1. Slide 1 of 24 From DC Douek, MD, at San Francisco, CA: March 24, 2013, IAS-USA. IAS–USA Daniel C. Douek, MD, PhD Bethesda, Maryland Immune Activation, HIV Persistence, and the Cure

2. Slide 2 of 24 From DC Douek, MD, at San Francisco, CA: March 24, 2013, IAS-USA. What We Talk About When We Talk About Immune Activation Normal innate response to viral infection in the acute phase of the infection A.R. Stacey et al JV 2009 Virus load Cytokine Immune activation occurs early in infection

3. Slide 3 of 24 From DC Douek, MD, at San Francisco, CA: March 24, 2013, IAS-USA. Cells: activated phenotype of Macrophages and Dendritic Cells Cytokines, chemokines: TNF, IL-1, IL-6, IL-8, IL-15, IL-10 Acute phase proteins: Serum Amyloid A, C-Reactive Protein Coagulation: D-dimers, Tissue Factor Fibrosis: Matrix Metalloprotease activation, collagen deposition Microbial sensors: Lipopolysaccharide Binding Protein, soluble CD14 Innate As viral load decreases, immune activation persists T cells: increased turnover, exhaustion, low thymic output, virus reservoir B cells: increased turnover, altered phenotypic profile, hyper-Ig-emia Frequency of activated T cells is a strong predictor of disease progression Adaptive What We Talk About When We Talk About Immune Activation

4. Slide 4 of 24 From DC Douek, MD, at San Francisco, CA: March 24, 2013, IAS-USA. Causes Of Chronic Immune Activation Raised cytokine and chemokine levels are a consequence of immune activation HIV-induced activation of innate immune system (N. Bhardwaj) –When virus load decreases after acute phase, immune activation remains elevated –Virus load alone is a poor predictor of disease progression (Rodriguez JAMA 2006) –Measures of immune activation predict disease progression independent of viral load (Giorgi, Deeks...) –Elite controllers who progress have increased activated CD38 + T cells (Hunt JID 2008) –When virus load is suppressed with ART immune activation still persists and predicts progression Increased antigen load, bacterial overgrowth, herpes viruses (S. Deeks, P. Hunt) Translocation of proinflammatory mediators across mucosae

5. Slide 5 of 24 From DC Douek, MD, at San Francisco, CA: March 24, 2013, IAS-USA. Consequences of HIV Infection in GI Tract Healthy Gut Tight epithelial junctions, mucus Anti-microbial peptides, Abs, cells Majority of CD4 T cells in body Cross-talk between microbes and epithelial cells and immune cells Mucus HIV-Infected Gut Massive loss of CD4 T cells Enteropathy 2-10x increased permeability Translocation of microbial products Systemic immune activation CD4 T cell loss Loss of tight junctions Enterocyte apoptosis Microbial products

6. Slide 6 of 24 From DC Douek, MD, at San Francisco, CA: March 24, 2013, IAS-USA. HIV immune activation gut CD4 depletion enteropathy Tcm Tem gut

7. Slide 7 of 24 From DC Douek, MD, at San Francisco, CA: March 24, 2013, IAS-USA. HIV immune activation gut CD4 depletion enteropathy Tcm Tem CMV low thymic output LT fibrosis T/B cell dysfunction inflammation tissue damage coagulopathy non-AIDS morbidity and mortality immune deficiency ???

8. Slide 8 of 24 From DC Douek, MD, at San Francisco, CA: March 24, 2013, IAS-USA. Tcm Tem gut Tcm Tem low thymic output LT fibrosis T/B cell dysfunction inflammation tissue damage coagulopathy non-AIDS morbidity and mortality immune deficiency CMV ??? HIV CD4 depletion enteropathy ART immune activation

9. Slide 9 of 24 From DC Douek, MD, at San Francisco, CA: March 24, 2013, IAS-USA. T cell activation declines during long-term ART, but remains elevated, even after many years of viral suppression Hunt, et al. J Infect Dis. 2003, 2008 and unpublished observations ART and T Cell Immune Activation HIV – (n=132) HIV + HAART (n=65) HIV + Untreated (n=82) 0 20 40 60 80 % CD38 + DR + CD8 T cells P<0.001

10. Slide 10 of 24 From DC Douek, MD, at San Francisco, CA: March 24, 2013, IAS-USA. Hunt, CROI 2012 SOCA/SCOPE cohorts Markers of Inflammation and GI Dysfunction Predict Mortality Odds of Mortality (4 th vs 1 st Quartile) Markers of inflammation and gut barrier dysfunction predict mortality independently of CD4 count and virus load

11. Slide 11 of 24 From DC Douek, MD, at San Francisco, CA: March 24, 2013, IAS-USA. ART, Immune Activation and CD4 T Cell Recovery Reduced CD4 T cell recovery associated with immune activation Hunt, et al. J Infect Dis. 2003 and unpublished observations

12. Slide 12 of 24 From DC Douek, MD, at San Francisco, CA: March 24, 2013, IAS-USA. When Immune Activation Turns To The Dark Side Good Anti-viral innate immune response Restoration of memory CD4 T cells Bad Target cell generation HIV replication Thymic dysfunction T and B cell exhaustion Macrophage/DC activation Cytokine/Chemokine secretion Lymph node fibrosis Generalized tissue fibrosis Coagulation cascade activation … … …

13. Slide 13 of 24 From DC Douek, MD, at San Francisco, CA: March 24, 2013, IAS-USA. P < 0.0001 P = 0.060 P < 0.0001 P = 0.266 P = 0.010 Llibre, Buzón, Massanella et al. Antiv Ther 2011 %CD38 + memory CD8 T cells Control Intensified Raltegravir intensification reduces immune activation significantly more than conventional therapy Raltegravir Intensification

14. Slide 14 of 24 From DC Douek, MD, at San Francisco, CA: March 24, 2013, IAS-USA. Raltegravir Intensification Raltegravir intensification in 9 subjects resulted in decrease in IUPM and CD8 T cell activation

15. Slide 15 of 24 From DC Douek, MD, at San Francisco, CA: March 24, 2013, IAS-USA. No association between plasma measures of viral persistence and T cell activation in blood Raltegravir Intensification

16. Slide 16 of 24 From DC Douek, MD, at San Francisco, CA: March 24, 2013, IAS-USA. Hunt, Yukl and Wong Virus and Immune Activation in Tissues Stronger association between cell-based measures of viral persistence and T cell activation in gut tissues r = 0.65 P = 0.012 Sheth, Muc Imm 2012

17. Raltegravir intensification reduces immune activation and HIV RNA levels in gut tissue sites Virus and Immune Activation in Tissues Slide 17 of 36

18. Slide 18 of 24 From DC Douek, MD, at San Francisco, CA: March 24, 2013, IAS-USA. Evidence against ongoing HIV replication on ART Increasing evidence in favor of ongoing replication Evidence it is associated with immune activation The source of the sample is key (blood vs tissues) The assay used to measure virus is critical Ongoing HIV Replication During ART? Although complete inhibition of viral replication is unlikely to be curative, all cure strategies are based on first having achieved complete suppression

19. Slide 19 of 24 From DC Douek, MD, at San Francisco, CA: March 24, 2013, IAS-USA. Saez-Cirion et al, in press 14 subjects who started therapy very early after infection They remained on cART for many years and then therapy was stopped HIV-DNA (log 10 copies/10 6 PBMC) AcuteChroniccARTElite Controllers Post-Rx Controllers 1 2 3 4 5 6 VISCONTI — Activation and Reservoir PTC did not rebound when cART was stopped Like elite controllers they had low cell-associated HIV DNA But, in contrast, they had very low T cell activation

20. Slide 20 of 24 From DC Douek, MD, at San Francisco, CA: March 24, 2013, IAS-USA. On suppressive ART, strong HIV specific T cell responses in the gut mucosa are associated with lower levels of PBMC viral DNA r = - 0.56, P = 0.01 CD4CD8 r = - 0.37, P = 0.12 Hatano JID 2011 HIV-Specific Immunity and HIV Persistence

21. Slide 21 of 24 From DC Douek, MD, at San Francisco, CA: March 24, 2013, IAS-USA. Tcm Tem gut Tcm Tem low thymic output LT fibrosis T/B cell dysfunction inflammation tissue damage coagulopathy non-AIDS morbidity and mortality immune deficiency CMV ??? ART Tem immune activation

22. Slide 22 of 24 From DC Douek, MD, at San Francisco, CA: March 24, 2013, IAS-USA. immune activation low thymic output lymphoid fibrosis poor CD4 T cell renewal T/B cell dysfunction mucosal damage target cell generation infected cell proliferation virus transcription virus production new infection events

23. Slide 23 of 24 From DC Douek, MD, at San Francisco, CA: March 24, 2013, IAS-USA. Chemokine receptor inhibitors: –maraviroc, TB-652 Anti-infective therapy: –CMV, EBV, HSV, HCV/HBV Microbial translocation: –sevelamer, colostrum, rifaximin Enhance T cell renewal: –Growth Hormone, IL-7 Anti-fibrotic drugs: –pirfenidone, ACEi, ARBs, KGF Anti-aging: –caloric restriction, sirtuin activators, vitamin D, omega-3 fatty acids, rapamycin, diet, exercise Anti-inflammatory drugs: –Chloroquine, HCQ –Minocycline –NSAIDs (COX-2i, aspirin) –Statins –Methotrexate –Thalidomide, lenalidomide, pentoxyfylline (weak TNF inhibitors) –Biologics (e.g., TNF inhibitors, IL-6 inhibitors, anti-IFN , anti-PD1 Anti-coagulants: -low dose warfarin, dabigatran, aspirin, clopidogrel Combination therapy may be necessary Therapeutic Interventions in Development

24. Slide 24 of 24 From DC Douek, MD, at San Francisco, CA: March 24, 2013, IAS-USA. Multiple mechanisms account for HIV persistence, all of which are being addressed therapeutically The unifying theme is to reduce HIV reservoir size –Reduce inflammation –Increase immune function –Early ART and ART intensification –Gene therapy to reduce reservoir size –Stem cell transplants can reduce reservoir size –Drugs with biologic activity against latent virus exist –Vaccines may enhance host-clearance mechanisms In The Context of The Cure Combination therapy may be necessary