Interactome screening of Human Cytomegalovirus and the Complement system & Investigating the pre-clinical immunogenicity of a novel SARS-CoV-2 Envelope (E) protein vaccine

Human Cytomegalovirus (HCMV) is a ubiquitous virus that is a leading cause of both congenital infections in neonates and opportunistic infections in immunodeficient persons such as HIV/AIDS patients and solid-organ transplant recipients. Few human viruses compare to the complexity, broad cell tropism, and life-long persistence observed in HCMV infections. HCMV has one of the largest genomes of any known human viral pathogen with an approximate size of 230kbp of DNA that encodes roughly 175 canonical genes. The genes encoded by HCMV have been characterized by forward genetics using a single-gene deletion approach to understand the essential or dispensable nature of each HCMV gene necessary for viral replication in vitro, however the precise biological functions of most HCMV genes remain incompletely understood. HCMV encodes an unparalleled number of proteins that subvert both innate and adaptive immunity, yet currently the role of the human complement system in HCMV infection and immunity remain poorly understood even though this important branch of innate immunity has been well characterized in other related human herpesviruses./nPrevious studies have reported that HCMV virions incubated in serum (a source of complement) are not neutralized but are nevertheless coated with activated complement proteins that normally proceed the direct lysis of microorganisms. This observation suggests that HCMV virions not only activate one or more pathways of complement, but are able to inhibit complement-mediated neutralization perhaps by a viral encoded complement regulating protein. In this dissertation, we screened 6 human complement proteins (CD55, Factor H, Mannose Binding Lectin 2, Mannan-Associated Serine Protease-1, Properdin, and C1q Binding Protein) against an HCMV genomic library in yeast containing 167 viral open reading frames. Out of 1,002 possible HCMV-Complement interactions tested, we identified 121 (8.2%) positive-protein interactions by yeast-two-hybrid among all six complement proteins tested and validated a subset of these novel HCMV-Complement interactions in human cells by co-immunoprecipitation. To date, our study is the largest, most comprehensive, and systematic investigation of interactions between HCMV the complement system, and provides a framework to further investigate HCMV-Complement interactions that may underlie undiscovered mechanisms of innate immune evasion by HCMV and potentially inform the development of novel drugs and vaccines for the treatment and prevention of HCMV infections./nDuring our studies, a novel coronavirus known as Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) emerged and spread rapidly in humans, which led to a pandemic and significant loss of life globally. In response to the pandemic, SARS-CoV-2 vaccines were developed but their efficacy has been undermined by the increasing emergence of SARS-CoV-2 isolates with mutations in the Spike (S) protein which is the primary vaccine antigen in all licensed SARS-CoV-2 vaccines. These observations underscore the significant need to identify additional antigens for possible inclusion in the next-generation of SARS-CoV-2 vaccines. To address this significant unmet medical need, we investigated the vaccine potential of a highly conserved 30 amino acid transmembrane domain epitope of the SARS-CoV-2 Envelope (E) protein in mice. To increase immunogenicity, we conjugated the E-protein transmembrane domain to the immunogenic carrier protein Keyhole Limpet Hemocyanin (KLH) to generate a vaccine antigen herein referred to as KLH-E. Mice were immunized with one or two doses of KLH-E vaccine or aluminum control vaccine intramuscularly in thirty-day intervals. The KLH-E vaccine elicited serum IgG antibodies and antigen-specific T-cells to each subunit of the vaccine (KLH and E), and to our surprise induced anti-E IgG antibodies and T-cells that recognize the native E protein from the genetically divergent but related human coronavirus 229E (HCoV-229E). Furthermore, pooled sera from mice had neutralizing activity against SARS-CoV-2 pseudovirions expressing S proteins from emerging variants of concern (Beta, Delta, Omicron, XBB), and had neutralizing activity against HCoV-229E which shares only a modest 32% E protein sequence homology to the vaccine antigen. Taken together, these results suggest that the SARS-CoV-2 E protein transmembrane domain contains important epitopes that are conserved across different human coronaviruses and demonstrates that immunization with the E protein transmembrane domain from SARS-CoV-2 can induce cross-reactive immune responses that may confer protection against emerging SARS-CoV-2 variants and other genetically diverse human coronaviruses.

Advisor: Liu, Fenyong