ANTI-CAPSID MONOCLONAL ANTIBODIES: TOOLS TO ASSESS HPV NEUTRALIZATION AND CAPSID STRUCTURE
Open Access
- Author:
- Bywaters Sylvester, Stephanie Marie
- Graduate Program:
- Microbiology and Immunology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 17, 2018
- Committee Members:
- Neil David Christensen, Dissertation Advisor/Co-Advisor
Neil David Christensen, Committee Chair/Co-Chair
Craig Matthew Meyers, Committee Member
Susan Hafenstein, Committee Member
Laura Carrel, Outside Member
Rebecca C Craven, Committee Member - Keywords:
- HPV
monoclonal antibody
capsid
structure
L2
competition - Abstract:
- Human Papillomavirus (HPV) is the known causative agent of cervical cancer in addition to other pathologies including anogenital and oropharyngeal cancers. The replication cycle of the virus is uniquely associated with the differentiation program within stratified epithelium. HPV strategically restricts expression of the antigenic capsid protein to the most suprabasal layer, complicating immunological detection of infection. Current vaccine strategies rely upon the antibody response to assembled, non-infectious HPV VLPs formed from only L1, the major capsid protein. HPV type-specific, conformation-sensitive antibodies elicited by the prophylactic vaccine bind and neutralize the virus upon subsequent natural infection. Although it is known that the antibody response is responsible for the protection against HPV infection, it is not well-understood how the antibodies neutralize the virus. The objective of this thesis was to examine how antibodies interact with the HPV capsid. Further, we wanted to determine the mechanisms of antibody-mediated HPV neutralization. We specifically wanted to define the epitopes of neutralizing monoclonal antibodies (mAbs) on the capsid of HPV. Previously, neutralizing epitopes have been mapped to the hypervariable loops of L1. Here, we specifically defined the amino acid footprint of five mouse anti-L1 mAbs. Using cryo-electron microscopy, we localized binding of four mAbs (V5, 1A, 14J, and 263.A2) to the vertices of L1 capsomers while one (U4) uniquely bound within the inter-capsomeric spaces. Our epitope mapping combined with observed changes in the capsid structure and biological assays allowed us to hypothesize how these antibodies neutralize HPV and we have implicated a combination of neutralizing mechanisms including capsid stabilization and virion crosslinking. While there exists a consensus on the structure of the HPV L1 VLP, the placement of the minor capsid protein (L2) remains controversial. Studies have suggested that there are anywhere from 12-72 L2 proteins incorporated into the HPV capsid. In contrast to L1, the L2 sequence is more conserved across the different HPV types. Therefore, the ability of L2 to generate neutralizing cross-reactive antibodies has been examined. Several studies have been successful in generating anti-L2 antibodies, but many have been polyclonal. Other highly cross-reactive mAbs such as RG-1 have been developed but this N-terminal epitope is believed to only be exposed on the capsid after interactions with cellular components. In order to help gain a better understanding of the virus capsid topology and neutralizing L2 epitopes, we developed and characterized a panel of anti-L2 mAbs. The panel detects both conformational and buried epitopes and several were cross-reactive. Although some of the mAbs in our panel recognize epitopes defined by historical mAbs described in previous studies, others uniquely recognize portions of L2 further downstream of the N-terminus. Therefore, these probes should be beneficial in future studies to further define the lifecycle of HPV as well as the structural properties of L2. In an effort to better describe the incorporation of L2 within the HPV capsid, we utilized two newly characterized anti-L2 mAbs (L2.1A and L2.2E) from our panel. Although previous studies have demonstrated that their targeted amino acids are not accessible on virions in solution, we showed that 1A and 2E are able to bind solution-phase capsids. Combined with our knowledge of the precise anti-L1 mAb epitopes, we used the anti-L2 mAbs and anti-L1 mAbs in a series of competition binding studies to approximate the location of L2. Similar to the U4 mAb, our studies suggest that the 1A and 2E epitopes are located between capsomers. We further quantitated L2 with anti-L1 and anti-L2 mAbs. Our studies support a subdominant incorporation of L2 with an average incorporation of 50 L2 monomers per capsid.