What drives persistent immune activation/inflammation in cART-treated HIV-1? Giulia Marchetti, MD, PhD Dept of Health Sciences, Clinic of Infectious Diseases - University of Milan, San Paolo Hospital, Italy

The revolution of cART Hammer, S et al. NEJM 1997; Palella, F et al. NEJM 1998 CD4+HIV-RNA

Full restoration of health upon cART?

Samji et al. ART-Cohort Collaboration. PlosOne 2013 Life expectancy in cART-treated patients ART-Cohort Collaboration individuals (82,022 person- year) At age 20

Serious non-AIDS events during long-term virologically suppressive cART Tenorio et al. JID 2014

HIV as an inflammatory disease Acute HIV associated with rapid/intense release of pro-inflammatory cytokines (IL-6, IP-10, TNF-a) and dramatic increase of activated innate immune cells T-, B-cells Chronic HIV: T-cell activation steady state

High proportion of activated CD8+ T-cells in HIV Giorgi, J et al. J Immunol 1993

CD8 T-cell activation predicts CD4+ T-cell count over time Deeks et al. Blood 2004

Shorter survival is associated with T- lymphocyte activation Giorgi, J et al. JID 1999

In untreated infection T-cell activation is associated to HIV viral replication Deeks S Blood 2004

What happens upon cART?

CD8+ T cell activation fails to fully normalize during effective cART Hunt PW, et al. J Infect Dis. 2003;187:

Chronic inflammation is a much more important determinant of mortality in treated HIV Kuller L PLOS Medicine 2008; also Hunt et al. AIDS 2011; Lok et al, AIDS 2013; Hunt et al. JID 2014; Tenorio et al JID 2014; …..

What drives persistent immune activation/inflammation in cART-treated disease?

Gut epithelial barrier dysfunction, microbiome and microbial translocation Co-infections (CMV et al….) Residual HIV replication Thymic dysfunction and residual defects in adaptive immune responses Lack of immunoregulatory responses- Lymphoid fibrosis Co-morbid conditions (metabolic syndrome, central adiposity)

What drives persistent immune activation/inflammation in cART-treated disease? Gut epithelial barrier dysfunction, microbiome and microbial translocation Co-infections (CMV et al….) Residual HIV replication Thymic dysfunction and residual defects in adaptive immune responses Lack of immunoregulatory responses- Lymphoid fibrosis Co-morbid conditions (metabolic syndrome, central adiposity)

Gut epithelial barrier dysfunction, microbiome and microbial translocation

The GI tract as a site of HIV pathogenesis Sandler & Douek, Nat Reviews 2012 Brenchley et al. Nat Med 2006

Persistent Depletion of CD4+ T cells in the GI Tract despite Normalization in the Peripheral Blood Mehandru S, Plos Med HIV+ patients (acute)

Persistent damage to the gut tight epithelial barrier despite cART HIV negative HIV+ cART-treated Tincati C et al. CROI 2014 Chung; Plos. Path. 2014; (see also Somsouk AIDS 2015)

Altered gut tight junctions associate with microbial translocation Brenchley J et al. Nat Med 2006; also Jiang et al. J Infect Dis 2009 Chung et al. Plos. Path. 2014; (see also Somsouk AIDS 2015)

(Altered) gut tight epithelial barrier as driver of inflammation?

Altered gut tight junctions associate with immune activation Chung et al Plos. Path. 2014; (see also Somsouk AIDS 2015) Tincati C et al. CROI 2014

Altered gut microbioma in SIV/HIV Brenchley Nat Med 2006; in humans: Gori et al. JCM 2008

Only partial recovery of gut microbioma upon successful cART 50 HIV+ patients before (T0) and after 12 months cART (T12)

(Altered) intestinal microbioma as driver of inflammation?

Greater representation of proinflammatory/inflammation- thriving class-level bacteria Correlation between gut microbioma and systemic immune activation Ellis et al. JAIDS 2011, also Dillon et al. Mucosal Immunol 2014

Persistent microbial translocation during cART Brenchley J et al. Nat Med 2006; also Jiang et al. J Infect Dis 2009

Microbial translocation hampers CD4+ T-cell recovery upon cART Marchetti G et al. AIDS 2008; Brenchley J et al. Nat Med 2006; also Jiang et al. J Infect Dis 2009

Microbial translocation and immune activation: what is the cause what is the effect?

Microbial translocation causes immune activation: colocalization of E.coli and IFN-  in colon Estes J et al. PLoS Pathogens 2010

Microbial translocation is associated to immune activation Brenchley J et al. Nat Med 2006 Marchetti G et al. AIDS 2008; Jiang et al. J Infect Dis 2009

Exogenous LPS administration enhances immune activation and HIV replication Pandrea et al J Immunol 2008; Pandrea et al. Blood 2012

Bacterial products drive monocyte expression of thrombosplastin Funderburg N et al Blood 2010

In vitro LPS stimulation of monocyte-derived macrophages: cytokine/chemokine expression of genes involved in the TLR pathway Merlini E et al ICI, International Congress of Immunology HIV+ cART- treated

Stimulation of peripheral blood cells by TLR ligands increases expression of CD38 on CD4+ and CD8+ T-lymphocytes - HIV-negative Funderburg N et al. PLoS One 2008

In vitro LPS stimulation on PBMC: CD4 and CD8 T- cell activation, proliferation and apoptosis Merlini E et al ICI, International Congress of Immunology HIV+ cART- treated

Sevelamer treatment reduces MT during early SIVsab infection of PTMs LNs stained for LPS core antigen (brown) Kristoff J, JCI, 2014: 124 (6)

Sevelamer treatment reduces immune activation/inflammation during early SIV infection in PTMs Kristoff J, J Clin Invest 2014

Sevelamer does not reduce LPS and sCD14 in chronic early-stage untreated HIV Sandler N, J Infect Dis 2014 Should we test sevelamer in cART- treated HIV?

Altered balance of gut immunoregulatory cells (e.g. Th17/Th22, gut- homing T-cells) as driver of inflammation?

Only partial recovery of gut-homing T-cells upon cART 20 HIV+ before and at 12 months cART 20 HIV+ cART- treated Mavigner et al. JCI 2012 Basilissi M ICAR 2015

Only partial recovery of gut-homing and Th17/Th22 T-cells upon cART 20 HIV+ before and at 12 months cART Basilissi M ICAR 2015

Low Th17/Treg ratio despite cART Favre et al Science Transl Med HIV+ cART- treated

Low Th17/Treg ratio is associated to immune activation Favre et al Science Transl Med HIV+ cART- treated

What drives persistent immune activation/inflammation in cART-treated disease? Gut epithelial barrier dysfunction, microbiome and microbial translocation Co-infections (CMV et al….) Residual HIV replication Thymic dysfunction and residual defects in adaptive immune responses Lack of immunoregulatory responses- Lymphoid fibrosis Co-morbid conditions (metabolic syndrome, central adiposity)

Higher non-AIDS morbidity/mortality in HIV+/CMV-Ab+ patients 6111 HIV+ (5119 CMV-Ab+), 12% cART-treated Lichtner M et al. J Infect Dis 2015

Naeger D et al. PlosOne 2010

cART-treated asymptomatic CMV seminal shedders present higher T- cell activation/proliferation Gianella S et al. J Virol HIV+ cART- treated

cART-treated asymptomatic CMV seminal shedders present higher T-cell expression of PD-1 45 HIV+ cART- treated Dan J et al. CROI 2015

Vita S et al. CROI 2015 Higher innate immunity markers in HIV/CMV co-infected patients on cART 69 HIV+ cART- treated (46/69 CMV Ab+)

Reduction of CD8 T-cell activation by valganciclovir…… Hunt et al., JID, HIV+, 70% cART-treated

……but not valacyclovir Yi TJ et al., CID, HIV+/HSV2+ cART-treated

HCV coinfection was associated with increased risk of developing an ADI (adjusted relative rate [ARR], 2.61; 95% confidence interval [CI], 1.88–3.61) *ARR, 3.15 *ARR, 3.87 *ARR, 2.68

127 HIV-infected hepatitis viruses co-infected patients (118 HCV, 9 HBV) - ART naïve, CD4 cell count >200/μl - known date of prior HIV neg/pos tests →immune activation (IA): IL-6,TNFα →microbial translocation (MT): LPS, sCD14

Gonzalez et al et al. J Virol : 14 HCV+/HIV+ cART-treated; 11 HCV+; 9 HIV+ treated

Hampered T-cell dynamics in HIV/HCV co-infected patients 356 HIV+ cART- treated : 130 HCV co-infected Zaegel-Fauchel O et al. AIDS 2015

Hunt et al. JID 2003; also Greub G Lancet 2000 HCV co-infection is associated to higher T- lymphocyte activation on cART

HCV treatment reduces immune activation ?

Reduction of T-cell activation by anti-HCV treatment Gonzalez et al et al. J Virol 2009; also Massanella M et al. Antiviral Therapy HIV+ cART-treated : 130 HCV co- infected

What drives persistent immune activation/inflammation in cART-treated disease? Gut epithelial barrier dysfunction, microbiome and microbial translocation Co-infections (CMV et al….) Residual HIV replication Thymic dysfunction and residual defects in adaptive immune responses Lack of immunoregulatory responses- Lymphoid fibrosis Co-morbid conditions (metabolic syndrome, central adiposity)

Adapted from Deeks S – International Congress on Drug Therapy in HIV Infection, Glasgow UK 2-6 Nov 2014 Despite cART, HIV viremia persists indefinitely at very low level

Immune activation does not correlate with residual plasma viremia…… Case: 123 HIV+ cART-treated with transient low level viremia (>50 <400 cp/ml) Control: HIV+ cART-treated RNA<50cp/ml Taiwo B et al. JAIDS 2013; also Chun TW et al. JID 2011 (including C-reactive protein, D-dimer, IL-6, soluble TNF receptor I); Steel A et al. Antiviral Therapy 2007…..

Steel A et al. Antiviral Therapy 200.

…but may associate with residual plasma viremia in the setting of poor immune recovery on cART…. Mauvigner M et al. PlosOne 2009 Marchetti G et al. AIDS 2006

….Immune activation (and senescence) does associate with cell-associated HIV-DNA/RNA in peripheral blood …. 190 HIV+ cART- treated Hatano H et al. JID 2012; also Stone SF HIV Med 2005

….and in tissues 23 HIV+ cART- treated Sheth PM et al. Mucosal Immuno 2008; also Yukl SA JID 2010; d’Ettorre G et al. Curr HIV Res 2011

Hypothesis: if residual HIV replication sustains immune activation upon cART, then cART intensification should lower immune activation

Any benefit by maraviroc intensification? Wilkin et al., JID 2012

Any benefit by maraviroc intensification? Hunt et al., Blood, 2013 Rusconi et al., PLOSOne, HIV+ cART- treated with low CD4+ immune recovery 45 HIV+ cART-treated with low CD4+ immune recovery

Any benefit by integrase inhibitors intensification? 30 HIV+ cART-treated with low CD4+ immune recovery Hatano H et al., JID, 2011; also Hatano H et al JAIDS 2012 Peripheral blood Gut

Raltegravir induced a specific reduction of CD38 expression in CD8 T cells Massanella et al., AIDS, 2012; also Vallejo A et al. AIDS 2012 Buzon MJ et al. Nat Med 2010; 69 HIV+ cART-treated

Any differences in the effect on immune activation by diverse cART class?

CD4 231/uL; n=76 AZT+ddI or AZT+3TC+ ABC or EFV or IDV/r Rizzardini et al., HIV Clin Trials 2006 Advanz Study CD4<100/uL; n=65 AZT+3TC+EFV or LPV/r Mirò et al. AIDS Res and Human Retrov 2010 Advanz 3 Study CD4<100/uL; n=89 TDF+FTC+ EFV or ATZ/r or LPV/r Mirò et al. JAIDS, 2015 Immuno Study CD4 250/uL; n=35 TDF+FTC+ EFV or DRV 800mg/r Tincati et al. under review Data are presented as median values

Advanz 3 Study CD4<100/uL; n=89 TDF+FTC+ EFV or ATZ/r or LPV/r Mirò et al. JAIDS, 2015 Immuno Study CD4 250/uL, n=35 TDF+FTC+ EFV or DRV 800mg/r Tincati et al. under review Data are presented as median values See also: McComsey, AIDS, 2012

Similar reduction of T-cell activation by different cART class 318 HIV+ starting first cART (170 PI; 128 NNRTI; 20 INI)

What drives persistent immune activation/inflammation in cART-treated disease? Gut epithelial barrier dysfunction, microbiome and microbial translocation Co-infections (CMV et al….) Residual HIV replication Thymic dysfunction and residual defects in adaptive immune responses Lack of immunoregulatory responses- Lymphoid fibrosis Co-morbid conditions (metabolic syndrome, central adiposity)

Isgro’ et al. CID 2008 Bellistrì et al. PlosOne 2010 Bone marrow alterations upon cART 23 HIV+ cART- treated cART

Douek et al. Nature 1998

What drives persistent immune activation/inflammation in cART-treated disease? Gut epithelial barrier dysfunction, microbiome and microbial translocation Co-infections (CMV et al….) Residual HIV replication Thymic dysfunction and residual defects in adaptive immune responses Lack of immunoregulatory responses- Lymphoid fibrosis Co-morbid conditions (metabolic syndrome, central adiposity)

In untreated HIV: hyper-inflamed cytokine milieu → Treg response → TGF-β → collagen deposition → Fibrosis → Reduced IL- 7 → Reduced T cell regeneration → inflammation

Zeng et al. PlosPathogens 2012 Recovery of collagen deposition according to the stage of cART start

Schaker et al. JID 2002 Collagen deposition in lymphoid tissues before cART substantially impacts the dynamics of T- lymphocyte reconstitution Zeng et al. PlosPathogens 2012 LN Asmuth et al. AIDS 2015 GUT

Tissue fibrosis as driver of immune activation?

Increased CD90+TLR4+ activated myofibroblast in HIV+ duodenal mucosa (  - SMA+FAP+) Asmuth et al. AIDS 2015

Pinchuck IV et al. Curr Gatroenterol Rep 2010

LPS stimulation of cultured intestinal myofibroblast from HIV+ patients upregulates pro-fibrotic mediators Asmuth et al. AIDS LPS

Klatt et al. Immunol Rev 2013

Thanks *Dept of Health Sciences- Clinic of Infectious Diseases- Univ of Milan, San Paolo H Esther Merlini Camilla Tincati Elvira S Cannizzo Giuseppe Ancona Giusi M Bellistrì Francesca Bai Matteo Basilissi Antonella d’Arminio Monforte ***all the patients and staff *Dept of Health Sciences- Pathology Dept- Univ of Milan, S Paolo Ho Delfina Tosi, Solange Romagnoli (now Roche Diagnostics, Germany) Alessandro Cozzi-Lepri, Miriam Lichtner, Antonella d’Arminio Monforte *Clinic of Infect Dis Univ of Milan, L Sacco H Stefano Rusconi, Massimo Galli