Association between HIV-1 Nef-mediated MHC-I downregulation and the maintenance of the replication-competent latent viral reservoir in individuals with virally suppressed HIV-1 in Uganda: an exploratory cohort study

Abstract

Background: The persistence of a replication-competent latent viral reservoir (RC-LVR) during antiretroviral therapy (ART) is a barrier to the development of a cure for HIV-1, but the role of viral genes in influencing RC-LVR size is unclear. We aimed to assess whether the magnitude by which the HIV-1 accessory protein Nef evades the adaptive immune response by downregulating MHC-I or CD4, or both, from the surface of infected cells is associated with the rate at which the RC-LVR in people with HIV-1 changes during long-term ART (>1 year).

Methods: We conducted an exploratory cohort study in which nef genes were sequenced from outgrowth viruses derived from the quantitative viral outgrowth assay (QVOA) for a group of people with ART-suppressed HIV-1 in Uganda between 2015 and 2020. Study participants were selected from the Rakai Health Sciences Program (RHSP) LVR cohort, a cohort of 90 adults (aged ≥18 years) who were HIV-1 positive, receiving ART, and had maintained viral suppression for at least 1 year at the time of study enrolment. For this study, participants were required to have available p24⁺ QVOA wells that contained a single viral outgrowth isolate, as assessed by next-generation sequencing. In cases where further sequencing identified wells containing multiple viral clones, all sequenced nef variants were included for functional analysis. The unique isolated nef variants were used to generate pseudoviruses, which were employed to measure cell surface CD4 and MHC-I downregulation in infected CD4⁺ Sup-T1 cells via flow cytometry. The size and rate of change of the RC-LVR in participants was estimated using previous QVOA results and a Bayesian model. We then assessed whether a correlation existed between the extent to which the Nef proteins downregulated cell surface MHC-I and CD4 and the calculated RC-LVR rate of change during the study period.

Findings: 14 (15%) of 90 participants from the RHSP cohort met the inclusion criteria and were enrolled in this study. 49 nef sequences were isolated from these participants. We observed variability in participant-derived Nef-mediated cell surface MHC-I downregulation (median 114·88% [IQR 104·93-121·51] of the downregulation capacity of NL4-3 Nef) and CD4 downregulation (94·50% [84·05-100·16] of NL4-3 Nef). The estimated rate of change of the RC-LVR was positive for four participants. For one donor, the rate of change was significantly positive (7·4 × 10^-4 logit infectious units per million [IUPM] per day [95% credibility interval 3·2 × 10^-4 to 1·2 × 10^-3]) over the course of the study period (2015-20). The estimated rate of change of the RC-LVR for the remaining ten participants was negative, and significantly negative in four donors (-1·1 × 10^-3 logit IUPM per day [95% credibility interval -1·8 × 10^-3 to -3·7 × 10^-4]; -1·4 × 10^-3 [-2·0 × 10^-3 to -8·5 × 10^-4]; -7·0 × 10^-4 [-1·3 × 10^-3 to -1·6 × 10^-4]; and -2·0 × 10^-3 [-2·9 × 10^-3 to -1·1 × 10^-3]). A significant relationship between Nef-mediated MHC-I downregulation and the RC-LVR rate of change during the 5-year study period (r=0·6088 [95% CI 0·2366 to 0·9810]; p=0·023) was found, in which less efficient MHC-I downregulation correlated with faster RC-LVR decay during long-term ART. By contrast, Nef-mediated CD4 downregulation was not associated with RC-LVR rate of change during the 5-year study period (-0·1604 [-0·7311 to 0·4102]; p=0·58).

Interpretation: Nef-mediated MHC-I downregulation might contribute to HIV-1 persistence during long-term ART. Strategies to inhibit Nef-mediated MHC-I downregulation could represent a viable therapeutic avenue to reduce the size of the latent reservoir in vivo, improving treatment outcomes in people with HIV-1.

Funding: Canadian Institutes of Health Research, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, and the REACH Martin Delaney Collaboratory.

Figure 1:. Maximum likelihood phylogeny relating primary HIV-1 nef nucleotide sequences used in the present study

The maximum likelihood phylogenetic tree relating the aligned primary nef nucleotide sequences was constructed with IQ-TREE using a TPM2u base substitution rate model with standard non-parametric bootstrap analysis with 10 000 replicate samples. The scale bar represents the estimated number of nucleotide substitutions per site. Internal nodes of the Newick output tree from IQ-TREE were annotated with bootstrap support values as percentiles; nodes with ≥70% support are shown accordingly. The individual primary nef isolates are coloured according to the participant from which the sequences were derived. HIV-1 subtypes were identified via the Recombinant Identification Program tool hosted by Los Alamos National Laboratory and are listed after the participant identifiers. The shape of the individual tips corresponds to the timepoint from which the nef sequences were collected. The phylogenetic tree is additionally rooted on the HIV subtype B reference strain NL4-3 nef. *nef variant (sequence identifier 17_4_1M13) encoding a premature stop codon and predicted to not express full-length Nef.

Figure 2:. Gating schematic used to quantify cell surface MHC-I and CD4 levels within infected cells

Sup-T1 cells were infected with vesicular stomatitis virus protein G-pseudotyped viruses encoding primary Nef isolates. Cell-surface CD4 and MHC-I levels were quantified on infected cells via flow cytometry. (A) Gating schematic used for identifying infected Sup-T1 cells (based on FMO control): initial gating by FSC-A by SSC-A; FSC-A by FSC-H to exclude doublets; viability dye to exclude dead cells; and eGFP to identify infected cells. (B–E) Representative plots for cells infected with pseudoviruses that were Nef-deficient (ΔNef negative control), pseudoviruses that expressed NL4-3 Nef (positive control), and pseudoviruses that encoded two participant-derived Nef samples: 17_0_1M12 and 17_4_1M13 (representing efficient and inefficient downregulation, respectively). Specifically: pseudocolour plots for MHC-I (B) and CD4 (C) and histograms (with gMFIs shown) for MHC-I (D) and CD4 (E). AF647=Alexa Fluor 647. eGFP=enhanced green fluorescent protein. FMO=fluorescence minus one. FSC-A=forward scatter area. FSC-H=forward scatter height. gMFI=geometric mean fluorescence intensity. NIR=near-infrared. PE=phycoerythrin. SSC-A=side scatter area. *The plots for Nef-deficient (ΔNef) and NL4-3 Nef pseudoviruses were derived from the first technical replicate of the given experiment.

Figure 3:. Participant-derived Nef-mediated downregulation of cell surface CD4 and MHC-I relative to NL4-3 Nef

Box plots show cell surface MHC-I (A) and CD4 downregulation (B)—compared with the mean Nef-deficient (ΔNef) negative control—as a percentage of mean NL4-3 Nef for each primary Nef variant (median with IQR displayed, n=4). The red dashed line indicates the functional output of NL4-3 Nef normalised to 100%. *Significant differences in CD4 and MHC-I downregulation between each isolate and the NL4-3 Nef positive control are denoted by an asterisk (p≤0·05); exact p values and the functional medians are provided in appendix 3 (pp 4-5).

Figure 4:. Correlation between the level of Nef-mediated MHC-I and CD4 downregulation and changes in RC-LVR size

The output for both functions is represented as cell-surface MHC-I or CD4 downregulation compared with the mean Nef-deficient (ΔNef) negative control followed by normalisation to mean NL4-3 Nef, as described in figure 3. For donors with more than one retrieved nef sequence, the median Nef functional output was selected for subsequent correlation analysis, and each dot represents an individual. The 17_4_1M13 sample with the premature stop codon located within the core region of the Nef protein was included in the median Nef functional output calculation. Correlation analysis comparing the slope of the change in reservoir size for each participant with HIV-1 involved in the study to median MHC-I cell-surface downregulation (A) and median CD4 cell-surface downregulation (B). To assess whether these correlations were significant, a Spearman correlation test was used to compare cell surface MHC-I or CD4 downregulation with the slope of the change in reservoir size, where a two-tailed p value of 0·05 or less was considered statistically significant. The Spearman correlation coefficients (r) are listed along with the 95% CIs. Linear regression best-fit lines and 95% CIs were plotted. Study participants with significant estimated RC-LVR rates of change are denoted in the colour key with an asterisk. An RC-LVR rate of change was deemed significantly positive or negative if the 95% credibility intervals were both positive or negative, respectively. ART=anti-retroviral therapy. IUPM=infectious units per million. RC-LVR=replication-competent latent viral reservoir.