1. HSV-2 is associated with HIV acquisition among both placebo & vaccine recipients in the Step Study Ruanne V Barnabas, MBChB DPhil HIV Vaccine Trials Network, Seattle 5 th IAS Conference on HIV Pathogensis, Treatment & Prevention 19-22 July 2009, Cape Town

2. Background: HSV-2-HIV synergy HSV-2: most frequent cause of GUD, majority subclinical HSV-2 and HIV –HSV-2 associated with increased risk of HIV acquisition 1 Men:RR=2.8 (95% CI: 2.1-3.7) Women:RR=3.4 (95% CI: 2.4-4.8) MSM:RR=1.6 (95% CI: 1.2-2.0) HSV-2 increases HIV VL (0.3-0.55log 10 ) 2,3 1 Glynnn JR, et al. (2009) Aids 2 Duffus W, et al (2005) IJ STDs & AIDS 3 Schacker T, et al (2002) JID

3. Background: Step study Step study –MRKAd5 HIV vaccine –3000 high-risk HIV uninfected men & women in 34 sites Primary outcome among 1,836 men 1 –No difference in HIV acquisition –No difference in viral load setpoint –Subgroup analyses: increased risk of HIV acquisition among vaccinated, uncircumcised men & vaccinated Ad5 exposed men 1 Buchbinder SP et al. (2008) Efficacy assessment of a cell-mediated immunity HIV-1 vaccine (the Step Study): a double-blind, randomised, placebo- controlled, test-of-concept trial. Lancet

4. Research questions 1.What is the impact of HSV-2 seropositivity on HIV acquisition? (Analysis in placebo recipients; N=922) 2.What is the impact of HSV-2 seropositivity on the effect of the vaccine on HIV acquisition? (Analysis in placebo and vaccine recipients) 3.What is the effect of HSV-2 seropositivity on HIV VL? (Analysis in HIV seroconverters; N=87) 4.What is the effect of HSV-2 seropositivity on time to ART initiation? (Analysis in HIV seroconverters; N=87)

5. Methods Laboratory methods –HSV: Western blot (UWDVL) 1 Statistical analysis –1,836 men, 88 HIV cases, infected prior to Oct 17 th, 2007 –Kaplan Meier curves: HSV-2 as a risk factor for HIV acquisition & time to ART initiation –Cox proportional hazards regression: HSV-2 as a risk factor for HIV acquisition & time to ART initiation (stepwise selection of risk behavior variables) –Linear regression models: HSV-2 as a predictor of early pre-ART VL 1 Ashley RL, et al (1988) Comparison of Western blot (immunoblot) and glycoprotein G- specific immunodot assay for detecting antibodies to HSV types 1 and 2 in human sera. J Clin Microbiol 26: 662-667.

6. Q1: Impact of baseline HSV-2 seropositivity on HIV acquisition among placebo recipients; Kaplan Meier curves: Baseline HSV-2 seropositivity increased acquisition of HIV among placebo recipients Events observed HSV-2 negative: 18 HSV-2 positive: 17 *Log-rank test: p= 0.019

7. Q1: Impact of baseline HSV-2 seropositivity on HIV acquisition Cox multivariate analysis results: HSV-2 seropositivity & age<30 increased risk of HIV acquisition among placebo recipients Risk factorHR95% HR CIp HSV-2 (seropositive)3.31.6 - 6.9<0.001 Age (<30 years)2.70.5 - 2.00.008 Region (N. America & Australia)2.90.7 - 12.90.16 Male circumcision (circumcised)2.50.7 - 8.70.15 Ad5 titer (>18)1.00.5 - 2.00.92 Unprotected insertive anal sex in the past 6 months at baseline 1.80.8 - 3.90.15 Unprotected receptive anal sex in the past 6 months at baseline 2.10.96 - 4.60.06

8. Q2: Impact of baseline HSV-2 seropositivity on HIV acquisition among placebo and vaccine recipients Kaplan Meier curves: HSV-2 increases risk of HIV acquisition among placebo & vaccine recipients Events observed HSV-2 negative: 50 HSV-2 positive: 36 *Log-rank test: p= 0.009

9. Q2: Impact of baseline HSV-2 seropositivity on effect of vaccine on HIV acquisition Cox Proportional Hazards Model Strong HSV-2 effect in univariate analysis: HR=1.7 (95% CI: 1.1- 2.7) Adjusted for demographic, pre-specified & risk behavior variables (significant when added in a stepwise manner) No significant interaction between HSV-2 & vaccine Strong interaction between circumcision & vaccine (p=0.013)

10. Q2: Impact of baseline HSV-2 seropositivity on HIV acquisition Cox results: HSV-2 positivity, N.America & Australia region, age<30, URAS £, UIAS £, Speed use in last 6 mnth^ & vaccine if uncirc’ed increased risk of HIV £ UI/RAS: Unprotected insertive/receptive anal sex; ^prior to infection or last visit; * Vaccine x Circumcision Interaction significant (p=0.013) Risk factorHR95% HR CIp HSV-2 (seropositive)2.21.4-3.5<0.001 Intervention (Vaccine)* Vaccine effect among uncircumcised Vaccine effect among circumcised 3.9 1.0 1.6 – 9.7 0.6 – 1.8 0.003 Male circumcision (circumcised) * Circ effect among placebo recipients Circ effect among vaccinees 2.1 0.5 0.8 – 5.4 0.3 – 1.1 0.140 Region (N. America & Australia)3.81.8 – 8.3<0.001 Age (<30 years)2.21.3 – 3.50.002 Ad5 titer (>18)1.20.8 – 2.00.380 Unprotected receptive anal sex1.91.2 – 3.10.008 Unprotected insertive anal sex1.81.1-3.00.016 Speed use within 6 mnth^3.21.8 - 5.8<0.001 UIAS with HIV-ve ptn within 6 mnth^0.60.3 – 1.00.064 URAS HIV unknown pnt within 6 mnth^1.60.9 – 2.60.1

11. Q3: Impact of baseline HSV-2 seropositivity on HIV VL (all volunteers) Non-significant increase in HIV VL among HSV-2 seropositive compared to HSV-2 seronegative volunteers Linear regression Adjusted for vaccine, circumcision status, age, race, Ad5 immunity, & region: 0.3 log 10 higher mean set point in HSV-2 seropositives (95% CI, 0.1 lower to 0.7 higher)

12. Q4: Impact of baseline HSV-2 seropositivity on time to ART initiation; Kaplan-Meier Curves: No significant association between HSV-2 & time to ART initiation among placebo cases Cox HR for ART initiation by HSV-2 status stratified for region and circumcision status: HR=1.4 (0.5-4.5) unadjusted HR=1.5 (0.4-5.5) adjusted for age, race & Ad5 Months since infection diagnosis Num. at risk (HSV-2 negative) 18 14 (1) 12 (1) 8 (1) 1 (1) 1 0 Num. at risk (HSV-2 positive) 16 11 (3) 11 4 (3) 2 0 0

13. Limitations Risk behavior data –Self-reported risk behavior potentially subject to social desirability bias &/or recall bias –HSV-2 may be marker for increased behavioral (as well as biological) risk for HIV acquisition VL, CD4 and ART initiation data are post- unblinding. Current analysis among men only – future analyses will look at HSV-2 & HIV among women, but power will be limited

14. Conclusions HSV-2 infection increased HIV acquisition more than 2 fold No significant interaction detected between HSV-2 & vaccine or circumcision HIV viral load was not higher among HSV-2 seropositive compared to HSV-2 seronegative seroconverters No significant association between HSV-2 serpositivity & HIV progression

15. Thank you! STEP Volunteers STEP Trial Sites Staff and Community Members University of Washington Diagnostic Virology Laboratory –Rhoda Ashley Morrow, Stacy Selke & Anne Cent SCHARP –Yunda Huang, Holly Janes, Maggie Wang, Alicia Sato, Liza Noonan, Peter Gilbert & Steve Self HVTN+ –Susan Buchbinder, Jonathan Fuchs, Karen Mark, Martin Casapia, Jorge Sanchez, Ann Duerr & Larry Corey Merck –Mike Robertson & Devan Mehrotra University of Washington –Judy Wasserheit HSV-2 manuscript team Step study sites

16. Q1: Risk factors for HSV-2 seropositivity at baseline Logistic regression (multivariate analysis): HSV-2 seropositivity associated with race/ethnicity, age>30, partners>4, UIAS & URAS Baseline risk factor *Over the last six months Odds ratio (95% CI) P Male circumcision (circumcised)0.7 (0.5 - 1.0)0.070 Race/Ethnicity: White (reference)1.0 Black2.0 (1.3 – 2.9)<0.001 Hispanic1.7 (1.1 – 2.5)0.012 Mestizo2.5 (1.6 – 4.0)<0.001 Multi-racial2.0 (1.0 – 3.8)0.052 Other1.1 (0.6 – 2.2)0.718 Region (North America and Australia)0.7 (0.4 – 1.1)0.095 Age (>30 years)0.3 (0.2 – 0.4)<0.001 Ad5 titer (>18)1.1 (0.9 – 1.5)0.233 Number of male partners (>4)1.6 (1.2 – 2.1)0.001 Unprotected insertive anal sex0.8 (0.6 – 0.99)0.038 Unprotected receptive anal sex1.5 (1.1 – 2.0)0.005