HIV DNA Levels Research Articles

HIV DNA Levels in Persons Who Acquired HIV in the Setting of Long-Acting Cabotegravir for HIV Prevention: Analysis of Cases from HPTN 083 and 084.

We evaluated HIV DNA levels in individuals who received long-acting cabotegravir (CAB-LA) or tenofovir disoproxil fumarate/emtricitabine (TDF/FTC) pre-exposure prophylaxis in the HPTN 083 and 084 trials and had HIV DNA testing performed to help determine HIV status. HIV DNA testing was performed using peripheral blood mononuclear cell (PBMC) samples collected after a reactive HIV test was obtained at a study site. DNA was quantified using droplet digital PCR (lower limit of detection [LLOD]: 4.09 copies/million PBMCs). Final HIV status and the timing of the first HIV-positive visit were determined by an independent adjudication committee based on HIV test results from real-time site testing and retrospective testing at a centralized laboratory. HIV DNA testing was performed for 133 participants [21 HIV-positive (7 CAB-LA arm, 14 TDF/FTC arm) and 112 HIV-negative; 1-6 tests/person]. For persons with HIV, the time between the first HIV-positive visit and collection of the first sample for DNA testing was a median of 81 days for those receiving CAB-LA (range 41-246) and 11 days for those receiving TDF/FTC (range 3-127). Four (57.1%) of the seven CAB-LA cases with infection had a low initial DNA result [three detected <LLOD; one near the LLOD (4.2 copies/106 PBMCs); in 2/4 cases, the DNA level was still <10 copies/106 PBMCs ≥40 weeks after the first HIV-positive visit. In contrast, only 3/14 (21.4%) of the TDF/FTC cases had a low or negative initial DNA test result (one not detected; two <10 copies/106 PBMCs). In this study, the time between the first HIV-positive visit and the first DNA test was longer in the CAB-LA cases than the TDF/FTC cases. Despite this difference, low or undetectable DNA levels were more frequently observed in the CAB-LA cases. This suggests that CAB-LA exposure may limit seeding of the HIV reservoir in early infection.

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.

HIV reservoir and premature aging: risk factors for aging-associated illnesses in adolescents and young adults with perinatally acquired HIV.

Despite receiving antiretroviral therapy (ART), an increasing number of adolescents and young adults with perinatally acquired HIV (PHIVAYA) are at risk of developing premature senescence and aging-associated illnesses, including cancer. Given this concern, it is crucial to assess aging biomarkers and their correlation with the HIV reservoir in order to comprehensively characterize and monitor these individuals. Fifty-five PHIVAYA (median age: 23, interquartile range [IQR]: 20-27 years, and 21 [18-23] years on ART at the time of study sampling) were studied along with 23 age-matched healthy controls. The PHIVAYA exhibited significantly higher percentages of activated, senescent, exhausted CD4 and CD8 T cells, shorter telomeres, reduced thymic output, and higher levels of circulating inflammatory markers (PAMPs, DAMPs, and pro-inflammatory cytokines IL-6, IL-8, and TNFα) as well as denervation biomarkers (neural cell adhesion molecule 1 [NCAM1] and C-terminal Agrin fragment [CAF]), compared to controls. HIV-DNA levels positively correlated with activated, senescent, exhausted CD4 and CD8 T cells, circulating biomarkers levels, and inversely with regulatory T and B cells and telomere length. According to their viremia over time, PHIVAYA were subgrouped into 14 Not Suppressed (NS)-PHIVAYA and 41 Suppressed (S)-PHIVAYA, of whom 6 who initiated ART within one year of age and maintained sustained viral suppression overtime were defined as Early Suppressed (ES)-PHIVAYA and the other 35 as Late Suppressed (LS)-PHIVAYA. ES-PHIVAYA exhibited significantly lower HIV-DNA reservoir, decreased percentages of senescent and exhausted CD4 and CD8 T cells, reduced levels of circulating inflammatory and denervation biomarkers, but longer telomere compared to LS- and NS-PHIVAYA. They differed significantly from healthy controls only in a few markers, including higher percentages of regulatory T and B cells, and higher levels of DAMPs. Overall, these results underscore the importance of initiating ART early and maintaining viral suppression to limit the establishment of the viral reservoir and to counteract immune and cellular premature aging. These findings also suggest new approaches for minimally invasive monitoring of individuals at high risk of developing premature aging and age-related illnesses.

HIV-1-DNA/RNA and immunometabolism in monocytes: contribution to the chronic immune activation and inflammation in people with HIV-1

Among people living with HIV-1 (PHIV), immunological non-responders (INR) experience incomplete immune recovery despite suppressive antiretroviral treatment (ART), facing more severe non-AIDS events than immunological responders (IR) due to higher chronic immune activation and inflammation (cIA/I). We analyzed the HIV-1 reservoir and immunometabolism in monocytes as a source of cIA/I. Cross-sectional study in which 110 participants were enrolled: 25 treatment-naïve; 35 INR; 40 IR; and 10 healthy controls. Cell-associated HIV-1-DNA (HIV-DNA) and -RNA (HIV-RNA) were measured in FACS-isolated monocytes using digital droplet PCR. Intact, 5' deleted, and 3' deleted proviruses were quantified by the intact proviral DNA assay. Systemic inflammation, monocyte immunophenotype, and immunometabolism were characterized by immunoassays, flow cytometry, and real-time cellular bioenergetics measurements, respectively. Monocytes from INR harbor higher HIV-RNA and HIV-DNA levels than IR. HIV-RNA was found in 14/21 treatment-naïve [2512 copies/106 TBP (331-4666)], 17/33 INR [240 (148-589)], and 15/28 IR [144 (15-309)], correlating directly with sCD163, IP-10, GLUT1high cells and glucose uptake, and inversely with the CD4+/CD8+ ratio. HIV-DNA was identified in all participants with detectable HIV-RNA, with intact provirus in 9/12 treatment-naïve [13 copies/106 monocytes (7-44)], 8/14 INR [46 (18-67)], and 9/13 IR [9 (7-24)]. INR presented glucose metabolism alterations and mitochondrial impairment; decreased coupling efficiency and BHI, and increased mitochondrial dysfunction inversely correlating with the CD4+/CD8+ ratio. HIV-RNA, more than HIV-DNA, in monocytes and their altered metabolism are factors associated with the higher cIA/I that characterize INR. This work was supported by the European Regional Development Fund, ISCIII, grant PI20/01646. Other funding sources:Instituto de Salud Carlos III through the Subprogram Miguel Servet (CP19/00159) to AGV, PFIS contracts (FI19/00304) to EMM, (FI21/00165) to ASA, and (FI19/00083) to CGC, and a mobility grant (MV21/00103) to EMM, from the Ministerio de Ciencia e Innovación, Spain. AJM was granted by a CSL Centenary Award.

Analysis of the effect of HDAC inhibitors on the formation of the HIV reservoir.

The HIV reservoir is more dynamic than previously thought with around 70% of the latent reservoir originating from viruses circulating within 1 year of the initiation of antiretroviral therapy (ART). In an ex vivo model system of HIV latency, it was reported that early exposure to class I histone deacetylase (HDAC) inhibitors might prevent these more recently infected cells from entering a state of stable viral latency. This finding raises the possibility that co-administration of HDAC inhibitors at the time of ART initiation may prevent the establishment of much of the HIV reservoir. Here, we tested the effects of the HDAC inhibitors suberoylanilide hydroxamic acid (SAHA) and panobinostat co-administered at the time of ART initiation on the formation of the viral reservoir in HIV-infected humanized mice. As previously shown, SAHA and panobinostat were well tolerated in humanized mice. Unexpectedly, co-administration of SAHA resulted in an increase in the frequency of CD4+ cells carrying HIV DNA but did not alter the frequency of cell-associated HIV RNA in HIV-infected, ART-treated humanized mice. Co-administration of panobinostat did not alter levels of cell-associated HIV DNA or RNA. Our in vivo findings indicate that co-administration of HDAC inhibitors initiated at the same time of ART treatment does not prevent recently infected cells from entering latency.IMPORTANCECurrent antiretroviral therapy (ART) does not eradicate cells harboring replication-competent HIV reservoir. Withdrawal of ART inevitably results in a rapid viremia rebound. The HIV reservoir is more dynamic than previously thought. Early exposure to class I histone deacetylase (HDAC) inhibitors inhibit these more recently infected cells from entering a state of stable viral latency in an ex vivo model of latency, raising the possibility that co-administration of HDAC inhibitors at the time of ART initiation may reduce much of the HIV reservoir. Here, we tested the effects of the HDAC inhibitors suberoylanilide hydroxamic acid or panobinostat during ART initiation on the formation of the viral reservoir in HIV-infected humanized mice. Our in vivo study indicates that in contrast to in vitro observations, the co-administration of HDAC inhibitors at the same time of ART initiation does not prevent recently infected cells from entering latency.

Machine learning approaches identify immunologic signatures of total and intact HIV DNA during long-term antiretroviral therapy.

Understanding the interplay between the HIV reservoir and the host immune system may yield insights into HIV persistence during antiretroviral therapy (ART) and inform strategies for a cure. Here, we applied machine learning (ML) approaches to cross-sectional high-parameter HIV reservoir and immunology data in order to characterize host-reservoir associations and generate new hypotheses about HIV reservoir biology. High-dimensional immunophenotyping, quantification of HIV-specific T cell responses, and measurement of genetically intact and total HIV proviral DNA frequencies were performed on peripheral blood samples from 115 people with HIV (PWH) on long-term ART. Analysis demonstrated that both intact and total proviral DNA frequencies were positively correlated with T cell activation and exhaustion. Years of ART and select bifunctional HIV-specific CD4 T cell responses were negatively correlated with the percentage of intact proviruses. A leave-one-covariate-out inference approach identified specific HIV reservoir and clinical-demographic parameters, such as age and biological sex, that were particularly important in predicting immunophenotypes. Overall, immune parameters were more strongly associated with total HIV proviral frequencies than intact proviral frequencies. Uniquely, however, expression of the IL-7 receptor alpha chain (CD127) on CD4 T cells was more strongly correlated with the intact reservoir. Unsupervised dimension reduction analysis identified two main clusters of PWH with distinct immune and reservoir characteristics. Using reservoir correlates identified in these initial analyses, decision tree methods were employed to visualize relationships among multiple immune and clinical-demographic parameters and the HIV reservoir. Finally, using random splits of our data as training-test sets, ML algorithms predicted with approximately 70% accuracy whether a given participant had qualitatively high or low levels of total or intact HIV DNA . The techniques described here may be useful for assessing global patterns within the increasingly high-dimensional data used in HIV reservoir and other studies of complex biology.

Machine learning approaches identify immunologic signatures of total and intact HIV DNA during long-term antiretroviral therapy

Understanding the interplay between the HIV reservoir and the host immune system may yield insights into HIV persistence during antiretroviral therapy (ART) and inform strategies for a cure. Here, we applied machine learning (ML) approaches to cross-sectional high-parameter HIV reservoir and immunology data in order to characterize host–reservoir associations and generate new hypotheses about HIV reservoir biology. High-dimensional immunophenotyping, quantification of HIV-specific T cell responses, and measurement of genetically intact and total HIV proviral DNA frequencies were performed on peripheral blood samples from 115 people with HIV (PWH) on long-term ART. Analysis demonstrated that both intact and total proviral DNA frequencies were positively correlated with T cell activation and exhaustion. Years of ART and select bifunctional HIV-specific CD4 T cell responses were negatively correlated with the percentage of intact proviruses. A leave-one-covariate-out inference approach identified specific HIV reservoir and clinical–demographic parameters, such as age and biological sex, that were particularly important in predicting immunophenotypes. Overall, immune parameters were more strongly associated with total HIV proviral frequencies than intact proviral frequencies. Uniquely, however, expression of the IL-7 receptor alpha chain (CD127) on CD4 T cells was more strongly correlated with the intact reservoir. Unsupervised dimension reduction analysis identified two main clusters of PWH with distinct immune and reservoir characteristics. Using reservoir correlates identified in these initial analyses, decision tree methods were employed to visualize relationships among multiple immune and clinical–demographic parameters and the HIV reservoir. Finally, using random splits of our data as training-test sets, ML algorithms predicted with approximately 70% accuracy whether a given participant had qualitatively high or low levels of total or intact HIV DNA . The techniques described here may be useful for assessing global patterns within the increasingly high-dimensional data used in HIV reservoir and other studies of complex biology.

Implications of accumulation of clonally expanded and senescent CD4+GNLY+ T cells in immunological non-responders of HIV-1 infection

ABSTRACT Increased CD4+GNLY+ T cells have been confirmed to be inversely associated with CD4+ T cell count in immunological non-responders (INRs), however, the underlying mechanisms are unknown. This study aimed to elucidate the characteristics of CD4+GNLY+ T cells and their relationship with immune restoration. Single-cell RNA sequencing, single-cell TCR sequencing, and flow cytometry were used to analyze the frequency, phenotypes, and function of CD4+GNLY+ T cells. Moreover, Enzyme linked immunosorbent assay was performed to detect plasma cytokines production in patients. CD4+GNLY+ T cells were found to be highly clonally expanded, characterized by higher levels of cytotoxicity, senescence, P24, and HIV-1 DNA than CD4+GNLY− T cells. Additionally, the frequency of CD4+GNLY+ T cells increased after ART, and further increased in INRs, and were positively associated with the antiretroviral therapy duration in INR. Furthermore, increased IL-15 levels in INRs positively correlated with the frequency and senescence of CD4+GNLY+ T cells, suggesting that CD4+GNLY+ T cells may provide new insights for understanding the poor immune reconstitution of INRs. In conclusion, increased, highly clonally expanded, and senescent CD4+GNLY+ T cells may contribute to poor immune reconstitution in HIV-1 infection.

Machine learning approaches identify immunologic signatures of total and intact HIV DNA during long-term antiretroviral therapy.

Understanding the interplay between the HIV reservoir and the host immune system may yield insights into HIV persistence during antiretroviral therapy (ART) and inform strategies for a cure. Here, we applied machine learning approaches to cross-sectional high-parameter HIV reservoir and immunology data in order to characterize host-reservoir associations and generate new hypotheses about HIV reservoir biology. High-dimensional immunophenotyping, quantification of HIV-specific T cell responses, and measurement of genetically intact and total HIV proviral DNA frequencies were performed on peripheral blood samples from 115 people with HIV (PWH) on long-term ART. Analysis demonstrated that both intact and total proviral DNA frequencies were positively correlated with T cell activation and exhaustion. Years of ART and select bifunctional HIV-specific CD4 T cell responses were negatively correlated with the percentage of intact proviruses. A Leave-One-Covariate-Out (LOCO) inference approach identified specific HIV reservoir and clinical-demographic parameters, such as age and biological sex, that were particularly important in predicting immunophenotypes. Overall, immune parameters were more strongly associated with total HIV proviral frequencies than intact proviral frequencies. Uniquely, however, expression of the IL-7 receptor alpha chain (CD127) on CD4 T cells was more strongly correlated with the intact reservoir. Unsupervised dimension reduction analysis identified two main clusters of PWH with distinct immune and reservoir characteristics. Using reservoir correlates identified in these initial analyses, decision tree methods were employed to visualize relationships among multiple immune and clinical-demographic parameters and the HIV reservoir. Finally, using random splits of our data as training-test sets, machine learning algorithms predicted with approximately 70% accuracy whether a given participant had qualitatively high or low levels of total or intact HIV DNA. The techniques described here may be useful for assessing global patterns within the increasingly high-dimensional data used in HIV reservoir and other studies of complex biology.

Molecular Transmission Network and Drug Resistance in Treatment-Naive HIV-1-Infected Patients in the Liangshan District, China.

This study aimed to investigate the molecular transmission network and drug resistance in treatment-naive HIV-1-infected patients in the Liangshan District, China. The research subjects for this study were HIV-1-infected patients who did not receive any antiretroviral therapy (ART) in the Liangshan District between January 2022 and July 2023. Peripheral venous whole-blood samples were collected from the research subjects. Two milliliters of blood was used for CD4+ T lymphocyte counting detection. Ten milliliters of blood was centrifuged to separate the plasma and blood cells for quantitative detection of HIV-1 RNA and DNA and drug resistance testing of HIV-1. A total of 156 participants were included in this study (88 males and 68 females). The median age of the participants was 37 years. The findings revealed a positive correlation between the HIV-1 DNA and the HIV-1 RNA levels (r = 0.478, p < 0.001). However, a negative correlation was observed between the HIV-1 DNA levels and CD4+ T lymphocyte counts (r = -0.186, p = 0.020). Of the 156 participants, 145 were successfully tested for drug resistance of HIV-1 RNA and HIV-1 DNA simultaneously. Four cases failed the HIV-1 RNA drug resistance testing, and another two failed the HIV-1 DNA drug resistance testing. The most common HIV-1 subtype was the CRF07_BC recombinant. In this study, the overall incidence of transmitted drug resistance (TDR) was 8.33%. The resistance rates of non-nucleoside reverse transcriptase inhibitor (NNRTI) and protease inhibitor (PI) were 7.69% and 0.64%, respectively. In addition, 32 participants were found to have drug-resistant mutations. The primary drug-resistant mutations were K103N, V179D, E157Q, and A128T, mainly against efavirenz (EFV) and nevirapine (NVP) resistance. The drug resistance of HIV-1-infected ART-naive patients in the Liangshan District cannot be ignored. HIV-1 drug resistance testing is recommended before initiating ART.

No change in HIV cellular burden and lower ADCC despite increased immune activation after combined immunotherapy with peg-IFN-α2b and two broadly neutralizing antibodies in suppressed HIV+ subjects

Abstract Background: Pegylated interferon α2b (peg-IFN-α2b) and the broadly neutralizing antibodies (bNabs) 3BNC117 and 10-1074 were individually shown to increase HIV suppression during antiretroviral therapy (ART) interruption (ATI). We evaluated the anti-HIV and immune effect of combined immunotherapy with peg-IFN-α2b+bNAbs in persons with HIV infection (PWH). Methods: Peripheral blood mononuclear cells and plasma from 14 ART-suppressed PWH were assessed at baseline (ART), ART+4 weeks of peg-IFN-α2b, and ATI+26 weeks of peg-IFN-α2b+bNAbs immunotherapy for: 1) intact HIV DNA, 2) NK constitutive, and in vitro gp120 or IFN-α-induced antibody-dependent cell-mediated cytotoxicity (ADCC) and direct cytotoxicity, and 3) immune activation. Results: Immunotherapy did not alter cell-associated intact HIV DNA levels nor IFN-α-induced NK direct cytotoxicity. Yet, it decreased gp120-induced ADDC, and the percentage (%) of CD56lo/+CD16+ on CD56+ while increasing the % of CD56hi and CD56lo/+CD16- on CD56+ suggesting that decreased expression of CD16 (Fc receptor) on NK could result to lower ADCC. Immunotherapy also decreased CD57 expression on CD56lo/+CD16+, while increased Tetherin, CD38, HLA-DR, NKp46, and CD169 on immune cells, and pro-inflammatory bisected GlcNAc glycans on bulk IgG. Conclusion: Decreased ADCC and circulating CD16+ NK cell subsets despite increased immune activation may explain the lack of change in HIV cell burden after Peg-IFN-α2b+bNAbs administration in PWH.

Increase in HIV reservoir and T cell immune response after CoronaVac vaccination in people living with HIV

IntroductionCoronaVac, an inactivated vaccine developed by Sinovac Life Sciences, has been widely used for protection against Coronavirus Disease 2019 (COVID-19). This study investigates its effect on the HIV reservoir and T cell repertoires in people living with HIV (PLWHs). MethodsBlood samples were collected from fifteen PLWHs who were administered at least two doses of CoronaVac between April 2021 and February 2022. The levels of cell-associated HIV RNA (CA HIV RNA) and HIV DNA, as well as the T cell receptor (TCR) repertoire profiles, TCR clustering and TCRβ annotation, were studied. ResultsA significant increase was observed in CA HIV RNA at 2 weeks (431.5 ± 164.2 copies/106 cells, P = 0.039) and 12 weeks (330.2 ± 105.9 copies/106 cells, P = 0.019) after the second dose, when compared to the baseline (0 weeks) (73.6 ± 23.7 copies/106 cells). Various diversity indices of the TCRβ repertoire, including Shannon index, Pielou's evenness index, and Hvj Index, revealed a slight increase (P < 0.05) following CoronaVac vaccination. The proportion of overlapping TCRβ clonotypes increased from baseline (31.9 %) to 2 weeks (32.5 %) and 12 weeks (40.4 %) after the second dose. We also found that the breadth and depth of COVID-19-specific T cells increased from baseline (0.003 and 0.0035) to 12 weeks (0.0066 and 0.0058) post the second dose. ConclusionsOur study demonstrated an initial increase in HIV reservoir and TCR repertoire diversity, as well as an expansion in the depth and breadth of COVID-19-specific T-cell clones among CoronaVac-vaccinated PLWHs. These findings provide important insights into the effects of COVID-19 vaccination in PLWHs.

Differential susceptibility of cells infected with defective and intact HIV proviruses to killing by obatoclax and other small molecules.

Some drugs that augment cell-intrinsic defenses or modulate cell death/survival pathways have been reported to selectively kill cells infected with HIV or Simian Immunodeficiency Virus (SIV), but comparative studies are lacking. We hypothesized that these drugs may differ in their ability to kill cells infected with intact and defective proviruses. To investigate this hypothesis, drugs were tested ex vivo on peripheral blood mononuclear cells (PBMC) from nine antiretroviral therapy (ART)-suppressed individuals. We tested drugs currently in clinical use or human trials, including auranofin (p53 modulator), interferon alpha2A, interferon gamma, acitretin (RIG-I inducer), GS-9620/vesatolimod (TLR7 agonist), nivolumab (PD-1 blocker), obatoclax (Bcl-2 inhibitor), birinapant [inhibitor of apoptosis proteins (IAP) inhibitor], bortezomib (proteasome inhibitor), and INK128/sapanisertib [mammalian target of rapamycin mTOR] [c]1/2 inhibitor). After 6 days of treatment, we measured cell counts/viabilities and quantified levels of total, intact, and defective HIV DNA by droplet digital PCR (Intact Proviral DNA Assay). Obatoclax reduced intact HIV DNA [median = 27-30% of dimethyl sulfoxide control (DMSO)] but not defective or total HIV DNA. Other drugs showed no statistically significant effects. Obatoclax and other Bcl-2 inhibitors deserve further study in combination therapies aimed at reducing the intact HIV reservoir in order to achieve a functional cure and/or reduce HIV-associated immune activation.

Adaptive NK Cells Rapidly Expand during Acute HIV Infection and Persist Despite Early Initiation of Antiretroviral Therapy.

HIV is associated with NK cell dysfunction and expansion of adaptive-like NK cells that persist despite antiretroviral therapy (ART). We investigated the timing of NK cell perturbations during acute HIV infection and the impact of early ART initiation. PBMCs and plasma were obtained from people with HIV (PWH; all men who have sex with men; median age, 26.0 y) diagnosed during Fiebig stages I, II, III, or IV/V. Participants initiated ART a median of 3 d after diagnosis, and immunophenotyping was performed at diagnosis and longitudinally after ART. Anti-CMV Abs were assessed by ELISA. Samples from matched HIV-uninfected males were also analyzed. Proportions of adaptive NK cells (A-NKs; defined as Fcε-Receptor-1γ-) were expanded at HIV diagnosis at all Fiebig stages (pooled median 66% versus 25% for controls; p < 0.001) and were not altered by early ART initiation. Abs to CMV immediate early protein were elevated in PWH diagnosed in Fiebig stages III and IV/V (p < 0.03 for both). Proportions of A-NKs defined as either Fcε-Receptor-1γ- or NKG2C+/CD57+ were significantly associated with HIV DNA levels at diagnosis (p = 0.046 and 0.029, respectively) and trended toward an association after 48 wk of ART. Proportions of activated HLA-DR+/CD38+ NK cells remained elevated in PWH despite early ART initiation. NK cell activation and A-NK expansion occur very early after HIV transmission, before T cell activation, and are not altered by ART initiation during acute infection. A-NKs may contribute to HIV control and thus be useful for HIV cure.

Impact of influenza and pneumococcal vaccines on HIV persistence and immune dynamics during suppressive antiretroviral therapy.

We sought to determine if standard influenza and pneumococcal vaccines can be used to stimulate HIV reservoirs during antiretroviral therapy (ART). A prospective, randomized, double-blinded, placebo-controlled, crossover trial of two clinically recommended vaccines (influenza and pneumococcal). Persons with HIV on ART ( N = 54) were enrolled in the clinical trial. Blood was collected at baseline and days 2,4,7,14, and 30 postimmunizations. Levels of cellular HIV RNA and HIV DNA were measured by ddPCR. Expression of immunological markers on T cell subsets was measured by flow cytometry. Changes in unspliced cellular HIV RNA from baseline to day 7 postinjection between each vaccine and placebo was the primary outcome. Forty-seven participants completed at least one cycle and there were no serious adverse events related to the intervention. We observed no significant differences in the change in cellular HIV RNA after either vaccine compared with placebo at any timepoint. In secondary analyses, we observed a transient increase in total HIV DNA levels after influenza vaccine, as well as increased T cell activation and exhaustion on CD4 + T cells after pneumococcal vaccine. Clinically recommended vaccines were well tolerated but did not appear to stimulate the immune system strongly enough to elicit significantly noticeable HIV RNA transcription during ART.Clinicaltrials.gov identifier: NCT02707692.

Impact of antiretroviral therapy during acute or early HIV infection on virologic and immunologic outcomes: results from a multinational clinical trial.

To assess how antiretroviral therapy (ART) initiation during acute or early HIV infection (AEHI) affects the viral reservoir and host immune responses. Single-arm trial of ART initiation during AEHI at 30 sites in the Americas, Africa, and Asia. HIV DNA was measured at week 48 of ART in 5 million CD4 + T cells by sensitive qPCR assays targeting HIV gag and pol . Peripheral blood mononuclear cells were stimulated with potential HIV T cell epitope peptide pools consisting of env , gag , nef, and pol peptides and stained for expression of CD3, CD4, CD8, and intracellular cytokines/chemokines. From 2017 to 2019, 188 participants initiated ART during Fiebig stages I ( n = 6), II ( n = 43), III ( n = 56), IV ( n = 23), and V ( n = 60). Median age was 27 years (interquartile range 23-38), 27 (14%) participants were female, and 180 (97%) cisgender. Among 154 virally suppressed participants at week 48, 100% had detectable HIV gag or pol DNA. Participants treated during Fiebig I had the lowest HIV DNA levels ( P < 0.001). Week 48 HIV DNA mostly did not correlate with concurrent CD4 + or CD8 + T cell HIV-specific immune responses (rho range -0.11 to +0.19, all P > 0.025). At week 48, the magnitude, but not polyfunctionality, of HIV-specific T cell responses was moderately reduced among participants who initiated ART earliest. Earlier ART initiation during AEHI reduced but did not eliminate the persistence of HIV-infected cells in blood. These findings explain the rapid viral rebound observed after ART cessation in early-treated individuals with undetectable HIV DNA by less sensitive methods.

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.

Characterization of HIV-1 Reservoirs in Children and Adolescents: A Systematic Review and Meta-Analysis Toward Pediatric HIV Cure

ObjectiveTo conduct a comprehensive, systematic review of the profile of HIV-1 reservoirs in children and adolescents with perinatally-acquired HIV infection. Study designRandomized and non-randomized trials, cohort studies, and cross-sectional studies on HIV reservoirs in pediatric populations, published between 2002 and 2022, were included. Archived-drug resistance mutations (ADRM) and the size of reservoirs were evaluated. Subgroup analyses were performed to characterize further the data, and the meta-analysis was done through random effect models. ResultsOverall, 49 studies from 17 countries worldwide were included, encompassing 2356 perinatally infected participants (48.83% females). There are limited data on the quantitative characterization of viral reservoirs in sub-Saharan Africa (SSA), with sensitive methodologies such as ddPCR rarely employed. The overall prevalence of ADRMs was 37.80% [95%CI: 13.89–65.17], with 48.79% [95%CI: 0–100] in Africa, 42.08% [6.68-82.71] in America, 23.88% [95%CI: 14.34–34.90] in Asia, and 20.00% [95%CI: 10.72–31.17] in Europe, without any difference between infants and adolescents (p=0.656). Starting antiretroviral therapy (ART) before 2 months of age limited the levels of HIV-1 DNA (p=0.054). Participants with long suppressed viremia (>5 years) had lower levels of HIV-1 DNA (p=0.027). Ppre- and post-ART CD4 ≤29% and pre-ART viremia ≥5Log were all found associated with higher levels of HIV-1 DNA (p=0.038, p=0.047 and p=0.041, respectively). ConclusionThe pooled prevalence of ADRMs is high in perinatally infected pediatric population, with larger proviral reservoir size driven by delayed ART initiation, a shorter period of viral suppression, and immuno-virological failures. Thus, strategies for pediatric HIV functional cure should target children and adolescents with very early ART initiation, immunocompetence, and long-term viral suppression.

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.

Validation of digital droplet PCR assays for cell-associated HIV-1 DNA, HIV-1 2-LTR circle, and HIV-1 unspliced RNA for clinical studies in HIV-1 cure research

BackgroundCell-associated HIV-1 DNA, HIV-1 2-LTR circle, and HIV-1 unspliced RNA (usRNA) are important virological parameters for monitoring HIV-1 persistence and activation of latent HIV-1. Assays fully validated by CLIA and/or GCLP standards are needed for future clinical trials that seek to evaluate treatments directed towards HIV-1 cure. ObjectivesTo determine performance characteristics of sensitive, moderate-throughput, digital droplet PCR (ddPCR) assays for cell-associated HIV-1 DNA, HIV-1 2-LTR circle, and HIV-1 usRNA that can detect a broad range of HIV-1 M-group subtypes. Study DesignTo evaluate linearity, limit of detection, precision, and accuracy of each assay, contrived specimens were analyzed in a background of uninfected PBMC. Detection breadth was evaluated by in silico analysis of primer and probes sets and analysis of material harvested from PBMC infected in vitro with various HIV-1 subtypes. A cohort of clinical specimens from viremic and virologically suppressed individuals was analyzed to demonstrate applicability to clinical research. ResultsThe empirically determined limit of detection of these assays was 29, 7, and 60 copies per million PBMC for HIV-1 DNA, HIV-1 2-LTR circle, and HIV-1 usRNA, respectively. The assays detect a broad range of HIV-1 M-group subtypes. Finally, analysis of clinical specimens demonstrate that these assays can detect low levels of cell-associated HIV-1 DNA, HIV-1 usRNA, and HIV-1 2-LTR circle and correlate with clinical histories and viral loads of untreated and antiretroviral treated individuals. ConclusionsWe report the clinical validation of three HIV reservoir assays with broad HIV-1 coverage for future cure studies.