The multifaceted nature of HIV tissue reservoirs.

PURPOSE OF REVIEW: To underline the complexity and the heterogeneity of the HIV reservoir. RECENT FINDINGS: While lymphoid tissues (spleen, lymph nodes, gut-associated lymphoid tissue) harbor specific subsets of specialized CD4 +  T cells enriched in HIV-infected cells, non-CD4 +  T cell reservoirs such as tissue-resident macrophages and dendritic cells have also been implicated to contribute to viral persistence. Moreover, studies have applied highly sensitive tools to detect transcriptional activity within HIV-infected cells during prolonged ART and revealed a broader spectrum of transcriptional activity for proviruses than previously thought. Finally, while a combination of factors might be involved in the regulation of HIV persistence within different tissues and remains to be fully elucidated, recent results from autopsy samples of HIV-infected ART suppressed individuals indicate extensive clonality of HIV reservoirs in multiple tissues and suggest that the recirculation of HIV-infected cells and their local expansions in tissues may also contribute to the complexity of the HIV reservoirs in humans. SUMMARY: HIV persistence in blood and multiple tissues despite long-standing and potent therapy is one of the major barriers to a cure. Given that the HIV reservoir is established early and is highly complex based on its composition, viral diversity, tissue distribution, transcriptional activity, replication competence, migration dynamics and proliferative potential across the human body and possible compartmentalization in specific tissues, combinatorial therapeutic approaches are needed that may synergize to target multiple viral reservoirs to achieve a cure for HIV infection.

Lymph node dendritic cells harbor inducible replication-competent HIV despite years of suppressive ART.

Although gut and lymph node (LN) memory CD4 T cells represent major HIV and simian immunodeficiency virus (SIV) tissue reservoirs, the study of the role of dendritic cells (DCs) in HIV persistence has long been limited to the blood due to difficulties to access lymphoid tissue samples. In this study, we show that LN migratory and resident DC subpopulations harbor distinct phenotypic and transcriptomic profiles. Interestingly, both LN DC subpopulations contain HIV intact provirus and inducible replication-competent HIV despite the expression of the antiviral restriction factor SAMHD1. Notably, LN DC subpopulations isolated from HIV-infected individuals treated for up to 14 years are transcriptionally silent but harbor replication-competent virus that can be induced upon TLR7/8 stimulation. Taken together, these results uncover a potential important contribution of LN DCs to HIV infection in the presence of ART.

Host Molecule Incorporation into HIV Virions, Potential Influences in HIV Pathogenesis.

During the last phase of HIV viral production, nascent HIV virions acquire a fraction of the cellular lipid membrane to create the external lipid envelope, a process by which cellular proteins present on the surface of the infected cell can be incorporated along with Env trimers. Interestingly, several studies indicated that these incorporated host molecules could conserve their biological activity and consequently contribute to HIV pathogenesis either by enhancing the infectivity of HIV virions, their tissue tropism or by affecting immune cell functions. The following review will describe the main approaches used to characterize membrane bound host molecule incorporation into HIV virions, the proposed mechanisms involved, and the role of a non-exhaustive list of incorporated molecules.

Elite and posttreatment controllers, two facets of HIV control.

PURPOSE OF REVIEW: The quest for HIV-1 cure could take advantage of the study of rare individuals that control viral replication spontaneously (elite controllers) or after an initial course of antiretroviral therapy (posttreatment controllers, PTCs). In this review, we will compare back-to-back the immunological and virological features underlying viral suppression in elite controllers and PTCs, and explore their possible contributions to the HIV-1 cure research. RECENT FINDINGS: HIV-1 control in elite controllers shows hallmarks of an effective antiviral response, favored by genetic background and possibly associated to residual immune activation. The immune pressure in elite controllers might select against actively transcribing intact proviruses, allowing the persistence of a small and poorly inducible reservoir. Evidence on PTCs is less abundant but preliminary data suggest that antiviral immune responses may be less pronounced. Therefore, these patients may rely on distinct mechanisms, not completely elucidated to date, suppressing HIV-1 transcription and replication. SUMMARY: PTCs and elite controllers may control HIV replication using distinct pathways, the elucidation of which may contribute to design future interventional strategies aiming to achieve a functional cure.

Active PD-L1 incorporation within HIV virions functionally impairs T follicular helper cells.

The limited development of broadly neutralizing antibodies (BnAbs) during HIV infection is classically attributed to an inadequate B-cell help brought by functionally impaired T follicular helper (Tfh) cells. However, the determinants of Tfh-cell functional impairment and the signals contributing to this condition remain elusive. In the present study, we showed that PD-L1 is incorporated within HIV virions through an active mechanism involving p17 HIV matrix protein. We subsequently showed that in vitro produced PD-L1high but not PD-L1low HIV virions, significantly reduced Tfh-cell proliferation and IL-21 production, ultimately leading to a decreased of IgG1 secretion from GC B cells. Interestingly, Tfh-cell functions were fully restored in presence of anti-PD-L1/2 blocking mAbs treatment, demonstrating that the incorporated PD-L1 proteins were functionally active. Taken together, the present study unveils an immunovirological mechanism by which HIV specifically exploits the regulatory potential of PD-L1 to suppress the immune system during the course of HIV infection.

Inducible HIV-1 Reservoir Quantification: Clinical Relevance, Applications and Advancements of TILDA.

The presence of a stable HIV-1 reservoir persisting over time despite effective antiretroviral suppression therapy precludes a cure for HIV-1. Characterizing and quantifying this residual reservoir is considered an essential prerequisite to develop and validate curative strategies. However, a sensitive, reproducible, cost-effective, and easily executable test is still needed. The quantitative viral outgrowth assay is considered the gold standard approach to quantify the reservoir in HIV-1-infected patients on suppressive ART, but it has several limitations. An alternative method to quantify the viral reservoir following the reactivation of latent HIV-1 provirus detects multiply-spliced tat/rev RNA (msRNA) molecules by real-time PCR [tat/rev induced limiting dilution assay (TILDA)]. This article provides a perspective overview of the clinical relevance, various applications, recent advancements of TILDA, and how the assay has contributed to our understanding of the HIV-1 reservoir.

HIV persistence in lymph nodes.

PURPOSE OF REVIEW: HIV persists in distinct cellular and anatomical compartments in the body including blood, Central nervous system, and lymphoid tissues (spleen, lymph nodes [LNs], gut-associated lymphoid tissue) by diverse mechanisms despite antiretroviral therapy. Within LNs, human and animal studies have highlighted that a specific CD4 T cell subset - called T follicular helper cells locating in B cell follicles is enriched in cells containing replication-competent HIV as compared to extra-follicular CD4 T cells. Therefore, the objective of the present review is to focus on the potential mechanisms allowing HIV to persist within LN microenvironment. RECENT FINDINGS: The combination of factors that might be involved in the regulation of HIV persistence within LNs remain to be fully identified but may include - the level of activation, antiretroviral drug concentrations, presence of cytolytic mechanisms and/or regulatory cells, in addition to cell survival and proliferation propensity which would ultimately determine the fate of HIV-infected cells within LN tissue areas. SUMMARY: HIV persistence in blood and distinct body compartments despite long-standing and potent therapy is one of the major barriers to a cure. Given that the HIV reservoir is established early and is highly complex based on composition, viral diversity, distribution, replication competence, migration dynamics across the human body and possible compartmentalization in specific tissues, combinatorial therapeutic approaches are needed that may synergize to target multiple viral reservoirs to achieve a cure for HIV infection.

Blood and Lymph Node Dissemination of Clonal Genome-Intact Human Immunodeficiency Virus 1 DNA Sequences During Suppressive Antiretroviral Therapy.

The majority of cells with latent human immunodeficiency virus 1 infection are located in lymphoid tissues that are difficult to access. In the current study, we used single-genome near-full-length proviral sequencing to evaluate intact and defective proviruses in blood and lymph node CD4 T cells enriched for specific functional polarizations. We observed minor variations between the frequencies of proviral sequences within individual CD4 T-cell subsets and across tissue compartments. However, we noted multiple clonal clusters of identical intact or defective proviral sequences from distinct compartments and CD4 T-cell subpopulations, suggesting frequent interchanges between viral reservoir cells in blood and tissues.

T-cell exhaustion in HIV infection.

The T-cell response is central in the adaptive immune-mediated elimination of pathogen-infected and/or cancer cells. This activated T-cell response can inflict an overwhelming degree of damage to the targeted cells, which in most instances leads to the control and elimination of foreign invaders. However, in conditions of chronic infection, persistent exposure of T cells to high levels of antigen results in a severe T-cell dysfunctional state called exhaustion. T-cell exhaustion leads to a suboptimal immune-mediated control of multiple viral infections including the human immunodeficiency virus (HIV). In this review, we will discuss the role of T-cell exhaustion in HIV disease progression, the long-term defect of T-cell function even in aviremic patients on antiretroviral therapy (ART), the role of exhaustion-specific markers in maintaining a reservoir of latently infected cells, and exploiting these markers in HIV cure strategies.

Lymph node migratory dendritic cells modulate HIV-1 transcription through PD-1 engagement.

T-follicular helper (Tfh) cells, co-expressing PD-1 and TIGIT, serve as a major cell reservoir for HIV-1 and are responsible for active and persistent HIV-1 transcription after prolonged antiretroviral therapy (ART). However, the precise mechanisms regulating HIV-1 transcription in lymph nodes (LNs) remain unclear. In the present study, we investigated the potential role of immune checkpoint (IC)/IC-Ligand (IC-L) interactions on HIV-1 transcription in LN-microenvironment. We show that PD-L1 (PD-1-ligand) and CD155 (TIGIT-ligand) are predominantly co-expressed on LN migratory (CD1chighCCR7+CD127+) dendritic cells (DCs), that locate predominantly in extra-follicular areas in ART treated individuals. We demonstrate that TCR-mediated HIV production is suppressed in vitro in the presence of recombinant PD-L1 or CD155 and, more importantly, when LN migratory DCs are co-cultured with PD-1+/Tfh cells. These results indicate that LN migratory DCs expressing IC-Ls may more efficiently restrict HIV-1 transcription in the extra-follicular areas and explain the persistence of HIV transcription in PD-1+/Tfh cells after prolonged ART within germinal centers.

TLR3 agonist and CD40-targeting vaccination induces immune responses and reduces HIV-1 reservoirs.

Activation of HIV-1 reservoirs and induction of anti-HIV-1 T cells are critical to control HIV-1 rebound after combined antiretroviral therapy (cART). Here we evaluated in humanized mice (hu-mice) with persistent HIV-1 infection the therapeutic effect of TLR3 agonist and a CD40-targeting HIV-1 vaccine, which consists of a string of 5 highly conserved CD4+ and CD8+ T cell epitope-rich regions of HIV-1 Gag, Nef, and Pol fused to the C-terminus of a recombinant anti-human CD40 antibody (αCD40.HIV5pep). We show that αCD40.HIV5pep vaccination coadministered with poly(I:C) adjuvant induced HIV-1-specific human CD8+ and CD4+ T cell responses in hu-mice. Interestingly, poly(I:C) treatment also reactivated HIV-1 reservoirs. When administrated in therapeutic settings in HIV-1-infected hu-mice under effective cART, αCD40.HIV5pep with poly(I:C) vaccination induced HIV-1-specific CD8+ T cells and reduced the level of cell-associated HIV-1 DNA (or HIV-1 reservoirs) in lymphoid tissues. Most strikingly, the vaccination significantly delayed HIV-1 rebound after cART cessation. In summary, the αCD40.HIV5pep with poly(I:C) vaccination approach both activates replication of HIV-1 reservoirs and enhances the anti-HIV-1 T cell response, leading to a reduced level of cell-associated HIV-1 DNA or reservoirs. Our proof-of-concept study has significant implication for the development of CD40-targeting HIV-1 vaccine to enhance anti-HIV-1 immunity and reduce HIV-1 reservoirs in patients with suppressive cART.

CD32+ and PD-1+ Lymph Node CD4 T Cells Support Persistent HIV-1 Transcription in Treated Aviremic Individuals.

A recent study conducted in blood has proposed CD32 as the marker identifying the "elusive" HIV reservoir. We have investigated the distribution of CD32+ CD4 T cells in blood and lymph nodes (LNs) of HIV-1-uninfected subjects and viremic untreated and long-term-treated HIV-1-infected individuals and their relationship with PD-1+ CD4 T cells. The frequency of CD32+ CD4 T cells was increased in viremic compared to treated individuals in LNs, and a large proportion (up to 50%) of CD32+ cells coexpressed PD-1 and were enriched within T follicular helper (Tfh) cells. We next investigated the role of LN CD32+ CD4 T cells in the HIV reservoir. Total HIV DNA was enriched in CD32+ and PD-1+ CD4 T cells compared to CD32- and PD-1- cells in both viremic and treated individuals, but there was no difference between CD32+ and PD-1+ cells. There was no enrichment of latently infected cells with inducible HIV-1 in CD32+ versus PD-1+ cells in antiretroviral therapy (ART)-treated individuals. HIV-1 transcription was then analyzed in LN memory CD4 T cell populations sorted on the basis of CD32 and PD-1 expression. CD32+ PD-1+ CD4 T cells were significantly enriched in cell-associated HIV RNA compared to CD32- PD-1- (averages of 5.2-fold in treated individuals and 86.6-fold in viremics), CD32+ PD-1- (2.2-fold in treated individuals and 4.3-fold in viremics), and CD32- PD-1+ (2.2-fold in ART-treated individuals and 4.6-fold in viremics) cell populations. Similar levels of HIV-1 transcription were found in CD32+ PD-1- and CD32- PD-1+ CD4 T cells. Interestingly, the proportion of CD32+ and PD-1+ CD4 T cells negatively correlated with CD4 T cell counts and length of therapy. Therefore, the expression of CD32 identifies, independently of PD-1, a CD4 T cell population with persistent HIV-1 transcription and coexpression of CD32 and PD-1, the CD4 T cell population with the highest levels of HIV-1 transcription in both viremic and treated individuals.IMPORTANCE The existence of long-lived latently infected resting memory CD4 T cells represents a major obstacle to the eradication of HIV infection. Identifying cell markers defining latently infected cells containing replication-competent virus is important in order to determine the mechanisms of HIV persistence and to develop novel therapeutic strategies to cure HIV infection. We provide evidence that PD-1 and CD32 may have a complementary role in better defining CD4 T cell populations infected with HIV-1. Furthermore, CD4 T cells coexpressing CD32 and PD-1 identify a CD4 T cell population with high levels of persistent HIV-1 transcription.

Blood CXCR3+ CD4 T Cells Are Enriched in Inducible Replication Competent HIV in Aviremic Antiretroviral Therapy-Treated Individuals.

We recently demonstrated that lymph nodes (LNs) PD-1+/T follicular helper (Tfh) cells from antiretroviral therapy (ART)-treated HIV-infected individuals were enriched in cells containing replication competent virus. However, the distribution of cells containing inducible replication competent virus has been only partially elucidated in blood memory CD4 T-cell populations including the Tfh cell counterpart circulating in blood (cTfh). In this context, we have investigated the distribution of (1) total HIV-infected cells and (2) cells containing replication competent and infectious virus within various blood and LN memory CD4 T-cell populations of conventional antiretroviral therapy (cART)-treated HIV-infected individuals. In the present study, we show that blood CXCR3-expressing memory CD4 T cells are enriched in cells containing inducible replication competent virus and contributed the most to the total pool of cells containing replication competent and infectious virus in blood. Interestingly, subsequent proviral sequence analysis did not indicate virus compartmentalization between blood and LN CD4 T-cell populations, suggesting dynamic interchanges between the two compartments. We then investigated whether the composition of blood HIV reservoir may reflect the polarization of LN CD4 T cells at the time of reservoir seeding and showed that LN PD-1+ CD4 T cells of viremic untreated HIV-infected individuals expressed significantly higher levels of CXCR3 as compared to CCR4 and/or CCR6, suggesting that blood CXCR3-expressing CD4 T cells may originate from LN PD-1+ CD4 T cells. Taken together, these results indicate that blood CXCR3-expressing CD4 T cells represent the major blood compartment containing inducible replication competent virus in treated aviremic HIV-infected individuals.

Targeted Immune Interventions for an HIV-1 Cure.

Combination antiretroviral therapy (cART) induces durable suppression of virus replication but is unable to eradicate HIV. Invariably, virus rebound follows treatment interruption and life-long cART is thus required. Advances have been made in our understanding of HIV latency, identification of HIV cell reservoirs, regulation of HIV-specific immune responses, as well as in the development of broad neutralizing antibodies and putative therapeutic vaccines. These have provided a scientific basis to explore alternative strategies that achieve durable suppression of viremia in the absence of cART, the so-called functional cure. Single intervention strategies have shown promise, albeit with limited efficacy. Consequently, a combination of interventions aiming to stimulate the immune response and prevent new rounds of viral infection and spreading may render the HIV functional cure a feasible goal.

PD-1(+) and follicular helper T cells are responsible for persistent HIV-1 transcription in treated aviremic individuals.

The mechanisms responsible for the persistence of HIV-1 after many years of suppressive antiretroviral therapy (ART) have been only partially elucidated. Most of the studies investigating HIV-1 persistence have been performed with blood, although it is well known that germinal centers (GCs) within lymph nodes (LNs) serve as primary sites for HIV-1 replication. We sought to identify the memory CD4 T cell populations in blood and LNs that are responsible for the production of replication-competent and infectious HIV-1, as well as for active and persistent virus transcription in ART-treated (for 1.5-14.0 years), aviremic (<50 HIV RNA copies/ml) HIV-infected individuals. We demonstrate that LN CD4 T cells that express programmed cell death 1 (PDCD1; also known as PD-1), which are composed of about 65% T follicular helper cells as defined by the expression of the cell surface receptors CXCR5 and PD-1, are the major source of replication-competent HIV-1 and of infectious virus, as compared to any other (CXCR5(-)PD-1(-) and CXCR5(+)PD-1(-)) blood or LN memory CD4 T cell populations. LN PD-1(+) cells accounted for 46% and 96% of the total pools of memory CD4 T cells containing inducible replication-competent or infectious virus, respectively. Notably, higher levels of cell-associated HIV-1 RNA were present in LN PD-1(+) cells after long-term (up to 12 years) ART than in other memory CD4 T cell subpopulations. These results indicate that LN PD-1(+) cells are the major CD4 T cell compartment in the blood and LNs for the production of replication-competent and infectious HIV-1, and for active and persistent virus transcription in long-term-ART-treated aviremic individuals. Thus, these cells may represent a major obstacle to finding a functional cure for HIV-1 infection.

Current approaches to assess HIV-1 persistence.

PURPOSE OF REVIEW: The persistence of HIV within long-lived HIV-infected CD4 T cells is the primary obstacle towards HIV eradication and numerous strategies are currently being evaluated to target and kill HIV-infected cells to ultimately find a cure. HIV reservoirs are classically quantified by standard methods such as integrated HIV DNA (Alu PCR) and/or quantitative viral outgrowth assay; however, recent technical advances may offer new opportunities to comprehensively assess the impact of clinical interventions. RECENT FINDINGS: Digital droplet PCR, tat/rev-induced limiting dilution analysis, enhanced quantitative viral outgrowth assay, and whole genome sequencing technologies offer increased precision and/or higher sensitivity to quantify and characterize HIV reservoirs in antiretroviral therapy-treated HIV-infected patients. SUMMARY: The objective of this review is to highlight the characteristics and limits of recent technical advances that may help to monitor the impact of clinical interventions in antiretroviral therapy-treated patients.

In Vitro Reactivation of Replication-Competent and Infectious HIV-1 by Histone Deacetylase Inhibitors.

UNLABELLED: The existence of long-lived HIV-1-infected resting memory CD4 T cells is thought to be the primary obstacle to HIV-1 eradication. In the search for novel therapeutic approaches that may reverse HIV-1 latency, inhibitors of histone deacetylases (HDACis) have been tested to reactivate HIV-1 replication with the objective of rendering HIV-1-infected cells susceptible to elimination either by HIV-specific CD8 T cells or through virus-mediated cytopathicity. In the present study, we evaluated the efficiency of HDACis to reactivate HIV-1 replication from resting memory CD4 T cells isolated from aviremic long-term-treated HIV-1-infected subjects. We demonstrate that following prolonged/repeated treatment of resting memory CD4 T cells with HDACis, HIV-1 replication may be induced from primary resting memory CD4 T cells isolated from aviremic long-term-treated HIV-1-infected subjects. More importantly, we demonstrate that HIV-1 reactivated in the cell cultures was not only replication competent but also infectious. Interestingly, givinostat, an HDACi that has not been investigated in clinical trials, was more efficient than vorinostat, panobinostat, and romidepsin in reversing HIV-1 latency in vitro. Taken together, these results support further evaluation of givinostat as a latency-reversing agent (LRA) in aviremic long-term-treated HIV-1-infected subjects. IMPORTANCE: The major barrier to HIV cure is the existence of long-lived latently HIV-1-infected resting memory CD4 T cells. Latently HIV-1-infected CD4 T cells are transcriptionally silent and are therefore not targeted by conventional antiretroviral therapy (ART) or the immune system. In this context, one strategy to target latently infected cells is based on pharmacological molecules that may force the virus to replicate and would therefore render HIV-1-infected cells susceptible to elimination either by HIV-specific CD8 T cells or through virus-mediated cytopathicity. In this context, we developed an experimental strategy that would allow the evaluation of latency-reversing agent (LRA) efficiency in vitro using primary CD4 T cells. In the present study, we demonstrate that HDACis are potent inducers of replication-competent and infectious HIV-1 in resting memory CD4 T cells of long-term ART-treated patients and identify givinostat as the most efficient LRA tested.

CD160-associated CD8 T-cell functional impairment is independent of PD-1 expression.

Expression of co-inhibitory molecules is generally associated with T-cell dysfunction in chronic viral infections such as HIV or HCV. However, their relative contribution in the T-cell impairment remains unclear. In the present study, we have evaluated the impact of the expression of co-inhibitory molecules such as 2B4, PD-1 and CD160 on the functions of CD8 T-cells specific to influenza, EBV and CMV. We show that CD8 T-cell populations expressing CD160, but not PD-1, had reduced proliferation capacity and perforin expression, thus indicating that the functional impairment in CD160(+) CD8 T cells may be independent of PD-1 expression. The blockade of CD160/CD160-ligand interaction restored CD8 T-cell proliferation capacity, and the extent of restoration directly correlated with the ex vivo proportion of CD160(+) CD8 T cells suggesting that CD160 negatively regulates TCR-mediated signaling. Furthermore, CD160 expression was not up-regulated upon T-cell activation or proliferation as compared to PD-1. Taken together, these results provide evidence that CD160-associated CD8 T-cell functional impairment is independent of PD-1 expression.