Effects of Antigen Dose and Immunization Regimens on Antibody Responses to a Cytomegalovirus Glycoprotein B Subunit Vaccine
- Sharon E. Frey1,
- Christopher Harrison2,
- Robert F. Pass3,
- Ellen Yang1,
- Daniel Boken2,
- Rose E. Sekulovich4,
- Sandra Percell4,
- Allen E. Izu4,
- Sue Hirabayashi4,
- Rae Lyn Burke4 and
- Anne-Marie Duliège4
- 1Division of Infectious Diseases and Immunology, Saint Louis University Health Sciences Center, St. Louis, Missouri
- 2Pediatric Infectious Diseases Division, Creighton University Medical Center, Omaha, Nebraska
- 3Department of Pediatrics, University of Alabama at Birmingham, Alabama
- 4Chiron Corporation, Emeryville, California
- Reprints or correspondence: Dr. Sharon E. Frey, Saint Louis University Health Sciences Center, Division of Infectious Diseases, 3635 Vista Ave. at Grand Blvd., FDT-8N, St. Louis, MO 63110-0250.
-
Presented in part: 6th International CMV Conference, Perdido Beach, Alabama, 5–8 March 1997.
Abstract
The purpose of this phase I study was to evaluate the safety and immunogenicity of 2 doses of cytomegalovirus glycoprotein B (CMV gB)/MF59 vaccine at 3 different immunization schedules. Ninety-five volunteers were randomized to 6 groups. Antibodies to gB represent the majority of the CMV-specific neutralizing response. Three groups received 5 µg of gB antigen combined with MF59 (a proprietary adjuvant) and 3 groups received a 30-µg dose at 0, 1, and 2 months; 0, 1, and 4 months; or 0, 1, and 6 months. The vaccine was well tolerated, and there was no significant difference in antibody production between the 2 doses. The vaccine induced highest antibody titers when given at 0, 1, and 6 months. A low dose of CMV gB/MF59 may be the preferred dose for future studies.
Cytomegalovirus (CMV) is a β-herpesvirus that infects 40%–100% of the population, depending on socioeconomic class [1, 2]. Although infection with CMV is common, overt or recognized disease is not. The most devastating disease occurs in congenitally infected children and in immunocompromised hosts, that is, transplant patients and patients with AIDS [3–6]. CMV congenital infection is most often asymptomatic. However, it can cause CMV congenital disease, which is associated with severe morbidity and mortality. Finally, some children who are asymptomatic at birth can develop long-term neurologic sequelae such as sensorineural hearing loss and learning disabilities. CMV is the most common cause of virus-induced mental retardation and deafness in the United States [7–9].
Efforts are being made to develop a vaccine that could be used to immunize women of childbearing years to protect the fetus and neonate, and to decrease the incidence and severity of infection in individuals at risk of developing serious disease from CMV infection. A CMV vaccine has been developed that is a recombinant molecule based on subunit gB (glycoprotein B) expressed in Chinese hamster ovary cells (CHO) (Chiron, Emeryville, CA). The majority of the antibody response to the vaccine is directed to the ADI region of the gB, which is the principal neutralizing domain [10]. It has been shown that gB monoclonal antibodies directed against the ADI region can block virion entry into cells, cell-to-cell transmission of infection, and fusion of infected cells. Therefore, the use of gB as a vaccine may elicit protective responses in CMV-seronegative individuals.
As of 1998, this CMV gB (adjuvanted with alum or MF59) has been administered to approximately 600 CMV-seronegative and 100 CMV-seropositive adults enrolled in 10 CMV vaccine studies and has proven to be well tolerated. MF59 has been administered to more than 8000 individuals in a variety of vaccine studies; it is an oil in water emulsion in which squalene constitutes the oil phase. Previous phase I trials demonstrated that MF59, when administered with recombinant CMV, herpes simplex virus, or human immunodeficiency virus (HIV) antigens, induces antibody titers that are significantly higher than those induced by antigens administered with alum [11]. Although transient pain and tenderness is reported by most immunized subjects, MF59 is generally well tolerated [11].
On the basis of results from previous phase I dose-ranging studies, 5-µg and 30-µg doses were used in this study. We conducted a randomized, double-blind (to dose only), parallel, multicenter, phase I trial of Chiron Corporation CMV gB vaccine adjuvanted with MF59 in adults by using 2 different doses of antigen and 3 immunization schedules. The goal was to select a safe and tolerable dose of CMV gB/MF59 and a schedule that induces optimal immunogenicity in the shortest period of time.
Materials and Methods
Vaccines
Chiron Corporation CMV gB is produced in recombinant CHO cells. The adjuvant, MF59, is an oil-in-water microfluidized emulsion consisting of 5% squalene, 0.5% polysorbate 80, and 0.5% sorbitan trioleate. A deep intramuscular injection of 0.5–mL CMV gB is delivered in the deltoid muscle.
Volunteers
This study was approved by the institutional review board of each institution. The vaccines were administered to healthy adult volunteers. Volunteers were CMV-seronegative, as screened by CMV ELISA Test (Abbott Laboratories, Abbott Park, IL) within 30 days of the first immunization. Volunteers were included if they were aged between 18 and 50 years, gave informed consent, and were judged to be in good health from their medical history, physical examination, and screening laboratory parameters.
Criteria that excluded volunteers from participating in the protocol were a history of immunodeficiency, serious chronic disease, use of immunosuppressive drugs within 6 months of the study, unwillingness to practice acceptable contraception until at least 1 month after the third injection, being at high risk of acquiring CMV infection, acute or chronic infection, serious vaccine reaction in the past, substance abuse within the past 6 months, and a psychological disorder within the past year that would impede adherence to the protocol. Assessable volunteers satisfied the admission criteria, received at least 1 immunization, and completed at least the first 2-week follow-up visit.
Vaccination and study design
Ninety-five volunteers were randomly divided into 6 groups with approximately 15 individuals per group. Three groups received 5 µg of CMV gB, and 3 groups received 30 µg of antigen per injection. One group per each dose was assigned to 1 of 3 immunization schedules: 0, 1, and 2 months; 0, 1, and 4 months; or 0, 1, and 6 months. Screening and enrollment lasted 8 weeks, and all volunteers were followed up for 6 months after the last immunization.
Clinical assessment
Volunteers were observed for 30 min after each immunization and returned for follow-up at 48 h to assess local and systemic reactions. Diary cards describing local and systemic reactions were completed by the volunteers for 7 days after each immunization. Local injection site reactions included pain, erythema, induration, tenderness, temperature, and swelling. Systemic reactions included chills, nausea, malaise, myalgia, arthralgia, headache, rash, and fever ⩾38°C (100.4°F). Volunteers were asked to note the impact of these signs and symptoms on their usual work and social pattern. Adverse events were reported as mild (events that do not require a therapeutic intervention), moderate (events that require pharmacologic or other therapeutic interventions but the volunteer is still able to perform routine daily functions), and severe (the volunteer is unable to perform routine daily functions).
Clinical laboratories
Complete blood counts, differentials, platelet counts, alanine transferase, creatinine, and urinalysis were completed prior to the first and third immunization, 2 weeks after the third immunization, and 6 months after the third immunization. Urine pregnancy tests were completed on the same day prior to each immunization.
Serologic assays
Serum antibody responses to CMV gB were evaluated by ELISA and plaque-reduction assay on blood drawn prior to the first, second, and third immunizations, 2 weeks after the second and third immunizations, and 3 and 6 months after the third immunization. An ELISA assay developed and performed at Chiron Vaccines was used to measure IgG antibody to CMV gB; the CMV gB used in the vaccine was used as the coat antigen in this assay. The lower limit of the assay was a titer of 100, and endpoint titers were determined by use of 2-fold serial dilutions. The neutralizing antibody titers were measured by use of a standard plaque-reduction technique; the assay endpoint was calculated as the inverse of the serum dilution that produced a 50% reduction in plaque count in the stock pool of Towne CMV relative to the control wells. Sera from healthy seronegative adults and from those with naturally acquired CMV infection were used for negative and positive controls with each antibody assay; in addition, a CMV-seropositive serum was used as a reference on each plate.
Statistical methods
Clinical safety was determined by evaluating local and systemic reactions, adverse events, and clinical laboratory results on all volunteers receiving at least 1 injection of vaccine. The number and percentage of local and systemic reactions reported by volunteers within 7 days after immunization were calculated for the severity of each symptom. Differences among the groups were assessed by use of a χ2 test. The original terms used by investigators to identify adverse events were translated into COSTART terms and then grouped into frequency tables according to body system. The number and percentage of volunteers experiencing adverse events were tabulated by body system/preferred term and vaccine group. Clinical laboratory results were evaluated at baseline and at each follow-up visit by vaccine group and were presented as descriptive statistics. For continuous laboratory parameters, we used the Kruskal-Wallis test to compare mean change from baseline to each follow-up visit among the vaccine groups.
Immunogenicity was evaluated by calculating geometric mean titers (GMT) and 95% confidence intervals for CMV gB ELISA and CMV plaque-reduction assay by vaccine group and time. Because of the skewness of the titer data, a log transformation (base 10) was used. Differences among the 3 vaccine schedules and between the 2 dose levels at 2 weeks, 3 months, and 6 months after the third immunization in mean log10 CMV gB ELISA and CMV plaque-reduction (GMT) were analyzed by use of a 3-way analysis of variance (ANOVA) with factors for immunization schedule, dose level, center, and their interactions. The equivalence of the 2 dose-level concentrations (5/MF59 and 30/MF59) and the 3 vaccination schedules (months 0, 1, 2; 0, 1,4; and 0, 1,6) were assessed by use of 95% equivalence intervals (i.e., 90% confidence intervals) or GMTs at 2 weeks, 3 months. and 6 months after the third immunization. All statistical tests were conducted 2-sided at the 5% significance level.
Results
Volunteers
Fifty-one women and 44 men were enrolled. Ninety volunteers were white, 4 were “other,” and 1 was Asian. The median age was 25.8 years (range, 22–47.2 years). Seven volunteers withdrew prior to completion of the study: 1 as a result of inappropriate enrollment, 3 from adverse events not related to the vaccine, and 3 because of withdrawal of consent.
Clinical assessments
The incidences of pain at the injection site, warmth at the injection site, and myalgia were statistically significantly higher (P ⩽s .05) in the 30/MF59 group than in the 5/MF59 group; however, the incidence was similar for each schedule. During the first week after any injection, symptoms frequently reported were injection site pain (84%), headache (42%), and myalgia (29%). Symptoms infrequently reported were erythema >10 mm (14%), induration >10 mm (4%), chills (6%), nausea (13%), malaise (12%), arthralgia (8%), and fever ⩾38°C (4%). These symptoms were self-limited, and mild or moderate in most cases. No rash was reported. There was only 1 report of severe pain and 2 reports of severe headache.
Clinical laboratories
There was no significant change in any laboratory measurement, including complete blood count, liver enzymes, or renal function when the vaccine groups were compared.
Serologic assays
No statistically significant difference in ELISA anti-CMV gB/MF59 GMT was seen between the 5/MF59 and 30/MF59 dose groups after the third immunization (table 1). However, at 6 months after the third immunization, the geometric mean CMV gB ELISA antibody was higher in the group on the 0, 1, 6 month schedule (P = .049) than in the groups on the 0, 1, 2 month schedule or the 0, 1, 4 month schedule.
Table 1
Cytomegalovirus glycoprotein B (CMV gB) ELISA in previously seronegative healthy adults after third immunization with CMV gB and adjuvant MF59.
The CMV plaque-reduction GMT was significantly higher in the group of individuals on the 0, 1, and 6 month schedule at 2 weeks (P = .0004) after the third immunization than in the group of individuals on the 0, 1, 2 month schedule or the 0, 1, 4 month schedule. This statistically significant difference was not observed at 6 months after the third immunization (P = .20). No significant difference between the 5 µg and the 30 µg dose groups was seen (table 2).
Table 2
Cytomegalovirus (CMV) plaque-reduction assay in previously seronegative healthy adults after third immunization with CMV glycoprotein B and adjuvant MF59.
Discussion
This study investigates the difference in antibody responses between 2 different doses and 3 different schedules of CMV gB vaccine in healthy, CMV-seronegative adults. All groups had similar demographic characteristics. The vaccine was safe and well tolerated in this group of adults. A statistically significant difference was seen in the reactogenicity profile between the 5-µg and the 30-µg groups; that is, more pain and warmth was seen at the injection site. There was also increased myalgia. Increases in plaque-reduction titers were the largest in the 0, 1, 6 month group when compared with the other groups 2 weeks after the third immunization, but was lost by 6 months. Increase in titers has been observed with other subunit vaccines, and it highlights the importance of a rest period for maximum response of the immune system [12]. In addition, the lower dose of antigen induced similar antibody responses in the plaque-reduction assays, which probably reflects the effect of the MF59 adjuvant. These results suggest that a low dose of CMV gB/MF59 given at 0, 1, and 6 months may be the preferred regimen for future studies.
One potential use of this vaccine is to administer it to CMV-seronegative boys and girls during the adolescent years before they become sexually active, or during the childbearing years to prevent disease in the fetus and neonate [13, 14]. This would decrease the incidence of serious sequelae such as mental retardation and deafness secondary to CMV. Immunization of children during infancy may lead to a significant decrease in infection or modification of infection, resulting in decreased duration of shedding and thereby further decreasing the “reservoir” of infection for susceptible mothers. In a toddler study, the mean antibody GMT by ELISA was 82,051 two weeks after the third dose of 20 µg CMV gB/MF59, which is significantly higher than that seen in adults. This suggests a difference in response on the basis of age [15]. Further studies are needed to test the safety and immunogenicity of this vaccine in infants for potential administration shortly after birth (prior to attending day care centers), and in other persons at risk of serious infection, such as potential transplant donors and recipients and persons who are HIV seropositive. This CMV vaccine may also have potential value in boosting the immune system of those seropositive, immunocompromised individuals at risk for reactivated CMV disease.
Footnotes
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Informed consent was obtained from all patients, and human experimentation guidelines were followed in accordance with US Department of Health and Human Services and our institutional review board.
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Financial support: This work was supported by a grant from Chiron Corporation.
- Received January 11, 1999.
- Revision received July 12, 1999.
- © 1999 by the Infectious Diseases Society of America
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