CpG dinucleotide enrichment in the influenza A virus genome as a live attenuated vaccine development strategy

Abstract

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

Understanding off-target growth defects introduced to influenza A virus by synonymous recoding

“…In support of this, we saw that while NP production was dramatically impaired during minireplicon assays, this did not impact polymerase activity. This highlights another previously unreported but important consideration for CpG-based recoding designs, as more robust phenotypes may be evident when targeting process-limiting transcripts (14).…”

“…CpG enrichment has gained traction as an attractive model for the development or augmentation of live attenuated vaccines (13,14). Here, we argue that understanding the mechanism of attenuation imparted by synonymous recoding is imperative as a safety feature of this technology.…”

“…A549 KHNYN-/-cells were a gift from Dr Chad Swanson (38). The knockout status of these cell lines was previously verified by our lab (14). Cells were checked monthly for mycoplasma contamination using Lonza MycoAlert kit.…”

“…Northern blotting. Northern blotting was performed as previously described (14). Briefly, A549 cells infected at MOI 3 for 8 hours.…”

“…Adding CpGs into viral genomes is a now well-characterised attenuation mechanism that has been proposed by ourselves and others to have potential application in live attenuated vaccine design (2,(6)(7)(8)(9)(10)(11)(12)(13), because adding CpGs limits viral replication but is unlikely to affect antigen conformation. Using influenza A virus (IAV) as a tractable model system in which to test this, we recently reported that increasing the CpG content of segment 1 of IAV resulted in a ZAPmediated attenuation that caused CpG-high transcript turnover, but not type I interferon induction (14).…”