Biology subjects

Dobrowolski, C.

Publications and source records attributed to Dobrowolski, C..

3 recordsLinked to original sources

bioRxiv2022

Reduction of the HIV-1 reservoir in T cells from persons with HIV-1 on suppressive antiretroviral therapy using expanded natural killer cells ex vivo

Treatment with latency-reversing agents (LRAs) alone has been ineffective in reducing HIV-1 reservoirs in persons with HIV-1 (PWH) on antiretroviral therapy (ART), due to inefficiencies in reservoir reactivation and adaptive immune responses. However, NK cells that are activated with cytokines may be able to target HIV-1 reservoirs more efficiently. To study the therapeutic potential of NK cells, we expanded blood NK cells from multiple donors ex vivo into CD56brightCD16+ "eNK" cells using artificial antigen presenting cells (aAPCs) expressing membrane-bound IL21. eNK cells express multiple activating receptors and are highly cytotoxic against specific target cells. eNK cells can also kill HIV-infected CD4 T cells via antibody dependent cell-mediated cytotoxicity (ADCC) using broadly neutralizing antibodies against HIV-1 Env gp120/gp41. Importantly, eNK cells from PWH on ART efficiently killed autologous HIV-1+ T cells reactivated by a combination of vorinostat (SAHA) and IL-15 or an IL-15 superagonist (N-803), as detected by declines in proviral load, inducible HIV-1 mRNA, and virus release. Adoptive immunotherapy with eNK cells is therefore a promising approach to reduce the latent HIV-1 reservoir in PWH when combined with LRA treatment. Author SummarySuccessful antiretroviral therapy (ART) eliminates progression to AIDS by reducing HIV to nearly indetectable levels by routine clinical measurements of blood samples. However, more sensitive DNA and RNA measurements show that most persons on ART retain a reservoir of long lived latently infected cells, which remain undetected by the immune system while no HIV is being produced. In nearly all cases, ART interruption results in a rebound of HIV production and spread, requiring an immediate return to ART. Currently the goal of HIV eradication is to achieve a "functional cure", where HIV reservoirs are reduced to the point where ART can be interrupted indefinitely, and low levels of infected cells remaining can be controlled by the immune system. Our eradication strategy combines HIV latency-reversing agents (LRAs), ex vivo expansion of natural killer (NK) cells, and enhancement of specificity and killing of infected cells with broadly neutralizing antibodies against HIV. In this study, we have demonstrated that NK cells from person living with HIV can be isolated and expanded ex vivo into "eNK" cells that kill HIV-infected cells without killing uninfected cells, especially when broadly neutralizing antibodies are present, and can significantly reduce HIV reservoirs after LRA treatment.

immunology↗

bioRxiv2022

Detecting sources of immune activation and viral rebound in HIV infection

Antiretroviral therapy (ART) generally suppresses HIV replication to undetectable levels in peripheral blood, but immune activation associated with increased morbidity and mortality is sustained during ART, and infection rebounds when treatment is interrupted. To identify drivers of immune activation and potential sources of viral rebound, we modified RNAscope in situ hybridization to visualize HIV-virus producing cells as a standard to compare the following assays of potential sources of immune activation and virus rebound following treatment interruption: 1) EDITS (envelope detection by induced transcription-based sequencing) assay; 2) HIV-Flow; and 3) Flow-FISH assays that can scan tissues and cell suspensions to detect rare cells expressing env mRNA, gag mRNA/Gag protein and p24 respectively; and 4) an ultrasensitive immunoassay that detects p24 in cell/tissue lysates at subfemtomolar levels. We show that the sensitivity of these assays is sufficient to detect a rare HIV-producing/env mRNA+/p24+ cell in a million uninfected cells. These high-throughput technologies thus provide contemporary tools to detect and characterize rare cells producing virus and viral antigens as potential sources of immune activation and viral rebound. ImportanceAnti-retroviral therapy (ART) has greatly improved the quality and length of life for people living with HIV, but immune activation does not normalize during ART, and persistent immune activation has been linked to increased morbidity and mortality. We report a comparison of assays of two potential sources of immune activation during ART: rare cells producing HIV virus or the virus major viral protein, p24, benchmarked on a cell model of active and latent infections and a method to visualize HIV-producing cells. We show that assays of HIV Envelope mRNA (EDITS assay) and gag mRNA and p24 (Flow-FISH, HIV-Flow and ultrasensitive p24 immunoassay) detect HIV-producing cells and p24 at sensitivities of one infected cell in a million uninfected cells, thus providing validated tools to explore sources of immune activation during ART in the lymphoid and other tissue reservoirs.

immunology↗

bioRxiv2021

Inhibition of the H3K27 demethylase UTX enhances the epigenetic silencing of HIV proviruses and induces HIV-1 DNA hypermethylation but fails to permanently block HIV reactivation

One strategy for a functional cure of HIV-1 is "block and lock", which seeks to permanently suppress the rebound of quiescent HIV-1 by epigenetic silencing. For the HIV LTR, both histone 3 lysine 27 tri-methylation (H3K27me3) and DNA methylation are associated with viral suppression, while H3K4 tri-methylation (H3K4me3) is correlated with viral expression. However, H3K27me3 is readily reversed upon activation of T-cells through the T-cell receptor. To suppress latent HIV-1 in a stable fashion, we depleted the expression or inhibited the activity of UTX/KDM6A, the major H3K27 demethylase, and investigated its impact on latent HIV-1 reactivation in T cells. Inhibition of UTX dramatically enhanced H3K27me3 levels at the HIV LTR and were associated with increased DNA methylation. In latently infected cells from patients, GSK-J4, which is a potent dual inhibitor of the H3K27me3/me2-demethylases JMJD3/KDM6B and UTX/KDM6A, effectively suppressed the reactivation of latent HIV-1 and induced DNA methylation at specific sites in the 5LTR of latent HIV-1 by the enhanced recruitment of DNMT3A to HIV-1. Nonetheless, suppression of HIV-1 through epigenetic silencing required the continued treatment with GSK-J4 and was rapidly reversed after removal of the drug. Thus, epigenetic silencing by itself appears to be insufficient to permanently silence HIV-1 proviral transcription. Author SummaryThe "block and lock" strategy for a functional HIV-1 cure is based on the premise that permanent inactivation of the HIV-1 can be achieved by epigenetic silencing of the proviral DNA. For cellular genes, long-term silencing is achieved during cell differentiation by the induction of specific epigenetic modifications involving histone and DNA methylation. During HIV-1 silencing, histone methylation and DNA methylation are observed, but both sets of modifications can be reversed upon activation of T-cells through the T-cell receptor or potent latency reversing agents. In an attempt to enhance silencing of HIV-1 transcription, we used an inhibitor of H3K27 demethylases to increase H3K27 methylation. This in turn led to enhanced DNA methylation of HIV-1. Unfortunately, although the treatment effectively silenced HIV-1 and prevented viral reactivation, the silencing effects were short-lived and quickly reversed after removal of the drug.

microbiology↗