The HIV latency reversing agent HODHBt inhibits the phosphatases PTPN1 and PTPN2.

Nonreceptor tyrosine phosphatases (NTPs) play an important role in regulating protein phosphorylation and have been proposed as attractive therapeutic targets for cancer and metabolic diseases. We have previously identified that 3-Hydroxy-1,2,3-benzotriazin-4(3H)-one (HODHBt) enhanced STAT activation upon cytokine stimulation, leading to increased reactivation of latent HIV and effector functions of NK and CD8 T cells. Here, we demonstrate that HODHBt interacted with and inhibited the NTPs PTPN1 and PTPN2 through a mixed inhibition mechanism. We also confirm that PTPN1 and PTPN2 specifically controlled the phosphorylation of different STATs. The small molecule ABBV-CLS-484 (AC-484) is an active site inhibitor of PTPN1 and PTPN2 currently in clinical trials for advanced solid tumors. We compared AC-484 and HODHBt and found similar effects on STAT5 and immune activation, albeit with different mechanisms of action leading to varying effects on latency reversal. Our studies provide the first specific evidence to our knowledge that enhancing STAT phosphorylation via inhibition of PTPN1 and PTPN2 is an effective tool against HIV.

HIV-1 latency reversal and immune enhancing activity of IL-15 is not influenced by sex hormones.

The role of different biological variables including biological sex, age, and sex hormones in Human immunodeficiency virus (HIV) cure approaches is not well understood. The γc-cytokine IL-15 is a clinically relevant cytokine that promotes immune activation and mediates HIV reactivation from latency. In this work, we examined the interplay that biological sex, age, and sex hormones 17ß-estradiol, progesterone, and testosterone may have on the biological activity of IL-15. We found that IL-15-mediated CD4+ T cell activation was higher in female donors than in male donors. This difference was abrogated at high 17ß-estradiol concentration. Additionally, there was a positive correlation between age and both IL-15-mediated CD8+ T cell activation and IFN-γ production. In a primary cell model of latency, biological sex, age, or sex hormones did not influence the ability of IL-15 to reactivate latent HIV. Finally, 17ß-estradiol did not consistently affect reactivation of translation-competent reservoirs in CD4+ T cells from people living with HIV who are antiretroviral therapy (ART) suppressed. Our study has found that biological sex and age, but not sex hormones, may influence some of the biological activities of IL-15. Understanding how different biological variables may affect HIV cure therapies will help us evaluate current and future clinical trials aimed toward HIV cure in diverse populations.

An Ultrasensitive p24 Assay to Measure HIV-1 in Diverse Biological Matrixes.

Assays to study HIV persistence are crucial to evaluate therapeutic strategies aimed toward an HIV cure. Several assays have been developed to date that rely on the measurement of nucleic acids. In recent years, the advancement of ultrasensitive technologies for the detection of proteins has improved our understanding of the role of translation-competent reservoirs in HIV persistence. In this chapter, we describe the development of an ultrasensitive p24 ELISA that uses planar array technology. This assay allows for the detection of HIV-1 p24 in the low fg/ml range in different biological matrixes, including cell lysates. This assay can be used to investigate the efficacy of latency reversing agents to reactivate HIV or to evaluate the persistence of translation-competent reservoirs in people living with HIV (PWH) in cells or diverse biological fluids.

AP-1/c-Fos supports SIV and HIV-1 latency in CD4 T cells infected in vivo.

Persistent HIV-1 reservoirs of infected CD4 T cells are a major barrier to HIV-1 cure, although the mechanisms by which they are established and maintained in vivo remain poorly characterized. To elucidate host cell gene expression patterns that govern virus gene expression, we analyzed viral RNA+ (vRNA) CD4 T cells of untreated simian immunodeficiency virus (SIV)-infected macaques by single-cell RNA sequencing. A subset of vRNA+ cells distinguished by spliced and high total vRNA (7-10% of reads) expressed diminished FOS, a component of the Activator protein 1 (AP-1) transcription factor, relative to vRNA-low and -negative cells. Conversely, FOS and JUN, another AP-1 component, were upregulated in HIV DNA+ infected cells compared to uninfected cells from people with HIV-1 on suppressive therapy. Inhibiting c-Fos in latently infected primary cells augmented reactivatable HIV-1 infection. These findings implicate AP-1 in latency establishment and maintenance and as a potential therapeutic target to limit HIV-1 reservoirs.

IL-15 and N-803 for HIV Cure Approaches.

In spite of the advances in antiretroviral therapy to treat HIV infection, the presence of a latent reservoir of HIV-infected cells represents the largest barrier towards finding a cure. Among the different strategies being pursued to eliminate or reduce this latent reservoir, the γc-cytokine IL-15 or its superagonist N-803 are currently under clinical investigation, either alone or with other interventions. They have been shown to reactivate latent HIV and enhance immune effector function, both of which are potentially required for effective reduction of latent reservoirs. In here, we present a comprehensive literature review of the different in vitro, ex vivo, and in vivo studies conducted to date that are aimed at targeting HIV reservoirs using IL-15 and N-803.

Aminobisphosphonates reactivate the latent reservoir in people living with HIV-1.

Antiretroviral therapy (ART) is not curative due to the existence of cellular reservoirs of latent HIV-1 that persist during therapy. Current research efforts to cure HIV-1 infection include "shock and kill" strategies to disrupt latency using small molecules or latency-reversing agents (LRAs) to induce expression of HIV-1 enabling cytotoxic immune cells to eliminate infected cells. The modest success of current LRAs urges the field to identify novel drugs with increased clinical efficacy. Aminobisphosphonates (N-BPs) that include pamidronate, zoledronate, or alendronate, are the first-line treatment of bone-related diseases including osteoporosis and bone malignancies. Here, we show the use of N-BPs as a novel class of LRA: we found in ex vivo assays using primary cells from ART-suppressed people living with HIV-1 that N-BPs induce HIV-1 from latency to levels that are comparable to the T cell activator phytohemagglutinin (PHA). RNA sequencing and mechanistic data suggested that reactivation may occur through activation of the activator protein 1 signaling pathway. Stored samples from a prior clinical trial aimed at analyzing the effect of alendronate on bone mineral density, provided further evidence of alendronate-mediated latency reversal and activation of immune effector cells. Decay of the reservoir measured by IPDA was however not detected. Our results demonstrate the novel use of N-BPs to reverse HIV-1 latency while inducing immune effector functions. This preliminary evidence merits further investigation in a controlled clinical setting possibly in combination with therapeutic vaccination.

The latency-reversing agent HODHBt synergizes with IL-15 to enhance cytotoxic function of HIV-specific T cells.

IL-15 is under clinical investigation toward the goal of curing HIV infection because of its abilities to reverse HIV latency and enhance immune effector function. However, increased potency through combination with other agents may be needed. 3-Hydroxy-1,2,3-benzotriazin-4(3H)-one (HODHBt) enhances IL-15-mediated latency reversal and NK cell function by increasing STAT5 activation. We hypothesized that HODHBt would also synergize with IL-15, via STAT5, to directly enhance HIV-specific cytotoxic T cell responses. We showed that ex vivo IL-15 + HODHBt treatment markedly enhanced HIV-specific granzyme B-releasing T cell responses in PBMCs from antiretroviral therapy-suppressed (ART-suppressed) donors. We also observed upregulation of antigen processing and presentation in CD4+ T cells and increased surface MHC-I. In ex vivo PBMCs, IL-15 + HODHBt was sufficient to reduce intact proviruses in 1 of 3 ART-suppressed donors. Our findings reveal the potential for second-generation IL-15 studies incorporating HODHBt-like therapeutics. Iterative studies layering on additional latency reversal or other agents are needed to achieve consistent ex vivo reservoir reductions.

Deep characterization of human γδ T cell subsets defines shared and lineage-specific traits.

Under non-pathological conditions, human γδ T cells represent a small fraction of CD3+ T cells in peripheral blood (1-10%). They constitute a unique subset of T lymphocytes that recognize stress ligands or non-peptide antigens through MHC-independent presentation. Major human γδ T cell subsets, Vδ1 and Vδ2, expand in response to microbial infection or malignancy, but possess distinct tissue localization, antigen recognition, and effector responses. We hypothesized that differences at the gene, phenotypic, and functional level would provide evidence that γδ T cell subpopulations belong to distinct lineages. Comparisons between each subset and the identification of the molecular determinants that underpin their differences has been hampered by experimental challenges in obtaining sufficient numbers of purified cells. By utilizing a stringent FACS-based isolation method, we compared highly purified human Vδ1 and Vδ2 cells in terms of phenotype, gene expression profile, and functional responses. We found distinct genetic and phenotypic signatures that define functional differences in γδ T cell populations. Differences in TCR components, repertoire, and responses to calcium-dependent pathways suggest that Vδ1 and Vδ2 T cells are different lineages. These findings will facilitate further investigation into the ligand specificity and unique role of Vδ1 and Vδ2 cells in early immune responses.

Antibody and T-cell responses to coronavirus disease 2019 vaccination in common variable immunodeficiency and specific antibody deficiency.

BACKGROUND: Clinical trials of the mRNA coronavirus disease 2019 (COVID-19) vaccines excluded individuals with primary antibody deficiencies. OBJECTIVE: To evaluate whether antibody and T-cell responses to mRNA COVID-19 vaccination in patients with common variable immunodeficiency (CVID) and specific antibody deficiency (SAD) were comparable to those in healthy controls. METHODS: We measured antibody responses against the spike glycoprotein and the receptor-binding domain (RBD) in addition to severe acute respiratory syndrome coronavirus 2 specific T-cell responses using peripheral blood mononuclear cells 2 to 8 weeks after the subjects completed the primary 2-dose vaccine series. RESULTS: The study comprised 12 patients with CVID, 7 patients with SAD, and 10 controls. Individuals with CVID had lower immunoglobulin (Ig) G and Ig A levels against spike glycoprotein than did both individuals with SAD (P = .27 and P = .01, respectively) and controls (P = .01 and P = .004, respectively). The CVID group developed lower IgG titers against the RBD epitope than did the control group (P = .01). Participants with CVID had lower neutralizing titers than did the control group (P = .002). All participants with SAD developed neutralizing titers. All 3 groups (SAD, CVID, and control) developed antigen-specific CD4+ and CD8+ T-cell responses after vaccination. CONCLUSION: Our results suggest that patients with CVID may have impaired antibody responses to COVID-19 vaccination but intact T-cell responses, whereas patients with SAD would be expected to have both intact antibody and T-cell responses to vaccination.