Abstract Body

We are developing a novel HIV eradication strategy that combines proviral reactivation, ex vivo stimulation, expansion of natural killer (NK) cells, and enhancement of NK cell killing specificity and cytotoxicity via antibody-dependent cell-mediated cytotoxicity (ADCC). Our main hypothesis isthat MHC Class I downregulation induced by the expression of Nef creates an opportunity for activated NK cells to target and kill latently infected primary T cells. We further hypothesized that broadly neutralizing antibodies (bnAb) recognizing the HIV envelope would be capable of mediating ADCC to target a wide range of HIV proviruses. Novel assays for NK cell effector function have been developed using both primary cell models of HIV latency and patient cells. For the primary cell models loss of HIV GFP expression and delivery of granzyme B are measured by flow cytometry. For patient cells loss of inducible cell-associated HIV mRNA and proviral DNA is measured using highly sensitive next generation sequencing readouts. When NK cells were cocultured with cells expressing IL-21, we observed dramatic NK cell expansion and enhancement of cytotoxicity against reactivated latently infected primary T cells. Using this protocol we are able to expand large numbers of highly cytotoxic NK cells that maintain their specificity for killing of infected cells and their ability to mediate ADCC. Importantly, this protocol is able to efficiently expand and activate NK cells from HIV+ donors. We have identified a combination of the histone deacetylase inhibitor (HDACi) Panobinostat with the PKC agonist Bryostatin or the combination of IL-15 with the HDACi SAHA as regimens that can optimally reactivate HIV‑1 while inducing minimal changes in cell surface expression profiles. Coculture of NK cells with infected primary T cells led to preferential killing of reactivated cells. We have identified at least one anti-Env antibody that can strongly mediate ADCC and enhance selectivity of killing reactivated HIV-1-infected CD4+ T cells while sparing uninfected cells. This antibody demonstrates ADCC activity at both low antibody and low NK cell to target cell ratios. We have also identified three more anti-Env antibodies that can enhance ADCC. Enhancing NK cell activity may represent an important new approach to virus eradication. We are optimizing protocols that can be rapidly translated into the clinic that involve proviral and NK cell reactivation and enhancement of killing by ADCC.