Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Master of Science

Program

Microbiology and Immunology

Supervisor

Creuzenet, C.

2nd Supervisor

Arts, E.

Co-Supervisor

Abstract

The great heterogeneity of HIV populations and richness of surface glycan clouds makes it difficult to locate a conserved and exposed protein epitope as an effective vaccine target. However, more than 80% new infections result from single transmitted founder (T/F) viruses. We set out to design a workflow to study the traits of T/Fs that allow for their superior infectivity, specifically, the glycosylation patterns of gp120, a subunit of HIV envelope protein responsible for binding to host cell receptors. Our main research methods include Western blot and mass spectrometry. Our current understanding of the mass spectrometry data indicates that our T/F and chronic HIV strains have differential distributions of glycan density at several key N-sites throughout the gp120 peptide backbones, which may be related to the differential transmission fitness of the two strains and potentially used as novel glycopeptide-based HIV vaccine targets.

Summary for Lay Audience

One of the greatest challenges in HIV vaccine development is that the viral populations are highly diverse, making it difficult to find a universal vaccine target that works on all viruses. Despite the large genetic diversity, more than 80% of new HIV infections result from the transmission of a single virus, known as a transmitted founder (T/F) virus. This indicates that usually only one T/F out of the pool of viruses from the HIV donor is able to establish a stable infection in a new recipient. Understanding the unique features of T/Fs will provide novel strategies for vaccines and ultimately prevent the spread of HIV worldwide.

We set out to design a workflow to study one of the major factors believed to give T/Fs a selective advantage during transmission: gp120 glycosylation. Gp120 is a protein on the surface of HIV particles and initiates the infection of a human host cell. Glycosylation is a network of sugar chains (or glycans) attached to the protein backbone at specific points known as N-linked sites. As HIV undergoes frequent genetic mutations, viruses evolve to have different numbers and locations of N-linked sites and different types of sugar chains. The goal of my project is to compare the gp120 glycosylation profiles of a T/F strain and a strain derived from chronic stage of untreated HIV-1 infection. The major tool we use is mass spectrometry, which breaks down gp120 into small fragments of peptides, sugars, and glycopeptides. This allows us to identify the types of the glycans present in the sample and at which N-linked sites they are attached. We then generate a matrix of N-linked sites and types of glycans for both strains. In this thesis, our findings show that the two strains have differential distributions of glycan density throughout the gp120 peptide and that they express distinct compositions of glycans.

This project is a proof-of-principle study that provides tools for larger-scale studies to include more strains of T/F and chronic HIV viruses. Ultimately, this workflow will help unveil the key glycopeptide signatures accountable for HIV transmission that can be used as novel vaccine candidates.

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