Development of an Attenuated Tat Protein as a Highly-effective Agent to Specifically Activate HIV-1 Latency

Although combined antiretroviral therapy (cART) successfully decreases plasma viremia to undetectable levels, the complete eradication of human immunodeficiency virus type 1 (HIV-1) remains impractical because of the existence of a viral reservoir, mainly in resting memory CD4⁺ T cells. Various cytokines, protein kinase C activators, and histone deacetylase inhibitors (HDACi) have been used as latency-reversing agents (LRAs), but their unacceptable side effects or low efficiencies limit their clinical use. Here, by a mutation accumulation strategy, we generated an attenuated HIV-1 Tat protein named Tat-R5M4, which has significantly reduced cytotoxicity and immunogenicity, yet retaining potent transactivation and membrane-penetration activity. Combined with HDACi, Tat-R5M4 activates highly genetically diverse and replication-competent viruses from resting CD4⁺ T lymphocytes isolated from HIV-1-infected individuals receiving suppressive cART. Thus, Tat-R5M4 has promising potential as a safe, efficient, and specific LRA in HIV-1 treatment.     

Small-molecule screening using a human primary cell model of HIV latency identifies compounds that reverse latency without cellular activation

The development of highly active antiretroviral therapy (HAART) to treat individuals infected with HIV-1 has dramatically improved patient outcomes, but HAART still fails to cure the infection. The latent viral reservoir in resting CD4⁺ T cells is a major barrier to virus eradication. Elimination of this reservoir requires reactivation of the latent virus. However, strategies for reactivating HIV-1 through nonspecific T cell activation have clinically unacceptable toxicities. We describe here the development of what we believe to be a novel in vitro model of HIV-1 latency that we used to search for compounds that can reverse latency. Human primary CD4⁺ T cells were transduced with the prosurvival molecule Bcl-2, and the resulting cells were shown to recapitulate the quiescent state of resting CD4⁺ T cells in vivo. Using this model system, we screened small-molecule libraries and identified a compound that reactivated latent HIV-1 without inducing global T cell activation, 5-hydroxynaphthalene-1,4-dione (5HN). Unlike previously described latency-reversing agents, 5HN activated latent HIV-1 through ROS and NF-κB without affecting nuclear factor of activated T cells (NFAT) and PKC, demonstrating that TCR pathways can be dissected and utilized to purge latent virus. Our study expands the number of classes of latency-reversing therapeutics and demonstrates the utility of this in vitro model for finding strategies to eradicate HIV-1 infection.     

IL-7 as a potential therapy for HIV-1-infected individuals

Highly active antiretroviral therapy (HAART), although effective in ameliorating the quality of life of HIV-1-infected individuals and their survival, has not been able to eradicate HIV-1. In fact, when HAART is interrupted, HIV-1 plasma viral load rebounds from viral reservoirs such as resting CD4⁺ T lymphocytes, monocytes and macrophages, remaining a major obstacle in attempting HIV eradication. Different therapeutic strategies have been attempted, such as structured treatment interruption (STI), immunotherapy (interleukin [IL]-2 and anti-CD3 antibodies [e.g., OKT3]), to try to stimulate HIV-1 out of latency along with antiretroviral intensification therapy. IL-7, a pleiotropic cytokine, bears diverse immune properties and plays a major role in T cell homeostasis. Moreover, IL-7 has recently been investigated as a possible immune adjuvant as well as a viral strain-specific inducer of HIV-1 replication. In fact, IL-7 was shown not only to be more effective than IL-2 in stimulating HIV-1 replication from resting CD4⁺ T lymphocytes ex vivo, but also to selectively induce a specific HIV-1 viral strain as compared with IL-2, suggesting the potential need for different viral inducers if complete eradication is to be achieved. In this present review, different immunological and virological properties of IL-7 are discussed, along with the possibility of its use as part of a combined antiretroviral-immune rationally based HIV-1 eradication approach.     

Zinc-finger Nuclease Editing of Human cxcr4 Promotes HIV-1 CD4+ T Cell Resistance and Enrichment

HIV-1-infected individuals can harbor viral isolates that can use CCR5, as well as CXCR4, for viral entry. To genetically engineer HIV-1 resistance in CD4⁺ T cells, we assessed whether transient, adenovirus delivered zinc-finger nuclease (ZFN) disruption of genomic cxcr4 or stable lentiviral expression of short hairpin RNAs (shRNAs) targeting CXCR4 mRNAs provides durable resistance to HIV-1 challenge. ZFN-modification of cxcr4 in CD4⁺ T cells was found to be superior to cell integrated lentivirus-expressing CXCR4 targeting shRNAs when CD4⁺ T cells were challenged with HIV-1s that utilizes CXCR4 for entry. Cxcr4 disruption in CD4⁺ T cells was found to be stable, conferred resistance, and provided for continued cell enrichment during HIV-1 infection in tissue culture and, in vivo, in peripheral blood mononuclear cell transplanted NSG mice. Moreover, HIV-1-infected mice with engrafted cxcr4 ZFN-modified CD4⁺ T cells demonstrated lower viral levels in contrast to mice engrafted with unmodified CD4⁺ T cells. These findings provide evidence that ZFN-mediated disruption of cxcr4 provides a selective advantage to CD4⁺ T cells during HIV-1 infection.     

T cell receptor–targeted immunotherapeutics drive selective in vivo HIV- and CMV-specific T cell expansion in humanized mice

To delineate the in vivo role of different costimulatory signals in activating and expanding highly functional virus-specific cytotoxic CD8⁺ T cells, we designed synTacs, infusible biologics that recapitulate antigen-specific T cell activation signals delivered by antigen-presenting cells. We constructed synTacs consisting of dimeric Fc-domain scaffolds linking CD28- or 4-1BB–specific ligands to HLA-A2 MHC molecules covalently tethered to HIV- or CMV-derived peptides. Treatment of HIV-infected donor PBMCs with synTacs bearing HIV- or CMV-derived peptides induced vigorous and selective ex vivo expansion of highly functional HIV- and/or CMV-specific CD8⁺ T cells, respectively, with potent antiviral activities. Intravenous injection of HIV- or CMV-specific synTacs into immunodeficient mice intrasplenically engrafted with donor PBMCs markedly and selectively expanded HIV-specific (32-fold) or CMV-specific (46-fold) human CD8⁺ T cells populating their spleens. Notably, these expanded HIV- or CMV-specific CD8⁺ T cells directed potent in vivo suppression of HIV or CMV infections in the humanized mice, providing strong rationale for consideration of synTac-based approaches as a therapeutic strategy to cure HIV and treat CMV and other viral infections. The synTac platform flexibility supports facile delineation of in vivo effects of different costimulatory signals on patient-derived virus-specific CD8⁺ T cells, enabling optimization of individualized therapies, including HIV cure strategies.     

Dual-Affinity Re-Targeting proteins direct T cell–mediated cytolysis of latently HIV-infected cells

Enhancement of HIV-specific immunity is likely required to eliminate latent HIV infection. Here, we have developed an immunotherapeutic modality aimed to improve T cell–mediated clearance of HIV-1–infected cells. Specifically, we employed Dual-Affinity Re-Targeting (DART) proteins, which are bispecific, antibody-based molecules that can bind 2 distinct cell-surface molecules simultaneously. We designed DARTs with a monovalent HIV-1 envelope-binding (Env-binding) arm that was derived from broadly binding, antibody-dependent cellular cytotoxicity–mediating antibodies known to bind to HIV-infected target cells coupled to a monovalent CD3 binding arm designed to engage cytolytic effector T cells (referred to as HIVxCD3 DARTs). Thus, these DARTs redirected polyclonal T cells to specifically engage with and kill Env-expressing cells, including CD4⁺ T cells infected with different HIV-1 subtypes, thereby obviating the requirement for HIV-specific immunity. Using lymphocytes from patients on suppressive antiretroviral therapy (ART), we demonstrated that DARTs mediate CD8⁺ T cell clearance of CD4⁺ T cells that are superinfected with the HIV-1 strain JR-CSF or infected with autologous reservoir viruses isolated from HIV-infected–patient resting CD4⁺ T cells. Moreover, DARTs mediated CD8⁺ T cell clearance of HIV from resting CD4⁺ T cell cultures following induction of latent virus expression. Combined with HIV latency reversing agents, HIVxCD3 DARTs have the potential to be effective immunotherapeutic agents to clear latent HIV-1 reservoirs in HIV-infected individuals.     

IL-15 promotes activation and expansion of CD8+ T cells in HIV-1 infection

In HIV-1–infected patients, increased numbers of circulating CD8⁺ T cells are linked to increased risk of morbidity and mortality. Here, we identified a bystander mechanism that promotes CD8 T cell activation and expansion in untreated HIV-1–infected patients. Compared with healthy controls, untreated HIV-1–infected patients have an increased population of proliferating, granzyme B⁺, CD8⁺ T cells in circulation. Vβ expression and deep sequencing of CDR3 revealed that in untreated HIV-1 infection, cycling memory CD8 T cells possess a broad T cell repertoire that reflects the repertoire of the resting population. This suggests that cycling is driven by bystander activation, rather than specific antigen exposure. Treatment of peripheral blood mononuclear cells with IL-15 induced a cycling, granzyme B⁺ phenotype in CD8⁺ T cells. Moreover, elevated IL-15 expression in the lymph nodes of untreated HIV-1–infected patients correlated with circulating CD8⁺ T cell counts and was normalized in these patients following antiretroviral therapy. Together, these results suggest that IL-15 drives bystander activation of CD8⁺ T cells, which predicts disease progression in untreated HIV-1–infected patients and suggests that elevated IL-15 may also drive CD8⁺ T cell expansion that is linked to increased morbidity and mortality in treated patients.     

The HIV-1 proviral landscape reveals that Nef contributes to HIV-1 persistence in effector memory CD4+ T cells

Despite long-term antiretroviral therapy (ART), HIV-1 persists within a reservoir of CD4⁺ T cells that contribute to viral rebound if treatment is interrupted. Identifying the cellular populations that contribute to the HIV-1 reservoir and understanding the mechanisms of viral persistence are necessary to achieve an effective cure. In this regard, through Full-Length Individual Proviral Sequencing, we observed that the HIV-1 proviral landscape was different and changed with time on ART across naive and memory CD4⁺ T cell subsets isolated from 24 participants. We found that the proportion of genetically intact HIV-1 proviruses was higher and persisted over time in effector memory CD4⁺ T cells when compared with naive, central, and transitional memory CD4⁺ T cells. Interestingly, we found that escape mutations remained stable over time within effector memory T cells during therapy. Finally, we provided evidence that Nef plays a role in the persistence of genetically intact HIV-1. These findings posit effector memory T cells as a key component of the HIV-1 reservoir and suggest Nef as an attractive therapeutic target.     

A stable latent reservoir for HIV-1 in resting CD4(+) T lymphocytes in infected children

HIV-1 persists in a latent state in resting CD4⁺ T lymphocytes of infected adults despite prolonged highly active antiretroviral therapy (HAART). To determine whether a latent reservoir for HIV-1 exists in infected children, we performed a quantitative viral culture assay on highly purified resting CD4⁺ T cells from 21 children with perinatally acquired infection. Replication-competent HIV-1 was recovered from all 18 children from whom sufficient cells were obtained. The frequency of latently infected resting CD4⁺ T cells directly correlated with plasma virus levels, suggesting that in children with ongoing viral replication, most latently infected cells are in the labile preintegration state of latency. However, in each of 7 children who had suppression of viral replication to undetectable levels for 1–3 years on HAART, latent replication-competent HIV-1 persisted with little decay, owing to a stable reservoir of infected cells in the postintegration stage of latency. Drug-resistance mutations generated by previous nonsuppressive regimens persisted in this compartment despite more than 1 year of fully suppressive HAART, rendering untenable the idea of recycling drugs that were part of failed regimens. Thus the latent reservoir for HIV-1 in resting CD4⁺ T cells will be a major obstacle to HIV-1 eradication in children.     

HIV-infected individuals receiving effective antiviral therapy for extended periods of time continually replenish their viral reservoir

The persistence of latently infected, resting CD4+ T cells is considered to be a major obstacle in preventing the eradication of HIV-1 even in patients who have received effective antiviral therapy for an average duration of 5 years. Although previous studies have suggested that the latent HIV reservoir in the resting CD4+ T cell compartment is virologically quiescent in the absence of activating stimuli, evidence has been mounting to suggest that low levels of ongoing viral replication persist and in turn, prolong the overall half-life of HIV in patients receiving antiviral therapy. Here, we demonstrate the persistence of replication-competent virus in CD4+ T cells in a cohort of patients who had received uninterrupted antiviral therapy for up to 9.1 years that rendered them consistently aviremic throughout that time. Surprisingly, substantially higher levels of HIV proviral DNA were found in activated CD4+ T cells when compared with resting CD4+ T cells in the majority of patients we studied. Phylogenetic analyses revealed evidence for cross infection between the resting and activated CD4+ T cell compartments, suggesting that ongoing reactivation of latently infected, resting CD4+ T cells and spread of virus by activated CD4+ T cells may occur in these patients. Such events may allow continual replenishment of the CD4+ T cell reservoir and resetting of the half-life of the latently infected, resting CD4+ T cells despite prolonged periods of aviremia.     

Potent inhibition of HIV-1 by TRIM5-cyclophilin fusion proteins engineered from human components

New World monkeys of the genus Aotus synthesize a fusion protein (AoT5Cyp) containing tripartite motif-containing 5 (TRIM5) and cyclophilin A (CypA) that potently blocks HIV-1 infection. We attempted to generate a human HIV-1 inhibitor modeled after AoT5Cyp, by fusing human CypA to human TRIM5 (hT5Cyp). Of 13 constructs, 3 showed substantial HIV-1–inhibitory activity when expressed in human cell lines. This activity required capsid binding by CypA and correlated with CypA linkage to the TRIM5a capsid-specificity determinant and the ability to form cytoplasmic bodies. CXCR4- and CCR5-tropic HIV-1 clones and primary isolates were inhibited from infecting multiple human macrophage and T cell lines and primary cells by hT5Cyp, as were HIV-2_ROD, SIV_AGMtan, FIV_PET, and a circulating HIV-1 isolate previously reported to be AoT5Cyp resistant. The anti–HIV-1 activity of hT5Cyp was surprisingly more effective than that of the well-characterized rhesus TRIM5α, especially in T cells. hT5Cyp also blocked HIV-1 infection of primary CD4⁺ T cells and macrophages and conferred a survival advantage to these cells without disrupting their function. Extensive attempts to elicit HIV-1 resistance to hT5Cyp were unsuccessful. Finally, Rag2^–/–γc^–/– mice were engrafted with human CD4⁺ T cells that had been transduced by optimized lentiviral vectors bearing hT5Cyp. Upon challenge with HIV-1, these mice showed decreased viremia and productive infection in lymphoid organs and preserved numbers of human CD4⁺ T cells. We conclude that hT5Cyp is an extraordinarily robust inhibitor of HIV-1 replication and a promising anti–HIV-1 gene therapy candidate.     

A simple SCID mouse model for HIV research

The engraftment in SCID mice of human cells susceptible to HIV-1 infection was studied. Repopulation of various tissues by CD4⁺ human cells in transplanted SCID recipients was monitored via FACS, PCR, and immunohistology. Injection into SCID mice resulted in extensive proliferation of CD4⁺ cells in the peritoneal cavity, secondary lymphoid organs and other organs. The SCIDCD4⁺ mouse may provide a simple model system for in vivo research purposes such as HIV-1 infection, anti-HIV drug screening, and vaccine development.     

Beneficial ex vivo immunomodulatory and clinical effects of clarithromycin in COVID-19

IntroductionMacrolide antibiotics have immunomodulatory properties which may be beneficial in viral infections. However, the precise effects of macrolides on T cell responses to COVID, differences between different macrolides, and synergistic effects with other antibiotics have not been explored.MethodsWe investigated the effect of antibiotics (amoxicillin, azithromycin, clarithromycin, and combined amoxicillin with clarithromycin) on lymphocyte intracellular cytokine levels and monocyte phagocytosis in healthy volunteer PBMCs stimulated ex vivo with SARS-CoV-2 S1+2 spike protein. A retrospective cohort study was performed on intensive care COVID-19 patients.ResultsCo-incubation of clarithromycin with spike protein-stimulated healthy volunteer PBMCs ex vivo resulted in an increase in CD8⁺ (p = 0.004) and CD4⁺ (p = 0.007) IL-2, with a decrease in CD8⁺ (p = 0.032) and CD4⁺ (p = 0.007) IL-10. The addition of amoxicillin to clarithromycin resulted in an increase in CD8⁺ IL-6 (p = 0.010), decrease in CD8⁺ (p = 0.014) and CD4⁺ (p = 0.022) TNF-alpha, and decrease in CD8⁺ IFN-alpha (p = 0.038). Amoxicillin alone had no effect on CD4⁺ or CD8⁺ cytokines. Co-incubation of azithromycin resulted in increased CD8⁺ (p = 0.007) and CD4⁺ (p = 0.011) IL-2. There were no effects on monocyte phagocytosis. 102 COVID-19 ICU patients received antibiotics on hospital admission; 62 (61%) received clarithromycin. Clarithromycin use was associated with reduction in mortality on univariate analysis (p = 0.023), but not following adjustment for confounders (HR = 0.540; p = 0.076).ConclusionsClarithromycin has immunomodulatory properties over and above azithromycin. Amoxicillin in addition to clarithromycin is associated with synergistic ex vivo immunomodulatory properties. The potential benefit of clarithromycin in critically ill patients with COVID-19 and other viral pneumonitis merits further exploration.     

HIV-1 Selection by Epidermal Dendritic Cells during Transmission across Human Skin

Macrophage tropic HIV-1 is predominant during the initial viremia after person to person transmission of HIV-1 (Zhu, T., H. Mo, N. Wang, D.S. Nam, Y. Cao, R.A. Koup, and D.D. Ho. 1993. Science. 261:1179–1181.), and this selection may occur during virus entry and carriage to the lymphoid tissue. Human skin explants were used to model HIV-1 selection that may occur at the skin or mucosal surface. Macrophage tropic, but not T cell line tropic strains of HIV-1 applied to the abraded epidermis were recovered from the cells emigrating from the skin explants. Dermis and epidermis were separated by dispase digestion after virus exposure to determine the site of viral selection within the skin. Uptake and transmission to T cells of all HIV-1 isolates was found with the dermal emigrant cells, but only macrophage tropic virus was transferred by emigrants from the epidermis exposed to HIV-1, indicating selection only within the epidermis. CD3⁺, CD4⁺ T cells were found in both the dermal and epidermal emigrant cells. After cell sorting to exclude contaminating T cells, macrophage tropic HIV-1 was found in both the dermal emigrant dendritic cells and in dendritic cells sorted from the epidermal emigrants. These observations suggest that selective infection of the immature epidermal dendritic cells represents the cellular mechanism that limits the initial viremia to HIV-1 that can use the CCR5 coreceptor.     

Histone Deacetylase Inhibitor Romidepsin Inhibits De Novo HIV-1 Infections

Adjunct therapy with the histone deacetylase inhibitor (HDACi) romidepsin increases plasma viremia in HIV patients on combination antiretroviral therapy (cART). However, a potential concern is that reversing HIV latency with an HDACi may reactivate the virus in anatomical compartments with suboptimal cART concentrations, leading to de novo infection of susceptible cells in these sites. We tested physiologically relevant romidepsin concentrations known to reactivate latent HIV in order to definitively address this concern. We found that romidepsin significantly inhibited HIV infection in peripheral blood mononuclear cells and CD4⁺ T cells but not in monocyte-derived macrophages. In addition, romidepsin impaired HIV spreading in CD4⁺ T cell cultures. When we evaluated the impact of romidepsin on quantitative viral outgrowth assays with primary resting CD4⁺ T cells, we found that resting CD4⁺ T cells exposed to romidepsin exhibited reduced proliferation and viability. This significantly lowered assay sensitivity when measuring the efficacy of romidepsin as an HIV latency reversal agent. Altogether, our data indicate that romidepsin-based HIV eradication strategies are unlikely to reseed a latent T cell reservoir, even under suboptimal cART conditions, because romidepsin profoundly restricts de novo HIV infections.     

Antibody-mediated depletion of viral reservoirs is limited in SIV-infected macaques treated early with antiretroviral therapy

The effectiveness of virus-specific strategies, including administered HIV-specific mAbs, to target cells that persistently harbor latent, rebound-competent HIV genomes during combination antiretroviral therapy (cART) has been limited by inefficient induction of viral protein expression. To examine antibody-mediated viral reservoir targeting without a need for viral induction, we used an anti-CD4 mAb to deplete both infected and uninfected CD4⁺ T cells. Ten rhesus macaques infected with barcoded SIVmac239M received cART for 93 weeks starting 4 days after infection. During cART, 5 animals received 5 to 6 anti-CD4 antibody administrations and CD4⁺ T cell populations were then allowed 1 year on cART to recover. Despite profound CD4⁺ T cell depletion in blood and lymph nodes, time to viral rebound following cART cessation was not significantly delayed in anti-CD4–treated animals compared with controls. Viral reactivation rates, determined based on rebounding SIVmac239M clonotype proportions, also were not significantly different in CD4-depleted animals. Notably, antibody-mediated depletion was limited in rectal tissue and negligible in lymphoid follicles. These results suggest that, even if robust viral reactivation can be achieved, antibody-mediated viral reservoir depletion may be limited in key tissue sites.     

NLRP3 inflammasome induces CD4+ T cell loss in chronically HIV-1–infected patients

Chronic HIV-1 infection is generally characterized by progressive CD4⁺ T cell depletion due to direct and bystander death that is closely associated with persistent HIV-1 replication and an inflammatory environment in vivo. The mechanisms underlying the loss of CD4⁺ T cells in patients with chronic HIV-1 infection are incompletely understood. In this study, we simultaneously monitored caspase-1 and caspase-3 activation in circulating CD4⁺ T cells, which revealed that pyroptotic and apoptotic CD4⁺ T cells are distinct cell populations with different phenotypic characteristics. Levels of pyroptosis and apoptosis in CD4⁺ T cells were significantly elevated during chronic HIV-1 infection, and decreased following effective antiretroviral therapy. Notably, the occurrence of pyroptosis was further confirmed by elevated gasdermin D activation in lymph nodes of HIV-1–infected individuals. Mechanistically, caspase-1 activation closely correlated with the inflammatory marker expression and was shown to occur through NLRP3 inflammasome activation driven by virus-dependent and/or -independent ROS production, while caspase-3 activation in CD4⁺ T cells was more closely related to T cell activation status. Hence, our findings show that NLRP3-dependent pyroptosis plays an essential role in CD4⁺ T cell loss in HIV-1–infected patients and implicate pyroptosis signaling as a target for anti–HIV-1 treatment.     

HIV-1 Variation Diminishes CD4 T Lymphocyte Recognition

Effective long-term antiviral immunity requires specific cytotoxic T lymphocytes and CD4⁺ T lymphocyte help. Failure of these helper responses can be a principle cause of viral persistence. We sought evidence that variation in HIV-1 CD4⁺ T helper epitopes might contribute to this phenomenon. To determine this, we assayed fresh peripheral blood mononuclear cells from 43 asymptomatic HIV-1⁺ patients for proliferative responses to HIV-1 antigens. 12 (28%) showed a positive response, and we went on to map dominant epitopes in two individuals, to p24 Gag restricted by human histocompatibility leukocyte antigen (HLA)-DR1 and to p17 Gag restricted by HLA-DRB52c. Nine naturally occurring variants of the p24 Gag epitope were found in the proviral DNA of the individual in whom this response was detected. All variants bound to HLA-DR1, but three of these peptides failed to stimulate a CD4⁺ T lymphocyte line which recognized the index sequence. Antigenic variation was also detected in the p17 Gag epitope; a dominant viral variant present in the patient was well recognized by a specific CD4⁺ T lymphocyte line, whereas several natural mutants were not. Importantly, variants detected at both epitopes also failed to stimulate fresh uncultured cells while index peptide stimulated successfully. These results demonstrate that variant antigens arise in HIV-1⁺ patients which fail to stimulate the T cell antigen receptor of HLA class II–restricted lymphocytes, although the peptide epitopes are capable of being presented on the cell surface. In HIV-1 infection, naturally occurring HLA class II–restricted altered peptide ligands that fail to stimulate the circulating T lymphocyte repertoire may curtail helper responses at sites where variant viruses predominate.