Suppressive ART Research Articles

Alterations in circulating immunoregulatory proteins discriminate poor CD4 T lymphocyte trajectories in people with HIV on suppressive antiretroviral therapy.

It is essential to track immune perturbations related to insufficient CD4 T-cell recovery in PWH on suppressive ART as those with incomplete reconstitution are at a greater risk of non-AIDS-related morbidity and mortality. Several inflammatory soluble mediators have associated with poor immune reconstitution and adverse morbid outcomes in PWH, yet their implementation into routine clinical care to guide management remains inconsistent. Circulating immune checkpoint proteins have been linked to dysregulated immune pathways during suppressive ART and may serve as improved surrogate markers of clinical relevance. Here we investigate soluble lymphocyte-associated immunoregulatory proteins in virally suppressed PWH with no reported co-morbid outcomes and varying CD4 T-cell counts, to reveal underlying pathways that remain perturbed despite ART. This novel signature of immunoregulatory markers pertaining to poor CD4 T-cell trajectories uncover previously overlooked immune checkpoints as important targets for clinical monitoring of PWH in the setting of durable viral suppression by ART.

The risk of non-AIDS defining events is lower in ART-naive HIV controllers than in normal progressors on suppressive ART.

We aimed to compare the non-AIDS events (nADE) risk between normal progressors using ART (NP-ART) and people with HIV (PWH) that naturally control HIV infection (HIV controllers), as well as the outcomes after ART in HIV controllers on nADE. The primary endpoint was major nADE defined as the composite of cardiovascular disease, non-AIDS malignancy or all-cause mortality, whichever came first.. The role of ART in HIV controllers was assessed as a time-varying covariate. We included 1007 ART-naive HIV controllers (of which 60 elite controllers), 1510 Early-ART (<6 months after negative HIV test) and 15437 NP-ART (reference group), contributing 3813, 11,060 and 160,050 years of follow-up, respectively. HIV controllers had lower risk of the primary endpoint (HR 0.55, 95%CI 0.38-0.81, P = 0.0023), all-cause mortality (Adjusted Hazard ratio [aHR]: 0.45, 95% confidence interval [CI] 0.25-0.79, P = 0.0054), cardiovascular disease (aHR 0.47, 95%CI 0.22-0.99, P = 0.046) , but not non-AIDS malignancy (aHR 0.74, 95%CI 0.41-1.35, P = 0.33) than NP-ART. Among HIV controllers, each log10 lower baseline viral load further decreased the risk of nADE (aHR 0.54, 95% CI 0.29-0.99, P = 0.045). ART in HIV controllers did not reduce the risk of any nADE (aHR 1.22, 95% CI 0.66-2.29, P = 0.53). We found a lower risk of nADE in HIV controllers than NP-ART, especially in those with low plasma viral loads. Initiation of ART did not alter the nADE risk in HIV controllers. Our findings help clinicians to decide on prescribing ART in HIV controllers.

Chronic immune activation and accelerated immune aging among HIV-infected adults receiving suppressive antiretroviral therapy for at least 12 years in an African cohort

BackgroundHIV-associated alterations innate and adaptive immune cell compartments are reminiscent of the process of immune aging. ObjectivesWe described immune aging phenotypes among ART-treated HIV-infected adults relative to age-matched HIV-negative counterparts. MethodsIn a cross-sectional comparative study of HIV-infected adults with CD4≥500 cells/μl after at least 12 years of suppressive ART and age-and-gender-matched HIV-negative individuals, immune activation and immune aging phenotypes were measured, using multi-color flowcytometry. ResultsART-treated HIV-infected individuals had higher body mass index (P = 0.004), waist-hip circumference (P = 0.041), hip circumference (P < 0.001), and diastolic blood pressure (P = 0.012) and immune activation (CD4+CD38+HLADR+; median 4.15,IQR(1.030,14.6)] relative to the HIV-negative age-matched individuals [median 3.14,IQR(1.030, 6.68)]; P=0.0034. Immune aging markers [CD4+CD57+T-cells; median 13.00 IQR (0.45,64.1)] were higher among HIV-infected ART-treated adults<50 years relative to HIV-negative<50 years[median 8.020,IQR(0.004,21.2)]; P=0.0010. Naïve CD4 T-cells, Central memory CD4 T-cells, Terminal Effector Memory T cells (TEMRA: CD27−CD45RA + CCR7-) and immune senescence CD4/CD8+CD28-/CD57+ T-cells were similar among ART-treated HIV-infected individuals<45 years relative to 60 years-and-older HIV-negative counterparts≥; p = 0.0932, p = 0.05357, p = 0.0950 and p = 0.5714 respectively. ConclusionART-treated adults are immunologically two decades older than their HIV-negative counterparts. Accelerated immune aging among individuals aging with HIV underscores the need for an HIV cure to avert the unprecedented complications of accelerated immune senescence and the associated NCD risk in African settings with protracted exposure to endemic co-infections.

Autologous neutralizing antibody responses after antiretroviral therapy in acute and early HIV-1.

BACKGROUNDEarly antiretroviral therapy initiation (ARTi) in HIV-1 restricts reservoir size and diversity while preserving immune function, potentially improving opportunities for immunotherapeutic cure strategies. For antibody-based cure approaches, the development of autologous neutralizing antibodies (anAbs) after acute/early ARTi is relevant but is poorly understood.METHODSWe characterized antibody responses in a cohort of 23 participants following ARTi in acute HIV (<60 days after acquisition) and early HIV (60-128 days after acquisition).RESULTSPlasma virus sequences at the time of ARTi revealed evidence of escape from anAbs after early, but not acute, ARTi. HIV-1 envelopes representing the transmitted/founder virus(es) (acute ARTi) or escape variants (early ARTi) were tested for sensitivity to longitudinal plasma IgG. After acute ARTi, no anAb responses developed over months to years of suppressive ART. In 2 of the 3 acute ARTi participants who experienced viremia after ARTi, however, anAbs arose shortly thereafter. After early ARTi, anAbs targeting those early variants developed between 12 and 42 weeks of ART and continued to increase in breadth and potency thereafter.CONCLUSIONResults indicate a threshold of virus replication (~60 days) required to induce anAbs, after which they continue to expand on suppressive ART to better target the range of reservoir variants.TRIAL REGISTRATIONClinicalTrials.gov NCT02656511.FUNDINGNIH grants U01AI169767, R01AI162646, UM1AI164570, UM1AI164560, U19AI096109, K23GM112526, T32AI118684, P30AI045008, P30AI027763, R24AI067039; Gilead Sciences grant INUS2361354; Viiv Healthcare grant A126326.

GITR activation ex vivo impairs CD8 T cell function in people with HIV on antiretroviral therapy

Glucocorticoid-induced tumor necrosis factor related protein (GITR) is a co-stimulatory immune checkpoint molecule constitutively expressed on regulatory Tcells (Tregs) and on activated T conventional cells (Tconv). In blood collected from PWH on suppressive ART, GITR expression was reduced in multiple activated CD4 and CD8 Tcell subsets but was increased in Tregs. HIV specific CD8 Tcells expressed higher levels of GITR and programmed cell death protein 1 (PD-1) compared to total CD8 Tcells. Following stimulation with HIV peptides and GITR-ligand (L), we demonstrated a significant decrease in killing by HIV specific CD8 Tcells and an increased exhausted profile. Tcell receptor co-stimulation with GITR-L abrogated Treg suppression and induced expansion of CD4 Tconv. We conclude that GITR activation is an additional factor contributing to an impaired HIV immune response in PWH on ART and that GITR agonist antibodies should not be pursued for HIV cure strategies.

Life expectancy of people with HIV on antiretroviral therapy in Spain.

To estimate life expectancy of people with HIV (PWH) and describe causes of death. Antiretroviral therapy (ART)-naive adults from the CoRIS cohort starting ART in 2004-2019. We calculated life expectancy at age 40 for men and women according to their ART initiation period, and stratified by transmission category, CD4 + cell count and AIDS diagnosis. We estimated life expectancy in 10-year age bands using life tables constructed from mortality rates, estimated through Poisson models. Life expectancy increased from 65.8 [95% confidence interval (CI) 65.0-66.6] in 2004-2008 to 72.9 (72.2-73.7) in 2014-2019 in men [general population comparators (GPC): 79.1 and 81.2 years, respectively] and from 65.8 (65.0-66.6) to 72.5 (71.8-73.3) in women (GPC: 84.9 and 86.4, respectively). Non-AIDS-related deaths accounted for 68% of deaths among men and 78% among women. Life expectancy was longer when starting ART with higher CD4 + cell counts and without AIDS. For men acquiring HIV through sex with men, starting ART in 2014-2019 without AIDS, life expectancy was 75.0 (74.2-75.7) with CD4 + cell count less than 200 cells/μl, rising to 78.1 (77.5-78.8) with CD4 + cell count at least 350 cells/μl. Corresponding figures were 70.1 (69.4-70.9) and 76.0 (75.3-76.7) for men acquiring HIV heterosexually (HTX) and 61.5 (60.7-62.3) and 69.0 (68.2-69.8) for those acquiring HIV through injection drug use (IDU). For women starting ART from 2014 without AIDS, life expectancy increased from 71.7 (71.0-72.4) to 77.3 (76.7-77.9) among HTX and from 63.7 (62.9-64.5) to 70.7 (70.0-71.5) among IDU. Our findings confirm the progressive improvement of life expectancy in PWH in Spain over the last decades, supporting the insurability of PWH on suppressive ART in our current setting and time.

Oestradiol concentrations in trans women with HIV suppressed on unboosted integrase inhibitor regimens versus trans women without HIV taking oral oestradiol: a pilot study.

Feminizing hormone therapy (FHT) is essential to many trans women. Concern about negative drug interactions between FHT and ART can be an ART adherence barrier among trans women with HIV. In this single-centre, parallel group, cross-sectional pilot study, we measured serum oestradiol concentrations in trans women with HIV taking FHT and unboosted integrase strand transfer inhibitor (INSTI)-based ART versus trans women without HIV taking FHT. We included trans women with and without HIV, aged ≥18 years, taking ≥2 mg/day of oral oestradiol for at least 3 months plus an anti-androgen. Trans women with HIV were on suppressive ART ≥3 months. Serum oestradiol concentrations were measured prior to medication dosing and 2, 4, 6 and 8 h post-dose. Median oestradiol concentrations were compared between groups using Wilcoxon rank-sum tests. Participants (n = 8 with HIV, n = 7 without) had a median age of 32 (IQR: 28, 39) years. Among participants, the median oral oestradiol dose was 4 mg (range 2-6 mg). Participants had been taking FHT for a median of 4 years (IQR: 2, 8). Six trans women with HIV were taking bictegravir/emtricitabine/tenofovir alafenamide and two were taking dolutegravir/abacavir/lamivudine. All oestradiol concentrations were not significantly different between groups. Eleven (73%) participants had target oestradiol concentrations in the range 200-735 pmol/L at C4h (75% among women with HIV, 71% among those without HIV). Oestradiol concentrations were not statistically different in trans women with HIV compared with those without HIV, suggesting a low probability of clinically relevant drug-drug interactions between FHT and unboosted INSTI-based ART.

Combined effects of loneliness and inflammation on depression in people with HIV

ObjectiveLoneliness is prevalent in people with HIV (PWH) and associated with adverse health-related consequences, including depression. Chronic inflammation has been linked to depression in PWH, though its association with loneliness is less well established. Simultaneous examination of inflammation, loneliness and depression is needed to clarify these relationships. This study investigated the relationship between loneliness and inflammation, and the effects of loneliness and inflammation on depression in PWH. Methods82 PWH who were on suppressive ART (mean age [SD] = 53.2 [9.0]) completed the UCLA Loneliness Scale-Version 3 and the Center for Epidemiologic Studies Depression Scale as part of a comprehensive evaluation. Biomarkers of systemic inflammation (CRP, IL-6, CCL2/MCP-1, sCD14) and coagulation (D-dimer) were measured in blood using commercial immunoassays.ResultsMultivariable linear regression analyses revealed that higher D-dimer, CCL2/MCP-1, and sCD14 were significant predictors of loneliness (ps < .05) while accounting for relevant covariates. Stepwise multiple linear regression models that included loneliness, biomarkers, and their interactions as predictors of depressive symptoms revealed significant main effects of loneliness and CCL2/MCP-1 levels (ps < .05), and a significant loneliness by D-dimer interaction (p < .05) whereby higher D-dimer was associated with increased depressive symptoms only at higher levels of loneliness. ConclusionsIncreased coagulation activity is associated with loneliness, and in the context of loneliness, may increase risk for depression. Increased inflammation was associated with depression suggesting potentially dissociable underlying biological processes. To the extent that these processes are modifiable, such findings could have important implications in the treatment of loneliness and depression in PWH.

Prospective pilot study on the relationship between seminal HIV-1 shedding and genital schistosomiasis in men receiving antiretroviral therapy along Lake Malawi

Male genital schistosomiasis (MGS) is hypothesized to increase seminal shedding of HIV-1. This prospective pilot study assessed seminal HIV-1 RNA shedding in men on long-term ART with and without a diagnosis of MGS. Study visits occurred at 0, 1, 3, 6 and 12 months. MGS was diagnosed by egg positivity on semen microscopy or PCR of seminal sediment. After optimization of the HIV-RNA assay, we examined 72 paired plasma and semen samples collected from 31 men (15 with and 16 without MGS) over 12 months. HIV-1 RNA was detected in 7/72 (9.7%) seminal samples and 25/72 (34.7%) plasma samples. When comparing sample pairs, 5/72 (6.9%) showed HIV-1 RNA detection only in the seminal sample. Overall, 3/31 (9.7%) participants, all with MGS, had detectable HIV-1 RNA in semen while plasma HIV-1 RNA was undetectable (< 22 copies/mL), with seminal levels ranging up to 400 copies/mL. Two participants showing HIV-1 RNA in seminal fluid from the MGS-negative group also had concomitant HIV-1 RNA detection in plasma. The findings suggest that MGS can be associated with low-level HIV-1 RNA shedding despite virologically suppressive ART. Further studies are warranted to confirm these observations and assess its implications.

HIV DNA positively correlates with HLA-DR+CD8+ T lymphocytes over 8-year suppressive ART.

Despite long-term suppressive cART, the activation level of T lymphocytes remains significantly high HIV-infected individuals. This study aims to unravel the relationship between CD8+ T cell activation and HIV DNA reservoir. In this retrospective study, 82 HIV-infected patients receiving suppressive cART for ≥8 years were included. Total HIV-1 DNA and expression of CD38 and HLA-DR in CD8+ T cell were quantified repeatedly during long-term follow-up. Longitudinal correlation between HIV-1 DNA and CD8+ T cell activation level was analysed using generalized estimating equation (GEE) model. Significant decrease of both total HIV-1 DNA and CD8+ T cell activation level were observed after combined antiretroviral therapy (cART) initiation. However, the expression level of HLA-DR in CD8+ T cells remained abnormally higher than normal range. GEE analysis revealed that HLA-DR expression was positively correlated with total HIV-1 DNA over long-term suppressive cART. HIV DNA reservoir may be closely related to CD8+ T cell activation and the inflammatory state in HIV-infected patients despite long-term cART.

Both N- and C-terminal domains of galectin-9 are capable of inducing HIV reactivation despite mediating differential immunomodulatory functionalities.

The shock-and-kill strategy for HIV cure requires the reactivation of latent HIV followed by the killing of the reactivated cellular reservoir. Galectin-9, an immunomodulatory protein, is shown to induce HIV reactivation as well as contribute to non-AIDS- and AIDS-defining events. The protein is prone to cleavage by inflammatory proteases at its linker region separating the N- and C-terminal carbohydrate-binding domains (N- and C-CRDs) which differ in their binding specificities. It is important to study the activity of its cleaved as well as uncleaved forms in mediating HIV reactivation and immunomodulation in order to understand their role in HIV pathogenesis and their further utilization for the shock-and-kill strategy. The PBMCs of HIV patients on virally suppressive ART (n = 11) were stimulated using 350 nM of the full-length protein and N- and C-CRDs of Gal-9. HIV reactivation was determined by analyzing gag RNA copies using qPCR using isolated CD4 cells and intracellular P24 staining of PBMCs by flow cytometry. Cytokine responses induced by the full-length protein and N- and C-CRDs of Gal-9 were also assessed by flow cytometry, Luminex, and gene expression assays. Changes in T helper cell gene expression pattern after the stimulation were also determined by real-time PCR array. Both N- and C-CRDs of galectin-9 induced HIV reactivation in addition to the full-length galectin-9 protein. The two domains elicited higher cytokine responses than the full-length protein, possibly capable of mediating higher perturbations in the immune system if used for HIV reactivation. N-CRD was found to induce the development of Treg cells, whereas C-CRD inhibited the induction of Treg cells. Despite this, both domains elicited IL-10 secretory response although targeting different CD4 cell phenotypes. N- and C-CRDs were found to induce HIV reactivation similar to that of the full-length protein, indicating their possible usefulness in the shock-and-kill strategy. The study indicated an anti-inflammatory role of N-CRD versus the proinflammatory properties of C-CRD of galectin-9 in HIV infection.

PP 1.15 – 00095 Proviruses persisting during the initial years of suppressive ART are relatively stable in terms of genetic diversity, clonal composition, and inferred integration date distribution

PP 1.15 – 00095 Proviruses persisting during the initial years of suppressive ART are relatively stable in terms of genetic diversity, clonal composition, and inferred integration date distribution

P 4.2 – 00029 Innate Immune Correlates of Cell-Associated HIV RNA And DNA During Long-Term Suppressive ART

P 4.2 – 00029 Innate Immune Correlates of Cell-Associated HIV RNA And DNA During Long-Term Suppressive ART

In vivo killing of primary HIV-infected cells by peripheral-injected early memory-enriched anti-HIV duoCAR T cells.

HIV-specific chimeric antigen receptor-T cell (CAR T cell) therapies are candidates to functionally cure HIV infection in people with HIV (PWH) by eliminating reactivated HIV-infected cells derived from latently infected cells within the HIV reservoir. Paramount to translating such therapeutic candidates successfully into the clinic will require anti-HIV CAR T cells to localize to lymphoid tissues in the body and eliminate reactivated HIV-infected cells such as CD4+ T cells and monocytes/macrophages. Here we show that i.v. injected anti-HIV duoCAR T cells, generated using a clinical-grade anti-HIV duoCAR lentiviral vector, localized to the site of active HIV infection in the spleen of humanized mice and eliminated HIV-infected PBMCs. CyTOF analysis of preinfusion duoCAR T cells revealed an early memory phenotype composed predominantly of CCR7+ stem cell-like/central memory T cells (TSCM/TCM) with expression of some effector-like molecules. In addition, we show that anti-HIV duoCAR T cells effectively sense and kill HIV-infected CD4+ T cells and monocytes/macrophages. Furthermore, we demonstrate efficient genetic modification of T cells from PWH on suppressive ART into anti-HIV duoCAR T cells that subsequently kill autologous PBMCs superinfected with HIV. These studies support the safety and efficacy of anti-HIV duoCAR T cell therapy in our presently open phase I/IIa clinical trial (NCT04648046).

Immunogenicity to COVID-19 mRNA vaccine third dose in people living with HIV

In order to investigate safety and immunogenicity of SARS-CoV-2 vaccine third dose in people living with HIV (PLWH), we analyze anti-RBD, microneutralization assay and IFN-γ production in 216 PLWH on ART with advanced disease (CD4 count <200 cell/mm3 and/or previous AIDS) receiving the third dose of a mRNA vaccine (BNT162b2 or mRNA-1273) after a median of 142 days from the second dose. Median age is 54 years, median CD4 nadir 45 cell/mm3 (20–122), 93% HIV-RNA < 50 c/mL. In 68% of PLWH at least one side-effect, generally mild, is recorded. Humoral response after the third dose was strong and higher than that achieved with the second dose (>2 log2 difference), especially when a heterologous combination with mRNA-1273 as third shot is used. In contrast, cell-mediated immunity remain stable. Our data support usefulness of third dose in PLWH currently receiving suppressive ART who presented with severe immune dysregulation.

CD8 lymphocytes mitigate HIV-1 persistence in lymph node follicular helper T cells during hyperacute-treated infection

HIV persistence in tissue sites despite ART is a major barrier to HIV cure. Detailed studies of HIV-infected cells and immune responses in native lymph node tissue environment is critical for gaining insight into immune mechanisms impacting HIV persistence and clearance in tissue sanctuary sites. We compared HIV persistence and HIV-specific T cell responses in lymph node biopsies obtained from 14 individuals who initiated therapy in Fiebig stages I/II, 5 persons treated in Fiebig stages III-V and 17 late treated individuals who initiated ART in Fiebig VI and beyond. Using multicolor immunofluorescence staining and in situ hybridization, we detect HIV RNA and/or protein in 12 of 14 Fiebig I/II treated persons on suppressive therapy for 1 to 55 months, and in late treated persons with persistent antigens. CXCR3+ T follicular helper cells harbor the greatest amounts of gag mRNA transcripts. Notably, HIV-specific CD8+ T cells responses are associated with lower HIV antigen burden, suggesting that these responses may contribute to HIV suppression in lymph nodes during therapy. These results reveal HIV persistence despite the initiation of ART in hyperacute infection and highlight the contribution of virus-specific responses to HIV suppression in tissue sanctuaries during suppressive ART.

Predictive value of CD8+ T cell and CD4/CD8 ratio at two years of successful ART in the risk of AIDS and non-AIDS events.

SummaryBackgroundWhile increased CD8 counts and low CD4/CD8 ratio during treated HIV correlate with immunosenescence, their additional predictive values to identify individuals with HIV at higher risk of clinical events remain controversial.MethodsWe selected treatment-naive individuals initiating ART from ACTG studies 384, 388, A5095, A5142, A5202, and A5257 who had achieved viral suppression at year 2. We examined the effect of CD8+ T cell counts and CD4/CD8 at year 2 on the probability of AIDS and serious non-AIDS events in years 3−7. We used inverse probability weighting methods to address informative censoring, combined with multivariable logistic regression models.FindingsWe analyzed 5133 participants with a median age of 38 years; 959 (19%) were female, pre-ART median CD4 counts were 249 (Q1-Q3 91–372) cell/µL. Compared to participants with CD8 counts between 500/µL and 1499/µL, those with >1500/µL had a higher risk of clinical events during years 3−7 (aOR 1.75; 95%CI 1.33−2.32). CD4/CD8 ratio was not predictive of greater risk of events through year 7. Additional analyses revealed consistent CD8 count effect sizes for the risk of AIDS events and noninfectious non-AIDS events, but opposite effects for the risk of severe infections, which were more frequent among individuals with CD8 counts <500/µL (aOR 1.70; 95%CI 1.09−2.65).InterpretationThe results of this analysis with pooled data from clinical trials support the value of the CD8 count as a predictor of clinical progression. People with very high CD8 counts during suppressive ART might benefit from closer monitoring and may be a target population for novel interventions.FundingThis research was supported by NIH/NIAID awards UM1 AI068634, UM1 AI068636, and UM1 AI106701 and Carlos III Health Institute and FEDER funds (BA21/00017 and BA21/00022).

HIV-specific CD8 T cells from elite controllers have an epigenetic imprint that preserves effector functions

Abstract CD8 T cells from HIV infected ‘elite controllers’ maintain long-lived protective properties including enhanced cytokine production and proliferative capacity over many years. With the advent of adoptive cellular therapy to treat chronic diseases it has become even more vital to understand the intrinsic cellular mechanisms of elite controller HIV specific CD8 T cells that enable lasting antiviral functionality. To identify epigenetic programs that regulate their functional capacity we performed genome-wide DNA methylation analysis of MHC Class I multimer+ CD8 T cells sorted from aviremic elite controllers compared to aviremic non-controllers on suppressive ART. Principle component analysis of the CpG sites broadly distinguished EC and ART HIV specific CD8 T cells, while deeper investigation of the differentially methylated region analysis revealed enrichment of pathways that support a multipotent differentiation state, cytokine signaling, and a long-lived effector cell fate in HIV-specific CD8 T cells from elite controllers. We also observed DNA methylation programs at the transcription factor binding sites of the stem-associated factors TCF-1 and LEF1 that delineate HIV-specific CD8 T cells from elite controllers versus ART-treated individuals. These findings show that HIV-specific CD8 T cells from elite controllers have DNA methylation programs that maintain developmental potential and in turn enable long-term survival, proliferative potential, and effector capacity. These data also provide new insights into the relationship between stem-associated transcription factors and stable epigenetic restriction of T cell developmental capacity. Supported by grants from NIH (R01AI114442 and R01CA237311 to BY, K23AI134327 to RLR), loan repayment program and National comprehensive Cancer Network Young Investigator Award (CZ), ASSISI foundation support (BY) and American Lebanese Syrian Associated Charities (ALSAC to BY)

Antiretroviral drug exposure in lymph nodes is heterogeneous and drug dependent.

IntroductionHIV reservoirs and infected cells may persist in tissues with low concentrations of antiretrovirals (ARVs). Traditional pharmacology methods cannot assess variability in ARV concentrations within morphologically complex tissues, such as lymph nodes (LNs). We evaluated the distribution of six ARVs into LNs and the proximity of these ARVs to CD4+ T cells and cell‐associated RT‐SHIV viral RNA.MethodsBetween December 2014 and April 2017, RT‐SHIV infected (SHIV+; N = 6) and healthy (SHIV–; N = 6) male rhesus macaques received two selected four‐drug combinations of six ARVs over 10 days to attain steady‐state conditions. Serial cryosections of axillary LN were analysed by a multimodal imaging approach that combined mass spectrometry imaging (MSI) for ARV disposition, RNAscope in situ hybridization for viral RNA (vRNA) and immunohistochemistry for CD4+ T cell and collagen expression. Spatial relationships across these four imaging domains were investigated by nearest neighbour search on co‐registered images using MATLAB.ResultsThrough MSI, ARV‐dependent, heterogeneous concentrations were observed in different morphological LN regions, such as the follicles and medullary sinuses. After 5–6 weeks of infection, more limited ARV penetration into LN tissue relative to the blood marker heme was found in SHIV+ animals (SHIV+: 0.7 [0.2–1.4] mm; SHIV–: 1.3 [0.5–1.7] mm), suggesting alterations in the microcirculation. However, we found no detectable increase in collagen deposition. Regimen‐wide maps of composite ARV distribution indicated that up to 27% of SHIV+ LN tissue area was not exposed to detectable ARVs. Regions associated with B cell follicles had median 1.15 [0.94–2.69] ‐fold reduction in areas with measurable drug, though differences were only statistically significant for tenofovir (p = 0.03). Median co‐localization of drug with CD4+ target cells and vRNA varied widely by ARV (5.1–100%), but nearest neighbour analysis indicated that up to 10% of target cells and cell‐associated vRNA were not directly contiguous to at least one drug at concentrations greater than the IC50 value.ConclusionsOur investigation of the spatial distributions of drug, virus and target cells underscores the influence of location and microenvironment within LN, where a small population of T cells may remain vulnerable to infection and low‐level viral replication during suppressive ART.

Measuring Proviral HIV-1 DNA: Hurdles and Improvements to an Assay Monitoring Integration Events Utilising Human Alu Repeat Sequences.

Integrated HIV-1 DNA persists despite antiretroviral therapy and can fuel viral rebound following treatment interruption. Hence, methods to specifically measure the integrated HIV-1 DNA portion only are important to monitor the reservoir in eradication trials. Here, we provide an up-to-date overview of the literature on the different approaches used to measure integrated HIV-1 DNA. Further, we propose an implemented standard-curve free assay to quantify integrated HIV-1 DNA, so-called Alu-5LTR PCR, which utilises novel primer combinations. We tested the Alu-5LTR PCR in 20 individuals on suppressive ART for a median of nine years; the results were compared to those produced with the standard-free Alu-gag assay. The numbers of median integrated HIV-1 DNA copies were 5 (range: 1–12) and 14 (5–26) with the Alu-gag and Alu-5LTR, respectively. The ratios between Alu-gag vs Alu-5LTR results were distributed within the cohort as follows: most patients (12/20, 60%) provided ratios between 2–5, with 3/20 (15%) and 5/20 (25%) being below or above this range, respectively. Alu-5LTR assay sensitivity was also determined using an “integrated standard”; the data confirmed the increased sensitivity of the assay, i.e., equal to 0.25 proviruses in 10,000 genomes. This work represents an improvement in the field of measuring proviral HIV-1 DNA that could be employed in future HIV-1 persistence and eradication studies.