EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment.
ABSTRACT: Suppression of HIV replication by antiretroviral therapy (ART) or host immunity can prevent AIDS but not other HIV-associated conditions including neurocognitive impairment (HIV-NCI). Pathogenesis in HIV-suppressed individuals has been attributed to reservoirs of latent-inducible virus in resting CD4+ T cells. Macrophages are persistently infected with HIV but their role as HIV reservoirs in vivo has not been fully explored. Here we show that infection of conventional mice with chimeric HIV, EcoHIV, reproduces physiological conditions for development of disease in people on ART including immunocompetence, stable suppression of HIV replication, persistence of integrated, replication-competent HIV in T cells and macrophages, and manifestation of learning and memory deficits in behavioral tests, termed here murine HIV-NCI. EcoHIV established latent reservoirs in CD4+ T lymphocytes in chronically-infected mice but could be induced by epigenetic modulators ex vivo and in mice. In contrast, macrophages expressed EcoHIV constitutively in mice for up to 16 months; murine leukemia virus (MLV), the donor of gp80 envelope in EcoHIV, did not infect macrophages. Both EcoHIV and MLV were found in brain tissue of infected mice but only EcoHIV induced NCI. Murine HIV-NCI was prevented by antiretroviral prophylaxis but once established neither persistent EcoHIV infection in mice nor NCI could be reversed by long-acting antiretroviral therapy. EcoHIV-infected, athymic mice were more permissive to virus replication in macrophages than were wild-type mice, suffered cognitive dysfunction, as well as increased numbers of monocytes and macrophages infiltrating the brain. Our results suggest an important role of HIV expressing macrophages in HIV neuropathogenesis in hosts with suppressed HIV replication.
Project description:OBJECTIVE:Almost half of HIV-positive people on antiretroviral therapy have demonstrable mild neurocognitive impairment (HIV-NCI), even when virologically suppressed. Intranasal insulin therapy improves cognition in Alzheimer's disease and diabetes. Here we tested intranasal insulin therapy in a model of HIV-NCI in EcoHIV-infected conventional mice. DESIGN AND METHODS:Insulin pharmacokinetics following intranasal administration to mice was determined by ELISA. Mice were inoculated with EcoHIV to cause NCI; 23 days or 3 months after infection they were treated daily for 9 days with intranasal insulin (2.4?IU/mouse) and examined for NCI in behavioral tests and HIV burdens by quantitative PCR. Some animals were tested for hippocampal neuronal integrity by immunostaining and expression of neuronal function-related genes by real time-quantitative PCR. The effect of insulin treatment discontinuation on cognition and neuropathology was also examined. RESULTS:Intranasal insulin administration to mice resulted in ?IU/ml levels of insulin in cerebrospinal fluid with a half-life of about 2?h, resembling pharmacokinetic parameters of patients receiving 40?IU. Intranasal insulin treatment starting 23 days or 3 months after infection completely reversed NCI in mice. Murine NCI correlated with reductions in hippocampal dendritic arbors and downregulation of neuronal function genes; intranasal insulin reversed these changes coincident with restoration of cognitive acuity, but they returned within 24?h of treatment cessation. Intranasal insulin treatment reduced brain HIV DNA when started 23 but not 90 days after infection. CONCLUSION:Our preclinical studies support the use of intranasal insulin administration for treatment of HIV-NCI and suggest that some dendritic injury in this condition is reversible.
Project description:While HIV kills most of the cells it infects, a small number of infected cells survive and become latent viral reservoirs, posing a significant barrier to HIV eradication. However, the mechanism by which immune cells resist HIV-induced apoptosis is still incompletely understood. Here, we demonstrate that while acute HIV infection of human microglia/macrophages results in massive apoptosis, a small population of HIV-infected cells survive infection, silence viral replication, and can reactivate viral production upon specific treatments. We also found that HIV fusion inhibitors intended for use as antiretroviral therapies extended the survival of HIV-infected macrophages. Analysis of the pro- and anti-apoptotic pathways indicated no significant changes in Bcl-2, Mcl-1, Bak, Bax or caspase activation, suggesting that HIV blocks a very early step of apoptosis. Interestingly, Bim, a highly pro-apoptotic negative regulator of Bcl-2, was upregulated and recruited into the mitochondria in latently HIV-infected macrophages both in vitro and in vivo. Together, these results demonstrate that macrophages/microglia act as HIV reservoirs and utilize a novel mechanism to prevent HIV-induced apoptosis. Furthermore, they also suggest that Bim recruitment to mitochondria could be used as a biomarker of viral reservoirs in vivo.
Project description:HIV-associated cerebrovascular events remain highly prevalent even in the current era of antiretroviral therapy (ART). We hypothesize that low-level HIV replication and associated inflammation endure despite antiretroviral treatment and affect ischemic stroke severity and outcomes. Using the EcoHIV infection model and the middle cerebral artery occlusion as the ischemic stroke model in mice, we present in vivo analysis of the relationship between HIV and stroke outcome. EcoHIV infection increases infarct size and negatively impacts tissue and functional recovery. Ischemic stroke also results in an increase in EcoHIV presence in the affected regions, suggesting post-stroke reactivation that magnifies pro-inflammatory status. Importantly, ART with a high CNS penetration effectiveness (CPE) is more beneficial than low CPE treatment in limiting tissue injury and accelerating post-stroke recovery. These results provide potential insight for treatment of HIV-infected patients that are at risk of developing cerebrovascular disease, such as ischemic stroke.
Project description:HIV associated neurocognitive impairment afflicts roughly half of infected individuals on antiretroviral therapy. This disease currently has no treatment. We have previously shown that type I interferon is induced by and partially controls infection and neuropathogenesis in mice infected by chimeric HIV, EcoHIV. Here we investigate the intentional ligation of the pattern recognition receptor Toll-like receptor 3 (TLR3) by polyinosinic-polycytidylic acid (poly I:C) for its ability to prevent or control infection and associated cognitive disease in EcoHIV infected mice. We tested topical, injection, and intranasal application of poly I:C in mice during primary infection through injection or sexual transmission or in established infection. We measured different forms of HIV DNA and RNA in tissues by real-time PCR and the development of HIV-associated cognitive disease by the radial arm water maze behavioral test. Our results indicate that poly I:C blocks primary EcoHIV infection of mice prior to reverse transcription and reduces established EcoHIV infection. Prevention or control of viral replication by poly I:C prevents or reverses HIV associated cognitive disease in mice. These findings indicate that poly I:C or other innate immune agonists may be useful in control of HIV cognitive disease.
Project description: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.
Project description:Eradication of latent human immunodeficiency virus (HIV) infection is a global health challenge. Reactivation of HIV latency and killing of virus-infected cells, the so-called "kick and kill" or "shock and kill" approaches, are a popular strategy for HIV cure. While antiretroviral therapy (ART) halts HIV replication by targeting multiple steps in the HIV life cycle, including viral entry, integration, replication, and production, it cannot get rid of the occult provirus incorporated into the host-cell genome. These latent proviruses are replication-competent and can rebound in cases of ART interruption or cessation. In general, a very small population of cells harbor provirus, serve as reservoirs in ART-controlled HIV subjects, and are capable of expressing little to no HIV RNA or proteins. Beyond the canonical resting memory CD4<sup>+</sup> T cells, HIV reservoirs also exist within tissue macrophages, myeloid cells, brain microglial cells, gut epithelial cells, and hematopoietic stem cells (HSCs). Despite a lack of active viral production, latently HIV-infected subjects continue to exhibit aberrant cellular signaling and metabolic dysfunction, leading to minor to major cellular and systemic complications or comorbidities. These include genomic DNA damage; telomere attrition; mitochondrial dysfunction; premature aging; and lymphocytic, cardiac, renal, hepatic, or pulmonary dysfunctions. Therefore, the arcane machineries involved in HIV latency and its reversal warrant further studies to identify the cryptic mechanisms of HIV reservoir formation and clearance. In this review, we discuss several molecules and signaling pathways, some of which have dual roles in maintaining or reversing HIV latency and reservoirs, and describe some evolving strategies and possible approaches to eliminate viral reservoirs and, ultimately, cure/eradicate HIV infection.
Project description:Despite the efficient suppression of HIV-1 replication that can be achieved with combined antiretroviral therapy (cART), low levels of type I interferon (IFN-I) signaling persist in some individuals. This sustained signaling may impede immune recovery and foster viral persistence. Here we report studies using a monoclonal antibody to block IFN-?/? receptor (IFNAR) signaling in humanized mice (hu-mice) that were persistently infected with HIV-1. We discovered that effective cART restored the number of human immune cells in HIV-1-infected hu-mice but did not rescue their immune hyperactivation and dysfunction. IFNAR blockade fully reversed HIV-1-induced immune hyperactivation and rescued anti-HIV-1 immune responses in T cells from HIV-1-infected hu-mice. Finally, we found that IFNAR blockade in the presence of cART reduced the size of HIV-1 reservoirs in lymphoid tissues and delayed HIV-1 rebound after cART cessation in the HIV-1-infected hu-mice. We conclude that low levels of IFN-I signaling contribute to HIV-1-associated immune dysfunction and foster HIV-1 persistence in cART-treated hosts. Our results suggest that blocking IFNAR may provide a potential strategy to enhance immune recovery and reduce HIV-1 reservoirs in individuals with sustained elevations in IFN-I signaling during suppressive cART.
Project description:Although antiretroviral therapy (ART) greatly suppresses HIV replication, lymphoid tissues remain a sanctuary site where the virus may replicate. Tracking the earliest steps of HIV spread from these cellular reservoirs after drug cessation is pivotal for elucidating how infection can be prevented. In this study, we developed an in vivo model of HIV persistence in which viral replication in the lymphoid compartments of humanized mice was inhibited by the HIV reverse transcriptase inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) to very low levels, which recapitulated ART-suppression in HIV-infected individuals. Using a combination of RNAscope in situ hybridization (ISH) and immunohistochemistry (IHC), we quantitatively investigated the distribution of HIV in the lymphoid tissues of humanized mice during active infection, EFdA suppression, and after drug cessation. The lymphoid compartments of EFdA-suppressed humanized mice harbored very rare transcription/translation-competent HIV reservoirs that enable viral rebound. Our data provided the visualization and direct measurement of the early steps of HIV reservoir expansion within anatomically intact lymphoid tissues soon after EFdA cessation and suggest a strategy to enhance therapeutic approaches aimed at eliminating the HIV reservoir.
Project description:The human nuclear envelope proteins emerin and lamina-associated polypeptide 2alpha (LAP2alpha) have been proposed to aid in the early replication steps of human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). However, whether these factors are essential for HIV-1 or MLV infection has been questioned. Prior studies in which conflicting results were obtained were highly dependent on RNA interference-mediated gene silencing. To shed light on these contradictory results, we examined whether HIV-1 or MLV could infect primary cells from mice deficient for emerin, LAP2alpha, or both emerin and LAP2alpha. We observed HIV-1 and MLV infectivity in mouse embryonic fibroblasts (MEFs) from emerin knockout, LAP2alpha knockout, or emerin and LAP2alpha double knockout mice to be comparable in infectivity to wild-type littermate-derived MEFs, indicating that both emerin and LAP2alpha were dispensable for HIV-1 and MLV infection of dividing, primary mouse cells. Because emerin has been suggested to be important for infection of human macrophages by HIV-1, we also examined HIV-1 transduction of macrophages from wild-type mice or knockout mice, but again we did not observe a difference in susceptibility. These findings prompted us to reexamine the role of human emerin in supporting HIV-1 and MLV infection. Notably, both viruses efficiently infected human cells expressing high levels of dominant-negative emerin. We thus conclude that emerin and LAP2alpha are not required for the early replication of HIV-1 and MLV in mouse or human cells.
Project description:Despite the success of antiretroviral therapy (ART), eradication of HIV-1 from brain reservoirs remains elusive. HIV-1 brain reservoirs include perivascular macrophages that are behind the blood-brain barrier and difficult to access by ART. Macrophages express transcription factor FOXO3a and the TNF superfamily cytokine TRAIL, which are known to target HIV-1-infected macrophages for viral inhibition. ONC201 is a novel and potent FOXO3a activator capable of inducing TRAIL. It can cross the blood-brain barrier, and has shown antitumor effects in clinical trials. We hypothesized that activation of FOXO3a/TRAIL by ONC201 will inhibit HIV-1 replication in macrophages. Using primary human monocyte-derived macrophages, we demonstrated that ONC201 dose-dependently decreased replication levels of both HIV-1 laboratory strain and primary strains as determined by HIV-1 reverse transcriptase activity assay. Consistent with data on HIV-1 replication, ONC201 also reduced intracellular and extracellular p24, viral RNA, and integrated HIV-1 DNA in infected macrophages. Blocking TRAIL or knockdown of FOXO3a with siRNA reversed ONC201-mediated HIV-1 suppression, suggesting that ONC201 inhibits HIV-1 through FOXO3a and TRAIL. The anti-HIV-1 effect of ONC201 was further validated in vivo in NOD/scid-IL-2Rgcnull mice. After intracranial injection of HIV-1-infected macrophages into the basal ganglia, we treated the mice daily with ONC201 through intraperitoneal injection for six days. ONC201 significantly decreased p24 levels in both the macrophages and the brain tissues, suggesting that ONC201 suppresses HIV-1 in vivo. Therefore, ONC201 can be a promising drug candidate to combat persistent HIV-1 infection in the brain.