1. Anti-HIV Antibody Responses Reflect the Quantifiable HIV Reservoir Size Sulggi Lee, M.D., Ph.D. Assistant Professor of Medicine University of California, San Francisco July 20, 2015

2. Chronic/Latent Viral Infections Lead to Prolonged Antibody Responses VZV Antibody Half-Life ~ 50 Years Amanna NEJM 2007

3. 0.0001 0.001 0.01 0.1 1 10 100 1000 10000 01234567 Time on HAART (years) Latently Infected Cells (Infectious Units per 10 6 cells) The Size of the HIV Reservoir is Also Stable During Long-Term ART Time to eradication > 73.4 years 0.00001 Siliciano Nat Med 2003

4. Anti-HIV Antibody Levels May Distinguish Individuals With Different HIV Reservoir Sizes Burbelo JID 2014 Antibody Levels Light Units (LU) Anti-gp41 Levels * * * *

5. Anti-HIV Antibody Levels May Be Sensitive Measures of the Reservoir Size Over Time Longitudinal data from Burbelo JID 2014 Anti –gp41 Levels Median Years of ART Antibody Levels Light Units (LU) Pre-ART 0

6. Hypothesis: Antibody Levels in Blood at Steady-State Reflect Total Body Reservoir Size Y Y Y Y Y Y Y Y Y Y Y Y Y CD4+ Primary Infection Establishment of Infection in Lymphoid Tissue Antibody Production

7. Current Issues in Measuring HIV Reservoir How to measure the success of HIV cure treatments if no scalable, precise assay of the reservoir? The HIV reservoir is much larger in tissue than in blood – A reliable measure that reflects the HIV reservoir in tissue? Most measures of the HIV reservoir are virologic assays, not cell-based assays measuring host response to virus Eriksson PLoS Pathogens 2013, Ho Cell 2013, Chun JID 2008, Yukl JID 2010

8. Study Design Aim: Determine whether anti-HIV antibody levels reflect the size of the latent reservoir Participants: 61 HIV+ individuals who – Initiated ART during chronic infection – Have been ART-suppressed for at least 3 years

9. P I V Q N I Q G Q M V H Q A I S P R T L N A W V K V V E E K A F S P E V I P M F S A L S E G A T P Q D L N T M L N T V G G H Q A A M Q M L K E T I N E E A A E W D R V H P V H A G P I A P G Q M R E P R G S D I A G T T S T L Q E Q I G W M T N N P P I P V G E I Y K R W I I L G L N K I V R M Y S P T S I L D I R Q G P K E P F R D Y V D R F Y K T L R A E Q A S Q E V K N W M T E T L L V Q N A N P D C K T I L K A L G P A A T L E E M M T A C Q G V G G P G H K A R V L * Luciferase One letter code for amino acids Y Y Y Y Y Y Y C C C C L L L L = linear epitope C= conformational epitopes Novel Luciferase Immunoprecipitation Systems (LIPS) Assay Detects Conformational Antibodies

10. Question: Are HIV Reservoir Measures Associated with Anti-HIV Antibody Levels? gp120 gp41 reverse transcriptase integrase protease matrix p24 12 Measures of the HIV Reservoir 7 Anti-HIV Antibody Levels Erikkson PLoS Pathogens 2013, Lewin J Virol 1999, Burbelo JID 2014 Permutation Testing To test the association between reservoir and antibody measures, adjusted for multiple comparisons Total HIV DNA CD4+ T cells (rtPCR) PBMCs (ddPCR) Resting CD4+ T cells (ddPCR) Integrated HIV DNA CD4+ T cells (rtPCR) PBMCs (aluPCR) Resting CD4+ T cells (aluPCR) 2-LTR HIV DNA CD4+ T cells (rtPCR) PBMCs (ddPCR) Resting CD4+ T cells (ddPCR) Unspliced HIV RNA CD4+ T cells (rtPCR) Plasma HIV RNA Plasma (rtPCR single copy assay) Infectious Units per Million Resting CD4+ T cells (VOA)

11. Study Participant Characteristics *Number (%) Median and Interquartile Range shown

12. Measures of the HIV DNA (Total, Integrated) are Correlated with Anti-HIV Antibody Levels gp120gp41RTINTPRMAp24 Total HIV DNA CD4+ T cells (rtPCR)0.16-0.030.160.180.170.11-0.08 PBMCs (ddPCR)0.440.480.300.40 0.230.16 Resting CD4+ T cells (ddPCR)0.510.280.620.570.600.540.29 Integrated HIV DNA CD4+ T cells (rtPCR)0.210.070.200.110.270.06 PBMCs (aluPCR)0.800.730.760.700.540.530.41 Resting CD4+ T cells (aluPCR)0.500.720.820.500.250.340.25

13. Measures of the “Active” or Replication-Competent Reservoir are Weakly Correlated with Anti-gp120 Levels gp120gp41RTINTPRMAp24 2-LTR HIV DNA CD4+ T cells (rtPCR)0.05-0.12-0.10 0.140.17-0.07 PBMCs (ddPCR)0.470.260.330.560.460.390.01 Resting CD4+ T cells (ddPCR)-0.050.140.090.010.020.09-0.14 Unspliced HIV RNA CD4+ T cells (rtPCR)0.400.150.090.120.050.070.10 Plasma HIV RNA Plasma (rtPCR SCA)-0.03-0.24-0.15-0.10-0.16-0.06-0.01 Infectious Units (Per 10 6 ) Resting CD4+ T cells (VOA)0.450.430.200.310.300.250.02

14. Overall, HIV Reservoir Measures Strongly Correlate with Anti-gp120, -gp41, and -RT Levels gp120gp41RTINTPRMAp24 All HIV Reservoir Measures Correlation (R)0.800.730.820.700.600.540.41 P 0.0090.040.0070.050.200.340.68 * Adjustment for age, nadir CD4+ T cell count, proximal CD4+ T cell count, years of ART suppression, pre-ART viral load did not significantly alter these results.

15. Conclusions We observed a strong association between measures of the latent HIV reservoir and anti-HIV antibody levels Overall, anti-gp120, -gp41, and -RT were most strongly correlated with HIV reservoir size Total and integrated HIV DNA were strongly associated with antibody levels against Env and Pol proteins Measures of the “active” (CA-US RNA, 2-LTR DNA) and replication-competent (VOA) reservoir were weakly associated with anti-gp120 levels

16. Remaining Questions Can viral RNA or proteins be produced in cells with defective proviruses? – And elicit a host immune response? Is there preferential B cell response? – Relative “importance” of Env > Pol > Gag antigens? Why are HIV CA-DNA and CA-RNA but not plasma RNA correlated with anti-HIV antibody levels? – Do antibody levels do a good job of quantifying the HIV tissue reservoir?

17. Can Anti-HIV Antibody Levels Monitor Response to HIV Cure Strategies? Antibody Levels Pre-TreatmentAntibody Levels Post-Treatment

18. Acknowledgements UCSF – Steven Deeks – Saunak Sen NIH/NIDCR – Peter Burbelo Johns Hopkins – Robert Siliciano – Janet Siliciano University of Melbourne – Sharon Lewin UCSD – Douglas Richman – Matthew Strain University of Pennsylvania – Una O’Doherty University of Sydney – Sarah Palmer University of Montreal – Nicolas Chomont – Remi Fromentin