Cell- and Tissue-based Measures of Viral Persistence Are Associated with Immune Activation and PD-1-Expressing CD4 T

1.  Cell- and Tissue-based Measures of Viral Persistence Are Associated with Immune Activation and PD-1-Expressing CD4+ T cells H Hatano1, V Jain1, PW Hunt1, JN Martin1, TH Lee2, E Sinclair1, JM McCune1, F Hecht1, MP Busch1,2, SG Deeks1 1University of California, San Francisco, CA, USA 2Blood 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) We initiated a series of analyses to assess significance of each of these factors. Target cell availability Ferre, Blood 2009: Control of HIV replication associated with high levels of mucosal HIV-specific total responses (IL-2 or TNFa or IFNg or CD107 � producing cells)We initiated a series of analyses to assess significance of each of these factors. Target cell availability Ferre, Blood 2009: Control of HIV replication associated with high levels of mucosal HIV-specific total responses (IL-2 or TNFa or IFNg or CD107 � producing cells)

3. Mucosal HIV-specific T cell responses may be controlling the size of the cellular reservoir Ferre: Control of HIV replication associated with high levels of mucosal HIV-specific total responses (IL-2 or TNFa or IFNg or CD107 � producing cells)Mucosal HIV-specific T cell responses may be controlling the size of the cellular reservoir Ferre: Control of HIV replication associated with high levels of mucosal HIV-specific total responses (IL-2 or TNFa or IFNg or CD107 � producing cells)

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-1high 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 Transcription-Mediated Amplification Targets LTR Adapted for CARNA Cell-associated RNA and proviral DNA was measured from PBMCs. The Transcription Mediated Amplification (TMA) assay (Aptima�, Gen-Probe) was used to measure cell-associated RNA. The TMA assay is a nucleic acid-amplification test that has been FDA-approved for the early detection of HIV infection in plasma for screening blood donors and has also been validated for clinical diagnostic use [23]. A modified approach of previously published methods for PBMC extraction and TMA amplification of cell-associated HCV was used [24, 25]. The output is a signal:cutoff (S/Co) ratio (range 0-30), with S/Co <1.0 considered HIV RNA �negative� and S/Co =1.0 considered �positive.� All S/Co ratios were normalized to the input number of CD4. � Total proviral DNA was extracted from PBMCs using modifications of previously-described methods [25, 26]. This assay has an overall sensitivity of 1 copy/3 ?g of input DNA, equivalent to approximately 450,000 PBMCs [27, 28]. All proviral DNA levels were normalized to the input number of CD4. Transcription-Mediated Amplification Targets LTR Adapted for CARNA Cell-associated RNA and proviral DNA was measured from PBMCs. The Transcription Mediated Amplification (TMA) assay (Aptima�, Gen-Probe) was used to measure cell-associated RNA. The TMA assay is a nucleic acid-amplification test that has been FDA-approved for the early detection of HIV infection in plasma for screening blood donors and has also been validated for clinical diagnostic use [23]. A modified approach of previously published methods for PBMC extraction and TMA amplification of cell-associated HCV was used [24, 25]. The output is a signal:cutoff (S/Co) ratio (range 0-30), with S/Co <1.0 considered HIV RNA �negative� and S/Co =1.0 considered �positive.� All S/Co ratios were normalized to the input number of CD4. � Total proviral DNA was extracted from PBMCs using modifications of previously-described methods [25, 26]. This assay has an overall sensitivity of 1 copy/3 ?g of input DNA, equivalent to approximately 450,000 PBMCs [27, 28]. All proviral DNA levels were normalized to the input number of CD4.

7. Baseline Characteristics

8. Median 0 copies/mL Rho=-0.04, P=0.67 Rho=-0.02, P=0.81 Rho=-0.06, P=0.55 Rho=0.10, P=0.27Median 0 copies/mL Rho=-0.04, P=0.67 Rho=-0.02, P=0.81 Rho=-0.06, P=0.55 Rho=0.10, P=0.27

9. Rho values are low, suggesting other important factors are contributing to this relationship.Rho values are low, suggesting other important factors are contributing to this relationship.

11. Gut-associated lymphoid tissue (GALT) samples were obtained from 14 subjects MVC HIV RNA/DNA ratio (marker of HIV production and/or replication)Gut-associated lymphoid tissue (GALT) samples were obtained from 14 subjects MVC HIV RNA/DNA ratio (marker of HIV production and/or replication)

12. What is the relationship between treatment response and�-Viral Persistence? -T Cell Activation/Dysfunction?

13. Divide cohort into Low CD4 (<350) and High CD4 (>350) group. Ultrasensitive plasma HIV RNA levels (Roche v2.0, < 5 copies/mL assay) 0 copies/mL (0 Low vs 0 High) Divide cohort into Low CD4 (<350) and High CD4 (>350) group. Ultrasensitive plasma HIV RNA levels (Roche v2.0, < 5 copies/mL assay) 0 copies/mL (0 Low vs 0 High)

14. Both surrogate measurements of size of latent reservoir CARNA: -704 S/Co / mil CD4 (878 Low vs 620 High, p=0.008) Proviral DNA: -361 copies/mil CD4 (600 Low vs 204 High, p=0.001)Both surrogate measurements of size of latent reservoir CARNA: -704 S/Co / mil CD4 (878 Low vs 620 High, p=0.008) Proviral DNA: -361 copies/mil CD4 (600 Low vs 204 High, p=0.001)

15. Even when adjusted for DurationVLsup and CD4nadir The low CD4+ group had an expansion of total CD4+ T cells expressing markers of T cell activation/dysfunction (CD38, HLA-DR, CCR5, and/or PD-1) (p<0.0001); this effect was most consistently observed in the central memory compartment (p<0.0001).� Even when adjusted for DurationVLsup and CD4nadir The low CD4+ group had an expansion of total CD4+ T cells expressing markers of T cell activation/dysfunction (CD38, HLA-DR, CCR5, and/or PD-1) (p<0.0001); this effect was most consistently observed in the central memory compartment (p<0.0001).�

16. The low CD4+ group had an expansion of total CD4+ T cells expressing markers of T cell activation/dysfunction (CD38, HLA-DR, CCR5, and/or PD-1) (p<0.0001); this effect was most consistently observed in the central memory compartment (p<0.0001).� **Also look at total numbers of these cells (not frequencies)The low CD4+ group had an expansion of total CD4+ T cells expressing markers of T cell activation/dysfunction (CD38, HLA-DR, CCR5, and/or PD-1) (p<0.0001); this effect was most consistently observed in the central memory compartment (p<0.0001).� **Also look at total numbers of these cells (not frequencies)

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)

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/mm3 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 A5301 is a single-arm pilot study to evaluate the safety, pharmacokinetic profile, and effects of a single dose of anti-PD-1 antibody in chronically HIV-infected patients receiving effective antiretroviral therapy (ART).A5301 is a single-arm pilot study to evaluate the safety, pharmacokinetic profile, and effects of a single dose of anti-PD-1 antibody in chronically HIV-infected patients receiving effective antiretroviral therapy (ART).

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. Suggesting thatSuggesting that

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) Most of latent reservoir resides in GALT (Anton, AIDS �03; Chun, JID �08; Yukl, JID �10) Yukl: Raltegravir intensification reduced activation and HIV RNA/DNA ratio in ileum but not in blood or other gut sitesMost of latent reservoir resides in GALT (Anton, AIDS �03; Chun, JID �08; Yukl, JID �10) Yukl: Raltegravir intensification reduced activation and HIV RNA/DNA ratio in ileum but not in blood or other gut sites

22. Acknowledgements UCSF/SFGH/PHP UCSF/SFGH/DEM Funding Vivek Jain Elizabeth Sinclair NIAID K23AI075985 Peter Hunt Joseph M. McCune Ma Somsouk Jeffrey Martin VGTI Florida Frederick Hecht Nicolas Chomont Steven Deeks Rafick Sekaly UCSF/SFVAMC Roche, Inc. Steven Yukl Tri Do Joseph Wong UCSF/BSRI Tzong-Hae Lee Michael Busch Vaccine and Gene Therapy InstituteVaccine and Gene Therapy Institute