Mann, Jaclyn Kelly.Dlamini , Siyamthemba Oswald.2026-05-292026-05-2920262026https://hdl.handle.net/10413/24416Masters Degree. University of KwaZulu-Natal, Durban.The persistence of latent, replication-competent viral reservoirs in people living with HIV (PLWH) on suppressive antiretroviral therapy (ART) remains the principal barrier to a cure. Broadly neutralizing antibodies (bNAbs), which target Env, represent a promising immunotherapeutic strategy to clear infected cells and block viral rebound. A key unresolved question is whether the viral variants that populate these anatomical reservoirs, particularly within lymphoid tissues and peripheral blood, harbour genetic differences that could confer altered sensitivity to bNAbs. Understanding this is important for the design of effective antibody-based interventions, as mutations in variants persisting in the reservoir could allow reservoir viruses to evade bNAb therapy. To address this, we conducted a comparative analysis of HIV-1 env sequences and bNAb neutralization sensitivity for viral variants derived from pre-ART plasma (PL), and post-ART peripheral blood mononuclear cells (PBMCs) and lymph node (LN) reservoirs in individuals living with HIV-1 subtype C. The study cohort comprised 11 adults from Durban, South Africa, on suppressive ART. Bulk env sequences were generated from pre-therapy plasma, and from PBMC and LN mononuclear cells sampled after one year of therapy and next generation sequencing was performed on env amplicons from pre-therapy plasma. Sequences were assessed for unique genetic differences in the reservoir variants. For a subset of six participants, selected to represent a range of genetic differences, functional recombinant Env viruses were generated for each compartment from bulk participant-derived env amplicons and a replication-competent env-deleted NL4-3 plasmid backbone. Infectious virus stocks were produced via transfection of env amplicons and plasmid backbone into HEK293T cells, followed by further passaging of stocks in CEMderived GXR25 T cells. Neutralization sensitivity of viruses was determined using a standardized TZM-bl cell assay and a panel of nine clinically relevant bNAbs targeting major epitopes - the CD4-binding site (VRC01, VRC07-523LS, N6), the V3-glycan supersite (10- 1074, PGT121), the V2-glycan apex (PGDM1400, CAP256-VRC26.25), the gp120-gp41 interface (PGT151), and the membrane-proximal external region (MPER) (10E8). Phylogenetic analysis confirmed participant-specific clustering of sequences and excluded cross-contamination. Analysis of sequencing data revealed a median of 6 (inter-quartile range [IQR] 3–22.5) and 19 (IQR 7–49) amino acid differences between pre-therapy plasma sequences and LN and PBMC reservoirs, respectively. Notably, 63% of LN and 73% of PBMC reservoir mutations were unique to the reservoir. However, only a median of 0 (LN) and 2 (PBMC) of these changes occurred at positions known to affect bNAb sensitivity. Neutralization assays using viruses from six participants against nine bNAbs showed that no consistent pattern of increased or decreased sensitivity was observed in one compartment over another - sensitivity was highly variable and dependent on the participant, viral compartment, and specific bNAb. Overall, most viruses were sensitive to the CD4 binding site antibodies regardless of compartment (IC50 median = 0.11 μg/ml; IQR = 0.05 – 0.17). Similarly, all viruses, with the exception of one with markedly reduced sensitivity (20 μg/ml), were sensitive to the interface antibody (median = 0.1; IQR = 0.07 – 0.18). On the other hand, resistance to V2 and V3 antibodies was more common, and varied between compartments, with IC50s >10 ug/ml in 80% (CAP256), 13.3% (PGDM1400) 73.3% (PGT121), and 80% (10-1074) of the viruses. There were mixed results for the MPER antibody, where most of the viruses were sensitive to the MPER with the exception of two viruses. These findings show that HIV-1 subtype C reservoir Env variants harbour genetic differences from pre-therapy plasma variants, but that this variation was seldom at sites reported to affect bNAb sensitivity and also did not result in consistent, compartment-specific differences in neutralization sensitivity to a broad panel of bNAbs. While clear genotype–phenotype associations were evident for some epitopes (notably the V3-glycan supersite), sequence data alone did not fully predict neutralization outcomes. The neutralization sensitivity results underscored the suitability of the CD4bs and interface as promising therapeutic targets forbN Ab therapy and reinforced the rationale for combination bNAb regimens. Together, these results highlight the importance of integrating genetic and functional analyses when assessing reservoir susceptibility and provide data that may inform the rational design of combination bNAb strategies for subtype C infection.enCentral nervous system.Envelope glycoprotein.Fetal bovine serum.Natural killer cells.Toll-like receptor.Thesis