IAS 2015 Towards an HIV Cure symposium Vancouver CTLA-4-expressing CD4 T cells are critical contributors to SIV viral persistence Colleen McGary Paiardini lab Emory University
HIV reservoir prevents eradication of HIV infection Treatment interruption Deeks S, et al. Nat Rev Imm 2012 Viral rebound ART is unable to eliminate virally infected cells (Finzi D, et al. 1997; Wong JK, et al. 1997; Strain MC, et al. 2005) Viral reservoir is established early during infection (Finzi D, et al. 1997; Chun T.W., et al. 1998; Whitney JB, et al. 2014) Resting memory CD4+ T cells are the best characterized viral reservoir (Siliciano JD, et al. 2003) Central and transitional memory CD4 (Chomont N, et al. 2009; Soriano-Sarabia N, et al. 2014) Stem cell memory CD4 (Buzon MJ, et al. 2014; Jaafoura S., et al. 2014) Frequency of latently infected resting CD4+ T cells is 1 in 10 6 (Chun T.W., et al. 1997)
Understanding the immunophenotype and anatomic location of latently infected cells Study Objective: to determine the relationship between the expression of co-inhibitory receptors and the level of latent viral infection
Co-inhibitory receptors and HIV/SIV Negatively regulate T cell activation and proliferation Several co-inhibitory receptors are upregulated on CD4 and CD8 T cells following HIV-1 infection PD-1, CTLA-4, TIM-3, LAG-3, CD160 Upregulation associated with T cell dysfunction and exhaustion (Day CL, et al. Nature 2006; Trautmann L. et al, Nat Med, 2006; Kaufmann DE, et al. Nat Imm, 2007; Jones RB, et al. J Ex Med, 2008; Petrovas C, et al. J Ex Med, 2006) T cell dysfunction unable to be restored by ART (Betts MR, et al. Blood 2006) PD-1+ cells are significantly enriched in HIV DNA in ART-treated HIV+ subjects (Chomont N, et al. Nat Med 2009) Larsson M, et al. Retrovirology 2013 CTLA-4+ CD4+ T cells are highly permissive to HIV in vitro and are preferentially infected in ART-naïve HIV+ individuals (El-Far M, et al. J Gen Virol. 2015)
Advantages to using NHP models in studies of HIV reservoir/cure 1. Ability to determine and control for clinical parameters that are virtually impossible to control in humans (Identity, dose, and route of virus challenge, time of infection, duration of ART, etc). 2. Ability to perform longitudinal collections in multiple anatomic locations (PB, LN, RB, BM) and elective necropsy: comprehensive cellular and anatomic characterization of virus reservoirs. 3. cart is able to achieve effective suppression of viral replication. (Shytaj IL, Norelli S, et al. PLoS Path. 2012; Del Prete GQ, et al. Antimicrob Agents Chemother. 2014; Del Prete GQ, et al. AIDS Res Hum Retroviruses 2015) 4. Ability to test novel and risky interventions for HIV eradication (i.e. combination therapy, cell depletion, stem cell-based interventions, etc.).
Study Design 10 RMs (4-10 yr old); 3 A01+, all B08- &B17- SIVmac251 1000 TCID50, i.v. Initiate ART Maraviroc 5-drug ART initiated (7) (PMPA, FTC, raltegravir, darunavir/ritonavir) Necropsy Month 1 2 3 4 5 6 7 8 9 Day -20 0 14 52 90 122 168 186 200 240 270 Blood/RB collected at time points indicated; LN collected D14, 52, 90, 200 5-drug ART regimen: Tenofovir/PMPA NRTI 20-25 mg/kg/d Emtricitabine/FTC NRTI 30-50 mg/kg/d Raltegravir Integrase inhibitor 100-150 mg/bid Darunavir Protease inhibitor 400-700 mg/bid Ritonavir (boost) PI (P-gp inhibitor) 50 mg/bid Maraviroc [7 RMs] CCR5 antagonist 100-150 mg/bid Sorts performed for cell-associated SIV DNA/RNA from CD4+ co-inhibitory subsets: MidART (D200+): PBMCs, LN Necropsy (D270+): PBMCs, LN, Spleen, Gut mucosa
Five-drug ART regimen effectively suppressed viral replication in SIVmac251-infected RMs Average time to initial suppression: 151 days Limit of detection: 60 copies/ml
Enhanced co-inhibitory receptor staining after stimulation 3 hr PMA/Ionomycin stimulation: (surface staining) TIM-3 PD-1 CTLA-4
PD-1+CTLA-4+ CD4+ T cells comprise the majority of memory CD4+ T cells PBMCs SIV(-) Pre-ART Necrops y RB LN
Sorting Gating Strategy SIV+ RM LN ART-treated Stimulated 3 hr PMA + Iono Necropsy: 1 st sort: PD-1 (+) and (-) PD-1(-) 2 nd sort: CTLA-4, TIM3 combinations PD-1(+) MidART: 4 population sort 7 5 1 3 8 6 2 4 Necropsy: 8 population sort
SIV DNA enrichment is only found in the lymph node after short duration of virus suppression MidART PBMCs LN MidART: 1 month undetectable plasma viremia (80±40 d)
Necropsy CTLA-4-expressing memory CD4 T cells harbor high levels of SIV DNA PBMCs LN Spleen Mucosa Necropsy: 3 months post undetectable plasma viremia (125±77d) TIM3+ cells represented by open symbols
Levels of CA-SIV DNA in blood decrease faster in memory CD4 T cells lacking co-inhibitory expression PBMCs MidART: 1 month undetectable plasma viremia (80±40 d) Necropsy: 3 months post undetectable plasma viremia (125±77d)
PD-1 and CTLA-4-expressing CD4 T cells contribute significantly more to the SIV DNA pool PBMCs LN Spleen Mucosa Contributions to SIV DNA pool at necropsy:
PD-1 and CTLA-4-expressing CD4 T cells contribute significantly more to the SIV DNA pool Rationale to test efficacy of PD-1/CTLA-4 dual blockade in setting of ART Targeting between 76-88% of infected CD4+ T cells Reinvigorate dysfunctional antiviral CD8+ T cells
CTLA-4 Blockade in an HIV+ Patient Fiona Wightman, Ajantha Solomon, Sanjeev S. Kumar, Nicolas Urriola, Kerri Gallagher, Bonnie Hiener, Sarah Palmer, Catriona Mcneil, Roger Garsia, and Sharon R. Lewin Ipilimumab produced greater changes in CA-US HIV RNA than what has been previously reported for HDAC inhibitors (vorinostat, panobinostat) or disulfiram CTLA-4 blockade resulted in 19.6-fold increase in cell-associated unspliced HIV RNA
Conclusions Five-drug ART regimen effectively suppresses plasma viremia Undetectable plasma viremia (<60 copies/ml) in 9 of 10 RMs Frequencies of PD-1+CTLA-4+ CD4+ memory T cells increase during SIV infection, despite ART suppression of plasma viremia CTLA-4(+) CD4+ T cells are significantly enriched in SIV DNA Enrichment persists over time Enrichment found across multiple tissue compartments: In PBMCs, LN, spleen, and mucosa at necropsy In LN compared to DP (double positive) cells at midart Limited to cell-associated DNA and RNA quantification Pursuing in situ hybridization confirmation and SGA sequencing for further characterization PD-1 and CTLA-4-expressing cells are major contributors to the SIV DNA pool within memory CD4+ T cells Rationale to target these two pathways in combination with ART
Future Dual Blockade Study To determine the efficacy of blocking the PD-1 and CTLA-4 pathways in targeting the persistent viral reservoir in ART-treated, SIV-infected RMs Group 1: SIVmac239 (i.v.) actla-4/pd-1 cart actla-4/pd-1 3-drug ART (PMPA, FTC, DTG) actla-4/pd-1 cart Interruption 4 months Necropsy Week -3 0 2 6 10 14 22 26 30 46 54 62 Group 2: apd-1 apd-1 apd-1 Group 1: receive actla-4/pd-1 Abs, s.c. (12 RMs) Group 2: receive anti-pd-1 Ab, s.c. (8 RMs) Group 3: receive no Ab, ART alone (8 RMs)
Acknowledgements Paiardini lab Sara Paganini Luca Micci Emily Ryan Justin Harper Yerkes Flow Cytometry Core Barbara Cervasi John Altman Kiran Gill Ragon Institute Mathias Lichterfeld Maria Buzon Sean Harrington Xu Yu CFAR Virology Core Thomas Vanderford Benton Lawson Melon Nega NCI Jeff Lifson Michael Piatak Jake Estes Emory- YNPRC Guido Silvestri Ann Chahroudi Diane Carnathan Ankita Chowdhury Jakob Habib Elizabeth Strobert Stephanie Ehnert Chris Souder Sherrie Jean NIH/NIAID Jason Brenchley Case Western Rafick Sekaly Drugs Daria Hazuda (Merck) Romas Geleziunas (Gilead) Guenter Kraus (Janssen) Supported by NIH R21/R33 AI104278, RO1 AI116379 PHS RR000165/OD011132