Elucidating the mechanisms of HIV viral reservoir establishment and maintenance
Creators: name: GARTNER, Matthew
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310706
subject: Virology

description: Human immunodeficiency virus type 1 (HIV-1) remains a significant global health problem with 38 million people living with HIV-1 (PLWH) globally. Fortunately, combination antiretroviral therapy (ART) has remarkably reduced the onset of AIDS, reduced the likelihood of transmission and restored life expectancy of PLWH. However, ART is not a cure for HIV-1 infection and if therapy is interrupted viral rebound occurs rapidly within two weeks. Viral rebound occurs following ART discontinuation due to the establishment and persistence of a stable reservoir of latently infected cells. The persistence of viral reservoirs necessitates the requirement for life-long ART for PLWH, which presents a considerable economic and public health burden.<br><br>

In PLWH on suppressive ART, proviral DNA is present in various CD4+ T cell subsets including naïve, stem cell memory (TSCM), central memory (CM), transitional memory (TM), effector memory (EM), and terminally differentiated (TD) CD4+ T cells during ART. These subsets differ in phenotypic properties, including effector function, activation status and expression of coreceptors for viral entry. Importantly, TSCM and CM cells are suggested to be highly stable reservoirs for HIV-1 during suppressive ART. This thesis aimed to determine which CD4+ T cell subsets are infected during early stages of disease and whether cellular tropism changed over time for different subsets in untreated PLWH.  Furthermore, the research reported in this thesis aimed to understand how different memory CD4+ T cell subsets contribute to the persistence of the HIV-1 reservoir during suppressive ART, and whether CD4+ T cell subsets found within different subsets demonstrate differential phenotypic properties.<br><br>

To understand how viral reservoirs are established in different memory CD4+ T cell subsets during untreated infection, subtype C (C-HIV) Env clones were amplified from the plasma of five C-HIV infected women from three untreated time-points; &lt;2 months, one-year and three-years post-infection. A total of 50 C-HIV envs were cloned and screened for functionality in pseudovirus infection assays. Coreceptor usage assays showed all viruses tested used CCR5 to mediate entry, with three Envs also weakly using CXCR4. In vitro infection assays showed TM and EM cells were more frequently infected (median: 46% and 25% of total infected CD4+ T cells respectively) than naïve, TSCM, CM and TD. This was not due to these subsets contributing a higher proportion of the CD4+ T cell pool, rather these subsets were more susceptible to infection (median: 5.38% EM and 2.15% TM cells infected), which was associated with higher CCR5 expression. Surprisingly, no inter- or intra-participant changes in CD4+ T cell subset tropism were observed across the three-time points despite phylogenetic data showing evolution in Env sequences over time.  Overall, this data suggests that CD4+ T cell subsets that express more CCR5 were more susceptible to infection with C-HIV Envs, suggesting that these may be the major cellular targets during the first three years of infection. Moreover, C-HIV Envs from acutely infected individuals showed a robust infection of CM cells in vitro, suggesting an avenue for early reservoir establishment via direct infection of CM cells.<br><br>

To understand how the reservoir persists in memory CD4+ T cell subsets over time in PLWH on suppressive ART, clinical samples were obtained from eight males living with subtype B (B-HIV) treated during chronic infection (median of 8 years before sampling) with undetectable viral loads (&lt;40 copies/ml). Single genome amplification (SGA) and Sanger sequencing of env was performed on gDNA from memory CD4+ T cell subsets at two time-points, separated by 2-4 years. Phylogenetic trees, sequence diversity, and genetic compartmentalization tests demonstrated no detectable evolution of viral sequences between time-points, suggesting residual replication was unlikely to be driving reservoir persistence over time. Instead, multiple clusters of identical sequences were observed within phylogenetic trees, some of which persisted across both time-points and multiple T cell subsets. Furthermore, analysis of identical sequences within different memory CD4+ T cell subsets revealed EM cells represented the bulk of identical sequences, suggesting cellular proliferation and differentiation are key drivers of HIV-1 persistence during suppressive ART.<br><br>

To understand whether Envs obtained from different CD4+ T cell subsets are phenotypically distinct, 108 B-HIV Envs generated by SGA were cloned into an expression vector to generate Env-pseudovirus particles. In vitro CD4+ T cell tropism assays showed dual-tropic viruses had an expanded tropism for resting CD4+ T cells as well as naïve and CM cells when compared to CCR5-using Envs, suggesting that CXCR4-using Envs may be more likely to directly infect less differentiated cells. Neutralisation assays showed dual-tropic viruses were more sensitive to the monoclonal antibodies VRC01, PGT121 and 10E8, which target different Env epitopes, suggesting differential neutralisation sensitivities of Envs can be influenced by coreceptor usage.<br><br>

Overall, the data presented suggests that viral reservoirs may be established early in TSCM and CM cells through direct infection with CCR5-using viruses. Coreceptor switching to CXCR4-usage improves tropism for naïve and CM cells, which may increase the likelihood of reservoir establishment in these subsets. Latently infected cells predominantly persist during suppressive ART through cellular proliferation and differentiation, and interventions that reduce cellular proliferation may significantly reduce reservoir size. Finally, although coreceptor usage influences neutralising sensitivity to clinical bNAbs, CD4bs and MPER antibodies demonstrate sufficient breadth and potency to effectively neutralise the diverse repertoire of Envs present in the reservoir.