Limiting Hiv Target Cells in the Female Genital Tract by Inducing Immune Quiescence

Abstract

Introduction and Objectives: In excess of 1.5 million new HIV-1 infections and over 800,000 HIV/AIDS-related deaths still occur annually across the globe despite much headway being made in HIV-1 management. Prevention of HIV-1 acquisition is therefore a priority, especially for populations that may be at higher risk of infection. Over the last decade, several avenues for HIV-1 infection have been explored, most targeting deactivation of the virus. The Immune quiescence (I.Q) phenotype, characterised by a low number of HIV target (CD4+CCR5+), a low inflammatory state and reduced T-cell activation in the host, as is observed among HIV Highly exposed sero-negative (HESN) cohorts, has been suggested to be protective against HIV-1 acquisition. Our study aimed to explore the hypothesis that the host immune system can be modulated to induce this I.Q phenotype using oral administration of available, non-stigmatising administered non-steroidal anti-inflammatory drugs, aspirin/acetylsalicylic acid (ASA) and hydroxychloroquine (HCQ) thereby adding to the arsenal available in the HIV-1 prevention toolkit, that does not target the virus. We therefore assessed the change in proportion of HIV target cells and level of T-cell activation among a healthy population after daily oral administration of ASA or HCQ. Study Design and Methodology: HIV negative, healthy women who were non-sex workers from the Pumwani Mother to Child cohort and Baba Dogo clinics in Nairobi, Kenya, were screened and recruited to a randomised, interventional, open labelled trial. In total, 162 participants were screened with being 91 randomised to either study arm of receiving a daily oral dose of either HCQ (200mg/day) or ASA (81mg/day) over a period of six weeks. Peripheral blood and cervical cytobrush samples from the female genital tract (FGT) from which mononuclear cells were extracted, as well as plasma and cervico-vaginal lavage samples were collected at baseline (0

week), at 2 weeks and 6 weeks from initiation either drug. Changes in proportion and activation of systemic and mucosal T-cells were analysed by flow cytometry on BD LSR II® and FlowJo® single cell analysis software; changes in cytokines expression analysed on a Luminex® platform; and statistical analyses done using STATA®.v15. Results & Discussion: Hydroxychloroquine Arm: Intention-to-treat analyses showed that there was no observed change in proportion of CD4+CCR5+ T-cells over the 6 week drug period in the systemic compartment [p=0.138] nor the mucosal compartment [p=0.617]; although the intensity of CCR5, expressed as median fluorescence intensity (MFI), on CD4+ cells was downregulated in blood [p=0.0002] though only trended to downregulation in CD4+ cells of the mucosal compartment [p=0.052]. The proportion of Th17 cells was similarly unaltered [PBMC p=0.505; CMC p=0.946]; but blood CCR5+Th17 cells decreased in proportion [p=0.051] and MFI [p=0.0001] while only MFI of mucosal CCR5+Th17 was downregulated [p=0.045] but not proportion [p=0.550]. The proportion of early activated of CD4+ cells was not significantly altered CD69+CD4+ [PBMC p=0.834; CMC p=0.171] although MFI downregulation was observed in the blood compartment [p=0.003]. The proportion of HLA-DR+CD4+ T cells was not significantly changed in either compartment over the 6 weeks, but proportion of systemic HLA-DR+CCR5+CD4+ cells increased [p=0.002] with no change in MFI [p=0.764]. Six weeks of HCQ intake did not significantly alter cytokine levels in blood or the CVL. Taken together, the results indicate that though HCQ downregulated the intensity of expression of T-cell activation markers, it did not decrease the number of HIV target cells in either blood or the FGT.

Acetylsalicylic (ASA) Arm: Intention-to-treat analyses showed a decrease in the proportion of CD4+CCR5+ T-cells was observed in both in the systemic compartment [p=0.017] and mucosal compartment [p=0.0005]; as well as a downregulation of the MFI of CCR5 on CD4+ cells [PBMC p=0.0001; CMC p=0.001] after 6-week ASA intake. The proportion of CCR5+Th17 cells decreased, as did the MFI both in the blood compartment [p=0.024; MFI p=0.0002] and the cervical compartment [p=0.019; MFI p=0.010]. The proportion of activated of CD4+ cells was not significantly altered neither in the systemic nor mucosal compartment, but MFI of CD69+CD4+ cells was downregulated in the blood compartment [p=0.001]. The proportion of activated of target cells (CD69+CCR5+CD4+ or HLA-DR+CCR5+CD4+) was not significantly altered over the 6 weeks of ASA in blood or mucosal compartment, but HLA-DR MFI on mucosal target cells was downregulated [p=0.033]. No change was observed in cytokine expression in plasma or CVL with 6-week ASA intake. Together, these findings suggest that ASA decreased the number of HIV target cells in both the FGT and in blood. Additionally, ASA did not deleteriously affect T-cell activation. Conclusion: The decrease in HIV-1 target cell numbers and downregulation of the HIV-1 co-receptor, CCR5 using ASA is an indication that induction of I.Q is indeed possible using safe, affordable, available non-stigmatising anti-inflammatory drugs such as low dose aspirin. This is the first in vivo trial showing the effects of an anti-inflammatory drug on human HIV-1 target cells and is a stepping stone for further clinical studies to explore re-purposing of drugs such as aspirin as an addition pharmaceutical tool for prevention of HIV-1 acquisition by inducing the immune quiescence phenotype. Such a preventive tool would indeed be beneficial among vulnerable and key populations