Novel H5N1 Avian Influenza Vaccines Based On Truncation Of NS1 And Modification Of HA

Highly pathogenic avian influenza (HPAI) is an acute and highly contagious respiratory disease, causing a great economic loss to poultry industry. Meanwhile, H5N1 HPAIVs could step across the species barrier to infect humans and then pose a significant public health threat. Since it was isolated from a sick goose flock during an outbreak in Guangdong Province, China, in 1996, the H5 HPAIV has undergone a series of evolutionary, resulting in multiple clades (clade 7.2,2.3.2.1 and 2.3.4.4) and multiple NA subtype (H5N1, H5N2, H5N5, H5N6, and H5N8) of H5 viruses co-circulate in China currently. Therefore, there is an urgent need to develop broad-spectrum vaccines allowing utilization of a DIVA (differentiating infected from vaccinated animals) strategy for the control of H5N1 avian influenza. In this study, an H5N1 HPAIV named as A/Mallard/Huadong/S/2005 (clade 2.3.4) was employed for construction of recombinant viruses harboring the truncated NS1 protein and modified HA protein through a reverse genetic system, and the biological characteristics, pathogenicity, and immunogenicity of recombinant viruses were evaluated.1. Truncation of NSl protein contributed to the attenuation of H5N1 HPAIV and its mechanismThe NS1-truncated viruses of S-NS48, S-NS70, S-NS73, and S-NS99 expressing 48 aa,70 aa, 73 aa, and 99 aa respectively in the N-terminal of NS1 protein of A/Mallard/Huadong/S/2005 were constructed. Growth kinetic analysis revealed that NS1-truncated viruses except for S-NS48 exhibited the comparable replication capacity with the parental virus rS in chicken eggs and CEF cells. Animal experiments utilizing S-NS70, S-NS73, and S-NS99 showed that NS1-truncated viruses could temperately replicate in lungs and be detected in cloacal and tracheal swabs on days 3 and 5 post-infection, indicating that the truncation of NSl significantly impaired the virus virulence for chickens. The NS variants were also significantly attenuated in BALB/c mice with the MLD50>106.5EID50 and locally replicated in lungs with low viral load. The differentially transcriptional profile in lungs of rS-and S-NS99-infected SPF chickens were analyzed by high-throughput sequencing. Among the top 10 up-regulated genes in rS-infected chickens, chemokine gene CCL19, CCL4, interferon related gene OAS*A and cytokine IL-6 gene were listed, while the chemokine gene CCL19, CCL4, and CCLI10, interferon related gene IFN-β, IFN-λ., IRG1, Mx, and OAS*A genes did that in S-NS99-infected ones. Functional enrichment analysis was performed through Gene Ontology database. The results showed that immune response, chemokine activity, cytokine activity, inflammatory response, defense response to virus, defense response, and response to virus were different between rS- and mock-infected chickens, and inflammatory response, defense response to virus, response to virus, and immune response were different between S-NS99-and mock-infected animals. Pathway analysis through KEGG revealed that relative to healthy controls, many differentially expressed genes affected by rS involved in the regulation of immune responses and were mapped to cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway, JAK-STAT signaling pathway, NOD-like receptor signaling pathway, p53 signaling pathway, and RIG-I-like receptor signaling pathway. However, differentially expressed genes affected by S-NS99 were limited in Toll-like receptor signaling pathway, indicating that truncation of NS1 protein of H5N1 virus contributed the attenuation of influenza virus by impairing the capacity to trigger expression of genes involved in related pathways and "cytokine storm".2. Development of NS1-truncated live attenuated vaccines against H5N1 avian influenzaA panel of NS1-truncated and low pathogenic HA-modified variants, S-HALo/NS48, S-HALo/NS70, S-HALo/NS73, and S-HALo/NS99, were rescued by using a reverse genetic system. The growth kinetics and pathogenicity in animals of NS1-truncated viruses expressing low pathogenic HA protein presented the similar scenario with that of NS1 mutants with highly pathogenic counterparts described above. The relative mRNA levels of IFN-β in the lungs of chickens inoculated with NS1-truncated viruses were significantly higher (p< 0.05) than those infected with full-length NS1 protein version, S-HALo/NSFu, on day 3 post-infection. Then, IFN-β levels decreased quickly, resulting in no significant difference (p> 0.05) between the NS1-truncated groups and S-HALo/NSFu group on days 6 and 14 post-infection. The CD4+/CD8+ ratios of peripheral blood mononuclear cells of chickens inoculated with NSl-truncted viruses were lower than that of mock-inoculated ones due to the CD8+ T-cell counts significantly increased, and S-HALo/NS73 was the extreme one. All chickens vaccinated with NS1-truncted viruses showed a 100% protection without any clinical signs after challenge with HPAIV S strain. Of note, viral shedding from the trachea and cloaca was not detected in any chickens vaccinated with S-HALo/NS73 virus, but low viral titers in S-HALo/NS70-and S-HALo/NS99-vaccinated chickens. During the period of the challenge experiments with heterologous H5 viruses of DT (clade 7.2), WX (clade 2.3.2.1), or CZG (clade 2.3.4.4), the chickens vaccinated with S-HALo/NS73 showed a 100% protection with low viral shedding in the trachea and cloaca on day 3 post-challenge. However, the chickens vaccinated with S-HALo/NS70 and S-HALo/NS99 showed 60-80% and 80-100% protection against heterologous H5N1 HPA1 viruses, respectively, along with higher viral shedding in the trachea and cloaca. Therefore, these findings suggested that S-HALo/NS73 was suitable candidate for develop attenuated vaccine against H5 HPAIVs of different clades.3. Development of DIVA vaccine of H5N1 avian influenza by introducing heterologous HA stalkRecombinant viruses expressing the chimeric HA, cH5head/H3stalk-YCD, cH5head/H3stalk-SB and cH5head/H3stalk-YZ were generated and the biological characteristics, pathogenicity and immunogenicity of recombinant viruses were evaluated. The results showed that cH5/H3 viruses cH5head/H3stalk-YCD, cH5head/H3stalk-SB, and cH5head/H3stalk-YZ shared the similar growth property with wild-type S-HALo in chicken embryos. SPF chickens vaccinated with cH5head/H3stalk-YCD were protected against H5N1HPAIV with the protection rate of 80%. To develop a serologic method for differentiating between infected and vaccinated animals during the application of chimeric HA vaccine, HA2 subunit of H5 HA was prokaryotic expressed and then an indirect ELISA to detect antibodies against H5 HA2 was established, with optimal coating antigen of 1.6μg/ml and serum dilution of 1:160. Minor cross-reactivity of antibodies against IBV, NDV, ILTV, H9 subtype virus, H3 subtype virus, E.coli and Salmonella was found by indirect ELISA with recombinant antigen of rH5-HA2. A higher sensitivity of indirect ELISA with a detection limit of 13200 than indirect immunofluorescence assay (1:800) indicated the acceptable sensitivity of the developed ELISA could support the practice of DIVA. Serum samples from all cH5/H3-vaccinated chickens, but not S-HALo-vaccinated birds, were negative at 3 weeks post-vaccination according to the HA2-ELISA; however, negative chicken sample of anti-HA2 antibody turned positive after challenging with H5N1 HPAIV.4. Cross-clade protective immune protection of H5N1 avian influenza vaccine with chimeric heterologous HA headRecombinant virus cHl head/H5 stalk expressing the chimeric H1/H5 was rescued and the biological characteristics and immunogenicity of recombinant virus were evaluated. The results showed that cHl head/H5stalk possessed the similar growth property with S-HALo in chicken embryos. Based on the vaccination with S-HALo, the chickens were boosted by S-HALo and cH1head/H5stalk separately. After challenging with homologous S strain, the group boosted by S-HALo and cH1head/H5stak conferred a strong protection with a protection rate of 100% and no viral shedding. However, in the heterologous challenges with DT, WX, and CZG, the chickens boosted with cHlhead/H5stalk showed a 100% protection with lower viral shedding than did with S-HALo.