Pediatric Infectious Disease Journal:
Antibody and Cell-mediated Immunity to Pertussis 4 Years After Monovalent Acellular Pertussis Vaccine at Birth
Wood, Nicholas MB BS, FRACP, PhD*†‡; Marshall, Helen MB BS, MD, DCH, MPH§¶; White, Olivia J. MSc, PhD‖**; Holt, Patrick G. DSc, FAA‖**; McIntyre, Peter MB BS, FRACP, FAFPHM, PhD*†
From the *National Centre for Immunisation Research and Surveillance of Vaccine Preventable Diseases, The Children’s Hospital at Westmead and the University of Sydney, New South Wales; †The Children’s Hospital, Westmead, Sydney; ‡The University of Sydney, New South Wales, Australia; §Discipline of Paediatrics, School of Paediatrics and Reproductive Health and the Robinson Institute, The University of Adelaide; ¶Vaccinology and Immunology Research Trials Unit, Women’s and Children’s Hospital, Women’s and Children’s Health Network, South Australia; ‖Telethon Institute for Child Health Research and Centre for Child Health Research, Faculty of Medicine and Dentistry, The University of Western Australia, Perth; and **Queensland Children’s Medical Research Institute, University of Queensland, Brisbane, Queensland, Australia,
Accepted for publication December 3, 2013.
This study was supported by a grant from the Financial Markets Foundation for Children, Australia (S112 – 2004) and the Women’s and Children’s Hospital Foundation, South Australia. H. Marshall received support from the National Health and Medical Research Council of Australia: Career Development Fellowship (1016272). The authors have no other funding or conflicts of interest to disclose.
Address for Correspondence: Nicholas Wood, MB BS, FRACP, PhD, National Centre for Immunisation Research and Surveillance of Vaccine Preventable Diseases, Locked Bag 4001, Westmead, NSW 2145, Australia. E-mail: firstname.lastname@example.org.
In a previous study, we found that monovalent acellular pertussis (aP) vaccine at birth and 1 month achieves higher IgG antibody (Ab) levels to pertussis toxoid (PT), filamentous hemagglutinin (FHA) and pertactin by 8 weeks, when compared with controls. Here, we report antibody and cell-mediated immune responses to 4 years of age.
IgG Ab to PT, filamentous hemagglutinin and pertactin, diphtheria (D) and tetanus (T) was measured in the 3 groups (aP vaccine at birth and 1 month, aP birth only and no aP) at 2 years of age and before and after DTaP-inactivated polio vaccine (DTaP-IPV) at 4 years of age. Cell-mediated immune responses to pertussis vaccine antigens were measured at 2 years of age. Adverse events following DTaP-IPV were recorded.
Of 74 subjects, 52 (70%) were available for follow up. Overall, 11 (21%) had detectable PT IgG at 2 years, decreasing to 10% before 4-year-old booster compared with 100% at 8 months of age. After the 4-year booster, pertussis antigen IgG levels were similar, but there was a trend to lower PT IgG levels in birth aP infants (geometric mean concentrations: 28.7 EI.U/mL) compared with controls (geometric mean concentrations: 53.6 EI.U/mL). The cytokine responses to pertussis antigen stimulation were higher in aP recipients at 2 years of age. There was no difference in injection site reactions among groups following the DTaP-IPV booster at 4 years of age.
In the longest reported follow-up of infants who received aP vaccine at birth, we found a trend to lower PT IgG antibodies post booster compared with receipt of first dose of aP-containing vaccine at 8 weeks of age. Short- and long-term antibody responses with and without prior maternal pertussis vaccination are crucial for further evaluation of this strategy for preventing severe early pertussis.
In the past decade, a resurgence of pertussis has been identified in Australia, the United States and more recently the United Kingdom, all countries with long-established pertussis immunization programs. This resurgence is focused on older children (presumptive waning immunity) and infants under the age of 3 months (too young to have received >1 dose of pertussis-containing vaccine).1–3 Unimmunized infants are most likely to develop severe disease requiring hospitalization, frequently complicated by apnea, seizures, encephalopathy or death.3–5 Strategies to reduce the risk of severe pertussis in infants too young to be immunized include: indirect protection through “cocooning” (immunization of parents and other close contacts of the infant), direct protection through maternal immunization during pregnancy, as has been recently introduced in the United States and the United Kingdom and immunization of the infant as soon as possible after birth.5 To date, 4 studies have examined immune responses following administration of monovalent acellular pertussis (aP) vaccine6–8 or diphtheria-tetanus-acellular pertussis (DTaP)9 vaccine at birth. In all 3 studies where newborns received monovalent aP vaccine, higher pertussis antibody responses were achieved earlier than control infants vaccinated at 6–8 weeks of age.6–8 Despite concerns about immune tolerance from earlier studies where whole cell pertussis vaccine was given to neonates,10,11 these studies did not find any decrease in antibody responses to pertussis antigens after the third dose of scheduled pertussis-containing vaccine, but 2 studies found some evidence of interference with responses to Hib and hepatitis B antigens.7,8,12
Antibody levels in the second year of life and booster response following receipt of pertussis-containing vaccines at birth have been reported with divergent findings recipients of DTaP versus aP at birth. Following DTaP at birth,9 there was significant blunting of antibody responses to pertussis and some concomitant antigens; however, no blunting of pertussis antibody responses was found following monovalent aP.13 The aim of this study was to describe the persistence of pertussis antibodies in children who previously received aP vaccine at birth ± 1 month of age and responses to pertussis and other antigens after a booster dose of DTaP-inactivated polio vaccine (DTaP-IPV) vaccine. We aimed to explore in this small study whether there was a difference in pertussis antibody levels post receipt of a booster dose of DTaP-IPV vaccine in children who received aP vaccine at birth compared with controls.
This long-term, follow-up study was conducted from 2007 to 2010 at the Children’s Hospital at Westmead, Sydney, and The Women’s and Children’s Hospital, Adelaide.
Participants in the primary vaccine study were randomized into 3 groups. Group 1 received aP vaccine and hepatitis B (HBV) vaccine within 4 days of birth and an additional aP vaccine at 4 weeks of age, Group 2 received aP and HBV vaccines at birth and Group 3 received only HBV at birth, as routinely recommended in Australia and the United States. All infants received DTaP-HBV-IPV/Hib [Infanrix Hexa; GlaxoSmithKline (GSK) Biologicals, Belgium] and 7-valent pneumococcal conjugate vaccine (Prevenar 7, Wyeth Pharmaceuticals Inc, Philadelphia, PA) at 2, 4 and 6 months of age. By 8 months of age, subjects in groups 1, 2 and 3 received 5, 4 and 3 doses, respectively, of an acellular pertussis-containing vaccine. At 4 years of age, participants received DTaP-IPV (Infanrix IPV; GSK Biologicals) and Measles-Mumps-Rubella vaccine (Priorix; GSK Biologicals), as is routine in the Australian National Immunization Program. A blood sample was collected at 3 time points: at 2 years of age, immediately before administration of scheduled 4-year immunizations and 4–6 weeks following. Serum samples were stored frozen for batch analysis. Peripheral blood mononuclear cells (PBMC) were isolated and stored in liquid nitrogen for batch analysis. The trial was registered with the Australian New Zealand Clinical Trials Registry (ACTRN012605000013662).
Assessment of Humoral Immunity
Titres of antibody to pertussis antigens [pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN)] were measured by GSK Biologicals using a standard enzyme linked immunosorbent assay (cut-off 5 EI.U/mL) developed for licensure of DTaP vaccines. Anti-diphtheria (D; cut-off 0.1 IU/mL) and anti-tetanus (T) (cut-off 0.1 IU/mL) were measured by enzyme linked immunosorbent assay at 2 and 4 years of age (before and after booster).
Assessment of Cell-mediated Immunity
PBMC were cultured in the presence of a mixture of pertussis vaccine antigens as described14 Cell culture supernatants were assessed for the production of cytokines (IL-5, IL-13, IL-6, IL-9, interferon-γ and tumor necrosis factor-α) by a multiplex assay as described elsewhere.14,15 In this study, we have not performed T cell proliferation assays or fluorescence-activated cell sorting and have only included measurement of the above cytokines.
Assessment of Reactogenicity
After administration of DTaP-IPV and measles, mumps, and rubella vaccines children were observed for 30 minutes. Parents were asked to record temperature using a supplied thermometer and any solicited adverse reactions observed at 3 and 6 hours after injection and at bedtime each evening for 7 days on a diary card. Solicited adverse reactions included: fever, drowsiness (unusually sleepy or inactive), irritability, anorexia, vomiting, redness and swelling at the vaccination site (each measured in millimetres) and pain. All unsolicited adverse events occurring within 30 days of vaccination were recorded by parent/guardian and/or study physician.
The investigators were responsible for study design and conduct and performed all statistical analyses on individual patient data. For pertussis antigens, antibody geometric mean concentrations (GMC) with 95% confidence intervals (CI) were calculated from the anti-log of the mean of the log-transformed values. Values below the laboratory assay cut-off were assigned a value half of the cut-off value to calculate the GMC. Numbers and proportions of subjects with combinations of both PT and PRN levels above and below detectable were compared between groups at 2 and 4 years of age. For diphtheria and tetanus, serologic response was defined as any level above the lower limit for detection in the assay used for each antibody (0.1 IU/mL and 0.15 μg/mL, respectively). Comparisons of antibody responses between groups were made using log-transformed data by the independent samples t test. Differences in cytokine responses between groups were calculated using Mann-Whitney U test. Comparisons between groups was considered statistically significant if P < 0.05 for all tests performed. No adjustment was calculated for multiple comparisons. This study was not powered to detect noninferiority, inferiority and/or equivalence between groups.
Of 76 neonates enrolled and vaccinated in the primary vaccination study, samples for antibody measurement were available at 2 years of age from 52 subjects (70.4%; Group 1, n = 22; Group 2, n = 16; Group 3, n = 14) and cell-mediated immune responses from 27 subjects (Table 1). At 4 years of age, samples were available for antibody measurement from 49 participants (64.5%; Group 1, n = 20; Group 2, n = 15; Group 3, n = 14; Table 2). There was no loss to follow up between pre- and post-booster vaccine in 4-year-old participants.
Persistence of Pertussis Antibodies and Response to 4-year DTaP-IPV Booster Vaccine
PT, FHA or PRN antibody levels at 2 years or before and following the 4-year DTaP-IPV immunization were similar between groups (Tables 1 and 2 and Fig. 1). At 2 years of age, anti-PT levels were low, with only 13–23% above the limit of detection of 5 EI.U/mL, compared with anti-FHA (71–86% >5 EI.U/mL) and anti-PRN (63–79% >5 EI.U/mL; Table 1). Despite Group 1 having higher anti-pertussis antibody levels at 8 months of age, post completion of the primary vaccine series, levels in all 3 groups had decreased to similar levels at 2 years of age (Fig. 1).
At 4 years of age, before a booster dose, the proportion with anti-PT levels >5 EI.U/mL had decreased to 7–14%, again substantially lower than for anti-FHA (73–95%) or anti-PRN (45–71%; Table 2). Notably, at 8 months of age, 1 month following their last vaccine dose, nearly all infants had detectable pertussis antibody levels to both PT and PRN, compared with 15–18% at 2 years of age and 9–14% immediately before the DTaP-IPV booster at 4 years of age (Table 3). In contrast, the proportions with detectable FHA IgG remained stable from 2 to 4 years of age (Tables 1 and 2).
In all 3 groups, there was a robust response to the DTaP-IPV booster vaccine at 4 years of age as shown in Table 2. Four-fold increases in PT IgG and in PRN IgG were seen in 78%, 92% and 93% and 94%, 92% and 92% of Groups 1, 2 and 3 subjects, respectively. One month following the booster vaccine dose, all participants had PT, FHA and PRN antibody levels above detectable. There was a trend to lower PT IgG levels post booster in those given aP at birth (GMC 28.7 EI.U/mL) compared with controls (GMC 53.6 EI.U/mL; Table 2).
Antibody Responses to Diphtheria and Tetanus Antigens
At 2 years of age, proportions above detectable levels for diphtheria and tetanus antigens were similar, with the exception of tetanus in Group 1 vs Groups 2 and 3 (59% vs. 75% and 77%; Table 1) Following DTaP-IPV vaccine at 4 years of age, diphtheria and tetanus antibodies were detectable in samples from all participants and at least a 4-fold rise was observed compared with prevaccination tires (Table 2).
Cytokine Responses to Pertussis Vaccine Antigens at 2 Years of Age
In vitro cytokine responses to a mixture of the pertussis antigens (PT, FHA and PRN) were measured in a subset of study subjects at 2 years of age. Cells from infants who had received birth aP (Groups 1 and 2) had statistically significantly higher Th2 cytokine levels (IL-5, IL-9 and IL-13) compared with infants in Group 3 (Fig. 2). There were no differences in the levels of Th1 cytokines (interferon-γ, IL-6 or tumor necrosis factor-α) between groups (Fig. 2).
Reactogenicity Following DTaP-IPV Vaccine at 4 Years of Age
The proportion that developed an injection site reaction was not significantly different between the groups, nor was there a dose-related trend evident for number of previous doses received; however, assessment was limited by the available study numbers (Fig. 3). Swelling or erythema at the injection site (>10 mm) and pain (≥ grade 2) occurred most commonly in Group 2 children compared with Groups 1 and 3, and only 2 subjects from any group had a recorded fever ≥38.5°C within 72 hours post booster vaccination. Five participants, all of whom had received aP vaccine at birth, had erythema or swelling >50 mm within 72 hours of vaccination (3 in Group 2, 2 in Group 1); all were confined to the injection site and resolved within 5 days. No serious adverse events were reported following receipt of the DTaP-IPV booster dose at 4 years of age.
In this study, infants who received aP vaccine at birth had similar pertussis antibody levels at 2 years of age to control infants, despite additional earlier aP doses and nonsignificantly higher levels at 8 months of age following completion of the primary vaccine series. Hallander et al16,17 describe a rapid decay of anti-PT in the first 8–9 months post completion of the 3 dose primary infant vaccine series, followed by a slower decay, with 10% having anti-PT levels above detectable by 6 years after dose 3, similar to levels in our study. Less than 20% of our participants had detectable IgG antibody to both PT and PRN at 2 years of age, declining to <15% before DTaP-IPV booster at 4 years of age. This is of potential clinical significance, as having detectable IgG antibody to both PT and PRN was shown to correlate with protection against pertussis infection in household contact studies in Sweden.18,19 PT IgG antibody decayed much more rapidly than PRN, and especially FHA at both 2 and 4 years. The reason for this differential rate of decay is not known. In the United States, a booster dose of DTaP is recommended in the second year of life; however, in the United Kingdom and Australia, a DTaP booster following the primary course is not recommended until 3 years and 4 months and 4 years of age, respectively. In the 2008–2011 pertussis epidemic in Australia, preschool aged children (2–5 year olds), had a markedly higher incidence of reported pertussis than in the previous epidemic in 2001, when a booster dose at 18 months was still recommended for this age group. The rapid waning of antibody we demonstrated by 2 years of age is consistent with this much higher incidence among children <5 years of age. Antibody levels to all 3 pertussis antigens at 2 years of age are lower in our study than those reported among infants who received aP vaccine at birth at the age of 18 months.13 This is likely to be largely related to the later age of measurement in our study, as both the vaccines received and the laboratory performing antibody assays were the same in both studies (GSK Biologicals, Belgium). Similarly, the antibody levels we observed are lower than those reported by studies of 18-month-old infants given 3 doses of DTaP-HBV-IPV/Hib at 2, 4 and 6 months of age and the reason for this is also not clear.20
Knuf et al.13 measured immune responses before and after DTaP booster vaccine at 18 months of age in infants who had been primed with neonatal aP vaccine and no evidence for immune hyporesponsiveness to pertussis vaccine antigens was observed (Table 4). In contrast, Halasa et al9 found pertussis immune responses to a booster DTaP vaccine, particularly PT and PRN, were blunted in toddlers who had received DTaP vaccine at birth compared with controls (Table 4). In our study, all participants showed strong booster responses to DTaP-IPV vaccine at 4 years of age, with pertussis antibody levels post booster not significantly different between aP at birth recipients and those who received the first aP-containing vaccine at 2 months of age. All participants achieved both anti-PT and anti-PRN levels above detectable post booster. This is consistent with known booster responses at 3–6 years of age21 and suggests that any effect of birth aP vaccine on subsequent pertussis antibody booster responses is minimal by 4 years of age. There was a trend to reduced PT levels post booster with receipt of birth aP vaccine (Group 1 < Groups 2 and 3); however, PRN and FHA responses were identical. This differential is similar to the lesser decay of PRN and FHA before 4 years of age.
The production of Th2 cytokines at 2 years of age from PBMC in T-helper-cell dependent memory responses to pertussis vaccine antigens was higher in the birth aP vaccine cohorts. This may be a consequence of 2 factors: first, the extra doses of aP vaccine they received, and second, the fact that the immune system in healthy neonates is known to be maximally Th2-polarised at birth when they received their first priming dose and is thus likely to favor the generation of strong Th2 memory. The higher Th2 response (IL-5 and IL-13) in birth aP vaccine groups seen post completion of the primary vaccine series at 8 months of age14 remained significantly higher than the control group at 2 years of age. In contrast, there was no statistically significant difference in Th1 (interferon-γ) responses, or in IL-6 and tumor necrosis factor-α levels between groups. Knuf et al13 similarly observed increasing lymphoproliferative responses over time in the aP group. The concern of Th2-polarised immune responses in early infancy is the potential to antagonize development of Type-1-dependent protective immunity, as well as increased reactogenicity with boosters due to excessive proinflammatory Type-2 cytokines in the resultant memory response.22 However, a recent whole genome network study describes a dynamic equilibrium in Th1 and antimicrobial gene signatures as well as Th2 signature in response to pertussis vaccine antigens, suggesting that the counterbalance provides protection from aberrant Th2 immunity.15 In our study, we did not detect any significant increase in reactogenicity seen following the 4 year of age booster in birth aP recipients; however, all subjects with erythema or swelling >50 mm had received aP vaccine at birth and we had limited power to detect any difference. Knuf et al13 also noted a nonsignificant trend toward higher reactogenicity in children who had received birth aP vaccine following DTaP booster in second year of life. Due to the small sample size of both this study and Knuf et al13 formal conclusions regarding whether birth aP vaccination results in higher rates of reactogenicity following booster doses cannot be made.
The generalizability of this study is limited by small numbers (n = 54), and we were only able to enrol 70% of our original sample into this long-term, follow-up study. This study was not powered to detect significant differences in pertussis antibody levels between groups and was an opportunistic follow up of children enrolled into the initial pilot study. However, this is the first study to report on long-term follow up of children who received acellular pertussis vaccine at birth. The results of this study are also not generalizable to the United States where although the infant vaccine schedule is similar, an 18 month of age DTaP booster dose is routine in the United States and not given in Australia.
Longer term theoretical concerns of neonatal pertussis vaccination include reduced responsiveness to booster doses of pertussis (immune hyporesponsiveness), interference with concomitant antigen responses and increased reactogenicity following booster doses.4 The increased reactogenicity may potentially be related to Th2-polarised cellular immune responses following infant vaccination. In this study, pertussis antibody levels waned significantly by 2–4 years of age, with <20% having detectable antibodies to both PT and PRN, thought to equate with protection, and this has implication for the timing of the booster dose of DTaP vaccines, particularly in Australia where a booster dose is no longer given in the second year of life. We found a nonsignificant trend to lower PT IgG antibodies post the 4-year booster compared with receipt of first dose of aP-containing vaccine at 8 weeks of age. We also found higher levels of Th2 cytokines at 2 years of age from PBMC in T-helper-cell-dependent memory responses to pertussis vaccine antigens in the birth aP vaccine cohorts. Newborn aP vaccination has been shown in 3 small studies to be immunogenic early in infancy and may prove to be an effective way to protect infants earlier than current immunization schedules that commence no earlier than 6 weeks of age; however, the possibility of prolonged immune tolerance to pertussis antigens, increased reactogenicity to booster doses and negative bystander interference on concomitantly administered antigens needs further investigation. Short- and long-term antibody responses with and without prior maternal pertussis vaccination are crucial for further evaluation of this strategy for preventing severe early pertussis. A large multicentre trial is currently underway in Australia to more definitively address these questions, examining humoral, cellular immune, interference of preexisting maternal antibody and safety responses following birth aP vaccine with subsequent DTaP combination vaccine at 6 weeks of age compared with control infants.
The authors acknowledge and thank the families for their participation in this study and the VIRTU staff including Diana Weber, Louise DeGaris, Michelle Clarke, Jane Tidswell, Susan Lee, Jan Walker, Sue Evans and Rachel Chen. We are grateful to GSK Biologicals for performing all serologic assays. Engerix and Infanrix Hexa are trademarks of the GlaxoSmithKline group of companies; Prevenar is a trademark of Wyeth Pharmaceuticals Inc.
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birth; acellular pertussis vaccine; immunogenicity
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