HIV Subtype and Nef-Mediated Immune Evasion Function Correlate with Viral Reservoir Size in Early-Treated Individuals

Abstract

The HIV accessory protein Nef modulates key immune evasion and pathogenic functions, and its encoding gene region exhibits high sequence diversity. Given the recent identification of early HIV-specific adaptive immune responses as novel correlates of HIV reservoir size, we hypothesized that viral factors that facilitate the evasion of such responses-namely, Nef genetic and functional diversity-might also influence reservoir establishment and/or persistence. We isolated baseline plasma HIV RNA-derived nef clones from 30 acute/early-infected individuals who participated in a clinical trial of early combination antiretroviral therapy (cART) (<6 months following infection) and assessed each Nef clone's ability to downregulate CD4 and human leukocyte antigen (HLA) class I in vitro We then explored the relationships between baseline clinical, immunological, and virological characteristics and the HIV reservoir size measured 48 weeks following initiation of suppressive cART (where the reservoir size was quantified in terms of the proviral DNA loads as well as the levels of replication-competent HIV in CD4⁺ T cells). Maximal within-host Nef-mediated downregulation of HLA, but not CD4, correlated positively with post-cART proviral DNA levels (Spearman's R = 0.61, P = 0.0004) and replication-competent reservoir sizes (Spearman's R = 0.36, P = 0.056) in univariable analyses. Furthermore, the Nef-mediated HLA downregulation function was retained in final multivariable models adjusting for established clinical and immunological correlates of reservoir size. Finally, HIV subtype B-infected persons (n = 25) harbored significantly larger viral reservoirs than non-subtype B-infected persons (2 infected with subtype CRF01_AE and 3 infected with subtype G). Our results highlight a potentially important role of viral factors-in particular, HIV subtype and accessory protein function-in modulating viral reservoir establishment and persistence.IMPORTANCE While combination antiretroviral therapies (cART) have transformed HIV infection into a chronic manageable condition, they do not act upon the latent HIV reservoir and are therefore not curative. As HIV cure or remission should be more readily achievable in individuals with smaller HIV reservoirs, achieving a deeper understanding of the clinical, immunological, and virological determinants of reservoir size is critical to eradication efforts. We performed a post hoc analysis of 30 participants of a clinical trial of early cART who had previously been assessed in detail for their clinical, immunological, and reservoir size characteristics. We observed that the HIV subtype and autologous Nef-mediated HLA downregulation function correlated with the viral reservoir size measured approximately 1 year post-cART initiation. Our findings highlight virological characteristics-both genetic and functional-as possible novel determinants of HIV reservoir establishment and persistence.

Keywords: CD4 downregulation; HIV reservoir; HIV-1; HLA downregulation; Nef; viral pathogenesis.

FIG 1

Maximum likelihood phylogeny relating participant bulk and clonal plasma HIV RNA Nef sequences. HIV nef isolates are colored by subtype, and bulk and clonal sequences are indicated. Defective Nef clones either encoded internal stop codons or lacked a stop codon. Participants who initiated cART very early (within an estimated 141 days of seroconversion), as determined by a multiassay algorithm (MAA⁺), are also indicated. Two participant pairs who shared highly genetically similar viruses, one pair infected with subtype B and the other pair infected with subtype G, are indicated by vertical lines. The phylogeny is rooted on the HIV subtype B reference strain HXB2. The scale is in the estimated number of nucleotide substitutions per site.

FIG 2

CD4 and HLA-I downregulation functions of participant Nef clones. (A) Representative flow plots demonstrate surface CD4 and HLA-I expression following transfection of CEM cells with the empty vector (PSEL_GFP), positive-control Nef (PSEL_SF2), and example participant Nef clones. The median fluorescence intensity (MFI) of receptor staining in the GFP⁻ (untransfected) and GFP⁺ (Nef-transfected) cells are displayed in the upper left and right corners of each gate, respectively. The normalized in vitro function of each Nef control and participant clone relative to that of Nef_SF2 is indicated as a percentage in the bottom right corner. (B) The normalized CD4 downregulation function of each participant Nef clone is shown and is expressed as the mean (box height) and standard deviation (error bars) from a minimum of triplicate independent experiments. Clones are colored by HIV subtype, as described in the key in Fig. 1. Defective Nef clones are indicated by a cross; these clones encoded either internal stop codons or lacked a stop codon. The dotted horizontal line indicates the population median. (C) The normalized HLA-I downregulation function of each participant Nef clone is shown, with all labeling being as described for panel B.

FIG 3

Relationship between Nef-mediated CD4 and HLA-I downregulation functions and Nef steady-state protein expression. (A) Spearman's correlation between the maximal within-host Nef-mediated HLA-I and CD4 downregulation function. Nef isolates are colored according to HIV subtype. (B) Results of Western blot analyses of Nef and cellular β-actin are shown for assay controls and 30 participant clones that exhibited maximal intraindividual HLA downregulation activity. Sample identifiers are colored according to HIV subtype. Mw, molecular weight.

FIG 4

Established and novel correlates of HIV reservoir size. (A to G) The correlations between log₁₀ proviral DNA levels measured at 48 weeks post-cART and either baseline proviral DNA levels (A), baseline log₁₀ plasma viral load (B), baseline CD4⁺ T cell counts (C), baseline granzyme B responses (D), timing of cART (E), baseline maximal Nef-mediated CD4 downregulation (F), or baseline maximal Nef-mediated HLA-I downregulation (G) are displayed. (H) The correlation between Nef-mediated HLA-I downregulation and replication HIV reservoir size measured at 48 weeks post-cART is shown. (I) The correlation between Nef-mediated HLA-I downregulation and baseline HIV-specific granzyme B responses is shown. (J) Week 48 proviral DNA levels are shown, stratified by HIV subtype. (K) Week 48 replication-competent reservoir sizes, assessed as the number of infectious units per million CD4⁺ T cells (IUPM), are shown, stratified by HIV subtype. Statistical significance was assessed using Spearman’s correlation or Mann-Whitney U tests. The Nef isolates in all panels are colored according to HIV subtype.

FIG 5

Confirmation of identified correlates in HIV subtype B infections only. (A to F) Correlations between log₁₀ proviral DNA levels measured at 48 weeks post-cART and either baseline proviral DNA levels (A), baseline CD4⁺ T cell counts (B), baseline log₁₀ plasma viral load levels (C), timing of cART (D), baseline granzyme B responses (E), or maximal baseline Nef-mediated HLA downregulation function (F) are shown, where all analyses are restricted to subtype B infections only. (G) A heat map summarizing Spearman's rho values (reported in boxes) and P values (depicted as colors) for the 15 possible pairwise comparisons between correlates of reservoir size in subtype B infections only is shown. NA, not applicable.