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University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh
Primary Physicians Research, Pittsburgh, Pennsylvania
MedImmune Vaccines, Inc., Mountain View
UCLA Center for Vaccine Research, Research and Education Institute at Harbor-UCLA, Torrance, California
Children's Memorial Hospital, Chicago, Illinois
Center for Immunization Research, Johns Hopkins University, Baltimore, Maryland
Background.
A phase 2 trial was conducted to assess in young infants the safety, tolerability, infectivity, and immunogenicity of multiple doses of an intranasal vaccine using bovine parainfluenza virus type 3 (bPIV3).
Methods.
One hundred ninety-two healthy 2-month-old infants were randomized 1 : 1 : 1 to receive 1 × 105 median tissue culture infective dose (TCID50) bPIV3 vaccine, 1 × 106 TCID50 bPIV3 vaccine, or placebo at 2, 4, 6, and 1215 months of age. Safety information was collected by use of diary sheets and telephone interviews. Nasal wash and serum specimens were collected for assessment of infectivity and immunogenicity.
Results.
The safety profiles of both dosages of bPIV3 were similar to that of placebo, with the exception of fever with temperature of 38.1°C after dose 2 only, occurring in 34% of the 1 × 105 TCID50 group, 35% of the 1 × 106 TCID50 group, and 12% of the placebo group (P < .01). No vaccine-related serious adverse events were reported. The cumulative vaccine infectivity (isolation of bPIV3 and/or bPIV3 seroconversion) after dose 3 was similar in the 2 vaccine groups (87% in the 1 × 105 TCID50 group and 77% in the 1 × 106 TCID50 group) (P = .46). Seroconversion rates after dose 3, assessed by means of hemagglutination inhibition assay, after adjustment for decrease in maternal antibody titers, were 67% in the 1 × 105 TCID50 group, 57% in the 1 × 106 TCID50 group, and 12% in the placebo group (P < .01). Isolation of bPIV3 was common after dose 1, dose 2, or dose 3, but only 1 of 51 participants in the vaccine groups had bPIV3 isolated after dose 4.
Conclusions.
Multiple doses of bPIV3 vaccine were well tolerated and immunogenic in young infants.
Human parainfluenza virus type 3 (hPIV3) is an important respiratory pathogen in young children and accounts for 11% of pediatric hospitalizations for acute respiratory tract illnesses [12]. hPIV3 is second only to respiratory syncytial virus (RSV) as a leading cause of bronchiolitis and pneumonia in young infants [24]. Because of the burden of disease associated with hPIV3 in young infants, developing a safe and effective vaccine is an important public health priority.
Bovine parainfluenza virus type 3 (bPIV3) has been in clinical development for use in a live intranasal vaccine to protect against hPIV3 disease. The rationale for this approach is that (1) replication of bPIV3 is attenuated in nonhuman primates, presumably because of host-range restriction; (2) bPIV3 and hPIV3 share neutralization epitopes [5]; (3) vaccination of primates with bPIV3 induces cross-protective immunity against challenge with hPIV3 [67]; and (4) intranasally administered viral vaccines may offer the advantage of inducing local mucosal immunity, which may correlate with protection against hPIV3 disease. Furthermore, current advances in genetic engineering techniques have presented the possibility of using a suitably attenuated bPIV3 vaccine as a vector to deliver hPIV3 antigens and/or RSV antigens to protect against infection with hPIV3 and/or RSV [8, 9].
In phase 1 studies, 1 or 2 doses of bPIV3, administered by intranasal drops, were demonstrated to be safe and immunogenic in limited numbers of adults, children, and infants [1012]. The present study was designed to assess the safety, tolerability, infectivity, and immunogenicity of multiple doses of bPIV3 vaccine, administered by intranasal spray, to young infants and to select an optimal dose and schedule for a primary immunization series.
SUBJECTS, MATERIALS, AND METHODS
Study population.
Healthy infants 2 months of age were eligible to participate. Potential subjects were excluded from participation in the event of (1) immunodeficiency or immunosuppressive therapy of the child or a household member, (2) febrile or upper respiratory tract illness 3 days before enrollment into the study, (3) use of intranasal medications 1 week before enrollment into the study, or (4) previous receipt of blood products 2 months before enrollment into the study or expected receipt of blood products at any time during the study period.
Study design.
The study was conducted at 4 clinical study centers in the United States. Enrollment began in the fall of 1997, and participation was completed in the spring of 1999. The protocol was approved by accredited institutional review boards responsible for each center, and signed informed consent was obtained from each child's parent or guardian. The human-experimentation guidelines of the US Department of Health and Human Services were followed in the conduct of the clinical research. Subjects were randomly assigned in equal proportions, by use of computer-generated numbers, to 1 of 3 groups receiving either intranasal placebo (tissue culture medium) or 1 × 105 TCID50 or 1 × 106 TCID50 of the bPIV3 Kansas/15626/84 strain grown in fetal rhesus monkey lung cells. MedImmune Vaccines, Inc. (Mountain View, CA) acquired the rights to the bPIV3 vaccine from the National Institutes of Health (NIH) and used the same vaccine materials that were previously tested in the NIH phase 1 and phase 2 studies [1012]; the vaccine was retested for potency and packaged into nasal sprayers. Each dose was 0.2 mL (0.1 mL/nostril) and was administered by intranasal spray by use of the Becton Dickinson Accuspray that is identical to that used to administer the live attenuated influenza vaccine (FluMist; MedImmune Vaccines) [13]. A total of 4 doses of vaccine or placebo, depending on group assignment, were administered at 2, 4, 6, and 1215 months of age concurrently with routine pediatric vaccines, following the American Academy of Pediatrics recommendations (i.e., diphtheria, tetanus, acellular pertussis , Haemophilus influenzae type b conjugate , inactivated or oral polio virus [IPV or OPV], hepatitis B, measles, mumps, rubella, and varicella vaccines) (table 1). Parents/guardians and study staff were blinded to the administered treatment. Parents/guardians recorded potential adverse events on diary cards for 14 days after each dose of the vaccine and were contacted by telephone 14 and 30 days after each dose of the vaccine, to capture additional adverse events. Five serum specimens were collected from the participants: at baseline immediately before dose 1, at 2 months of age; before and 1 month after dose 3, at 6 months of age; and before and 1 month after dose 4 (booster), at 1215 months of age. At 3 of the 4 investigative sites, a single nasal wash specimen, for isolation of bPIV3, was collected from each subject 57 days after each dose. Virus culture specimens were collected for outpatient illnesses during the safety assessment periods (28 days after each dose), and these specimens were not collected to assess vaccine efficacy.
Virological assays.
Undiluted nasal wash specimens were stored frozen at -70°C until analyzed. Isolation of bPIV3 was performed by use of Madin-Darby bovine kidney cells. Viral isolates were identified as bPIV3 (vaccine) or hPIV3 (wild-type) by use of indirect immunofluorescence assay with specific monoclonal antibodies [1012].
Serological assays.
Hemagglutination inhibition (HI) antibodies against bPIV3 and hPIV3 antigens were measured by use of 0.5% guinea pig red blood cells and 4 hemagglutination units of hPIV3/Washington/1957 strain or bPIV3/Kansas/15626/84 strain viruses. The antibody titer was defined as the reciprocal of the highest serum dilution that completely inhibited hemagglutination, according to the standard procedure [14]. The starting serum dilution was 1 : 4, and the cutoff level of seropositivity was set at 4 [15, 16]. Titers of hPIV3 HI antibodies found at 2 months of age, considered to be of maternal origin, were used as the baseline to define hPIV3 seroconversion at 6, 7, 1215, and 1316 months of age, after adjustment of hPIV3 HI antibody titers for a 51-day biological half-life of maternal hPIV3 HI antibody titers [15, 16]. bPIV3 HI antibodies were also detected at 2 months of age, because of cross-reactivity between maternal hPIV3 antibodies and bPIV3 antigens. Therefore, bPIV3 seroconversion was calculated after adjustment for maternal bPIV3 HI antibody titers by use of the same adjustment method. bPIV3 seroconversion was defined as a 4-fold increase in bPIV3 HI antibody titers after adjustment for maternal bPIV3 HI antibody titers.
Statistical analysis.
Rates of adverse events were compared overall for the 3 treatment groups by use of 2-tailed Fisher's exact test. If statistical significance (P .05) or marginal significance was observed, pairwise comparisons between the groups were conducted. Seroconversion rates and rates of isolation of bPIV3 between treatment groups were also compared by use of 2-tailed Fisher's exact test. Exact 95% confidence intervals were obtained for these rates. hPIV3 and bPIV3 seroconversion rates within the same treatment group were compared by use of McNemar's test. A dose-response relationship was tested by use of the Mantel-Haenszel 2 test for trend. Statistical analyses were performed by use of SAS software (version 6.12; SAS Inc.) and StatXact software (version 3.1; Cytel Software).
RESULTS
Demographic characteristics.
A total of 192 participants64 in the 1 × 105 TCID50 group, 62 in the 1 × 106 TCID50 group, and 66 in the placebo groupwere enrolled. The 3 treatment groups were similar with respect to mean age at dose 1 of the vaccine (9.1, 9.2, and 9.4 weeks, respectively), sex (56%, 42%, and 52% male, respectively), and race/ethnicity (48%, 39%, and 44% Hispanic, respectively; 27%, 32%, and 36% white, respectively).
Safety.
Incidence rates of solicited adverse events after each dose of the vaccine are shown in table 2. After dose 1, the most frequently reported solicited adverse events in all 3 groups were runny nose/nasal congestion, irritability, and cough. In general, these events occurred at comparable rates in all groups after all 4 doses except after dose 2, when a significantly higher incidence of fever with temperature 38.1°C was found in the vaccine groups compared with the placebo group (P < .01, for each vaccine group compared with the placebo group) (table 2). The elevated temperatures occurred primarily on day 0 or day 1 after dose 2. There were no significant differences between the treatment groups in incidence of fever at higher temperature thresholds (39.0°C) after dose 2 (table 2). Interestingly, the placebo group had a significantly higher incidence of fever 14 days after dose 3 than the 1 × 105 TCID50 group (41% vs. 18%) (P = .012, by Fisher's exact test), and the placebo group had a significantly higher incidence of fever 14 days after dose 4 than both vaccine groups (55% in the placebo group vs. 30% in the 1 × 105 TCID50 group and 28% in the 1 × 106 TCID50 group) (P < .05, by Fisher's exact test) (table 2). After dose 2, cough occurred in 25% of the 1 × 105 TCID50 group and in 27% of the 1 × 106 TCID50 group, compared with 12% of the placebo group, but these differences were not statistically significant (P = .097 and P = .057, respectively, compared with the placebo group; P = .084, for 3 groups overall, by Fisher's exact test) (table 2). The cough episodes were evenly distributed over the 14 days. Rates of cough were comparable in the vaccine groups and the placebo group after dose 1, dose 3, and dose 4.
It is possible that high maternal bPIV3 HI antibody titers protected 2-month-old infants against fever with temperature 38.1°C and/or cough after dose 1 and that, by 4 months of age, the titers decreased sufficiently for the rates of these adverse events to increase after dose 2. To evaluate this possibility, we categorized bPIV3 HI antibody titers at 2 months of age into 4 groups (<4, 4, 8, and 16). No correlation between bPIV3 HI antibody titers at 2 months of age and incidence of fever with temperature 38.1°C and/or cough after dose 1 or dose 2 was observed in either vaccine group (table 3), which supports the hypothesis that the higher incidence of fever or cough after dose 2 was not related to the vaccine.
Eight serious adverse events, all of which resulted in hospitalizations, were reported, and 0 were classified as being vaccine-related. These included 4 events in 4 children in the 1 × 105 TCID50 group (parainfluenza type 1 culture-positive croup 8 days after dose 1, Streptococcus pneumoniae bacterial meningitis 30 days after dose 1, otitis media 12 days after dose 2, and elective surgery for correction of syndactyly 20 days after dose 4), 2 events in 2 children in the 1 × 106 TCID50 group (retropharyngeal abscess 11 days after dose 2 and acute otitis media and rotavirus culture-positive gastroenteritis in 1 child 29 days after dose 2), and 2 events in 1 child in the placebo group (bronchiolitis due to RSV at 38 days after dose 1 and an abdominal mass subsequently diagnosed as neuroblastoma presenting at 27 days after dose 2).
Infectivity.
The infectivity of the bPIV3 vaccine was assessed by measurement of the proportion of study participants who either had bPIV3 isolated in nasal wash specimens or had a 4-fold increase in bPIV3 HI antibody titers (table 4). This analysis was restricted to participants at the 3 sites at which isolation of bPIV3 was assessed. After dose 1, at 2 months of age, 38% of the 1 × 105 TCID50 group and 57% of the 1 × 106 TCID50 group had bPIV3 isolated (P = .20, by Fisher's exact test). After dose 2, at 4 months of age, 37% of the 1 × 105 TCID50 group and 34% of the 1 × 106 TCID50 group had bPIV3 isolated. After dose 3, a similar proportion of participants in each vaccine group (25%) had bPIV3 isolated. After dose 4, only 1 participant in the 1 × 106 TCID50 group had bPIV3 isolated. After dose 3, 0 participants in the placebo group had bPIV3 isolated, and 1 participant had hPIV3 isolated at day 6. The cumulative proportion of those in the vaccine groups who had bPIV3 isolated after dose 1, dose 2, or dose 3 was the same (79%). After dose 3, bPIV3 seroconversion rates in the vaccine groups were similar (67% in the 1 × 105 TCID50 group and 57% in the 1 × 106 TCID50 group) (P = .40, by Fisher's exact test). When rates of isolation of bPIV3 or of seroconversion were combined, after dose 3, the cumulative infectivity rate was 87% in the 1 × 105 TCID50 group and 77% in the 1 × 106 TCID50 group (P = .46, by Fisher's exact test). After dose 4, only 13% of participants in the vaccine groups developed increases in bPIV3 HI antibody titers.
To evaluate the correlation between passively acquired maternal bPIV3 HI antibodies and isolation of bPIV3 after dose 1 or dose 2, we categorized bPIV3 HI antibody titers at 2 months of age into 4 groups (<4, 4, 8, and 16). No correlation between bPIV3 HI antibody titers at 2 months of age and isolation of bPIV3 after dose 1 or dose 2 was observed in either vaccine group (table 3). The correlation between preexisting bPIV3 HI antibody titers and development of bPIV3 seroconversion after dose 1 or dose 2 cannot be evaluated, because serum specimens were not collected between dose 1 and dose 2. However, we were able to assess the impact of bPIV3 HI antibody titers on bPIV3 seroconversion after dose 3. Participants in the vaccine groups who had lower preexisting bPIV3 HI antibody titers had significantly higher bPIV3 seroconversion rates than participants in the vaccine groups who had higher preexisting HI bPIV3 antibody titers (P = .002, for the 1 × 105 TCID50 group; P = .013, for the 1 × 106 TCID50 group, by Mantel-Haenszel 2 test for trend) (table 5).
Immunogenicity.
bPIV3 and hPIV3 seroconversion rates are shown in table 6. At all 4 time points, bPIV3 seroconversion rates were significantly higher than hPIV3 seroconversion rates in the vaccine groups, indicating that an antibody response to the bPIV3 vaccine was more likely to be detected by the bPIV3 HI assay than by the hPIV3 HI assay. bPIV3 seroconversion rates increased steadily from 45% before dose 3, at 6 months of age, to 86% after dose 4, at 1316 months of age, in the 1 × 105 TCID50 group, and from 48% to 100%, respectively, in the 1 × 106 TCID50 group; these rates in the vaccine groups were significantly higher than those in the placebo group (9% before dose 3, at 6 months of age, and 50% after dose 4, at 1316 months of age) (P < .05, by Fisher's exact test). No vaccine was given between 7 months of age and 1215 months of age, but hPIV3 seroconversion rates during the interval increased from 16% to 47% in the placebo group, from 25% to 61% in the 1 × 105 TCID50 group, and from 21% to 46% in the 1 × 106 TCID50 group, indicating that exposure to hPIV3 was likely during the interval (table 6).
DISCUSSION
The present study showed that the bPIV3 vaccine was well tolerated and immunogenic in young infants. In a previous phase 1 study of 19 infants 26 months of age, the incidence of fever was not significantly higher in the 1 × 105 TCID50 group after dose 1, compared with that in the placebo group [11]. Although our data after dose 1 are consistent with those in the previous study [11], a higher incidence of low-grade fever after dose 2 in the vaccine groups was observed in the present study. This higher incidence of low-grade fever may be related to the combined effects of concurrent vaccination with multiple other vaccines (e.g., DTaP, Hib, IPV, or OPV) or may have occurred by chance. Similarly, the higher incidence of fever in the placebo group after dose 3 and dose 4 may be due to chance alone. Larger sample sizes will be required in future studies, to clarify any cause-effect relationship.
After dose 3, 82% (37/45) of those in the vaccine groups were infected with bPIV3, as measured by rates of isolation of bPIV3 or bPIV3 seroconversion, whereas, after dose 4, only low proportions of those in the vaccine groups had increased bPIV3 HI antibody titers (12/92; 13%) or shed virus (1/51; 2%) (table 4, data combined from the 1 × 105 TCID50 and 1 × 106 TCID50 groups). These data support the hypothesis that 3 primary doses likely provide sufficient immunity against hPIV3. Although data on seroconversion was combined with data on isolation of bPIV3 for the purpose of detecting vaccine infectivity in the present study, these rates may still underestimate the vaccine's infectivity, because viral cultures were obtained only once after each dose. The lower rates of isolation of bPIV3 after dose 1 in the present study (38%57% in the 2 vaccinated groups) is in contrast to those in a previous study of this vaccine, in which 11 of 12 vaccinees (92%) (26 months of age; mean age, 4.3 months) had bPIV3 isolated after dose 1 [11]. This difference may be explained by less frequent collection of nasal wash specimens (only once in the week after vaccination in the present study, compared with 4 specimens obtained 11 days after each dose of vaccine in the previous study).
Previous studies and the present study have shown bPIV3 to be safe and immunogenic in young infants [1012]. Recent in vitro and animal studies have confirmed the potential of bPIV3 vaccine to serve as a vector to express antigens of hPIV3 and RSV [8, 9]. Future development of the bPIV3 vaccine as a vector for hPIV3, RSV, and other respiratory virus antigens in humans is warranted.
Acknowledgment
We thank the study coordinators, referring pediatricians, and parents of participating infants. Karen Smail, Janet Mugger, Jill Schwalb, Nancy Lodell, Julie Iwashita, Iksung Cho, Stephen Chan, Denise Dawson, Tim Kang, Katie Komaroff, Sharon Mathie, Sandra Holmes, and Mengshu Zhao also contributed to this study.
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