Safety and Immunogenicity of a Recombinant Parvovirus B19 Vaccine Formulated with MF59C.1

Parvovirus B19 produces mild, self-limiting illness in immunocompetent, hematologically normal individuals; however, it can cause a variety of more serious conditions, including erythema infectiosum in children, transient aplastic crisis in patients with hemolytic anemia, chronic red blood cell aplasia and anemia in immunodeficient patients, persistent arthropathy in adults, and hydrops fetalis or abortion in pregnant women [1 –3]. Antiviral drugs are not available for the treatment of parvovirus B19 infection, and no vaccines for the virus are currently approved

A recombinant human parvovirus B19 vaccine (MEDI-491; MedImmune) composed of VP1 and VP2 was expressed in a baculovirus system in which the 2 capsid proteins self-assemble into viruslike particles devoid of viral DNA. Multiple linear neutralizing epitopes are present in the VP1 unique and VP1/VP2 junction regions. A recombinant peptide corresponding to aa 60–100 of the VP1 unique region, including a sequence with known neutralizing activity, reacted with IgG in 86% of serum samples from human volunteers with documented signs of past B19 infection [4]

In a previous study, MEDI-491 formulated with aluminum hydroxide was found to be safe and well tolerated but failed to induce virus-neutralizing antibodies (J. Balsley, unpublished data). Subsequent preclinical studies identified MF59C.1 (Chiron), an oil-in-water emulsion (squalene) in citrate buffer, as a potent adjuvant for MEDI-491 [5]. MF59C.1 adjuvant has been extensively tested in humans [6] and was selected as the adjuvant in the present study

Materials and methodsParvovirus B19 empty capsid particles consisting of ∼25% VP1 and ∼75% VP2 were produced using a recombinant baculovirus system, as described elsewhere [7]. Surfactant-stabilized emulsion adjuvant (MF59C.1) was supplied by Chiron. MF59C.1 is an oil-in-water formulation that contains surfactants (Tween 80 and Span 85), along with squalene emulsified under high-pressure conditions [8]

Twenty-four healthy, parvovirus B19–seronegative adults aged 18–45 years were randomly assigned in a double-blind fashion to receive either 2.5 or 25 μg of MEDI-491 in 0.5 mL of MF59C.1 intramuscularly at 0, 1, and 6 months. Safety was assessed through collection of solicited (days 0–7 after each dose) and unsolicited (days 0–28 after each dose) information about adverse events. Reports of serious adverse events were collected through 28 days after the final dose. Blood was collected for hematologic testing and serum chemical analysis and urinalysis were performed 1 week after each dose of vaccine

Parvovirus B19–specific IgG in serum samples was measured by ELISA. In brief, Immulon II plates (Dynex) were coated with PBS containing 0.25 μg/mL parvovirus B19 capsids and incubated overnight at 4°C. Plates were washed in PBS containing 0.1% Tween 20 and incubated at room temperature with blocking buffer. After washing, serum diluted in blocking buffer (total volume, 100 μL) was added, and plates were incubated at room temperature. The plates were washed and incubated with goat anti–human horseradish peroxidase–conjugated IgG (BioSource International). The plates were washed and developed with peroxidase substrate (Kirkegaard & Perry), and absorbance was read at 405 nm using a plate reader (Dynex). The end-point titer was the highest serial dilution that yielded an optical density greater than twice the optical density of the negative control serum at the same dilution. Seroconversion was defined as a 4-fold increase in the end-point titer over the baseline value. For study day 0 samples, seronegativity was defined as an optical density for a 1:100 dilution of serum that was <3 times the optical density of the blank

Parvovirus B19–neutralizing antibody was measured using a reverse-transcriptase polymerase chain reaction (RT-PCR) assay. Megakaryocytoblastic UT-7 cells adapted to growth in erythropoietin (UT-7/Epo cells) were provided by N. Komatsu (Jichi Medical School, Tochigi, Japan) [9, 10] and maintained in RPMI 1640 medium containing 10% fetal calf serum and recombinant erythropoietin (R&D Systems). For neutralization assays, 5×10⁵ UT-7/Epo cells/well were seeded in a 24-well plate (Falcon/VWR). Test serum was added in serial dilutions of 1:200 to 1:10,000, and viremic serum was simultaneously added at a final dilution of 1:25,000. Infected cultures were incubated at 37°C for 36 h, after which cells were harvested and total cellular RNA was collected (RNAgents kit; Promega). Parvovirus infection was demonstrated by RT-PCR detection of spliced parvovirus mRNA species [11]. Cellular β-actin mRNA served as a control; primers designed to overlap splice donor/acceptor sites were used

Total cellular RNA was diluted in nuclease-free distilled water and treated with RQ1 RNase-free DNase (Promega). After DNase treatment, RNA samples were heated to inactivate added DNase and to denature RNA. Heat-denatured RNA then was stored on ice. Transcription of cDNA was primed with the ACT-4 primer (5′-GGAGCAATGATCTTGATCTTC-3′) and the VP-B primer (5′-AGCATCAGGAGCTATACTTCC-3′) in a reaction mix containing 1 μL of a 2.5 μM solution of each primer, 2 μL of a 25 mM stock of dNTPs, 5 μL of 5× RT buffer, 2 μL of 0.1 M dithiothreitol, 0.5 μL of RNase inhibitor, and 0.5 μL of murine leukemia virus RT (Promega) in a 25-μL reaction volume. Each reaction was incubated for 60 min at 37°C and then heat-inactivated at 65°C for 5 min. An equal volume (4 μL) of each RT reaction product was amplified in a 50-μL reaction mix containing 2 μL of a 25 μM stock of each primer, 2 μL of a 2.5 mM dNTP stock, 10 μL of 5× PCR buffer, 10 μL of 25 mM MgCl₂, and 0.5 μL of Taq polymerase (Perkin-Elmer). Upstream primers for the PCR were ACT-3 (5′-GATGACCCAGATCATGTTTG-3′) and VP-A (5′-AAGTTTGCCGGAAGTTCCCG-3′). Thirty-five cycles of PCR were carried out: 94°C for 30 s, 58°C for 30 s, and 70°C for 2 min. A final extension step at 72°C for 10 min was performed before analysis of product amplimers by 2% agarose/ethidium bromide gel electrophoresis. Neutralization was reported as the highest dilution of serum that yielded no virus-specific RT-PCR products. All RT-PCR assays were performed in duplicate

All volunteers who received at least 1 immunization were included in the safety summaries. All volunteers who received 3 immunizations and had immunogenicity measurements were included in the immunogenicity summaries. One volunteer who received 2 immunizations (study days 0 and 28) was only included in immunogenicity summaries through study day 168. Immunogenicity parameters were summarized using 95% confidence intervals for geometric means. Single sample t distribution confidence intervals were first calculated for log-transformed data and then transformed back to obtain confidence intervals for the geometric means. Log-transformed geometric means were analyzed for differences between groups, using a 2-sample t test (assuming equal variances) and the Wilcoxon&amp;rank sum test on each study day. Group means were analyzed for boosting (4-fold increase) after the third dose, using a 1-sample t test and the Wilcoxon signed-rank test

ResultsTwo-thirds of the volunteers were female, and the majority of the subjects (79%) were non-Hispanic white. Ages ranged from 19 to 44 years, with a mean age of 31 years. Sex, race/ethnicity, and age were balanced across treatment groups

Twenty-three of 24 volunteers received 3 immunizations with MEDI-491. One volunteer in the 25-μg–dose group became pregnant after the second dose and did not receive a third dose. Twenty-two volunteers completed the study. Two volunteers in the 2.5-μg–dose group were considered to be lost to follow-up at study day 365

Ten of 12 volunteers in the 2.5-μg–dose group and all 12 volunteers in the 25-μg–dose group made a solicited report of at least 1 adverse event within 7 days of immunization (table 1). The rate and intensity of these adverse events did not increase with vaccine dose or number of immunizations. Mild or moderate injection site pain was reported by 75% and 92% of the volunteers in the 2.5-μg– and 25-μg–dose groups, respectively. Fever, headache, gastrointestinal symptoms, and fatigue related to vaccination occurred in 3–6 volunteers in each dose group and were mild or moderate, except for 1 volunteer who reported severe fatigue for 6 days after the first immunization. Resolution of solicited adverse events occurred within 48 h for 92% of events. There was no trend toward longer duration of the events with higher dose or repeated immunization

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Figure 1

Geometric mean ELISA and neutralizing antibody titers for volunteers who received the vaccine MEDI-491 against parvovirus B19. ELISA end-point titers for the 2.5-μg–dose (circles) and 25-μg–dose (squares) groups are shown as solid lines. ELISA detects IgG bound to captured B19 capsids. Neutralizing antibody end-point titers are shown as dashed lines. End-point neutralization titers are the highest dilution of serum that yielded no parvovirus B19–specific reverse-transcriptase polymerase chain reaction products. Vertical bars show 95% confidence intervals

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Table 1

Number of volunteers with solicited reports of adverse events after vaccination with MEDI-491 against parvovirus B19, by association with study vaccine

One or more unsolicited reports of adverse events were made by 12 subjects in the 2.5-μg–group and by 10 subjects in the 25-μg–dose group. These adverse events were mild or moderate, except for a severe corneal abrasion. All adverse events from unsolicited reports, except for 2 episodes of myalgia, were considered to be unrelated to study vaccine. One volunteer in the 25-μg–dose group became pregnant after receiving the second dose of study vaccine and discontinued the vaccination program. She later delivered a healthy, full-term baby. The child was not tested for the presence of antibodies against parvovirus B19. No clinically significant changes were observed in mean hematologic, serum chemistry, or urinalysis values in either dose group. There were no deaths or serious adverse events during the study

All volunteers seroconverted to parvovirus B19 positive after receiving at least 2 doses of vaccine. ELISA end-point titers and neutralization titers for both dose groups are presented in figure 1. Geometric mean ELISA titers increased after the first immunization, peaked at 28 days after the second immunization, and decreased before the third immunization. After the third immunization, titers increased further and continued to increase for 27 days, after which they began to decrease. ELISA titers were significantly higher in the 25-μg–dose group at study days 28, 56, 168, and 196 (P<.05). Boosting after the third dose (4-fold increase on study day 196, relative to titers on study day 56) was observed in 7 of 11 volunteers in each group. All volunteers developed virus-neutralizing antibodies. Peak titers of neutralizing antibodies occurred after the third immunization, and levels remained high for at least 1 year after the first immunization

DiscussionMEDI-491, a recombinant parvovirus B19 vaccine formulated with MF59C.1, was found to be safe, well tolerated, and highly immunogenic. Mild-to-moderate pain at the injection site and mild-to-moderate general symptoms, including headache, low-grade fever, gastrointestinal complaints, and fatigue, were reported at rates that were consistent with those reported for other subunit vaccines administered with MF59C.1 [12 –14]. All subjects in both vaccine dose groups developed virus-neutralizing antibodies that were sustained for at least 6 months after the third dose. The immune responses to MEDI-491 formulated with MF59C.1 were much greater than those observed in an earlier phase 1 study of MEDI-491 that used aluminum hydroxide as an adjuvant (J. Balsley, unpublished data). In that study, neutralizing antibody titers were detected in only 1 volunteer, and the titer was low. MF59C.1 was, therefore, an effective adjuvant for MEDI-491 and enhanced the production of high titers of neutralizing antibodies against parvovirus B19. Although this was not specifically evaluated in the trial described here, it is highly unlikely that administration of the adjuvant alone would stimulate production of parvovirus B19–neutralizing antibodies

When adjusted for total concentration of IgG, the serum neutralizing antibody titers reported here were similar to neutralization titers measured in convalescent-phase serum samples from individuals naturally infected with parvovirus B19 and higher than those measured in commercial gamma globulin preparations [8]. Interestingly, such preparations have been successfully used in placental-exchange transfusions to prevent fetal hydrops due to parvovirus B19 infection during pregnancy [15]. The vaccine, therefore, induced neutralizing antibody titers that may be sufficient to mediate protection against parvovirus B19 disease. Children with sickle cell disease are at particularly high risk for significant morbidity following parvovirus B19 infection, and targeted immunization of these children could potentially reduce the risk of life-threatening parvovirus B19–mediated disease, including transient aplastic crises

• Received February 22, 2002.
• Revision received October 30, 2002.

• © 2003 by the Infectious Diseases Society of America