The Step Study opened in December 2004 to study participants with Ad5 titers ≤200 at screening, and was amended to include participants with Ad5 titers >200 at screening in July 2005. Three thousand participants were enrolled through March 2007 at 34 sites in North America, the Caribbean, South America, and Australia. Protocol adherence was excellent, with 94% of the vaccine and placebo groups receiving all 3 study injections (). Overall, 6.5% and 5.8% of vaccine and placebo recipients, respectively, had discontinued follow-up in the study. Baseline demographic and risk characteristics are shown in , stratified by gender and baseline Ad5 titer. Overall, the study cohort was diverse and reported substantial levels of HIV risk. More than 75% of each stratum reported multiple male sex partners of unknown HIV serostatus, while a substantial proportion of men also reported having known HIV- positive male partners; only 68 men were exclusively heterosexual. Pregnancy rates in female vaccine and placebo participants were 12.8% and 9.9%, respectively, at the time of the interim analysis, indicating substantial levels of unprotected vaginal sex. Within pre-specified Ad5 strata, vaccine and placebo recipients were well-matched on demographic and risk characteristics at baseline. However, there were substantial differences in several important demographic and HIV risk factors between individuals in the low vs. high Ad5 strata. For example, men with baseline Ad5 titers >200 were significantly more likely to have been enrolled outside of North America, to be non-white, and to be uncircumcised. Men with high Ad5 titers were also less likely to have known HIV-positive partners or to use recreational drugs.
Figure 1 Trial profile. Discontinued includes study participants who were unwilling or unable to continue follow-up in the trial at the time the dataset was frozen. All participants who received at least one dose of vaccine or placebo were included in the safety (more ...)
Baseline characteristics, stratified by gender and baseline Ad5 antibody titer
Side effects from the vaccine were similar to those reported earlier (18
). Injection site pain (70% of vaccinees and 34% of placebo recipients) and headache (32% of vaccinees and 27% of placebo recipients) were most common. There were no clinically significant differences in safety laboratory results between vaccine and placebo recipients. Of 40 serious adverse events reported by blinded study investigators, only 2 (fever, rigors) were reported in the vaccine group that were deemed related to study vaccine.
Among the 25% pre-specified random sample of study volunteers evaluated for IFN-y ELISPOT responses at the week 8 time-point, 75% of vaccinees responded to one or more HIV antigens, with geometric mean titers of several hundred (). Response rates were higher among those with baseline Ad5 titers ≤200 than those with Ad5 >200; overall responses did not differ between men and women.
IFN-gamma ELISPOT summaries at week 8 for the vaccine group
Interim efficacy results
As pre-specified in the protocol, an interim analysis of HIV incidence and early HIV-1 viral load was conducted when there were 30 per-protocol events in the Ad5 ≤200 stratum. Results of this interim analysis are presented in . Overall HIV-1 seroincidence at the time of the interim analysis in the modified intention-to-treat (MITT) population was 3.6% per year in men (95% CI 2.6 – 4.8) and 0.2% per year in women (95% CI 0.0 - 1.3). HIV infection rates and mean viral load setpoint were no different or slightly higher in vaccine than placebo recipients in both the PP and MITT analysis subsets. The p-values for a beneficial effect exceeded 0.5 for both primary endpoints, thereby meeting the pre-specified futility criteria. Based on these results, the Step Study Protocol Team immediately halted all additional immunizations in the trial, and began notifying the study investigators, study participants, and the general public of the trial results within 72 hours of the DSMB meeting. After extensive discussions with study investigators, staff, and community representatives about the benefits of continuing blinded versus unblinded follow-up, the Step Study Protocol team decided to unblind study participants in November 2007.
Results of Pre-specified Interim Analysis for the Ad5 ≤ 200 Subgroup (Infection and Viral Load (VL)# Endpoints)
Exploratory efficacy analyses in male participants
Because the study unexpectedly met the pre-specified futility boundaries at the first interim analysis, additional analyses were initiated to explore reasons for the vaccine’s lack of efficacy. The interim data were expanded to include an additional 8 HIV-1 infections in participants with Ad5 titers ≤200 and 30 in participants with Ad5 titers >200 accrued through October 17, 2007. Because only 1 HIV-1 infection had occurred in a female participant, all subsequent analyses are limited to male participants in the MITT population.
In this expanded analysis of data through October 17, 2007, 49 of the 914 male vaccine recipients became HIV infected (annual HIV incidence 4.6%, 95% CI 3.4 to 6.1) and 33 of the 922 male placebo recipients became HIV infected (annual incidence 3.1%, 95% CI 2.1 to 4.3). The overall treatment effect hazard ratio from the univariate Cox model was 1.5 (95% CI 0.97 to 2.3, p=0.07). As randomization occurred within each of 4 pre-specified Ad5 strata, data are presented for each stratum (). Although HIV acquisition rates were similar in vaccine and placebo recipients with baseline Ad5 titers ≤18 (Ad5 seronegative participants), surprisingly, rates appeared to be more than twice as high in vaccinees compared with placebo recipients in Ad5 strata >18, (overall HIV acquisition rate 5.1% vs. 2.3%/year, unadjusted two-tailed p-value 0.013). There was also evidence that the hazard ratio increased with increasing log10(Ad5) (univariate Cox model trend test p-value = .06).
Figure 2 Kaplan Meier plots of HIV infection for male vaccine and placebo groups by A) baseline Ad5 ≤18; B) baseline Ad5 >18 and ≤200; C)baseline Ad5 >200 and ≤1000; and D) baseline Ad5 >1000. Each hazard ratio (HR) (more ...)
Viral load setpoints were not materially different between vaccine and placebo recipients in either the Ad5 seronegative or Ad5 seropositive stratum (, p>.25 for all comparisons).
Figure 3 Early plasma viral load (VL) at ~ 3 months after detection of infection in male study participants by A) baseline Ad5 ≤ 18; B) baseline Ad5 >18; and C) all participants. The bar in each panel denotes the geometric mean titers of the plasma (more ...)
Factors associated with HIV infection risk
Univariate Cox proportional hazard analyses were conducted to evaluate if vaccine effects on HIV acquisition rates were different for different subgroups of participants (). The hazard ratio (HR) of HIV acquisition in vaccine versus placebo recipients was consistently close to 1.5 among all subgroups defined by age, race, unprotected receptive anal sex, unprotected insertive anal sex, drug use, and number of male sex partners. The elevated risk of HIV acquisition seen in Ad5 seropositive men appeared absent in Ad5 seronegative men (HR 2.3 versus 1.0 respectively, interaction test p = .08). Similarly, the HR was elevated in uncircumcised men, but not in circumcised men (HR = 3.8 vs. 1.0, interaction test p = .01). The univariate results did not materially change after adjusting for other baseline and demographic covariates in multivariate models (data not shown).
Hazard Ratios of HIV Infection for Male Subgroups Defined by Demographic and Baseline Behavioral Risk Factors (Univariate Cox Model Analyses)
To evaluate whether Ad5 serostatus or circumcision status were independent risk factors for HIV infection in the placebo group alone, we applied the Cox model adjusting for baseline demographic and risk variables. The adjusted hazard ratio was 1.6 (95% CI 0.7 to 3.6, p=0.23) for Ad5 >18 versus Ad5 ≤18 placebo recipients, and 2.5 (95% CI 0.7 to 8.7, p=0.14) for circumcised versus uncircumcised placebo recipients. These results do not support either variable as a significant independent predictor of HIV infection; however, they must be interpreted with caution because the study did not randomize participants to Ad5 or circumcision groups, only to vaccine or placebo.
Because circumcision rates were substantially higher in the Ad5 seronegative than Ad5 seropositive participants (77.6% vs. 40.4%, p<0.001), hazard ratios were calculated for 4 different subpopulations of men in the trial, based on Ad5 and circumcision status. The unadjusted hazard ratio for risk of HIV acquisition among vaccinees compared with placebo recipients was highest among uncircumcised, Ad5 seropositive men (n= 620, HR 3.9, 95% CI 1.3 – 11.9). Risk was intermediate among uncircumcised, Ad5 seronegative men (n=168, HR 3.3, 95% CI 0.7 – 15.8) and circumcised, Ad5 seropositive men (n=421, HR 1.6, 95% CI 0.7 – 3.8). Risk did not appear to be elevated in men who were both circumcised and Ad5 seronegative (n=578, HR 0.7, 95% CI 0.3 – 1.4). These results did not change significantly when using adjusted hazard ratios from any of several multivariate Cox models.
To evaluate whether the increased hazard of HIV acquisition seen within these subgroups occurred only in peri-vaccination periods or persisted over time, the relative HIV incidence (vaccine:placebo) was evaluated during 3 semi-annual periods from the time of enrollment (). Overall and within subgroups, HIV incidence was approximately constant over time for both vaccinees and placebo recipients through 78 weeks of follow-up.
Figure 4 HIV incidence during 6-month time intervals for male vaccine and placebo groups by A) baseline Ad5 titer ≤18; B) baseline Ad5 >18; C) overall; D) circumcised; and E) uncircumcised. Each 2-tailed p-value (p) is from a univariate Cox regression (more ...)
If the vaccine increased the risk of HIV acquisition in uncircumcised male participants, a likely mechanism would be through insertive anal sex exposures and therefore, relative hazards should be particularly high in men reporting this risk. Therefore, the relative hazard of HIV infection was compared between men who had and had not reported unprotected insertive anal sex with HIV positive or unknown serostatus partners at baseline. Among uncircumcised men, the hazard ratios appeared to be even higher in men who reported unprotected insertive anal sex at baseline than in men who did not report this risk (HR 6.1 vs. 2.5 respectively). No such relationship was seen with unprotected receptive anal sex with HIV positive or unknown partners; the hazard ratio for uncircumcised men who reported this risk at baseline was lower than in uncircumcised men who did not report this risk (HR 3.7 vs. 5.7 respectively). Among circumcised men, hazard ratios were consistently near 1.0, regardless of reported baseline risk.
The difference in infection rates between vaccine and placebo recipients could be attributed to differences in risk practices between vaccine and placebo groups, particularly if any substantial levels of unblinding had occurred. Risk data were compared between vaccine and placebo recipients through 18 months of follow-up. For both vaccine and placebo recipients, the proportion of study participants reporting risk declined substantially during the first 6 months of the study, and then remained relatively level throughout follow-up (data not shown). The frequency of all measured risk behavior variables was similar for Ad5 seropositive vaccine and placebo recipients over time, and for uncircumcised vaccine and placebo recipients over time (all p-values > 0.20 data not shown).