Passive immunity is acquired in utero by placental transfer of maternal antibodies to the developing fetus, which peaks in the third trimester of pregnancy.1–4 Immunization of pregnant women during this period facilitates passive immunity in the neonate, offering direct protection from birth. Maternal immunization against tetanus has been implemented globally where indicated for more than 50 years and recently national recommendations for routine vaccination with influenza (during influenza season) and combined tetanus–diphtheria–acellular pertussis have become common.5,6
At birth, neonates have a developing immune system and are particularly vulnerable to infection by vertically acquired and postnatally acquired pathogens such as group B streptococci (GBS). Many countries recommend intrapartum antibiotic prophylaxis to women who test positive after screening for GBS colonization during pregnancy; because approximately 25–30% of women are carriers of GBS, this results in large amounts of intrapartum antibiotic prophylaxis being prescribed to otherwise healthy pregnant women.7–9 Although this approach has been associated with a lower incidence of early-onset cases of neonatal group B streptococcal disease,10 an associated decline in late-onset disease (after the first week of age), or in premature neonates11,12 has not been observed. A vaccine against group B streptococcal disease is considered to have the most potential for prevention of disease where antibiotics have limited or no effect or in settings where antibiotics are impractical or cannot be administered and to further reduce the incidence of early-onset disease without the need for intrapartum antibiotic prophylaxis administration.13
In this study, we evaluated the safety and immunogenicity of an investigational trivalent glycoconjugate group B streptococcal vaccine in pregnant women and their neonates in Belgium and Canada.
MATERIALS AND METHODS
This phase 2, randomized, observer-blind, multicenter study was conducted in Belgium (two sites) and Canada (three sites) between September 2011 and October 2013. The primary objective of this study was to evaluate the placental transfer of GBS-specific antibodies to newborns born to pregnant women administered an investigational trivalent CRM197-conjugated group B streptococcal vaccine or a placebo. Secondary immunogenicity objectives included evaluation of kinetics of GBS serotype-specific antibodies up to 3 months after delivery in immunized women and their infants and antibody concentrations in women 30 days postvaccination. In addition, infant responses to antidiphtheria vaccination were measured 1 month after the third vaccination to evaluate potential interference with routine vaccination. As an exploratory objective, the effect of time from vaccination to delivery on maternal and infant antibody concentrations at delivery and birth was assessed. Vaccine safety and tolerability in the maternal participants were evaluated by local and systemic reactogenicity, reporting of adverse events and obstetric outcome, and was evaluated in the infants by developmental outcome and serious adverse event reporting.
The trial was conducted in accordance with the Declaration of Helsinki and the principles of Good Clinical Practice. The protocol was reviewed and approved by the appropriate ethics review committees and institutional review boards of all involved study centers (Appendix 1, available online at http://links.lww.com/AOG/A746), and informed written consent was obtained from all women on behalf of themselves and their infants before enrollment in the trial. The trial was registered at www.clinicaltrials.gov (NCT01446289).
Healthy pregnant women aged 18–40 years at 24–35 weeks of gestation were enrolled. Women were excluded from participation if they had a history of allergic reaction or hypersensitivity to previous vaccines or vaccine components; known or suspected impairment or alteration of immune function; high-risk pregnancy, as determined by the investigator (eg, gestational diabetes, [pre]eclampsia, risk of preterm labor, history of pregnancy complications including stillbirth); receipt of any other investigational agent or intervention anticipated during the study; any acute, chronic, or progressive disease; any condition associated with prolonged bleeding; cognitive impairment or psychiatric disease that could interfere with the participant's ability to take part in the study; severe neurologic or seizure disorder; or human immunodeficiency virus infection.
Women were allocated to receive either the group B streptococcal vaccination or placebo injection in a 3:2 ratio (Fig. 1). A validated web-based randomization system assigned participants to treatment arms at the specified ratio. Blood samples (20 mL) were taken by venipuncture before vaccination on day 1, 30 days postvaccination (day 31), at delivery, and at 13 weeks postpartum (day 91). For analysis of neonatal immunogenicity objectives, blood samples were taken from cord blood at delivery (10 mL) to estimate transfer ratios and at 3 months of age (0.5 mL) to measure levels of anti-GBS antibody. If insufficient cord blood was available, heel-stick or venipuncture was performed on the newborn (0.5 mL). One month after the last diphtheria-containing vaccination (month 5 in Belgium and month 7 in Canada), blood was taken to test immune response to diphtheria in infants born to women in both the GBS-vaccinated and placebo groups.
One 0.5-mL, 5-microgram dose of the group B streptococcal trivalent investigational vaccine reconstituted with 0.9% sodium chloride contained 5 micrograms of each of capsular polysaccharides of serotypes Ia, Ib, and III, individually conjugated to CRM197. A single placebo injection consisted of 0.5 mL isotonic saline solution (0.9% sodium chloride). All vaccines and placebo were administered intramuscularly, into the deltoid muscle of the nondominant arm. Routine infant diphtheria vaccination was given to all infants at 2, 3, and 4 months of age in Belgium and at 2, 4, and 6 months of age in Canada, and all other recommended childhood vaccines (eg, Prevenar 13) were administered to infants as per local recommendations.
Participants were observed for the first 30 minutes after vaccination for any immediate reactions; they were provided with a diary card to record solicited adverse reactions for 7 days postvaccination and any unsolicited adverse events, that is, any untoward medical occurrence in a participant that does not necessarily have to have a causal relationship with the treatment. Adverse events were recorded up to delivery, and any adverse events requiring a nonroutine physician visit or leading to study withdrawal were recorded throughout the study. Serious adverse events were recorded throughout the study (ie, to 5 [Belgium] or 7 [Canada] months of age among infants). Solicited local adverse reactions were induration, swelling, ecchymosis, erythema, and pain, and systemic adverse reactions were chills, nausea, malaise, myalgia, arthralgia, headache, fatigue, rash, and fever (38°C or greater). Infant safety was assessed in terms of effect on infant development using Apgar scores (birth) and Denver Development Assessment II (1 month after final diphtheria vaccination) and reports of serious adverse events. Adverse events and serious adverse events were categorized by the investigator according to their likelihood of being related to vaccination.
For immunogenicity analysis, sera were collected, stored at −20°C and every 24 hours in batches transported on dry ice to the GSK Clinical Sciences Laboratory in Marburg, Germany. To assess GBS-specific antibody concentrations, 96-well plates were coated with 1 microgram/mL human serum albumin-conjugated GBS polysaccharide (Ia, Ib, or III). After washing and blocking with buffer containing 2% bovine serum albumin, the plates were dried and stored at 4°C until use. Serially diluted serum samples were incubated on the coated plates for 1 hour at 37°C and then the plates were washed three times. Detection was done using an alkaline phosphatase-conjugated goat antihuman immunoglobulin G secondary antibody for 90 minutes at 37°C. After three washes, 100 microliter SeramunGelb pNPP was added to the plates and incubated 30 minutes at room temperature and the reaction was stopped by adding 100 microliter SeramunGelb stopp. Optical density values at 405 nm were measured using a BEP III ELISA processor. Antibody concentrations were calculated from a standard curve using Mikrowin 2000. Group B streptococci serotype–specific antibody responses were reported as geometric mean concentrations in micrograms/mL. The mean of the logarithmically (base 10) transformed antibody concentrations was exponentiated to obtain the geometric mean concentration. Analysis of maternal antibody levels was also performed on subgroups of women stratified by baseline antibody levels: at or above the lower limit of quantification, or below the lower limit of quantification. The lower limit of quantification refers to the lowest antibody concentration that could be detected by the assay. Maternal antibody transfer rate was calculated as the paired ratio of the GBS-specific antibody concentration measured in the cord blood of the neonate and in maternal sera at birth among participants with quantifiable antibody levels. Diphtheria antibody levels were measured using standard enzyme-linked immunosorbent assay tests at Focus Diagnostic Laboratory in California.
No formal statistical hypotheses were tested in this study because the study was not powered to detect a difference between vaccine and placebo. With 60 participants in the vaccine group, the probability of observing at least one adverse event was 90% if the actual rate of the event was 3.8%; this formed the justification for the chosen sample size. All immunogenicity analyses were performed on the per-protocol data set, which consisted of women who correctly received the study vaccine or placebo, provided evaluable sera at all relevant time points, and had no major protocol deviations. The same criteria applied for infants but in addition their mother must have correctly received the study vaccine or placebo and had no major protocol deviations. Maternal antibody transfer ratios were modeled using an analysis of variance model with vaccine group and country as qualitative factors and presented as adjusted ratios with two-sided 95% confidence intervals (Clopper Pearson) calculated based on least square mean estimates from the model. Values for women with a lower limit of quantification at delivery were set to half the lower limit of quantification for analysis. Maternal antibody concentrations were modeled using an analysis of covariance model with vaccine group and country as qualitative factors and a common slope representing prevaccination antibody concentration. Infant antibody concentrations (GBS and diphtheria) were modeled as described for the antibody transfer ratios. In addition, trends in the effect of time from vaccination to delivery on maternal and neonatal antibody concentrations at delivery and birth were analyzed using linear regression. Safety data and infant developmental outcomes were evaluated descriptively. Safety data are presented for the safety data set, which included all women and infants who provided postvaccination safety data. All statistical analysis was performed using SAS 9.1.
A total of 86 pregnant women and their newborns were enrolled. The investigational group B streptococcal vaccine was administered to 51 women, and 35 women received placebo in accordance with randomization (Fig. 1). One woman (group B streptococcal vaccine group) and her infant did not complete the study after withdrawal of consent. Baseline characteristics were similar between groups (Table 1).
Three participants (one in the placebo group and two in the vaccine group) were excluded from the reactogenicity analysis (to day 7) because they did not provide any solicited adverse reaction data. Among the remaining participants, rates of reporting of solicited adverse reactions were very similar across the two groups; 54% (confidence interval [CI] 39.3–68.2%) of women in the vaccine group reported at least one solicited reaction compared with 53% (CI 35.1–70.2%) in the placebo group (Table 2). Rates of systemic reactions were also comparable in the two groups; however, more participants in the vaccine group reported local adverse reactions (40%) than in the placebo group (24%). No severe local or systemic reactions were reported among the vaccine group with 0–6% among the placebo group reporting severe reactions. The most commonly reported reactions were pain, headache, and fatigue.
Unsolicited adverse events were reported by 32 of 51 (63% [95% CI 48.1–75.9%]) and 26 of 35 (74% [95% CI 56.7–87.5%]) of the women in the vaccine and placebo groups, respectively. Of these, rates of at least possibly related adverse events, serious adverse events, and medically attended adverse events were comparable across the two groups. No single adverse event predominated with no more that four participants (11%) in either group experiencing the same adverse event (headache, hemorrhoids, nasopharyngitis, bronchitis, urinary tract infection). None of the participants withdrew from the study as a result of adverse events and there were no deaths. All women gave birth to single, liveborn neonates. Obstetric outcomes were similar between the vaccine and placebo groups (Appendix 2, available online at http://links.lww.com/AOG/A747).
Serious adverse events were reported in 24% and 31% of infants in the vaccine and placebo groups, respectively, with one event (neonatal asphyxia occurring 28 days after maternal vaccination) assessed by investigators as possibly related to group B streptococcal vaccination. No infant deaths occurred during the study and no infant adverse event caused withdrawal from the study. One- and 5-minute Apgar scores were comparable between groups (Appendix 2, http://links.lww.com/AOG/A747), and the percentages of infants with “normal” development scores were 71–96% and 93–100% for the infants tested at 5 and 7 months, respectively. There were no consistent differences in percentages of infants testing normal between the vaccine and placebo groups.
Maternal GBS-specific geometric mean concentrations were low at baseline and increased at subsequent time points postvaccination for each serotype. By day 91 postpartum, geometric mean concentrations had increased 19-fold, 33-fold, and 28-fold from baseline against serotypes Ia, Ib, and III, respectively (Table 3). Across the placebo and vaccine groups combined, 76%, 56%, and 64% of women had baseline antibody concentrations below the lower limit of quantification for Ia, Ib, and III serotype, respectively (lower limit of quantification: 0.326 micrograms/mL for serotype Ia, 0.083 micrograms/mL for serotype Ib, and 0.080 micrograms/mL for serotype III). When stratified by baseline lower limit of quantification status, geometric mean concentrations were substantially higher postvaccination for women who were at or above the lower limit of quantification at baseline compared with those at the lower limit of quantification at baseline and had higher geometric mean ratios at all time points postvaccination. Geometric mean ratios in the lower limit of quantification group were still considerably higher postvaccination than either placebo group at all tested time points (Appendix 3, available online at http://links.lww.com/AOG/A748). All the vaccinated women who were at or above the lower limit of quantification at baseline had antibody concentrations 0.5 micrograms/mL or greater at all tested time points postvaccination. There was no notable changes from baseline in percentages of women attaining different threshold antibody concentrations in either of the placebo groups (less than or at or above the lower limit of quantification).
Group B streptococci–specific antibody ratios between mother and infant were in the range 0.68–0.81 across the three serotypes for the neonates born to women who received the investigational group B streptococcal vaccine (serotype Ia: 0.81 [95% CI 0.72–0.91], serotype Ib: 0.77 [95% CI 0.62–0.97], and serotype III: 0.68 [95% CI 0.59–0.75]). Transfer ratios were artificially high for the placebo group (0.97 [95% CI 0.85–1.11], 1.44 [95% CI 1.06–1.94], and 0.99 [95% CI 0.83–1.18] for serotypes Ia, Ib, and III, respectively), which was an artifact of very low maternal serotype-specific antibody levels making the transfer ratio estimates unreliable.
In infants, persistence of maternally transferred GBS-specific antibody was measured 3 months after birth (Fig. 2). Antibody concentrations decreased after birth and by day 91 were 22–25% of the levels measured at birth and were still 5-fold to 8.5-fold higher than those observed in the placebo group. Geometric mean concentrations to infant diphtheria vaccine were 1.38 international units/mL (95% CI 1.13–1.69) for the investigational group B streptococcal vaccine group and 1.49 international units/mL (1.17–1.90) for the placebo group (P=.8).
There was no clear relationship between time from vaccination to delivery and maternal or neonatal antibody concentrations at delivery or birth for any of the serotypes (data not shown).
A maternally administered vaccine has been proposed as potentially the most effective preventative measure against group B streptococcal disease across the entire period of vulnerability in newborns.13 Previous phase 1 and 1b research on the investigational trivalent group B streptococcal vaccine tested in this study demonstrated that the vaccine was well-tolerated in nonpregnant women (unpublished data: Madhi SA, Lerouz-Roels G, Koen A, Jose L, Cutland C, Maes C, et al, presented at the 31st Annual Meeting of the European Society for Pediatric Infectious Diseases, 2013, Milan, Italy). In this study, we observed levels of antibody transfer of 66–79%, which are not dissimilar to transfer ratios seen after maternal immunization with other polysaccharide vaccines such as Haemophilus influenzae type b and meningococcal vaccines.2,14–17
Because a correlation between GBS-specific antibody concentration and vulnerability to infant group B streptococcal disease has been demonstrated,18 a robust antibody response has the potential to help provide high levels of protection of infants from group B streptococcal disease. Responses to group B streptococcal vaccination in the subgroup of women without detectable antibodies at enrollment were considerably lower than those who already had detectable concentrations before receiving the investigational group B streptococcal vaccine. Because the cutoff value for antibody concentration that correlates with protection against invasive group B streptococcal disease is not yet clearly established, we do not know whether the lower maternal antibody response to the vaccine in women without pre-existing antibodies is sufficient to protect their neonates. Still, women in the vaccine group who did not have detectable pre-existing antibodies reached postvaccination geometric mean concentrations comparable with those of the women in the placebo group who did have pre-existing antibodies, whereas women in the placebo group who did not have pre-existing antibodies had no measurable response.
Antibody levels in vaccinated mothers continued to rise until at least 3 months after giving birth, indicating that timing of vaccination in pregnancy may be important. Because we found that there was no relationship between the time from vaccination to delivery and the antibody concentrations in women or neonates at delivery, administration of a dose earlier in pregnancy may be immunologically feasible. Alternatively, although operationally challenging, it could be envisioned to administer a single dose during adolescence as a “priming dose” followed by a booster administered during pregnancy or even during adolescence before pregnancy provided long-lasting immunity could be demonstrated.
In a previous study trialing the investigational vaccine in nonpregnant women in Belgium, a much higher rate of solicited reactions and unsolicited adverse events was reported with the same dosage and schedule of the vaccine (100% and 80% reported at least one solicited reaction or adverse event, respectively) (Madhi et al). Thus, the difference observed between this study and previous studies may be specific to the study participants and highlights the importance of the inclusion of an appropriate control group in each of the studies as a comparison. As expected as a result of the exclusion of patients with risk factors for preterm birth, the observed rates of preterm births across groups (2.3%) were considerably lower than those generally reported in Belgium and Canada.19
Because the tested investigational group B streptococcal vaccine contained CRM197, there was a hypothetical possibility that this inclusion could lead to interference with the infant response to routine diphtheria vaccination. We found no observable effect on response to the course of diphtheria vaccinations, which is in line with previous findings both with this and other CRM197-containing glycoconjugate vaccines.20,21 Similarly, no adverse effects were observed on infant development or on immediate outcome at birth. One newborn experienced perinatal asphyxia, which was considered possibly related to vaccination because no other obvious cause was present.
In summary, administration of the investigational trivalent group B streptococcal vaccine to pregnant women had no concerning safety signals in the women or their infants or interference with responses to routine diphtheria immunization schedules in the infants. Antibody responses were higher in vaccinated women who had detectable antibody concentrations at baseline, but there was a less pronounced response in women with undetectable levels at baseline. Maternal transfer ratios were similar to those observed in other polysaccharide vaccines, and antibodies persisted for at least 91 days postnatally in infants of vaccinated women. Further research is needed to address the needs of women with undetectable antibody concentrations before vaccination.
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