An Investigation of the SHIV reservoirs in the lymphoid organs of HIV-vaccinated rhesus monkeys after SHIV challenge

Benn, Kealan Timothy

An Investigation of the SHIV reservoirs in the lymphoid organs of HIV-vaccinated rhesus monkeys after SHIV challenge

Master Thesis

2022

Abstract

The development of an effective vaccine against HIV-1 is thought to be a key component of combating the current HIV epidemic. This study aimed to investigate viral reservoirs in the blood and lymphoid tissues after HIV vaccination and virus challenge using the rhesus macaque model. In addition, the study investigated the envelope sequences of the virus located in the potential reservoir sites in an attempt to understand viral variability. Cryopreserved peripheral blood mononuclear cells (PBMC) and cells isolated from lymphoid tissues obtained at termination of vaccine group (P4, P11, P25, P47, P52) and control group (P67, P70, P71, P72) macaques were processed for determination of viral RNA and proviral DNA loads, and HIV-1 Env amino acid sequences using single genome amplification (SGA) sequencing method. In addition, cryopreserved plasma obtained at key time points pre- and post- vaccination and SHIV challenge were used to measure the levels of HIV Env and SIV Gag antibodies by Western blotting, and HIV-1 Env gp140 ELISA. RNA viral loads were low for all animals with the median of the averages of the viral load for PBMCs being 3.82 copies/107 cells and a range of < 1 copies/107 cells to 61.15 copies/107 cells. For the control group, only P67 had a detectable viral load in the PBMC. The median of the averages for proviral DNA load for PBMCs was 1903.98 copies/107 cells and a range of 696.93 copies/107 cells to 28 663.88 copies/107 cells. Proviral DNA levels were higher than the RNA viral loads indicating a larger amount of integrated provirus and potentially the presence of reservoirs within the macaques. While the low viral load values could mean there are few actively replicating viruses present in the PBMCs. The median of the averages for the RNA viral loads in the inguinal tissue cells was 58.08 copies/107 cells with a range of 8.85 copies/107 cells to 911.61 copies/107 cells. The median of the average for proviral DNA load in the inguinal tissue cells was 3160.36 copies/107 cells with a range of 604.67 copies/107 cells to 73 140.68 copies/107 cells. Proviral DNA levels in the inguinal tissues were higher than the RNA viral load levels which indicates a larger amount of integrated provirus present in the cells of the inguinal tissues and the presence of a potential reservoir. This also indicates that there is less circulating virus. The Western blots showed that the macaques developed binding antibodies to both HIV Env and SIV Gag, however, non-specific binding of antibodies to proteins other than HIV Env and SIV Gag was also seen. The ELISA effectively showed that both the vaccine group and control group macaques were able to produce binding antibodies. Macaques P4, P47, and P52 had the highest endpoint titre for the vaccine group of 5120. Macaque P71 had the highest overall endpoint titre of 20 480. Macaques P11 and P25 of the vaccine group had an endpoint titre of 1280 which was the same endpoint titre for control group macaques P67 and P70, while macaque P72 had an endpoint titre of 5120. Single genome amplification and sequencing showed that there were very few amino acid changes between sequences found in macaques. There was minimal variation within the vaccine group and between the vaccine and control groups with amino acid changes in the variable region 1 (V1), glycan N276 in the conserved region 2 (C2) and variable region 4 (V4) resulting in potential N-glycosylation sites (PNGS) being shifted as well as new glycosylation sites being formed while other lost. This study demonstrates that RNA viral loads and proviral DNA loads were detectable in both macaque groups with no significant difference being present in the viral RNA and proviral DNA loads between the two macaque groups. The lack of changes seen in the sequences of the inguinal and PBMC tissues are due to the early virus seeding the reservoir. Once the reservoir is seeded active replication does not occur in the reservoir resulting in the viruses having similar sequences.

Keywords

Medicine

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