1. Inflammation biomarkers in HIV infection Laurence WEISS

2. Predictive biomarkers in HIV infection 1.Reflect The extent of immune deficiency The level of HIV replication and of HIV reservoir The level of chronic immune activation/inflammation The strength and quality of HIV-specific immune responses 2.Type of biomarkers Virologic, immunologic, activation/inflammatory and genetic biomarkers (HLA, CCR5, KIR) Predictive biomarkers for HIV progression in untreated and/or treated patients Predictive biomarkers for the occurrence of co morbidities in patients with ART-mediated viral suppression

3. Predictive biomarkers in HIV infection 1.Reflect The extent of immune deficiency The level of HIV replication and of HIV reservoir The level of chronic immune activation/inflammation The strength and quality of HIV-specific immune responses 2.Type of biomarkers Virologic, immunologic, activation/inflammatory and genetic biomarkers (HLA, CCR5, KIR) Surrogate markers for HIV progression in untreated and/or treated patients Surrogate markers for the occurrence of co morbidities in patients with ART-mediated viral suppression

4. - Membrane expression of the activation markers HLA-DR (MHC class II), CD38, CD25, CD70 - Intranuclear expression of Ki-67 (cell cycle) T-cell activation markers in the chronic phase of HIV infection Giorgi, JID 99 Leng et al, J.AIDS 2001 Hazenberg et al, AIDS 2003 HLA-DR CD38 CD4 CD8 Healthy donorSurvival > 18 MSurvival < 6 M T-cell activation is associated with mortality

5. PROINFLAMMATORY CYTOKINE NETWORK COAGULATION TISSUE FACTOR  D-dimer T-cell activation  sCD14  sCD163 Innate immunity Mono/M ,DC, NK cell activation Adaptative immunity INFLAMMATION PROINFLAMMATORY CYTOKINE NETWORK COAGULATION TISSUE FACTOR 1234560 IL-6 C-Reactive Protein Haptoglobin  1-glycoprotein acid  D-dimer  APP HIV Other viruses (CMV, HCV...) Bacterial products (LPS…) T-cell activation  sCD14  sCD163

7. PROINFLAMMATORY CYTOKINE NETWORK COAGULATION TISSUE FACTOR  D-dimer T-cell activation  sCD14  sCD163 Innate immunity Mono/M ,DC, NK cell activation Adaptative immunity INFLAMMATION PROINFLAMMATORY CYTOKINE NETWORK COAGULATION TISSUE FACTOR 1234560 IL-6 C-Reactive Protein Haptoglobin  1-glycoprotein acid  D-dimer  APP HIV Other viruses (CMV, HCV...) Bacterial products (LPS…) T-cell activation  sCD14  sCD163  sCD14  sCD163 … T-cell activation

8. Soluble activation biomarkers – β2- microglobulin, neopterin – hsCRP: acute phase protein – IL-6, IL-1RA, sTNFR: Cytokines and CR of innate immunity – D dimer: marker of procoagulant activity – sCD14: acute phase protein, monocyte activation – sCD163: secreted by activated monocytes/macrophages – ICAM, VCAM: endothelial activation/dysfunction – IP-10: chimiokine produced in response to IFN Fahey, NEJM 1990, Kuller, PloS Med 2008, Sandler, JID 2011, Burdo, JID 2011

9. Predictive biomarkers in primary HIV infection

10. Deeks, Blood 2004 Immune activation set point during early HIV infection: predictive of subsequent CD4 T-cell changes independently of viral load CD38-MFI on CD8 T cells Time (weeks) proportion with CD4 > 350 Time to a CD4 T-cell count less than 350 cells/mm 3 68 recently HIV-infected adults before ART CD8 T-cell activation set point

11. The early level of double negative CD4 - CD8 - T cells predicts the level of T-cell activation at set point R= -0.76 p= 0.004 R= -0.60 p= 0.035 Petitjean, Chevalier et al, AIDS 2012  DN T cells might play a role in the control of the harmful systemic immune activation DN T cells produce anti-inflammatory cytokines

12. BaselineM6 plasma IL-1RA (log pg/mL) IL-1RA An innate immune setpoint ? plasma sCD14 at baseline (ng/mL) plasma IL-1RA at baseline (log pg/mL) % CD38 + HLA-DR + CD8 T cells at M6 % CD38 + HLA-DR + CD8 T cells at M6 % CD38 + HLA-DR + CD8 T cells at M6 log Th17/Treg ratio at baseline Acute HIV baseline Acute HIV M6 Chronic HIV (untreated) Sepsis (S. aureus) n=27 n=25n=20n=10 plasma 16S rDNA (copies/µL) Chevalier, Plos Path 2013 Innate immune activation set point plasma sCD14 (ng/mL) BaselineM6 sCD14

13. Predictive biomarkers in untreated chronic infection

14. T-cell activation, predictive marker of AIDS progression independent of VL Proportion of CD70+CD4+ T cells > median (—) < median (- - -) Giorgi, JAIDS 1998 Hatzenberg, AIDS 2003

15. Relationship between T-cell activation and HIV reservoir The proportion of HLA-DR+ CD38+/ CD8+ T cells before TI predicts the increase in total HIV- DNA levels between baseline and M12 of TI (ANRS 116 SALTO) (r= 0.552; p = 0.004) Weiss, PlosOne 2010 Positive relationship between total HIV-DNA and CD8 and CD4 T-cell activation at 12 months of TI See poster Weiss et al MOPDA0106

16. Predictive values of soluble biomarkers in cohort studies C Reactive Protein Level: associated with AIDS - free survival Multivariate model VariableRelative Time (95% CI) P Value CRP, mg/L <1.2 1.3-2.3 >2.3 1.0 0.86 (0.68-1.09) 0.63 (0.51-0.79) 0.21 <0.001 CD4+ cell count1.12 (1.08-1.16)<0.001 HIV RNA (log10)0.34 (0.29-0.39)<0.001 Hemoglobin (g/dL)1.14 (1.06-1.23)<0.001 Lau, Arch Intern Med 2006 N= 513 patients randomly selected from the MACS population Median CD4: 532 (IQR 342;721) Median HIV-RNA: 18450 (IQR 5359; 63741) 62%  AIDS event 2709 person-years of follow-up

17. Fibrinogen and CRP, independent predictors of mortality in the FRAM study Tien, JAIDS 2010 922 HIV-infected participants > 85% on cART (past or present) 70% with history of AIDS  50% HIV-RNA BLD 20% HCV+ 5-year mortality risk

18. Biomarker levels associated with progression in RCT All-cause mortality: higher for patients with CD4> 350 randomly assigned to CD4- guided interruption of ART (DC) than continuous ART (VS) Most common causes of death: non AIDS-malignancy, CVD Case control study (85 cases and 170 matched controls) ; a study to compare DC and VS participants for biomarker changes (249 DC and 250 VS) Baseline IL-6, hsCRP and D-dimer associated with all cause mortality Higher BL IL-6, D-dimer and hsCRP: related to CVD IL-6 and D-dimer ↗ at 1 mo in the DC group. Increases related to HIV-RNA levels BL or latest IL-6 or hsCRP: predictive of OI Elevated pre-ART levels of hsCRP, IL-6 and D-dimer: strongly associated with early mortality after ART initiation Ledwaba, PlosOne 2012 Other markers associated with disease progression (case control ACTG 384 and 5015) : sTNFR-1, sCD27 and sCD40L Kalayjian, JID 2010 Kuller Plos Medicine 2008 Rodger JID 2008 Duprez PlosOne 2012 SMART PHIDISA (South Africa trial)

19. IP10 and sCD163 levels predict subsequent CD4 counts Predictive inflammatory biomarkers in HIV controllers Lambotte, IAS 2014, MoAA0102

20. Predictive biomarkers in patients with chronic infection and ART-mediated viral suppression Residual immune activation and inflammation under ART

21. Hunt, JID, 2003 Hunt, JID 2008 Persistence of residual chronic T-cell activation in ART- treated patients n = 30 HIV+ with CV < 75 c/mL

22. Despite long-term viral suppression, soluble inflammatory biomarkers remain higher in patients compared to the general population % Diff. from General Population (MESA) Neuhaus JID 2010

23. Inflammation with ongoing viral replication Inflammation with ongoing viral replication Inflammation under ART Inflammation under ART Inflammation HIV- controls Inflammation HIV- controls MonocytespDCs Innate Immunity NK Adaptative immunity

24. Nested case-control study in SMART 74 deaths + 120 CV events + 100 AIDS events (20 NA) / N= 5472 2 controls/case Most patients under ART with VL < 400 cp/mL Baseline plasma sCD14, IFABP, LPS, EndoCAB SCD14: marker of monocyte activation (acute phase protein) not necessarily indicative of microbial translocation Sandler, JID 2011 Plasma levels of sCD14: independent predictor of mortality in the SMART study

25. Can we improve prediction of mortality by adding inflammatory biomarkers? Veterans Aging Cohort Study: 1 302 veterans under ART; 70% with VL <500 c/mL; 154 deaths RI= age/CD4/VL VACS index= RI + Hb/ FIB4 index  age, transa, platelets  /HCV/eGFR: better prediction of mortality than any biomarker or than RI (p< 0.001) VACS+ inflammatory biomarkers (IL-6, D-dimer, sCD14) Justice CID 2012

26. Cardio- Vascular Diseases Cognitive disorders Inflammation CANCER CHRONIC VIRAL INFECTIONS (HIV) Osteoporosis

27. Cardiovascular comorbidities In HIV infection : Alteration in biomarkers known or potentially associated with CVD in non-HIV infected individuals HDL cholesterol depletion Chronic inflammation (  CRP,  IL-6,  sCD14) Endothelial activation/dysfunction (  VCAM, ICAM) Activation of coagulation (  D-dimer) Only partially normalized during sucessfull ART Atherosclerotic plaque

28. Association of soluble CD163 with arterial inflammation Hypothesis: Arterial inflammation -> HIV + pts compared with non-HIV FRS-matched controls -Correlated to mono/Mф activation (CD163) 81 participants. 27 HIV pts under ART (CD4 nadir  100) FDG-PET (activated Mф : high metabolic rate) VariablesHIV+ (N= 27) FRS-matched HIV- (N= 27) HIV- atheroscl (N= 27) P value Mean FDG uptake (95% CI) 2.23 (2.07-2.40)1.89 (1.80-1.97)2.13 (2.03-2.23)<0.001 CAC score median (IQR) 24.4 (0.92-6)0 (0-4.8) [N= 24]425.2 (88.8-1234) [N= 16] < 0.001 Subramanian JAMA 2012 Lack of association between aortic FDG uptake and CRP or D-dimer in the HIV+ population FDG uptake

29. Soluble markers of inflammation & coagulation, but not T-Cell activation, predict non-AIDS defining events during suppressive ART Case-control study of ALLRT subjects (ACTG studies) (ART-naïve at BL and ART-suppressed during FU) Cases : non-AIDS death, MI, stroke, non- AIDS cancer, or serious non-AIDS bacterial infection Controls (2 - 3/case) Greater CD4 change at yr 1 associated with a decreased risk for non-AIDS event (OR per 100 cells increase= 0.81, p= 0.007) High T-cell activation: not consistently associated with a non-AIDS-related event Higher IL-6,sCD14, sTNFR-I, sTNFR-II, and D-dimer prior to ART independently associated with non AIDS events Tenorio CROI 2013

30. Soluble markers of inflammation & coagulation, but not T-Cell activation, predict non-AIDS defining events during suppressive ART Case-control study of ALLRT subjects (ACTG studies) (ART-naïve at BL and ART-suppressed during FU) Cases : non-accidental non-AIDS death, MI, stroke, non-AIDS cancer, or serious non-AIDS bacterial infection Controls (2 - 3/case) Greater CD4 change at yr 1 associated with a decreased risk of non-AIDS event (OR per 100 cells increase= 0.81, p= 0.007) High T-cell activation: not consistently associated with a non-AIDS-related event Higher IL-6,sCD14, sTNFR-I, sTNFR-II, and D-dimer prior to ART independently associated with non AIDS event Tenorio CROI 2013

31. Suboptimal CD4 T-cell gains Immune senescence Comorbidities (Accelerated atherosclerosis, arterial inflammation, cognitive disorders, chronic renal disease, osteoporosis: « Inflam-Aging », cancers) ↗ Risk of mortality Persistent Inflammation under ART  CRP, IL-6, IL-1RA, sCD14, sTNFR  D-dimer  Fg

32. Summary Levels of immune activation (T-cell activation and inflammation) predict HIV disease progression in untreated patients with primary and chronic infection Soluble biomarkers of inflammation and coagulation, but not T-cell activation, predict non-AIDS defining events during suppressive ART Unresolved issues Value of these biomarkers to improve CV risk prediction in the HIV-infected population ? Significance: just an association or role in pathogenesis ? Help to identify cellular pathways important in the pathogenesis of HIV disease Interventions that target these pathways (e.g. IL-6) and/or the mechanisms involved in low levels of chronic activation/inflammation (e. g. ongoing low- level viral replication, MT, coinfections..) are warranted (or already in progress) with the ultimate goal to decrease the incidence of non-AIDS related morbidity in HIV-infected patients on virally suppressive ART

33. All the patients included in the studies Hôpital Européen G. Pompidou Christophe Piketty Maria Manea Erika Bourzam Hôpital Saint-Antoine Pierre-Marie Girard Pauline Campa Nelly Desplanques Hôpital Tenon Laurence Slama Gilles Pialoux CHU Carémeau Nîmes Jean-Philippe Lavigne Catherine Dunyach Mathieu Chevalier Gaël Petitjean Céline Didier Daniel Scott-Algara Françoise Barré-Sinoussi …and all the physicians that included patients INSERM U 1018 Laurence Meyer Christiane Deveau Feriel Tibaoui Remerciements U943 Dominique Costagliola Lambert Assoumou the ANRS 116 SALTO study group EA 3620 Christine Rouzioux