Methodologies to target conserved viral epitopes for vaccine and therapeutic development

The eradication of smallpox signaled a paradigmatic shift in vaccinology. Eradication of the red plague, as it has been called, solidified the lifesaving potential of vaccines. Spurred on from this were massive advances in the fields of vaccinology and virology. These technological advances carried with them new vaccines. Live viral attenuation, reverse vaccinology, computational protein engineering, and most recently mRNA vaccination, are examples of methodologies that improved vaccine efficacy and activity. It is almost certain that future technological advances will drive the development of vaccines capable of protecting against the pathogens still intractable to modem vaccines (e.g. influenza and HIV-1). In this thesis we lay out several methodologies, primarily in vaccine design but also in therapeutic development, that aim to further develop and improve our ability to prevent or treat diseases. In chapter 1 we lay the groundwork for methods termed "immunofocusing" which seek to overcome inherent limitations in current vaccine approaches. Methods like cross strain boosting, mosaic display, protein dissection, epitope scaffolding, and epitope masking are addressed. We evaluate the methods and educate on their applications. Chapter 2 introduces a novel immunofocusing method, protect, modify, deprotect (PMD), which aims to improve upon current immunofocusing methods. We show that PMD, which uses an antibody against a conserved epitope as a molecular stencil, produces a vaccine against influenza, capable of providing improved binding, neutralization, and lethal challenge protection against an array of influenza variants. We further extend this work by providing the foundation for an in solution PMD method, which could be applied to future whole-virus or nanoparticle-based vaccines. Chapter 3 introduces a novel method for therapeutic development utilizing non-neutralizing antibodies which target conserved epitopes. Receptor binding conserved non-neutralizing antibodies (ReconnAbs), leverage the binding activity of non-neutralizing antibodies to deliver a weakly neutralizing component. We demonstrate that ReconnAbs against SARS-CoV-2 provide broad-spectrum protection against an array of SARS-CoV-2 variants of concern. In chapter 4 we develop a novel vaccine microparticle scaffold from unnatural-amino acid incorporated bacterial sacculi. We demonstrate the utility and versatility of this novel microparticle platform using two different subunit antigens (sfGFP and SARS-CoV-2 receptor binding domain) as well as two different animal models (guinea pigs and mice). Collectively, these novel nanoparticles are immunogenically equivalent to the commonly used carrier protein keyhole limpet hemocyanin (KLH) while being easier to purify and more stable. In chapter 5 we use protein engineering to develop a novel purification strategy for glycoprotein modified ferritin nanoparticles. We show that the strategic incorporation of poly-histidine tags into flexible loops on the surface of ferritin enables robust metal-cation based purification of whole nanoparticles. Following purification these proteins elicit similar immune responses to their non-tagged counterparts. Such installations may be useful for both purification or installation of T-cell epitopes for downstream applications. Chapter 6 outlines collaborative work developing a novel, ferritin nanoparticle-based SARS-CoV-2 vaccine. We show that this vaccine confers long-term protection against an array of variants of concern. Moreover, we show that variant specific vaccines do not improve the overall efficacy of the nanoparticle vaccine -- a result which has been since supported in literature about variant specific vaccines and complicates the future of SARS-CoV-2 vaccines. Chapter 7 introduce a novel concept in immunofocusing termed epitope-agnostic immunofocusing. Using the SARS-CoV-2 spike protein we produce 15 different glycosylation variants exposing different epitopes for attempted immunofocusing. We aim to target epitopes for which no known broadly neutralizing antibodies (bnAbs) have been identified. We find a protein with a glycan hole at the fusion peptide elicits better variant-specific protection. This result is surprising given the lack of known bnAbs which target this epitope. These results demonstrate the utility of producing an array of potential immunofocusing vaccine candidates in future immunofocusing efforts. In chapter 8 we utilize gold nanoparticles (AuNPs) to attempt to produce an anti-HIV N-heptad repeat (NHR) vaccine. We optimize conjugation of a mimetic of the NHR to the surface of gold nanoparticles. We demonstrate that these complexes alter AuNP behavior and lead us to a novel strategy for conjugating CpG DNA to the surface of the AuNPs. Immunogenicity of these AuNP conjugates is amplified by the addition of CpG DNA to AuNP surface. Finally, chapter 9 is a summary of the methods utilized throughout this thesis

Keywords: Chemistry.
Advisor: Kim Peter
Bertozzi Carolyn R.
Khosla Chaitan