Short-term Addition of Efavirenz to Induce CARD8-mediated Reduction of Persistent Nonsuppressible HIV Viremia in People With High Adherence to ART.

Background:

Combination antiretroviral therapies (cART) have transformed HIV into a chronic manageable health condition for many PLWH.

Despite potent cART, the vast majority of PLWH have residual of viremia that remains below the limit of detection of current clinical assays of 20 copies/ml plasma but can often be detected by highly sensitive assays. A number of studies report that PLWH compliant with cART, without drug resistance mutations, have ongoing detectable low level viremia (also referred to as nonsuppressible viremia, NSV), usually from 20-400 copies/ml, ranging in frequency from 1 in 250 to 9% and 10%. In Ontario, over the past two years, 11.9% of PLWH had two or more consecutive values of low level viremia (LLV) (personal communication Vanessa Tran, Ontario Public Health labs). Although low level viremia on compliant cART is felt not to reflect treatment failure, there is evidence that it may not be clinically insignificant. Recent studies of large cohorts predict a 2-3 fold chance of virologic failure and increased all cause mortality and non-AIDS events with LLV. White et. al. showed that detectable LLV is due to expansion of clones containing both defective and replication competent virus, that are proliferating likely in response to antigens. Thus, individuals with LLV, likely have very large populations of proliferated clones in their reservoirs. Whether this higher level of daily virus production sustains inflammation and immune activation is poorly understood. Moreover, the clinical short-term significance of these findings is unclear, however, these individuals represent a unique group to further study cure strategies that decrease the reservoir size.

Recent observations, suggest that some NNRTIs (especially efavirenz) could be repurposed to kill these virus-producing cells. The HIV protease enzyme is part of a larger gag-pol protein precursor that is produced as an inactive monomer in the cytoplasm of infected cells after HIV transcription. In order for the HIV protease to be active, it must dimerize, and this dimerization only happens within the virus particle that has budded off the cell, which results in mature, infectious viral particles. It has been shown that some NNRTIs such as efavirenz can induce intracellular Gag-Pol dimerization and premature protease activation in the cytosol prior to budding, which can trigger the CARD8 (Caspase recruitment domain protein 8) inflammasome, resulting in death of HIV-1-producing cells via pyroptosis. This process occurs when the compound binds to the RT domain of the Gag-Pol polyprotein, leading to a conformational change that induces its dimerization within the cell cytoplasm rather than in the virus particle, and the consequent autocatalytic activity of the protease enzyme. Thus EFV can induce Gag-pol dimerization in the cytoplasm, which triggers CARD8 sensing and inflammasome activation Intra-cytoplasmic protease-CARD8 activation would be blocked by the presence of protease inhibitors (eg. indinavir). Preliminary data from the Simonetti lab (one of the co-investigators) using CD4+ T cells from people on cART experiencing non-suppressive, but low level viremia, have shown that micromolar concentrations of efavirenz (EFV) can significantly reduce virus production upon strong T cell activation by CD3/CD28 stimulation, due to CARD8 sensing of HIV protease activity . In other words, these virus infected cells were killed in the presence of EFV during immune activation. In control experiments, with the addition of the protease inhibitor lopinavir, the effect of EFV is completely abrogated. Experiments were conducted with EFV at 5uM, which can be reached in plasma in a large fraction of individuals with the recommended full dose (600mg q.d.). Previous pharmacokinetics studies showed that EFV reaching plasma concentration above the EC50 required for its killing effect (1uM) and reaches even higher concentrations in tissues. Recent work reported NNRTI-like molecules with comparable antiviral activity to EFV but significantly higher potency in inducing dimerization and CARD8-mediated killing. However, such compounds are still in the early stages of pre-clinical investigation and their pharmacokinetic profile and activity in vivo has not been explored. This effect, of killing HIV infected cells by pyroptosis after CARD8 sensing has been termed TACK (targeted activator of cell killing). EFV represents the best TACK molecule to test whether the CARD8-inflammasome can be harnessed as a therapeutic option in people living with HIV on ART EFV is recognized as the most promising and potent FDA approved NNRTI compound for this purpose. In the proposed study, cART intensification with EFV is used to induced CARD8 sensing and not to block viral replication.

Hypothesis: