Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Microbiology and Immunology

Supervisor

Mann, Jamie F.S.

Abstract

Introduction:

Since Human Immunodeficiency Virus (HIV)-1 was determined to be the etiological agent behind acquired immunodeficiency syndrome (AIDS) in 1983, numerous attempts at a cure have been made; however, none have been effective. One of the primary roadblocks in achieving a cure is a transcriptionally-silent latent reservoir of memory CD4+ T cells harboring HIV provirus. Combined antiretroviral therapy (cART) inhibits actively replicating virus by interfering with various stages of the replication cycle. Therefore, non-replicative viruses–like the proviruses found in latently infected cells–are hidden from the actions of continued antiretroviral therapy. As a result, cART discontinuation or treatment holidays can result in rapid viral recrudescence within days to weeks. It is thought that latency is established when HIV infects CD4 T cells that are transitioning from effector to memory status, which effectively traps the virus in an environment that is not conducive to replication due to the low metabolic activity characteristic of these cells at G0. During latency reversal, it is believed that the provirus undergoes transcriptional reactivation, allowing HIV-1 to replicate and produce infectious progeny virus that can be targeted by cART. However, a significant hurdle against the development of a vaccine against HIV is the immense viral diversity within each infected individual. A vaccine preparation should therefore encompass a near-complete repertoire of viral quasispecies to increase antigen coverage for maximal reactivation. Based on this, our group has developed a genetically diverse virus-like particle (VLP)-based vaccine, which is thought to be capable of activating a diverse array of cognate HIV-specific CD4+ T cells. These VLPs are morphologically and enzymatically identical to wild-type HIV-1; however, it contains several mutations that abrogate reverse transcription, integration, and viral RNA packaging.

Methodology:

VP and VLP antigenicity was determined as a function of i) TNFα secretion measured in cell culture supernatant of HIV– PBMC and ii) NF-κB and interferon regulatory factor (IRF) activation. Latency reversal was performed through an ex vivo DC-T-cell co-culture assay where induced HIV RNA from budded virus was quantified in cell culture supernatants following latency reversal and using an in-house developed qRT-PCR assay specific for the 5’ region of the HIV RNA. In addition, T cell activation was measured as a function of IFN-γ secretion using a commercially available ELISpot kit. To determine if immunostimulatory RNA could be preferentially packaged into VLP, 293T cells were co-transfected with a VLP-encoding plasmid and plasmids encoding for immunostimulatory RNAs. The adjuvanted VLPs were then concentrated by a combination of filtration and ultra-centrifugation with purified VLPs assayed for p24 and RNA content. Immunostimulatory potential of these RNA were measured as a function of NF-κB and IRF activation in THP-1 cells, and TNF-α and IFN-α secretion in HIV– PBMC.

Results:

VPs induced greater TNFα secretion than VLPs in HIV– PBMC, supporting observations of a VPs inducing greater increases in NF-κB and IRF activation compared to VLP. In addition, these VPs induced greater latency reversal in HIV+ PBMC, as a function of increased CD4+ T cell activation and increased HIV RNA in culture supernatant following latency reversal. We therefore engineered immunostimulatory RNA that can be packaged into VLPs, and found that these RNAs augmented VLP antigenicity in PBMC and THP-1 cells in a manner that was dependent on RNA recognition by intracellular PRRs.

Summary for Lay Audience

Since Human Immunodeficiency Virus (HIV)-1 was determined to be the etiological agent behind acquired immunodeficiency syndrome (AIDS) in 1983, numerous attempts at a cure have been made; however, none have been effective. One of the primary roadblocks in achieving a cure is a transcriptionally-silent latent reservoir of memory CD4+ T cells harboring HIV provirus. Combined antiretroviral therapy (cART) inhibits actively replicating virus by interfering with various stages of the replication cycle. Therefore, non-replicative viruses–like the proviruses found in latently infected cells–are hidden from the actions of continued antiretroviral therapy. As a result, cART discontinuation or treatment holidays can result in rapid viral recrudescence within days to weeks. Our group has developed a genetically diverse virus-like particle (VLP)-based vaccine, which is thought to be capable of activating this reservoir. These VLPs are morphologically and enzymatically identical to wild-type HIV-1; however, it contains several mutations that abrogate reverse transcription, integration, and viral RNA packaging. In this thesis, we show that virally packaged RNA within these particles can augment VLP antigenicity and latency reversal.

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