Mechanistic approaches for reactivation and elimination of latent HIV-1

Although HIV-1 can be adequately controlled by combination antiretroviral therapy (cART), it is still an incurable disease due in part to the latent reservoir that persists in resting CD4+ T cells (rCD4s). Latently infected rCD4s do not actively express HIV-1, allowing them to evade immune surveillance, but their ability to reinitiate viral expression maintains this barrier to a cure. Shock-and-kill, a prominent strategy for HIV-1 eradication, requires the reactivation of latent HIV-1 gene expression. Global T cell activation is a well-characterized means of inducing HIV-1 transcription, but is considered too toxic for clinical applications. Here, we explore a strategy involving a combination of immune activation and the immunosuppressive mTOR inhibitor rapamycin. We show that rapamycin downregulates markers of toxicity, including pro-inflammatory cytokine release and cellular proliferation, induced by potent T cell activating agents. Using an ex vivo assay for HIV-1 mRNA in rCD4s from infected individuals on antiretroviral therapy, we demonstrate that despite this immunomodulatory effect, rapamycin does not affect HIV-1 gene expression induced by T cell activation. In contrast the immunosuppressant cyclosporin, a calcineurin inhibitor, robustly inhibits HIV-1 reactivation. Importantly, cytotoxic T lymphocyte (CTL) recognition and killing of infected cells is also not impaired by rapamycin treatment. These findings are also being extended to HIV-positive individuals requiring solid organ transplant, which acts an in vivo immune activating stimulus without the need for additional intervention. A previous study has shown that a group of these patients who received rapamycin for immunosuppressive maintenance had lower levels of HIV-1 DNA in peripheral blood cells when compared with patients receiving a calcineurin inhibitor such as cyclosporin. This previous finding is in support of our findings, and suggests that rapamycin may allow for shock-and-kill in vivo. Here we compare pre- and post- transplant reservoir size using a novel assay, the quantitative viral induction assay (QVIA), and discuss the surprising result of an increase in the size of the HIV-1 latent reservoir post-transplant. Alternatively, compounds that may not elicit T cell activation phenotypes, and are not necessarily thought to act via any of the same pathways as T cell activation, may effectively stimulate HIV-1 transcription. Several latency reversing agents (LRAs) have activity in primary rCD4s isolated from patients on cART, including PKC agonists, HDAC inhibitors (HDACi), and bromodomain and extra-terminal (BET) bromodomain inhibitors. Of particular interest are HDACi, as there are several drugs in this class approved for clinical use. Although the activity of HDACi on HIV-1 expression is well documented; they act synergistically with other LRAs in primary cells and have high activity alone in several cell models for latency; these drugs have a broad scope of activities and their mechanism of action in this context is controversial. If a mechanism can be identified, a more targeted approach to reactivate latent HIV-1 may be developed. We therefore used a combination of gene expression and transcription factor array studies in the context of both primary and model cells to further understand how HDACi may be acting upon HIV-1 transcription. Using specific small molecule inhibitors we have identified a potential role for p21 in HDACi activity on latent HIV-1