Study Design
Full details on the design of this phase Ib/IIa, study have previously been reported [13]. In brief, the study which included HIV+ subjects, aged 18–55 years was conducted at The Ottawa Hospital Clinical Investigation Unit, Ottawa, Canada. The study was approved by The Ottawa Hospital Research Ethics Board. Subjects were on highly active antiretroviral therapy (HAART) for a minimum of six months, with CD4 T lymphocyte counts ≥ 200 cells/μL and HIV RNA < 50 copies/mL for a minimum of three months. Subjects included in this component of the study all had anti-HBs titres <10 mIU/mL, half being vaccine naïve and half having failed a previous course of 3 or more doses with a commercial HBV vaccine. Subjects were anti-HBc, HBsAg and HBV DNA negative. These susceptible subjects were randomized to receive Engerix-B (GlaxoSmithKline, Rixensart BE) or Engerix-B admixed with CPG 7909.
Vaccines and Control Injections
All subjects were dosed at 0, 1 and 2 months and received two intramuscular injections, one into each deltoid, of an adult dose of Engerix-B, thus a total of 40 μg HBsAg adsorbed to alum. Experimental vaccine recipients also received 0.5 mg CPG 7909 in 100 μl mixed with each injection of vaccine for a total dose of 1 mg CPG 7909. Control vaccine subjects received 100 μl of saline added to each vaccine injections. In all cases, the volume injected into each arm was 1.1 mL. CPG 7909, a B-Class CpG ODN of the human optimized sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' was synthesized with a wholly phosphorothioate backbone (Coley Pharmaceutical Group, Wellesley MA).
Immunological Evaluations
Lymphocyte proliferation assay (LPA)
To assess cell-mediated immune responses, whole blood was collected from all subjects at baseline and at 4, 8, 12, 24 and 48. Peripheral blood mononuclear clear cells (PBMC) were isolated from whole blood by Ficoll-Paque™ Plus (Pharmacia Fine Chemicals, Piscataway, NJ) gradient separation, washed twice in PBS and resuspended in RPMI medium 1640 (Invitrogen, Auckland, NZ) supplemented with penicillin-streptomycin (Invitrogen, Auckland, NZ) and 5% AB serum (Sigma, St. Louis, MO). PBMC were plated in triplicate at 3 × 105 cells/well and stimulated with various antigens. Vaccine antigen specific responses were assessed using recombinant yeast-derived HBsAg (2.5 mg/ml; subtype ad; International Enzymes Inc, Fallbrook, CA). Antigen used to assess HIV specific responses was HIV-1 gag p24 (5 mg/ml) (Immunodiagnostics, Inc, Woburn, MA). Mitogens, irrelevant antigens, and negative controls included: 1) pokeweed mitogen (PWM) (0.1 mg/ml; Sigma, St. Louis, MO), 2) tetanus toxoid (2 LFU/ml; Massachusetts Public Health Biologic Laboratories, Boston, MA), 3) Candida albicans antigen (10 mg/ml; Greer laboratories, Lenoir, NC), 4) cytomegalovirus (CMV), CF antigen strain AD169 (1:100; Biowhittaker, Walkersville, MD) 5) varicella-zoster virus (VZV) (1:100; Biowhittaker), 6) Keyhole limpet hemocyanin (KLH) (50 mg/ml; Sigma) and 7) no antigen. Cells were incubated for 6 days at 37°C prior to pulsing with 3H-thymidine (1 mCi/well) for a further 6 hours. Plates were harvested on glass fibre filters and counted in a 1450 microbeta Trilux scintillation counter (Wallac, Boston, Ma). Stimulation index (SI) was calculated by dividing the counts per minute (cpm) in the stimulated wells by the cpm from control unstimulated wells.
Whole blood immunophenotyping
Peripheral blood samples were collected in a 5 ml vacutainer containing EDTA. Whole blood was incubated with BSA-Cy5 (Amersham Biosciences, Piscataway, NJ) for 10 minutes prior to staining with antibodies conjugated to fluorescein isothyocyanate (FITC), phycoerythrin (PE) or phycoerythrin-cyanin 5.1 (PC5) for 25 minutes in the dark. The antibodies specific for cell surface markers that were utilized were anti-CD3 (UCHT1), CD4 (13B8.2), CD8 (B9.11), HLA-DR (Immu-357), CD38 (T16), CD28 (CD28.2), CD45RA (ALB11), CD45RO (UCHL1), CD62L (DREG56), (all from Beckman coulter) and anti-CD95 (DX2) (BD Pharmingen, Mississauga, Canada). Lysing and fixing of cell preparations were performed using the ImmunoPrep™ reagent system in a Coulter Multi-Q Prep (Beckman-Coulter, Inc., Fullerton, CA). Ten thousand events were acquired on a Beckman Coulter ALTRA flow cytometer.
Statistical Analysis
The number of patients in this study was chosen to identify important differences in measures of safety between subjects receiving CPG 7909 and those receiving placebo with their Engerix-B vaccines. Immunologic measures were summarized using group-wise means and 95% CI. Potential differences between the two treatment groups were evaluated using a repeated measures analysis of variance. The SAS mixed procedure was used with an autoregressive covariance structure. Since this method requires equal time intervals between measurements, two analyses were done for each dependent measure – one using weeks 4, 8 and 12 and a second using weeks 24 and 48. Three effects were modeled: treatment group, time and the treatment group by time interaction. The proportion of subjects with a positive proliferative responses (SI>5) in each group was compared using Chi square test.