Association between Interferon Response and Protective Efficacy of NS1-Truncated Mutants as Influenza Vaccine Candidates in Chickens

Abstract

Influenza virus mutants that encode C-terminally truncated NS1 proteins (NS1-truncated mutants) are attractive candidates for avian live attenuated influenza vaccine (LAIV) development because they are both attenuated and immunogenic in chickens. We previously showed that a high protective efficacy of NS1-truncated LAIV in chickens corresponds with induction of high levels of type I interferon (IFN) responses in chicken embryonic fibroblast cells. In this study, we investigated the relationship between induction of IFN and IFN-stimulated gene responses in vivo and the immunogenicity and protective efficacy of NS1-truncated LAIV. Our data demonstrates that accelerated antibody induction and protective efficacy of NS1-truncated LAIV correlates well with upregulation of IFN-stimulated genes. Further, through oral administration of recombinant chicken IFN alpha in drinking water, we provide direct evidence that type I IFN can promote rapid induction of adaptive immune responses and protective efficacy of influenza vaccine in chickens.

Fig 1. Serum antibody response in chickens following vaccination with LAIV candidates or infection with rgWT virus.

Serum was collected at 8 and 14 days post-vaccination/infection (dpv/dpi) and tested for the presence of influenza virus A/TK/OR/71 specific hemagglutination inhibition (HI) antibodies. HI titers are presented as circles (rgWT), squares (pc2-LAIV), and triangles (pc4-LAIV). The thick horizontal lines represent median titers of the groups.

Fig 2. Comparison of virus replication in trachea.

The EID₅₀ equivalent titers were interpolated from qRT-PCR Ct values of tracheal swab viral RNA as described in Materials and Methods. Statistical significance, *p<0.05. EID₅₀, median egg infectious doses. n/n, number of virus positive birds/total number of birds in the group.

Fig 3. IFN and ISG responses after vaccination with LAIV candidates or infection with rgWT virus.

See Materials and Methods for details on fold change calculation, statistical analysis and data normalization. dpv/dpi, days post vaccination/infection. All groups were included in the statistical analysis where the unvaccinated (uninfected) group was used as the reference. Error bars, mean ± S.D. Statistical significance, *p<0.05, **p<0.001.

Fig 4. Oral rChIFN-α treatment in chickens.

(A) Antibody response to vaccination with inactivated vaccine (IV) with or without rChIFN-α (IFN) treatment at 8 and 14 days post vaccination (dpv). (B) ISG and IFN gene responses in spleens of unvaccinated chickens at 1 and 3 days post treatment (dpt). (C) Comparison of ISG and IFN gene responses in spleens of chickens vaccinated with IV with or without rChIFN-α treatment at 3dpt. All groups were included in the statistical analysis where the untreated control group was used as the reference. Statistical significance, *p<0.05, **p<0.001.

Fig 5. Pre-challenge antibody responses.

The development of antibody response was monitored at 8 and 13 days post-vaccination (dpv). Horizontal bars represent mean antibody titer for the group (n = 7).

Fig 6. Replication and shedding of heterologous challenge virus.

Top: Viral titers expressed as median (50%) egg-infectious dose equivalent by qRT-PCR (see Materials and Methods) [27, 28]. Bottom: viral titers re-isolated in MDCK cells. Horizontal bars represent mean antibody titer for the group (n = 7). dpc, days post challenge. TCID₅₀, median (50%) tissue culture infective dose. *p<0.05. **p<0.001.