Size Of HIV Reservoir Research Articles

Differences in HIV-1 reservoir size, landscape characteristics and decay dynamics in acute and chronic treated HIV-1 Clade C infection.

Background Persisting HIV reservoir viruses in resting CD4 T cells and other cellular subsets are the main barrier to cure efforts. Antiretroviral therapy (ART) intensification by early initiation has been shown to enable post-treatment viral control in some cases but the underlying mechanisms are not fully understood. We hypothesized that ART initiated during the hyperacute phase of infection before peak will affect the size, decay dynamics and landscape characteristics of HIV-1 subtype C viral reservoirs. Methods We studied 35 women at high risk of infection from Durban, South Africa identified with hyperacute HIV infection by twice weekly testing for plasma HIV-1 RNA. Study participants included 11 who started ART at a median of 456 (297-1203) days post onset of viremia (DPOV), and 24 who started ART at a median of 1 (1-3) DPOV. We used peripheral blood mononuclear cells (PBMC) to measure total HIV-1 DNA by ddPCR and to sequence reservoir viral genomes by full length individual proviral sequencing (FLIP-seq) from onset of detection of HIV up to 1 year post treatment initiation. Results Whereas ART in hyperacute infection blunted peak viremia compared to untreated individuals (p<0.0001), there was no difference in total HIV-1 DNA measured contemporaneously (p=0.104). There was a steady decline of total HIV DNA in early treated persons over 1 year of ART (p=0.0004), with no significant change observed in the late treated group. Total HIV-1 DNA after one year of treatment was lower in the early treated compared to the late treated group (p=0.02). Generation of 697 single viral genome sequences revealed a difference in the longitudinal proviral genetic landscape over one year between untreated, late treated, and early treated infection: the relative contribution of intact genomes to the total pool of HIV-1 DNA after 1 year was higher in untreated infection (31%) compared to late treated (14%) and early treated infection (0%). Treatment initiated in both late and early infection resulted in a more rapid decay of intact (13% and 51% per month) versus defective (2% and 35% per month) viral genomes. However, intact genomes were still observed one year post chronic treatment initiation in contrast to early treatment where intact genomes were no longer detectable. Moreover, early ART reduced phylogenetic diversity of intact genomes and limited the seeding and persistence of cytotoxic T lymphocyte immune escape variants in the reservoir. Conclusions Overall, our results show that whereas ART initiated in hyperacute HIV-1 subtype C infection did not impact reservoir seeding, it was nevertheless associated with more rapid decay of intact viral genomes, decreased genetic complexity and immune escape in reservoirs, which could accelerate reservoir clearance when combined with other interventional strategies.

Soluble markers of viral rebound and post-treatment HIV control.

We focus on the different classes of biological molecules measurable in easily accessible bodily fluids that have the potential to serve as biomarkers for the HIV post-treatment controller (PTC) phenotype and/or the timing of viral rebound after stopping antiretroviral therapy (ART). Various viral components and host factors measurable in body fluids can play crucial roles in understanding and predicting the PTC phenotype. We review recent findings linking viral components, the quantitative and qualitative features of antibodies (including autologous HIV-specific antibodies), markers of inflammation and tissue damage, other host proteins (including hormones such as sex hormones), as well as metabolites, extracellular vesicles, and cell-free DNA to HIV control post-ART interruption. Several of these molecules can or have the potential to predict the time and probability of viral rebound after stopping ART and are biologically active molecules that can directly or indirectly (by modulating immune pressures) impact the size and activity of HIV reservoirs during and post-ART interruption. A comprehensive model combining multiple markers is needed to predict the PTC phenotype. This model can be leveraged to predict and understand the PTC phenotype, which can guide novel curative interventions to replicate this phenotype in post-treatment non-controllers.

Sirolimus reduces T cell cycling, immune checkpoint marker expression, and HIV-1 DNA in people with HIV

Key HIV cure strategies involve reversing immune dysfunction and limiting the proliferation of infected Tcells. We evaluate the safety of sirolimus, a mammalian target of rapamycin (mTOR) inhibitor, in people with HIV (PWH) and study the impact of sirolimus on HIV-1 reservoir size and HIV-1-specific immunity in a single-arm study of 20weeks of treatment in PWH on antiretroviral therapy (ART). Sirolimus treatment does not impact HIV-1-specific CD8 Tcell responses but leads to a significant decrease in CD4+ Tcell-associated HIV-1 DNA levels at 20weeks of therapy in the primary efficacy population (n= 16; 31% decline, p=0.008). This decline persists for at least 12weeks following cessation of the study drug. Sirolimus treatment also leads to a significant reduction in CD4+ Tcell cycling and PD-1 expression on CD8+ lymphocytes. These data suggest that homeostatic proliferation of infected cells, an important mechanism for HIV persistence, is an intriguing therapeutic target.

Specific quantification of inducible HIV-1 reservoir by RT-LAMP

BackgroundStrategies toward HIV-1 cure aim to clear, inactivate, reduce, or immunologically control the virus from a pool of latently infected cells such that combination antiretroviral therapy (cART) can be safely interrupted. In order to assess the impact of any putative curative interventions on the size and inducibility of the latent HIV-1 reservoir, robust and scalable assays are needed to precisely quantify the frequency of infected cells containing inducible HIV-1.MethodsWe developed Specific Quantification of Inducible HIV−1 by RT-LAMP (SQuHIVLa), leveraging the high sensitivity and specificity of RT-LAMP, performed in a single reaction, to detect and quantify cells expressing tat/rev HIV-1 multiply spliced RNA (msRNA) upon activation. The LAMP primer/probe used in SQuHIVLa was designed to exclusively detect HIV-1 tat/rev msRNA and adapted for different HIV-1 subtypes.ResultsUsing SQuHIVLa, we successfully quantify the inducible viral reservoir in CD4+ T cells from people living with HIV-1 subtypes B and C on cART. The assay demonstrates high sensitivity, specificity, and reproducibility.ConclusionsSQuHIVLa offers a high throughput, scalable, and specific HIV-1 reservoir quantification tool that is amenable to resource-limited settings. This assay poses remarkable potential in facilitating the evaluation of potential interventional strategies toward achieving HIV-1 cure.

Impact of interrupting antiretroviral therapy started during primary HIV-1 infection on plasma neurofilament light chain protein, a marker of neuronal injury: The SPARTAC trial

ObjectiveAntiretroviral therapy (ART)-conferred suppression of HIV replication limits neuronal injury and inflammation. ART interruption tests efficacy in HIV cure trials and viral rebound after ART interruption may induce neuronal injury. We investigated the impact of protocol-defined ART interruption, commenced during primary HIV-1 infection (PHI) on a biomarker of neuro-axonal injury (neurofilament light protein (NfL)), and its associations with inflammation (D-dimer and interleukin-6 (IL-6)) and HIV-1 reservoir size (total HIV-1 DNA). DesignRetrospective study measuring plasma NfL in 83 participants enrolled in SPARTAC randomised to receive 48-weeks ART initiated during PHI, followed by ART interruption. MethodsNfL (Simoa immunoassay, Quanterix™) was measured before ART, after 48 weeks on ART, and 12 weeks after stopping ART. Plasma D-dimer and IL-6, and total HIV-1 DNA in peripheral CD4+ T-cells results were available in a subset of participants. Longitudinal NfL changes were assessed using mixed models, and associations with clinical and laboratory parameters using linear regression. ResultsNfL decreased following 48-weeks ART (geometric mean 6.9 to 5.8 pg/mL, p = 0.006) with no further significant change up to 12-weeks post-stopping ART despite viral rebound in the majority of participants (median 1.7 to 3.9 plasma HIV-1 RNA log10 copies/mL). Higher baseline NfL was independently associated with higher plasma HIV-1 RNA (p = 0.020) and older age (p = 0.002). While NfL was positively associated with D-dimer (n = 48; p = 0.002), there was no significant association with IL-6 (n = 48) or total HIV-1 DNA (n = 51). ConclusionsUsing plasma NfL as a surrogate marker, a decrease in neuro-axonal injury was observed in a cohort of participants following ART initiation during PHI, with no evidence of neuro-axonal injury rebound following ART interruption for up to 12 weeks, despite viral rebound in the majority of participants.

Evaluation of HIV-1 DNA levels among adolescents living with perinatally acquired HIV-1 in Yaounde, Cameroon: A contribution to paediatric HIV cure research in Sub-Saharan Africa

BackgroundWith the advent of antiretroviral therapy (ART), most children living with HIV in sub-Saharan Africa (SSA) are growing toward adolescence, with scarcity of evidence on the size of viral reservoirs to enhance paediatric cure research strategies. This study aims to compare HIV-1 proviral DNA levels according to virological response among adolescents living with perinatally acquired HIV-1 (ALPHIV) and identify associated-factors in the Cameroonian context. MethodsIn this observational cohort study, HIV-1 RNA viremia and CD4+ T-cell count were assessed through RT-PCR and flow cytometry respectively at three time-points over 18 months of observation. At the third time-point, 80 randomly-selected participants were classified as with viremia (≥50 HIV-1 copies/mL; n = 40) or without viremia (<50 HIV-1 copies/mL; n = 40); immune-competent (≥500 CD4+ T cells/mm3) or immunocompromised (<500 CD4+ T cells/mm3). Among these participants, total HIV-1 DNA load was quantified through droplet digital PCR using Bio-Rad QX200. ResultsOf the 80 randomly-selected adolescents, median [IQR] age was 15 (13-17) years, 56.2% were female, duration on ART was 9.3 [5.4–12.2] years. Among the 40 viremic ones (median viremia 7312 [283–71482]) HIV-1 copies/ml, 75.0% (30/40) were in virological failure (≥1000 HIV-1 copies/ml), while median of CD4 T cells were 494 [360–793] cell/mm3 with 48.8% (39/80) immunocompromised. No significant variation in HIV-1 RNA viremia and CD4 T cell count was observed between the three time-points, and 13.7% (11/80) adolescents remained aviremic and immune-competent throughout (stable adolescents). A positive and moderate correlation (r = 0.59; p < 0.001) was found between HIV-1 DNA levels and HIV- 1 RNA viremia. Regarding the CD4 T cell count, a negative and weak correlation (r = −0.28; p = 0.014) was found with HIV-1 DNA loads only among adolescents with viremia. Starting ART within the first year of life, ART for over 9 years and aviremia appear as predictors of low HIV-1 DNA loads. ConclusionAmong ALPHIV, high HIV-1 RNA indicates an elevated viral reservoir size, representing a drawback to cure research. Interestingly, early ART initiation and longer ARTduration lead to sustained viral control and limited HIV-1 reservoir size. As limited size of viral reservoir appears consistent with viral control and immune competence, adolescents with sustained viral control (about 14% of this target population) would be candidates for analytical ART interruptions toward establishing paediatric post-treatment controllers in SSA.

Effects of COVID-19 mRNA vaccination on HIV viremia and reservoir size.

The immunogenic nature of coronavirus disease 2019 (COVID-19) mRNA vaccines led to some initial concern that these could stimulate the HIV reservoir. We analyzed changes in plasma HIV loads (pVL) and reservoir size following COVID-19 mRNA vaccination in 62 people with HIV (PWH) receiving antiretroviral therapy (ART), and analyzed province-wide trends in pVL before and after the mass vaccination campaign. Longitudinal observational cohort and province-wide analysis. Sixty-two participants were sampled prevaccination, and one month after their first and second COVID-19 immunizations. Vaccine-induced anti-SARS-CoV-2-Spike antibodies in serum were measured using the Roche Elecsys Anti-S assay. HIV reservoirs were quantified using the intact proviral DNA assay; pVL were measured using the cobas 6800 (lower limit of quantification: 20 copies/ml). The province-wide analysis included all 290 401 pVL performed in British Columbia, Canada between 2012 and 2022. Prevaccination, the median intact reservoir size was 77 [interquartile range (IQR): 20-204] HIV copies/million CD4 + T-cells, compared to 74 (IQR: 27-212) and 65 (IQR: 22-174) postfirst and -second dose, respectively (all comparisons P > 0.07). Prevaccination, 82% of participants had pVL <20 copies/ml (max: 110 copies/ml), compared to 79% postfirst dose (max: 183 copies/ml) and 85% postsecond dose (max: 79 copies/ml) ( P > 0.4). There was no evidence that the magnitude of the vaccine-elicited anti-SARS-CoV-2-Spike immune response influenced pVL nor changes in reservoir size ( P > 0.6). We found no evidence linking the COVID-19 mass vaccination campaign to population-level increases in detectable pVL frequency among all PWH in the province, nor among those who maintained pVL suppression on ART. We found no evidence that COVID-19 mRNA vaccines induced changes in HIV reservoir size nor plasma viremia.

Comparison of HIV-1 DNA load measurements in blood and in relation to successful proviral sequencing

ObjectiveHIV DNA sequencing is now routinely used for HIV-infected individuals on antiretroviral therapy (ART) with or without partial genotypic history. Successful amplification of HIV pol gene has yet to be correlated with HIV DNA levels. Here, we assessed the relationship between HIV DNA load and sequencing results. MethodsWe analyzed three different qPCR measurements of total (LTR and LTR-gag) and integrated (Alu-LTR) HIV DNA in blood samples collected from viremic as well as virally suppressed HIV-infected individuals on ART. HIV DNA levels were compared to HIV DNA Sanger sequencing and clinical and therapeutic parameters. ResultsAmong the 135 individuals analyzed for HIV DNA measurements and sequencing, all three HIV DNA measurements were associated with HIV DNA Sanger sequencing results. A threshold of around 2 and 1.5 log copies/million leukocytes of total HIV DNA was identified for LTR and LTR-gag qPCRs, respectively. Integrated HIV DNA positivity was also associated with successful sequencing. We further compared HIV DNA measurement techniques in an extended cohort of 312 individuals and showed that all measurements correlated between the different techniques, regardless of the HIV-1 subtypes analyzed. However, higher detection rates were observed with LTR (96%) compared to LTR-gag (86%) and Alu-LTR (59%) qPCRs. Duration of virological control on ART and CD4 nadir were the main determinants of HIV reservoir size. ConclusionsHIV DNA measurement is associated with Sanger sequencing success, regardless of the technique used. In a clinical setting, Application of HIV DNA quantification before sequencing should be further evaluated.

HIV-1 reservoir size after neonatal antiretroviral therapy and the potential to evaluate antiretroviral-therapy-free remission (IMPAACT P1115): a phase 1/2 proof-of-concept study

Infants born with HIV-1 require lifelong antiretroviral therapy (ART). We aimed to assess whether very early ART in neonates might restrict HIV-1 reservoirs, an important step towards ART-free remission. IMPAACT P1115 is an ongoing, phase 1/2, proof-of-concept study in which infants were enrolled at 30 research clinics in 11 countries (Brazil, Haiti, Kenya, Malawi, South Africa, Tanzania, Thailand, Uganda, the USA, Zambia, and Zimbabwe) into two cohorts. Infants at least 34 weeks' gestational age at high risk for in-utero HIV-1 with either untreated maternal HIV-1 (cohort 1) or who were receiving pre-emptive triple antiretroviral prophylaxis outside of the study (maternal ART permissible; cohort 2) were included. All infants initiated treatment within 48 h of life. Cohort 1 initiated three-drug nevirapine-based ART, and cohort 2 initiated three-drug nevirapine-based prophylaxis then three-drug nevirapine-based ART following HIV diagnosis by age 10 days. We added twice-daily coformulated oral ritonavir 75 mg/m2 and lopinavir 300 mg/m2 from 14 days of life and 42 weeks postmenstrual age. We discontinued nevirapine 12 weeks after two consecutive plasma HIV-1 RNA levels below limit of detection. We tracked virological suppression, safety outcomes, and meeting a predetermined biomarker profile at age 2 years (undetectable RNA since week 48, HIV-1 antibody-negative, HIV-1 DNA not detected, and normal CD4 count and CD4 percentage) to assess qualification for analytical treatment interruption. This study is registered with ClinicalTrials.gov, NCT02140255. Between Jan 23, 2015, and Dec 14, 2017, 440 infants were included in cohort 1 and 20 were included in cohort 2. 54 of these infants (34 from cohort 1 and 20 from cohort 2) had confirmed in-utero HIV-1 and were enrolled to receive study ART. 33 (61%) of 54 infants were female and 21 (39%) were male. The estimated probability of maintaining undetectable plasma RNA through to 2 years was 33% (95% CI 17-49) in cohort 1 and 57% (28-78) in cohort 2. Among infants maintaining protocol-defined virological control criteria through to study week 108, seven of 11 (64%, 95% CI 31-89) in cohort 1 and five of seven (71%, 29-96) in cohort 2 had no detected HIV-1 DNA. Ten of 12 (83%, 52-100) in cohort 1 and all seven (100%, 59-100) in cohort 2 tested HIV-1 antibody-negative at week 108. Among 54 infants initiated on very early ART, ten (19%; six in cohort 1 and four in cohort 2) met all criteria for possible analytical treatment interruption. Reversible grade 3 or 4 adverse events occurred in 15 (44%) of 34 infants in cohort 1 and seven (35%) of 20 infants in cohort 2. Very early ART for in-utero HIV-1 can achieve sustained virological suppression in association with biomarkers indicating restricted HIV-1 reservoirs by age 2 years, which might enable potential ART-free remission. National Institute of Allergy and Infectious Diseases, the Eunice Kennedy Shriver National Institute of Child Health and Human Development, and the National Institute of Mental Health.

A cohort-based study of host gene expression: tumor suppressor and innate immune/inflammatory pathways associated with the HIV reservoir size.

The major barrier to an HIV cure is the HIV reservoir: latently-infected cells that persist despite effective antiretroviral therapy (ART). There have been few cohort-based studies evaluating host genomic or transcriptomic predictors of the HIV reservoir. We performed host RNA sequencing and HIV reservoir quantification (total DNA [tDNA], unspliced RNA [usRNA], intact DNA) from peripheral CD4+ T cells from 191 ART-suppressed people with HIV (PWH). After adjusting for nadir CD4+ count, timing of ART initiation, and genetic ancestry, we identified two host genes for which higher expression was significantly associated with smaller total DNA viral reservoir size, P3H3 and NBL1, both known tumor suppressor genes. We then identified 17 host genes for which lower expression was associated with higher residual transcription (HIV usRNA). These included novel associations with membrane channel (KCNJ2, GJB2), inflammasome (IL1A, CSF3, TNFAIP5, TNFAIP6, TNFAIP9, CXCL3, CXCL10), and innate immunity (TLR7) genes (FDR-adjusted q<0.05). Gene set enrichment analyses further identified significant associations of HIV usRNA with TLR4/microbial translocation (q = 0.006), IL-1/NRLP3 inflammasome (q = 0.008), and IL-10 (q = 0.037) signaling. Protein validation assays using ELISA and multiplex cytokine assays supported these observed inverse host gene correlations, with P3H3, IL-10, and TNF-α protein associations achieving statistical significance (p<0.05). Plasma IL-10 was also significantly inversely associated with HIV DNA (p = 0.016). HIV intact DNA was not associated with differential host gene expression, although this may have been due to a large number of undetectable values in our study. To our knowledge, this is the largest host transcriptomic study of the HIV reservoir. Our findings suggest that host gene expression may vary in response to the transcriptionally active reservoir and that changes in cellular proliferation genes may influence the size of the HIV reservoir. These findings add important data to the limited host genetic HIV reservoir studies to date.

Impact of predicted HLA class I immunopeptidome on viral reservoir in a cohort of people living with HIV in Italy.

The class I HLA genotype has been widely recognized as a factor influencing HIV disease progression in treatment-naïve subjects. However, little is known regarding its role in HIV disease course and how it influences the size of the viral reservoir once anti-retroviral therapy (ART) is started. Here, leveraging on cutting-edge bioinformatic tools, we explored the relationship between HLA class I and the HIV reservoir in a cohort of 90 people living with HIV (PLWH) undergoing ART and who achieved viral suppression. Analysis of HLA allele distribution among patients with high and low HIV reservoir allowed us to document a predominant role of HLA-B and -C genes in regulating the size of HIV reservoir. We then focused on the analysis of HIV antigen (Ag) repertoire, by investigating immunogenetic parameters such as the degree of homozygosity, HLA evolutionary distance and Ag load. In particular, we used two different bioinformatic algorithms, NetMHCpan and MixMHCpred, to predict HLA presentation of immunogenic HIV-derived peptides and identified HLA-B*57:01 and HLA-B*58:01 among the highest ranking HLAs in terms of total load, suggesting that their previously reported protective role against HIV disease progression might be linked to a more effective viral recognition and presentation to Cytotoxic T lymphocytes (CTLs). Further, we speculated that some peptide-HLA complexes, including those produced by the interaction between HLA-B*27 and the HIV Gag protein, might be particularly relevant for the efficient regulation of HIV replication and containment of the HIV reservoir. Last, we provide evidence of a possible synergistic effect between the CCR5 ∆32 mutation and Ag load in controlling HIV reservoir.

The latent HIV reservoir: current advances in genetic sequencing approaches.

Multiple cellular HIV reservoirs in diverse anatomical sites can undergo clonal expansion and persist for years despite suppressive antiretroviral therapy, posing a major barrier toward an HIV cure. Commonly adopted assays to assess HIV reservoir size mainly consist of PCR-based measures of cell-associated total proviral DNA, intact proviruses and transcriptionally competent provirus (viral RNA), flow cytometry and microscopy-based methods to measure translationally competent provirus (viral protein), and quantitative viral outgrowth assay, the gold standard to measure replication-competent provirus; yet no assay alone can provide a comprehensive view of the total HIV reservoir or its dynamics. Furthermore, the detection of extant provirus by these measures does not preclude defects affecting replication competence. An accurate measure of the latent reservoir is essential for evaluating the efficacy of HIV cure strategies. Recent approaches have been developed, which generate proviral sequence data to create a more detailed profile of the latent reservoir. These sequencing approaches are valuable tools to understand the complex multicellular processes in a diverse range of tissues and cell types and have provided insights into the mechanisms of HIV establishment and persistence. These advancements over previous sequencing methods have allowed multiplexing and new assays have emerged, which can document transcriptional activity, chromosome accessibility, and in-depth cellular phenotypes harboring latent HIV, enabling the characterization of rare infected cells across restrictive sites such as the brain. In this manuscript, we provide a review of HIV sequencing-based assays adopted to address challenges in quantifying and characterizing the latent HIV reservoir.

Therapeutic vaccination following early antiretroviral therapy elicits highly functional T cell responses against conserved HIV-1 regions

‘Kick and kill’ cure strategies aim to induce HIV protein expression in latently infected cells (kick), and thus trigger their elimination by cytolytic T cells (kill). In the Research in Viral Eradication of HIV Reservoirs trial (NCT02336074), people diagnosed with primary HIV infection received immediate antiretroviral therapy (ART) and were randomised 24 weeks later to either a latency-reversing agent, vorinostat, together with ChAdV63.HIVconsv and MVA.HIVconsv vaccines, or ART alone. This intervention conferred no reduction in HIV-1 reservoir size over ART alone, despite boosting virus-specific CD4+ and CD8+ T cells. The effects of the intervention were examined at the cellular level in the two trial arms using unbiased computational analysis of polyfunctional scores. This showed that the frequency and polyfunctionality of virus-specific CD4+ and CD8+ T cell populations were significantly increased over 12 weeks post-vaccination, compared to the ART-only arm. HIV-specific IL-2-secreting CD8+ T cells also expanded significantly in the intervention arm and were correlated with antiviral activity against heterologous HIV in vitro. Therapeutic vaccination during ART commenced in primary infection can induce functional T cell responses that are phenotypically similar to those of HIV controllers. Analytical therapy interruption may help determine their ability to control HIV in vivo.

Higher HIV-1 Env gp120-Specific Antibody-Dependent Cellular Cytotoxicity (ADCC) Activity Is Associated with Lower Levels of Defective HIV-1 Provirus.

A cure for HIV-1 (HIV) remains unrealized due to a reservoir of latently infected cells that persist during antiretroviral therapy (ART), with reservoir size associated with adverse health outcomes and inversely with time to viral rebound upon ART cessation. Once established during ART, the HIV reservoir decays minimally over time; thus, understanding factors that impact the size of the HIV reservoir near its establishment is key to improving the health of people living with HIV and for the development of novel cure strategies. Yet, to date, few correlates of HIV reservoir size have been identified, particularly in pediatric populations. Here, we employed a cross-subtype intact proviral DNA assay (CS-IPDA) to quantify HIV provirus between one- and two-years post-ART initiation in a cohort of Kenyan children (n = 72), which had a median of 99 intact (range: 0-2469), 1340 defective (range: 172-3.84 × 104), and 1729 total (range: 178-5.11 × 104) HIV proviral copies per one million T cells. Additionally, pre-ART plasma was tested for HIV Env-specific antibody-dependent cellular cytotoxicity (ADCC) activity. We found that pre-ART gp120-specific ADCC activity inversely correlated with defective provirus levels (n = 68, r = -0.285, p = 0.0214) but not the intact reservoir (n = 68, r = -0.0321, p-value = 0.800). Pre-ART gp41-specific ADCC did not significantly correlate with either proviral population (n = 68; intact: r = -0.0512, p-value = 0.686; defective: r = -0.109, p-value = 0.389). This suggests specific host immune factors prior to ART initiation can impact proviruses that persist during ART.

Identification of system-level features in HIV migration within a host.

Identify system-level features in HIV migration within a host across body tissues. Evaluate heterogeneity in the presence and magnitude of these features across hosts. Using HIV DNA deep sequencing data generated across multiple tissues from 8 people with HIV, we represent the complex dependencies of HIV migration among tissues as a network and model these networks using the family of exponential random graph models (ERGMs). ERGMs allow for the statistical assessment of whether network features occur more (or less) frequently in viral migration than might be expected by chance. The analysis investigates five potential features of the viral migration network: (1) bi-directional flow between tissues; (2) preferential migration among tissues in the same biological system; (3) heterogeneity in the level of viral migration related to HIV reservoir size; (4) hierarchical structure of migration; and (5) cyclical migration among several tissues. We calculate the Cohran's Q statistic to assess heterogeneity in the magnitude of the presence of these features across hosts. The analysis adjusts for missing data on body tissues. We observe strong evidence for bi-directional flow between tissues; migration among tissues in the same biological system; and hierarchical structure of the viral migration network. This analysis shows no evidence for differential level of viral migration with respect to the HIV reservoir size of a tissue. There is evidence that cyclical migration among three tissues occurs less frequent than expected given the amount of viral migration. The analysis also provides evidence for heterogeneity in the magnitude that these features are present across hosts. Adjusting for missing tissue data identifies system-level features within a host as well as heterogeneity in the presence of these features across hosts that are not detected when the analysis only considers the observed data. Identification of common features in viral migration may increase the efficiency of HIV cure efforts as it enables targeting specific processes.

Adaptation of Droplet Digital PCR-Based HIV Transcription Profiling to Digital PCR and Association of HIV Transcription and Total or Intact HIV DNA.

In most people living with HIV (PLWH) on effective antiretroviral therapy (ART), cell-associated viral transcripts are readily detectable in CD4+ T cells despite the absence of viremia. Quantification of HIV RNA species provides insights into the transcriptional activity of proviruses that persist in cells and tissues throughout the body during ART ('HIV reservoir'). One such technique for HIV RNA quantitation, 'HIV transcription profiling', developed in the Yukl laboratory, measures a series of HIV RNA species using droplet digital PCR. To take advantage of advances in digital (d)PCR, we adapted the 'HIV transcription profiling' technique to Qiagen's dPCR platform (QIAcuity) and compared its performance to droplet digital (dd)PCR (Bio-Rad QX200 system). Using RNA standards, the two technologies were tested in parallel and assessed for multiple parameters including sensitivity, specificity, linearity, and intra- and inter-assay variability. The newly validated dPCR assays were then applied to samples from PLWH to determine HIV transcriptional activity relative to HIV reservoir size. We report that HIV transcriptional profiling was readily adapted to dPCR and assays performed similarly to ddPCR, with no differences in assay characteristics. We applied these assays in a cohort of 23 PLWH and found that HIV reservoir size, based on genetically intact proviral DNA, does not predict HIV transcriptional activity. In contrast, levels of total DNA correlated with levels of most HIV transcripts (initiated, proximally and distally elongated, unspliced, and completed, but not multiply spliced), suggesting that a considerable proportion of HIV transcripts likely originate from defective proviruses. These findings may have implications for measuring and assessing curative strategies and clinical trial outcomes.

Immune activation and exhaustion marker expression on T-cell subsets in ART-treated adolescents and young adults with perinatal HIV-1 infection as correlates of viral persistence.

HIV-1 infection in memory CD4+ T cells forms a latent reservoir that is a barrier to cure. Identification of immune biomarkers that correlate with HIV-1 reservoir size may aid with evaluating efficacy of HIV-1 eradication strategies, towards ART-free remission and cure. In adults living with non-perinatal HIV-1, the immune exhaustion marker PD-1 on central memory CD4+ T cells (Tcm) correlates with measures of HIV-1 reservoir size. Immune correlates of HIV-1 are less defined in adolescents and young adults with perinatal HIV-1. With multi-parameter flow cytometry, we examined immune activation (CD69, CD25, HLA-DR), and exhaustion (PD-1, TIGIT, TIM-3 and LAG-3) markers on CD4+ T cell subsets (naïve (Tn), central memory (Tcm), and the combination (Ttem) of transitional (Ttm) and effector memory (Tem) cells, in 10 adolescents and young adults living with perinatal HIV-1 (median age 15.9 years; median duration of virologic suppression 7.0 years), in whom HIV-1 reservoir size was measured with the Intact Proviral HIV-1 DNA Assay (IPDA) and an enhanced Tat/Rev limiting dilution assay (TILDA). RNA-seq was also performed on the unstimulated CD4+ T cells. The median total HIV-1 DNA concentration in memory CD4+ T cells was 211.90 copies per million CD4+ T cells. In the 7 participants with subtype B HIV-1 infection, the median intact proviral DNA load was 7.96 copies per million CD4+ T cells. Levels of HLA-DR and TIGIT on the Ttem were correlated with total HIV-1 DNA (r=0.76, p=0.015) and (r=0.72, p=0.023), respectively, but not with intact proviral load or induced reservoir size. HIV-1 DNA load was also positively correlated with transcriptional clusters associated with HLA-DR expression by RNA-seq. In contrast, PD-1 expression on Tcm was inversely correlated with total HIV-1 DNA (r=-0.67, p=0.039). Reservoir size by IPDA and TILDA were correlated (r=0.81, p=0.036). Thus, in this cohort of youths with long-standing treated perinatal infection, HLA-DR and TIGIT on Ttem were the key correlates of HIV-1 infected cell frequencies by total HIV-1 DNA, and not PD-1. Total HIV-1 DNA was negatively correlated with PD-1 expressing Tcm. These differences in longstanding perinatal HIV-1 infection compared with adult infection requires further study in larger cohorts.

Targeted plasma proteomics identifies MICA and IL1R1 proteins associated with HIV-1 reservoir size

HIV-1 reservoir shows high variability in size and activity among virally suppressed individuals. Differences in the size of the viral reservoir may relate to differences in plasma protein concentrations. We tested whether plasma protein expression levels are associated with levels of cell-associated (CA) HIV-1 DNA and RNA in 211 virally suppressed people living with HIV (PLHIV). Plasma concentrations of FOLR1, IL1R1, MICA, and FETUB showed a positive association with CA HIV-1 RNA and DNA. Moreover, SNPs in close proximity to IL1R1 and MICA genes were found to influence the levels of CA HIV-1 RNA and DNA. We found a difference in mRNA expression of the MICA gene in homozygotes carrying the rs9348866-A allele compared to the ones carrying the G allele (p<0.005). Overall, our findings pinpoint plasma proteins that could serve as potential targets for therapeutic interventions to lower or even eradicate cells containing CA HIV-1 RNA and DNA in PLHIV.

HLA-B*57 and B*58 Associate with Predictors of Reservoir Size in an Acutely Treated HIV Cohort.

Much has been learnt about the role of human leukocyte antigen (HLA) alleles during natural infection of HIV-1, but far less is known about their role in people living with HIV (PLWH) on suppressive antiretroviral therapy (ART). In this study we used variable selection to identify predictors of HIV reservoir size, as measured by total HIV DNA in 192 participants in an acute HIV infection (AHI) cohort. Baseline clinical data including pre-ART CD4 T cell counts and plasma viral load (VL) were available from all participants along with longitudinal measurements after ART initiation during AHI. Time to VL suppression, time to CD4 reconstitution, and pre-ART viremia were the strongest predictors of undetectable total HIV DNA at 24 weeks after ART initiation. We next performed HLA typing in 526 participants from the same cohort and investigated associations with the three predictors of reservoir size. HLA-B*57 and B*58 both associated significantly with time to VL suppression, which was one of the predictors of the size of the HIV reservoir. These findings are significant in PLWH and have to be considered in the context of therapeutic intervention when conducting analytic treatment interruption studies as participants with these alleles could impact clinical findings given the small sizes of these studies.

OP 7.3 – 00139 Pre-treatment Interruption Plasma Metabolites and Glycans Correlate with Time to HIV Rebound and Reservoir Size in ACTG A5345

OP 7.3 – 00139 Pre-treatment Interruption Plasma Metabolites and Glycans Correlate with Time to HIV Rebound and Reservoir Size in ACTG A5345