Development of a conditionally-attenuated cytomegalovirus (CMV) vaccine vector platform, and initial investigations into use of CMV as a vaccine against pandemic influenza A virus
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Abstract Cytomegalovirus (CMV)-based vectors are a promising vaccine platform with an ability to induce high levels of durable, immediate, effector memory T (TEM) cell responses against their heterologous encoded pathogen target antigen. The primary focus of this thesis is centered on a hypothesis that targeting of essential CMV tegument proteins by using regulatable protein-destabilization is suitable as a conditional vaccine attenuation strategy. CMVs are generally benign, however in individuals whose immune systems are immature or weakened, CMV can be a significant pathogen causing substantial morbidity and mortality. Previous studies have shown that replication-defective versions of CMV made by conventional permanent deletion of essential genes are not compromised in terms of their immunogenicity. However, such non-conditional attenuation requirs parallel development of complementing cell lines, which is technically difficult, expensive and not suitable for use in many situations and environments, in particular low and middle-income countries (LMICs). Recently, protein destabilizing domain (DD) technology has been developed that has the potential to be brought against this issue. DDs are conditionally unstable protein domains that can provide regulatable degradation of a desired protein by genetic fusion to the targeted protein. Addition of a small-molecule binding ligand stabilizes the DD, thereby increasing levels of the targeted protein in a rapid and dose-dependent manner. Using the murine CMV (MCMV) system, we hypothesized that fusion of a DD to essential tegument proteins of MCMV would be capable of regulating the stability of the tegument proteins resulting in generation of conditionally-attenuated MCMV vectors. Part one of this thesis details construction and in vitro characterization of recombinant MCMVs using this DD-based destabilization strategy, and identifies this approach as able to provide a means for production of CMV-based vaccines that differ in their levels of attenuation based on the specific tegument protein targeted. The second part of the thesis is concerned with initial development of CMV as a vaccine vector against pandemic influenza A (IA) virus infection. IA virus is a respiratory pathogen that despite the availability of vaccines continues to have an enormous impact on population health and world economy. Standard seasonal (epidemic) IA vaccines provide only ‘homosubtypic’ immunity. The omnipresent potential for emergence of pandemic IA subtypes, for which the human population possesses no immunity, makes IA a major global health concern. The hypothesis being tested in these studies is that targeting of more conserved IA virus proteins with CD8+ TEM cell-based immunity by using CMV-based vectors as a quintessential inducer of such ‘effector’ memory responses, will provide the desired heterotypic immunity capable of preventing pandemic IA. This initial study determined the capacity of a MCMV-based vaccine expressing the conserved IA proteins nucleoprotein (NP), polymerase (PA) and non-structural protein 2 (NS2) to induce immunity in mice as a strategy to prevent pandemic IA emergence.