Vaccine and adjuvant: The vaccine is a recombinant parvovirus B19 vaccine composed of the VP1 and VP2 proteins of the virus [11
]. VP1 and VP2 were expressed in a baculovirus system in which the 2 capsid proteins self-assemble into virus like particles (VLP), as previously described [10
]. The capsid preparation was approximately 22.6% VP1 and about 77.4% VP2, meeting pre-specified specifications. Vaccine was supplied by Meridian Life Science, Inc. (fdba Viral Antigens, Inc.,Memphis, Tennessee) and has a designation of VAI-VP705 (referred to as parvovirus B19 vaccine in the remainder of the text). Tests for the presence of contaminating Sf9 host cell DNA was evaluated by quantitative PCR at the unprocessed bulk stage (the stage most likely to contain such contaminants) and was found to be below the detectable limit of the assay. The concentration of VLP proteins was determined by bicinchoninic acid assay (BCA). Purity of the preparation was evaluated by SDS-PAGE and silver staining and the ratio of VP1 to VP2 proteins comprising the VLPs was determined by SDS-PAGE and silver staining.
MF-59 (Novartis) is an oil (squalene)-in-water emulsion that was used at a concentration of 10 mg of squalene/dose. The vaccine and the adjuvant were available in separate vials, and were mixed prior to administration because the combined product was not stable for long periods of time. A diluent (20 mM tris buffer with 5% sucrose and 0.005% tween-80, pH 7.5) was used instead of the adjuvant for subjects randomized to the non-adjuvanted arm of the study. Placebo recipients received saline.
Subjects were healthy 18 to 45 year old adults recruited at Cincinnati Children's Hospital Medical Center and Baylor College of Medicine. Subjects were screened for health status by history, vital signs and laboratory studies including negative tests for active HIV, hepatitis B and C infection, and normal hemoglobin, white blood counts and alanine transaminase (ALT). Only subjects, who were seronegative for antibodies to parvovirus B19, as assessed by a commercial ELISA assay, were enrolled into the study.
The study was approved by the Institutional Review Board at each site. Written informed consent was obtained before performing any study procedures. A Safety Monitoring committee (SMC) comprised of 3 Independent Safety monitors periodically reviewed and evaluated the accumulated study safety data in a blinded fashion.
This study was a Phase I/II randomized, placebo-controlled, double-blind clinical trial evaluating the safety and immunogenicity of 2 dose levels of parvovirus B19 vaccine. Eighty-nine parvovirus B19 seronegative healthy adults were to be randomized to 1 of 4 groups: 26 to each vaccine group (parvovirus B19 2.5 μg with the adjuvant MF59, 25 μg with the adjuvant MF59, 25 μg without the adjuvant MF59) and 11 subjects to a placebo group (saline control). Each participant was to receive 3 separate vaccinations (at 0, 1, and 6 months).
Study participants were followed to evaluate safety and immune response. Safety was assessed by clinical evaluation and laboratory parameters on days 7 and 28 after each injection. Specific solicited injection site and generalized adverse events (AEs) were assessed for 7 days following vaccination beginning on the day of vaccination using a memory aid completed daily by the subject. The severity of solicited events was graded on a scale of 1-3, corresponding to mild (symptom present but does not interfere with activity), moderate (interferes somewhat with activity) and severe (incapacitating). Antibody responses were to be measured at 28 days after the administration of each vaccine and at the 6-month follow-up visit.
A commercial parvovirus B19 IgG ELISA (Biotrin International, Dublin, Ireland) was performed according to the manufacturer's instructions to screen all subjects for antibody and to assess for seroconversion after the first and second dose. Results are reported as positive or negative. The absorbance (optical density) is also provided as a semi quantitative measure of activity. Two subjects who had a negative result on screening were found to have detectable antibody when retested just prior to vaccination. These subjects were not included in the analysis of immunogenicity.
The neutralization assay was performed in triplicate using methods similar to those previously described [15
] . Briefly, 10 μL of virus (2 × 107
genome equivalents/ml) and 10 μL of serum dilutions of 10, 100, 1000) were pre-incubated in 96-well plates for 1 hour at room temperature. Then, ~2×104
UT7/Epo-S1 cells in 10 μL were added and allowed to incubate at 4°C for 2 h. After the incubation, 70 μL of culture media was added to the well, bringing the final volume to 100 μL. The cells were incubated at 37°C with 5% CO2
and harvested for analysis on day 3.
RNA was then extracted from 25 μL of cells using the TurboCapture 96 mRNA Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocols. mRNA was converted to cDNA using MMLV-RT (Invitrogen, Carlsbad, CA, USA) and 10 ng/μL Random Primers (Invitrogen) following manufacturer's protocol scaled up to a 50 μL reaction volume. Parvovirus B19 RNA transcripts were quantitated by real-time RT-PCR designed to amplify products in the NS regions using the PerfeCTa™ Multiplex qPCR SuperMix (Quanta Biosciences Inc., purchased through VWR Scientifics, West Chester, PA, USA). In a final reaction volume of 25 μL, the PerfeCTa™ Multiplex qPCR SuperMix was combined with 5 μL of the RT reaction, 0.4 μM of the NS amplification primers (5’- GGGCAGCATGTGTTAAAGTGGA - 3’ and 5’- TGGCCATTGCCAAGTTTGT - 3’, and 0.2 μM of the NS probe TYE665-TTATGGGCCGCCAAGTACAGGAAA-IB RQ. After an initial activation step of 3 min at 95°C, 45 cycles of 15s at 94°C and 60s at 60°C were performed, the number of transcripts was determined by comparison against a standard curve created using serial dilutions of pYT103, a plasmid containing the parvovirus B19 coding region. To confirm extraction of RNA and to normalize the number of transcripts per cell, quantitative RT-PCR (qRT-PCR) was performed using the same amplification conditions, but with primers β-actin F (5’- GGCACCCAGCACAATGAAG - 3’), β -actin R (5’- GCCGATCCACACGGAGTACT - 3’) and β-actin probe (MAX550-TCAAGATCATTGCTCCTCCTGAGCGC-IB FQ). An actin standard curve was obtained from serial dilutions of a plasmid containing an extended region of the actin coding sequence. All probes and primers were purchased from Integrated DNA Technologies (Coralville, IA, USA). Inhibition of at least 50% virus transcript copies compared to the negative control serum was defined as a positive response. The neutralization titer was defined as the highest serum dilution that met the definition of a positive response. For the calculation of GMT a value of 5 was used for samples that were not positive at the lowest dilution tested (1:10)
The sample size for this study of this newly manufactured vaccine was selected to provide preliminary information on safety and immunogenicity. The sample size chosen (26 subjects per vaccine group and 11 placebo recipients) had at least 72% power to observe 1 or more SAEs of a particular type in a single active vaccine group when the underlying probability was 5% or greater. Combining the vaccine groups, this power is increased to 92%. Due to the adverse events discussed below, full enrollment was not achieved. Forty-three subjects received the first dose and were available for safety analyses and twenty-eight were available for the main immunogenicity analyses distributed across four groups; substantially reducing the power to detect statistically significant differences. Student's T-test was used to compare ELISAs and log transformed neutralization results. Chi-square tests and McNemar's test were performed to compare response rate and response concordance. However, descriptive statistics should be viewed as the principal summary of the data.