L10 and its role on HIV reservoir maintenance
ART has reduced HIV-related morbidities and mortality for the majority of infected individuals, however a therapeutic regimen able to eradicate or functionally cure HIV infection does not currently exist. Thus, lifelong administration of ART is necessary, placing an inordinate burden on individuals and public health systems. Furthermore, while the benefits of ART are unquestionable, long-term treated HIV-infected subjects continue to have significantly shorter life spans. For these reasons, eradicating or at least functionally curing HIV infection would have an extraordinary impact on global health. Major obstacles to cure HIV infection include low levels of continuous viral replication, particularly in tissues, the presence of a small pool of long-lived latently infected CD4 T cells including the recently identified Tfh reservoir in B cell sanctuaries and Tregs, and functional exhaustion of antiviral T cells due in part to Tregs, which are unable to clear the virus. Importantly, these contributors may be triggering and sustaining each other, thus creating a vicious cycle that maintains HIV persistence during ART. The goal of this project is to define the mechanisms of action that lead to the strong positive correlation between IL-10 and the HIV reservoir. Definitive demonstration that IL-10 regulates HIV persistence will be provided by examining the impact of IL-10 blockade on virus persistence in ART-treated, SIV-infected rhesus macaques (RMs). The rationale for this intervention comes from preliminary results showing that IL-10 1) enhances the survival of infected cells leading to the persistence of cells producing the virus, 2) enhances the survival of activated T cells leading to the persistence of target cells for HIV infection, 3) upregulates PD-1 expression and other immune checkpoint blockers (ICB) which we have shown to be involved in the establishment of latency, 4) upregulates the expression of Histone Deacetylases (HDAC) also known to have a direct impact on the induction of HIV latency 5) impedes T cell function leading to immunosenescence. Lastly, plasma IL-10 titers directly correlate with latent HIV reservoir size in ART-treated SIV infected RMs as well as in humans. Based on these findings, the project hypothesis is that IL-10 produced by B cells, innate immune cells, and type-1 regulatory (Tr1) cells trigger T follicular helper cell (Tfh) differentiation leading to the establishment of HIV/SIV persistence in B cell follicles – thereby compromising the anti-HIV immune response required to eliminate virus producing cells.
HIV pathogenesis outcomes (CD4 counts and HIV reservoir) and associated immune parameters new therapeutic targets: Despite durable suppression of HIV replication, anti-retroviral therapy (ART) is not curative and the virus persists indefinitely. ART fails to attain immune reconstitution in many people (“immunologic non-responders”, or INRs); this failure is associated with increased risk of non-AIDS morbidity and mortality. Current therapeutic interventions to eradicate latent HIV (“reservoir”) and restore immune function have been unsuccessful, and the mechanisms governing these outcomes remain unidentified. On this project, a comprehensive systems biology approach is used to identify cells, molecular pathways and therapeutic targets for reconstituting immunity and eradicating HIV. So far, senescence and inflammation characterized two INR groups, and independently were associated to poor CD4 T cell reconstitution. A gene-based classifier predicted each group with 80% accuracy. Senescent INRs had higher frequency of memory CD4 T cells with inducible multi-spliced-HIV RNA (“inducible-HIV”) compared to inflammatory INRs and immunologic responders (IRs). Bacterial metabolites that regulate differentiation of TGF-β producing Tregs3, pathways downstream of PD-1/TGF-β signaling and the transcription factors (TFs) FOXO3A, FOXO4 and IRF-3, known to regulate senescence, were correlated with low CD4 T cell counts and increased inducible-HIV in senescent INRs. Pathways associated with inflammation, apoptosis and which are targets of the pro-inflammatory TFs HIF-1α, NF-κB and IRF-7 were correlated with low CD4 T cell counts and increased inducible-HIV in inflammatory INRs. Inducible-HIV was driven by IL-1 signaling and metabolites of the glycolytic pathway - a hallmark of inflammation in IRs. Our results identify interventions specific to each INR group targeting senescence (PD- 1 and TGF-) or inflammation (IL1) to restore immunity in ART-treated HIV infected subjects. These strategies have shown safety, partial efficacy in cancer and may prove to be crucial for HIV eradication.
NLRX1 and its mechanisms leading to HIV infection susceptibility: In a prior work by the group, show that virus can rapidly disseminate following mucosal SIV infection of rhesus monkeys and trigger components of the inflammasome, both at the site of inoculation and at early sites of distal virus spread. A proinflammatory signature was observed 24h following inoculation and this signature lacked antiviral restriction factors. The early innate response included expression of NLRX1, which inhibits antiviral responses, and activation of the TGF-β pathway, which negatively regulates adaptive immune responses was observed, suggesting that the virus triggers specific host mechanisms that suppress the generation of antiviral innate and adaptive immune responses in the first few days of infection, facilitating its own replication. In the current project we have screened the cytokines triggered early on after HIV infection as well as later cytokines, secreted by innate and adaptive cells and analyzed NLRX1 induction in different cell subsets, as well overall anti-viral responses and impacts on T cell metabolic status. Based on the impact of the cytokines on the functional status of the cells, they were later evaluated on their ability to promote or decrease HIV infection. The ongoing multiparametric analysis can bring important targets in how to interfere with HIV infection and HIV spreading.
TFH cells as a major HIV reservoir compartment: Effective antiretroviral therapy (ART) has prevented the progression to AIDS and reduced HIV-related morbidities and mortality for the majority of infected individuals. However, a lifelong administration of ART is necessary, placing an inordinate burden on individuals and public health systems. Therefore, discovering therapeutic regimens able to eradicate or functionally cure HIV infection of great importance. ART interruption leads to viral rebound highlighting the establishment and maintenance of a latent viral reservoir compartment even under long-term treatment. Follicular helper CD4 T cells (TFH) have been reported as a major cell compartment contributing to viral persistence, consequent to their susceptibility to infection and ability to release replication competent new virions. BCL6, the master transcription factor of TFHs is known to suppress the expression of restriction factors making these cells more susceptible to infection aside preserving intact provirus by the lack of restriction factors. In this project we hypothesize that CRISPR-CAS9 of BLC6 will make T cells resistant to HIV infection, and the ones that get. Infected will harbor defective provirus.
Ex vivo reservoir measurement (intact x defective provirus) and reactivation HIV cure: HIV virus reservoir is established early on after infection. ART interruption leads to virus rebound in most of the cases proving the persistence of cells harboring intact provirus. So far, no established technique is able to deeply evaluated the quality of the HIV reservoir on HIV infected patients, neither to identify which subset contributes the most for its persistence. In this project, we developed a flow cytometry assay that is able to detect reactivated virus in different cell subsets in cells isolated from HIV infected donors. Additionally, by coupling the reactivation based on gag expression with HIV-envelope detection, we can imply which reservoir is indeed able to release new virions. The usage of Envelope antibodies with different posttranslational modifications, will allow us to imply the integrity of the provirus, that along with QVOA assay, will bring a deeper understanding of the HIV reservoir on these subjects, and guide for new therapeutic strategies to eliminate the virus from specific cells.
In vitro screening for T cell differentiation components and HIV reactivation to expunge HIV reservoir - shock and kill strategies:HIV-1 persists as a latent reservoir in individuals on ART in central (TCM), transitional (TTM) and effector memory (TEM) CD4+ T cells. Phenotypic analysis of latency reversal in CD4+ T cells from virally suppressed individuals identified the TEM subset as the largest contributor to the inducible reservoir. Differential responses of memory CD4+ T cell subsets to latency reversing agents (LRAs) demonstrated heightened expression of transcriptional pathways associated with differentiation, acquisition of effector function and cell cycle entry correlated with HIV gene expression. In vitro modeling of the HIV latent reservoir in memory CD4+ T cell subsets can help us to identify LRAs that impact on the differentiation status of T cells and make them reactivable. Additionally, using compounds that can induce the metabolic status of effector memory cells can also lead to reservoir reactivation and reach and HIV functional cure.
Opioid antagonism, anti-viral immunity rescue and HIV reservoir decay: HIV-infection is a public health crisis, with 25-57% of HIV+ subjects dependent on opioids. Among people living with HIV (PLHIV) treated with highly active antiretroviral therapy (ART), illicit drug use increases risk of opportunistic infections and progression to AIDS and death, independent from ART adherence. These epidemiologic findings strongly suggest a detrimental effect of opioids on immune function in PLHIV. The interplay between opioids and HIV may accelerate intestinal epithelial damage to promote lipopolysaccharide (LPS) translocation and exacerbate HIV-associated immune dysregulation, chronic inflammation and coagulopathy, and predispose to non-AIDS related mortality and morbidities such as cardiovascular disease and stroke. Opioids may also drive HIV disease progression by interfering with anti-viral mechanisms to facilitate HIV replication. The scientific premise to support that opioid use disorders (OUD), or opioid medications used to treat OUD, promote immune dysregulation among PLHIV by increasing the burden of LPS-mediated immune activation and disrupting the TLR-4 pathway, include data illustrating that opioids directly bind and activate the TLR-4 complex. In addition to direct activation of the TLR-4 pathway, opioids increase the burden of LPS-mediated immune activation by: 1) damaging intestinal mucosa to promote translocation of enteric gram-negative bacteria; and 2) interfering with phagocytic clearance of LPS. Opioids, including morphine, methadone, and heroin, may also drive HIV-disease progression by suppressing type-I interferons and antiviral miRNAs that normally limit viral replication. Opioid-agonists, such as methadone or buprenorphine, are used to treat OUD among PLHIV, and these therapies could exacerbate HIV-associated immune dysregulation and impede viral control. In this project, we will address a primary question of whether or not two different treatment approaches to OUD, long-term opioid antagonism with extended release naltrexone (XR-NTX), versus treatment-as-usual (TAU) with opioid agonists, differentially impact immune dysregulation and viral control among PLHIV. We propose that the long acting opioid-antagonist XR-NTX, a highly effective treatment for OUD, will be associated with reductions in gut permeability, immune activation, exhaustion, and coagulopathy, and gains in T cell anti-viral capacity and will promote early viral control and reduction in the inducible HIV reservoir among PLHIV with OUD (HIV+/OUD+) as they undergo ART combined with OUD therapy.