Enrollment and vaccination of 324 volunteers commenced in May 2006 and was completed in October 2006 with 144, 120 and 60 participants at the Ugandan, Kenyan and Tanzanian sites, respectively. The average age was 28 years (range 18–49) and 103 (32%) participants were female. Three hundred and eleven (96%) participants completed the 12-month study and 13 (4%) terminated the study before the final visit. Nineteen (6%) participants did not complete the vaccination schedule (8 placebo and 11 vaccine recipients). The most common reason for discontinuation of vaccination was pregnancy (N = 8). Two individuals prematurely stopped vaccination due to adverse events (one vaccine recipient for urticaria, and one placebo recipient for generalized itching). No deaths occurred during the 12 months of the study. A single HIV infection was observed during the study period in a vaccine recipient. All traceable volunteers were offered counseling and HIV testing following public disclosure of the Merck STEP study results and 264 (81%) attended briefing and VCT sessions. A total of 4 additional HIV infections have been identified in extended follow-up (one vaccine recipient and 3 placebo recipients) yielding an overall infection rate of 0.611 per 100 person-years observation. The infection rate observed by MHRP in community cohort studies at or near these research sites have ranged from 0.77 to 1.8 per 100 person-years observation (M. Hoelscher, personal communication;[20
Vaccinations were well tolerated (supplemental figure 1
). Solicited local and systemic adverse events were commonly reported by both vaccine (88% for both) and placebo recipients (80% for both). The frequency and severity of local and systemic solicited adverse reactions was not different between the high and low dose of rAd5 either alone (p=0.440 and 0.367 for local and systemic adverse events, respectively) or in the prime-boost regimen (p=0.458 and 0.803 for local and systemic adverse events, respectively). The presence and titer of pre-existing antibody to Ad5 did not alter the frequency or severity of solicited local or systemic adverse events among vaccine recipients (data not shown, p=0.767 and p=0.938 respectively). There were no differences between vaccine and placebo recipients in the frequency or severity of routinely collected clinical laboratory measures (data not shown).
A total of 1446 AE were reported among 309 of 324 participants. Infectious diseases were the predominate adverse events with upper respiratory tract infections most common followed by malaria, urinary tract infections and gastroenteritis. Blood disorders were the second most common diagnoses led by neutropenia and thrombocytopenia. Adverse event rates were similar among placebo and vaccine participants. 62 adverse events were graded as severe or life-threatening and malaria and neutropenia were the most common diagnoses. The proportion of severe or life-threatening adverse events among vaccine and placebo participants were similar (4.9% versus 3.4%, p =0.159). Four of these, all occurring in vaccine recipients, were considered possibly or probably related to vaccine. These were two cases of generalized urticaria, an episode of neutropenia and an episode of malaise. There was no relationship between the frequency or severity of adverse events and the titer of pre-existing adenovirus antibody for either placebo or vaccine recipients (data not shown).
Immunogenicity Assessment Results
Responses to HIV antigens were measured using IFN-γ ELISpot prior to and 6 weeks after either a single rAd5 injection or the DNA/rAd5 regimen. Results from groups 3 and 5 were combined (3/5) as they evaluated the same regimen. Fifteen study participants who did not complete vaccination as assigned per protocol were excluded from analysis and in some cases, insufficient cells for analysis resulted in further missing data points.
IFN-γ ELISpot response frequencies post-vaccination for each peptide pool and for any positive response are shown for placebo and vaccine recipients by study group in . False positive rates from placebo (7/121;6%) and baseline samples were low for all peptide pools evaluated together (9/275; 3%, data not shown). The response rates to any peptide pool for the low and high dose rAd5 alone were 74% and 52% respectively (p=0.221). Similarly, response rates for DNA prime and low or high dose rAd5 boost were 63% and 60% respectively (p=0.806). Positive IFN-γ ELISpot responses to any Env, Gag or Pol peptide pool at 6 weeks following rAd5 alone (groups 1 and 2) and DNA/rAd5 (groups 3, 4 and 5) were 29 of 46 (63%) and 78 of 125 (62%), respectively and were not significantly different. Response rates for each Env peptide pool were not different between the Ad5 alone and prime-boost groups. The Env pools matching the vaccine amino acid sequence elicited better response rates compared to the subtype C Env peptide pool regardless of study group. The frequency of Gag responses in the rAd5 alone group was significantly less than that in the DNA/rAd5 groups, (3/45 versus 41/124; p < 0.001). Pol responses were more common in the rAd5 alone groups compared to the DNA/rAd5 groups, (15/45 versus 19/120; p=0.018).
Frequency (percentage) of subjects with detectable T cell responses to HIV antigens by treatment group, as measured by interferon-gamma enzyme-linked immunospot (ELISpot) assay in CD4+ or CD8+ T cells, 6 weeks after immunization.
There was no difference in the magnitude of response measured by IFN-γ ELISpot among the four regimens examined in the study (, p=0.254). The median (range) responses were 127 (60–493), 141 (70–463), 163 (55–2103) and 236 (58–1007) SFC/106 PBMC in Groups 1, 2, 3/5 and 4, respectively. There was no difference between low and high dose rAd5 alone, (arm 1 versus arm 2) or in a prime/boost format between arms 3/5 and 4 in terms of response frequency or magnitude. Analyzing these data with stratification for pre-existing Adenovirus antibody titers shows no significant differences but these results must be interpreted with caution given the small numbers of such a comparison (data not shown). The proportions of volunteers with responses to combinations of Env, Gag and/or Pol peptides are shown in . The frequency of positive responses was predominantly to Env, followed by Pol or Gag, regardless of immunization regimen. Among the four regimen groups, roughly equal proportions recognized two antigens (20%–26%). Group 4, with the high dose rAd5 boost had the highest frequency of responders to all three antigens tested (20%, p = 0.137).
Figure 1 The maximum magnitude of T cell responses measured by interferon-γ ELISpot expressed as spot forming units (SFU) per one million peripheral blood mononuclear cells (PBMC) in vaccinees to either Env, Pol or Gag by immunization regimen 6 weeks after (more ...)
Figure 2 Breadth of T cell responses to HIV antigens. Frequencies of vaccinated subjects with detectable T cell responses to HIV antigens 6 weeks after immunization. Responses were measured by the interferon (IFN)-γ ELISpot assay. A positive response was (more ...)
Ad5 Nab was present at baseline in 175 of 186 vaccine recipients (94%) with a median titer of 2385. After substituting a value of 8750 for titers reported as >8748, the corresponding median and geometric mean were 3521 and 1407, respectively. Positive IFN-γ ELISpot response rates to HIV Env, Pol or Gag peptide pools among vaccine recipients only, stratified by Ad5 Nab titer, are shown in . The frequency of response to any HIV antigen after rAd5 immunization alone was 3 of 4 (75%) for participants with negative Ad5 Nab titers, 14 of 18 (78%) for participants with Ad5 Nab titers of <1000, 7 of 12 (58%) for those with titers 1000 to 5000, and 5 of 12 (42%) for those with titers > 5000 (p=0.048). Pre-existing immunity to Ad5 had no effect (p=0.252) on HIV-specific response rates in prime and boost recipients where 6 of 7 (86%) with negative Ad5 Nab titers had a positive IFN-γ ELISpot response, compared with 24 of 39 (62%) with Ad5 Nab < 1000, 27 of 41 (66%) of those with titers 1000 to 5000, and 21 of 38 (55%) of those with titers over 5000. The magnitude of IFN-γ ELISpot responses for groups 1 and 2 combined and groups 3, 4 and 5 combined and stratified by baseline Ad5 Nab titer is shown in . A modest reduction in magnitude with increasing Ad5 Nab titer is apparent, though not statistically significant for both single rAd5 vaccination (p=0.184) and the prime boost regimens (p=0.595).
Effect of pre-existing Adenovirus 5 neutralizing antibody titer on HIV-1 specific interferon-gamma ELISpot reactivity
Figure 3 The maximum magnitude of IFN-γ ELISpot responses stratified by baseline Adenovirus type 5 neutralizing antibody titer of <12, 12–1000, 1000–5000 and > 5000. Results are expressed as spot forming units (SFU) per (more ...)