Advances Toward a Cure for HIV Janet M

Advances Toward a Cure for HIV Janet M. Siliciano, Ph. D Associate Professor of Medicine Johns Hopkins University Baltimore, Maryland 1

Financial Relationships With Commercial Entities Dr Siliciano has served on the scientific advisory board for Gilead Sciences, Inc. Her lab has received a grant from Gilead Sciences, Inc. (Updated 08/3/20) Slide 2 of 50

Learning Objectives After attending this presentation, learners will be able to: • Describe basic mechanisms that allow HIV to persist despite antiretroviral therapy (ART) • Recognize how proliferation of latently infected resting CD 4+ T cells contributes to viral persistence • Describe 1 current approach for achieving an HIV cure Slide 3 of 50

Magnitude of the problem 38, 000 infected 23, 000 on ART 3 people cured Slide 4 of 50

HIV replication dynamics Plasma HIV-1 RNA (copes/ml) 10, 000 v Set point 1, 000 + ART Intensify 100, 000 † 10, 000 100 10 R 0 = 10 Limit of detection t 1/2 = 1 d t 1/2 = 14 d Residual viremia 1 Time (months) Slide 5 of 50 Wei et al. Nature 1995 Ho et al, Nature 1995 Perelson et al, Nature 1997 Finzi et al, Nature Med 1999 Dornadula et al, JAMA 1999 Dinoso et al, PNAS, 2009 Robb and Ananworanich, COHA, 2016

Memory Naive Physiology of resting and activated CD 4+ T cells Slide 6 of 50

Naive Response of resting T cells to antigen Ag † † † Memory † Slide 7 of 50

Naive Recall response of memory T cells to antigen † † Ag † Memory † Ag † † † Slide 8 of 50

Infection of activated and resting CD 4+ T cells Ag HIV Naive † Ag HIV Memory † Slide 9 of 50 HIV †

Naive Establishment and maintenance of a latent reservoir Ag † † † Memory † Slide 10 of 50 HIV

HIV gene expression depends on inducible host factors NFκB IκB p 50 P NFAT p 65 P NFAT Cytoplasm Nucleus NFAT p 50 p 65 PP P Ac Ac HATs HDACs p 50 Nuc-0 Ac Ac Ac Slide 11 of 50 p 65 P CDK 9 Cyclin T 1 SP 1 HIV LTR P P P Ac RNA Pol II Ac P Nuc-1 Ac Ac CDK 9 Cyclin T 1 Tat P RNA Pol II P-TEFb Nabel and Baltimore, Nature 1987 Tong-Starksen et al, PNAS. 1987 Duh et al, PNAS 1989 Kinoshita et al, Immunity 1997

Naive A stable latent reservoir for HIV Ag † † † Memory † Slide 12 of 50 HIV

Naive Reactivation of latent HIV Ag † † † Memory † Slide 13 of 50 Ag † HIV

Quantitative viral outgrowth assay purified resting CD 4+ T cells T cell activation e co gat nt ive ro l N 10 x 6 1. 8 x 10 3 4 10 x 4 2 x 10 5 6 10 x 1 5 x 10 6 2 180 -200 ml blood 1/1, 000 p 24 Ag d 2: add CD 4+ lymphoblasts from HIVdonors Slide 14 of 50 d 7: add CD 4+ lymphoblasts from HIVdonors Chun et al, Nature Med 1995 Chun et al, Nature 1997 Finzi et al, Science 1997

Slow decay of latently infected CD 4+ T cells Frequency (per 106 cells) 10000 100 t 1/2= 3. 7 years 10 1 0. 01 - 0. 001 0. 00001 0 1 2 3 4 5 6 7 Time on ART (years) Slide 15 of 50 Finzi et al. , Nature Med. , 1999 Siliciano et al. , Nature Med. , 2003

e co gat nt ive ro l N 10 x 6 1. 8 x 10 3 4 10 x 4 2 x 10 5 6 x 1 5 purified resting CD 4+ T cells 10 6 10 PCR for proviral DNA x 180 -200 ml blood 2 Quantitative viral outgrowth assay T cell activation p 24 Ag d 2: add CD 4+ lymphoblasts from HIVdonors Slide 16 of 50 d 7: add CD 4+ lymphoblasts from HIVdonors Chun et al, Nature Med 1995 Chun et al, Nature 1997 Finzi et al, Science 1997

Comparison of assays for the latent reservoir Assay Viral outgrowth Cell/tissue Resting CD 4 Total HIV DNA PBMC Resting CD 4 Total HIV DNA Integrated HIV DNA PBMC Resting CD 4 Rectal CD 4 2 LTR circles Residual viremia PBMC Plasma Infected cell frequency (per 106) rho = 0. 19 p = 0. 31 1, 000 100 10 10 1 1 r = 0. 38 p = 0. 28 0. 1 Cohort Slide 17 of 50 rho = 0. 07 p = 0. 71 Chronic Acute r = 0. 70 p < 0. 01 r = 0. 41 p = 0. 13 r = 0. 05 p = 0. 86 Chronic Acute Plasmas HIV RNA (copies/ml) 10, 000 0. 1 Chronic Acute Eriksson et al, PLOS Pathogens, 2013

Most HIV proviruses are defective Slide 18 of 50 Intact provirus Ho et al, Cell, 2013; Bruner et al, Nature Med 2016

Digital droplet assay for intact proviruses Slide 19 of 50 Bruner et al, Nature 2019

ψ fluorescence Intact proviral DNA assay DNA in droplets Slide 20 of 50 AAAA Q 1 Q 2 Q 3 Q 4 env fluorescence Bruner et al, Nature 2019

Large scale IPDA analysis in pts on ART Frequency (per 106 CD 4+ T cells) 10000 • The IPDA measures the N = 400 reservoir, not active viral replication • The IPDA measures viral DNA 1000 in infected CD 4+ T cells, not free virus particles in the plasma. 100 • The IPDA is almost always 10 1 not detected ct a t In Slide 21 of 50 / d d d e te t e l ta e e d u d 5′ 3′ erm p y h To l ta positive even in people on ART who have plasma virus levels below the limit of detection. • The clinical utility of the IPDA is in analysis of curative interventions as it measures the cells that are a barrier to cure Simonetti et al, PNAS in press

Intact proviruses decay more rapidly that defective proviruses Intact • Cells with intact proviruses t 1/2 = 44 months decay more rapidly than cells with defective proviruses • This may indicate some Defective immune pressure on infected cells during ART. • This decay is slow! In first 7 years, t 1/2 is close to 44 months. Subsequently, even slower Slide 22 of 50 Peluso et al, JCI Insight 2020

Naive Response of resting T cells to antigen Ag † † † Memory † Slide 23 of 50

Naive Infected cells can also proliferate † † Ag Memory † Slide 24 of 50 †

Proliferation of infected cells Time post entry (d) Plasma Proviral sequences from resting CD 4+ T cells Activated Residual viremia Slide 25 of 50 Tobin et al, J Virol 2005 Bailey et al, J Virol 2006

Clonal nature of residual viremia Time post entry (d) Plasma Proviral sequences from resting CD 4+ T cells Activated • These sequences reflect extensive proliferation of a clone of infected cells • The cells we want to eliminate can proliferate in vivo! Slide 26 of 50 Tobin et al, J Virol 2005 Bailey et al, J Virol 2006

Clonal expansion detected by integration site analysis Slide 27 of 50 Maldarelli et al, Science 2014 Wagner et al, Science 2014 Cohn et al, Cell 2015

The latent reservoir Slide 28 of 50

Clones of latently infected cells Slide 29 of 50

Clones of latently infected cells Slide 30 of 50

Naive Infected cells can also proliferate † † Ag Memory † Slide 31 of 50 †

Slow decay of the latent reservoir + in resting CD 4 T cells Frequency (per 106 cells) 10000 Half-life: 44 months Time to eradication: > 73. 4 years 1000 10 1 0. 01 - 0. 001 0. 00001 0 Slide 32 of 50 1 2 3 4 5 Time on ART (years) 6 7 Finzi et al, Nature Med 1999 J. Siliciano et al, Nature Med 2003

Clones of latently infected cells wax and wane Slide 33 of 50

Clones of latently infected cells wax and wane Slide 34 of 50

Clones of latently infected cells wax and wane Slide 35 of 50

Clones of latently infected cells wax and wane Slide 36 of 50

Clones of latently infected cells wax and wane Slide 37 of 50

Clones of latently infected cells wax and wane Slide 38 of 50

Clones of latently infected cells wax and wane Slide 39 of 50

The “shock and kill approach” to HIV cure TLR 7 agonists PKC agonists SMAC mimetics CTL T cell activation NFκB † IκB p 50 P NFAT p 65 P NFAT Cytoplasm Nucleus NFAT p 50 Ac Ac HATs HDACs p 50 Nuc-0 † CTL Ac Ac Ac Slide 40 of 50 PP P † LRAs p 65 P CDK 9 Cyclin T 1 SP 1 HIV LTR Histone deacetylase inhibitors: Vorinostat Romidepsin Panobinostat P P P Ac RNA Pol II Ac P Nuc-1 Ac Ac CDK 9 Cyclin T 1 Tat P RNA Pol II P-TEFb

Assessing LRA efficacy Plasma HIV-1 RNA (copies/ml) 10, 000 1, 000 • Might cause blips LRA † Set point ART 100, 000 LRA 10, 000 100 Limit of detection 10 1 Time (months) Slide 41 of 50 Stop ART

Assessing LRA efficacy Plasma HIV-1 RNA (copies/ml) 10, 000 1, 000 LRA † Set point ART 100, 000 LRA 10, 000 100 Limit of detection 10 1 Time (months) Slide 42 of 50 Stop ART • Might cause blips • Should reduce reservoir as determined by QVOA or IPDA

Assessing LRA efficacy Plasma HIV-1 RNA (copies/ml) 10, 000 1, 000 LRA † Set point Stop ART 100, 000 LRA 10, 000 100 Limit of detection 10 11 1 Time (months) Slide 43 of 50 • Might cause blips • Should reduce • reservoir as determined by QVOA or IPDA Should delay rebound on interruption of ART

Clinical trail of romidepsin Slide 44 of 50 Courtesy of O. Søgaard, Aarhus University

Broadly neutralizing antibodies PGT 121 VRC 01 Slide 45 of 50 • Neutralize diverse HIV isolates • Arise slowly, generally after virus has already escaped • Can be administered passively as infusion or with AAV vectors • Block infection and target infected cells for killing Bonsignori et al, Imm Rev 2017

Effects of antibodies Neutralization of free virus particles NK cell Killing of infected cells Slide 46 of 50

Clinical trials of b. NABs QVOA . VRC 01 QVOA . Months • b. NAb VRC 01 infused just prior to and during analytical treatment interruption (ATI) Slide 47 of 50 Bar et al NEJM 2016 Salantes et al JCI 2018

Clinical trials of b. NABs Slide 48 of 50 • b. NAb VRC 01 was safe but caused only a slight delay in rebound as compared to historical controls • Pre-existing or newly arising viral variants resistant to VRCO 1 monotherapy were selected during rebound • Reservoir size was not changed indicating that b. NAbs alone do not decrease the latent reservoir and that short ATIs do not increase reservoir. Bar et al NEJM 2016 Salantes et al JCI 2018

Conclusions • The latent reservoir in resting CD 4+ T cells is the major barrier to cure • Accurate measurement of the reservoir is important for evaluating cure interventions and requires distinguishing intact proviruses from defective ones. This can be done with a novel assay, the IPDA. • The reservoir is maintained by the proliferation of infected cells in response to antigens. This is a serious problem for cure efforts. • Eliminating the reservoir through the “shock and kill” strategy will require finding better ways to turn on latent HIV and better ways to induce the killing of productively infected cells • Broadly neutralizing antibodies are of great interest in the HIV vaccine field and may also be useful to enhance killing of infected cells. Slide 49 of 50

Thank you Collaborators Steve Deeks Rebecca Hoh Greg Laird Katie Bar Pablo Tebas Funding Gilead (unrestricted research grant) Howard Hughes Medical Institute NIH: Martin Delaney Collaboratories Bill and Melinda Gates Foundation Martin Delaney Collabortories Slide 50 of 50

Question-and-Answer Session 51