CpG enrichment as a live attenuated vaccine development strategy for influenza A virus

Synonymous recoding of RNA virus genomes is a promising approach for generating attenuated viruses to 11 use as vaccines. However, such recoding typically hinders virus growth. A potential solution to this is CpG 12 dinucleotide enrichment. CpGs are recognised by cellular zinc-finger antiviral protein (ZAP), leading to viral 13 RNA degradation and/or type I interferon stimulation and consequently reduced viral yields. Therefore, in 14 principle, removing the ZAP CpG sensor from a virus propagation system will reverse attenuation of a CpG- 15 enriched virus. Using influenza A viruses (IAVs) engineered for increased CpG content in different genome 16 regions, we show that ZAP mediated virus attenuation is dependent on the viral segment to which CpGs are 17 added. In ZAP-expressing human cells, CpG introduction invariably impaired transcript and protein production, 18 but ZAP-sensitivity did not convey type I interferon activation. Importantly for vaccine development, CpG- 19 enriched viruses were genetically stable during serial passage. Furthermore, the ZAP-sensitive virus was fully 20 replication competent in MDCK cells and in embryonated hens’ eggs, both of which are used to propagate live 21 attenuated influenza vaccines. Thus, ZAP-sensitive CpG enriched viruses that are defective in human systems 22 can yield high titre in vaccine propagation systems, providing a realistic, economically viable platform for live 23 attenuated vaccine development.