Impact of tetanus-diphtheria-acellular pertussis immunization during pregnancy on subsequent infant immunization seroresponses: follow-up from a large randomized placebo-controlled trial

https://doi.org/10.1016/j.vaccine.2019.10.104 0264-410X/ 2019 GlaxoSmithKline Biologicals S.A. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Abbreviations: AE, adverse event; ATP, according-to-protocol; CI, confidence interval; CPS, capsular polysaccharide; DTaP-HepB-IPV/Hib, diphtheria-tetanuspertussis-hepatitis B virus-inactivated poliovirus and Haemophilus influenzae type b vaccine; ECL, electrochemiluminescence; ELISA, enzyme-linked immunosorbe FHA, filamentous hemagglutinin; HBs, hepatitis B surface antigen; GMC, geometric mean concentration; GMT, geometric mean titer; Hib, Haemophilus influenzae type lower limit of quantitation; PCV13, 13-valent pneumococcal conjugate vaccine; PRN, pertactin; PRP, polyribosylribitol phosphate; PT, pertussis toxoid; RCT, ran controlled trial; SAE, serious adverse event; Tdap, diphtheria-tetanus-acellular pertussis vaccine; TVC, total vaccinated cohort. ⇑ Corresponding author at: GSK, Avenue Fleming 20, 1300 Wavre, Belgium. E-mail addresses: kirsten.perrett@rch.org.au (K.P. Perrett), scott.halperin@dal.ca (S.A. Halperin), t.nolan@unimelb.edu.au (T. Nolan), alfonsocarmona@ihppedia (A. Carmona Martínez), federico.martinon.torres@sergas.es (F. Martinón-Torres), jgarcia-sicilia@telefonica.net (J. García-Sicilia), miia.virta@staff.uta.fi (M ovanderk@ucalgary.ca (O.G. Vanderkooi), gianvincenzo.zuccotti@unimi.it (G.V. Zuccotti), lusine.x.kostanyan@gsk.com (L. Kostanyan), nadia.x.meyer@gsk.com (N maria-angeles.x.ceregido@gsk.com (M.A. Ceregido), brigitte.cheuvart@gsk.com (B. Cheuvart), sherine.o.kuriyakose@gsk.com (S.O. Kuriyakose), z.stranak@se (Z. Stranak), mjose.cilleruelo@salud.madrid.org (M.J. Cilleruelo Ortega), mariano.miranda@andaluciajunta.es (M. Miranda-Valdivieso), barias@sanitas.es (B. Arias josetomas.ramos@salud.madrid.org (J.T. Ramos Amador), pediatra@manuelbaca.com (M. Baca), paola.marchisio@unimi.it (P.G. Marchisio), narcisa.x.mesaros@ (N. Mesaros). Kirsten P. Perrett , Scott A. Halperin , Terry Nolan , Alfonso Carmona Martínez , Federico Martinón-Torres , Jose García-Sicilia , Miia Virta , Otto G. Vanderkooi , Gian Vincenzo Zuccotti , Paolo Manzoni , Lusine Kostanyan , Nadia Meyer , Maria Angeles Ceregido , Brigitte Cheuvart , Sherine O. Kuriyakose , Zbynek Stranak, Jose M. Merino Arribas , María José Cilleruelo Ortega , Mariano Miranda-Valdivieso , Begoña Arias Novas , Jose Tomas Ramos Amador , Felix Omeñaca , Manuel Baca , Paola Giovanna Marchisio , Narcisa Mesaros k,⇑


Introduction
Despite comprehensive global infant immunization programs, pertussis (Bordetella pertussis) continues to cause high morbidity and mortality among infants <2 months of age who are too young to be vaccinated [1,2]. Several strategies to optimize pertussis control and protection during this susceptible period were pursued [3]; vaccination of pregnant women is the most commonly implemented.

Study design and participants
This phase IV, multi-center, open-label, non-randomized trial with two parallel groups was conducted between 22 January 2016 and 7 March 2018 in Australia, Canada, Czech Republic, Finland, Italy and Spain. The trial (ClinicalTrials.gov: NCT02422264) was performed according to the principles of Good Clinical Practice, the Declaration of Helsinki and applicable regulations. The centers' Institutional Review Boards and/or Ethics Committees (Supplementary material) approved the protocol, informed consent form and other study-related documents. An independent data monitoring committee oversaw the participants' safety.
We enrolled healthy infants 6-14 weeks old whose mothers had participated in a phase IV, observer-blind, randomized, placebo-controlled maternal immunization trial (NCT02377349) in which they received reduced-antigen-content Tdap vaccine or placebo at 27 0/7 -36 6/7 weeks' gestation and crossover administration within 72 h postpartum [24]. Infants born prematurely (<37 weeks' gestation, but after 27 weeks' gestation) could be enrolled if they were medically stable. Exclusion criteria included a history of diphtheria, tetanus, pertussis, hepatitis B, polio, Hib or pneumococcal diseases; vaccination against any of these diseases since birth (except hepatitis B vaccination); administration of long-acting immune-modifying drugs, any chronic drug therapy or immunoglobulins and/or blood products; and immunosuppressive conditions. The Supplementary methods provide detailed inclusion and exclusion criteria. The parent(s) or legally acceptable representative(s) of each participant provided written informed consent before enrollment.
The infants' group allocation was determined by the intervention their mothers received during pregnancy (Tdap or placebo) in the maternal immunization trial [24]. The study had an open-label design because all infants received the same vaccines, but investigators and study staff involved in the infants' care and responsible for evaluating the study endpoints and laboratory testing remained blinded to the treatment allocation of the infants' mothers.
At each vaccination visit, the infants' parents or legally acceptable representatives (LARs) received diary cards to record solicited local (injection site pain, redness, swelling) and general (drowsiness, irritability, loss of appetite, fever) adverse events (AEs) within 4 days and unsolicited AEs within 31 days after each vaccination. Diary cards were returned at the next visit, during which the investigators asked the parents/LARs about any other possible AEs (or serious AEs [SAEs]) occurring since the previous visit. SAEs were collected from the first DTaP-HepB-IPV/Hib dose until study end. The total safety follow-up time depended on the vaccination schedule: approximately 5 months for infants receiving a 3-dose schedule at 2, 4 and 6 months of age and approximately 3 months for infants receiving the other schedules. The investigators assessed the intensity of all AEs and their causal relation to infant vaccination. Solicited local AEs were all considered vaccinationrelated.

Objectives
The primary objective was to assess the immune response to DTaP-HepB-IPV/Hib in terms of anti-diphtheria, anti-tetanus, anti-HBs, anti-poliovirus types 1-3 and anti-PRP seroprotection; and anti-FHA, anti-PRN and anti-PT vaccine responses 1 month post-primary vaccination. Vaccine response was defined as a post-vaccination antibody concentration at least as high as the LLoQ for infants with a pre-vaccination concentration <LLoQ; and a post-vaccination antibody concentration at least as high as the pre-vaccination concentration for infants with a pre-vaccination concentration LLoQ. Because of the expected decline in maternally transferred pertussis antibodies between the pre-and postvaccination time points, a post-vaccination concentration equal to the pre-vaccination concentration would correspond to at least a 2-fold increase in infant-induced antibodies.
Secondary immunogenicity objectives were to assess antibody concentrations or titers against all DTaP-HepB-IPV/Hib antigens, seropositivity for pertussis, and serotype-specific antipneumococcal antibody concentrations 1 month after the last DTaP-HepB-IPV/Hib primary dose; and the persistence of maternally transferred antibodies to all Tdap antigens in terms of concentrations and seroprotection/seropositivity before the first DTaP-HepB-IPV/Hib infant primary dose.
The reactogenicity and safety of DTaP-HepB-IPV/Hib and PCV13 primary vaccination were assessed as secondary objective in terms of the occurrence of solicited AEs, unsolicited AEs and SAEs.

Statistical analyses
The sample size was based on the number of mothers enrolled in the maternal immunization trial. The primary immunogenicity analyses were performed on the according-to-protocol (ATP) cohort for immunogenicity, including all eligible participants who received at least 1 dose of the study vaccines per protocol, complied with study procedures and intervals, were born full term (37 weeks' gestation) and had immunogenicity results available for at least one of the study vaccines' antigens. Seroprotection, seropositivity and vaccine response rates were calculated with exact 95% confidence intervals (CIs). Geometric mean antibody concentrations and titers (GMCs and GMTs) were calculated with 95% CIs by taking the anti-log of the mean of the log10 concentration or titer transformations. Antibody concentrations or titers  Only for infants receiving a 3-dose DTaP-HepB-IPV/Hib primary schedule. Infants who received a 2-dose DTaP-HepB-IPV/Hib primary schedule did not attend visit 3. d 3-dose DTaP-HepB-IPV/Hib primary schedule at 2, 3 and 4 months of age administered in Czechia. e 3-dose DTaP-HepB-IPV/Hib primary schedule at 2, 4 and 6 months of age administered in Australia, Canada and Spain. f PCV13 was co-administered as a 2-dose or 3-dose primary vaccination schedule; in some countries, infants received a 3-dose DTaP-HepB-IPV/Hib schedule but a 2-dose PCV13 schedule (at 2 and 4 months of age). below the assay cut-offs were given arbitrary values of half the cutoffs for the GMC and GMT calculations.
We also performed exploratory subgroup analyses by dose schedule (2-dose vs 3-dose primary schedule).
The primary safety analyses were performed on the total vaccinated cohort (TVC), including all participants who received at least 1 study vaccine dose. Percentages of participants for whom solicited or unsolicited AEs were reported were calculated with exact 95% CIs. SAEs were described in detail.
Congenital anomalies which became apparent once the maternal immunization trial [24] ended were reported as pre-existing medical condition in the present study and analyzed post-hoc.
All endpoints were descriptive. Analyses were performed using SAS version 9.2.

Study population
601 infants were enrolled and included in the TVC: 296 whose mothers were randomized to receive Tdap (Tdap group) and 305 whose mothers were randomized to receive placebo (control group) during pregnancy in the maternal immunization trial. 592 infants (98.5%) completed the study and 542 (90.2%) were included in the ATP cohort for immunogenicity (Fig. 2). Baseline characteristics were comparable between the two groups (Table 1). Most infants (~88%) received a 3-dose primary DTaP-HepB-IPV/Hib schedule (mainly at 2, 4 and 6 months of age); of those who received a 2-dose primary schedule, nearly all received their doses at 3 and 5 months of age (Table 1).

Response to the DTaP-HepB-IPV/Hib primary series
The percentages of infants who reached the seroprotective thresholds for anti-diphtheria, anti-tetanus, anti-HBs, antipoliovirus types 1-3 and anti-PRP 1 month post-primary vaccination were similar between groups: 100% in both groups for antidiphtheria and anti-tetanus, 98.5% for anti-HBs, 95.9% for anti-poliovirus and 94.5% for anti-PRP (Table 2). Vaccine response rates against the three pertussis antigens were lower in the Tdap than in the control group (39.6% vs 94.8% for anti-FHA, 37.5% vs 90.0% for anti-PRN and 77.1% vs 99.2% for anti-PT; seropositive for all pertussis antibodies but antibody GMCs were lower in the Tdap than in the control group (68.5 vs 103.5 IU/mL for anti-FHA, 60.5 vs 92.0 for anti-PRN, and 32.7 vs 54.7 for anti-PT; Table 3). Post-primary anti-diphtheria antibody GMCs were also lower in the Tdap than in the control group. For the other DTaP-HepB-IPV/Hib antigens, antibody GMCs were similar between the two groups ( Table 2).

Response to the PCV13 primary series
One month post-primary vaccination, the percentages of infants with serotype-specific anti-pneumococcal antibody concentrations 0.35 mg/mL were similar in both groups for each PCV13 serotype (Table 4). Serotype-specific antibody GMCs were in the same range in both groups for most PCV13 serotypes; minimal differences in GMCs were observed for serotypes 4 and 19F (marginally lower in the Tdap group) ( Table 4).

Persistence of maternally transferred antibodies to Tdap antigens
Before the first primary dose (i.e., at 2 or 3 months of age depending on the schedule the infant received), more infants in the Tdap group were seroprotected against diphtheria (82.6% in Tdap vs 43.7% in control); against tetanus (99.2% in Tdap vs 88.9% in control); and also more were seropositive for the three pertussis antigens (90.1%-100% in Tdap vs 34.8%-83.0% in control) ( Table 5). Antibody GMCs at the pre-primary time point in the Tdap group were significantly higher than those in the control group for all Tdap antigens (Table 5).

Subgroup analysis by dose schedule
We evaluated the immune response to the DTaP-HepB-IPV/Hib and PCV13 primary series separately in infants who received a 2dose schedule (nearly all at 3 and 5 months of age) and in infants who received a 3-dose schedule (predominantly at 2, 4 and 6 months of age). Generally, the trends we described for the overall population were observed for both schedules (Supplementary  tables 1-3). However, interpretation of these results is limited by the small sample size of the 2-dose subgroup.

Reactogenicity and safety
Solicited AEs were reported at similar rates in both groups, with redness being the most commonly reported local AE at the DTaP-HepB-IPV/Hib and PCV13 injection sites (Table 6) and irritability the most commonly reported general AE (Table 6). Most solicited AEs in both groups were mild or moderate. Unsolicited AEs were reported for 54.4% and 56.7% of infants in the Tdap and control groups, respectively, most being mild or moderate (Table 6). Upper respiratory tract infection was the most common unsolicited AE (Tdap: 12.2%, 95% CI: 8.7-16.4; control: 10.8%, 7.6-14.9).   Abbreviations: %, percentage of infants with antibody concentrations 0.35 mg/mL; ATP, according-to-protocol; CI, confidence interval; GMC, geometric mean concentration; N, number of infants with available results; Tdap, reduced-antigen-content diphtheria-tetanus-acellular pertussis vaccine. a 1 month after DTaP-HepB-IPV/Hib primary vaccination, which is 1 month after PCV13 primary vaccination for most infants, but 3 months after PCV13 primary vaccination for those who received a 3-dose DTaP-HepB-IPV/Hib schedule at 2, 4 and 6 months of age and a 2-dose PCV13 schedule at 2 and 4 months of age. Abbreviations: %, percentage of infants who mounted a vaccine response or were seropositive (antibody concentration equal to or above the specified LLoQs); ATP, accordingto-protocol; CI, confidence interval; FHA, filamentous hemagglutinin; GMC, geometric mean concentration; IU, international unit; LLoQ, lower limit of quantitation; N, number of infants with pre-and post-vaccination results available; N', number of infants with post-vaccination results available; PRN, pertactin; PT, pertussis toxoid; Tdap, reduced-antigen-content diphtheria-tetanus-acellular pertussis vaccine. a Vaccine response to FHA, PRN and PT antigens is defined as a post-vaccination antibody concentration LLoQ for infants with a pre-vaccination concentration <LLoQ; a post-vaccination concentration at least as high as the pre-vaccination concentration for infants with a pre-vaccination concentration LLoQ. Abbreviations: %, percentage of infants with antibody concentrations greater than or equal to the specified seroprotection cut-offs (for diphtheria and tetanus) or LLoQs (for pertussis); ATP, according-to-protocol; CI, confidence interval; D, diphtheria; FHA, filamentous hemagglutinin; GMC, geometric mean concentration; IU, international unit; LLoQ, lower limit of quantitation; N, number of infants with available results; PRN, pertactin; PT, pertussis toxoid; T, tetanus; Tdap, reduced-antigen-content diphtheriatetanus-acellular pertussis vaccine.
Supplementary Fig. 1 depicts a plain language summary outlining the findings and highlighting their clinical relevance.

Discussion
The key rationale for pertussis immunization during pregnancy is to protect infants too young to be vaccinated from pertussis disease and death by achieving persistent high levels of maternally transferred pertussis antibodies in infants between birth and the first primary immunization dose. Our study-the largest RCT to date to investigate this-showed that administration of the reduced-antigen-content Tdap vaccine during the third trimester of pregnancy resulted in high levels of maternally transferred pertussis antibodies in infants up to the pre-primary vaccination time point (at 2 or 3 months of age). Infants of Tdap-immunized mothers presented significantly higher antibody levels for all Tdap antigens before the primary series compared to infants of control mothers. However, immunological interference was evident for pertussis (in terms of GMCs and vaccine response rates) and to a lesser extent for diphtheria (in terms of GMC but not seroprotection rate) after the primary DTaP-HBV-IPV/Hib series in infants born to Tdap mothers.
There was no evidence of maternally derived antibodies interfering with the infant immune response to tetanus, hepatitis B, poliovirus or Hib PRP in terms of seroprotection rates and GMCs after the primary DTaP-HBV-IPV/Hib series. Likewise, we observed no interference with the response to PCV13 in terms of percentages of infants achieving the 0.35 mg/mL threshold (shown to be equivalent to the threshold used for PCV licensure [33,34]). The minimal GMC differences observed for serotypes 4 and 19F between the two groups may be explained by the absence of statistical adjustments for multiple testing.
The concept of immune interference or blunting, where maternal antibodies reduce antibody generation to the infant primary series [35,36], resulting in lower post-primary antibody concentrations, is not new. Previous studies assessing the administration of Table 6 Solicited and unsolicited adverse events after primary vaccination (total vaccinated cohort). Abbreviations: AE, adverse event; CI, confidence interval; DTaP-HepB-IPV/Hib, diphtheria-tetanus-acellular pertussis-hepatitis B virus-inactivated poliovirus and Haemophilus influenzae type b vaccine; N, number of infants with at least one documented dose (for solicited AEs) or at least one administered dose (for unsolicited AEs); n/%, number/percentage of infants for whom the specified AE was reported at least once during the follow-up periods after any of the doses; Tdap, reduced-antigen-content diphtheria-tetanus-acellular pertussis vaccine. Grade 3 pain was defined as crying when the limb was moved or the limb being spontaneously painful; grade 3 irritability as crying that could not be comforted or irritability preventing normal activity; grade 3 drowsiness as drowsiness preventing normal activity; and grade 3 loss of appetite as not eating at all; unsolicited AEs were considered grade 3 if they prevented normal activity.
Tdap vaccines (three-or five-component pertussis vaccines) during pregnancy have reported significantly lower antibody responses to one or more pertussis antigens in infants born to Tdap-vaccinated mothers following completion of their primary series compared to infants whose mothers had not been Tdap-vaccinated [4][5][6][7][8]10,[20][21][22]. However, this effect was not consistently observed [4][5][6]10,21,22]. Similarly, several studies have shown that maternally transferred diphtheria antibodies can interfere with diphtheria and diphtheria-derived CRM-conjugated pneumococcal vaccine responses to the infant primary series following Tdap immunization during pregnancy [4,5,10,21,22,37]. In contrast to other studies [4,5,21,22] we did not see an enhancement of the immune response to tetanus post-primary series, despite higher preprimary anti-tetanus GMCs in infants of Tdap-vaccinated mothers than in controls. Maternal antibody interference with the immune response to infant vaccination has also been described as a natural phenomenon in the absence of maternal immunization in a recent meta-analysis of 32 clinical trials [38]. This meta-analysis showed that pre-existing antibodies inhibit infant immune responses to primary immunization for 20 of 21 measured antigens, and this in the absence of maternal vaccination [38]. Two-fold higher maternal antibody levels were estimated to result in 11% lower post-vaccination antibody levels for PT and FHA, 22% lower for PRN, 13% lower for tetanus and 24% lower for diphtheria [38]. Given that maternal antibodies decline rapidly over the first months of life, mathematical modeling indicated that the inhibitory effect of a 2-fold to 5-fold increase in maternal antibody concentrations in infants can be offset by a delay of 2.2-5.0 weeks in starting primary vaccination [38]. A recent RCT in the Netherlands investigated the effect of maternal Tdap immunization on pertussis antibody responses of infants starting primary vaccination at 3 months (instead of 2 months) and still found significant blunting [8]. In our subgroup analysis by dose schedule, nearly all infants in the 2-dose subgroup started their primary series at 3 months of age; interference with the pertussis response seemed to occur in this subgroup as well; however, the small sample size of this subgroup precludes any sound conclusions.
Importantly, the clinical relevance of the lower post-primary antibody levels in infants from Tdap-vaccinated mothers remains unknown, since there is no established correlate of protection for pertussis. To date, there is no evidence that the observed immunological interference is of clinical significance as there has been no increase in pertussis disease in infants born to Tdap-vaccinated mothers following primary series vaccinations in the first year of life [13,14,39]. However, this requires ongoing monitoring.
This study provides further evidence of the tolerability and safety of DTaP-HepB-IPV/Hib and PCV13 in infants whose mothers received Tdap vaccine during pregnancy. Rates of solicited and unsolicited AEs were similar between groups. SAEs were not reported more frequently in the Tdap group compared to the control group. Hence, the safety profile of DTaP-HepB-IPV/Hib and PCV13 in infants did not change depending on whether their mothers received Tdap during pregnancy.
The current study has several potential limitations. It was conducted in six high-income countries, and participants of the original study (NCT02377349) were mainly white Caucasian women and women with low-risk pregnancies. Hence, the results may not be generalizable to low-and middle-income countries, to infants of other ethnic groups or infants born from high-risk pregnancies. In addition, analyses were descriptive with no adjustment for multiplicity. We also did not assess the effect of breastfeeding on antibody levels before and after the infant primary series. Because mothers in the control group received Tdap postpartum, antibodies to Tdap antigens might have been transferred through breast milk to infants in the control group.
A follow-up study is ongoing in the same infant cohort to investigate the effect of maternal Tdap immunization on the persistence of antibodies induced by the primary DTaP-HepB-IPV/Hib and PCV13 series up to 11-18 months of age and the effect on the immune response to booster DTaP-HepB-IPV/Hib and PCV13 vaccination in the child's second year of life (NCT02853929).

Conclusion
Our study is the largest RCT to date providing evidence that immunization against pertussis during pregnancy leads to high levels of pertussis antibodies in young infants that persist until the start of the primary immunization series. These high levels of maternal pertussis antibodies can help to further close the susceptibly gap to severe pertussis disease and death in young infants but appear to interfere with the infant's immune response to the primary pertussis immunization series. The clinical significance of this interference remains unknown in the absence of a correlate of protection. Ongoing epidemiological surveillance of the maternal immunization strategy is required to further understand if there is a clinical impact. We found no evidence of maternally derived antibodies to Tdap antigens interfering with the infant immune response to tetanus, hepatitis B, poliovirus or Hib PRP and only minimal potential interference with the response to diphtheria and two pneumococcal serotypes. The reactogenicity and safety of DTaP-HepB-IPV/Hib and PCV13 in infants did not seem impacted by whether their mothers received Tdap or placebo during pregnancy. KPP wrote the manuscript and all authors have revised and approved the manuscript.

Funding
This work was supported by GlaxoSmithKline Biologicals S.A., which was the funding source, was involved in all stages of the study conduct and analysis and paid for all costs associated with the development and publishing of this manuscript.

Declaration of Competing Interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [BAN reports grant from the GSK group of companies (GSK) and personal fees from Pfizer, MSD and Sanofi Pasteur. BC, MAC, NMes, NMey and SOK are employees of GSK, and BC and NMes own GSK restricted shares. FMT, KPP, OGV, SAH and TN's institutions received grants from GSK during the conduct of the study. FMT's institution received financial support from GSK during the conduct of the study, as well as financial and non-financial support outside the submitted work; he also received personal fees from Pfizer, Novavax, MSD and Sanofi Pasteur; his institution also received financial support as trial fees from Ablynx, Jansen, Regeneron, Medimmune, Pfizer, MSD, Sanofi Pasteur, Novavax and Novartis, as well as non-financial support from Pfizer and MSD and grants from MSD and AstraZeneca. JMMA reports receiving fees and non-financial support from GSK during the conduct of the study, as well as fees from GSK, Pfizer and MSD outside the submitted work. LK is working as consultant for GSK. SAH is member of ad-hoc advisory committees for GSK and Sanofi Pasteur and he has a patent for novel triple adjuvant issued. ACM, FOT, GVZ, JGS, JTRA, MB, MJCO, MMV, MV, PGM, PM and ZS declare no conflicts of interest.].