1. Cell- and Tissue-based Measures of Viral Persistence Are Associated with Immune Activation and PD-1-Expressing CD4+ T cells H Hatano 1, V Jain 1, PW Hunt 1, JN Martin 1, TH Lee 2, E Sinclair 1, JM McCune 1, F Hecht 1, MP Busch 1,2, SG Deeks 1 1 University of California, San Francisco, CA, USA 2 Blood Systems Research Institute, San Francisco, CA, USA

2. What are the determinants of HIV persistence? Determinants of HIV persistence during long-term HAART remain unknown, but may include: –Ongoing viral replication (Buzon, Nat Med 2010) –Potency of HIV-specific responses Mucosal HIV-specific T cell responses (Hatano, JID 2011)

3. Higher Levels of GALT HIV-specific CD8+ T Cells Are Associated with Lower Levels of Proviral DNA rho = - 0.50, p = 0.03 CD4CD8 rho = - 0.42, p = 0.07 Hatano et al, JID 2011

4. What are the determinants of HIV persistence? Determinants of HIV persistence during long-term HAART remain unknown, but may include: –Persistent immune activation Immune activation levels remain elevated despite effective HAART-mediated viral suppression –Negative regulators that reverse activated state (PD-1) (Chomont, Nat Med 2009) PD-1 high CD4+ T cells have high levels of proviral DNA Triggering of PD-1 inhibits HIV transcription, and inhibiting the PD-1/PD-L1 interaction increases HIV production PD-1 expressing CD4+ T cells → Preferential reservoir for HIV Understanding the causes of persistent inflammation are important for preventing non- AIDS morbidity and for strategies towards cure

5. Study Objectives To assess the relationship between measurements of viral persistence and immune activation –Plasma RNA –Cell-associated RNA and proviral DNA –Tissue-associated RNA and proviral DNA To determine the relationship between treatment response and –T cell activation/dysfunction –Viral persistence To identify potential interventions to decrease HIV persistence

6. Methods 190 HAART-suppressed subjects identified from UCSF SCOPE/OPTIONS cohorts Ultrasensitive plasma HIV RNA –Modified Roche CAP/CTM v2.0 (LOD <5 copies/mL) Cell-associated RNA Proviral DNA Immune activation ( % CD4+ and CD8+ T cells) –CD38+HLA-DR+, PD-1 Gut-associated lymphoid tissue (GALT) samples were obtained from 14 subjects

7. Baseline Characteristics (n=190) CD4 count (cells/mm 3 )523 Age (years)51 Gender (% male)92% Duration VL suppression (months)31 Nadir CD4 count (cells/mm 3 )113

8. No Association Between Low-Level Plasma RNA Levels and T Cell Activation All p-values > 0.20

9. Modest Association Between Cell-Based Measures of Viral Persistence and T Cell Activation rho = 0.23, p = 0.014rho = 0.16, p = 0.057 rho = 0.22, p = 0.008rho = 0.14, p = 0.088

10. Highly Significant Association between Proviral DNA Levels and Frequency of PD-1 Expressing CD4+ T Cells rho = 0.28, p = 0.0005 These observations are consistent with PD-1 being a marker of latently infected CD4+ T cells (Chomont et al, Nature Med 2009)

11. Strong Association Between Viral Persistence in GALT and T Cell Activation

12. What is the relationship between treatment response and… -Viral Persistence? -T Cell Activation/Dysfunction? Will treated subjects with a low CD4+ T cell count require unique curative strategies?

13. Plasma RNA Levels Similar in Low and High CD4+ Groups p = NS

14. Cell-Associated RNA and Proviral DNA Levels Are Higher in Low CD4+ Group p = 0.008p = 0.001

15. CD4+ T Cell Activation and PD-1 Expression are Higher in Low CD4+ Group p < 0.0001

16. Higher Frequencies of PD-1 Expressing T CM CD4+ T Cells in Low CD4+ Group p < 0.0001

17. Conclusions I. No associations between ultrasensitive plasma HIV RNA levels and immune activation Cell-based measurements of viral persistence were modestly but consistently associated with markers of immune activation/dysfunction and frequency of PD-1 expressing CD4+ T cells Stronger positive correlation between tissue-based measurements of viral persistence and immune activation

18. Highly significant association between proviral DNA levels and frequency of PD-1 expressing CD4+ T Cells Phase I study of an anti-PD-1 monoclonal antibody aimed at clearing the latent reservoir is in development (ACTG 5301) Potential Eradication Strategies

19. ACTG 5301 Study Schema Study Design: Single arm, dose-finding study Population: –HIV-infected female and male subjects ≥ 18 years of age. Females of reproductive potential are excluded from the study. –Screening CD4+ T cell count > 350 cells/mm 3 –Plasma HV RNA < 75 copies/mL while taking HAART for previous 36 months Sample size: 40 (10 subjects in each dose cohort) Study duration: 16 weeks Intervention: Single IV dose of open-label MK3475 at dose of 0.1, 1, 3, or 10 mg/kg

20. Treated patients with a low CD4+ T cell count had: –Higher cell-based measures of viral persistence –Expansion of CD4+ T cells expressing PD-1 Most consistently observed in the central memory compartment Treated individuals with low CD4+ T cell counts may be more difficult to cure and/or will require unique interventions Conclusions II.

21. Implications Understanding the causes of viral persistence and inflammation in the setting of HAART are necessary to develop new strategies towards cure Future studies of viral persistence should focus on cell- and tissue-based measurements of viral persistence, not on plasma RNA (Chun, JID 2008; Yukl, JID 2010; Hatano, JID 2011)

22. Acknowledgements UCSF/SFGH/PHPUCSF/SFGH/DEMFunding Vivek JainElizabeth SinclairNIAID K23AI075985 Peter HuntJoseph M. McCune Ma Somsouk Jeffrey MartinVGTI Florida Frederick HechtNicolas Chomont Steven DeeksRafick Sekaly UCSF/SFVAMCRoche, Inc. Steven YuklTri Do Joseph Wong UCSF/BSRI Tzong-Hae Lee Michael Busch