Viral Vectors for Gene Therapy, Cancer, and SARS-CoV-2

Abstract

Viral vectors have become crucial to multiple fields as vehicles for gene therapy, vaccines, and reporter genes, among other uses. Two of the most prominent vectors were generated from adenoviruses and adeno-associated viruses (AAVs). Presently, adenoviral vectors have been clinically studied as vaccines for Zika, Ebola, and SARS-CoV-2 viruses. AAVs are now used in FDA approved gene therapy treatments. While vectors have already had an immense impact on the scientific and medical world, there remain many possible new applications as well as areas of improvement for these tools. In chapter two a new virome (SIV infected rhesus macaque) is explored allowing for the discovery of five novel AAV capsids. Structure guided analysis of the RhAAV4282 capsid to the closely related AAV7 capsid enabled the identification of a novel AAV capsid neutralization epitope named the Hypervariable Region IV Neutralizing Epitope (HRNE). Swapping the HRNE between RhAAV4282 and AAV7 capsids significantly reduced neutralizing antibody titers against the parental capsids. In chapter three the adenovirus serotype 26 (Ad26) vector platform is used to deliver neoantigen vaccines in a cancer model. Ad26 and long peptide neoantigen delivery were analyzed by traditional mechanistic immunology methods and scRNA-seq/TCR-seq. This analysis showed increased immune responses, upregulation of maturation and activation T cell pathways, clonal hyper-expansion, and protection by the Ad26 platform but no difference in tumor T cell infiltration. These results highlighted the importance of not just T cell quantity, but also T cell quality induced by a cancer vaccine. In chapter four the proteomes of 82 different sarbecoviruses were aligned to identify conserved regions, with the goal of generating a broadly protective sarbecovirus vaccine. A portion of the sarbecovirus RNA-dependent RNA polymerase and helicase were identified as highly conserved (93% perfect T cell epitope conservation) among sarbecoviruses. A rhesus adenovirus serotype 52 (RhAd52) vaccine carrying this immunogen was found to be immunogenic and protective against challenge by mouse adapted SARS-CoV-2. The data presented in this thesis generates new possibilities for AAV capsid engineering, furthers understanding of how to design neoantigen cancer vaccines, and identifies a highly conserved sarbecovirus immunogen. This research has implications for advances in viral vector applications, vaccines, cancer, and gene therapies.