Institute of Biomedical Sciences

Title

CTLs Eliminate Defective HIV Proviruses Without Impacting Infectious Latent Reservoirs

Poster Number

18

Document Type

Poster

Keywords

HIV cure strategies, latency reversal, reservoir eradication, CTLs,

Publication Date

4-2017

Abstract

Background: The “shock-and-kill” paradigm of combining latency-reversing agents (LRAs) to “shock” latent HIV reservoirs awake, then “kill” them with immune effectors is currently the predominant strategy in the field of HIV cure research. However, the majority of “shock-and-kill” studies have been performed using primary cell models of HIV latency, which are imperfect representations of natural viral reservoirs found in people living with HIV. Thus, a need remains for a rigorous investigation of the efficacy of this cure strategy in natural reservoirs. Here, we treat ex vivo CD4+ T-cells from HIV+ individuals on long-term ARV therapy (>5 years), with LRAs and autologous HIV-specific CTL clones (targeting non-escaped epitopes), and assessed the impact on total and intact-inducible proviruses.

Method: HIV-specific CTL clones targeting known HIV epitopes were isolated from ARV-treated subjects by limiting dilution, and killing activities were confirmed by flow cytometric assays. We developed an HIV eradication (HIVE) assay to test the abilities of these CTLs to reduce viral reservoirs in combination with HDACi’s, PKC activators, or an IL-15 super agonist with Pam3CSK4. In short, resting CD4+ T-cells from HIV+ leukapheresis samples are co-cultured with LRAs + CTLs for 5 days with ARVs, and activation/memory phenotypes are monitored. CD4+ T-cells are isolated after treatment, and total/intact-inducible reservoirs are measured by cell-associated HIV DNA (ddPCR) and by quantitative viral outgrowth assay (QVOA).

Results: Combinations of bryostatin and IL-15SA+Pam3CSK4 with HIV-specific CTL generally led to significant decreases in cell-associated HIV DNA, with the greatest effects observed for bryostatin (up to 50% reductions, p < 0.01). Critically, these decreases in HIV DNA were not associated with measurable reductions in intact-inducible virus, regardless of the CTL clone or LRA combination used (powered to detect ~50% reductions with 95% confidence). Even when combined with PMA/ionomycin, CTLs were unable to drive reductions in intact-inducible virus. CTLs degranulated (CD107a) in response to autologous activated CD4+ T-cells that had been infected with virus from positive QVOA wells, ruling out a role for immune escape in our observation.

Conclusions: Recently, it has been demonstrated that some defective proviruses can be expressed as antigens, enabling CTL recognition. Data from our ex vivo experiments are consistent with the preferential depletion of defective proviruses by CTLs, leading to reductions in HIV DNA without impacting intact proviruses. Understanding and overcoming the mechanisms limiting CTL against the intact-inducible reservoir may be key to successful CTL-based shock and kill interventions.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Open Access

1

Comments

To be presented at GW Annual Research Days 2017.

This document is currently not available here.

Share

COinS
 

CTLs Eliminate Defective HIV Proviruses Without Impacting Infectious Latent Reservoirs

Background: The “shock-and-kill” paradigm of combining latency-reversing agents (LRAs) to “shock” latent HIV reservoirs awake, then “kill” them with immune effectors is currently the predominant strategy in the field of HIV cure research. However, the majority of “shock-and-kill” studies have been performed using primary cell models of HIV latency, which are imperfect representations of natural viral reservoirs found in people living with HIV. Thus, a need remains for a rigorous investigation of the efficacy of this cure strategy in natural reservoirs. Here, we treat ex vivo CD4+ T-cells from HIV+ individuals on long-term ARV therapy (>5 years), with LRAs and autologous HIV-specific CTL clones (targeting non-escaped epitopes), and assessed the impact on total and intact-inducible proviruses.

Method: HIV-specific CTL clones targeting known HIV epitopes were isolated from ARV-treated subjects by limiting dilution, and killing activities were confirmed by flow cytometric assays. We developed an HIV eradication (HIVE) assay to test the abilities of these CTLs to reduce viral reservoirs in combination with HDACi’s, PKC activators, or an IL-15 super agonist with Pam3CSK4. In short, resting CD4+ T-cells from HIV+ leukapheresis samples are co-cultured with LRAs + CTLs for 5 days with ARVs, and activation/memory phenotypes are monitored. CD4+ T-cells are isolated after treatment, and total/intact-inducible reservoirs are measured by cell-associated HIV DNA (ddPCR) and by quantitative viral outgrowth assay (QVOA).

Results: Combinations of bryostatin and IL-15SA+Pam3CSK4 with HIV-specific CTL generally led to significant decreases in cell-associated HIV DNA, with the greatest effects observed for bryostatin (up to 50% reductions, p < 0.01). Critically, these decreases in HIV DNA were not associated with measurable reductions in intact-inducible virus, regardless of the CTL clone or LRA combination used (powered to detect ~50% reductions with 95% confidence). Even when combined with PMA/ionomycin, CTLs were unable to drive reductions in intact-inducible virus. CTLs degranulated (CD107a) in response to autologous activated CD4+ T-cells that had been infected with virus from positive QVOA wells, ruling out a role for immune escape in our observation.

Conclusions: Recently, it has been demonstrated that some defective proviruses can be expressed as antigens, enabling CTL recognition. Data from our ex vivo experiments are consistent with the preferential depletion of defective proviruses by CTLs, leading to reductions in HIV DNA without impacting intact proviruses. Understanding and overcoming the mechanisms limiting CTL against the intact-inducible reservoir may be key to successful CTL-based shock and kill interventions.