The primary objectives of dose-ranging V520 Protocol 007 and Protocol 012 were to assess the safety, tolerability, and immunogenicity of a three-dose regimen of Ad5-vectored HIV-1 vaccine candidates. The primary immunogenicity endpoint was specified per protocol as the proportion of subjects with a positive unfractionated Gag-specific interferon (IFN)-γ ELISPOT response to 15-mer peptides using peripheral blood mononuclear cells (PMBC) obtained at week 30 (4 weeks after the last dose).
Vector construction and vaccine composition
Two similar adenoviral vectors were studied. Each vector was based on a replication-defective adenovirus type 5 backbone in which the deleted E1 gene region had been replaced with an expression cassette for the gag gene from the CAM-1 strain of HIV-1 (GenBank Locus BAA00992). This HIV strain was chosen because its gag gene closely resembles the clade B consensus amino acid sequence. The gag construct was ‘‘optimized’’ using nucleotide base substitution to improve protein expression in mammalian cells. The vaccine was formulated in a sterile solution of 10 mM TRIS buffer with 10 mM histidine, 5% sucrose, 75 mM NaCl, 1 mM MgCl2, 0.1 mM EDTA, 0.5% ethanol, and 0.02% polysorbate 80, pH 7.4. Placebo consisted of an identical vehicle without the adenovirus component.
The first vector studied was simply designated Ad5 HIV-1 gag. In this vector, the adenovirus E3 gene region had also been deleted. In preclinical studies, this vaccine appeared to be immunogenic and to have a safety profile that was acceptable for moving into human trials, culminating in Protocol 007 to study this vector in humans. After the initiation of Protocol 007, studies designed to support large-scale manufacture of the Ad5 HIV-1 gag vaccine revealed a degree of genetic instability in later passages of the vector that would make the vector unsuitable for large-scale production.
In an attempt to improve the genetic stability of the vector, the same gag
transgene was incorporated into a related Ad5 vector with a modified backbone designated MRKAd5 HIV-1 gag.26
The key differences between the original and the modified vector included thr following: (1) the orientation of the HIV-1 transgene was changed from antiparallel to parallel, (2) intron A of the CMV promoter was removed, (3) the 5′ adenovirus packaging region was modified, and (4) the adenoviral E3 region was reinserted into this backbone. The MRKAd5 HIV-1 gag
vaccine was used in Protocol 012. This modified vector was subsequently chosen for further development because no significant genetic instability could be demonstrated.
Both studies were Phase 1, placebo-controlled, multicenter, double-blind, randomized, dose-escalating trials. Eligible subjects were healthy HIV-seronegative adults aged 18–50 years who denied engaging in behaviors that would put them at high risk of exposure to HIV. Subjects had to be in good health as determined by medical history and physical examination, and laboratory tests of blood chemistry, hematology, liver function, renal function, and clotting function had to be within normal limits. Pregnant or breastfeeding women were excluded. Female subjects of childbearing potential were required to have a negative urine pregnancy test immediately prior to each injection and were counseled to avoid pregnancy during trial participation. Ongoing HIV risk assessment and prevention counseling was offered to the study participants during the trials. Each protocol was approved by the institutional review board at the participating sites. Written informed consent was obtained from all subjects.
The studies enrolled volunteers in successive dose-escalating stages such that clinical and laboratory safety data for 12 subjects (nine vaccine and three placebo recipients) at each dose level were reviewed and found to be acceptable by a Safety Evaluation Committee prior to enrollment of subjects in the next higher dose group. After the highest dose level had been satisfactorily tested, additional subjects were randomized across all dose levels. In Protocol 007, the dose levels of the Ad5 HIV-1 gag vaccine tested were 1×108, 1×109, 1×1010, or 1×1011 vp/dose. In Protocol 012, the dose levels of the MRKAd5 HIV-1 gag vaccine tested were 1×109, 1×1010, or 1×1011 vp/dose. Randomization was not stratified by pre-existing Ad5 immunity, except for the final stage in Protocol 012 when subjects were randomized across all dose levels.
Enrolled subjects were randomized to receive 1.0-ml injections of placebo or vaccine into the deltoid muscle. Allocation schedules were generated by computer. Doses were to be given at day 1, week 4, and week 26. Investigators, subjects, clinical safety monitors, and laboratory personnel performing the biological assays were blinded to treatment assignments. The studies were designed to have a total duration of 5 years, but subjects could discontinue from the parent study after 1 year to participate in another Phase I HIV vaccine trial.
Safety and immunogenicity assessments
Clinic visits for safety evaluations were scheduled 1 and 2 weeks after each dose. For 5 days after each injection, subjects were to record the largest diameter of induration or erythema at the injection site with a standard ruler. Injection site pain and tenderness were to be rated as ‘‘none,’’ ‘‘mild,’’ ‘‘moderate,’’ or ‘‘severe.’’ Subjects were to complete a vaccine report card tracking daily temperatures and physical complaints for 29 days after each dose. All adverse events were graded according to prespecified severity scales. Fever was defined as a temperature >100°F at any time within the 29-day period following each injection. Routine laboratory tests including a complete blood count, urinalysis, and serum levels of creatinine, amylase, prothrombin and partial thromboplastin times, C-reactive protein, creatine phosphokinase, phosphorus, and liver function tests were to be performed at each safety follow-up visit. Surveillance for shedding of the vaccine Ad5 strain was to be performed during the 2-week period following each injection by collecting pharyngeal adenovirus culture from subjects developing symptoms compatible with a viral respiratory infection or influenza-like illness (manifested as fever, chills, fatigue, and/or myalgia) and by collecting urine cultures from subjects experiencing symptoms of urinary tract infection or with asymptomatic hematuria or pyuria on routine urinalysis. Ad5 isolates were compared to the vaccine strain.27
CMI responses were measured by an unfractionated HIV-1 Gag-specific IFN-γ ELISPOT assay.28,29
A positive ELISPOT response required both ≥55 spot-forming cells (SFCs)/106
peripheral blood mononuclear cells (PBMCs) and ≥4-fold increase over the nonantigen control result.29,30
Serum Ad5 titers were measured at baseline and ~4 weeks after each dose in a quantitative neutralization assay using a recombinant adenovirus type 5.31
Combining data from protocols 007 and 012. Because the vaccine vector was the only meaningful difference between the protocols and was not expected to differentially impact the safety or immunogenicity results, it was decided after completion of the studies to perform analyses on the combined dataset. No formal test of homogeneity across studies was performed.
All subjects receiving one or more doses were included in the safety analysis. The proportions of subjects with injection-site reactions, systemic adverse events, and laboratory adverse events within 29 days after each dose were summarized by treatment group separately for the two baseline Ad5 antibody strata as well as for the two strata combined. For the combined strata, summary statistics were calculated using a weighted average of the observed stratum-specific percentages, with weights proportional to the overall observed stratum sizes. The frequencies of specific types of injection-site reactions, systemic adverse events, and laboratory adverse events in the vaccine dose groups were compared to the corresponding frequency in the placebo group by the Cochran–Armitage trend test, starting with all the vaccine groups and sequentially excluding the highest remaining vaccine dose group as long as the difference versus the placebo group remained significant as judged by a one-tailed p
-value <0.025. Since multiple adverse events were being compared separately, p
-values were deemed statistically significant only if they remained <0.025 after applying a multiplicity adjustment.30
Small sample sizes precluded comparisons between treatment groups within baseline Ad5 strata.
Only subjects receiving all three scheduled doses of vaccine or placebo and without major protocol violations were included in the analysis of immunogenicity. Three patients who acquired HIV infection were removed from the analyses. ELISPOT results were summarized by treatment arm at each time point in a manner analogous to that used for the safety analyses. Summary statistics included the proportions of ELISPOT responders and the geometric means of the quantitative ELISPOT responses. Differences in the frequencies of postprime (week 8) and postboost (week 30) ELISPOT responders between a given vaccine dose group and the placebo group were analyzed using the Cochran–Armitage trend test as detailed above. One-tailed p
-values <0.025 were considered statistically significant; a multiplicity adjustment was not required because a given dose was form ally compared to placebo only if the higher doses statistically differed from placebo.33
An analogous sequential trend testing approach was used for comparison of geometric mean ELISPOT values between each vaccine dose and placebo. The effect of baseline Ad5 titers on ELISPOT responses was explored using standard linear regression techniques.