Files and links (1)
PDFOpen Access (License Unspecified), Open Access
Dissertation
Studies into the mechanism underlying the neutralization of HIV-1 by the monoclonal antibody, M18
Doctor of Philosophy (Ph.D.), Drexel University
Sep 2010
DOI:
https://doi.org/10.17918/00000775
Abstract
We investigated the interaction between HIV-1 gp120 and the neutralizing monoclonal antibody m18 in order to deepen our understanding of how this antibody inhibits viral entry into cells. Through surface plasmon resonance and isothermal titration calorimetry, we found that m18 binds gp120 with low nanomolar affinity and suppresses gp120 interactions with both soluble CD4 and the co-receptor surrogate, mAb 17b. The dual antagonism of both receptor binding sites in gp120 suggests that m18 is not functioning as a mimic of CD4. Interactions of m18 with various conformational mutants of gp120 were studied. Mutations to the co-receptor site had minimal effects on m18 binding, while mutations in the CD4 site had a strong impact on m18 binding. In contrast, we found that m18 could bind to gp120 saturated with the CD4 mimic, NBD-556, indicating that m18 does not require the Phe43 cavity and that the linkage between m18 and CD4 binding is non-competitive. Binding analysis with the functional outer domain construct of gp120 showed that m18 requires more than just this domain for interaction. In conjunction with gp120 variants, mutagenic analysis of m18 was performed to determine the contact residues required for gp120 interaction. Direct binding and competition analyses through SPR indicated that gp120 binding uses a broad paratope of residues in m18. Strikingly, several single site m18 mutants were identified that bound well to gp120 but did not interfere significantly with CD4 and 17b binding. A subset of these mutants did however neutralize viral infection. These results fit with a model in which m18 binding occurs by a two-phase process involving both m18-gp120 interaction and entrapment of gp120 into an inactivated conformational state that, by isothermal titration calorimetry, appears less ordered than the CD4-bound state Inhibition of viral infection may result from both receptor binding interference and disruption of the functional organization of the viral envelope spike. Further understanding of the structural components of m18 responsible for binding, conformational entrapment and spike organization offers the opportunity to design antibody-inspired allosteric inhibitors of HIV-1 entry and infection.
Metrics
20 File views/ downloads
20 Record Views
Details
- Title
- Studies into the mechanism underlying the neutralization of HIV-1 by the monoclonal antibody, M18
- Creators
- Syna Kuriakose Gift
- Contributors
- Irwin Chaiken (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xiii, 178 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Biochemistry and Molecular Biology; College of Medicine; Drexel University
- Other Identifier
- 991014970300804721