Pathogenesis Update Robert F. Siliciano, MD, PhD

Pathogenesis Update Robert F. Siliciano, MD, PhD Professor of Medicine and Molecular Biology and Genetics Johns Hopkins University School of Medicine Investigator, Howard Hughes Medical Institute

HIV-1 Eradication Strategies: Design, Assessment, and Clinical Consequences Disclosures: None

Viral dynamics in patients on Rx 1000000 100000 10000 1000 100 10 1 0.1 0.01 0.001 Start therapy a 0 100 Time on therapy (days) v + t 1/2 = ln2 / a = 1 day a Activated CD4+ T cells Ho et al, Nature, 1995; Wei et al, Nature, 1995

Viral dynamics with monotherapy 1000000 100000 10000 1000 100 10 1 0.1 0.01 0.001 Start therapy 0 100 200 300 Time on HAART (days)

Viral dynamics in patients on HAART 1000000 100000 10000 1000 100 10 1 0.1 0.01 0.001 m t 1/2 = 1 d Limit of detection (50 copies/ml) + t 1/2 = 14 d 0 100 200 300 Time on HAART (days) v + Eradication in 2 to 3 years Activated CD4 + T cells Hammer et al, NEJM, 1997; Gulick et al, NEJM, 1997; Perelson et al, Nature, 1997 a

Establishment of immunologic memory Naive Ag Memory Ag

HIV infection of activated and resting CD4+ T cells Naive Ag HIV a HIV Ag a Memory HIV

Establishment of the latent reservoir in resting CD4+ T cells Naive Ag HIV Memory

NFº B sites in the HIV LTR U 3 R U 5 Modulatory region Enhancer Core Cell DNA AP1 NFAT1 USF1 Ets1 LEF NFκB NFAT Sp1 TBP LBP1 Nabel et al, Nature, 1987;326:711; Tong-Starksen et al, PNAS, 1987;84:6845; Bohnlein et al, Cell, 1988;53:827; Duh et al, PNAS, 1989;86:5974

Reactivation of latent HIV Naive Ag HIV Memory Ag

Quantitative viral outgrowth assay for latent HIV-1 in resting CD4 cells 180-200 ml blood Purified resting PHA + irradiated allogeneic PBMC CD4+ T cells 1/10 6 Day 2: add CD4+ lymphoblasts from HIVdonors p24 Ag Day 7: add CD4+ lymphoblasts from HIVdonors Finzi et al, Science, 1997; Chun et al, PNAS, 1997

Slow decay of latently infected CD4+ T cells Frequency (per 10 6 cells) 10000 1000 100 10 1 0.1 0.01 0.001 0.0001 0.00001 0 1 2 3 4 5 6 7 Time on HAART (years) - Time to eradication > 73.4 years Finzi et al, Nature Med, 1999; Siliciano et al, Nature Med, 2003

Viral dynamics in patients on HAART 1000000 100000 10000 1000 100 10 1 0.1 0.01 0.001 m t 1/2 = 1 d + u t 1/2 = 14 d v Residual Eradication viremia in 2 to 3 years 0 100 200 300 Time on HAART (days) + a 400 Dornadula et al, JAMA, 1999; Palmer et al, PNAS, 2008

HIV cures The Berlin patient HSC transplantation with elimination of viral reservoirs by chemotherapy/radiation and GVH disease The Mississippi infant early treatment prior to establishment of the reservoir The Visconti cohort adults treated early after infection who control viral replication after discontinuation of Rx

The first cure Conditioning regimen Allogeneic bone marrow transplant NY Times, 11/28/11 Graft protected from HIV GVHD eliminates recipient immune cells

Cure of a 28-month-old perinatally infected child Born at 35 weeks of gestation (2.5 kg) Normal spontaneous vaginal delivery Not breast-fed Mother found to have positive rapid HIV test during labor No antiretroviral medications administered during labor (delivery was precipitous) Infant transferred to University of Mississippi at 30 hours and started on HAART Persaud et al, CROI 2013

Decay of viremia on HAART 19,812 c/ml 2,617 c/ml 516 c/ml 265 c/ml <48 c/ml AZT/3TC/NVP AZT/3TC/LPV/r 31 hours 7 days 7 days 18 months Persaud et al, CROI 2013

No rebound viremia after discontinuation of HAART Regimen #1: AZT/3TC/NVP Regimen #2: AZT/3TC/LPV/r Lost to follow-up; HAART discontinued by caretaker at age 18 months Persaud et al, CROI 2013

A latent reservoir for HIV in resting memory CD4+ T cells Naive Ag HIV Memory Ag

Development of T-cell memory >90% of CD3+ T-lymphocytes are naive in first weeks Adult proportion of memory T-lymphocytes achieved by 12 years of age

Visconti cohort 14 patients treated during primary HIV infection with prolonged control of viremia after interruption of Rx High HIV RNA and loss of CD4 cells during primary infection Relatively weak CTL responses Only 3 of 14 have protective HLA alleles common in elite suppressors Levels of HIV DNA in PBMCs only slightly lower than in patients on HAART Replication-competent virus can be isolated Saez-Cirion et al, PLoS Pathogens 2013

Visconti cohort This is not eradication. 6 of 14 have had episodes of detectable viremia. Appears to be control of viremia but may differ from spontaneous control observed in elite suppressors. May be control induced by early Rx. Mechanism unclear. Denominator unclear. Saez-Cirion et al, PLoS Pathogens 2013

Fundamental approach to HIV eradication v u a? a2` a Will resting cells die rapidly after reversal of latency? How do we identify small molecules that reverse latency without inducing T-cell activation?

Finding latency-reversing drugs 293 or Hela cells Epithelial Transformed Proliferating LTR-reporter constructs Transformed T-cell lines T cells Transformed Proliferating Proviruses Clonal Primary CD4+ T-cell models T cells Nontransformed G 0 Proviruses High frequency CD4+ T cells from patients T cells Nontransformed G 0 Proviruses Low frequency Screening throughput In vivo relevance

Multiple molecular mechanisms maintain HIV latency PKC activators Cytoplasm Iº B p50 p65 T-cell activation P NFAT Ca 2+ influx Nucleus HDAC inhibitors HMT inhibitors Suv39h1 HDACs CTIP-2 Me Nuc-0 CpG Island 1 p50 p50 SP1 SP1 SP1 TSS Nuc-1 CpG Island 2 Me Me DNMTs 7SK RNA CDK9 Cyclin T1 Hexim-1 ptefb activators DNMTs DNMT inhibitors

Finding latency-reversing drugs 293 or Hela cells Epithelial Transformed Proliferating LTR-reporter constructs Transformed T-cell lines T cells Transformed Proliferating Proviruses Clonal Primary CD4+ T-cell models T cells Nontransformed G 0 Proviruses High frequency CD4+ T cells from patients T cells Nontransformed G 0 Proviruses Low frequency Screening throughput In vivo relevance

A primary cell model for latent HIV Activate Transduce with bcl-2 Culture Primary resting CD4+ T cells Activate bcl-2-transduced primary CD4+ T cells Infect Culture Advantages Cells are fully quiescent Picks up all known activators Recapitulates memory cell generation in vivo Captures time-dependent mechanisms Activate Sort Yang et al, J Clin Invest, 2009

A primary cell model for latent HIV Activate Transduce with bcl-2 Culture Primary resting CD4+ T cells Activate bcl-2-transduced primary CD4+ T cells Infect Culture Advantages Cells are fully quiescent Picks up all known activators Recapitulates memory cell generation in vivo Captures time-dependent mechanisms Test cmpd Sort Yang et al, J Clin Invest, 2009

Screening for agents that reverse latency without T-cell activation 5-Hydroxy- naphthalene- 1,4-dione 5-Chloro-7- substituted quinolines Disulfiram: FDA approved to treat alcoholism Nitroxoline derivatives Yang et al, J Clin Invest, 2009; Xing et al, J Virol, 2011; Xing et al, J Antimicrob Chemother, 2011

Testing latency-reversing drugs 293 or Hela cells Epithelial Transformed Proliferating LTR-reporter constructs Screening throughput Transformed T-cell lines T cells Transformed Proliferating Proviruses Clonal Primary CD4+ T-cell models T cells Nontransformed G 0 Proviruses High frequency CD4+ T cells from patients T cells Nontransformed G 0 Proviruses Low frequency 1/10 6 In vivo relevance

Assay for reversal of latency using patient resting CD4+ T cells 500 x 10 6 resting CD4+ T cells Test compound Assay for production of viral RNA or infectious virus

Fundamental approach to HIV eradication v u a LRA?? a2` a Will resting cells die rapidly after reversal of latency?

Fate of infected CD4+ T cells after reversal of latency TCR pathway agonists HDACi Residual GFP+ cells (%) 120 100 80 60 40 20 0 0 HDACi ±CD3 + ±CD28 2 3 4 5 6 7 Days after reactivation Shan et al, Immunity, 2012

CTL killing of latently infected cells treated with an HDACi Normal donor 1 Surviving infected cells (%) 100 80 60 40 20 0 0 2 4 6 8 Time of coculture (days) Normal donor 2 Normal donor 3 Elite suppressor 1 Elite suppressor 2 Elite suppressor 3 HAART patient 1 HAART patient 2 HAART patient 3 HAART patient 4 HAART patient 5 HAART patient 6 Shan et al, Immunity, 2012

Measuring reductions in the reservoir in patients in eradication trials 180-200 ml blood Purified resting CD4+ T cells PHA + irradiated allogeneic PBMC Day 2: add CD4+ lymphoblasts from HIVdonors Day 7: add CD4+ lymphoblasts from HIVdonors p24 Ag

Comparison of reservoir assays Send to UCSD Digital droplet PCR UCSD

Assays for persistent HIV in patients on HAART Assay Cell/tissue Infected cell frequency (per 10 6 ) 10,000 1,000 100 10 1 0.1 Viral outgrowth Total HIV DNA Resting CD4 PBMC Resting CD4 Integrated HIV DNA Total HIV DNA PBMC Resting CD4 Rectal CD4 2 LTR circles PBMC Quantitative viral outgrowth assay Residual viremia Plasma 1 0,000 1, 000 100 10 1 0.1 Plasmas HIV RNA (copies/ml) Cohort Chronic Acute Chronic Acute Chronic Acute Chronic Acute Chronic Acute Chronic Acute Chronic Acute Chronic Acute Eriksson et al, PLOS Pathogens, 2013

Assays for persistent HIV in patients on HAART Assay Cell/tissue Infected cell frequency (per 10 6 ) 10,000 1,000 100 10 1 0.1 Viral outgrowth Total HIV DNA Resting CD4 PBMC Resting CD4 Integrated HIV DNA Total HIV DNA PBMC Resting CD4 Rectal CD4 2 LTR circles PBMC Residual viremia Plasma 1 0,000 1, 0 0 0 Droplet digital PCR 100 on PBMC 1 0 1 0.1 Plasmas HIV RNA (copies/ml) Cohort Chronic Acute Chronic Acute Chronic Acute Chronic Acute Chronic Acute Chronic Acute Chronic Acute Chronic Acute Eriksson et al, PLOS Pathogens, 2013

Correlation between culture and PCR assays HIV DNA (copies/10 6 PBMC) 1 0, 0 0 0 1, 0 0 0 1 0 0 1 0 1 0.01 0.1 1 10 100 Viral outgrowth (IUPM) r P Combined 0.20 0.32 Chronic -0.09 0.73 Acute 0.42 0.26 n 27 18 9 Eriksson et al, PLOS Pathogens, 2013

Ratio of infected cell frequencies by PCR and culture assays Ratio of HIV-1 DNA to Infectious Units 4000 3500 3000 2500 2000 1500 1000 500 0 * * 1079 1126 1234 2013 2021 2056 2104 2114 2147 3068 3178 2238 2263 2264 2277 2418 Chronic Patient ID Acute Eriksson et al, PLOS Pathogens, 2013

Assays for persistent HIV in patients on HAART Assay Cell/tissue Infected cell frequency (per 10 6 ) 10,000 1,000 100 10 1 0.1 Viral outgrowth Total HIV DNA Resting CD4 PBMC Resting CD4 r = 0.38 p = 0.28 Integrated HIV DNA PBMC Resting CD4 Rectal CD4 r = 0.70 p < 0.01 r = 0.41 p = 0.13 Total HIV DNA r = 0.05 p = 0.86 2 LTR circles PBMC rho = 0.19 p = 0.31 Residual viremia Plasma 1 0,000 1, 000 100 10 1 0.1 Plasmas HIV RNA (copies/ml) Cohort Chronic Acute Chronic Acute Chronic Acute Chronic Acute Chronic Acute Chronic Acute Chronic Acute Chronic Acute Eriksson et al, PLOS Pathogens, 2013

Residual viremia + 1000000 100000 10000 1000 100 10 1 0.1 0.01 0.001 v + r u a 0 100 200 300 Time on HAART (days) 400

Assays for persistent HIV in patients on HAART Assay Cell/tissue Infected cell frequency (per 10 6 ) 10,000 1,000 100 10 1 0.1 Viral outgrowth Total HIV DNA Resting CD4 PBMC Resting CD4 r = 0.38 p = 0.28 Integrated HIV DNA PBMC Resting CD4 Rectal CD4 r = 0.70 p < 0.01 r = 0.41 p = 0.13 Total HIV DNA r = 0.05 p = 0.86 2 LTR circles PBMC rho = 0.19 p = 0.31 Residual viremia Plasma rho = 0.07 p = 0.71 1 0,000 1, 000 100 10 1 0.1 Plasmas HIV RNA (copies/ml) Cohort Chronic Acute Chronic Acute Chronic Acute Chronic Acute Chronic Acute Chronic Acute Chronic Acute Chronic Acute Eriksson et al, PLOS Pathogens, 2013

Which assay should be used? Assay Cell/tissue Infected cell frequency (per 10 6 ) 10,000 1,000 100 10 1 0.1 Viral outgrowth Total HIV DNA Resting CD4 PBMC Resting CD4 r = 0.38 p = 0.28 300x Integrated HIV DNA PBMC Resting CD4 Rectal CD4 r = 0.70 p < 0.01 r = 0.41 p = 0.13 Total HIV DNA r = 0.05 p = 0.86 2 LTR circles PBMC rho = 0.19 p = 0.31 Residual viremia Plasma rho = 0.07 p = 0.71 1 0,000 1, 000 100 10 1 0.1 Plasmas HIV RNA (copies/ml) Cohort Chronic Acute Chronic Acute Chronic Acute Chronic Acute Chronic Acute Chronic Acute Chronic Acute Chronic Acute Eriksson et al, PLOS Pathogens, 2013

180-200 ml blood Noninduced proviruses Noninduced proviruses PHA + irradiated allogeneic PBMC Full-length, single genome analysis Noninduced ` Noninducible Day 2: CD4+ blasts from HIVdonors Day 7: CD4+ blasts from HIVdonors p24 Ag

Noninduced proviral clones (n=213) N H 2 O N H N Deletion in È O N O N Hypermutated 27.7% TGG Trp TAG Stop Large internal deletion 49.8.0% Nonsense MSD mutation Other mutation

Eradication strategies depend on production of viral proteins HDACi CTL CTL killing HDACi CTL Cells with defective proviruses may not be eliminated by eradication strategies.

Noninduced proviral clones (n=213) Deletion in È Intact 12.2% O N N H 2 N O H N O N Hypermutated 27.7% TGG Trp TAG Stop Large internal deletion 49.8.0% Nonsense MSD mutation Other mutation

What will eradication look like clinically? Will latency-reversing agents cause a transient increase in residual viremia? v u k2 k r r2 a LRA a2 Residual GFP+ cells (%) Virions/cell/day 10 4 120 SAHA 100 80 60 40 20 10 3 10 2 10 1 0 0 10 0 ±CD3+±CD28 2 3 4 5 6 7 Days Activated after reactivation Resting Peak fold increase in v H r 2k 2a r2k 2a2 Alison Hill, Daniel Rosenbloom, Martin Nowak

Increase in viremia with LRAs k2 1000000 100000 10000 1000 100 10 1 0.1 0.01 0.001 v u a r r2 LRA Peak fold increase in v H a2 r 2k 2a r2k 2a2 LRA 0 100 200 300 Time on HAART (days) 400 Alison Hill, Daniel Rosenbloom, Martin Nowak

What will eradication look like clinically? How do reductions in the size of the latent reservoir affect time until rebound after interruption of HAART? k v + u a r Alison Hill, Daniel Rosenbloom, Martin Nowak

Modeling eradication 80 60 40 20 very late rebounders Reduction in reservoir No LRA 1 log 2 log 3 log 4 log 5 log 6 log 10 days 100 days 1000 days (2.7 years) Time after stopping HAART 10,000 days (27 years) Alison Hill, Daniel Rosenbloom, Martin Nowak

Conclusions When latency is reversed without T-cell activation, the cells do not die and are not killed by CTLs from most patients on HAART. The CTL defect can be reversed by antigen stimulation. It may be necessary to combine latency-reversing strategies with therapeutic vaccination.?? v u

Conclusions Intact 12.7% Hypermutated 27.7% Large internal deletion 46.0% PCR assays give infected cell frequencies at least 2 logs higher than, and are poorly correlated with, the viral outgrowth assay. A large and variable fraction of proviruses detected by PCR have major defects that preclude replication. Cells with defective proviruses may not be eliminated by eradication strategies. PCR assays may not be appropriate for either cross-sectional or longitudinal analysis of the reservoir in eradication trials.

Conclusions Intact 12.7% Hypermutated 27.7% Large internal deletion 46.0% Although most proviruses are defective, the number of intact noninduced proviruses is still much greater than the number detected in viral outgrowth assays (40- to 50-fold). If these proviruses can be induced in vivo, then the reservoir may be 40- to 50-fold larger than estimated by the viral outgrowth assay. Latency-reversing agents should induce measurable transient increases in residual viremia. Reservoir reductions of >2 logs will be required for clinically significant delays in rebound, but time until rebound will be highly variable.

Thanks Janet Siliciano Andrew Yang Sifei Xing Adam Spivak Adriana Andrade

Thanks Ya-Chi Ho Liang Shan Greg Laird Alison Hill and Daniel Rosenbloom

Thanks Collaborators Steve Deeks Dave Margolis Joel Gallant Joe Cofrancesco Doug Richman Jon Karn Martin Nowak Matt Strain Sarah Palmer Una O Doherty Joe Wong Steve Yukl Funding Foundation for AIDS Research (amfar): ARCHE NIH: Martin Delaney Collaboratories CARE and DARE Johns Hopkins Center for AIDS Research Howard Hughes Medical Institute