Doherty Institute - Theses
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ItemThe role of antibody-dependent cellular cytotoxicity in curing latent HIV-1 infection( 2018)As of 2017, 36.9 million people worldwide were living with HIV-1, with approximately 21.7 million people treated with antiretroviral therapy (ART) and only a single individual cured of HIV-1. HIV-1 infection has proven difficult to cure as HIV-1 integrates into the genome of host cells and establishes a latent viral reservoir despite ART, necessitating lifelong therapy for the millions of people living with HIV-1. This continues to place a massive economic burden on the health sector, substantiating the need for an HIV-1 cure to end the HIV-1/AIDS pandemic. One of the most extensively studied cure strategies is the ‘shock and kill’ approach, which aims to reactivate HIV-1 expression from latently infected cells and subsequently eliminate the reactivated cells through immune-mediated mechanisms. We hypothesised that antibody Fc effector functions including antibody-dependent cellular cytotoxicity (ADCC) could be harnessed to eliminate the reactivated viral reservoir. This thesis first examined whether antibodies within HIV-1-infected individuals could recognise and eliminate cells reactivated from latency (Chapter 2). We found that reactivation of HIV-1 expression in the latently infected ACH-2 cell line elicited antibody-dependent natural killer (NK) cell activation but did not result in antibody-mediated killing. The lack of CD4 expression on ACH-2 cells likely resulted in the concealment of CD4-induced (CD4i) epitopes on HIV-1 envelope (Env) that are highly targeted by ADCC antibodies within HIV-1-infected individuals. We found that ex vivo-expanded primary CD4+ T cells from HIV-1-infected individuals were modestly susceptible to ADCC mediated by autologous serum antibodies and effector cells. Importantly, ADCC against these ex vivo-expanded CD4+ T cells could be enhanced following incubation with a small-molecule CD4 mimetic compound that exposes CD4i ADCC epitopes on Env. Since HIV-1-specific ADCC antibodies decline in individuals on long-term ART, this thesis next examined whether latency reversal with the histone deacetylase inhibitor panobinostat could provide sufficient antigenic stimulus to boost ADCC antibodies (Chapter 3). We found that in vivo treatment with panobinostat or a short analytical treatment interruption (ATI) of median 21 days was not sufficient to stimulate an increase in HIV-1-specific ADCC antibodies, despite viral rebound in all individuals who underwent the short ATI. In contrast, a longer ATI of 2 to 12 months robustly boosted HIV-1-specific Fc receptor-binding antibodies and ADCC against HIV-1-infected cells in vitro. These results show that there is a delay between viral recrudescence and the boosting of ADCC antibodies, which has implications for strategies aiming to utilise autologous ADCC responses against latently infected cells. Given that the CD4-bound conformation of Env is predominantly recognised by ADCC antibodies within HIV-1-infected individuals, this thesis next examined whether early-stage infected cells in the process of downregulating CD4 were more susceptible to ADCC compared to late-stage infected cells that have fully downregulated CD4 (Chapter 4). We found that both early- and late-stage infected cells were eliminated by antibodies within plasma from HIV-1-infected individuals, even though there was higher binding of plasma antibodies to the CD4-intermediate early-stage infected cells. Our results show that soluble Env and virions derived from the viral inoculum could sensitise early-stage infected cells to ADCC prior to de novo Env expression, resulting in artefactual in vitro ADCC measurements. Future studies will need to develop improved models to address the potential in vivo role for ADCC against cells with nascent HIV-1-infection. Collectively, our studies highlight a potential role for HIV-1-specific ADCC in curative HIV-1 strategies and in controlling HIV-1 infection. Our results suggest that sufficient expression of Env and the expression of appropriate epitopes will be needed for cells reactivated from latency to be eliminated by autologous ADCC antibodies. Future studies will need to optimise latency reversal in vivo and examine strategies to either boost or administer ADCC antibodies to ensure efficient elimination of the reactivated viral reservoir.
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ItemAn evaluation of charged Pam2Cys-based lipopeptides as novel adjuvants for subunit-based vaccines( 2017)The use of subunit antigens in modern vaccines generally requires the addition of an adjuvant due to the lack of immunostimulatory features that would otherwise allow them to induce strong immune responses. A problem facing the use of classical adjuvants such as Alum is that it is not known for inducing effective Th-1 responses that are important for clearing viral and intracellular bacterial infection. There is a shortage of adjuvants that are appropriate for use in animals including humans and as a consequence there is a wealth of research being carried out to discover and design novel and safe ways of delivering vaccines that will induce strong antibody- and/or cell-based immune responses. The TLR2 agonist-based adjuvant R4Pam2Cys is a synthetic adjuvant with a branched structure that has been shown to enhance humoral and also CD8+ T cell responses when formulated with soluble protein antigens. The studies presented in this thesis describe experiments that were designed to improve the economies of chemical synthesis, with a view to improving manufacturing conditions, and to better understand the role played by geometry of the final Pam2Cys-based adjuvant in its in vitro and in vivo biological activities. The results demonstrate that the efficiency of synthesis process could be improved without affecting biological activity by solid phase assembly of the lipid moiety prior to assembly of the R4 moiety. This also saved consumption, and hence costs, of reagents. The branched structure of the peptide portion of R4Pam2Cys was shown to confer resistance to trypsin hydrolysis and to play an important role in providing optimum binding to protein antigens. Finally R4Pam2Cys was compared with the clinically approved and commercially available hepatitis B virus (HBV) vaccine in which Alum is used to improve immunogenicity of the HBV antigen HBsAg. Clear immunological benefits were apparent using R4Pam2Cys over Alum; not only were better antibody titres obtained but robust CD8+ T cell responses were elicited by HBsAg-R4Pam2Cys complexes whereas the commercially available vaccine elicited few if any CD8+ T cells. The use of R4Pam2Cys also resulted in an improvement of protective efficacy in an animal model against HBV infection when used in prophylactic and therapeutic settings. These are promising findings and encourage the lipopeptide’s future application in development of an HBV therapeutic vaccine.