LIVE-ATTENUATED VACCINES AGAINST INFLUENZA VIRUSES AND ROLE OF NS1 IN HOST RESPONSE
Perez, Daniel R.
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The research interests in this dissertation thesis are to develop a better live attenuated influenza virus A (LAIV) vaccine which allows mass vaccination and further understand the phenotype changes of a quail-adapted influenza virus. The recurrent outbreaks of low pathogenic avian influenza (LPAI) and highly pathogenic avian influenza (HPAI) highlight the pressing need for the development of mass in-ovo vaccination strategies using LAIV. However, this method often results in a poor hatchability (unpublished data). Our study shows that after vaccination with a LAIV carrying the modified H7 and H9 hemagglutinin (HA) proteins by substituting the amino acids at the cleavage site for those found in the H6 HA subtype, the hatchability of egg embryos was significantly improved (≥90%). Furthermore, a single dose in-ovo vaccination in 19-day-old chicken embryo provided complete protection against homologous challenge with low pathogenic virus in ≥ 70% of chickens at 2 or 6 weeks post-hatching. Our findings indicate that the strategy of modifying the HA cleavage site in a LAIV backbone could be used for improving in-ovo vaccination. A good vaccine candidate should grow to high titer in eggs or cell culture. In this study, through egg adaptation, a LAIV (2 mouse-adapted(ma) Ca/04 H1N1:6WF10att) mounted a higher HA titre in eggs compare to its parental virus. Sequence comparison of the egg-adapted virus with its parental virus revealed only one amino acid substitution in PA protein (E59V). Using reverse genetics, E59V was introduced in PA protein of the parental virus, which renders the virus similar growth characteristics as the egg-adapted virus. Importantly, this mutation did not change the temperature sensitive (TS) phenotype of the attenuated virus. Furthermore, the E59A in PA also moderately enhanced both HA and viral titers in wild type WF10 and Turkey/04 att backbones. In contrast, E59V had no significant effect on that of PR8 or ca Ann Arbor/60 backbone viruses. Take together; these data suggest that single change in PA, E59V, is critical for some LAIV vaccines efficient growth in eggs or cell culture. A QA23 virus was previously generated through 23 serial passages of the H9N2 (A/duck/Hong Kong/702/79) virus in quail lung. Phenotypically, the QA23 viruses replicate and transmit more efficiently in both chickens and quail. Interestingly, QA23 NS1 protein was mainly distributed in the cytoplasm, which is remarkably different from the parent strain NS1 that was mainly accumulated in the nucleus. Although mutations occurred in the nuclear localization signal sequence, QA23 NS1 did not display a significant change in antagonizing IFN-β function. In addition, QA23 NS1 up-regulated the viral protein synthesis in DF1 cells and greatly reduced the apoptosis of the cell during the infection. However, there is no evidence showing that QA23 NS1 is associated the enhanced replication and transmission of the viruses in Japanese quail.