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Therapeutic strategies are being clinically tested either to eradicate latent HIV reservoirs or to achieve virologic control in the absence of antiretroviral therapy. Attaining this goal will require a consensus on how best to measure the numbers of persistently infected cells with the potential to cause viral rebound after antiretroviral-therapy cessation in assessing the results of cure-directed strategies in vivo. Current measurements assess various aspects of the HIV provirus and its functionality and produce divergent results. Here, we provide recommendations from the BEAT-HIV Martin Delaney Collaboratory on which viral measurements should be prioritized in HIV-cure-directed clinical trials.

We administered Ad26, modified vaccinia Ankara vectors containing mosaic HIV-1 antigens or placebo in 26 individuals who initiated antiretroviral therapy during acute human immunodeficiency virus infection as an exploratory study to determine the safety and duration of viremic control after treatment interruption. The vaccine was safe and generated robust immune responses, but delayed time to viral rebound compared to that in placebo recipients by only several days and did not lead to viremic control after treatment interruption (clinical trial NCT02919306).

HIV-1-specific broadly neutralising antibodies such as VRC01 could promote HIV remission by halting viral replication and clearing infected cells. We investigated whether VRC01 could promote sustained viral control off antiretroviral therapy (ART) in adults who initiated ART during acute HIV infection.

HIV persists in tissues during antiretroviral therapy (ART), but the relative contribution of different anatomical compartments to the viral reservoir in humans remains unknown. We performed an extensive characterization of HIV reservoirs in two men who donated their bodies to HIV cure research and who had been on suppressive ART for years. HIV DNA is detected in all tissues, with large variations across anatomical compartments and between participants. Intact HIV genomes represent 2% and 25% of all proviruses in the two participants and are mainly detected in secondary lymphoid organs, with the spleen and mediastinal lymph nodes harboring intact viral genomes in both individuals. Multiple copies of identical HIV genomes are found in all tissues, indicating that clonal expansions are common in anatomical sites. The majority (>85%) of these expanded clones are shared across multiple tissues. These findings suggest that infected cells expand, migrate, and possibly circulate between anatomical sites.

Sophisticated experimental approaches reveal cellular and immune-system mechanisms that enable rare HIV-infected cells to persist for decades in people who are taking antiretroviral drugs.

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Antiretroviral therapy during the earliest stage of acute HIV infection (Fiebig I) might minimize establishment of a latent HIV reservoir and thereby facilitate viremic control after analytical treatment interruption. We show that 8 participants, who initiated treatment during Fiebig I and were treated for a median of 2.8 years, all experienced rapid viral load rebound following analytical treatment interruption, indicating that additional strategies are required to control or eradicate HIV. Initiation of antiretroviral therapy in the first 2 weeks of HIV infection fails to prevent resurgence of virus after stopping treatment, indicating early establishment of a resilient viral reservoir.

Latent replication-competent HIV-1 persists in individuals on long-term antiretroviral therapy (ART). We developed the Full-Length Individual Proviral Sequencing (FLIPS) assay to determine the distribution of latent replication-competent HIV-1 within memory CD4+T cell subsets in six individuals on long-term ART. FLIPS is an efficient, high-throughput assay that amplifies and sequences near full-length (∼9 kb) HIV-1 proviral genomes and determines potential replication competency through genetic characterization. FLIPS provides a genome-scale perspective that addresses the limitations of other methods that also genetically characterize the latent reservoir. Using FLIPS, we identified 5% of proviruses as intact and potentially replication competent. Intact proviruses were unequally distributed between T cell subsets, with effector memory cells containing the largest proportion of genetically intact HIV-1 proviruses. We identified multiple identical intact proviruses, suggesting a role for cellular proliferation in the maintenance of the latent HIV-1 reservoir.

This protocol describes flow cytometric identification of viral translation-competent reservoirs, based on concurrent detection of cellular HIV GagpolmRNA by in situRNA hybridization combined with antibody staining for the HIV Gag protein.

IMPORTANCE: Human immunodeficiency virus (HIV) infection is associated with a high risk of cardiovascular disease and increased arterial inflammation. In HIV, inflammation is also increased within lymph nodes (LNs), tissues known to harbor the virus even among treated and suppressed individuals. OBJECTIVE: To test the hypothesis that arterial inflammation is linked to HIV disease activity and to inflammation within HIV-infected tissues (LNs). DESIGN, SETTING, AND PARTICIPANTS: For this case-control study, participants were recruited from the SCOPE (Observational Study of the Consequences of the Protease Inhibitor Era) cohort, a clinic-based cohort of individuals receiving care at San Francisco General Hospital and the San Francisco Veteran’s Affairs Medical Center. Arterial and LN inflammation were measured using 18F-fluorodeoxyglucose positron emission tomography. Detailed immunophenotyping was performed, along with measurement of viral activity/persistence and of circulating inflammatory biomarkers. MAIN OUTCOMES AND MEASURES: Arterial and LN inflammation. RESULTS: A total of 74 men were studied (45 HIV-infected men with a median age of 53 years [interquartile range, 49-59 years] and 29 uninfected male controls with a median age of 52 years [interquartile range, 46-56 years]). Lymph node inflammation was higher in HIV-infected individuals and correlated with markers of viral disease activity (viral load, CD8+ T cells, and CD4/CD8 ratio) and CD4+ T-cell activation. Uninfected controls had the lowest LN activity (mean [SD] maximum axillary LN standardized uptake value, 1.53 [0.56]), the elite controller and ART-suppressed groups had intermediate levels of LN (mean [SD] maximum axillary LN standardized uptake value, 2.12 [0.87] and 2.32 [1.79], respectively), and the noncontrollers had the highest activity (mean [SD] maximum axillary LN standardized uptake value, 8.82 [3.08]). Arterial inflammation was modestly increased in HIV-infected individuals and was positively correlated with circulating inflammatory biomarkers (high-sensitivity C-reactive protein and IL-6) and activated monocytes (CD14dimCD16+; nonclassical) but not with markers of HIV. While LN and arterial inflammation were increased in HIV, inflammatory activity in these tissues was not related (r = 0.09, P = .56). CONCLUSIONS AND RELEVANCE: While LNs and, to a lesser degree, the arterial wall are inflamed in HIV, inflammation in these tissues is not closely linked. Namely, measures of HIV disease activity are strongly associated with LN inflammation but not with arterial inflammation. These data suggest that LN and arterial inflammation do not share underlying pathways of immune activation and also that therapeutic interventions that reduce viral disease activity may not predictably reduce arterial inflammation in HIV or its downstream consequence (ie, cardiovascular disease).

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Antiretroviral therapy is not curative. Given the challenges in providing lifelong therapy to a global population of more than 35 million people living with HIV, there is intense interest in developing a cure for HIV infection. The International AIDS Society convened a group of international experts to develop a scientific strategy for research towards an HIV cure. This Perspective summarizes the group's strategy.

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Programmed cell death 1 (PD1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4) suppress CD4+T cell activation and may promote latent HIV infection. By performing leukapheresis (n = 21) and lymph node biopsies (n = 8) in people with HIV on antiretroviral therapy (ART) and sorting memory CD4+T cells into subsets based on PD1/CTLA4 expression, we investigate the role of PD1 and CTLA 4 in HIV persistence. We show that double-positive (PD1+CTLA4+) cells in blood contain more HIV DNA compared with double-negative (PD1−CTLA4−) cells but still have a lower proportion of cells producing multiply spliced HIV RNA after stimulation as well as reduced upregulation of T cell activation and proliferation markers. Transcriptomics analyses identify differential expression of key genes regulating T cell activation and proliferation with MAF, KLRB1, and TIGIT being upregulated in double-positive compared with double-negative cells, whereas FOS is downregulated. We conclude that, in addition to being enriched for HIV DNA, double-positive cells are characterized by negative signaling and a reduced capacity to respond to stimulation, favoring HIV latency.

Understanding the extent to which early antiretroviral therapy (ART) can limit the establishment and persistence of the HIV reservoir is an important step to designing interventions aimed at achieving HIV cure. We measured the markers of HIV persistence and HIV-specific immunity in early treated children.

HIV persists in latently infected memory CD4+ T cells during antiretroviral therapy (ART). When administered to HIV-infected subjects receiving suppressive ART, interleukin-7 (IL-7) increases the number of CD4+ T cells by promoting their survival and proliferation. However, little is known about the impact of IL-7 on HIV persistence during ART. By isolating large numbers of CD4+ T cells from HIV-infected subjects, we demonstrate that IL-7 enhances viral production in productively infected cells but does not disrupt viral latency in latently infected cells. When administered to virally suppressed subjects, IL-7 led to the rapid proliferation of memory CD4+ T cells, which resulted in a 70% increase in the absolute number of circulating CD4+ T cells harboring integrated HIV DNA 4 weeks after therapy. The genetic diversity of the viral reservoir increased transiently in the majority of the subjects studied before returning to baseline values. Altogether, our results indicate that IL-7 promotes the mechanisms of HIV persistence during ART by enhancing residual levels of viral production and inducing proliferation of latently infected cells, and suggest that IL-7 does not represent a suitable candidate therapeutic strategy for HIV eradication. This trial was registered at www.clinicaltrials.gov as #NCT00099671 (AIDS Clinical Trials Group protocol 5214).

Immediate-early host-virus interactions that occur during the first weeks after HIV infection have a major impact on disease progression. The mechanisms underlying the failure of HIV-specific CD8 T-cell response to persist and control viral replication early in infection are yet to be characterized. In this study, we performed a thorough phenotypic, gene expression and functional analysis to compare HIV-specific CD8 T cells in acutely and chronically infected subjects. We showed that HIV-specific CD8 T cells in primary infection can be distinguished by their metabolic state, rate of proliferation, and susceptibility to apoptosis. HIV-specific CD8 T cells in acute/early HIV infection secreted less IFN-γ but were more cytotoxic than their counterparts in chronic infection. Importantly, we showed that the levels of IL-7R expression and the capacity of HIV-specific CD8 T cells to secrete IL-2 on antigenic restimulation during primary infection were inversely correlated with the viral set-point. Altogether, these data suggest an altered metabolic state of HIV-specific CD8 T cells in primary infection resulting from hyperproliferation and stress induced signals, demonstrate the discordant function of HIV-specific CD8 T cells during early/acute infection, and highlight the importance of T-cell maintenance for viral control.

T cells actively migrate along reticular networks within lymphoid organs in search for cognate antigen, but how these behaviors impact HIV entry and infection is unclear. Here, we show that migratory T cells in 3D collagen matrix display significantly enhanced infection and integration by cell-free R5-tropic lab adapted and transmitted/founder molecular HIV clones in the absence of exogenous cytokines or cationic polymers. Using two different collagen matrices that either support or restrict T cell migration, we observe high levels of HIV fusion in migratory T cells, whereas non-motile T cells display low viral entry and integration. Motile T cells were less sensitive to combination antiretroviral drugs and were able to freely migrate into regions with high HIV densities, resulting in high infection rates. Together, our studies indicate that the environmental context in which initial HIV-T cell encounters occur modulates HIV-1 entry and integration efficiencies.

In people living with HIV (PLWH) on antiretroviral therapy (ART), virus persists in a latent form where there is minimal transcription or protein expression. Latently infected cells are a major barrier to curing HIV. Increasing HIV transcription and viral production in latently infected cells could facilitate immune recognition and reduce the pool of infected cells that persist on ART. Given that programmed cell death protein 1 (PD-1) expressing CD4+T cells are preferentially infected with HIV in PLWH on ART, we aimed to determine whether administration of antibodies targeting PD-1 would reverse HIV latency in vivo. We therefore evaluated the impact of intravenous administration of pembrolizumab every 3 weeks on HIV latency in 32 PLWH and cancer on ART. After the first infusion of anti–PD-1, we observed a median 1.32-fold increase in unspliced HIV RNA and 1.61-fold increase in unspliced RNA:DNA ratio in sorted blood CD4+T cells compared to baseline. We also observed a 1.65-fold increase in plasma HIV RNA. The frequency of CD4+T cells with inducible virus evaluated using the tat/revlimiting dilution assay was higher after 6 cycles compared to baseline. Phylogenetic analyses of HIV envsequences in a participant who developed low concentrations of HIV viremia after 6 cycles of pembrolizumab did not demonstrate clonal expansion of HIV-infected cells. These data are consistent with anti–PD-1 being able to reverse HIV latency in vivo and support the rationale for combining anti–PD-1 with other interventions to reduce the HIV reservoir.

ABSTRACTIn the present study, we assessed whether human immunodeficiency virus type 1 (HIV-1) genetic compartmentalization was associated with phenotypic CCR5 (R5) or CXCR4 (X4) coreceptor usage differences between the systemic and the genital viral populations. Four clinically asymptomatic and treatment-nai¨ve clade A HIV-1-infected patients were selected from a cohort of 274 African women, because they were free of all the biological cofactors known to modify the kinetics of viral production in the genital tract. HIV RNA envelope sequences (V1 to V3) derived from plasma and cervicovaginal secretions (CVS) were amplified, subcloned, and sequenced. CCR5 or CXCR4 coreceptor usage was determined by production of recombinant viral particles, followed by single-cycle infection assays of indicator cell lines, using the tropism recombinant test. In these four selected patients, CVS-derived sequences appeared to be genetically distinct from blood-derived sequences (P= 0.001). Two patients were found to harbor virus populations with only the R5 phenotype in both compartments, whereas viruses using CXCR4 in addition to CCR5 were detected in two other patients. In particular, one woman harbored genital virus populations with mixed R5 and X4 phenotypes associated with peripheral blood populations with only the R5 phenotype. These results demonstrate genetic compartmentalization of HIV between the plasma and genital secretions of clinically asymptomatic, treatment-nai¨ve, clade A-infected women. Also, for one patient, we report phenotypic coreceptor usage differences between the systemic (R5) and genital (R5/X4) viral populations. These features may be critical for the development of further mucosal vaccines, therapies, or new preventive strategies to block heterosexual transmission.

In a few people living with HIV, the virus remains under control without antiretroviral therapy. It emerges that, in these people, the viral DNA that is integrated into the host genome is in a deeply transcriptionally repressed state.

Enteroviruses (EVs) (Picornaviridae) in the female genital tract may constitute possible sources of antenatal or perinatal infection. The presence of EV genomes in the acellular part of cervicovaginal lavages of 119 non‐pregnant childbearing‐aged African women was determined using a semiquantitative RT‐PCR and hybridization detection assay. EV‐specific cervicovaginal IgA and IgG antibodies were also detected by immunocapture ELISA assays. Of 119 CVS samples tested, only 10 (8%) were positive for the detection of EV RNA, demonstrating an genital shedding of EVs in African woman. EV‐RNA positivity was not associated with the HIV serostatus or with the presence of semen traces in female genital secretion. The microwell hybridization assay of EV amplified RT‐PCR products indicated the presence of low levels of EV genomes, ranging from 50 to 100 RNA copies per ml of genital fluids. EV‐specific cervicovaginal IgA or IgG antibodies were detected only in two hemoglobin‐positive cervicovaginal secretions samples from women without genital EVs. The lack of EV specific IgA or IgG antibody secretion by the cervicovaginal mucosa supported the hypothesis of genital shedding of EVs without ongoing viral replication in the female genital tract. In conclusion, the findings demonstrated the presence of EV genomes in nearly 10% of childbearing‐aged women living in Central Africa, and provided the basis of possible antenatal or perinatal transmission of EV from mother‐to‐child. J. Med. Virol.78:591–597, 2006. © 2006 Wiley‐Liss, Inc.

ABSTRACTHuman immunodeficiency virus type 1 (HIV-1) RNA was detected by means of ultrasensitive reverse transcription-PCR assay of 19 cervicovaginal lavage, 21 cervical mucus, 18 vaginal wall, and 17 vaginal fornix paired samples from 25 asymptomatic HIV-1-infected women (76, 84, 72, and 68%, respectively; P> 0.5). Levels of HIV-1 RNA in cervicovaginal wash samples were highly correlated with those in paired endocervical mucus samples (r= 0.71; P= 0.0006), indicating that cervicovaginal washing and endocervical swabbing are equivalent collection procedures.

ABSTRACTTransmigration of human immunodeficiency virus (HIV)-infected mononuclear cells through the genital mucosa is one of the possible mechanisms of sexual transmission of HIV. Here, we investigated the transmigration of cell-associated R5-tropic HIV type 1 (HIV-1) through a tight monolayer of human epithelial cells in vitro. We show that this process is dependent on an initial interaction between aLß2 integrin CD11a/CD18 on infected monocytic cells and intercellular adhesion molecule 2 (ICAM-2; CD102) and ICAM-3 (CD50) on the apical membrane of epithelial cells. The CD50 and CD102 ligands were overexpressed on epithelial cells when the cells were activated by proinflammatory cytokines in the cellular microenvironment. An accumulation of proviral DNA was found in the transmigrated cells, clearly reflecting the preferential transepithelial migration of HIV-1-infected cells under proinflammatory conditions. Our observations provide new insights supporting the hypothesis that HIV-infected mononuclear cells contained in genital secretions from infected individuals may cross the epithelial genital mucosa of an exposed receptive sexual partner, particularly under inflammatory conditions of damaged genital tissue. Understanding the fundamental aspects of the initial HIV entry process during sexual transmission remains a critical step for preventing human infection and developing further vaccinal strategies and virucidal agents.

ABSTRACTWe report that both primary and laboratory-adapted infectious human immunodeficiency virus type 1 (HIV-1) isolates in a cell-free form are capable of transcytosis through a tight and polarized monolayer of human endometrial cells. Trancytosis of cell-free HIV occurs in a strain-selective fashion and appears to be dependent on interactions between HIV envelope glycoproteins and lectins on the apical membrane of the epithelial cells. These findings provide new insights into the initial events occurring during heterosexual transmission of the virus.

Although understanding the diversity of HIV-1 reservoirs is key to achieving a cure, their study at the single-cell level in primary samples remains challenging. We combine flow cytometric multiplexed fluorescent in situRNA hybridization for different viral genes with HIV-1 p24 protein detection, cell phenotyping, and downstream near-full-length single-cell vDNA sequencing. Stimulation-induced viral RNA-positive (vRNA+) cells from viremic and antiretroviral-therapy (ART)-suppressed individuals differ in their ability to produce p24. In participants on ART, latency-reversing agents (LRAs) induce a wide variety of viral gene transcription and translation patterns with LRA class-specific differences in reactivation potency. Reactivated proviruses, including in p24+cells, are mostly defective. Although LRAs efficiently induce transcription in all memory cell subsets, we observe induction of translation mostly in effector memory cells, rather than in the long-lived central memory pool. We identify HIV-1 clones with diverse transcriptional and translational patterns between individual cells, and this finding suggests that cell-intrinsic factors influence reservoir persistence and heterogeneity.

The greatest obstacle to an HIV cure is the persistence of latently infected cellular reservoirs in people living with HIV (PLWH) taking antiretroviral therapy (ART). However, no consensus exists on the direct link between local tissue inflammation and the HIV burden. Herein, we have compared the levels of local inflammation, epithelial integrity and HIV DNA between inflamed and non-inflamed colon tissue in a PLWH who underwent a colectomy due to ulcerative colitis. We have observed a 27-fold higher frequency of cells harboring HIV DNA in inflamed compared to non-inflamed colon tissue. Analysis of the expression of occludin-1 and claudin-3 confirmed our macroscopic characterization of inflamed and non-inflamed colon. Our results confirm that increased gut permeability and inflammation are associated with a higher frequency of infected cells and suggest that restoring gut barrier integrity may be used as a strategy to reduce inflammation and HIV persistence in the gut.

A randomized, open-label pilot study in individuals treated with antiretroviral therapy (ART) since acute HIV infection (AHI) with a regimen including a histone deacetylase inhibitor to induce HIV from latency and control HIV replication during subsequent treatment interruption (TI).

Although a large pool of cells is infected during acute HIV infection, most of these early targets are rapidly cleared upon ART initiation.

Latent proviruses persist in central (TCM), transitional (TTM), and effector (TEM) memory cells. We measured the levels of cellular factors involved in HIV gene expression in these subsets. The highest levels of acetylated H4, active nuclear factor κB (NF-κB), and active positive transcription elongation factor b (P-TEFb) were measured in TEM, TCM, and TTMcells, respectively. Vorinostat and romidepsin display opposite abilities to induce H4 acetylation across subsets. Protein kinase C (PKC) agonists are more efficient at inducing NF-κB phosphorylation in TCMcells but more potent at activating PTEF-b in the TEMsubset. We selected the most efficient latency-reversing agents (LRAs) and measured their ability to reverse latency in each subset. While ingenol alone has modest activities in the three subsets, its combination with a histone deacetylase inhibitor (HDACi) dramatically increases latency reversal in TCMcells. Altogether, these results indicate that cellular HIV reservoirs are differentially responsive to common LRAs and suggest that combination of compounds will be required to achieve latency reversal in all subsets.

Anti-α4β7 integrin therapy results in attrition of lymphoid aggregates within the gastrointestinal tract of HIV-1–infected individuals with IBD.

Potent CD8+T cells endowed with effector functions able to kill HIV-producing cells and reduce the seeding of the HIV reservoir are only detected at peak viremia in acute HIV infection.

A single infusion with broadly neutralizing antibody VRC01 resulted in lowered plasma virus load in HIV-1–infected subjects.

ABSTRACTAntiretroviral therapy (ART) inhibits HIV-1 replication, but the virus persists in latently infected resting memory CD4+T cells susceptible to viral reactivation. The virus-encoded early gene product Tat activates transcription of the viral genome and promotes exponential viral production. Here we show that the Tat inhibitor didehydro-cortistatin A (dCA), unlike other antiretrovirals, reduces residual levels of viral transcription in several models of HIV latency, breaks the Tat-mediated transcriptional feedback loop, and establishes a nearly permanent state of latency, which greatly diminishes the capacity for virus reactivation. Importantly, treatment with dCA induces inactivation of viral transcription even after its removal, suggesting that the HIV promoter is epigenetically repressed. Critically, dCA inhibits viral reactivation upon CD3/CD28 or prostratin stimulation of latently infected CD4+T cells from HIV-infected subjects receiving suppressive ART. Our results suggest that inclusion of a Tat inhibitor in current ART regimens may contribute to a functional HIV-1 cure by reducing low-level viremia and preventing viral reactivation from latent reservoirs.IMPORTANCEAntiretroviral therapy (ART) reduces HIV-1 replication to very low levels, but the virus persists in latently infected memory CD4+T cells, representing a long-lasting source of resurgent virus upon ART interruption. Based on the mode of action of didehydro-cortistatin A (dCA), a Tat-dependent transcription inhibitor, our work highlights an alternative approach to current HIV-1 eradication strategies to decrease the latent reservoir. In our model, dCA blocks the Tat feedback loop initiated after low-level basal reactivation, blocking transcriptional elongation and hence viral production from latently infected cells. Therefore, dCA combined with ART would be aimed at delaying or halting ongoing viral replication, reactivation, and replenishment of the latent viral reservoir. Thus, the latent pool of cells in an infected individual would be stabilized, and death of the long-lived infected memory T cells would result in a continuous decay of this pool over time, possibly culminating in the long-awaited sterilizing cure.