Eradication of HIV-1 by the “kick and kill” strategy requires reactivation of latent virus to cause death of infected cells by either HIV-induced or immune-mediated apoptosis. To date this strategy has been unsuccessful, possibly due to insufficient cell death in reactivated cells to effectively reduce HIV-1 reservoir size. As a possible cause for this cell death resistance, we examined whether leading latency reversal agents (LRAs) affected apoptosis sensitivity of CD4 T cells. Multiple LRAs of different classes inhibited apoptosis in CD4 T cells. Protein kinase C (PKC) agonists bryostatin-1 and prostratin induced phosphorylation and enhanced neutralizing capability of the anti-apoptotic protein BCL2 in a PKC-dependent manner, leading to resistance to apoptosis induced by both intrinsic and extrinsic death stimuli. Furthermore, HIV-1 producing CD4 T cells expressed more BCL2 than uninfected cells, both in vivo and after ex vivo reactivation. Therefore, activation of BCL2 likely contributes to HIV-1 persistence after latency reversal with PKC agonists. The effects of LRAs on apoptosis sensitivity should be considered in designing HIV cure strategies predicated upon the “kick and kill” paradigm.

The anti-apoptotic protein BCL2 inhibits death of HIV-infected cells. Previously, we have shown that the BCL2 inhibitor venetoclax selectively kills acutely HIV-infected cells and reduces HIV DNA in latently infected CD4 T cells ex vivo after reactivation with αCD3/αCD28. However, there is a need to identify a combination therapy with venetoclax and a clinically relevant latency reversal agent. Ixazomib is an oral proteasome inhibitor which we have shown reactivates latent HIV and predisposes reactivated cells to cell death. Here, we determined that the combination of venetoclax and ixazomib kills more latently HIV-infected cells and leads to greater reduction in HIV replication compared to either treatment alone in vitro in a T cell model. However, combination treatment of ex vivo CD4 T cells from ART-suppressed, HIV-positive participants resulted in unanticipated and unacceptable non-specific toxicity in primary cells. Therefore, while we show proof of concept that multiple agents can enhance selective killing of HIV infected cells, the combination of venetoclax and ixazomib has unacceptable toxicity in primary cells, and so further investigation is needed to identify a clinically relevant latency reversal agent to combine with venetoclax as a novel strategy to reduce the size of the HIV reservoir. IMPORTANCE: A cure for HIV would require eliminating cells that contain the virus in a latent form from the body. Current antiretroviral medications are unable to rid the body of latently infected cells. Here we show that a combination of investigational agents – ixazomib plus venetoclax- which reactivate latent virus, and predispose infected cells to apoptosis, may reduce latent virus in a T cell model, but at the expense of non-specific toxicity in primary cells.

Background Gastrointestinal (GI) motility is a complex physiological process that is critical for normal GI function. Disruption of GI motility frequently occurs in GI diseases or as side effects of therapeutics. Whole gut transit measurements, like carmine red leading‐edge transit, in mice form the cornerstone of in vivo preclinical GI motility studies. Method We have developed an easily achievable, labor‐saving method to measure whole gut transit time in mice. This approach uses inexpensive, commercially available materials to monitor pellet production over time via high definition cameras capturing time‐lapse video for offline analysis. Key Result We describe the assembly of our automated gut transit setup and validate this approach by comparing the results with loperamide to delay transit and conventional transit measurements. We demonstrate that compared to the control group, the loperamide group had slowed transit, evidenced by a decrease in total pellet production and prolonged whole gut transit time. The control group had an extended transit time compared with the results reported in the literature. Whole gut transit rates accelerated to times comparable to the literature by disrupting cages every 10‐15 min to imitate the conventional approach, suggesting that disruption affects the assay and supports the use of an automated approach. Conclusion & Inferences A novel automated, inexpensive, and easily assembled whole gut transit setup is labor‐saving and allows minimal disruption to animal behavior compared with the conventional approach.

This study is the first to examine the applicability of BCL-2 inhibition in the setting of active HIV infection in vivo . Furthermore, this study demonstrates that venetoclax significantly enhances target cell killing induced by Fas ligand, TRAIL, and perforin/granzyme B and synergistically enhances autologous NK and CD8 cells’ killing of target cells.