1 Advances and ChallengesHIV Cure Slide: HIV Cure: Advances and Challenges Advances and Challenges This activity is jointly provided by the University of Nebraska Medical Center, the University of Florida College of Pharmacy, and Practice Point Communications® Supported by an independent educational grant from Gilead Sciences Medical Affairs Simply Speaking® HIV “HIV Cure: Advances and Challenges” is Copyrighted 2017 by Practice Point Communications, unless otherwise noted.  All rights reserved.

2 Medical Director, Southern California Men’s Medical GroupEducator Anthony Mills, MD Medical Director, Southern California Men’s Medical Group Los Angeles, CA Disclosures Employment (includes retainer): insert here Industry funded research/investigator: insert here Consultant: insert here Speakers’ bureau, faculty, peer reviewer: insert here Advisory committee/board: insert here Stock/ownership: insert here Patent holder: insert here Other (please describe): insert here Plan to discuss investigational/off-label uses of drugs or devices? (Y/N) Slide: Educator

3 Sign-In Process Please clearly print all information on the sign-in sheet You must indicate your NAME, DEGREE, MAILING ADDRESS, , and SIGNATURE in order to attend this lecture You must indicate a unique identification number to attend this lecture: MD/DO/PA = NPI Number NP/RN = State License Number PharmD/RPh = NABP & Date of Birth Other = NPI or State License Number (if available) Completion is required for all healthcare providers Failure to provide complete information may disqualify you from participating in future lectures Slide: Sign-In Process

4 Accreditation and Disclosure InformationPlease refer to your program handouts to review the following: Accreditation statements Disclosure policy Disclosures of content faculty, reviewers, and planners Slide: Accreditation and Disclosure Information

5 Evaluation and Outcomes Measurement ProcessYou will receive an electronic initial evaluation to the address provided within 1 business day Reminder communications will be sent up to 5 days post lecture until the evaluation is completed Incomplete evaluations may preclude attendees from receiving their CME/CNE/CPE certificate & future communications about lectures in your area In addition, you will receive a long-term evaluation via 8 to 12 weeks after completing this course to measure competence, performance, and/or patient outcomes achieved as a result of your participation in this CME/CNE/CPE sponsored educational activity Slide: Evaluation and Outcomes Measurement Process (Please note: If you attended multiple Simply Speaking® lectures throughout the year, a separate initial and long-term evaluation will be sent to you for each lecture.)

6 Learning Objectives (CME/CNE/CPE)Upon completion of this educational activity, participants should be able to: Describe the benefits associated with successful treatment with current antiretroviral therapy (ART) Discuss the biology of retroviral persistence Outline investigative approaches for an HIV cure to reinforce current management practices for HIV patients Slide: Learning Objectives (CME/CNE/CPE)

7 Program Overview Benefits of ART and search for an HIV cureBiologic considerations in retroviral persistence Defining a cure Strategies in HIV cure research Challenges and future perspectives Slide: Program Overview

8 CDC: Estimated New HIV Infections in the US (2008-2014)Slide CDC: Estimated New HIV Infections in the US ( ) Change in Estimated New HIV Infections/Year ( ) Persons Living With Undiagnosed HIV in 2014 (%) Total (-3.6%) 15% MSM* (-0.7%) Number of Cases (thousands) 17% Slide: CDC: Estimated New HIV Infections in the US ( ) Singh and colleagues estimated HIV incidence, prevalence and percent of undiagnosed infections using data from the National HIV Surveillance System on HIV diagnoses.1 Overall, new HIV diagnoses decreased 3.6% from 2008 to All transmission groups had a decrease in HIV diagnoses except for MSM. The percentage of undiagnosed infections overall was 15%, and 17% among MSM.1 Reference Singh A, Song R, Johnson AS, et al. HIV incidence, prevalence, and undiagnosed infections in men who have sex with men. Program and abstracts from the 24th Conference on Retroviruses and Opportunistic Infection; February 13-16, 2017; Seattle, WA. Abstract 30. Heterosexual Contact (-7.3%) 16% PWID (-13.8) 6% *Among MSM, HIV incidence decreased among black and white MSM (-0.7% and -3.1%, respectively). In contrast, HIV incidence increased among MSM Hispanics (2.4%) and year olds (4.8%). In 2014, 52% of young MSM with HIV were undiagnosed. Singh S, et al. 24th CROI. Seattle, Abstract 30.

9 New AIDS Cases and AIDS-Related Deaths in the United StatesUpdated Slide Number of Cases New AIDS Cases Slide: New AIDS Cases and AIDS-Related Deaths in the United States The dramatic decline in annual AIDS-related mortality over the past 10 years has been largely attributed to use of ART.1 The greatest reduction in HIV mortality occurred from 1995 through Many of the first patients receiving ART had AIDS and often required regimens of high pill-burden for HIV.1 Reference Centers for Disease Control and Prevention. HIV Surveillance Report, Vol. 27. Atlanta: U.S. Department of Health and Human Services, Centers for Disease Control and Prevention; 2016: AIDS-Related Deaths Year CDC. HIV Surveillance Report, Published November 2016.

10 New Slide Narrowing the Gap in Life Expectancy Between HIV-Positive and Uninfected Persons ( ) Average Life Expectancy Remaining At Age 20 Years 65 63 HIV negative 60 12-Year Gap 13-Year Gap 53 44-Year Gap 47 Life Expectancy (years) HIV positive Slide: Narrowing the Gap in Life Expectancy Between HIV-Positive and Uninfected Persons ( ) Marcus and colleagues conducted a cohort study of HIV-infected adults who were members of Kaiser Permanente California during and HIV-uninfected members matched 10:1 on age, gender, medical center, and year. Deaths were comprehensively ascertained through 2011 from the electronic health record, California death certificates, and Social Security Administration datasets. Abridged life tables were used to estimate the average number of years of life remaining at age 20 years (“life expectancy at age 20”) in HIV-infected and HIV-uninfected individuals in and For the recent era, we estimated life expectancy at age 20 years by demographics and HIV risk group.1 Life expectancies at age 20 for HIV-uninfected individuals were 63 years in and 65 years in 2011, while increasing for HIV-infected individuals from 19 years in to 53 years. As such ,the life expectancy gap between HIV-positive and HIV-negative people has decreased from 44 to 12 years from to 2011, respectively.1 Reference Marcus JL, Chao CR, Leyden WA, et al. Narrowing the gap in life expectancy between HIV-infected and HIV-uninfected individuals with access to care. J Acquir Immune Defic Syndr. 2016;73:39-46. 19 96-97 98-99 00-01 02-03 04-05 06 07 08 09 10 2011 Calendar Year Kaiser Permanente Northern California (1996 to 2011): HIV-positive (n=25,768) and matched non-HIV-infected adults (n=257,600). Males (91%) and MSM (75%). Marcus JL, et al. JAIDS. 2016;73:39-46.

11 New Slide Average Life Expectancy Remaining At Age 20 Years by Demographic Characteristics Overall Women Men 65 HIV- 62 HIV- 62 64 62 HIV- 62 13 13 13 Life Expectancy (years) Life Expectancy (years) 28 25 Life Expectancy (years) 24 51 49 49 HIV+ HIV+ HIV+ 37 37 38 96-07 08-11 96-07 08-11 96-07 08-11 Calendar Era Calendar Era Calendar Era Slide: Average Life Expectancy Remaining At Age 20 Years by Demographic Characteristics Significant gains in survival for HIV-infected individuals were observed across all gender, race/ethnicity, and HIV-transmission risk groups from to (P<0.001).1 Reference Marcus JL, Chao CR, Leyden WA, et al. Narrowing the gap in life expectancy between HIV-infected and HIV-uninfected individuals with access to care. J Acquir Immune Defic Syndr. 2016;73:39-46. Black White Hispanic HIV- 68 63 65 HIV- 61 HIV- 61 59 16 11 28 13 36 Life Expectancy (years) Life Expectancy (years) 24 Life Expectancy (years) 52 50 46 HIV+ HIV+ HIV+ 37 39 35 96-07 08-11 96-07 08-11 96-07 08-11 Calendar Era Calendar Era Calendar Era P<0.001 for change in life expectancy ( versus ) in HIV-positive persons. Marcus JL, et al. JAIDS. 2016;73:39-46.

12 ART Cohort Collaboration: 50% of Deaths Due to Non-AIDS-Related Causes (1996-2006)Infections Cardiovascular Disease Non-AIDS Malignancies 8% 8% 12% Liver Disease 7% Violence or Drug-Related 8% 7% Other Slide: ART Cohort Collaboration: 50% of Deaths Due to Non-AIDS-Related Causes ( ) Mortality data were combined from 13 cohort studies in Europe and North America (ART Cohort Collaboration).1 A total of 1876 deaths were recorded among 39,272 patients (>15 years of age) who started triple antiretroviral therapy during 1996 to Causes of death were retrospectively assigned to 85% of the 1876 deaths. Combined mortality data from these cohorts show that 50% of deaths were due to non-AIDS-related causes.1 Reference The Antiretroviral Therapy Cohort Collaboration. Causes of death in HIV-1-infected patients treated with antiretroviral therapy, : collaborative analysis of 13 HIV cohort studies. Clin Infect Dis. 2010;50: AIDS-Related (50%) n=39,272 HIV-infected patients from 13 cohort studies in Europe and North America who were treated with ART. Causes of death were retrospectively assigned to 85% of total deaths (1597/1876). ART Collaboration. Clin Infect Dis. 2010;50:

13 Abnormal Levels of T-Cell Activation Despite ART-Mediated HIV RNA SuppressionActivated CD4+ T Cells Activated CD8+ T Cells Median Median Percentage Activated Percentage Activated P<0.001 Slide: Abnormal Levels of T-Cell Activation Despite ART-Mediated HIV RNA Suppression Hunt and colleagues evaluated patients who had maintained viral suppression for >3 months to determine if persistent T-cell activation affects immune reconstitution in HIV-infected patients with viral suppression during ART (n=99).1 Activated T-cell subsets were measured by flow cytometry. Plasma HIV RNA level <1000 copies/mL: median of 21 months while on ART. ART-treated patients had a lower median percentage of activated (CD38 HLA-DR) CD4 T cells than untreated HIV-infected participants (P<0.001), but a higher median percentage than HIV-uninfected volunteers (P<0.001).1 Similarly, the ART-treated patients had a lower median percentage of activated CD8 T cells than untreated HIV-infected participants (P<0.001), but a higher percentage than HIV-uninfected volunteers (P<0.001).1 These data illustrate that patients with ART-mediated viral suppression continued to have higher levels of both CD4 and CD8 T-cell activation than the HIV-uninfected control subjects, even when the analysis was restricted to the 58 patients who did not have HCV or the 68 patients who had maintained undetectable plasma HIV RNA levels in the year before immunophenotyping.1 Reference Hunt PW, Martin JN, Sinclair E, et al. T cell activation is associated with lower CD4+ T cell gains in human immunodeficiency virus-infected patients with sustained viral suppression during antiretroviral therapy. J Infect Dis. 2003;187: P<0.001 P<0.001 P<0.001 HIV-Infected No ART (n=13) HIV-Infected ART (n=99) Not HIV-Infected (n=5) HIV-Infected No ART (n=13) HIV-Infected ART (n=99) Not HIV-Infected (n=5) ART-treated patients with plasma HIV RNA <1000 copies/mL for >3 months before immunophenotyping. Median age 45 years; 90% male; pre-ART HIV RNA 4.4 log10 copies/mL; pre-ART CD4 210 cells/mm3; median duration of viral suppression 44 months. Hunt PW, et al. J Infect Dis. 2003;187:

14 HIV Cascade of Care: Missed Opportunities in the USHIV-Infected: >35 Years of Age (n=896,800) HIV-Infected: 13-29 Years of Age (n=78,949) ~88% ~73% HIV-Infected (%) HIV-Infected (%) Slide: HIV Cascade of Care: Missed Opportunities in the US Data from studies by Zanoni et al and Hall et al illustrate the significant age disparities that exist at each step of the continuum of care.1,2 References Zanoni BC, Mayer KH. The adolescent and young adult HIV cascade of care in the United States: exaggerated health disparities. AIDS Patient Care STDS. 2014;28: Hall HI, Frazier EL, Rhodes P, et al. Differences in human immunodeficiency virus care and treatment among subpopulations in the United States. JAMA Intern Med. 2013;173: ~40% 40% ~28% 25% 11% 6% Diagnosed Linked to Care Retained in Care Viral Suppression Diagnosed Linked to Care Retained in Care Viral Suppression Zanoni BC, et al. AIDS Patient Care STDS. 2014;28: Hall HI, et al. JAMA Intern Med. 2013;173:

15 Need for HIV Cure ResearchNeed for life-long ART Side effects and long-term toxicities Burden of life-long adherence Cost Sustainability Adverse effects of HIV-1 persistence Inappropriate immune activation Cardiovascular, CNS, and other end-organ damage Potential risk of transmission Ongoing stigma of HIV infection Slide: Need for HIV Cure Research Recent approaches to early diagnosis and use of current ART regimens have dramatically improved the clinical outcomes and life expectancies of patients with HIV infections. However, despite this success, the shortcomings of ART underscore the continued need for HIV cure research. Current ART can not eradicate the virus, which persists in reservoirs despite ART. As such, ART is a life-long commitment that is not without side ffects, long-term toxicities, the burden of life-long adherence, and cost. In addition, persistent HIV presence in reservoirs contributes to ongoing immune dysfunction and inflammation that increase the risk of non-AIDS morbidity and transmission.

16 Program Overview Biologic considerations in retroviral persistenceBenefits of ART and search for an HIV cure Biologic considerations in retroviral persistence Defining a cure Strategies in HIV cure research Challenges and future perspectives Slide: Program Overview

17 HIV Life Cycle Cytoplasm Nucleus Slide: HIV Life CycleThe first step is the attachment of the virus particle to receptors on the cell surface. The HIV RNA genome then enters the cytoplasm as part of a nucleoprotein complex. The viral RNA genome is reverse-transcribed into a collinear DNA duplex, which has terminal duplications known as long terminal repeats (LTRs) that are not present in HIV RNA. Once the viral DNA has been synthesized, the linear viral DNA molecule is incorporated into a preintegration complex that enters the nucleus.1 In the nucleus, unintegrated viral DNA is found in both linear and circular forms. The unintegrated circular forms of viral DNA have either 1 or 2 LTRs, are by-products of the integration process, and are found exclusively in the nucleus. The linear unintegrated viral DNA is the precursor of integrated proviral DNA, which is a stable structure that remains indefinitely in the host-cell genome and serves as a template for viral transcription.1 Transcription of the proviral DNA template and alternative mRNA splicing creates spliced viral mRNA species encoding the viral accessory proteins, including Tat, Rev, and Nef, and the unspliced viral mRNA encoding the viral structural proteins, including the gag–pol precursor protein. A shift in the transcriptional pattern from the expression of predominantly multiply spliced viral mRNA to predominantly unspliced viral mRNA is indicative of active viral replication.1 All the viral transcripts are exported into the cytoplasm, where translation and assembly and processing of the retroviral particle take place. The cycle is completed by the release of infectious retroviral particles from the cell.1 Reference Furtado MR, Callaway DS, Phair JP, et al. Persistence of HIV-1 transcription in peripheral-blood mononuclear cells in patients receiving potent antiretroviral therapy. N Engl J Med. 1999;340: Furtado MR, et al. N Engl J Med. 1999;340:

18 Latently Infected Cells (IUPM)Long-Term Persistence of Latently Infected CD4+ T Cells in Patients on ART HIV-1 infected adults on ART with ongoing HIV RNA suppression and no treatment failure (n=62) Mean time on ART: 45.4 months Half-life decay of reservoir: 44 months ( months) Time on ART to eradication of a reservoir size of 106 cells Whole cohort: 73.4 years With viral blips: 95.8 years Without blips: 51.2 years Frequency of Latently Infected CD4+ T Cells Harboring Replication-Competent HIV-1 10,000 100 Latently Infected Cells (IUPM) 1.0 Slide: Long-Term Persistence of Latently Infected CD4+ T Cells in Patients on ART Siliciano and colleagues demonstrated that latent HIV-1 persists in resting memory CD4+ T cells, even in patients receiving ART. These investigators conducted limiting dilution virus culture assays on highly purified resting CD4 cells from 62 HIV-1 infected adults on ART with ongoing HIV RNA suppression and no treatment failure for as long as 7 years (mean time on ART: 45.4 months).1 The half-life decay of the CD4 reservoir was 44 months ( months), which translates to the following time on ART needed to eradicate a reservoir size of 106 cells:1 Whole cohort: 73.4 years. With viral blips: 95.8 years. Without blips: 51.2 years These data illustrate that eradication of virus is unlikely even in ART-treated patients with no detectable plasma HIV RNA for as long as 7 years.1 Reference Siliciano JD, Kajdas J, Finzi D, et al. Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells. Nat Med. 2003;9: 0.01 0.0001 Time on ART (years) Siliciano JD, et al. Nat Med. 2003;9:

19 HIV Reservoir (T-Cell Subsets) Time After HIV Infection (days)HIV Acute Infection: Contribution of Each T-Cell Subset to the Establishment of the HIV Reservoir Reservoir Is Present in Predominately Transitional Memory Cells in the Very Earliest Stages of Infection, but Moves into Central Memory Cells During Chronic HIV Infection Eclipse Phase Fiebig I-III Fiebig IV-V Chronic HIV Infection Transitional memory cells Central memory cells Resting naïve cells Effector memory cells HIV Reservoir (T-Cell Subsets) Slide: HIV Acute Infection: Contribution of Each T-Cell Subset to the Establishment of the HIV Reservoir This illustration depicts HIV acute infection and the contribution of each T-cell subset to the establishment of the HIV reservoir.1 Early ART during the earliest stages of HIV infection (Fiebig stages I–III) may limit the number of infected cells and protect central memory cells from HIV infection.1 ART during later stages of HIV infection (Fiebig stages IV-V) may decrease the contribution of central memory cells to the reservoir due to low relative abundance of this cell subset. The frequency of central memory cells normalizes during chronic infection, increasing the contribution of these cells to the reservoir.1 Reference Passaes CP, Sáez-Cirión A. HIV cure research: advances and prospects. Virology. 2014; : Time After HIV Infection (days) Passaes CP, et al. Virology. 2014; :

20 HIV Transcription: Resting and Activated CD4+ T CellsLatent Provirus Slide: HIV Transcription: Resting and Activated CD4+ T Cells The transcriptional status of HIV is tightly coupled to the activation state of its host cell.1 In resting T cells, histone deacetylases and histone methyltransferases are recruited to the HIV promoter via p50 homodimers and CBF-1 bound to the NFkB sites and via the Sp1-binding CTIP-2 and the methylated DNA-binding MBD2 proteins.1 Deacetylation and indicated histone methylation on Nuc 0 and Nuc 1 induce a state of heterochromatin. Methylation of two CpG islands by DNA methyltransferases can further repress transcription. Moreover, the cyclin T1/CDK9-containing pTEFb is sequestered by the Hexim1/7SK RNA complex, whereas the active form of NFkB is kept by IkB in the cytoplasm, where the phosphorylated and unphosphorylated forms of NFAT and STAT5, respectively, are also retained. Upon T cell activation, a series of events inverts this flow of repressive inputs. In activated T cells, HATs and the cyclin T1/CDK9 complex are recruited to the viral promoter by NFkB p50-p65 and Sp1 bound to their cognate sites, and by Tat bound to the TAR (Tat responsive) sequence of nascent RNA transcripts.1 Histone acetylation of nearby nucleosomes and phosphorylation of the RNAPII C-terminal domain ensue, leading to more accessible chromatin conformation and increased transcriptional elongation, respectively. HIV gene expression is further stimulated by the binding of NFAT and phosphorylated STAT5 to their cognate sites. Reference Trono D, Van Lint C, Rouzioux C, et al. HIV persistence and the prospect of long-term drug-free remissions for HIV-infected individuals. Science. 2010;329: Activated Provirus Trono D, et al. Science ;329:

21 Relative Sizes of the Infected Resting CD4+ T-Cell Populations in 8 Patients on Suppressive ARTSize of Relevant Reservoir Is Unknown Approximately 90% of HIV-1 DNA is defective A small subset of replication-competent virus is induced in vitro Scale: 100/106 Resting CD4+ T Cells Cells with HIV-1 DNA Cells with intact, non-induced provirus Viral outgrowth assay (minimum size of latency reservoir) Slide: Relative Sizes of the Infected Resting CD4+ T-Cell Populations in 8 Patients on Suppressive ART Ho and colleagues compared the frequency of induced proviruses (defined using the viral outgrowth assay) and intact non-induced proviruses (quantitated as the product of total proviral DNA frequency and the fraction of non-induced proviruses that are intact) among the total pool of proviruses (measured by quantitative real-time PCR). Their statistical models indicated that the median frequency of intact non-induced proviruses was at least 60-fold higher than the frequency of induced proviruses detected in the viral growth assay. If the intact non-induced proviruses described by Ho and colleagues can be induced in vivo, then the size of the latent reservoir is much greater than previously thought.1 Reference Ho YC, Shan L, Hosmane NN, et al. Replication-competent noninduced proviruses in the latent reservoir increase barrier to HIV-1 cure. Cell. 2013;155: Ho Y-C, et al. Cell. 2013;155:

22 Approaches to Measure Persistent HIVLatent Infection (Cell) Productive Infection Extracellular HIV RNA No Yes HIV DNA 2-LTR circles Inducible provirus Cell-associated RNA (US RNA and MS RNA) Plasma HIV RNA (SCA) Slide: Approaches to Measure Persistent HIV There are multiple methods currently used to quantify different stages of the HIV life cycle in HIV-infected patients on ART.1,2 References Lewin SR, Rouzioux C. HIV cure and eradication: how will we get from the laboratory to effective clinical trials? AIDS. 2011;25: Rouzioux C, Richman D. How to best measure HIV reservoirs? Curr Opin HIV AIDS. 2013;8: US: unspliced; MS: multiple spliced. Lewin SR, et al AIDS. 2011;25: Rouzioux C, et al. Curr Opin HIV AIDS. 2013;8:

23 Accumulation of Defective Proviruses After Acute, Chronic, and Single Round of HIV InfectionAcute HIV infection (on ART) Hypermutation more prominent Chronic HIV infection (on ART) Proviruses are overwhelmingly defective (<5% are intact) Single round of infection Defective proviruses are readily detected Individuals initiating ART within 3 months of infection High percentage of defective proviruses (accumulation early after infection) Acute and chronic HIV (on ART) Expanded clones are readily detected and predominantly defective Percent of Proviruses by Course of HIV Infection Acute HIV, On ART Chronic HIV, Single Round Infection Intact proviruses 5 2 59 Defective proviruses Hypermutated 19 7 Hypermutated and deleted 18 8 -- Packaging signal deletion 4 Very large internal deletion 21 Deletion at 3’ end of genome 26 36 27 Deletion at 5’ end of genome 22 Slide: Accumulation of Defective Proviruses After Acute, Chronic, and Single Round of HIV Infection Bruner and colleagues characterized proviruses from patients treated in either acute or chronic infection and untreated viremic patients. They also performed an in vitro infection of CD4+ T cells to determine the fraction of defective proviruses after a single round of infection.1 Proviruses were analyzed by an unbiased, limiting dilution, full genome PCR and direct sequencing of PCR products. The number of intact proviruses was quantified as a percentage for each patient and compared to total HIV-1 DNA ddPCR values and QVOA IUPMs. Proviruses were also analyzed at the DNA level to predict the likelihood of making HIV-1 RNA or protein, as required for detection by current HIV-1 assays and for elimination by a shock and kill strategy. Main findings:1 Proviruses in individuals initiating ART during chronic HIV infection are overwhelmingly defective (<5% are intact). Defective proviruses are readily detected even after a single round of infection. Individuals initiating ART with 3 months of infection have a high percentage of defective proviruses, indicating accumulation early after infection. Hypermutation more prominent in acute infection. Expanded clones are readily detected and predominantly defective in patients who initiated ART during acute or chronic infection. Reference Bruner KM, Murray A, Pollock R, et al. Defective proviruses rapidly accumulate during acute HIV-1 infection. Nat Med. 2016;22: Acute HIV (n=6 subjects, 91 sequences). Chronic HIV (n=10 subjects, 152 sequences). Single round infection (n=22 sequences from in vitro infection of CD4+ T cells). Bruner KM, et al. Nat Med. 2016;22:

24 Main Characteristics of HIV-1 Reservoirs in the Major Anatomic SitesCSF HIV-infected macrophages are integral in causing neuronal damage Strong compartmentalization of HIV-1 variants Suboptimal penetration of ARV drugs Lymph Nodes Replication occurs in many independent sites (local spread of a genetically homogenous quasi-species) Highly efficient HIV-1 spread between cells favored by cell-to-cell transmission and the presence of follicular dendritic cells Suboptimal penetration of some ARV drugs GI Tract Effector memory cells (main HIV-1 cellular reservoir) Free exchange of HIV-infected cells between gut and blood Absence of de novo rounds of HIV-1 replication during ART Suboptimal penetration of ARV drugs in some regions (eg, ileum, rectum) Genital Tract Intermittent seminal HIV shedding occurs despite ART Viral compartmentalization with poor penetration of ARV drugs Virus replication stimulated by concurrent STI Genital inflammation Slide: Main Characteristics of HIV-1 Reservoirs in the Major Anatomic Sites This slide summarizes the role of HIV-1 reservoirs in different anatomic sites in the pathogenesis of HIV-1 infection. Developing therapeutic strategies to induce maximal virologic suppression will be based on an understanding of HIV-1 dynamics infection in each of these sites.1 Reference Svicher V, Ceccherini-Silberstein F, Antinori A, et al. Understanding HIV compartments and reservoirs. Curr HIV/AIDS Rep. 2014;11: Svicher V, et al. Curr HIV/AIDS Rep. 2014;11:

25 Program Overview Defining a cureBenefits of ART and search for an HIV cure Biologic considerations in retroviral persistence Defining a cure Strategies in HIV cure research Challenges and future perspectives Slide: Program Overview

26 Proposed Definitions of HIV Remission and CureImplications for Clinical Biomarkers Comprehensive Permanent remission of disease following cessation of therapy Adults Sustained Host-mediated control of HIV replication in the absence of ART and: Normalized, effective immune function Decreased HIV-related inflammation Reduced risk of HIV transmission Detectable replication-competent HIV by viral outgrowth assay Normalization of inflammatory biomarkers Sterilizing Complete elimination of replication- competent HIV Undetectable replication-competent HIV Children Pediatric remission Sustained virologic control in the absence of ART that includes: Normal CD4+ T-cell levels Preservation of immune responses to routine immunization Normalization of CD4+ T-cell percentages and absolute numbers Maintenance of vaccine-specific antibodies and cell-mediated immunity Slide: Proposed Definitions of HIV Remission and Cure The definition(s) of HIV cure continually evolve with advances in our understanding of the latent reservoir and HIV persistence.1-3 Comprehensive cure reflects the permanent remission of disease following cessation of therapy; however, it lacks specific benchmarks to indicate whether an individual would be capable of long-term remission. The difference between sustained or sterilizing cure definitions is the presence of replication-competent HIV in the reservoirs. However, the unpredictability of virologic rebound following ART cessation necessitates that the appropriate terminology for the majority of HIV-infected individuals is not cure but remission.1-3 References Fauci AS, Marston HD, Folkers GK. An HIV cure: feasibility, discovery, and implementation. JAMA. 2014;312: Katlama C, Deeks SG, Autran B, et al. Barriers to a cure for HIV: new ways to target and eradicate HIV-1 reservoirs. Lancet. 2013;381: Rainwater-Lovett K, Luzuriaga K, Persaud D. Very early combination antiretroviral therapy in infants: prospects for cure. Curr Opin HIV AIDS. 2015;10:4-11. Fauci AS, et al. JAMA. 2014;312: Katlama C, et al. Lancet. 2013;381: Rainwater-Lovett K, et al. Curr Opin HIV AIDS. 2015;10:4-11.

27 Program Overview Strategies in HIV cure researchBenefits of ART and search for an HIV cure Biologic considerations in retroviral persistence Defining a cure Strategies in HIV cure research Induce HIV expression from latently infected cells Hematopoietic stem cell transplantation Genetically modified CD4 T cells Immune-based interventions Broadly neutralizing antibodies Immediate initiation of ART Combination approaches Challenges and future perspectives Slide: Program Overview

28 HIV Chromatin Regulation“Closed” Nucleosome Transcription Repressed Deacetylated Slide: HIV Chromatin Regulation The provirus undergoes packaging into chromatin after integration into the host genome. Histones, which form the foundation of chromatin structure, associate with DNA to form highly organized and compact structures called nucleosome. The tight arrangement acts as a barrier for DNA-binding factors, which limits the ability of the virus to express genes. Thus, histones are important in the regulation of gene transcription.1 Upon HIV-1 integration into the host genome, the deposition of two nucleosomes, nuc-1 or closed, and nuc-0 or open, occurs at 2 different regions of the proviral long terminal repeat.1 The closed nucleosome plays an integral role in the suppression of HIV-1 gene expression. Histone deacetylases (HDACs) have been associated with HIV-1 transcriptional silencing and viral latency. This impairs the ability of RNA polymerase II to localize to the HIV-1 promoter and initiate transcription. Acetylation of histones produces transcriptional activation of the HIV-1 promoter. Acetylation primarily occurs at the lysine residues of the histone tails, which reduces the positive charge of the tails, lowering their affinity for DNA and ultimately causing partial or full disassociation with the DNA. Histone acetyltransferases have an opposite action to HDACs. Reference Groen JN, Morris KV. Chromatin, non-coding RNAs, and expression of HIV. Viruses. 2013;5: “Open” Nucleosome Transcription Enabled acetylated Courtesy of D. Margolis.

29 If HIV RNA >1000 copies/mL Weekly HIV RNA MonitoringSEARCH 019 Study: Impact of Vorinostat + Maraviroc + Hydroxychloroquine + ART on HIV Viremia Randomized, open label Diagnosed and treated acute HIV HIV RNA <50 copies/mL for >2 years Low HIV reservoir size ART Only (n=5) Study regimen Vorinostat: 400 mg/day, 14 days on/off, (3 cycles [10 weeks]) Hydroxychloroquine: 200 mg/dose, bid, 10 weeks Maraviroc: 600 mg/dose, bid, 10 weeks If HIV RNA >1000 copies/mL Weekly HIV RNA Monitoring VHM + ART (n=10) Week Stop VHM Begin Interruption Slide: SEARCH 019 Study: Impact of Vorinostat + Maraviroc + Hydroxychloroquine + ART on HIV Viremia In this randomized, open-label study, Kroon and colleagues evaluated the use of VHM (vorinostat/hydroxychloroquine/maraviroc) + ART versus ART, both followed by treatment interruption on the time to virologic rebound and size of the latent reservoir in acute HIV patients with HIV RNA <50 copies/mL.1 The VHM arm received 3 cycles of vorinostat 400 mg/day (14 days on/14 days off) plus hydroxychloroquine (400 mg/day) and maraviroc (1200 mg/day).1 Reference Kroon E, Ananworanich J, Eubanks K, et al. Effect of vorinostat, hydroxychloroquine and maraviroc combination therapy on viremia following treatment interruption in individuals initiating ART during acute HIV infection. JAIDS. 2016;19(suppl 5):28. Abstract TUAX0101LB. VHM: vorinostat, maraviroc, hydroxychloroquine. Endpoints of interest: Time to virologic rebound. Size of latent reservoir. Baseline demographics (study regimen + ART/ART only): Age: 28/26 years. ART duration: 224/155 weeks. CD4 count: 634/1079 cells/mm3. HIV RNA in PBMC: 44/27 copies/106 cells. Kroon E, et al. JAIDS. 2016;19(suppl 5):28. Abstract TUAX0101LB.

30 Weeks After InterruptionSEARCH 019 Study: HIV RNA After Interruption of Vorinostat + Maraviroc + Hydroxychloroquine + ART VHM + ART ART ART Resumed: HIV RNA >1000 copies/mL HIV RNA (copies/mL) Slide: SEARCH 019 Study: HIV RNA After Interruption of Vorinostat + Maraviroc + Hydroxychloroquine + ART Two individuals in the VHM arm had serious adverse events, and one withdrew from the study due to renal insufficiency and thrombocytopenia.1 Fourteen participants underwent treatment interruption (9 VHM + ART, 5 ART) and all experienced HIV RNA rebound with no difference between arms (median time: 22 days). One participant in the ART arm had viremic control for 11 weeks.1 In this proof-of-concept study, all individuals who initiated ART during acute HIV infection experienced HIV RNA rebound following treatment interruption regardless of VHM treatment. The investigators noted that alternative strategies to reduce or eliminate HIV reservoirs are needed.1 Reference Kroon E, Ananworanich J, Eubanks K, et al. Effect of vorinostat, hydroxychloroquine and maraviroc combination therapy on viremia following treatment interruption in individuals initiating ART during acute HIV infection. JAIDS. 2016;19(suppl 5):28. Abstract TUAX0101LB. Weeks After Interruption Median time to first HIV RNA detection: 22 days (range: 14 to 77 days). 2/8 had detected CSF HIV RNA during plasma viremia post interruption. Kroon E, et al. JAIDS. 2016;19(suppl 5):28. Abstract TUAX0101LB.

31 CLEAR Study: Panobinostat (HDAC Inhibitor) and Viral ReactivationPhase 1/2 trial Patients on ART (n=15) HIV RNA <50 copies/mL for >2 years CD4 >500 cells/mm3 No HBV or HCV coinfection No PI as part of ART Panobinostat 20 mg orally 3 times/week every other week for 8 weeks Primary endpoint Change from baseline in cell-associated HIV RNA (unspliced gag HIV RNA)/106 CD4+ T cells Baseline Characteristics Patients (n=15) Male (%) 100 White (%) Age (years) 47 Time in months Since HIV diagnosis HIV diagnosis and start of ART On ART HIV RNA <50 copies/mL 81.8 18 43.4 38 CD4 (cells/mm3) 935 Slide: CLEAR Study: Panobinostat (HDAC Inhibitor) and Viral Reactivation Rasmussen and colleagues conducted a phase 1/2 trial to evaluate the ability of the histone deacetylase (HDAC) inhibitor panobinostat to reverse HIV latency.1 HIV patients on ART (n=15). Oral panobinostat 20 mg 3 times/week every other week for 8 weeks. The primary endpoint: cell-associated unspliced HIV RNA (CA-US RNA) in total CD4+ T cells using semi-nested qPCR. Secondary endpoints included safety, plasma HIV-RNA, as well as total and integrated HIV DNA. Reference Rasmussen TA, Tolstrup M, Brinkmann CR, et al. Panobinostat, a histone deacetylase inhibitor, for latent-virus reactivation in HIV-infected patients on suppressive antiretroviral therapy: a phase 1/2, single group, clinical trial. Lancet HIV. 2014;1:e13-e21. HDAC: histone deacetylase. Rasmussen TA, et al. Lancet HIV. 2014;1:e13-e21.

32 CLEAR Study: Panobinostat (HDAC Inhibitor) and Viral ReactivationCell-Associated HIV-1 RNA Expression Histone H3 Acetylation Mean Fluorescence Intensity Fold Increased Slide: CLEAR Study: Panobinostat (HDAC Inhibitor) and Viral Reactivation Overall, panobinostat treatment resulted in substantial cyclic changes in histone H3 acetylation with increased acetylation detected during panobinostat treatment weeks. Mean histone H3 acetylation increased rapidly, by 2.5-fold (P<0.0001) 2 hours after the first dose of panobinostat.1 Levels of CA-US RNA increased significantly during panobinostat treatment (P<0.0001) with significant increases at time points on-treatment as compared to baseline. The median maximal fold-increase in CA-US RNA was 3.5 and remained elevated 4 weeks post-panobinostat (fold increase 1.60; P=0.003).1 Reference Rasmussen TA, Tolstrup M, Brinkmann CR, et al. Panobinostat, a histone deacetylase inhibitor, for latent-virus reactivation in HIV-infected patients on suppressive antiretroviral therapy: a phase 1/2, single group, clinical trial. Lancet HIV. 2014;1:e13-e21. Days Days Shaded bars are panobinostat dosing periods. Low-level viremia via single copy assay and TMA based. Rasmussen TA, et al. Lancet HIV. 2014;1:e13-e21.

33 CLEAR Study: Panobinostat (HDAC Inhibitor) and Viral ReactivationOnly 1/15 patients remained HIV RNA undetectable at all time points during panobinostat dosing 3 patients were HIV RNA positive at all time points Panobinostat cyclic dosing was well tolerated First proof of viral “kick” leading to consistent plasma release of viral particles Low-Level Viremia Patients (%) Slide: CLEAR Study: Panobinostat (HDAC Inhibitor) and Viral Reactivation Only 1/15 patients remained HIV RNA undetectable at all time points during panobinostat dosing. Three patients were HIV RNA positive at all time points.1 Overall, panobinostat cyclic dosing was well tolerated.1 This was the first proof of inducing viral “kick” leading to consistent plasma release of viral particles with repeat dosing of an HDAC inhibitor.1 Reference Rasmussen TA, Tolstrup M, Brinkmann CR, et al. Panobinostat, a histone deacetylase inhibitor, for latent-virus reactivation in HIV-infected patients on suppressive antiretroviral therapy: a phase 1/2, single group, clinical trial. Lancet HIV. 2014;1:e13-e21. Days Shaded bars are panobinostat dosing periods. Low-level viremia via single copy assay and TMA based. Rasmussen TA, et al. Lancet HIV. 2014;1:e13-e21.

34 Romidepsin (HDAC Inhibitor): Reactivation of Latent HIV-1 In VivoPhase 1/2, nonrandomized trial HIV-infected patients on suppressive ART (n=6) Median CD4: 760 cells/mm3 Mean age: 54 years Median duration of ART: 9.5 years None received ART during primary HIV infection Romidepsin 5 mg/m2 IV on days 0, 7, and 14 Primary endpoints Safety Activation of HIV-1 transcription and production (cell-associated HIV RNA in total CD4 cells and plasma HIV RNA) Slide: Romidepsin (HDAC Inhibitor): Reactivation of Latent HIV-1 In Vivo In a phase 1/2, nonrandomized trial, Sögaard and colleagues evaluated the effects of romidepsin on measures of viral transcription and plasma viremia in vivo in 6 HIV-infected patients on suppressive ART.1 Median CD4: 760 cells/mm3. Mean age: 54 years. Median duration of ART: 9.5 years. None received ART during primary HIV infection. All patients received romidepsin 5 mg/m2 IV on days 0, 7, and 14.1 The primary endpoints were safety and activation of HIV-1 transcription and production (cell-associated HIV RNA in total CD4 cells and plasma HIV RNA).1 Reference Søgaard OS, Graversen ME, Leth S, et al. The depsipeptide romidepsin reverses HIV-1 latency in vivo. PLoS Pathog. 2015;Sep 17;11:e HDAC: histone deacetylase. Søgaard OS, et al. PLoS Pathog. 2015;Sep 17;11:e

35 Romidepsin (HDAC Inhibitor): Reactivation of Latent HIV-1 In VivoCell-Associated Unspliced HIV RNA Plasma HIV RNA (Individual Subjects) Pretherapy Mean fold change from pretherapy for group (n=6) Romidepsin Romidepsin Romidepsin Romidepsin Romidepsin Romidepsin Plasma HIV RNA (copies/mL) HIV RNA (fold changes) Slide: Romidepsin (HDAC Inhibitor): Reactivation of Latent HIV-1 In Vivo Cell-associated, unspliced HIV-1-RNA levels increased significantly from baseline during treatment (2.1- to 3.9-fold after 2nd; P=0.03).1 Importantly, viral load increased from “undetectable” at baseline to readily quantifiable levels at multiple post-infusion time points in 5 of 6 patients (range copies/mL after 2nd infusion, P=0.007).1 The emergence of quantifiable plasma HIV-1 RNA corresponded directly with the cyclic romidepsin infusions.1 Reference Søgaard OS, Graversen ME, Leth S, et al. The depsipeptide romidepsin reverses HIV-1 latency in vivo. PLoS Pathog. 2015;Sep 17;11:e LOQ LOD Time (days) Time (days) Red arrows: romidepsin dose days. HDAC: histone deacetylase. Søgaard OS, et al. PLoS Pathog. 2015;Sep 17;11:e

36 Romidepsin (HDAC Inhibitor): Summary of ResultsCell-associated HIV RNA All patients had significant increases in cell-associated, unspliced HIV RNA Plasma HIV RNA Quantifiable plasma HIV RNA (n=5) No change in HIV DNA from baseline to day 21 Safety 36 treatment-emergent adverse events Most were mild and resolved spontaneously Most common: abdominal symptoms (nausea, borborygmi, abdominal pain, diarrhea, vomiting) and fatigue Slide: Romidepsin (HDAC Inhibitor): Summary of Results In summary, all patients had significant increases in cell-associated, unspliced HIV RNA and quantifiable plasma HIV RNA (5 of 6 subjects). There was no change in HIV DNA from baseline to day 21.1 Safety included 36 treatment-emergent adverse events that of mild severity and resolved spontaneously. The most common adverse events were abdominal symptoms (nausea, borborygmi, abdominal pain, diarrhea, vomiting) and fatigue.1 Reference Søgaard OS, Graversen ME, Leth S, et al. The depsipeptide romidepsin reverses HIV-1 latency in vivo. PLoS Pathog. 2015;Sep 17;11:e HDAC: histone deacetylase. Søgaard OS, et al. PLoS Pathog. 2015;Sep 17;11:e

37 Treatment With a TLR7 Agonist Induces Transient Viremia in SIV-Infected ART-Suppressed MonkeysExpressed in plasmacytoid dendritic cells and B lymphocytes Part of innate immune system linked to adaptive immunity Activation leads to stimulation of the latent virus reservoir via multiple mechanisms Increased antigen presentation Enhanced NK and CD8+ T-cell activation Activation of CD4+ T cells SIV rhesus macaque study design Intra-rectal SIVmac251 Dolutegravir, emtricitabine, and tenofovir DF administered on days TLR7 agonist (GS-9620) or placebo administered every other week (7 doses) 0.1, 0.2, 0.3 mg/kg Slide: Treatment With a TLR7 Agonist Induces Transient Viremia in SIV-Infected ART-Suppressed Monkeys The toll-like receptor 7 (TLR7) is expressed in plasmacytoid dendritic cells and B lymphocytes and is part of the innate immune system linked to adaptive immunity. Activation of TLR7 leads to stimulation of the latent virus reservoir via multiple mechanisms, including increased antigen presentation, enhanced NK and CD8+ T-cell activation, and activation of CD4+ T cells.1 Whitney and colleagues conducted a study in SIV-infected rhesus macaques on ART to determine if administration of an oral TLR7 (GS-9620) agonist would induce transient plasma viremia and reduce viral reservoirs.1 Intra-rectal SIVmac251. Dolutegravir, emtricitabine, and tenofovir DF were administered on days TLR7 agonist (GS-9620) or placebo was administered every other week (7 doses) at 0.1, 0.2, and 0.3 mg/kg. Reference Whitney JB, Lim S-Y, Osuna CE, et al. Treatment with a TLR7 agonist induces transient viremia in SIV-infected ART suppressed monkeys. Program and abstracts from the 22nd Conference on Retroviruses and Opportunistic Infection; February 23-26, 2015; Seattle, WA. Abstract 108. TLR7: toll-like receptor 7. Whitney J, et al. 22nd CROI. Seattle, Abstract 108.

38 Plasma SIV RNA (log10 copies/mL) Post SIV Infection (Days)TLR7 Agonist: Induces Transient Viremia and Lower Virus Set Point in SIV-Infected ART-Suppressed Monkeys ART Placebo (n=6) TLR agonist (n=4) Plasma SIV RNA (log10 copies/mL) TLR Agonist (mg/kg) Slide: TLR7 Agonist: Induces Transient Viremia and Lower Virus Set Point in SIV-Infected ART-Suppressed Monkeys The first 3 doses of TLR7 agonist administered to the SIV-infected ART-suppressed macaques had a limited effect on plasma viremia. However, doses 4 through 7 led to transient and consistent increases in plasma virus ( SIV RNA copies/mL) in all treated macaques with a return to a 10-fold reduction in plasma virus setpoint as compared with the placebo group.1 Reference Whitney JB, Lim S-Y, Osuna CE, et al. Treatment with a TLR7 agonist induces transient viremia in SIV-infected ART suppressed monkeys. Program and abstracts from the 22nd Conference on Retroviruses and Opportunistic Infection; February 23-26, 2015; Seattle, WA. Abstract 108. 0.1 0.2 0.3 0.3 0.3 0.3 0.3 Blips: 2/ /4 4/4 4/4 Post SIV Infection (Days) TLR7: toll-like receptor 7. Whitney J, et al. 22nd CROI. Seattle, Abstract 108.

39 Repeated TLR7 Agonist Administration Can Lead to Viral Remission in SIV-Infected ART-Suppressed Monkeys SIV rhesus macaque study design (n=11) Intra-rectal inoculation of SIVmac251 Dolutegravir, emtricitabine, and tenofovir DF administered on starting on day 65 On day 467 TLR7 agonists GS-9620 (0.05 and 0.15 mg/kg) or GS-986 (0.1 mg/kg) administered every other week for 10 doses, followed by a 3-month hiatus before administering the final 9 doses (n=9) Placebo (n=2) Plasma SIV RNA <50 copies/mL TLR7 agonists induced small blips after doses 3-10 No blips for doses 11-19 Lower doses of TLR7 agonists induced minimal to no plasma IFNα Cessation of ART TLR7 agonist groups: 2/9 had undetectable plasma SIV RNA up to 3-4 months after stopping ART Slide: Repeated TLR7 Agonist Administration Can Lead to Viral Remission in SIV-Infected ART-Suppressed Monkeys Reference Whitney JB, Lim S-Y, Osuna CE, et al. Repeated TLR7 agonist treatment of SIV+ monkeys on ART can lead to viral remission. Program and abstracts from the 23rd Conference on Retroviruses and Opportunistic Infection; February 22-25, 2016; Boston, MA. Abstract 95LB. Whitney JB, et al. 23rd CROI. Boston, Abstract 54.

40 Other Approaches to HIV InductionBromodomain inhibitors Prevent BRD4 competition with Tat for pTEFb binding SMYD2 inhibitors Block methylation of Tat Protein kinase C activators Bryostatin, prostratin Hydroxybenzotriazoles Increase HIV production in vitro without stimulating cell proliferation or activation Cytokines IL-7, IL-15 Slide: Other Approaches to HIV Induction Other investigative approaches to HIV induction include:1-3 Bromodomain inhibitors: prevent BRD4 competition with Tat for pTEFb binding. SMYD2 inhibitors: block methylation of Tat. Protein kinase C activators: examples include bryostatin, prostratin. Hydroxybenzotriazoles: increase HIV production in vitro without stimulating cell proliferation or activation. Cytokines IL-7, IL-15. References Mbonye U, Karn J. Transcriptional control of HIV latency: cellular signaling pathways, epigenetics, happenstance and the hope for a cure. Virology. 2014; : Archin NM, Sung JM, Garrido C, et al. Eradicating HIV-1 infection: seeking to clear a persistent pathogen. Nat Rev Microbiol. 2014;12: Passaes CP, Sáez-Cirión A. HIV cure research: advances and prospects. Virology. 2014; : Mbonye U, et al. Virology. 2014; : Archin NM, et al. Nat Rev Microbiol. 2014;12: Passes CP, et al. Virology. 2014; :

41 Program Overview Strategies in HIV cure researchBenefits of ART and search for an HIV cure Biologic considerations in retroviral persistence Defining a cure Strategies in HIV cure research Induce HIV expression from latently infected cells Hematopoietic stem cell transplantation Genetically modified CD4 T cells Immune-based interventions Broadly neutralizing antibodies Immediate initiation of ART Combination approaches Challenges and future perspectives Slide: Program Overview

42 Long-Term Control of HIV by CCR5 Δ32/Δ32 Stem-Cell TransplantationHIV-infected, 40-year-old, white man with newly diagnosed acute myeloid leukemia (FAB M4 subtype, with normal cytogenetic features) HIV diagnosis >10 years prior Efavirenz/emtricitabine/tenofovir DF for previous 4 years At time of AML diagnosis HIV RNA undetectable, CD4 415 cells/mm3 ART discontinued shortly during first course of consolidation chemotherapy, resumed on viral rebound, then discontinued 3 months later Remains off ART First stem cell transplant Grade 1 skin graft-versus-host disease (successfully treated) Second stem cell transplant after relapse of leukemia (still in remission) Liver and brain graft-versus-host disease (successfully treated) Slide: Long-Term Control of HIV by CCR5 Δ32/Δ32 Stem-Cell Transplantation HIV-infected, 40-year-old, white man with newly diagnosed acute myeloid leukemia (FAB M4 subtype, with normal cytogenetic features) who was diagnosed with HIV >10 years prior. The patient has been efavirenz/emtricitabine/tenofovir DF for the previous 4 years.1 At the time of AML diagnosis, HIV RNA was undetectable and CD4 count was 415 cells/mm3.1 ART was discontinued shortly during the first course of consolidation chemotherapy, resumed on viral rebound, then discontinued 3 months later. The patient remains off ART. First stem cell transplant, the patient had a grade 1 skin graft-versus-host disease (successfully treated).1 Second stem cell transplant after relapse of leukemia (still in remission). Liver and brain graft-versus-host disease (successfully treated). Reference Hütter G, Nowak D, Mossner M, et al. Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation. N Engl J Med. 2009;360: Hütter G, et al. N Engl J Med. 2009;360:

43 Days Before or After Stem Cell TransplantationFunctional Cure Following Stem Cell Transplantation From a CCR5 Δ32/Δ32 Donor AML Diagnosis AML Relapse First SCT 100% Chimerism Rectal Biopsy AML Relapse Second SCT 100% Chimerism HIV RNA 107 ART 106 105 104 Copies/mL 103 102 Undetectable Cx Cx Cx Cx/TBI Cx TBI ATG/MMF/Cs MMF/Cs CD4 Slide: Functional Cure Following Stem Cell Transplantation From a CCR5 Δ32/Δ32 Donor After 6 years of follow-up in the absence of ART, the patient showed no trace of HIV in blood and tissue samples, revealing no evidence for persistent HIV.1 Reference Hütter G, Nowak D, Mossner M, et al. Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation. N Engl J Med. 2009;360: 400 ART 300 Cells/mm3 200 100 Days Before or After Stem Cell Transplantation ATG: antithymocyte globulin; Cs: cyclosporine; Cx: chemotherapy; MMF: mycophenolate mofetil; TBI: total-body irradiation. Hütter G, et al. N Engl J Med. 2009;360:

44 Berlin Patient: Summary of Results From Follow-Up Tests for Viral Persistence (2011-2012)Testing Laboratories (Positive Results/Total Number) Consensus Typical Levels With ART Viral Suppression Fold Difference Plasma HIV RNA 2/4 ?Intermittent positive ?<1 copy/mL 1-2 copy/mL 2-20 PBMC HIV DNA HIV RNA 0/4 0/3 Negative (<1 in 106-7) 751/106 total PBMC 66/106 total PBMC 66-660 Sorted cells from blood 0/1 Negative Unknown -- Peripheral CD4+ T (IUPM) 0/2 Negative (<1 IU/107-9) 1/106 CD4+ T cell CSF HIV RNA CSF cells HIV DNA Lymph node 1-12 copies/100 ng <4 log10 copies/g Rectum (biopsy or cells) 1/2 777/106 total gut cells 21/106 total gut cells 780 21-210 Ileum (biopsy or cells) Negative (<1 in 106) 415/106 total gut cells 37/106 total gut cells 415 37 Slide: Berlin Patient: Summary of Results From Follow-Up Tests for Viral Persistence ( ) Yuki and colleagues applied a series of measurements of HIV persistence to the study of an HIV-infected adult who has exhibited evidence of cure after allogeneic hematopoietic stem cell transplant from a homozygous CCR5Δ32 donor. Samples from blood, spinal fluid, lymph node, and gut were analyzed in multiple laboratories using different approaches.1 No HIV DNA or RNA was detected in peripheral blood mononuclear cells (PBMCs), spinal fluid, lymph node, or terminal ileum, and no replication-competent virus could be cultured from PBMCs. However, HIV RNA was detected in plasma (2 laboratories) and HIV DNA was detected in the rectum (1 laboratory) at levels considerably lower than those expected in ART-suppressed patients.1 HIV antibody levels were readily detectable but declined over time; T-cell responses were largely absent.1 The occasional, low-level PCR signals raise the possibility that some HIV nucleic acid might persist, although they could also be false positives. Since HIV levels in well-treated individuals are near the limits of detection of current assays, more sensitive assays need to be developed and validated. The absence of recrudescent HIV replication and waning HIV-specific immune responses five years after withdrawal of treatment provide proof of a clinical cure.1 Reference Yukl SA, Boritz E, Busch M, et al. Challenges in detecting HIV persistence during potentially curative interventions: a study of the Berlin patient. PLoS Pathog. 2013;9:e Yukl SA, et al. PLoS Pathog. 2013;9:e

45 Berlin Patient: ConsiderationsWhat factors contributed to “functional cure” in this case? Myeloablative chemotherapy? Replacement of CCR5+ with CCR5 Δ32 hematopoietic stem cells? Graft-versus-host disease? Immunosuppressive therapy? Other? Can this outcome be replicated? Slide: Berlin Patient: Considerations What factors contributed to “functional cure” in this case? Myeloablative chemotherapy? Replacement of CCR5+ with CCR5 Δ32 hematopoietic stem cells? Graft-versus-host disease? Immunosuppressive therapy? Other? Can this outcome be replicated?

46 HIV-Infected Men Who Received Allogeneic Transplant From Stem-Cell Donor With CCR5 delta/32/delta Mutation Location of Transplant Recipient Age (years) Cancer Graft Outcome Post Transplant Berlin, Germany 40 AML HLA-matched unrelated Alive after 7 years No viral rebound, no ART Utrecht, The Netherlands 53 MS Combined haploidentical bridge with umbilical-cord blood Died from relapse of the myelodysplastic syndrome and pneumonia after 2 months Muster, Germany 51 NHL HLA-mismatched unrelated Died from infection after 4 months Essen, Germany 30 CXCR4-tropic HIV-1 rebound Died from relapse of NHL after 12 months Minneapolis, Minnesota 12 ALL Umbilical-cord blood Died from GVHD after 3 months Santiago, Chile 46 HLA-matched related Died from pneumonia shortly afterward Barcelona, Spain 37 Died from relapse of NHL after 3 months Slide: HIV-Infected Men Who Received Allogeneic Transplant From Stem-Cell Donor With CCR5 delta/32/delta Mutation Since the successful transplantation reported for the Berlin patient in 2007, there have been at least 6 patients who received a graft from a donor who was homozygous for CCR5 delta32/delta32 mutation. Unfortunately, survival of these patients was 1 year or less.1 The case from Essen, Germany experienced CXCR4-tropic HIV-1 rebound. The author suggested that:1 ART may need to be continued during the transplantation until a stable donor chimerism is achieved. Finding appropriate unrelated donors for transplantation and selecting an HLA match as well as an adult donor who is homozygous for the CCR5 delta32/delta32 mutation is worth careful consideration. Reference Hütter G. More on shift of HIV tropism in stem-cell transplantation with CCR5 delta32/delta32 mutation. N Engl J Med. 2014;371: AML: acute myeloid leukemia; MS: myelodysplastic syndrome; NHL: non-Hodgkin’s lymphoma; ALL: acute lyphoblastic leukemia. GVHD: graft-versus-host-disease. Hütter G. N Engl J Med. 2014;371:

47 Long-Term Reduction in Peripheral Blood HIV-1 Reservoirs Following RIC-alloHSCTPatient A Patient B Perinatally acquired HIV Heterozygous for CCR5 Δ32 mutation Donor: CCR5 wild-type RIC-alloHSCT after relapse for treatment of stage IV, nodular sclerosing Hodgkin lymphoma ART pre- and post-transplantation Efavirenz/emtricitabine/tenofovir DF Sexually acquired HIV Heterozygous for CCR5 Δ32 mutation Donor: CCR5 wild type RIC-alloHSCT after relapse for treatment of stage IV mixed cellularity Hodgkin disease ART pre- and post-transplantation Raltegravir/emtricitabine/tenofovir DF Slide: Long-Term Reduction in Peripheral Blood HIV-1 Reservoirs Following RIC-alloHSCT Henrich and colleagues previously reported the loss of detectable peripheral blood HIV-1 reservoirs in 2 individuals following reduced-intensity conditioning allogeneic hematopoietic stem cell transplantation (RIC-alloHSCT) from wild-type CCR5 donors. To understand further the impact of alloHSCT on viral reservoirs, Henrich and colleagues studied the longitudinal effects of HSCT on host microchimerism and HIV-specific cellular immunity, and tested rectal tissue and peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis for evidence of residual HIV-1 DNA or replication-competent proviruses up to 4.3 years post-transplantation.1 Patient A (perinatally acquired HIV) underwent RIC-alloHSCT after relapse for treatment of stage IV, nodular sclerosing Hodgkin lymphoma. The patient received efavirenz/emtricitabine/tenofovir DF pre- and post-transplantation. Patient B (sexually acquired HIV) underwent RIC-alloHSCT after relapse for treatment of stage IV mixed cellularity Hodgkin disease. This patient received raltegravir/emtricitabine/tenofovir DF pre- and post-transplantation. Evaluations included:1 Collection of PBMCs by leukapheresis for large-scale HIV-1 quantification of genomic DNA and viral co-culture from purified CD4+ T lymphocytes. HIV-1 DNA PCR on rectal tissue (one patient). Microchimerism studies of residual donor PBMCs. HIV-specific cellular immune function by ELISpot IFN-gamma screenings of total PBMCs involving comprehensive HLA-specific peptide panels. Reference Henrich TJ, Hanhauser E, Marty FM, et al. Antiretroviral-free HIV-1 remission and viral rebound after allogeneic stem cell transplantation: report of 2 cases. Ann Intern Med. 2014;161: RIC-alloHSCT: reduced-intensity allogeneic hematopoietic stem cell transplantation. Evaluations: Total HIV-1 DNA and replication-competent proviral load in PBMCs. HIV-1 DNA levels in rectal tissue. Longitudinal effects of HSCT on host microchimerism. HIV-specific cellular immunity before and after HSCT. Henrich TJ, et al. Ann Intern Med. 2014;161:

48 Patient A: Long-Term PBMC DNA and CD4 Count Following RIC-alloHSCT and Receiving ART(>3.3 log10 reduction) CD4 Count HSCT HIV DNA (copies/106 PBMC) CD4+ T Cells (per mm3) 100% Donor Lymphocyte Chimerism 4.3 Years Post-HSCT PBMC DNA Not Detected (<0.07 copies/106) Slide: Long-Term PBMC DNA and CD4 Count Following RIC-alloHSCT No HIV-1 DNA was detected from PBMCs from both previously reported RIC-alloHSCT patients, indicating at least a 3 to 4 log10 decrease in peripheral viral reservoir size post-transplantation. Both patients experienced a robust CD4 cell gain.1 Reference Henrich TJ, Hanhauser E, Marty FM, et al. Antiretroviral-free HIV-1 remission and viral rebound after allogeneic stem cell transplantation: report of 2 cases. Ann Intern Med. 2014;161: Post HSCT (days) Post HSCT (days) RIC-alloHSCT: reduced-intensity allogeneic hematopoietic stem cell transplantation. Evaluation at 4.3 years post HSCT: PBMC DNA (input 25 x 106 cells, qPCR for LTR/gag): not detected. Peripheral CD4+ T cells (input 150 x 106 cells, co-culture assay): not detected. Henrich TJ, et al. Ann Intern Med. 2014;161:

49 Patient B: Long-Term PBMC DNA and CD4 Count Following RIC-alloHSCT and Receiving ART(> log10 reduction) CD4 Count 100% Donor Lymphocyte Chimerism HIV DNA (copies/106 PBMC) CD4+ T Cells (per mm3) 2.6 Years Post-HSCT PBMC DNA Not Detected (<0.04 copies/106) Slide: Long-Term PBMC DNA and CD4 Count Following RIC-alloHSCT No HIV-1 DNA was detected from PBMCs from both previously-reported RIC-alloHSCT patients, indicating at least a 3 to 4 log10 decrease in peripheral viral reservoir size post-transplantation. Both patients experienced a robust CD4 cell gain.1 Reference Henrich TJ, Hanhauser E, Marty FM, et al. Antiretroviral-free HIV-1 remission and viral rebound after allogeneic stem cell transplantation: report of 2 cases. Ann Intern Med. 2014;161: HSCT Post HSCT (days) Post HSCT (days) RIC-alloHSCT: reduced intensity allogeneic hematopoietic stem cell transplantation. Evaluation at 2.6 years post HSCT: PBMC DNA (input 50 x 106 cells, qPCR for LTR/gag): not detected. Peripheral CD4+ T cells (input 150 x 106 cells, co-culture assay): not detected. Rectal biopsies (DNA from 1.3 x 103 cells, qPCR for LTR/gag): not detected. Henrich TJ, et al. Ann Intern Med. 2014;161:

50 Analytic ART Treatment Interruption Following RIC-alloHSCTART treatment interruption to assess the extent of reservoir depletion Full human research committee review and 2nd informed consent process Frequent patient monitoring during ATI Plasma HIV-1 RNA tested weekly (Cobas TaqMan v.2) PBMC HIV-1 DNA tested twice a month (clinical lab) Single-copy RNA assay and additional large-volume PBMC sampling every months Criteria for restarting ART HIV RNA >1000 copies/mL or confirmed >200 copies/mL HIV-1 Reservoir Status Prior to ATI Patient A B Time on ART after HSCT (years) 4.3 2.6 Total HIV-1 PBMC DNA Level (copies/106 cells) Total cells tested (x106 PBMCs) Positive wells/total wells <0.12 26 0/42 <0.13 24 Infectious virus by viral outgrowth assay Level (IUPM cells) Total cells tested (x106 CD4+) <0.007 150 0/30 <0.006 160 0/32 Rectal tissue Total cells tested (x106 nucleated cells) -- <2.4 1.23 0/22 Slide: Analytic ART Treatment Interruption Following RIC-alloHSCT Henrich and colleagues took steps to characterize HIV-1 reservoirs in blood and tissues and perform analytic antiretroviral treatment interruptions (ATI) to determine the potential for allogeneic HSCT to lead to sustained, antiretroviral-free HIV-1 remission. This approach underwent a full human research committee review and 2nd informed consent process.1 There was frequent patient monitoring during ATI that included:1 Plasma HIV-1 RNA tested weekly (Cobas TaqMan v.2). PBMC HIV-1 DNA tested twice a month (clinical lab). Single-copy RNA assay and additional large-volume PBMC sampling every 3 months. The criteria for restarting ART was when HIV RNA >1000 copies/mL or confirmed >200 copies/mL.1 Reference Henrich TJ, Hanhauser E, Marty FM, et al. Antiretroviral-free HIV-1 remission and viral rebound after allogeneic stem cell transplantation: report of 2 cases. Ann Intern Med. 2014;161: Henrich TJ, et al. Ann Intern Med. 2014;161:

51 Patient A (ATI): 84 Days of Remission and Subsequent Viral ReboundDRV/r RAL/ FTC/TDF HIV RNA EFV/ FTC/TDF 107 Fevers, headache, nausea Symptoms resolve 106 CSF HIV RNA <20 copies/mL 105 Copies/mL 104 103 102 DRV/r RAL/FTC/TDF 101 HBV DNA* Post ATI (days) CD4+ T Cell Slide: Patient A (ATI): 84 Days of Remission and Subsequent Viral Rebound HIV-1 was first detected 12 weeks after ATI in Patient A. Clinical symptoms of the acute retroviral syndrome followed a rapid increase in plasma viral load and resolved at the time of active ART reinitiation. A plasma RNA level of 20,202 copies/mL was recorded 226 days after ATI, but this may have been due to sample switching in the clinical laboratory. CD4 T-cell counts transiently declined during the time of peak viremia.1 Reference Henrich TJ, Hanhauser E, Marty FM, et al. Antiretroviral-free HIV-1 remission and viral rebound after allogeneic stem cell transplantation: report of 2 cases. Ann Intern Med. 2014;161: 1000 800 600 Cells/mm3 400 200 Post ATI (days) *HBV DNA (copies/106 PBMC). Shaded bars: on ART. Henrich TJ, et al. Ann Intern Med. 2014;161:

52 Patient B (ATI): 225 Days of Remission and Subsequent Viral ReboundHIV RNA 107 Fevers, malaise CSF HIV RNA 269 copies/mL 106 Plasma HIV RNA (<0.4 copies/mL) Plasma HIV RNA (<0.4 copies/mL) Symptoms resolve 105 HIV-1 DNA (<0.5 copies/106 PBMC) HIV-1 DNA (<0.2 copies/106 PBMC) Copies/mL 104 103 102 DRV + FTC/TDF 101 HBV DNA* Post ATI (days) CD4+ T Cell Slide: Patient B (ATI): 225 Days of Remission and Subsequent Viral Rebound HIV-1 was first detected 32 weeks after ATI in Patient B. In addition, no HIV-1 DNA or plasma RNA was detected by sensitive research assays 38 and 129 days after ART discontinuation. Clinical symptoms of the acute retroviral syndrome occurred approximately 7 days after the last negative viral load test result. Symptoms resolved with prompt initiation of ART and subsequent viral suppression.1 Reference Henrich TJ, Hanhauser E, Marty FM, et al. Antiretroviral-free HIV-1 remission and viral rebound after allogeneic stem cell transplantation: report of 2 cases. Ann Intern Med. 2014;161: 1000 800 600 Cells/mm3 400 200 Post ATI (days) *HBV DNA (copies/106 PBMC). Shaded bars: on ART. Henrich TJ, et al. Ann Intern Med. 2014;161:

53 Long-Term Reduction in Peripheral Blood HIV-1 Reservoirs Following RIC-alloHSCTAllogeneic HSCT resulted in Loss of detectable HIV-1 DNA from blood and rectal mucosa ART-free remission of variable duration Viral rebound occurred despite a >3 log10 reduction in reservoir size (HIV-1 DNA PBMC) Long-lived tissue reservoirs inaccessible to sampling may have contributed to viral rebound Predicting outcomes of treatment to eradicate the latent reservoir for HIV-1 Much higher efficacy will be required for eradication Modeling predicts that the latent reservoir would have to be depleted >5 log10 fold for a durable cure to be likely Slide: Long-Term Reduction in Peripheral Blood HIV-1 Reservoirs Following RIC-alloHSCT Allogeneic HSCT resulted in the loss of detectable HIV-1 DNA from blood and rectal mucosa in these 2 patients. ART-free remission of variable duration and viral rebound occurred despite a >3 log10 reduction in reservoir size (HIV-1 DNA PBMC).1 The long-lived tissue reservoirs inaccessible to sampling may have contributed to viral rebound.1 Predicting outcomes of treatment to eradicate the latent reservoir for HIV-1 may require much higher efficacy for eradication. Modeling predicts that the latent reservoir would have to be depleted >5 log10 fold for a durable cure to be likely.2 References Henrich TJ, Hanhauser E, Marty FM, et al. Antiretroviral-free HIV-1 remission and viral rebound after allogeneic stem cell transplantation: report of 2 cases. Ann Intern Med. 2014;161: Hill AL, Rosenbloom DI, Fu F, et al. Predicting the outcomes of treatment to eradicate the latent reservoir for HIV-1. Proc Natl Acad Sci U S A. 2014;111: Henrich TJ, et al. Ann Intern Med. 2014;161: Hill AL, et al. PNAS. 2014;111:

54 EpiStem Consortium: Allogeneic Stem Cell Transplantation in HIV-Infected PatientsViral Reservoir Analysis Patients >3 Years Follow-Up (post-transplant, ART continued) Patient 1 2 Patient 3 Graft versus host disease No Yes CCR5 donor status WT d32 Post-transplant (months) Single copy assay (HIV RNA copies/mL) 5 -- UD Total DNA (copies/106 CD4+) 25 Quantitative viral outgrowth assay (infectious units/million) 0.034 Ileum (CD4 cells) Trace Total HIV DNA (CD4+) HIV DNA (copies/106) Total HIV DNA (PBMC) Slide: EpiStem Consortium: Allogeneic Stem Cell Transplantation in HIV-Infected Patients In this observational cohort, 15 of the 24 HIV-infected persons with hematologic cancer received allogeneic stem cell transplant. Viral reservoir analysis: leukophoresis (multiple 500 mL blood draws [ddPCR and qVOA]).1 Preliminary analysis showed a systematic reduction of HIV-1 reservoirs to very low levels following allogeneic stem cell transplant. In 2 cases with long-term follow-up, infectious virus was not detected in blood independent of CCR5 status of donor. In tissues, only traces of HIV DNA could be detected.1 The authors hypothesized from these results that graft versus HIV-1 reservoir effect contributed to the clearance of the viral reservoir.1 Reference Wensing AM, Diez-Martin JL, Huetter G, et al. Allogeneic stem cell transplantation in HIV-1-infected individuals; the EPISTEM consortium. JAIDS. 2016;19(suppl 5): Abstract THAA0105. UD: undetectable. Years on ART Observational cohort with hematologic cancer (n=24, of whom 15 received allogeneic stem cell transplant (n=15). Viral reservoir analysis: leukophoresis (multiple 500 mL blood draws [ddPCR and qVOA]). Hypothesis: graft versus HIV-1 reservoir effect contributed to the clearance of the viral reservoir. Wensing A, et al. JAIDS. 2016;19(suppl 5): Abstract THAA0105.

55 Program Overview Strategies in HIV cure researchBenefits of ART and search for an HIV cure Biologic considerations in retroviral persistence Defining a cure Strategies in HIV cure research Induce HIV expression from latently infected cells Hematopoietic stem cell transplantation Genetically modified CD4 T cells Immune-based interventions Broadly neutralizing antibodies Immediate initiation of ART Combination approaches Challenges and future perspectives Slide: Program Overview

56 Disrupt the CCR5 Gene by Expressing an Engineered Zinc-Finger NucleaseZinc-finger nucleases 2 functional domains (DNA binding and cleaving) Recognize a unique hexamer sequence of DNA to create a single, specific double-strand break at the user-defined locus Cut modules are stitched together to form a zinc-finger protein Highly specific pair of genomic scissors CCR5-modified CD4 T cells Preclinical tests Produced functional normal CD4 cells in response to mitogens Protected against HIV Reduced HIV RNA levels in humanized mouse model of HIV infection Zinc-Finger Nuclease Cleaving Domain Slide: Disrupt the CCR5 Gene by Expressing an Engineered Zinc-Finger Nuclease Zinc-finger nucleases:1 2 functional domains (DNA binding and cleaving) that recognize a unique hexamer sequence of DNA to create a single, specific double-strand break at the user-defined locus. Cut modules are stitched together to form a zinc-finger protein with a highly specific pair of genomic scissors. CCR5-modified CD4 T cells in preclinical tests produced functional normal CD4 cells in response to mitogens, protected against HIV, and reduced HIV RNA levels in a humanized mouse model of HIV infection.1 Reference Tebas P, Stein D, Tang WW, et al. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl J Med. 2014;370: Binding Domain Tebas P, et al. N Engl J Med. 2014;370:

57 Baseline CharacteristicsGene Editing of CCR5 in Autologous CD4+ T Cells in HIV-Infected Patients Open-label, non-randomized, uncontrolled study in HIV patients controlled on ART (n=12) Cohort 1: adequate CD4 recovery on ART Cohort 2: inadequate CD4 recovery on ART Single, infused dose of SB-728-T Autologous CD4-enriched cells modified at the CCR5 gene locus Primary outcome: safety Secondary outcomes: CD4 count, persistence of modified cells, homing to gut mucosa, HIV RNA Total lymphocyte and CD4 counts significantly increased 1 week after infusion Progressively declined thereafter but remained above baseline values Increase in CD8 was moderate and transient Baseline Characteristics Cohort 1 (n=6) Cohort 2 Median age (years) 50 46 Male (number) 6 4 White/Black/Asian/Hispanic (number) 4/1/1/0 1/3/1/1 Duration of HIV (years) 12.3 14.9 Median CD4 (cells/mm3) 662 272 Median CD4:CD8 ratio 1.42 0.72 SB-728-T (median) Dose (x1010) CD3 (%) Cell modification (%) 1.00 97.8 16.5 0.85 95.8 26.0 Slide: Gene Editing of CCR5 in Autologous CD4+ T Cells in HIV-Infected Patients Tebas and colleagues conducted an open-label, non-randomized, uncontrolled study HIV patients who were virologically suppressed on ART to receive a single dose of zinc-finger nuclease (ZFN)-modified autologous CD4 T cells (n=12).1 Cohort 1: adequate CD4 recovery on ART. Cohort 2: inadequate CD4 recovery on ART. Both arms received a single, infused dose of SB-728-T with autologous CD4-enriched cells modified at the CCR5 gene locus. The primary outcome was safety. Secondary outcomes included CD4 count, persistence of modified cells, homing to gut mucosa, and HIV RNA.1 They found that total lymphocyte and CD4 counts significantly increased 1 week after infusion and then progressively declined thereafter but remained above baseline values. They reported an increase in CD8, which was moderate and transient.1 Reference Tebas P, Stein D, Tang WW, et al. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl J Med. 2014;370: Tebas P, et al. N Engl J Med. 2014;370:

58 Single-Dose Infusion of CCR5-Modified CD4 T Cells: Persistency in Plasma and TissuesCirculation: CCR5-Modified CD4 T Cells Rectal Mucosal Tissue: CCR5-Modified CD4 T Cells Median Cohort 1 Cohort 2 Cohort 1 Absolute Number/mm3 Absolute Number/106 Mucosal Cells Slide: Single-Dose Infusion of CCR5-Modified CD4 T Cells: Persistency in Plasma and Tissues The median absolute number of CCR5-modified circulating CD4 T cells was similar in participants with adequate CD4 T-cell recovery after ART (cohort 1) and in those with inadequate CD4 T-cell recovery after ART (cohort 2).1 Patients in cohort 1 underwent a rectal biopsy at baseline and on days 21 and 112, and those in cohort 2 underwent biopsies at baseline and on days 42 and 252. CCR5-modified CD4 T cells constituted a mean of 0.6%, 1.1%, 0.4%, and 0.4% (and a median of 0.8%, 0.4%, 0.2%, and 0.2%) of rectal mucosal mononuclear cells on days 21, 42, 112, and 252, respectively.1 Reference Tebas P, Stein D, Tang WW, et al. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl J Med. 2014;370: Cohort 2 Time (days) Time (days) Baseline number of CCR5-modified CD4 T cells in rectal biopsies at baseline was 0. Cohort 1 (n=6): adequate CD4 recovery on ART. Cohort 2 (n=6): inadequate CD4 recovery on ART. Tebas P, et al. N Engl J Med. 2014;370:

59 (Individual Patient Data) Historical Viral Load Set PointsSingle-Dose Infusion of CCR5-Modified CD4 T Cells: Change in HIV RNA Following ART ATI (Cohort 1) Analytic treatment interruption in Cohort 1 (n=6; adequate CD4 response on ART) Began 4 weeks after infusion of CCR5-modified CD4 T cells ATI stopping rule: HIV RNA >100K copies/mL HIV RNA became detectable in all 6 patients Early ART resumption (n=2) Median CD4 counts (total and CCR5-modified) declined HIV RNA and ATI (Individual Patient Data) Historical Viral Load Set Points 106 105 104 HIV RNA (copies/mL) Slide: Single-Dose Infusion of CCR5-Modified CD4 T Cells: Change in HIV RNA Following ART ATI (Cohort 1) Analytic treatment interruption in Cohort 1 (n=6; adequate CD4 response on ART) began 4 weeks after infusion of CCR5-modified CD4 T cells with an ATI stopping rule of HIV RNA >100K copies/mL.1 During ATI, HIV RNA became detectable in 4/6 patients. Early ART resumption was required for 2 patients. During ATI, median CD4 counts (total and CCR5-modified) declined.1 Reference Tebas P, Stein D, Tang WW, et al. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl J Med. 2014;370: 103 102 101 Time (days) Shaded area: analytic treatment interruption. Dotted lines: resumption of ART after/during ATI. Tebas P, et al. N Engl J Med. 2014;370:

60 Cohorts 1, 2, 4, 5: enriched total T cellsStudy 1101: Cyclophosphamide Enhancement of SB-728-T Engraftment to Improve Anti-HIV Response Open-label, CTX dose escalation, with single-dose SB-728-T infusion CTX 0.1, 0.5, 1.0, 1.5, 2.0 g/m2 1 to 3 days after CTX, patients receive a single infusion of SB-728-T (1-4 x 1010 cells) 6 weeks after SB-728-T infusion Begin 16-week ART interruption Extended if HIV RNA <10K copies/mL and CD4 >500 cells/mm3 Study 1101 Cohorts 1, 2, 4, 5: enriched total T cells Cohort 3: no enrichment Enrich CD4 Cells Apheresis SB-728 CCR5 ZFNs in Ad 5/35 Vector HIV-positive Aviremic on ART R5 tropic virus CD4 >500 cells/mm3 Platelets: >200K/mm3 PMN: >2500/mm3 Slide: Study 1101: Cyclophosphamide Enhancement of SB-728-T Engraftment to Improve Anti-HIV Response SB-728-T refers to autologous CD4+ enriched T cells that have been transduced ex vivo with SB-728, a replication deficient recombinant Ad5/35 viral vector encoding the CCR5-specific zinc finger nucleases (ZFNs) (SBS8196z and SBS8267), resulting in modification of the CCR5 gene.1 2 ZFNs bind to a composite 24-bp sequence found specifically in the region encoding the first transmembrane domain of the CCR5 gene, just upstream from the naturally occurring CCR5-Δ32 mutation. Expression of the CCR5-specific ZFNs induces a double-stranded break in the cell’s DNA which is repaired by cellular machinery, leading to random sequence insertions or deletions in ~25% of transduced cells. These insertions and deletions disrupt the CCR5 coding sequence, leading to frameshift mutation and termination of protein expression. CCR5-modified CD4 T cells could be tracked due to the acquisition of a unique chromosomal 5-nucleotide (pentamer) DNA sequence, CTGAT, in approximately 25% of the modified cells. Blick and colleagues conducted an open-label, cyclophosphamide (CTX) dose escalation with single-dose SB-728-T infusion: 1. CTX 0.1, 0.5, 1.0, 1.5, 2.0 g/m2; 2. 1 to 3 days after CTX, patients receive a single infusion of SB-728-T (1-4 x 1010 cells); 3. 6 weeks after SB-728-T infusion, patients begin a 16-week ART interruption period, which was extended if HIV RNA <10K copies/mL and CD4 >500 cells/mm3.1 Reference Blick G, Lalezari J, Hsu R, et al. Cytoxan enhancement of SB-728-T engraftment: a strategy to improve anti-HIV response. Program and abstracts from the 22nd Conference on Retroviruses and Opportunistic Infection; February 23-26, 2015; Seattle, WA. Abstract 434. CCR5 Gene Disruption Median CCR5 Modification ~25% Expand, Formulate, and Test SB-728-T Single infusion (1-4 x 1010 cells) CTX: cyclophosphamide. Blick G, et al. 22nd CROI. Seattle, Abstract 434.

61 Undetectable Patient (CTX 1.0 g/m2) Pentamer Duplication/µLStudy 1101: Cyclophosphamide Enhancement of SB-728-T Engraftment to Improve Anti-HIV Response SB-728 CD4/CD8 T cells (40.5%/46.9%) after CTX conditioning (n=3) Doubling of CD8 T cells ( cells/µL) Levels of CCR5-modified CD4 and CD8 T cells were high, reflecting expansion of CD8 T-cells Treatment interruption 1 patient undetectable 1 had controlled viremia, and another had delayed-onset viremia The large increase in CCR5- modified CD8 T cells may Mimic elite controllers Improve HIV control through CD8 HIV cytolytic T cells Undetectable Patient (CTX 1.0 g/m2) CD4 (27.6%): 5.58 x 109 CD8 (58.3%): x 109 107 106 105 104 103 102 101 ART Interruption 300 250 200 150 100 50 7000 6000 5000 4000 3000 2000 1000 HIV RNA Absolute CD4 Absolute CD8 Pentamer Viral RNA (copies/mL) Slide: Study 1101: Cyclophosphamide Enhancement of SB-728-T Engraftment to Improve Anti-HIV Response 3 patients were eligible to receive SB-728 CD4/CD8 T cells (40.5%/46.9%) after CTX conditioning. Patients experienced a doubling of CD8 T cells ( cells/µL). The levels of CCR5-modified CD4 and CD8 T cells were high, reflecting expansion of CD8 T cells.1 During the treatment interruption phase, 1 patient was undetectable (graph), 1 had controlled viremia, and another had delayed-onset viremia.1 The large increase in CCR5-modified CD8 T cells may mimic elite controllers and improve HIV control through CD8 HIV cytolytic T cells.1 Reference Blick G, Lalezari J, Hsu R, et al. Cytoxan enhancement of SB-728-T engraftment: a strategy to improve anti-HIV response. Program and abstracts from the 22nd Conference on Retroviruses and Opportunistic Infection; February 23-26, 2015; Seattle, WA. Abstract 434. Pentamer Duplication/µL CD4 and CD8 (cells/µL) Below LOQ Days From Baseline CTX: cyclophosphamide. Pentamer duplication: surrogate by product of modifications induced by the CCR5 ZFNs. Blick G, et al. 22nd CROI. Seattle, Abstract 434.

62 Program Overview Strategies in HIV cure researchBenefits of ART and search for an HIV cure Biologic considerations in retroviral persistence Defining a cure Strategies in HIV cure research Induce HIV expression from latently infected cells Hematopoietic stem cell transplantation Genetically modified CD4 T cells Immune-based interventions Broadly neutralizing antibodies Immediate initiation of ART Combination approaches Challenges and future perspectives Slide: Program Overview

63 Persistent Inflammation During ART: Multiple Mechanisms and ConsequencesSlide: Persistent Inflammation During ART: Multiple Mechanisms and Consequences Despite effective ART, many if not most HIV-infected adults have evidence of persistent inflammation and immune dysfunction. Root causes of inflammation include ongoing HIV production, high levels of other co-pathogens, irreversible damage to immunoregulation, and translocation of microbial products across damaged mucosal surfaces. This inflammatory environment causes end-organ damage through several potential pathways.1 Reference Deeks SG, Lewin SR, Havlir D. The end of AIDS: HIV infection as a chronic disease. Lancet. 2013;382: Deeks SG, et al. Lancet. 2013;382:

64 Potential Investigative OptionsDevelopmental Considerations for Reducing Persistent Immune Activation in HIV Potential Investigative Options Biologics Anti-PD-1 antibodies, anti-IFN-alfa antibodies TNF inhibitors, IL-6 inhibitors Enhance T-cell renewal Gamma-chain cytokines (IL-2, IL-7, IL-15), growth hormone Microbial translocation Rifaximin, sevelamer, prebiotics/probiotics, colostrum Chemokine receptor inhibitor Maraviroc, cenicriviroc Anti-fibrotic agents Angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, keratinocyte growth factor, pirfenidone Anti-coagulants Low-dose warfarin, dabigatran, aspirin, clopidogrel Anti-infective therapy CMV, HCV/HBV, HSV, EBV Anti-inflammatory agents Chloroquine, hydroxychloroquine NSAIDs (aspirin, mesalamine, COX-2 inhibitors) Statins Minocycline Methotrexate Thalidomide, lenalidomide, pentoxifylline Anti-aging Sirolimus, caloric restriction, omega-3 fatty acids, diet, exercise Slide: Developmental Considerations for Reducing Persistent Immune Activation in HIV There are a number of steps in the processes of underlying persistent immune activation that can serve as therapeutic targets. This table lists therapeutic interventions in development, which include biologics, drugs that may reduce microbial translocation, chemokine receptor inhibitors, anti-fibrotic agents, anti-coagulants, anti-infective therapies, and anti-aging strategies. It seems likely that combination approaches will be necessary to reduce persistent immune activation.1 Reference Douek DC. Immune activation, HIV persistence, and the cure. Top Antivir Med. 2013;21: Douek DC. Top Antivir Med. 2013;21:

65 Selected Results for Immunotherapy for HIVResponses Promoting immune responses IL-2 during ART treatment interruptions No evidence of improved HIV-1 specific immunity IL-7 in ART-treated patients Increases in CD4+ T cell numbers IL-21 in SIV-infected Rhesus macaques Increased CD8+ T-cell function and Th I7 cells Reduction in bacterial translocation and immune activation Immunomodulatory drugs Enhanced T-cell responses to dendritic cells (gag or NEF mRNA) Correcting immune activation and dysfunction Aspirin Reductions in CD38, HLA-DR, and sCD14 COX-2 inhibitor (celecoxib) Reduced expression of CD38 and inflammatory markers Anti-PD-1 Increased cytokine expression in HIV in vitro Anti-CLTA4 in SIV-infected macaques Decreased TGF-β, IDO, and viral RNA Maraviroc Reduction in CD38 HLA-DA expressing CD4+ T cells Normalized CD8+ T-cell skewing Symbiotic plus dietary fiber No alteration in bacterial translocation Prebiotic and probiotic Reduced bacterial DNA, CD4+ T cells, and IL-6 Slide: Selected Results for Immunotherapy for HIV There are numerous immunotherapeutic approaches under investigation. This table provides a brief overview of selected results.1-12 References INSIGHT-ESPRIT Study Group. Interleukin-2 therapy in patients with HIV infection. N Engl J Med. 2009;361: Pallikkuth S, Rogers K, Villinger F, et al. Interleukin-21 administration to rhesus macaques chronically infected with simian immunodeficiency virus increases cytotoxic effector molecules in T cells and NK cells and enhances B cell function without increasing immune activation or viral replication. Vaccine. 2011;29: Pallikkuth S, Micci L, Ende ZS, et al. Maintenance of intestinal Th17 cells and reduced microbial translocation in SIV-infected rhesus macaques treated with interleukin (IL)-21. PLoS Pathog. 2013;9:e Lévy Y, Sereti I, Tambussi G, et al. Effects of recombinant human interleukin 7 on T-cell recovery and thymic output in HIV-infected patients receiving antiretroviral therapy: results of a phase I/IIa randomized, placebo-controlled, multicenter study. Clin Infect Dis. 2012;55: De Keersmaecker B, Allard SD, Lacor P, et al. Expansion of polyfunctional HIV-specific T cells upon stimulation with mRNA electroporated dendritic cells in the presence of immunomodulatory drugs. Virology. 2012;86: O’Brien M, Montenont E, Hu L, et al. Aspirin attenuates platelet activation and immune activation in HIV-1-infected subjects on antiretroviral therapy: a pilot study. J Acquir Immune Defic Syndr. 2013;63: Pettersen FO, Torheim EA, Dahm AE, et al. An exploratory trial of cyclooxygenase type 2 inhibitor in HIV-1 infection: downregulated immune activation and improved T cell-dependent vaccine responses. J Virol. 2011;85: Porichis F, Hart MG, Zupkosky J, et al. Differential impact of PD-1 and/or IL-10 blockade on HIV-1-specific CD4 T cell and antigen-presenting cell functions. J Virol. 2013;88: Leng Q, Bentwich Z, Magen E, et al. CTLA-4 upregulation during HIV infection: association with anergy and possible target for therapeutic intervention. AIDS. 2002;16: Westrop SJ, Moyle G, Jackson A, et al. CCR5 antagonism impacts vaccination response and immune profile in HIV-1 infection. Mol Med. 2012;18: Schunter M, Chu H, Hayes TL, et al. Randomized pilot trial of a synbiotic dietary supplement in chronic HIV-1 infection. BMC Complement Altern Med. 2012;12:84. González-Hernández LA, Jave-Suarez LF, Fafutis-Morris M, et al. Synbiotic therapy decreases microbial translocation and inflammation and improves immunological status in HIV-infected patients: a double-blind randomized controlled pilot trial. Nutr J. 2012;11:90. INSIGHT-ESPRIT Study Group, et al. N Engl J Med. 2009;361: ; Pallikkuth S, et al. Vaccine. 2011;29: ; Pallikkuth S, et al. PLoS Pathog. 2013;9:e ; Levy Y, et al. Clin Infect Dis. 2012;55: ; De Keersmaecker B, et al. Virology. 2012;86: ; O’Brien M, et al. JAIDS. 2013;63: ; Pettersen FO, et al. J Virol. 2011;85: ; Porichis F, et al. J Virol. 2014;88: ; Leng Q, et al. AIDS. 2002;16: ; Westrop SJ, et al. Mol Med. 2012;18: ; Schunter M, et al. BMC Complement Altern Med. 2012;12:84; Gonzalez-Hernandez LA, et al. Nutr J. 2012;11:90.

66 Selected Therapeutic Vaccines Under InvestigationViral vector vaccines DNA vaccines Dendritic cell vaccines Subunit vaccines Slide: Selected Therapeutic Vaccines Under Investigation Viral vector vaccines, DNA vaccines, dendritic cell vaccines, and subunit vaccines.

67 Immunotherapy and Therapeutic Vaccination for HIVEnhance the Immune Response Gamma Chain Cytokines Immunomodulatory Agents Aspirin Immunomodulatory + Subunit Vaccine ? Cytokine + DNA Vaccine Slide: Immunotherapy and Therapeutic Vaccination for HIV Immunotherapy to enhance immune responses or inhibit immune dysfunction should be studied alongside therapeutic vaccination to develop a functional cure consisting of controlled immune responses and repressed viral replication. Inhibit Immune Dysfunction Therapeutic Vaccines ? Anti-CLA-4 Anti-PD1 Celecoxib Probiotics Viral vectors DNA Dendritic cells Peptide ?

68 Program Overview Strategies in HIV cure researchBenefits of ART and search for an HIV cure Biologic considerations in retroviral persistence Defining a cure Strategies in HIV cure research Induce HIV expression from latently infected cells Hematopoietic stem cell transplantation Genetically modified CD4 T cells Immune-based interventions Broadly neutralizing antibodies Immediate initiation of ART Combination approaches Challenges and future perspectives Slide: Program Overview

69 Antibody Responses to Envelope Glycoproteins in HIV InfectionAntibody responses to Env proteins Non-neutralizing Bind to Env epitopes not on Env trimer Highly strain-specific neutralization Bind to variable loops/other regions of gp120, Env trimers (autologous) Often leads to neutralization escape Broadly neutralizing Breadth of coverage (neutralize the majority of HIV-1 strains) Maximize potency (avoid neutralization escape and kill infected cells, ADCC or other Fc- mediated effector functions) Neutralizing Epitopes on Env Trimer ADCC: antibody-dependent cellular cytotoxicity. Figure: Peter Kwong, Jonathan Stuckley. Burton DB, et al. Nature Immunol. 2015;16: Mascola JR, et al. Immunol Rev. 2013;254:

70 Broadly Neutralizing AntibodiesAdvantages Do not interfere with DNA replication Long in vivo effect (potentially months) Many licensed monoclonal antibodies Block HIV entry to cell, different mechanism than current ART Limitations/challenges Each monoclonal antibody has gap in coverage (5% to 50%) Combination of 2 monoclonal antibodies: some viruses are only sensitive to 1 of the 2 monoclonal antibodies Parenteral route of administration

71 Week After AAV InoculationAAV-Expressed eCD4-Ig: Impact on Multiple SHIV-AD8 Challenges in Rhesus Macaques eCD4-Ig Fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide Binds avidly and cooperatively to the HIV-1 envelope glycoprotein; much more potent than existing neutralizing antibiody AAV vectors can deliver antibodies that prevent or treat HIV-1 infection Rhesus macaques (n=4) Inoculated with AAV1 that generates eCD4-Ig 6 SHIV-AD8 challenges (up to 16x the animal infectious dose) Results AAV-rh-eCD4-Ig protected all 4 monkeys for at least 1 year All 4 controls were infected Promising preliminary results Could potentially be adapted for vaccine or therapeutic purposes Viral RNA Levels SHIV-AD8 (pg) 2 20 200 400 400 800 108 107 106 105 104 103 102 101 Controls (n=4) AAV-eCD4-Ig (n=4) Viral RNA (copies/mL) Slide: AAV-Expressed eCD4-Ig: Impact on Multiple SHIV-AD8 Challenges in Rhesus Macaques eCD4-Ig is a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide that binds avidly and cooperatively to the HIV-1 envelope glycoprotein; and is much more potent than existing neutralizing antibody. AAV vectors can deliver antibodies that prevent or treat HIV-1 infection.1,2 Farzan and colleagues characterized eCD4-Ig and assessed the AAV delivery of eCD4-Ig as a means of stably protecting individuals from a new HIV-1 infection. Rhesus macaques inoculated with an AAV vector expressed µg/mL of fully functional rhesus eCD4-Ig over 60 weeks, and these macaques were protected from multiple challenges with SHIV-AD8 that efficiently infected control macaques. Moreover, eCD4-Ig was markedly less immunogenic than any of 4 well-characterized bNAbs.1,2 These promising preliminary results could potentially be adapted for vaccine or therapeutic purposes.1,2 References Farzan M. AAV-expressed eCD4-Ig protects rhesus macaques from multiple SHIV-AD8 challenges. Program and abstracts from the 22nd Conference on Retroviruses and Opportunistic Infection; February 23-26, 2015; Seattle, WA. Abstract 163. Gardner MR, Kattenhorn LM, Kondur HR, et al. AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges. Nature. 2015;519:87-91. Week After AAV Inoculation AAV: adeno-associated virus. Farzan M. 22nd CROI. Seattle, Abstract 163. Gardner MR, et al. Nature. 2015;519:87-91.

72 HIV RNA Levels (n=8, none on ART) Viral RNA (log copies/mL)Suppression of HIV RNA in HIV-Infected Patients by Broadly Neutralizing Antibody 3BNC117 Open-label phase 1 study, single infusion of 3BNC117 1, 3, 10, 30 mg/kg HIV-infected (2/17 on ART) Mean CD4: 655 cells/mm3 HIV RNA: 9420 copies/mL 3BNC mg/kg (n=8) Time to lowest level of viremia Median 7 days HIV RNA decline Range: log10 copies/mL Significantly reduced for 28 days HIV RNA did not return to day 0 pre- infusion levels in 4 of 8 3BNC117 Single Infusion: HIV RNA Levels (n=8, none on ART) Viral RNA (log copies/mL) Days After Infusion Caskey M, et al. Nature. 2015;522:

73 Updated Citation ACTG A5340: Impact of VRC01 on Viral Kinetics After Analytic Treatment Interruption Open label trial (n=14 males) On ART (HIV RNA <50 copies/mL for >6 months and CD4 >400 cells/mm3 VRC01 infusions: 40 mg/kg, q3 weeks ATI 1 week after VRC01 initiation, ART resumed after confirmed viral rebound (HIV RNA >1000 copies/mL) Results Safe, well-tolerated, and modest delay in return of viremia (versus historical controls) Does not maintain viral suppression in the majority of participants Future studies needed Efficacy of combination bNAbs, baseline susceptibility to neutralization, and in vivo mechanism of action HIV-1 RNA After ATI Slide: ACTG A5340: Impact of VRC01 on Viral Kinetics After Analytic Treatment Interruption Reference Bar KJ, Sneller MC, Harrison LJ, et al. Effect of HIV antibody VRC01 on viral rebound after treatment interruption. N Engl J Med. 2016;375: HIV RNA (copies/mL) Weeks After ATI Bar KJ, et al. N Engl J Med. 2016;375:

74 Days After ART InterruptionUpdated Citation VRC01 Infusion and HIV Rebound After ART Interruption in Patients With Chronic HIV Infection HIV-1 RNA After ATI Plasma VRC01 at 1st viremia: 142 to 583 ug/mL HIV RNA (copies/mL) Slide: VRC01 Infusion and HIV Rebound After ART Interruption in Patients With Chronic HIV Infection Bar and colleague conducted a single-arm, open-label study to evaluate the effect of VRC01 infusion on plasma viral rebound following discontinuation of ART in HIV-infected patients who initiated ART during the chronic phase of HIV infection and who suppressed plasma viremia >3 years with CD4 count >450 cells/mm3 at enrollment.1 10 patients received VRC01 (40 mg/kg) 3 days prior to and 14 and 28 days following interruption of ART, and monthly thereafter for up to 6 months. Mean duration of ART was 10.6 years. Mean baseline CD4 and CD8 counts: 796 and 768 cells/mm3, respectively. Multiple infusions of VRC01 were safe and well tolerated. ART reinitiation (any of the following criteria): >30% decline in baseline CD4 cell count; absolute CD4 cell count <350 cells/mm3; a sustained (>4 weeks) HIV RNA >1000 copies/mL; any HIV-related symptoms; or pregnancy. All 10 patients experienced plasma viral rebound (HIV RNA >40 copies/mL) between 11 and 86 days (median 39) following cessation of ART. The slide shows the 9 patients who subsequently reinitiated ART per protocol.1 While multiple infusions of VRC01 were safe and well-tolerated, the majority of patients experienced plasma viral rebound despite adequate levels of antibody in plasma. Therefore, therapeutic strategies involving passive transfer of broad neutralizing antibodies may require a combination of antibodies and/or resistance prescreening to achieve sustained virologic control in HIV-infected patients upon withdrawal of ART.1 Reference Bar KJ, Sneller MC, Harrison LJ, et al. Effect of HIV antibody VRC01 on viral rebound after treatment interruption. N Engl J Med. 2016;375: Days After ART Interruption VRC01 (40 mg/kg) 3 days prior to and 14 and 28 days following ART interruption (monthly thereafter up to 6 months) (n=10). 10/10 experienced HIV rebound; 9 reinitiated ART per protocol (either >30% decline in baseline CD4 cell count; absolute CD4 cell count <350 cells/mm3; a sustained [>4 weeks] HIV RNA >1000 copies/mL; any HIV-related symptoms; or pregnancy). Bar KJ, et al. N Engl J Med. 2016;375:

75 Clinical Potential of Broadly Neutralizing AntibodiesHelp guide vaccine design and therapeutic strategies for HIV Prevention High-risk young adults High-risk MSMs Discordant couples Breastfeeding infants Treatment Acute affect on viremia Treatment interruption/sparing Impact on viral reservoir Combined with ART (functional cure)

76 Program Overview Strategies in HIV cure researchBenefits of ART and search for an HIV cure Biologic considerations in retroviral persistence Defining a cure Strategies in HIV cure research Induce HIV expression from latently infected cells Hematopoietic stem cell transplantation Genetically modified CD4 T cells Immune-based interventions Broadly neutralizing antibodies Immediate initiation of ART Combination approaches Challenges and future perspectives Slide: Program Overview

77 Patient CharacteristicsVISCONTI Study: Post-Treatment HIV-1 Controllers Following Interruption of Early ART ART initiation during primary HIV infection (n=14) PI + 2 NRTIs (n=4); NNRTI + 2 NRTIs (n=1); or 3 NRTIs (n=2) PI + 2 or 3 NRTIs → 3 NRTIs (n=5) PI + 2 NRTIs → NNRTI + NRTI (n=1) 2 NRTIs → PI + 2 NRTIs (n=1) Post-treatment controllers versus spontaneous HIV controllers (PRIMO cohort) Higher HIV RNA levels and lower CD4 counts during primary HIV infection Time to HIV RNA undetectable: 3 months Following ART interruption Viral control persisted for a median of 89 months and CD4 T-cell counts remained stable Remained HIV RNA undetectable (n=8) Patient Characteristics Patients (n=14) Male (number) 10 Year of diagnosis* 1999 Fiebig ART initiation* V Symptomatic primary HIV infection (number) 12 Time on ART (months)* 36.5 Time since ART interruption (months)* 89 CD4 (cells/µL) First ART discontinuation Last 502 927 837 Last measurement HIV DNA (copies/106 PBMC) HIV RNA (copies/mL) 51.5 <20 Slide: VISCONTI Study: Post-Treatment HIV-1 Controllers Following Interruption of Early ART The VISCONTI study (Viro-Immunological Sustained CONtrol after Treatment Interruption) investigated whether the characteristics of 14 post-treatment controllers were similar to those observed in HIV controllers that spontaneously control HIV replication.1 ART duration after primary infection was 3 years, and after treatment interruption, post-treatment controllers presented sustained control for a median of 7 years.1 Reference Sáez-Cirión A, Bacchus C, Hocqueloux L, et al. Post-treatment HIV-1 controllers with a long-term virological remission after the interruption of early initiated antiretroviral therapy ANRS VISCONTI Study. PLoS Pathog. 2013;9:e *Median. Sáez-Cirión A, et al. PLoS Pathog. 2013;9:e

78 VISCONTI Study: HIV RNA and CD4 Post-Treatment HIV-1 Controllers Following Interruption of Early ARTSlide: VISCONTI Study: HIV RNA and CD4 Post-Treatment HIV-1 Controllers Following Interruption of Early ART Long-term control of viremia and stable CD4+ T-cell counts in 14 patients after interruption of ART initiated in primary HIV-1 infection. CD4+ T-cell counts and plasma HIV-1 RNA viral loads during the follow-up after PHI diagnosis in the 14 PTCs included in the study.1 Reference Sáez-Cirión A, Bacchus C, Hocqueloux L, et al. Post-treatment HIV-1 controllers with a long-term virological remission after the interruption of early initiated antiretroviral therapy ANRS VISCONTI Study. PLoS Pathog. 2013;9:e ● CD4 counts ● Plasma HIV RNA levels Gray shaded areas: time on ART Sáez-Cirión A, et al. PLoS Pathog. 2013;9:e

79 VISCONTI Study: Contribution of Long-Lived Resting CD4 T Cells to the HIV ReservoirHIV DNA Levels in CD4 Subsets CD4 Subset Contribution Median Below threshold of detection Detectable Median Cell-Associated HIV DNA (log10 copies/106 cells) Contribution to Resting HIV Reservoir (%) Slide: VISCONTI Study: Contribution of Long-Lived Resting CD4 T Cells to the HIV Reservoir There was weak contribution of long-lived resting CD4+ T cells to the HIV reservoir in the post-treatment controllers.1 Reference Sáez-Cirión A, Bacchus C, Hocqueloux L, et al. Post-treatment HIV-1 controllers with a long-term virological remission after the interruption of early initiated antiretroviral therapy ANRS VISCONTI Study. PLoS Pathog. 2013;9:e P=0.007 P=0.003 P=0.003 TN (Resting Naïve) TCM (Central Memory) TTM (Transitional Memory) TEM (Effector Memory) TN (Resting Naïve) TCM (Central Memory) TTM (Transitional Memory) TEM (Effector Memory) n=11 post-treatment HIV-1 controllers. Sáez-Cirión A, et al. PLoS Pathog. 2013;9:e

80 VISCONTI Study: Contribution of Long-Lived Resting CD4 T Cells to the HIV ReservoirPost-treatment HIV controllers Achieved control of infection through mechanisms that are, at least in part, different from those commonly observed in spontaneous HIV controllers Capacity to control infection is likely related to early ART In some cases, post-treatment HIV controllers were able to maintain (and in some cases further reduce) a weak viral reservoir after ART interruption Probability of maintaining viral control at 24 months post-early ART interruption: 15% (French Hospital Database for HIV [n=3538; ]) Higher than what is expected with spontaneous HIV controllers Slide: VISCONTI Study: Contribution of Long-Lived Resting CD4 T Cells to the HIV Reservoir Post-treatment HIV controllers achieved control of infection through mechanisms that are, at least in part, different from those commonly observed in spontaneous HIV controllers. The capacity to control infection is likely related to early ART.1 In some cases, post-treatment HIV controllers were able to maintain (and in some cases further reduce) a weak viral reservoir after ART interruption.1 The probability of maintaining viral control at 24 months post-early ART interruption was 15% (French Hospital Database for HIV [n=3538; ]) and higher than what is expected with spontaneous HIV controllers.1 Reference Sáez-Cirión A, Bacchus C, Hocqueloux L, et al. Post-treatment HIV-1 controllers with a long-term virological remission after the interruption of early initiated antiretroviral therapy ANRS VISCONTI Study. PLoS Pathog. 2013;9:e Sáez-Cirión A, et al. PLoS Pathog. 2013;9:e

81 Mississippi Baby: Very Early ART and Initial Control of HIV InfectionInfant born by spontaneous vaginal delivery at 35 weeks’ gestation Mother did not receive prenatal care, found to be HIV positive during labor (confirmed later by Western blot) Delivery completed before ART prophylaxis was administered ART started for infant at 30 hours of age Zidovudine + lamivudine + nevirapine (30 hours to 10 days of age) Zidovudine + lamivudine + lopinavir/r (11 days to 18 months of age) Child lost to follow-up from 18 to 23 months of age (mother reports ART discontinued at 18 months of age) Month 23 and 24: undetectable HIV RNA Month 24: HIV DNA PCR negative Mother/Infant Characteristics Mother HIV RNA (copies/mL) 24 hours after delivery 26 months after delivery CD4 (cells/mm3) 14 days after delivery 2423 6762 644 513 Infant HIV DNA at 30 hours of age 31 hours of age 29 days of age CD4 percentage at 8 days of age (%) Positive 19,812 <48 69 Slide: Mississippi Baby: Very Early ART and Initial Control of HIV Infection An example suggesting that very early treatment may lead to HIV remission is the report by Persaud and colleagues on the Mississippi baby. This infant born to a seropositive mother started receiving ART 30 hours after birth. HIV-1 RNA was detectable at 31 hours, days 6, 11, and 199, and reached undetectable levels at day 29. ART was discontinued sometime between months 18 and 23.1 Reference Persaud D, Gay H, Ziemniak C, et al. Absence of detectable HIV-1 viremia after treatment cessation in an infant. N Engl J Med. 2013;369: Persaud D, et al. N Engl J Med. 2013;369:

82 Mississippi Baby: Initial Control and Persistence of HIV InfectionART 30 hours-10 days: ZDV+3TC+NVP 11 days to 18 months: ZDV+3TC+LPV/r Month 24 26 Proviral DNA (copies/106 cells) PBMCs Resting CD4+ T cells PBMCs enriched for activated CD4+ T cells Monocyte-derived adherent cells <2.7 <3.5 <2.2 37.6 4.2 <2.5 <2.6 <11.5 Residual viremia (copies/mL) 1 <2 Infectious virus recovery (IUPM) -- <0.05 HIV RNA (copies/mL) Slide: Mississippi Baby: Initial Control and Persistence of HIV Infection At 36 months of age and after therapy withdrawal, HIV-1 RNA proviral DNA and HIV-1 antibodies remained consistently undetectable or extremely low in blood and tissues.1 Reference Persaud D, Gay H, Ziemniak C, et al. Absence of detectable HIV-1 viremia after treatment cessation in an infant. N Engl J Med. 2013;369: ART Stopped Pharmacy Records Undetectable Maternal Report Age (days) Age (months) Persaud D, et al. N Engl J Med. 2013;369:

83 Mississippi Baby: Long-Term Follow-upPlasma HIV-1 RNA and proviral DNA had been negative (<1.6 copies/mL) for 40 months HIV RNA rebound 27 months post-ART interruption HIV RNA 16,750 copies/mL Last sample obtained 4 weeks prior to rebound was positive for 2 long- term terminal repeat circles Single-copy assay: 9.9 copies/mL (retrospective testing) Rebound associated with seroconversion Western blot positive at time of rebound Slide: Mississippi Baby: Long-Term Follow-up Plasma HIV-1 RNA and proviral DNA had been negative (<1.6 copies/mL) for 40 months. However, HIV RNA rebound occurred 27 months post-ART interruption (HIV RNA 16,750 copies/mL).1 The last sample obtained 4 weeks prior to rebound was positive for 2 long-term terminal repeat circles (single-copy assay: 9.9 copies/mL via retrospective testing).1 Viral rebound was associated with seroconversion, with Western blot positive at time of rebound.1 Reference Luzuriaga K, Gay H, Ziemniak C, et al. Viremic relapse after HIV-1 remission in a perinatally infected child. N Engl J Med. 2015;372: Luzuriaga K, et al. N Engl J Med. 2015;372:

84 ANRS CO10 EPF Pediatric Cohort: HIV-1 Remission for >12 Years After Early ARTMother at delivery ddC since 13 weeks gestation HIV RNA 4.63 log10 copies/mL and CD4 81 cells/mm3 Infant (delivery 37 weeks, 5 days of gestation) At birth Zidovudine prophylaxis, HIV RNA undetectable at day 3 HIV DNA undetectable at days 3 and 14, detected at week 4 Week 6: zidovudine interrupted, HIV RNA rose sharply to 2.17 million copies/mL Month 3: initiate ART (zidovudine, didanosine, lamivudine, ritonavir) Loss to follow-up between 5.8 and 6.8 years ART interrupted by mother at approximately year 6.5 Remains HIV RNA undetectable after returning to care Unknown if HIV-1 remission in this child represents early treatment translating into long-term control or if long-term control is related to other factors Slide: ANRS CO10 EPF Pediatric Cohort: HIV-1 Remission for >12 Years After Early ART Frange and colleagues presented a case of a pediatric patient in HIV-1 remission for >12 years after early ART. At the time of delivery, the mother had received ddC since 13 weeks gestation and had an HIV RNA 4.63 log10 copies/mL and CD4 81 cells/mm3.1 The infant was delivered 37 weeks, 5 days of gestation. At birth, the infant received zidovudine prophylaxis and achieved undetectable HIV RNA levels at day 3 and HIV DNA undetectable levels at days 3 and 14, detected at week 4. At week 6, zidovudine prophylaxis was interrupted which led to a sharp increase in HIV RNA to 2.17 million copies/mL at month 3. ART was initiated (zidovudine, didanosine, lamivudine, ritonavir) and the child remained on therapy until lost to follow-up (between 5.8 and 6.8 years). A few months prior to returning the child to care, the mother noted she stopped the child’s ART. The child has remained HIV RNA undetectable after returning to care.1 It is unknown if HIV-1 remission in this child represents early treatment translating into long-term control or if long-term control is related to other factors.1 Reference Frange P, Faye A, Avettand-Fenoël V, et al. HIV-1 virological remission lasting more than 12 years after interruption of early antiretroviral therapy in a perinatally infected teenager enrolled in the French ANRS EPF-CO10 paediatric cohort: a case report. Lancet HIIV. 2016;3:e49-e54. Frange P, et al. Lancet HIV. 2016;3:e49-e54.

85 Seeding of the Viral Reservoir Prior to SIV Viremia in Rhesus MonkeysOld-world rhesus monkeys infected with SIVmac251 by intrarectal inoculation (n=20) ART initiated on days 3, 7, 10, and 14 after infection Tenofovir DF, emtricitabine, and dolutegravir daily, sc injection for 24 weeks Viral kinetics after ART ART day 3: blocked emergence of viral RNA and proviral DNA in peripheral blood; substantially reduced proviral levels in lymph nodes and GI mucosa ART days 7, 10, and 14: virus growth interrupted and achieved plasma viral RNA <50 copies/mL within 3-4 weeks Slide: Seeding of the Viral Reservoir Prior to SIV Viremia in Rhesus Monkeys Whitney and colleagues evaluated the impact of early ART on the viral reservoir by initiating suppressive ART at various time points after mucosal SIV infection of rhesus monkeys.1 ART initiated on days 3, 7, 10, and 14 after infection. Tenofovir DF, emtricitabine, and dolutegravir daily, sc injection for 24 weeks. Viral kinetics after ART:1 ART day 3: blocked emergence of viral RNA and proviral DNA in peripheral blood; substantially reduced proviral levels in lymph nodes and GI mucosa. ART days 7, 10, and 14: virus growth interrupted and achieved plasma viral RNA <50 copies/mL within 3-4 weeks. Reference Whitney JB, Hill AL, Sanisetty S, et al. Rapid seeding of the viral reservoir prior to SIV viraemia in rhesus monkeys. Nature. 2014;512:74-77. Whitney JB, et al. Nature. 2014;512:74-77.

86 Seeding of the Viral Reservoir Prior to SIV Viremia in Rhesus MonkeysProviral DNA (log10 copies/106 cells) Plasma SIV RNA (log10 copies/mL) ART on Day 3 Peripheral Blood MCs Lymph Node MCs GI MCs ART on Day 7 (Similar results with ART on days 10 and 14) Slide: Seeding of the Viral Reservoir Prior to SIV Viremia in Rhesus Monkeys Treatment with ART on day 3 blocked the emergence of viral RNA and proviral DNA in peripheral blood and also substantially reduced levels of proviral DNA in lymph nodes and gastrointestinal mucosa as compared with treatment at later time points.1 Reference Whitney JB, Hill AL, Sanisetty S, et al. Rapid seeding of the viral reservoir prior to SIV viraemia in rhesus monkeys. Nature. 2014;512:74-77. Time After SIV Infection (Days) MCs: mononuclear cells. Whitney JB, et al. Nature. 2014;512:74-77.

87 Seeding of the Viral Reservoir Prior to SIV Viremia in Rhesus MonkeysART discontinuation after 24 weeks All monkeys experienced viral rebound ART on day 3 exhibited a delayed viral rebound compared with ART on later days (P<0.004) Time to viral rebound correlated with Total viremia during acute infection Proviral DNA at time of ART discontinuation Conclusions Viral reservoir is seeded rapidly after intrarectal SIV infection in rhesus monkeys, occurring during the eclipse phase and before detectable viremia Viral Rebound After ART Discontinuation Median Days to Viral Rebound Slide: Seeding of the Viral Reservoir Prior to SIV Viremia in Rhesus Monkeys After discontinuation of ART following 24 weeks of fully suppressive therapy, virus rebounded in all animals, although the monkeys that were treated on day 3 exhibited a delayed viral rebound as compared with those treated on days 7, 10, and 14. The time to viral rebound correlated with total viremia during acute infection and with proviral DNA at the time of ART discontinuation.1 These data demonstrate that the viral reservoir is seeded rapidly after intrarectal SIV infection of rhesus monkeys, during the eclipse phase, and before detectable viremia. This early seeding of the refractory viral reservoir raises important challenges for HIV-1 eradication strategies.1 Reference Whitney JB, Hill AL, Sanisetty S, et al. Rapid seeding of the viral reservoir prior to SIV viraemia in rhesus monkeys. Nature. 2014;512:74-77. Day 3 Day 7 Day 10 Day 14 Day of ART Initiation After SIV Infection Whitney JB, et al. Nature. 2014;512:74-77.

88 Timing of ART Initiation in Primary Infection and HIV Reservoirs: Key LessonsHIV persistence Established early in primary HIV infection in long-lived memory CD4+ T cells and not eliminated by immune surveillance or ART ART initiated during primary HIV infection Can reduce the HIV reservoir size to a greater extent than when treatment is given in chronic HIV Infection persists in memory CD4+ T cells in most early treated individuals Treatment initiated in the earliest primary HIV infection stage (Fiebig I) May protect central memory CD4+ T cells from infection and skew the distribution of latently infected cells to the shorter-lived memory CD4+ T cells (eg, elite controllers and post-treatment controllers) Containing HIV reservoir seeding with ART in primary HIV infection (before use of other interventions aimed at eliminating all latently infected cells) May be a first critical step in achieving HIV remission Slide: Timing of ART Initiation in Primary Infection and HIV Reservoirs: Key Lessons HIV persistence is established early in primary HIV infection in long-lived memory CD4+ T cells and not eliminated by immune surveillance or ART.1 ART initiated during primary HIV infection can reduce the HIV reservoir size to a greater extent than when treatment is given in chronic HIV. Infection persists in memory CD4+ T cells in most early treated individuals.1 Treatment initiated in the earliest primary HIV infection stage (Fiebig I) may protect central memory CD4+ T cells from infection and skew the distribution of latently infected cells to the shorter-lived memory CD4+ T cells (eg, elite controllers and post-treatment controllers).1 The containment of the HIV reservoir seeding with ART in primary HIV infection (before use of other interventions aimed at eliminating all latently infected cells) may be a first critical step in achieving HIV remission.1 Reference Ananworanich J, Dubé K, Chomont N. et al. How does the timing of antiretroviral therapy initiation in acute infection affect HIV reservoirs? Curr Opin HIV AIDS. 2015;10:18-28. Ananworanich J, et al. Curr Opin HIV AIDS. 2015;10:18-28.

89 Program Overview Strategies in HIV cure researchBenefits of ART and search for an HIV cure Biologic considerations in retroviral persistence Defining a cure Strategies in HIV cure research Induce HIV expression from latently infected cells Hematopoietic stem cell transplantation Genetically modified CD4 T cells Immune-based interventions Broadly neutralizing antibodies Immediate initiation of ART Combination approaches Challenges and future perspectives Slide: Program Overview

90 HIV Cure Strategies Currently in Human TrialsMinimize Reservoir Early Treatment to Limit Reservoir ART Broadly neutralizing antibodies Shock Reactivate Latently Infected Cells HDAC inhibition Bromodomain and extra-terminal inhibition Activate toll-like receptors Activate protein kinase Kill Viral Clearance by Immune System Broadly neutralizing antibodies Therapeutic vaccines Anti-programmed death (PD) 1 Anti-PD ligand 1 Slide: HIV Cure Strategies Currently in Human Trials HIV-Resistant Cells Transfuse Cells Without CCR5 Gene Gene-editing therapy Bone marrow or cord blood transplantation

91 Program Overview Challenges and future perspectivesBenefits of ART and search for an HIV cure Biologic considerations in retroviral persistence Defining a cure Strategies in HIV cure research Induce HIV expression from latently infected cells Hematopoietic stem cell transplantation Genetically modified CD4 T cells Immune-based interventions Broadly neutralizing antibodies Immediate initiation of ART Combination approaches Challenges and future perspectives Slide: Program Overview

92 Clinical Trials for HIV Remission and CureIt is essential that promising leads be advanced rapidly into pilot human trials Capacity for phase 1, 2a/2b trials Assays to assess biological effects of interventions Prioritize combinations for testing in human trials Role for combinatorial testing in animal models? Analytical treatment interruptions Establishing parameters Safety Surrogate markers Slide: Clinical Trials for HIV Remission and Cure It is essential that promising leads be advanced rapidly into pilot human trials (capacity for phase 1, 2a/2b trials and assays to assess biological effects of interventions). Prioritize combinations for testing in human trials. Is there a role for combinatorial testing in animal models? Analytical treatment interruptions need to establish parameters, safety, and surrogate markers.

93 Unique Populations Newborns Acutely infected personsPatients on long-term suppressive ART HIV controllers Patients undergoing hematopoietic stem cell transplantation Slide: Unique Populations Newborns. Acutely infected persons. Patients on long-term suppressive ART. HIV controllers. Patients undergoing hematopoietic stem cell transplantation.

94 Towards a Cure for HIV: ConclusionsSuccessful approaches to SIV and HIV eradication CMV vector vaccine maintaining effector T-cell response Bone marrow transplantation Early ART Currently the most effective approach to limit the HIV reservoir HIV remission and cure will require a combination of approaches to reduce the HIV reservoir and boost the immune response to HIV Slide: Towards a Cure for HIV: Conclusions Successful approaches to SIV and HIV eradication include CMV vector vaccine maintaining effector T-cell response and bone marrow transplantation. Early ART is currently the most effective approach to limit the HIV reservoir. HIV remission and cure will require a combination of approaches to reduce the HIV reservoir and boost the immune response.

95 Evaluation and Outcomes Measurement ProcessYou will receive an electronic initial evaluation to the address provided within 1 business day Reminder communications will be sent up to 5 days post lecture until the evaluation is completed Incomplete evaluations may preclude attendees from receiving their CME/CNE/CPE certificate & future communications about lectures in your area In addition, you will receive a long-term evaluation via 8 to 12 weeks after completing this course to measure competence, performance, and/or patient outcomes achieved as a result of your participation in this CME/CNE/CPE sponsored educational activity Slide: Evaluation and Outcomes Measurement Process (Please note: If you attended multiple Simply Speaking® lectures throughout the year, a separate initial and long-term evaluation will be sent to you for each lecture.)