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The prevention of HIV infection and the stimulation of cells latently infected with HIV

  • Author(s): Fischer, Kathryn Elyse
  • Advisor(s): LiWang, Patricia
  • et al.
Abstract

Thirty-five years after its exposure, HIV is still a global health threat with millions of new infections annually. The goal of our research is to stop the global spread of HIV, first by preventing new HIV infections and then by working to cure existing infections. Chapter one reviews the history of HIV, the infection and transmission process and the current approaches for the prevention and cure of HIV so that the reader will have a thorough understanding of the molecular workings of HIV. I also address the origin, discovery and classification of HIV. In chapter 2 we investigated the anti-HIV lectin Griffithsin that inhibits HIV by binding to high mannose glycans on the viral envelope protein gp120. Mutational studies that removed or shifted glycans on gp120 showed that Griffithsin retains anti-HIV-1 potency despite changes in gp120 glycosylation. We found through HIV capture ELISAs that Griffithsin’s ability to alter the structure of gp120, exposing the CD4 binding site, correlated with the presence of glycosylation at N295 only in B clade strains, not C clade strains. We further demonstrated that Griffithsin can rescue the activity of broadly neutralizing antibodies PGT121 and PGT126 in the event of loss or shift of glycosylation at N332, where these broadly neutralizing antibodies suffer a drastic loss of potency. Chapter 3 reviews the purification and testing of a chimeric protein composed of the HIV transcriptional activator protein Tat, and a variant of a human chemokine, 5P14-RANTES, which binds to CCR5, a receptor that HIV utilizes during the infection process. This chimeric protein was designed with the theoretical purpose of targeting to and then activating cells latently infected with HIV. We attempted to produce and purify the 5P14-Linker-Tat protein as a full-length construct and by linking the two separate proteins with a sortase enzyme. Regardless of the method used, the protein was difficult to produce in large quantities and the final product contained protein contaminants. Unfortunately, when we tested our construct (in both orientations, 5P14-Linker-Tat and Tat-Linker-5P14) using a MAGI colorimetric assay, we found that it did not show targeting to CCR5 cells and little to no activation of the HIV LTR linked GFP.

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