CCR5-edited CD4+ T cells augment HIV-specific immunity to enable post-rebound control of HIV replication

Abstract

BackgroundWe conducted a phase I clinical trial that infused CCR5 gene-edited CD4+ T cells to determine how these T cells can better enable HIV cure strategies.MethodsThe aim of trial was to develop RNA-based approaches to deliver zinc finger nuclease (ZFN), evaluate the effect of CCR5 gene-edited CD4+ T cells on the HIV-specific T cell response, test the ability of infused CCR5 gene-edited T cells to delay viral rebound during analytical treatment interruption, and determine whether individuals heterozygous for CCR5 Δ32 preferentially benefit. We enrolled 14 individuals living with HIV whose viral load was well controlled by antiretroviral therapy (ART). We measured the time to viral rebound after ART withdrawal, the persistence of CCR5-edited CD4+ T cells, and whether infusion of 10 billion CCR5-edited CD4+ T cells augmented the HIV-specific immune response.ResultsInfusion of the CD4+ T cells was well tolerated, with no serious adverse events. We observed a modest delay in the time to viral rebound relative to historical controls; however, 3 of the 14 individuals, 2 of whom were heterozygous for CCR5 Δ32, showed post-viral rebound control of viremia, before ultimately losing control of viral replication. Interestingly, only these individuals had substantial restoration of HIV-specific CD8+ T cell responses. We observed immune escape for 1 of these reinvigorated responses at viral recrudescence, illustrating a direct link between viral control and enhanced CD8+ T cell responses.ConclusionThese findings demonstrate how CCR5 gene-edited CD4+ T cell infusion could aid HIV cure strategies by augmenting preexisting HIV-specific immune responses.REGISTRATIONClinicalTrials.gov NCT02388594.FundingNIH funding (R01AI104400, UM1AI126620, U19AI149680, T32AI007632) was provided by the National Institute of Allergy and Infectious Diseases (NIAID), the National Institute on Drug Abuse (NIDA), the National Institute of Mental Health (NIMH), and the National Institute of Neurological Disorders and Stroke (NINDS). Sangamo Therapeutics also provided funding for these studies.

Keywords: AIDS/HIV; Gene therapy; T cells.

Conflict of interest statement

Conflict of interest: YZ, BLL, CHJ, and JLR are founders of and hold equity in Tmunity Therapeutics. YZ and JLR receive research support from Tmunity Therapeutics. CHJ reports receiving grants from Tmunity Therapeutics and holds founders stock in Tmunity Therapeutics and DeCART Therapeutics. CHJ also receives personal income from Celldex Therapeutics, Viracta Therapeutics, and WIRB-Copernicus Group as well as royalties from Novartis. BLL is a consultant for Novartis, Terumo, and Lilly Asia Ventures and serves on the scientific advisory boards of Avectas, Patheon/Thermo Fisher Viral Vector Services, Immuneel Therapeutics, Incysus Therapeutics, Ori Biotech, and Vycellix. DA and GL were employees and equity holders of Sangamo Therapeutics.

Figures

Figure 1. Schematic outlining the timeline and sample collection points for NCT02388594.

The clinical study was divided into 4 steps. During step 1, participants underwent leukapheresis to collect cells for manufacturing, followed by a second leukapheresis and rectal biopsy to serve as baseline samples. In cohorts 2 and 3, participants were treated with a single dose of cyclophosphamide 2 days before receiving modified cells (day –2). Participants entered step 2 on the day of cell infusion (day 0), and the edited cells were allowed to engraft for either 4 weeks (cohorts 2 and 3) or 8 weeks (cohort 1) before the 16-week analytical treatment interruption was initiated in step 3. At the conclusion of the treatment interruption, the participants entered step 4 to be monitored for safety until HIV RNA levels fell below the limit of quantification. Safety laboratory values and HIV viral load were monitored at regular intervals throughout the study.

Figure 2. Time to viral rebound during an ATI.

(A) All individuals who received CCR5-edited CD4+ T cells were compared with historical controls for the time to reach a viral load of at least 200 copies/mL. The Peto-Peto P value is shown for the group comparison of the survival curves. (B) Each cohort within the clinical trial was compared with the ACTG control group. Participant 305 was excluded because of continued ART during the ATI. Pt, participant.

Figure 3. Analysis of CD4+ and CD8+ T cell counts, viral load, and percentage of CCR5 disruption within peripheral CD4+ T cells.

At the indicated time points, CD4+ and CD8+ T cell counts as well as viral load were measured. The percentage of CCR5 disruption was calculated as described in Methods. Individuals who participated in an extended ATI are indicated with a red subject ID.

Figure 4. Viral reservoir before and after infusion of CCR5-edited CD4+ T cells.

An IPDA was performed before and at least 6 months after the reinitiation ART. (A) Number of integrated HIV genomes with defective 5′ sequences per million CD4+ T cells. (B) Number of integrated HIV genomes with defective 3′ sequences per million CD4+ T cells. (C) Number of integrated HIV genomes with intact viruses per million CD4+ T cells. P values were calculated by paired t test. Blue indicates individuals with CCR5 Δ32 heterozygosity, and green represents CCR5 WT individuals.

Figure 5. Enhanced HIVGag-specific CD8+ T cell responses were observed after infusion of CCR5-deficient CD4+ T cells.

(A) Representative flow cytometric plots showing the IFN-γ response to Gag pool number 21 from cells collected from participant 303 during apheresis numbers 2 and 5. (B) Heatmap showing the change in the percentage of IFN-γ+ CD8+ T cells when exposed to the indicated peptide pool between apheresis number 2 and number 5. (C) Deconvolution of peptide pool data shows where within the Gag protein enhanced responses were observed. Patient ID, MHC haplotype, and epitope for each enhanced response greater than 0.5%. Ensemble score estimates peptide affinity for a particular HLA allele, with the highest-affinity allele identified. Green indicates that the allele affinity is under 500 µM, and yellow highlights alleles in which there is no predication model.

Figure 6. Immune escape was observed following a heightened immune response.

(A) Consensus amino acid sequences, derived by single-genome sequencing (the number of sequences is enumerated on the right), are shown for the predicted CD8+ T cell epitopes within Gag for participants 203, 301, and 303, at early and late time points. In participant 303, the fifth position of the first Gag T cell epitope (highlighted in yellow), changed from lysine (in 5 of 5 sequences) to phenylalanine in (6 of 6 sequences). (B) Highlighter plot of Gag amino acid single-genome sequences for participant 303, demonstrating a change in the CTL epitope from consensus (L) to F. (C) Both WT and L-to-F–mutated peptides from Gag pool number 21 were incubated with PBMCs isolated from participant 303, and intracellular IFN-γ production was measured.