Characterisation of the HIV reservoir in individuals living in South East Asia on ART

<p>Human immunodeficiency virus (HIV)/Acquired Immune Deficiency Syndrome (AIDS) is an ongoing global health burden, responsible for an estimated 32 million deaths since the start of the epidemic.  While the introduction of antiretroviral therapy has significantly reduced the mortality of HIV, interruption to therapy results in viral rebound, and treatment is thus lifelong. HIV establishes latent/dormant infection in CD4+ lymphocytes and macrophages, leading to its persistence despite suppressive therapy, and constituting a major barrier to cure/control strategies. While the majority of HIV research has focussed on viral subtypes B and C in Western and African countries respectively, circulating recombinant forms of the virus, such as CRF01-AE present in South East Asia, are less well defined. HIV subtype CRF01-AE has now spread globally, with reports of AE strains in Central and West Africa, North America, Europe, Asia and Australia; HIV subtype AE is responsible for 5.3% of global HIV infections as of 2019.  People living with HIV (PLWH) infected with subtype CRF01-AE have increased rates of CD4+ T cell decline, higher levels of HIV-1 DNA within peripheral blood mononuclear cells (PBMCs), and progress faster to AIDS.</p> <p>Latently HIV-infected resting memory CD4+ T cells are thought to respond to immune signals to persist and undergo homeostatic proliferation and may also be subject to antigen mediated or integration site driven clonal expansion. This is thought to be the major driver of HIV persistence on ART. Antigen mediated clonal expansion can be driven by specific common antigens, such as cytomegalovirus (CMV), Epstein Barr virus (EBV), or human papilloma virus (HPV). In South East Asia, a high prevalence of sexually transmitted diseases and human herpes viruses (4) is associated with increased immune activation of PLWH on ART. Increased antigen mediated clonal expansion due to a high pathogen burden may play a significant role in the persistence of the HIV-1 reservoir in PLWH from South East Asia.</p> <p>This research examines the impact of clonal expansion on the latent HIV reservoir in memory CD4+ T cell subsets from PLWH from South East Asia and compares both the contribution of each subset to the HIV reservoir as well as the frequency of clonally expanded HIV infected cells with that in PLWH from Western cohort. We hypothesised that 1) HIV persists in a range of CD4+ T cell subsets, with more differentiated subsets contributing to the majority of the viral reservoir, and; 2) that PLWH from South East Asia (predominately Clade AE virus and higher pathogen burden) harbour a higher proportion of clonally expanded HIV infected cells, contributing to a larger reservoir of HIV, when compared to PLWH from a Western country.</p> <p>The contribution of each CD4+ T cell memory subset (i.e. naïve, central memory, transitional memory, effector memory and terminally differentiated cells) to the total pool of HIV infected CD4+ T cells was determined for 12 PLWH recruited from South East Asia, defined the South East Asian HIV cohort and compared to published studies investigating PLWH from Western countries (Western HIV cohort). Participants of the South East Asian cohort were men at least 25 years old, who were virologically suppressed (HIV-1 RNA < 20 copies/mL of plasma) and on stable ART for at least 12 months prior to recruitment. The contribution of each CD4+ T cell subset to the total pool of infected cells was determined by isolation of each subset from peripheral blood mononuclear cells (PBMCs) through flow cytometry and subsequent quantification of total HIV DNA in that subset. Central memory CD4+ T cells from PLWH from the South East Asian cohort contributed more to the total pool of HIV infected CD4+ T cells than transitional, effector and terminally differentiated subsets (40.1% vs 10.5% (P = 0.0157), 40.1% vs 8.8% (P = 0.0131) and 40.1% vs 6.1% (P = 0.0023), respectively). In contrast to previous research using cohorts of Western PLWH, naïve CD4+ T cells contributed more to the total pool of HIV infected CD4+ T cells than further differentiated T cells.</p> <p>The proportion of clonally expanded HIV infected T cells was determined through single genome amplification of the V1-V3 loop of the env gene for selected participants of the cohort (n=6). Phylogenetic tree construction revealed clonal expansion in all participants with the frequency of identical sequences within each participant ranging from 14.3% to 62.4%. Over 50% of clonally expanded sequences appeared in two or more subsets, indicating that latently HIV infected cells can differentiate without reactivation.</p> <p>Sequences from 4 of 6 participants were identified as clade AE viruses, with a median of 31.9% and 65.2% of sequences predicted to be X4-using viruses through Geno2Pheno and Phenoseq programs, respectively. Variable regions characteristics analysis shows differences in the length, net charge and potential N-linked glycosylation sites in the V1 to V2 and V3 loops of CXCR4 - using vs CCR5 - using viruses.</p> <p>Taken together, the results of this thesis emphasise the importance of characterising the reservoir in PLWH in non-Western countries where non-B or C subtypes such as AE predominate, where both biological and socio-economic factors such as viral clade, pathogen burden and access to healthcare may influence the HIV reservoir. Therefore, future research must acknowledge the differential impacts of HIV subtype, a LMIC setting with poor health hygiene, higher pathological burden and increased discrimination, when conducting HIV remission research.</p>