Evaluation of neutralization sensitivity of HIV-1 subtype C between plasma, peripheral blood mononuclear cells and lymph node-derived viruses.

Abstract

Background: Genetic variations have been observed in the HIV-1 env single genome amplicons (SGA) derived from plasma, peripheral blood mononuclear cells (PBMCs) and lymph node samples. However, the impact of these amino acid variations on the sensitivity of broadly neutralizing antibodies is not known. Therefore, the aim of this study was to evaluate HIV-1 env sequence differences within bnAb epitopes and assess their neutralization sensitivity in HIV-1 subtype C between plasma (PL), PBMC and lymph node (LN)-derived clones. Methods: Full-length HIV-1 env gene were amplified from four HIV-1 subtype C-infected ART-naïve individuals selected from the FRESH cohort based in KwaZulu-Natal, South Africa. Single genome amplicons (SGAs) were produced from plasma, PBMC and LN samples, sequenced by Sanger sequencing and cloned into a TOPO 2.1 vector. The env pseudoviruses were prepared through co-transfection of an envelope plasmid with the pSG3 Env backbone into 293T cells using the TransGENE 9 reagent. The neutralization sensitivity was assessed using the TZM-bl neutralization assay against a panel of broadly neutralizing antibodies (bnAbs) targeting the CD4 binding site, V1V2 loop, V3 glycan, gp120/gp41 interface, MPER, fusion peptide and silent face epitopes. HIV-1 env sequences were analyzed within bnAb epitopes and the neutralisation sensitivity was compared between LN, PBMCs and plasma samples. Results: Phylogenetic analysis of HIV-1 env sequences across participants revealed consistent and strong participant-specific clustering, confirming that viral populations evolved independently within each individual. Plasma sequences tended to have a slightly longer V1 loop lengths and more potential N-linked glycosylation sites (PNGS) in the V4 loop, compared to PBMC and LN clones, suggesting a marginally denser glycan shield. A high frequency of discordant amino acid mutations was observed in all three compartments in the V2 apex and the V3 loops while few discordant amino acid mutations were observed in the conserved regions including the CD4 binding site, MPER and gp120-gp41 interface. The highest frequency of resistant mutations was observed in the plasma compartment followed by PBMCs and LN. Discordant mutations were associated with differences in neutralization sensitivity between viruses derived from LN, PBMCs and plasma. Conclusion: Our findings suggest that plasma viruses have a higher frequency of resistance mutations within bnAb epitopes, and lower neutralisation sensitivity compared to PBMC and lymph nodes. However, there was no evidence of compartmentalisation, and minor differences in variable loop length and potential N-linked glycosylation sites. These findings provide a preliminary insight on the ability of bnAbs to reduce the size of viral reservoirs in PL and LN compartments. These findings may also influence the selection of bnAbs in immunotherapy studies.