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-derivednefclones 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 Iin 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'sR = 0.61,P = 0.0004) and replication-competent reservoir sizes (Spearman'sR = 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.IMPORTANCEWhile 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 apost hocanalysis 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.

Structure, function, and inhibitor targeting of HIV-1 Nef-effector kinase complexes

“…As described above, Nef prevents cell-surface display of MHC-I complexes with HIV-1 antigenic peptides on infected cells, allowing escape from cytotoxic T lymphocytes. In this way, Nef inhibits clearance of the virus and may contribute to establishment and maintenance of the persistent viral reservoir (104). Mujib et al (102) showed that the direct acting Nef inhibitor B9, as well as several firstgeneration B9 analogs (103), restored MHC-I to the surface of HIV-infected CD4 T cells.…”

Longitudinal within-host evolution of HIV Nef-mediated CD4, HLA and SERINC5 downregulation activity: a case study

“…While the major genetic determinants of Nef function are genetically separable [55][56][57], previous studies of natural nef sequences have demonstrated modest correlations between certain Nef activities [33,35,46,47], suggesting the presence of secondary or shared genetic determinants. Consistent with this, Nefmediated CD4 and HLA downregulation functions of the 50 studied clones correlated weakly (Spearman R = 0.35; p = 0.013), as did Nef-mediated CD4 and SERINC5 downregulation functions (Spearman R = 0.24; p = 0.047) (data not shown).…”

“…Nef amplicons were cloned into a modified pSELECT-GFPzeo vector containing AscI and SacII restriction sites within its multiple cloning site [47]. As described in [47], nef amplicons were digested with AscI and SacII, ligated into cut pSELECT-GFPzeo (T4 ligase; Thermo Fisher ® ), and transformed into chemically competent E. coli (E. cloni 10G DUOs; Lucigen). Plasmid DNA from a minimum of three colonies per transformation was isolated, purified (Thermo Fisher ® OMEGA EZNA plasmid minikit) and re-sequenced to confirm identity.…”

“…Cell surface expression of CD4 and HLA were measured using flow cytometry (Millipore Guava 8HT). The CD4 and HLA downregulation functions of participant-derived Nef clones were normalized to those of the positive control, SF2 NEF , using the following equation [47]:…”

“…Though nef undergoes substantial within-host evolution [38][39][40][41], studies characterizing the relationships between primary nef sequences and the functions of their corresponding expressed proteins have predominantly been cross-sectional, with one or a few nef sequences evaluated per participant at a single timepoint [34,[42][43][44][45][46][47]. Few studies have simultaneously assessed withinhost genetic [48,49] and functional Nef evolution over long timescales [50,51], and none to our knowledge have investigated Nef-mediated SERINC5 downregulation longitudinally.…”