LAIV, and the pc4-LAIV may be synergistically used with other vaccines as an essential component of the prime-boost regimen.