Preexposure prophylaxis (PrEP) with the oral antiretroviral. Original Research

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1 Annals of Internal Medicine Original Research Daily Oral Tenofovir and Emtricitabine Tenofovir Preexposure Prophylaxis Reduces Herpes Simplex Virus Type 2 Acquisition Among Heterosexual HIV-1 Uninfected Men and Women A Subgroup Analysis of a Randomized Trial Connie Celum, MD, MPH; Rhoda A. Morrow, PhD; Deborah Donnell, PhD; Ting Hong, MD, PhD; Craig W. Hendrix, MD, PhD; Katherine K. Thomas, MS; Kenneth H. Fife, MD, PhD; Edith Nakku-Joloba, MBChB, PhD; Andrew Mujugira, MBChB, MPH; and Jared M. Baeten, MD, PhD, for the Partners PrEP Study Team* Background: Daily oral preexposure prophylaxis (PrEP) using the antiretroviral tenofovir disoproxil fumarate (TDF) alone or in combination with emtricitabine (FTC TDF) reduces the risk for HIV-1 acquisition. Tenofovir has in vitro activity against herpes simplex virus type 2 (HSV-2). Objective: To assess the efficacy of daily oral PrEP with tenofovir and FTC TDF in the prevention of HSV-2 acquisition. Design: Subgroup analysis of data from a randomized, placebocontrolled trial with concealed allocation. (ClinicalTrials.gov: NCT ) Setting: Multiple sites in Kenya and Uganda. Participants: Heterosexual men and women who were seronegative for HIV-1 and HSV-2 and at high risk for HIV-1 acquisition due to having an HIV-1 infected partner. Measurements: HSV-2 seroconversion. Results: A total of 131 participants seroconverted to HSV-2 (79 of 1041 assigned to tenofovir or FTC TDF PrEP [HSV-2 incidence, 5.6 per 100 person-years] and 52 of 481 assigned to placebo [HSV-2 incidence, 7.7 per 100 person-years]). The hazard ratio (HR) for HSV-2 acquisition with daily oral PrEP was 0.70 (95% CI, 0.49 to 0.99; P 0.047) compared with placebo, and the absolute risk reduction was 2.1 per 100 person-years. Among the 1044 participants with HSV-2 infected partners, the HR for PrEP was 0.67 (CI, 0.46 to 0.98; P 0.038) compared with placebo, and the absolute risk reduction was 3.1 per 100 person-years. Limitation: Randomization was not stratified by HSV-2 status, and diagnostic tests to exclude participants with acute HSV-2 at baseline are not available. Conclusion: Daily oral tenofovir-based PrEP significantly reduced the risk for HSV-2 acquisition among heterosexual men and women. Modest protection against HSV-2 is an added benefit of HIV-1 prevention with oral tenofovir-based PrEP. Primary Funding Source: Bill & Melinda Gates Foundation. Ann Intern Med. 2014;161: doi: /m For author affiliations, see end of text. * For a list of the members of the Partners PrEP Study Team, see the Appendix (available at Preexposure prophylaxis (PrEP) with the oral antiretroviral tenofovir disoproxil fumarate (TDF) alone or in combination with emtricitabine (FTC TDF) has been shown to significantly reduce the risk for HIV-1 acquisition in 4 randomized, placebo-controlled clinical trials in high-risk populations (1 4). In addition to its potent activity as a nucleotide inhibitor of HIV-1 reverse transcriptase, tenofovir has anti herpes simplex virus type 2 (HSV-2) activity in vitro, although less than the related compounds cidofovir and adefovir (5). In a randomized trial of pericoital dosing of intravaginal 1% tenofovir gel, HSV-2 incidence was reduced by 51% compared with placebo (6 8) and vaginal tenofovir levels were associated with protection against HIV-1 and HSV-2 (9). These results were consistent with in vitro studies showing antiherpetic activity against HSV-2 isolates at concentrations of 10 to 200 g/ml (10), levels that are in the range of those achieved with vaginal dosing of tenofovir gel (11). However, whether tenofovir concentrations achieved through oral dosing are sufficient for HSV-2 protection in humans is unknown. The efficacy of daily oral tenofovir-based PrEP for HSV-2 protection has not been assessed in heterosexual populations. In vitro data have shown that high concentrations of tenofovir are required for HSV-2 inhibition, and high adherence to oral PrEP may be necessary to achieve such levels. We performed a secondary analysis of HSV-2 acquisition among participants who were initially seronegative for HIV-1 and HSV-2 in a randomized trial of PrEP for HIV-1 prevention that showed high efficacy for HIV-1 protection (2). METHODS Study Population and Procedures Between July 2008 and November 2010, a total of 4747 heterosexual HIV-1 serodiscordant couples were enrolled in a randomized trial of PrEP for HIV-1 prevention (the Partners PrEP Study) and followed at 9 research sites in Kenya and Uganda (2, 12). The primary aims of the study were to determine the efficacy and safety of PrEP with tenofovir and FTC TDF, each compared with placebo, for prevention of HIV-1 acquisition. Eligibility criteria for HIV-1 seronegative partners included normal renal function and not being pregnant or breastfeeding. At enrollment, HIV-1 seronegative partners were assigned in 2014 American College of Physicians 11

2 Original Research HSV-2 Protection With Tenofovir-Based Preexposure Prophylaxis Context Herpes simplex virus type 2 (HSV-2) infection is common worldwide, can lead to complications, and has been associated with an increased risk for HIV-1 infection. Contribution In a secondary analysis of a large randomized clinical trial testing the ability of tenofovir-based preexposure regimens to prevent HIV-1 infection, participants receiving tenofovir were less likely to acquire HSV-2 than those receiving placebo. Caution The trial was not originally designed to study HSV-2 acquisition. Implication Protection against HSV-2 acquisition may be an added benefit of using tenofovir for HIV-1 preexposure prophylaxis. The Editors a 1:1:1 ratio to once-daily oral tenofovir, FTC TDF, or placebo; investigators and participants were blinded to randomization assignments. Randomization was by fixedsize blocks and was stratified by site but not by HSV-2 serostatus. Participants who were HIV-1 seronegative had monthly visits for up to 36 months that included HIV-1 testing, dispensation of study medication, and standardized assessment of sexual behavior and clinical and laboratory safety. Study medication was withheld from women who became pregnant or began breastfeeding. All participants received a comprehensive package of HIV-1 prevention services, including individual and couples risk-reduction counseling, screening for and treatment of sexually transmitted infections, condoms, and referral for medical male circumcision and postexposure prophylaxis according to national policies. Partners infected with HIV-1 were not eligible for antiretroviral therapy (ART) under the national policies of Kenya and Uganda at the time of their enrollment in the trial; they were followed in parallel with their HIV-1 uninfected partners and referred for initiation of ART when they became eligible. Participants with HIV-1 seroconversion were referred for care, but the standard of care in Kenya and Uganda is to not treat acute HIV-1 infection, although clinical teams referred pregnant women with acute HIV-1 infection for prompt ART initiation. At an interim review in July 2011, the trial s independent data and safety monitoring board recommended that the placebo group be discontinued early because of clear demonstration of PrEP efficacy for HIV-1 prevention (2). A secondary analysis of HSV-2 protection from oral tenofovir-based PrEP compared with placebo was planned after July 2010, when a randomized trial of pericoital dosing of 1% tenofovir gel showed efficacy against HSV-2 acquisition (6 8). This finding was not anticipated in the original design of trials of tenofovir-based PrEP but could be assessed among the subset of participants who were HSV-2 uninfected at baseline. All HSV-2 testing and determination of end points was concluded before unblinding of HSV-2 outcomes. To correspond with the period covered in the primary HIV-1 acquisition results from the trial, we included data through July 2011 in our analysis of HSV-2 acquisition. Because the objective of this secondary analysis of HSV-2 prevention was to determine the effect of daily oral tenofovir on HSV-2 incidence, this report includes data only on participants who did not have HSV-2 infection at baseline (Figure 1). The study protocol was approved by the University of Washington Human Subjects Review Committee and ethics review committees at each of the study sites. All participants provided written informed consent in English or their local language. Laboratory Procedures HSV-2 Serologic Tests For HIV-1 uninfected partners, we tested archived baseline sera for HSV-2 antibodies by using an HSV-2 specific enzyme immunoassay (EIA) (Focus Technologies, Cypress, California). On the basis of previous experience with this EIA in East Africa (13), index value results less than 0.9 were considered to represent HSV-2 uninfected status, values of 3.5 or greater signified HSV-2 infected status, and values of 0.9 to 3.4 were considered indeterminate. We used HSV-2 specific Western blot to clarify indeterminate results (14). For participants determined to be HSV-2 seronegative at baseline, serostatus at the final visit through July 2011 was then assessed with an HSV-2 specific EIA and Western blot using the same approach as for baseline samples. Finally, individuals found to have seroconverted to HSV-2 between baseline and their final follow-up visit had sera from study months 1, 3, 6, 12, 18, 24, 30, and 36 tested in batch by HSV-2 specific Western blot to determine the timing of seroconversion. An HSV-2 end point committee reviewed the serologic data in a blinded manner to adjudicate end points. We tested HSV-2 serostatus at baseline for the HIV-1 infected partners of HSV-2 seronegative participants. Index EIA values less than 0.9 signified HSV-2 uninfected status, values of 3.5 or greater signified HSV-2 infected status, and HSV-2 specific Western blot was used to confirm the status of EIA values between 0.9 and 3.4. We did not conduct longitudinal testing among HIV-1 infected partners who were HSV-2 seronegative because of the high baseline prevalence of HSV-2. Plasma Tenofovir Levels We used a case cohort design to select a random sample of participants from the active PrEP groups for assessment of plasma concentrations of tenofovir. Case participants were those with HSV-2 seroconversion in the active 12 1 July 2014 Annals of Internal Medicine Volume 161 Number 1

3 HSV-2 Protection With Tenofovir-Based Preexposure Prophylaxis Original Research Figure 1. Study flow diagram. Eligible couples randomly assigned (n = 4747) Randomly assigned to TDF (n = 1584) Randomly assigned to FTC TDF (n = 1579) Randomly assigned to placebo (n = 1584) Excluded (n = 1071) HSV-2 positive: 973 Indeterminate HSV-2 status: 56 Missing result: 42 Excluded (n = 1051) HSV-2 positive: 964 Indeterminate HSV-2 status: 56 Missing result: 31 Excluded (n = 1103) HSV-2 positive: 1012 Indeterminate HSV-2 status: 55 Missing result: 36 HSV-2 negative at enrollment (n = 513) HSV-2 negative at enrollment (n = 528) HSV-2 negative at enrollment (n = 481) Retention 6 mo: 495/511 (96.9%) 12 mo: 396/425 (93.2%) 24 mo: 199/219 (90.9%) 36 mo: 2/2 (100%) Retention 6 mo: 501/525 (95.4%) 12 mo: 420/453 (92.7%) 24 mo: 214/233 (91.9%) 36 mo: 3/3 (100%) Retention 6 mo: 463/479 (96.7%) 12 mo: 399/422 (94.6%) 24 mo: 207/224 (92.4%) 36 mo: 2/2 (100%) No follow-up (n = 8) No follow-up (n = 7) Indeterminate HSV-2 status at No follow-up (n = 8) last visit (n = 1) Analyzed (n = 506) HSV-2 positive: 42 HSV-2 negative: 464 Analyzed (n = 519) HSV-2 positive: 37 HSV-2 negative: 482 Analyzed (n = 473) HSV-2 positive: 52 HSV-2 negative: 421 Analytic sample Retention of partners who were seronegative for HIV-1 and HSV-2 was calculated as cumulative retention, which was defined as remaining in follow-up and having available HSV-2 serostatus information. A total of 131 participants seroconverted to HSV-2 (42 in the tenofovir group, 37 in the FTC TDF group, and 52 in the placebo group) and were included in the intention-to-treat analysis. FTC TDF emtricitabine in combination with tenofovir disoproxil fumarate; HSV-2 herpes simplex virus type 2; TDF tenofovir disoproxil fumarate. PrEP groups, and control participants were a random sample (selected in a 2:1 ratio compared with case participants) who were assigned to active PrEP and did not acquire HSV-2. We assessed plasma tenofovir levels in case and control participants at 1, 3, 6, 12, 18, 24, 30, and 36 months after enrollment, thus including testing at the visits at which HSV-2 seroconversion was detected for case participants. Plasma tenofovir concentrations were determined by previously described liquid chromatography mass spectrometry methods (15). Plasma HIV-1 RNA Levels and CD4 Cell Counts in HIV-1 Infected Partners We quantified CD4 cell counts by using standard flow cytometry and plasma HIV-1 RNA levels by using the COBAS AmpliPrep/COBAS TaqMan real-time HIV-1 RNA assay, version 1.0 (Roche Diagnostics, Indianapolis, Indiana), with a lower limit of quantification of 240 copies/ml. Statistical Analysis The primary outcome was HSV-2 seroconversion among persons who were HSV-2 seronegative at baseline, and the primary analysis was intention-to-treat. The only exception was that participants who acquired HIV-1 were censored after the first visit at which they were HIV-1 seropositive because the study drug was withheld at that time. Thus, those with HIV-1 seroconversion contributed follow-up time for assessment of HSV-2 up to and including the visit when HSV-2 seroconversion or HIV-1 seroconversion was detected, whichever occurred first. We compared both active PrEP groups (FTC TDF and tenofovir combined) with the placebo group given that tenofovir was the common active moiety against HSV-2 in both groups and emtricitabine is not known to have activity against HSV-2 (16). Assessment of HSV-2 was intervalcensored at the specified study visits, so discrete time proportional hazards models were used to estimate hazard ratios (HRs) for HSV-2 infection (17). We fit these models using the PROC LOGISTIC statement in SAS, version 9.3 (SAS Institute, Cary, North Carolina), with a complementary log-log link, including visit interval as a class variable without an intercept term, in addition to the covariate of interest (treatment group). The proportional hazards assumption was tested by including interactions between visit and treatment group. In a sensitivity analysis, we also stratified the proportional hazards model by site. Prespeci- 1 July 2014 Annals of Internal Medicine Volume 161 Number 1 13

4 Original Research HSV-2 Protection With Tenofovir-Based Preexposure Prophylaxis fied subgroup analyses were performed to explore the uniformity of treatment effects found in the overall analysis. These included baseline covariates of sex of HIV-1 uninfected partners; circumcision status of HIV-1 uninfected male participants; and plasma HIV-1 RNA level ( vs copies/ml), CD4 count ( vs cells/l), and HSV-2 serostatus of HIV-1 infected partners. In addition to the primary proportional hazards analysis, we conducted sensitivity analyses to examine the potential effect of confounding and bias that could have resulted from using the subset of the HSV-2 uninfected cohort from the randomized trial, loss to follow-up, and censoring of participants with HIV-1 seroconversion. A marginal structural model approach was used, adjusted for age, sex, unprotected sex at baseline, and weighting that accounted for time-dependent unprotected sex during follow-up. Logistic regression was used to estimate stabilized inverse weights (18), which were used to conduct an adjusted, weighted discrete proportional hazards analysis using the procedure described earlier. In a separate sensitivity analysis to assess the potential effects of early censoring of participants who acquired HIV-1, we conducted simulation modeling to estimate the HR if these participants had continued in follow-up at the observed HSV-2 risk and at higher risk. We performed a case cohort analysis of plasma tenofovir concentrations to assess the risk for HSV-2 acquisition associated with detection ( 0.3 ng/ml) versus nondetection, using the same approach as in the case cohort analysis of plasma tenofovir concentrations and HIV-1 protection in the Partners PrEP Study (2). The case cohort analysis was performed using drug concentrations in all selected participants, regardless of drug holds in the prior interval (for example, for pregnancy or clinical events). We used case cohort methods in a Cox proportional hazards model to estimate the HR of infection for detectable versus undetectable tenofovir in plasma (19). In this nonrandomized comparison, the model was adjusted for factors that might be associated with adherence to the study drug, including age, polygamous marriage, and unprotected sex as a time-dependent variable (20). This analysis used the 2-phase design approach to the case cohort analysis (21). All analyses were conducted using SAS, version 9.3, with the exception of the case cohort analysis, for which we used R, version (survey package and Hmisc package , R Foundation for Statistical Computing, Vienna, Austria). Role of the Funding Source The Bill & Melinda Gates Foundation funded the study but did not oversee the protocol. Gilead Sciences donated the study medication but had no role in data collection or analysis. RESULTS Of the 4747 HIV-1 uninfected partners enrolled in the trial, 4638 (97.7%) had baseline samples available for HSV-2 testing; of these, 1522 (32.8%) were HSV-2 seronegative (Figure 1), of whom 1498 (98.4%) had a final study visit sample for HSV-2 testing. The median age of the initially HSV-2 seronegative participants was 31 years, and 80% were men, 40% of whom were uncircumcised. At enrollment, 96% were married to their HIV-1 infected partner, 8% reported an outside partner, the median number of sex acts in the prior month was 4, and 27% reported unprotected sex (Table 1). A total of 237 HIV-1 infected partners (15.6%) initiated ART during follow-up; 25 (1.6% of all HIV-1 infected partners) reported receiving a tenofovir-containing regimen. Follow-up and Adherence Retention was greater than 90% for all groups during the study period (Figure 1), with a median follow-up of 18 months (interquartile range, 12 to 24 months; range, 0 to 36 months). Study medication was dispensed at 96% of attended visits. The most common reason for withholding study medication was pregnancy, which did not differ significantly by study group and accounted for 1.5% of follow-up time. Study medication interruptions due to safety-related reasons accounted for 0.5% of follow-up time. The primary measure of adherence was monthly pill counts of returned study tablets. Ninety-eight percent of dispensed study bottles were returned, and pill counts indicated that 96% of dispensed tablets were taken. When missed visits, other reasons for nondispensation of study medication, and nonadherence to dispensed study pills were taken into account, 90.9% of follow-up time for this analysis was covered by study medication. HSV-2 Incidence by Group A total of 131 participants had incident HSV-2 infection (79 in the active PrEP groups [incidence, 5.6 per 100 person-years] and 52 in the placebo group [incidence, 7.7 per 100 person-years]) (Table 2). The HR for HSV-2 acquisition with PrEP compared with placebo was 0.70 (95% CI, 0.49 to 0.99; P 0.047) (Figure 2), and the absolute risk reduction was 2.1 per 100 person-years. When the 2 PrEP groups were considered separately, 42 HSV-2 infections were observed among participants assigned tenofovir (incidence, 6.1 per 100 person-years) and 37 were seen among those assigned FTC TDF (incidence, 5.1 per 100 person-years), indicating an HR for HSV-2 acquisition of 0.76 (CI, 0.51 to 1.14; P 0.186) for tenofovir and 0.64 (CI, 0.42 to 0.98; P 0.041) for FTC TDF, each compared with placebo. The causal effect of PrEP, which we estimated using marginal structural modeling, was essentially the same as the result from the proportional hazards model (HR, 0.69 [CI, 0.49 to 0.99]; P 0.041). The magnitude of reduction in incident HSV-2 in the PrEP groups compared with placebo was 14 1 July 2014 Annals of Internal Medicine Volume 161 Number 1

5 HSV-2 Protection With Tenofovir-Based Preexposure Prophylaxis Original Research Table 1. Baseline Characteristics of Participants Who Were Dually Seronegative for HIV-1 and HSV-2 Variable Tenofovir (n 513) FTC TDF (n 528) Placebo (n 481) Total (n 1522) Demographic characteristics Men n (%) 408 (80) 422 (80) 389 (81) 1219 (80) Median age (IQR), y 30 (26 36) 30 (26 36) 31 (26 36) 31 (26 36) Median education (IQR), y 8 (5 12) 8 (5 11) 8 (5 11) 8 (5 11) Any monthly income, n (%) 435 (85) 433 (82) 398 (83) 1266 (83) Couple characteristics Married to study partner, n (%) 489 (95) 506 (96) 466 (97) 1461 (96) Median time living with study partner (IQR), y 4.7 ( ) 5.0 ( ) 4.5 ( ) 5.0 ( ) Sexual risk behavior Median sex acts in prior month (IQR), n 4 (3 8) 4 (3 8) 4 (3 9) 4 (3 8) Any unprotected sex acts in prior month, n (%) 150 (29) 145 (27) 113 (23) 408 (27) Any sex with outside partner in prior month, n (%) 48 (9) 39 (7) 36 (7) 123 (8) Clinical characteristics of HIV-1 seronegative partner Median CD4 count (IQR), 10 9 cells/l ( ) ( ) ( ) ( ) Median plasma HIV-1 RNA level (IQR), log 10 copies/ml* 3.8 ( ) 3.7 ( ) 3.9 ( ) 3.8 ( ) Clinical characteristics of HIV-1/HSV-2 seronegative participant, n (%) Uncircumcised (men only) 162 (40) 177 (42) 150 (39) 489 (40) Curable sexually transmitted infection 26 (5) 19 (4) 35 (7) 80 (5) FTC TDF emtricitabine in combination with tenofovir disoproxil fumarate; HSV-2 herpes simplex virus type 2; IQR interquartile range. * Data were not available for 16 HIV-1 infected partners. Neisseria gonorrhoeae, Chlamydia trachomatis, or Trichomonas vaginalis detected by nucleic acid amplification testing. Data were not available for 14 participants. similar in subgroup categories, including men and women, circumcised and uncircumcised HIV-1 infected men, and individuals whose HIV-1 infected partners had higher or lower plasma HIV-1 RNA levels. However, PrEP was more effective in reducing HSV-2 acquisition among individuals whose HIV-1 infected partner had a baseline CD4 count of at least cells/l than among those whose partner had a CD4 count below this level. Baseline HSV-2 serostatus was available for 1493 HIV-1 infected partners, of whom 1044 (69.9%) were HSV-2 seropositive. For HIV-1 uninfected persons with a partner co-infected with HIV-1 and HSV-2, incidence of HSV-2 was 10.1 per 100 person-years in the placebo group and 7.0 per 100 person-years in the PrEP groups (HR, 0.67 [CI, 0.46 to 0.98]; P 0.038). Participants whose HIV-1 infected partners were HSV-2 seronegative could have acquired HSV-2 from outside partners; among the 13 such participants with incident HSV-2 infection, none reported outside partners at study enrollment, but 6 reported an outside partner during follow-up (5 of the HIV-1 uninfected partners and 1 of the HIV-1 infected partners). Twenty-three participants seroconverted to HIV-1; of these, 5 seroconverted to HSV-2 before HIV-1 and 18 (11 in the placebo group and 7 in the PrEP groups) were HSV-2 seronegative at the last visit before HIV-1 seroconversion. A sensitivity analysis of the potential effect of informative censoring of participants with HIV-1 seroconversion that assumed increased risk for HSV-2 (1.5 times the placebo rate) and no protective effect (because the study drug was withheld at detection of HIV-1 seroconversion) produced a narrow range of HRs over 1000 simulations (median, [CI, to 0.998]) (Table 2), indicating little sensitivity to censoring. Tenofovir Exposure and HSV-2 Protection We compared plasma tenofovir levels among the 79 case participants with and 160 control participants without HSV-2 seroconversion randomly selected from the active PrEP groups. Overall, 76.8% of control participants had detectable tenofovir ( 0.3 ng/ml) compared with 75.9% of case participants at the visit where HSV-2 infection was first detected, and detection of tenofovir was not associated with HSV-2 protection (adjusted HR, 1.72 [CI, 0.86 to 3.44]; P 0.123). DISCUSSION In this secondary analysis within a randomized, placebo-controlled trial of PrEP for HIV-1 prevention among 4747 HIV-1 serodiscordant couples, oral tenofovir-based PrEP reduced HSV-2 acquisition by 30% compared with placebo among initially HSV-2 seronegative persons. In the subgroup analysis of participants with known exposure to HSV-2 due to having a partner co-infected with HIV-1 and HSV-2, oral tenofovir-based PrEP reduced HSV-2 seroincidence by 33%. This is, to our knowledge, the first evidence of efficacy of daily oral tenofovir-based PrEP in providing protection against HSV-2 in a study population with high adherence to daily oral PrEP. The efficacy of tenofovir to reduce the risk for HSV-2 acquisition found in this study is consistent with the 1 July 2014 Annals of Internal Medicine Volume 161 Number 1 15

6 Original Research HSV-2 Protection With Tenofovir-Based Preexposure Prophylaxis Table 2. HSV-2 Seroincidence, by Study Group Variable Combined Treatment Group (Tenofovir or FTC TDF) Placebo Participants, n Events, n Person-Years Incidence, events per 100 person-years Participants, n Events, n Person-Years Incidence, events per 100 person-years Overall Sex Male Female Circumcision among HIV-1/HSV-2 seronegative men Circumcised Uncircumcised Baseline plasma HIV-1 RNA level of HIV-1 infected partner copies/ml copies/ml Baseline HSV-2 status of HIV-1 infected partner Positive Negative Baseline CD4 count of HIV-1 infected partner cells/l cells/l FTC TDF emtricitabine in combination with tenofovir disoproxil fumarate; HR hazard ratio; HSV-2 herpes simplex virus type 2. * The HR was 0.69 (95% CI, ; P 0.041) in the Cox proportional hazards model and 0.70 (CI, ; P 0.053) in the marginal structural model when each model was stratified by site. To account for potential informative censoring of participants with HIV-1 seroconversion (n 18 censored at the time of seroconversion, with no additional HSV-2 testing after that point), we conducted simulation modeling to assess the effect of following participants after seroconversion. We simulated a conservative scenario 1000 times under the assumption that the risk for HSV-2 acquisition after HIV-1 seroconversion was elevated (1.5 times the HSV-2 seroconversion rate in the placebo group among those who were HIV-1 uninfected) and the same in the preexposure prophylaxis and placebo groups (HR, 1). In this scenario, the overall median HR was (CI, ) essentially the same as the HRs from the proportional hazards and marginal structural models suggesting that censoring participants at the time of HIV-1 seroconversion had little effect on the overall findings. Values in bold are statistically significant. Test for effect modification. HSV-2 protective effect of 51% observed with pericoital use of topical 1% tenofovir gel (6, 8). Topical use of tenofovir or high adherence to daily dosing of oral tenofovir may be needed to achieve sufficient levels to provide protection against HSV-2 given that the 90% effective concentration (EC 90 ) of tenofovir for HSV-2 is high (10, 11). We did not find a relationship between detection of tenofovir in plasma and protection against HSV-2, although similar analyses for HIV-1 protection among the HIV-1 uninfected participants in the Partners PrEP Study showed an association (2). The lack of relationship between detection of tenofovir in plasma and protection against HSV-2 may indicate either less ability to discern a concentration response relationship between tenofovir exposure and HSV-2 protection if the actual protective effect is modest or less precise timing of drug exposure and HSV-2 seroconversion due to assessing HSV-2 serostatus up to every 6 months (in contrast to HIV-1 serostatus, which was assessed monthly). In addition, 80% of the HSV-2 susceptible participants in this study were men; the biology of heterosexual HSV-2 transmission with respect to HSV-2 target cells and the bioavailability of oral tenofovir in penile epithelium are not well-characterized. Protection from HSV-2 with daily oral tenofovirbased PrEP is biologically plausible given recent in vitro data showing that tenofovir inhibits HSV-2 replication in epithelial raft cultures, a mouse model, and ex vivo human lymphoid and cervicovaginal tissue from persons monoinfected with HSV-2 and those co-infected with HIV-1 and HSV-2 (10). Steady-state tenofovir concentrations in the genital tract after daily oral FTC TDF are approximately 100 ng/ml (22), and in vitro studies indicate HSV suppression at tenofovir concentrations between 10 and 200 g/ml (10), which would only be achieved in the context of high adherence to daily oral dosing and accumulation of active metabolites in the genital tract. The lack of HSV-2 protection with oral FTC TDF among men who have sex with men in another PrEP trial was potentially due to lower adherence in that study (23); only about half of participants had objective evidence of PrEP adherence (1) compared with more than 80% in our study (2). Given the strong association between adherence and HIV-1 protec July 2014 Annals of Internal Medicine Volume 161 Number 1

7 HSV-2 Protection With Tenofovir-Based Preexposure Prophylaxis Original Research Table 2 Continued Proportional Hazards Model* HR vs. Placebo (95% CI) P Value Marginal Structural Model* HR vs. Placebo (95% CI) P Value 0.70 ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) tion with daily oral dosing of PrEP, new formulations for sustained delivery of PrEP products, such as an intravaginal ring containing tenofovir, may provide a less userdependent formulation with sustained release of tenofovir in the genital tract (24). Our results and those of others indicate that tenofovirbased PrEP may have prophylactic activity against other viruses in addition to HIV-1. Tenofovir-containing ART regimens have been shown to be associated with reduced incidence of hepatitis B infection in HIV-1 infected persons (25), which is consistent with in vitro and in vivo activity of tenofovir (and emtricitabine) in persons with chronic hepatitis B infection (26, 27). The efficacy of tenofovir-containing ART regimens has not been evaluated for HSV-2 protection in HIV-1 infected persons who are HSV-2 uninfected. Ongoing studies are evaluating the therapeutic efficacy of tenofovir in reducing HSV-2 shedding in persons with chronic HSV-2 infection with and without HIV-1 infection. One small study in persons coinfected with HIV-1 and HSV-2 found that tenofovircontaining ART was not associated with reduced rates of HSV-2 shedding, but the study had limited power to detect a difference (28). Interventions to reduce the risk for HSV-2 acquisition are a high priority. Incidence of HSV-2 is high in populations worldwide; annualized incidence was 20% among young South African women (6) and 15% among HIV-1 serodiscordant couples in Kenya who reported a high rate of condom use (29). In addition, observational data indi- Figure 2. Cumulative probability of HSV-2 seroconversion in the active PrEP (tenofovir and FTC TDF) versus placebo groups. Cumulative Probability of HSV-2 Seroconversion PrEP Placebo Months Since Start At risk, n PrEP Placebo FTC TDF emtricitabine in combination with tenofovir disoproxil fumarate; HSV-2 herpes simplex virus type 2; PrEP preexposure prophylaxis. 1 July 2014 Annals of Internal Medicine Volume 161 Number 1 17

8 Original Research HSV-2 Protection With Tenofovir-Based Preexposure Prophylaxis cate a 3-fold increased risk for HIV-1 acquisition due to prevalent HSV-2 infection and up to a 6-fold increase with incident HSV-2 infection (30). Interventions to reduce the risk for HSV-2 transmission and acquisition are limited. Condoms are estimated to reduce HSV-2 acquisition by 30% (31), which is substantially lower than the 80% efficacy of condoms in reducing the per-contact risk for HIV-1 acquisition (32), and medical circumcision provided modest protection against HSV-2 acquisition in HIV-1 uninfected men in 2 of 3 trials (33 35). Candidate preventive HSV-2 vaccines to date have not shown efficacy in preventing HSV-2 infection (36, 37). Widely used agents for treatment of HSV-2 infection have shown mixed results when used for prevention of HSV-2 infection, with 1 study finding that valacyclovir, 500 mg once daily, reduced HSV-2 transmission by 50% to partners of HSV-2 infected persons who were not infected with HIV-1 (38), but other studies found that acyclovir, 400 mg twice daily, did not reduce HSV-2 transmission to partners of persons co-infected with HIV-1 and HSV-2 (39) or HSV-2 acquisition in HIV-1 uninfected persons (40, 41). No studies are available of valacyclovir or acyclovir for primary prophylaxis against HSV-2 acquisition. Strengths of this study include the use of a blinded end point determination process based on the best available HSV-2 serologic testing algorithm and sufficient power with 131 end points to determine modest effectiveness. Subgroup analysis based on known exposure to an HSV-2 infected partner was feasible because of the couples design. The consistency of results between the proportional hazards and marginal structural models indicates that intention-to-treat assumptions were appropriate and that significant confounding was not present in the subsample of initially HSV-2 seronegative persons and was not induced by censoring at HIV-1 seroconversion or loss to follow-up. Limitations of this study include the inability to detect and exclude individuals with acute HSV-2 infection at baseline due to a lack of laboratory methods. Ten percent of incident HSV-2 infections occurred in partnerships where the HIV-1 infected partner was HSV-2 seronegative at baseline, but laboratory methods are not available for linkage of HSV-2 strains. Because of availability of testing and samples, we were unable to assess the timing of HSV-2 acquisition with greater precision than according to the schedule of follow-up testing we performed, which may have limited our ability to demonstrate an association between HSV-2 protection and plasma tenofovir detection. Finally, we censored participants after HIV-1 seroconversion because the study drug was discontinued and we could not determine HSV-2 seroconversion in these participants; however, sensitivity analyses indicated that the HRs for HSV-2 acquisition were relatively stable despite this potentially informative censoring. In summary, tenofovir-based PrEP provides modest protection against HSV-2. Incidence was 7.7 per 100 person-years in the placebo group of this study, nearly 4-fold greater than the HIV-1 incidence (2.0 per 100 person-years). Given the high prevalence of HSV-2 in HIV-1 infected persons, and given that HSV-2 is a significant risk factor for HIV-1 acquisition, identifying effective primary prevention strategies for HSV-2 is a public health priority. The HSV-2 protective effects of oral tenofovirbased PrEP add benefit to the high protection against HIV-1 in populations who are often at risk for HIV-1 and HSV-2. From University of Washington and Fred Hutchinson Cancer Research Center, Seattle, Washington; Johns Hopkins University, Baltimore, Maryland; Indiana University, Indianapolis, Indiana; and Makerere University, Kampala, Uganda. Note: All authors vouch for the completeness and accuracy of the data presented. Acknowledgment: The authors thank the study participants and the study team. Grant Support: By the Bill & Melinda Gates Foundation (OOP47674). Disclosures: Disclosures can be viewed at /icmje/conflictofinterestforms.do?msnum M Reproducible Research Statement: Study protocol and statistical code: Available from Dr. Celum ( , ccelum@uw.edu). Data set: Portions of the analytic data set are available to approved individuals through written agreement with Dr. Celum ( , ccelum@uw.edu). Requests for Single Reprints: Connie Celum, MD, MPH, University of Washington, 325 Ninth Avenue, UW Box , Seattle, WA Current author addresses and author contributions are available at References 1. Grant RM, Lama JR, Anderson PL, McMahan V, Liu AY, Vargas L, et al; iprex Study Team. Preexposure chemoprophylaxis for HIV prevention in men who have sex with men. N Engl J Med. 2010;363: [PMID: ] 2. Baeten JM, Donnell D, Ndase P, Mugo NR, Campbell JD, Wangisi J, et al; Partners PrEP Study Team. Antiretroviral prophylaxis for HIV prevention in heterosexual men and women. N Engl J Med. 2012;367: [PMID: ] 3. Choopanya K, Martin M, Suntharasamai P, Sangkum U, Mock PA, Leethochawalit M, et al; Bangkok Tenofovir Study Group. Antiretroviral prophylaxis for HIV infection in injecting drug users in Bangkok, Thailand (the Bangkok Tenofovir Study): a randomised, double-blind, placebo-controlled phase 3 trial. Lancet. 2013;381: [PMID: ] 4. Thigpen MC, Kebaabetswe PM, Paxton LA, Smith DK, Rose CE, Segolodi TM, et al; TDF2 Study Group. Antiretroviral preexposure prophylaxis for heterosexual HIV transmission in Botswana. N Engl J Med. 2012;367: [PMID: ] 5. Balzarini J, Holy A, Jindrich J, Naesens L, Snoeck R, Schols D, et al. Differential antiherpesvirus and antiretrovirus effects of the (S) and (R) enantiomers of acyclic nucleoside phosphonates: potent and selective in vitro and in vivo antiretrovirus activities of (R)-9-(2-phosphonomethoxypropyl)-2,6- diaminopurine. Antimicrob Agents Chemother. 1993;37: [PMID: ] 18 1 July 2014 Annals of Internal Medicine Volume 161 Number 1

9 HSV-2 Protection With Tenofovir-Based Preexposure Prophylaxis Original Research 6. Abdool Karim Q, Abdool Karim SS, Frohlich JA, Grobler AC, Baxter C, Mansoor LE, et al; CAPRISA 004 Trial Group. Effectiveness and safety of tenofovir gel, an antiretroviral microbicide, for the prevention of HIV infection in women. Science. 2010;329: [PMID: ] 7. Tan D. Potential role of tenofovir vaginal gel for reduction of risk of herpes simplex virus in females. Int J Womens Health. 2012;4: [PMID: ] 8. Centre for the AIDS Programme of Research in South Africa. Effectiveness & safety of vaginal microbicide 1% tenofovir gel for prevention of HIV infection in women. Congella, South Africa: Centre for the AIDS Programme of Research in South Africa; Accessed at _CAPRISA%20004%20Results.pdf on 7 May Karim SS, Kashuba AD, Werner L, Karim QA. Drug concentrations after topical and oral antiretroviral pre-exposure prophylaxis: implications for HIV prevention in women. Lancet. 2011;378: [PMID: ] 10. Andrei G, Lisco A, Vanpouille C, Introini A, Balestra E, van den Oord J, et al. Topical tenofovir, a microbicide effective against HIV, inhibits herpes simplex virus-2 replication. Cell Host Microbe. 2011;10: [PMID: ] 11. Schwartz JL, Rountree W, Kashuba AD, Brache V, Creinin MD, Poindexter A, et al. A multi-compartment, single and multiple dose pharmacokinetic study of the vaginal candidate microbicide 1% tenofovir gel. PLoS One. 2011;6: e [PMID: ] 12. Mujugira A, Baeten JM, Donnell D, Ndase P, Mugo NR, Barnes L, et al; Partners PrEP Study Team. Characteristics of HIV-1 serodiscordant couples enrolled in a clinical trial of antiretroviral pre-exposure prophylaxis for HIV-1 prevention. PLoS One. 2011;6:e [PMID: ] 13. Lingappa J, Nakku-Joloba E, Magaret A, Friedrich D, Dragavon J, Kambugu F, et al. Sensitivity and specificity of herpes simplex virus-2 serological assays among HIV-infected and uninfected urban Ugandans. Int J STD AIDS. 2010;21: [PMID: ] 14. 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No impact of oral tenofovir disoproxil fumarate on herpes simplex virus shedding in HIV-infected adults. AIDS. 2011;25: [PMID: ] 29. Muiru AN, Guthrie BL, Bosire R, Merkel M, Liu AY, Choi RY, et al. Incident HSV-2 infections are common among HIV-1-discordant couples. J Infect Dis. 2013;208: [PMID: ] 30. Freeman EE, Weiss HA, Glynn JR, Cross PL, Whitworth JA, Hayes RJ. Herpes simplex virus 2 infection increases HIV acquisition in men and women: systematic review and meta-analysis of longitudinal studies. AIDS. 2006;20: [PMID: ] 31. Martin ET, Krantz E, Gottlieb SL, Magaret AS, Langenberg A, Stanberry L, et al. A pooled analysis of the effect of condoms in preventing HSV-2 acquisition. Arch Intern Med. 2009;169: [PMID: ] 32. Hughes JP, Baeten JM, Lingappa JR, Magaret AS, Wald A, de Bruyn G, et al; Partners in Prevention HSV/HIV Transmission Study Team. Determinants of per-coital-act HIV-1 infectivity among African HIV-1-serodiscordant couples. J Infect Dis. 2012;205: [PMID: ] 33. Mahiane SG, Legeai C, Taljaard D, Latouche A, Puren A, Peillon A, et al. Transmission probabilities of HIV and herpes simplex virus type 2, effect of male circumcision and interaction: a longitudinal study in a township of South Africa. AIDS. 2009;23: [PMID: ] 34. Tobian AA, Serwadda D, Quinn TC, Kigozi G, Gravitt PE, Laeyendecker O, et al. Male circumcision for the prevention of HSV-2 and HPV infections and syphilis. N Engl J Med. 2009;360: [PMID: ] 35. Mehta SD, Moses S, Parker CB, Agot K, Maclean I, Bailey RC. Circumcision status and incident herpes simplex virus type 2 infection, genital ulcer disease, and HIV infection. AIDS. 2012;26: [PMID: ] 36. Belshe RB, Leone PA, Bernstein DI, Wald A, Levin MJ, Stapleton JT, et al; Herpevac Trial for Women. Efficacy results of a trial of a herpes simplex vaccine. N Engl J Med. 2012;366: [PMID: ] 37. Johnston C, Koelle DM, Wald A. Current status and prospects for development of an HSV vaccine. Vaccine. 2014;32: [PMID: ] 38. Corey L, Wald A, Patel R, Sacks SL, Tyring SK, Warren T, et al; Valacyclovir HSV Transmission Study Group. Once-daily valacyclovir to reduce the risk of transmission of genital herpes. N Engl J Med. 2004;350: [PMID: ] 39. Mujugira A, Magaret AS, Celum C, Baeten JM, Lingappa JR, Morrow RA, et al; Partners in Prevention HSV/HIV Transmission Study Team. Daily acyclovir to decrease herpes simplex virus type 2 (HSV-2) transmission from HSV- 2/HIV-1 coinfected persons: a randomized controlled trial. J Infect Dis. 2013; 208: [PMID: ] 40. Celum C, Wald A, Hughes J, Sanchez J, Reid S, Delany-Moretlwe S, et al; HPTN 039 Protocol Team. Effect of aciclovir on HIV-1 acquisition in herpes simplex virus 2 seropositive women and men who have sex with men: a randomised, double-blind, placebo-controlled trial. Lancet. 2008;371: [PMID: ] 41. Watson-Jones D, Weiss HA, Rusizoka M, Changalucha J, Baisley K, Mugeye K, et al; HSV trial team. Effect of herpes simplex suppression on incidence of HIV among women in Tanzania. N Engl J Med. 2008;358: [PMID: ] 1 July 2014 Annals of Internal Medicine Volume 161 Number 1 19

10 Annals of Internal Medicine Current Author Addresses: Drs. Celum, Donnell, Hong, and Baeten; Ms. Thomas; and Mr. Mujugira: University of Washington, 325 Ninth Avenue, UW Box , Seattle, WA Dr. Morrow: Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, LE-500, Seattle, WA Dr. Hendrix: Department of Medicine, Johns Hopkins University, 600 North Wolfe Street, Harvey 502, Baltimore, MD Dr. Fife: Department of Medicine, Indiana University, Emerson H/Room 435, 545 Barnhill Drive, Indianapolis, IN Dr. Nakku-Joloba: School of Public Health, Makerere University, PO Box 22418, Plot 106, Block 213, Bukoto, Kampala, Uganda. Author Contributions: Conception and design: C. Celum, D. Donnell, J.M. Baeten. Analysis and interpretation of the data: C. Celum, R.A. Morrow, D. Donnell, T. Hong, C.W. Hendrix, K.K. Thomas, K.H. Fife, J.M. Baeten. Drafting of the article: C. Celum, D. Donnell, J.M. Baeten. Critical revision of the article for important intellectual content: C. Celum, D. Donnell, K.H. Fife, E. Nakku-Joloba, J.M. Baeten. Final approval of the article: C. Celum, R.A. Morrow, D. Donnell, C.W. Hendrix, K.H. Fife, E. Nakku-Joloba, A. Mujugira, J.M. Baeten. Provision of study materials or patients: K.H. Fife, A. Mujugira, J.M. Baeten. Statistical expertise: D. Donnell, T. Hong, K.K. Thomas. Obtaining of funding: C. Celum, J.M. Baeten. Administrative, technical, or logistic support: E. Nakku-Joloba, A. Mujugira, J.M. Baeten. Collection and assembly of data: C. Celum, R.A. Morrow, D. Donnell, C.W. Hendrix, K.H. Fife, E. Nakku-Joloba, A. Mujugira, J.M. Baeten. APPENDIX: PARTNERS PREP STUDY TEAM University of Washington Coordinating Center and Central Laboratories, Seattle, Washington Connie Celum (principal investigator and protocol cochair), Jared M. Baeten (medical director and protocol co-chair), Deborah Donnell (protocol statistician), Robert W. Coombs (University of Washington Retrovirology Laboratory), Anne Cent (University of Washington Virology Laboratory), and Jairam Lingappa (University of Washington International Clinical Research Center Biorepository). Study Sites and Principal Investigators Eldoret, Kenya (Moi University and Indiana University): Kenneth Fife and Edwin Were; Kabwohe, Uganda (Kabwohe Clinical Research Center): Elioda Tumwesigye; Jinja, Uganda (Makerere University and University of Washington): Patrick Ndase and Elly Katabira; Kampala, Uganda (Makerere University): Elly Katabira and Allan Ronald; Kisumu, Kenya (Kenya Medical Research Institute and University of California, San Francisco): Elizabeth Bukusi and Craig Cohen; Mbale, Uganda (The AIDS Support Organization and Centers for Disease Control and Prevention Uganda): Jonathan Wangisi, James Campbell, and Jordan Tappero; Nairobi, Kenya (University of Nairobi and University of Washington): James Kiarie, Carey Farquhar, and Grace John-Stewart; Thika, Kenya (University of Nairobi and University of Washington): Nelly Rwamba Mugo; Tororo, Uganda (Centers for Disease Control and Prevention Uganda and The AIDS Support Organization): James Campbell, Jordan Tappero, and Jonathan Wangisi. Data management was provided by DF/Net Research (Seattle, Washington), and site laboratory oversight was provided by Contract Laboratory Services (University of the Witwatersrand, Johannesburg, South Africa). 1 July 2014 Annals of Internal Medicine Volume 161 Number 1