Antiretroviral therapy alone versus antiretroviral therapy with a kick and kill approach, on measures of the HIV reservoir in participants with recent HIV infection (the RIVER trial): a phase 2, randomised trial

A Randomized Comparison of Antiretroviral Therapy Alone Versus Antiretroviral Therapy with a 'Kick-and-Kill' Approach, on Measures of the HIV Reservoir Amongst Participants with Recent HIV Infection: The RIVER Trial

Dynamics of HIV DNA and Residual Viremia in Patients Treated With a Raltegravir-Containing Regimen

The latent HIV reservoir is a major impediment to curing HIV infection. The contribution of CD4(+) T cell activation status to the establishment and maintenance of the latent reservoir was investigated by enumerating viral DNA components in a cohort of 12 individuals commencing antiretroviral therapy (ART) containing raltegravir, an integrase inhibitor. Prior to ART, the levels of total HIV DNA were similar across HLA-DR(+) and HLA-DR(-) (HLA-DR(±)) CD38(±) memory CD4(+) T cell phenotypes; episomal two-long terminal repeat (2-LTR) HIV DNA levels were higher in resting (HLA-DR(-) CD38(-)) cells, and this phenotype exhibited a significantly higher ratio of 2-LTR to integrated HIV DNA (P = 0.002). After 1 year of ART, there were no significant differences across each of the memory phenotypes of any HIV DNA component. The decay dynamics of integrated HIV DNA were slow within each subset, and integrated HIV DNA in the resting HLA-DR(-) CD38(-) subset per mm(3) of peripheral blood exhibited no significant decay (half-life of 25 years). Episomal 2-LTR HIV DNA decayed relative to integrated HIV DNA in resting cells with a half-life of 134 days. Surprisingly, from week 12 on, the decay rates of both total and episomal HIV DNA were lower in activated CD38(+) cells. By weeks 24 and 52, HIV RNA levels in plasma were most significantly correlated with the numbers of resting cells containing integrated HIV DNA. On the other hand, total HIV DNA levels in all subsets were significantly correlated with the numbers of HLA-DR(+) CD38(-) cells containing integrated HIV DNA. These results provide insights into the interrelatedness of cell activation and reservoir maintenance, with implications for the design of therapeutic strategies targeting HIV persistence. It is generally believed that HIV is not cleared by extensive antiretroviral therapy (ART) due to the difficulty in eradicating the latent reservoir in resting CD4(+) T cells. New therapies that attempt to activate this reservoir so that immune or viral cytopathic mechanisms can remove those infected cells are currently being investigated. However, results obtained in this research indicate that activation, at least on some level, already occurs within this reservoir. Furthermore, we are the first to describe the dynamics of different HIV DNA species in resting and activated memory CD4+ T cell subsets that point to the role different levels of activation play in maintaining the HIV reservoir.

Birth diagnosis of HIV-1 infection offers an ideal opportunity for early antiretroviral therapy (ART) to limit HIV-1 reservoir size and limit disease progression. Although data on cellular HIV-1 DNA decay exist for children commencing treatment from 2 to 3 months of age, data are lacking for starting shortly after birth. We studied infants who initiated ART within 8 days after birth to assess HIV-1 DNA levels longitudinally. Children were recruited from public health clinics in Cape Town where birth diagnosis of HIV-1 coupled with early ART initiation occurred. Total cellular HIV-1 DNA levels were determined using a sensitive quantitative PCR targeting a conserved region in integrase. Of 11 infants diagnosed and beginning ART within 8 days of birth with detectable pre-ART HIV-1 DNA, three subsequently had undetectable HIV-1 DNA after 6 days, 3 months and 4 months on treatment, respectively. In seven who had virologic suppression (defined as a continuous downward trend in plasma HIV-1 RNA, and <100 copies/ml after 6 months) total HIV-1 DNA continued to decay over 12 months [mean half-life of 64.8 days (95% confidence interval: 47.9-105.7)]. In infants initiated on ART within 8 days of life the combination of maternal ART, and early ART for prophylaxis and treatment contribute to rapid decline of HIV-1 infected cells to low or undetectable levels. However, rapid decline of HIV-1 RNA and DNA may complicate definitive diagnosis when confirmatory testing is delayed.

BackgroundDespite the effective antiretroviral treatment (ART) of HIV-infected individuals, HIV persists in a small pool. Central memory CD4+ T cells (Tcm) make a major contribution to HIV persistence. We found that unlike HLA-DR, CD38 is highly expressed on the Tcm of HIV-infected subjects receiving ART for > 5 years. It has been reported that the half-life of total and episomal HIV DNA in the CD4+CD38+ T cell subset, exhibits lower decay rates at 12 weeks of ART. Whether CD38 contributes to HIV latency in HIV-infected individuals receiving long-term ART is yet to be addressed.MethodsPeripheral blood mononuclear cells (PBMCs) were isolated from the whole blood of HIV-infected subjects receiving suppressive ART. The immunophenotyping, proliferation and apoptosis of CD4+ T cell subpopulations were detected by flow cytometry, and the level of CD38 mRNA and total HIV DNA were measured using real-time PCR and digital droplet PCR, respectively. A negative binomial regression model was used to determine the correlation between CD4+CD38+ Tcm and total HIV DNA in CD4+ T cells.ResultsCD38 was highly expressed on CD4+ Tcm cells from HIV infected individuals on long-term ART. Comparing with HLA-DR−Tcm and CD4+HLA-DR+ T cells, CD4+CD38+ Tcm cells displayed lower levels of activation (CD25 and CD69) and higher levels of CD127 expression. The proportion of CD38+ Tcm, but not CD38− Tcm cells can predict the total HIV DNA in the CD4+ T cells and the CD38+ Tcm subset harbored higher total HIV DNA copy numbers than the CD38− Tcm subset. After transfected with CD38 si-RNA in CD4+ T cells, the proliferation of CD4+ T cells was inhibited.ConclusionThe current date indicates that CD4+CD38+ Tcm cells contribute to HIV persistence in HIV-infected individuals on long-term ART. Our study provides a potential target to resolve HIV persistence.

Understanding HIV persistence in treated patients is an important milestone toward drug-free control. We aimed at analyzing total HIV DNA dynamics and influencing factors in Japanese patients who received more than a decade of suppressive antiretroviral treatment (ART). A retrospective study including clinical records and 840 peripheral blood mononuclear cells samples (mean 14 samples/patient) for 59 patients (92% male) was performed. Subjects were divided into 2 groups: with and without hematological comorbidity (mainly hemophilia) plus hepatitis C virus coinfection. Total HIV DNA was measured in peripheral blood mononuclear cells by quantitative polymerase chain reaction. The dynamics, regression over time, and influence of antiretrovirals by group were estimated using a novel regression model (R software v 3.2.3). Total HIV DNA decreased on ART initiation, and subsequently, its dynamics varied between groups with previously undescribed fluctuations. If calculated by on-treatment, the mean total HIV DNA levels were similar. The comorbidity group had unstable levels showing different regression over time (P = 0.088/0.094 in year 1/after year 8 of ART) and significantly different treatment responses as shown by antiretroviral group switching estimates. Furthermore, curing hepatitis C virus in hemophiliacs did not significantly alter total HIV DNA levels or regression. Our data identified some effects of the long-term treatment on total HIV DNA levels and highlighted the partial influence of comorbidities and coinfections. Total HIV DNA monitoring contributed to therapy response estimates and HIV reservoir quantification. The results suggest that HIV DNA monitoring during ART might be useful as a persistence marker in both HIV-monoinfected patients and those with comorbidities and coinfections.

…. is sure design'd, by fraud or force: trust not their presents, nor admit the horse. Virgil, Aeneid Human immunodeficiency virus type 1 (HIV-1), the lymphotropic virus that causes AIDS, has infected more than 60 million people worldwide since its clinical appearance in 1981. Despite intensive prevention efforts, the HIV/AIDS epidemic continues to spread, particularly in developing countries in sub-Saharan Africa, southeast Asia and the Caribbean, as well as the developed world [1]. Although HIV can be transmitted very efficiently parenterally, the advent of routine blood screening prior to transfusion and harm reduction programs for injection drug users, have made this mode of transmission much less common than mucosal transmission. Most new HIV infections are attributable to mucosal transmission: through genital and rectal mucosae in the case of sexual transmission and through oral or gastrointestinal mucosae in the case of mother-to-child transmission [2]. Much has been learned about HIV pathogenesis and infection mechanisms at the molecular level, but the scientific community has yet to develop an effective vaccine or microbicide for HIV prevention. Many unanswered questions remain concerning HIV-1 sexual transmission. In 1983, barely 2 years into the AIDS epidemic, we hypothesized that the agent that was subsequently identified as HIV-1 may be sexually transmitted by infected ‘Trojan Horse’ leukocytes in semen [3]. This hypothesis was based on our knowledge at the time that human semen contains substantial numbers of T lymphocytes and macrophages, which could host a T-cell tropic virus, and the following assumptions: intracellular virus would be better protected than free virus from adverse effects of antiviral factors in the genital environment such as antiviral antibodies likely to be present in genital secretions of the virus-infected transmitter, as well as antimicrobial peptides that play an important role in genital innate immune defense; and virus-infected allogeneic cells could also escape early detection by major histocompatibility complex (MHC)-restricted cytotoxic T cells in a new host. Over the intervening 25+ years, others have also championed this cause [4,5], and convincing evidence has emerged from clinical research as well as in-vitro and animal studies that infected leukocytes indeed play a role in HIV transmission. Yet, most recent research on sexual HIV transmission has focused on cell-free HIV in genital secretions because of the wide availability of HIV RNA quantification assays. Furthermore, the majority of HIV vaccines and microbicides have been designed to block transmission of cell-free virus and have been tested in animal and in-vitro models that use cell-free virus as the only infectious inoculum. As the molecular events underlying cell-associated HIV transmission differ from those underlying cell-free virus transmission, many of the current vaccine and microbicide candidates might not be expected to protect against cell-associated HIV transmission. The failure of several recent vaccine and microbicide clinical trials may be due in part to this oversight. It should be possible to design strategies that block cell-associated HIV transmission as well as cell-free HIV transmission. In this article, we present an overview of research that has been conducted on cell-associated HIV mucosal transmission and recommendations for future research. We focus on sexual HIV transmission, but this review also has relevance for mother-to-child HIV transmission, which may occur through mucosal transmission of cell-associated HIV from maternal genital or mammary gland secretions [6–8]. We review published reports that describe and enumerate HIV-infected cells in genital secretions, and compelling evidence from clinical, animal and in-vitro studies demonstrating that such cells can transmit HIV across genital tract epithelial surfaces; potential molecular mechanisms underlying cell-associated HIV transmission that could be specifically targeted by future HIV prevention strategies; and in-vitro and animal cell-associated HIV transmission models currently used for studies on cell-associated HIV transmission mechanisms and for testing vaccine and microbicide candidates. Using this information as a foundation, we discuss the evidence and probability that various current microbicide and vaccine approaches prevent cell-associated HIV transmission, and suggest additional microbicide and vaccine concepts and experiments that will move this field forward.

A small pool of infected cells persists in HIV-infected individuals receiving antiretroviral therapy (ART). Here, we developed ultrasensitive assays to precisely measure the frequency of cells harboring total HIV DNA, integrated HIV DNA, and two long terminal repeat (2-LTR) circles. These assays are performed on cell lysates, which circumvents the labor-intensive step of DNA extraction, and rely on the coquantification of each HIV molecular form together with CD3 gene sequences to precisely measure cell input. Using primary isolates from HIV subtypes A, B, C, D, and CRF01_A/E, we demonstrate that these assays can efficiently quantify low target copy numbers from diverse HIV subtypes. We further used these assays to measure total HIV DNA, integrated HIV DNA, and 2-LTR circles in CD4(+) T cells from HIV-infected subjects infected with subtype B. All samples obtained from ART-naive subjects were positive for the three HIV molecular forms (n = 15). Total HIV DNA, integrated HIV DNA, and 2-LTR circles were detected in, respectively, 100%, 94%, and 77% of the samples from individuals in which HIV was suppressed by ART. Higher levels of total HIV DNA and 2-LTR circles were detected in untreated subjects than individuals on ART (P = 0.0003 and P = 0.0004, respectively), while the frequency of CD4(+) T cells harboring integrated HIV DNA did not differ between the two groups. These results demonstrate that these novel assays have the ability to quantify very low levels of HIV DNA of multiple HIV subtypes without the need for nucleic acid extraction, making them well suited for the monitoring of viral persistence in large populations of HIV-infected individuals. Since the discovery of viral reservoirs in HIV-infected subjects receiving suppressive ART, measuring the degree of viral persistence has been one of the greatest challenges in the field of HIV research. Here, we report the development and validation of ultrasensitive assays to measure HIV persistence in HIV-infected individuals from multiple geographical regions. These assays are relatively inexpensive, do not require DNA extraction, and can be completed in a single day. Therefore, they are perfectly adapted to monitor HIV persistence in large cohorts of HIV-infected individuals and, given their sensitivity, can be used to monitor the efficacy of therapeutic strategies aimed at interfering with HIV persistence after prolonged ART.

We determined predictors of both intact (estimate of replication-competent) and total (intact and defective) HIV DNA in the reservoir among children with HIV. HIV DNA in the reservoir was quantified longitudinally in children who initiated antiretroviral therapy (ART) at less than 1 year of age using a novel cross-subtype intact proviral DNA assay that measures both intact and total proviruses. Quantitative PCR was used to measure pre-ART cytomegalovirus (CMV) viral load. Linear mixed effects models were used to determine predictors of intact and total HIV DNA levels (log 10 copies/million). Among 65 children, median age at ART initiation was 5 months and median follow-up was 5.2 years; 86% of children had CMV viremia pre-ART. Lower pre-ART CD4 + percentage [adjusted relative risk (aRR): 0.87, 95% confidence intervals (95% CI): 0.79-0.97; P = 0.009] and higher HIV RNA (aRR: 1.21, 95% CI: 1.06-1.39; P = 0.004) predicted higher levels of total HIV DNA during ART. Pre-ART CD4 + percentage (aRR: 0.76, 95% CI: 0.65-0.89; P < 0.001), CMV viral load (aRR: 1.16, 95% CI: 1.01-1.34; P = 0.041), and first-line protease inhibitor-based regimens compared with nonnucleoside reverse transcriptase-based regimens (aRR: 1.36, 95% CI: 1.04-1.77; P = 0.025) predicted higher levels of intact HIV DNA. Pre-ART immunosuppression, first-line ART regimen, and CMV viral load may influence establishment and sustainment of intact HIV DNA in the reservoir.

Controversy continues regarding the extent of ongoing viral replication in HIV-1-infected patients on effective antiretroviral therapy (ART). Adding an additional potent agent, such as raltegravir, to effective ART in patients with low-level residual viremia may reveal whether there is ongoing HIV-1 replication. We previously reported the outcome of a randomized placebo-controlled study of raltegravir intensification in patients on ART with HIV-1 RNA <50 copies per milliliter that showed no effect on residual viremia measured by single copy assay. We now report the effects of raltegravir intensification in that trial on other potential measures of ongoing HIV-1 replication as follows: 2-LTR HIV-1 circles, total cellular HIV-1 DNA, and T-cell activation. Of 50 patients tested, 12 (24%) had 2-LTR circles detected at baseline. Patients who were 2-LTR-positive had higher plasma HIV-1 RNA and HIV-1 DNA levels than 2-LTR-negative individuals. At week 12 of raltegravir intensification, there was no change from baseline in 2-LTR circles, in total HIV-1 DNA or in the ratio of 2-LTR circles to total HIV-1 DNA. There was also no change in markers of T-cell activation. In HIV-1-infected individuals on effective ART, we find no evidence of ongoing viral replication in the blood that is suppressible by raltegravir intensification. The results imply that raltegravir intensification alone will not eradicate HIV-1 infection.

Long-lived latently infected resting CD4+ T cells are the main reason why current antiretroviral therapy (ART) is unable to cure HIV infection [1]. Recent work has suggested that the expression of immune checkpoint markers, such as programmed death-1 (PD1), may play a role in viral persistence on ART via either suppression of virus transcription and/or reduced HIV-specific T cell activity [2,3], but the role of cytotoxic T lymphocyte antigen 4 (CTLA-4 or CD152) in HIV persistence on ART is not clear. Ipilimumab (Yervoy, Bristol-Myers Squibb, New York, New York) is a human immunoglobulin G1 antibody to CTLA-4 that inhibits binding of CTLA-4, expressed on activated T cells and regulatory T cells (Tregs), to its ligands CD80 and CD86. The drug is used to treat metastatic melanoma and has been associated with multiple changes in immune function thought to enhance antitumor T cell function [4]. In HIV-infected individuals, CTLA-4 expression on CD4+ T cells correlates with HIV disease progression [5], and loss of HIV-specific CD4+ T cell function can be reversed in vitro by CTLA-4 blockade [5–7]. In a simian immunodeficiency virus (SIV) macaque model, CTLA-4 blockade led to an increase in T-cell activation and viral replication [8]. Here, we describe changes in the HIV reservoir in an HIV-infected patient on ART who received ipilimumab for the treatment of metastatic melanoma. At initiation of ipilimumab treatment in October 2013 for disseminated melanoma, the patient was a 51-year-old man diagnosed with HIV in 1986 and with a CD4+ nadir of 159 cells/μl in 1995. He was on ART since 1996 and plasma HIV RNA was less than 400 copies/ml from 2004 and less than 20 copies/ml from July 2012 (Fig. 1a). He received four doses of ipilimumab 3 mg/kg given at three-weekly intervals. Fig. 1 Clinical details and changes and impact of ipilimumab on virological and immunological parameters Whilst receiving ipilimumab, there was no overall change in plasma HIV RNA as measured by the Roche viral load assay [lower limit of detection (LLOD) = 20 copies/ml; Fig. 1c]. Using a sensitive single-copy HIV RNA assay (SCA) (LLOD = 0.3 copies/ml) [9], there was a cyclical decrease in plasma HIV RNA following each infusion and an overall decline from 60 to 5 copies/ml (Fig. 1c). Given more frequent sampling was performed with the SCA, we believe that longitudinal changes over time were best assessed with this assay. There was an increase in CD4+ T cells after each infusion (overall change from 610 to 900 cells/μl) (Fig. 1b). This increase was predominantly in total memory (Fig. 1d) and effector memory CD4+ T cells (Fig. 1e). Postinfusion increases in CD4+ T-cell activation were seen as measured by human leukocyte antigen-DR and CD38 and CCR5 expression (Fig. 1f). There were transient increases in CD8+ T cells following the second and third infusions, but no overall change in CD8+ T cell activation (Fig. 1g). Cell-associated unspliced HIV RNA in sorted CD4+ T cells was quantified with increases observed following the first and second infusions, with a maximum change from baseline of 19.6-fold (Fig. 1h). The changes in cell-associated unspliced HIV RNA was greater than those recently reported, following the administration of the histone deacetylase inhibitors vorinostat [10,11] or panobinostat [12], or following disulfiram [13]. There was no change in cell-associated HIV DNA (Fig. 1i), but any change in the small proportion of cells with HIV DNA containing inducible proviruses [14] may not have been detectable with the assays used here. Acknowledging the limitations deriving from this being a single case, we speculate the increase in cell-associated unspliced RNA could have been due to mechanisms, including an increase in HIV RNA transcription secondary to blocking the inhibitory effects of CTLA-4 on T cell transcription, similar to that described following ex-vivo anti-PD1 treatment of CD4+ T cells from HIV-infected patients on ART [15]; redistribution or expansion of effector memory CD4+ T cells that may have a higher ratio of cell-associated HIV RNA to HIV DNA [16] (Satish Pillai, San Francisco, UCSF, San Francisco, California, personal communication); or redistribution or expansion of activated T cells including Tregs. The increase in cell-associated unspliced HIV RNA and decline in SCA was intriguing, perhaps mediated by elimination of latently infected CD4+ T cells that were induced to express viral antigens. But the rapidity of the decline in SCA makes this somewhat unlikely. Blockade of CTLA-4 with ipilimumab in an HIV-infected patient on ART had significant effects on the total number and phenotype of CD4+ T cells and induced a profound increase in cell-associated unspliced HIV RNA with onset after the first dose and was associated with subsequent decline in plasma HIV RNA. Further studies are warranted to determine if ipilimumab could play a role in eliminating latently infected cells in HIV-infected patients on ART.

Effect of therapeutic intensification followed by HIV DNA prime and rAd5 boost vaccination on HIV-specific immunity and HIV reservoir (EraMune 02): a multicentre randomised clinical trial

The dynamics of latent HIV is linked to infection and clearance of resting memory CD4+ T cells. Infection also resides within activated, non-dividing memory cells and can be impacted by antigen-driven and homeostatic proliferation despite suppressive antiretroviral therapy (ART). We investigated whether plasma viral level (pVL) and HIV DNA dynamics could be explained by HIV’s impact on memory CD4+ T cell homeostasis. Median total, 2-LTR and integrated HIV DNA levels per μL of peripheral blood, for 8 primary (PHI) and 8 chronic HIV infected (CHI) individuals enrolled on a raltegravir (RAL) based regimen, exhibited greatest changes over the 1st year of ART. Dynamics slowed over the following 2 years so that total HIV DNA levels were equivalent to reported values for individuals after 10 years of ART. The mathematical model reproduced the multiphasic dynamics of pVL, and levels of total, 2-LTR and integrated HIV DNA in both PHI and CHI over 3 years of ART. Under these simulations, residual viremia originated from reactivated latently infected cells where most of these cells arose from clonal expansion within the resting phenotype. Since virion production from clonally expanded cells will not be affected by antiretroviral drugs, simulations of ART intensification had little impact on pVL. HIV DNA decay over the first year of ART followed the loss of activated memory cells (120 day half-life) while the 5.9 year half-life of total HIV DNA after this point mirrored the slower decay of resting memory cells. Simulations had difficulty reproducing the fast early HIV DNA dynamics, including 2-LTR levels peaking at week 12, and the later slow loss of total and 2-LTR HIV DNA, suggesting some ongoing infection. In summary, our modelling indicates that much of the dynamical behavior of HIV can be explained by its impact on memory CD4+ T cell homeostasis.

The Benefits of Early Antiretroviral Therapy for HIV Infection: How Early is Early Enough?

To measure proviral HIV-1 DNA in patients treated with effective antiretroviral therapy (ART) during recent and chronic HIV-1 infection, and in long-term non-progressors (LTNP). We quantified HIV-1 DNA in peripheral blood samples from 39 HIV-1-infected subjects; 26 patients initiated non-nucleoside reverse transcriptase inhibitor (NNRTI) based ART at two different stages of infection: 16 during recent infection (RI) (HIV-1 exposure >60 days <1 year), and 10 during chronic infection (CI) (infected >2 years). The results were compared with those seen in 13 LTNP (infected >8 years, therapy naïve, and controlled viremia). Thirty-six weeks after initiation of ART, HIV-1-proviral DNA levels decreased from baseline in the RI group (P < 0.005) to levels comparable to LTNP. HIV-1 DNA also declined in the CI group (P = 0.053) but it remained significantly higher than in RI (P < 0.002) and LTNP (P < 0.02). However, plasma HIV-1 RNA levels become undetectable in 80% of CI patients 12 weeks post initiation of ART, compared to 41.2% in the RI group. All patients reached undetectable viremia by week 36 of therapy. These data indicate that initiation of NNRTI based ART during recent HIV-1 infection reduces HIV-1 DNA to levels comparable to those seen in LTNP, which is not apparent if therapy is started during chronic infection, and suggests an association between timing of initiation of ART and decay of the HIV-1 reservoir.