A quantitative approach for measuring the reservoir of latent HIV-1 proviruses.

A stable latent reservoir for HIV-1 in resting CD4+ T cells is the principal barrier to a cure1-3. Curative strategies that target the reservoir are being tested4,5 and require accurate, scalable reservoir assays. The reservoir was defined with quantitative viral outgrowth assays for cells that release infectious virus after one round of T cell activation1. However, these quantitative outgrowth assays and newer assays for cells that produce viral RNA after activation6 may underestimate the reservoir size because one round of activation does not induce all proviruses7. Many studies rely on simple assays based on polymerase chain reaction to detect proviral DNA regardless of transcriptional status, but the clinical relevance of these assays is unclear, as the vast majority of proviruses are defective7-9. Here we describe a more accurate method of measuring the HIV-1 reservoir that separately quantifies intact and defective proviruses. We show that the dynamics of cells that carry intact and defective proviruses are different in vitro and in vivo. These findings have implications for targeting the intact proviruses that are a barrier to curing HIV infection

Understanding Antigen Processing of HIV-1 for Improved Vaccine Design and Cure Efforts

The HIV-1 pandemic continues to be a major healthcare crisis for which there are no preventative vaccines or cure. The human immune system is unable to clear HIV-1 due to the extensive genetic variability of the virus and its ability to undergo latency. Combination antiretroviral therapy (cART) blocks viral replication but does not eradicate HIV-1, due to the persistence of proviral DNA in a stable reservoir of long-lived memory CD4+ T cells (described in Chapter 1). A preventative or therapeutic vaccine to HIV-1 requires the induction of protective CD4+T cell responses, which are induced by the appropriate pMHC-II. Determining HIV-1 epitopes optimal for viral control requires profiling potential immunodominant epitopes of a given protein antigen from the perspective of antigen processing. In Chapter 2 of this dissertation, we demonstrate how a minimalist cell-free antigen processing system that utilizes key components of the class II pathway can correctly identify abundantly-presented, immunodominant epitopes across the whole HIV-1 proteome. Remarkably, this system revealed novel epitopes and glycopeptides that were not previously identified in studies utilizing overlapping HIV-1 peptides. Chapter 3 of this dissertation builds on the importance of antigen density in designing immunotherapies. We identified HIV-1 pMHC-I epitopes presented on the surface of infected cells and used a diverse phage display library to pan for phage bearing scFv against the relevant HIV pMHC-I. Sequences from pMHC-specific phage were used to compose bispecific antibody reagents containing one domain against CD3 and another domain against particular pMHC complexes. Binding of the bispecific to CD3 on the effector cell and the HIV-1 pMHC on the infected cell tethers the effector to the target to form an immune cytolytic synapse. We constructed highly specific and potent bispecific antibodies against HIV-1 pMHC-I complexes that could induce killing of infected cells. The efficacy of the reagents depended on pMHC-I antigen density. These results underscore the importance of not just targeting highly conserved epitopes, which has been a longstanding goal of the field, but also abundantly-presented epitopes. Altogether, our findings outline the importance of understanding the peptide landscape of infected cells in order to guide effective vaccine and immunotherapy development

A cell-free antigen processing system informs HIV-1 epitope selection and vaccine design

Distinct CD4+ T cell epitopes have been associated with spontaneous control of HIV-1 replication, but analysis of antigen-dependent factors that influence epitope selection is lacking. To examine these factors, we used a cell-free antigen processing system that incorporates soluble HLA-DR (DR1), HLA-DM (DM), cathepsins, and full-length protein antigens for epitope identification by LC-MS/MS. HIV-1 Gag, Pol, Env, Vif, Tat, Rev, and Nef were examined using this system. We identified 35 novel epitopes, including glycopeptides. Epitopes from smaller HIV-1 proteins mapped to regions of low protein stability and higher solvent accessibility. HIV-1 antigens associated with limited CD4+ T cell responses were processed efficiently, while some protective epitopes were inefficiently processed. 55% of epitopes obtained from cell-free processing induced memory CD4+ T cell responses in HIV-1+ donors, including eight of 19 novel epitopes tested. Thus, an in vitro processing system utilizing the components of Class II processing reveals factors influencing epitope selection of HIV-1 and represents an approach to understanding epitope selection from non-HIV-1 antigens

A quantitative approach for measuring the reservoir of latent HIV-1 proviruses.

A stable latent reservoir for HIV-1 in resting CD4+ T cells is the principal barrier to a cure1-3. Curative strategies that target the reservoir are being tested4,5 and require accurate, scalable reservoir assays. The reservoir was defined with quantitative viral outgrowth assays for cells that release infectious virus after one round of T cell activation1. However, these quantitative outgrowth assays and newer assays for cells that produce viral RNA after activation6 may underestimate the reservoir size because one round of activation does not induce all proviruses7. Many studies rely on simple assays based on polymerase chain reaction to detect proviral DNA regardless of transcriptional status, but the clinical relevance of these assays is unclear, as the vast majority of proviruses are defective7-9. Here we describe a more accurate method of measuring the HIV-1 reservoir that separately quantifies intact and defective proviruses. We show that the dynamics of cells that carry intact and defective proviruses are different in vitro and in vivo. These findings have implications for targeting the intact proviruses that are a barrier to curing HIV infection