Pertussis seroprevalence among adults of reproductive age (20-39 years) in fourteen European countries.

The reported incidence of pertussis in European countries varies considerably. We aimed to study specific Bordetella pertussis seroprevalence in Europe by measuring serum IgG antibody levels to pertussis toxin (anti-PT IgG). Fourteen national laboratories participated in this study including Belgium, Denmark, Finland, Greece, Hungary, Italy, Lithuania, Malta, Norway, Poland, Portugal, Romania, Spain, and Sweden. Each country collected approximately 250 samples (N = 7903) from the age groups 20-29 years (N = 3976) and 30-39 years (N = 3927) during 2010-2013. Samples were anonymous residual sera from diagnostic laboratories and were analyzed at the national laboratories by a Swedish reference method, a commercial ELISA kit, or were sent to Sweden for analysis. The median anti-PT IgG concentrations ranged from 4 to 13.6 IU/mL. The proportion of samples with anti-PT IgG ≥100 IU/mL, indicating a recent infection ranged from 0.2% (Hungary) to 5.7% (Portugal). The highest proportion of sera with anti-PT IgG levels between 50 and <100 IU/mL, indicating an infection within the last few years, was found in Portugal (12.3%) and Italy (13.9%). This study shows that the circulation of B. pertussis is quite extensive in adults, aged 20-39 years, despite well-established vaccination programs in Europe.

Further investigations of the IgE response to tetanus and diphtheria following covaccination with acellular rather than cellular Bordetella pertussis.

BACKGROUND: It has previously been shown in an uncontrolled study that the IgE response to vaccine antigens is downregulated by co-vaccination with cellular Bordetella pertussis vaccine. METHODS: In the present study, we compared in a controlled trial the humoral immune response to diphtheria toxoid (D) and tetanus toxoid (T) in relation to co-vaccinated cellular or acellular B pertussis vaccine. IgE, IgG4, and IgG to D and T were analyzed at 2, 7, and 12 months of age in sera of children vaccinated with D and T (DT, N = 68), cellular (DTPw, N = 68), 2- or 5-component acellular B pertussis vaccine (DTPa2, N = 64; DTPa5, N = 65). RESULTS: One month after vaccination, D-IgE was detected in 10% sera of DTPw-vaccinated children, whereas vaccination in the absence of whole-cell pertussis resulted in 50%-60% IgE positivity. Six months after vaccination, the IgE antibody levels were found to be more persistent than the IgG antibodies. These diphtheria findings were mirrored by those for tetanus. Only minor differences between vaccine groups were found with regard to D-IgG and T-IgG. No immediate-type allergic reactions were observed. CONCLUSION: Cellular (but not acellular) B pertussis vaccine downregulates IgE to co-vaccinated antigens in infants. We assume that the absence of immediate-type allergic reactions is due to the high levels of IgG antibodies competing with IgE antibodies.

Pertactin-deficient Bordetella pertussis isolates: evidence of increased circulation in Europe, 1998 to 2015.

IntroductionPertussis outbreaks have occurred in several industrialised countries using acellular pertussis vaccines (ACVs) since the 1990s. High prevalence of pertactin (PRN)-deficient Bordetella pertussis isolates has been found in these countries.AimsTo evaluate in Europe: (i) whether proportions of PRN-deficient strains increased in consecutive collections of B. pertussis clinical isolates; (ii) if the frequency of PRN-deficient strains in countries correlated with the time since ACV introduction; (iii) the presence of pertussis toxin (PT)-, filamentous haemagglutinin (FHA)- or fimbriae (Fim)-deficient isolates.MethodsB. pertussis clinical isolates were obtained from different European countries during four periods (EUpert I-IV studies): 1998 to 2001 (n = 102), 2004 to 2005 (n = 154), 2007 to 2009 (n = 140) and 2012 to 2015 (n = 265). The isolates' selection criteria remained unchanged in all periods. PRN, PT, FHA and Fim2 and Fim3 expression were assessed by ELISA.ResultsIn each period 1.0% (1/102), 1.9% (3/154), 6.4% (9/140) and 24.9% (66/265) of isolates were PRN-deficient. In EUpert IV, PRN-deficient isolates occurred in all countries sampled and in six countries their frequency was higher than in EUpert III (for Sweden and the United Kingdom, p < 0.0001 and p = 0.0155, respectively). Sweden and Italy which used ACVs since the mid 1990s had the highest frequencies (69%; 20/29 and 55%; 11/20, respectively) while Finland, where primary immunisations with ACV containing PRN dated from 2009 had the lowest (3.6%). Throughout the study, no PT- or FHA-deficient isolate and one Fim2/3-deficient was detected.ConclusionResults suggest that the longer the period since the introduction of ACVs containing PRN, the higher the frequency of circulating PRN-deficient isolates.

Surveillance of Circulating Bordetella pertussis Strains in Europe during 1998 to 2015.

One reason for increased pertussis incidence is the adaptation of Bordetella pertussis to vaccine-induced immunity by modulating its genomic structure. This study, EUpert IV, includes 265 isolates collected from nine European countries during 2012 to 2015 (n = 265) and compares the results to previous EUpert I to III studies (1998 to 2009). The analyses included genotyping, serotyping, pulsed-field gel electrophoresis (PFGE), and multilocus variable-number tandem-repeat analysis (MLVA). Genotyping results showed only small variations among the common virulence genes of B. pertussis The frequencies of serotypes Fim2 and Fim3 varied among the four collections. Genomic analyses showed that MLVA type 27 increased to 80% between the periods of 1998 to 2001 and 2012 to 2015. Two PFGE profiles, BpSR3 (29.4%) and BpSR10 (27.2%), constituted more than 50% of the circulating isolates in the present collection. Our study indicates that the European B. pertussis population is changing and became more homogenous after the introduction of acellular pertussis vaccines.

Pertussis-specific memory B-cell and humoral IgG responses in adolescents after a fifth consecutive dose of acellular pertussis vaccine.

In order to impede the increase in pertussis incidence in the adolescent group, a school-leaving booster dose administered at the age of 14 to 16 years will be introduced in Sweden in 2016. Preceding this introduction, an open-label, randomized, multicenter, clinical trial without a control group and with blinded analysis was performed, investigating both safety and immunogenicity. Reported here are the memory B-cell and serological responses detected in a smaller cohort (n = 34) of the 230 subjects recruited to the study. All subjects had received primary vaccination consisting of three doses of diphtheria-tetanus-5-component pertussis (DTaP5) vaccine, at 3, 5, and 12 months of age, and a tetanus-low-dose diphtheria-5-component pertussis (Tdap5) vaccine booster at 5.5 years. In this study, the subjects were randomly assigned and received either a Tdap1 or Tdap5 booster. Of the 230 participants, 34 subjects had samples available for evaluation of IgG-producing memory B-cell responses. Both vaccine groups had significant increases in pertussis toxin-specific serum IgG levels, but only the 1-component group showed significant increases in pertussis toxin-specific memory B cells. The 5-component group had significant increases in filamentous hemagglutinin- and pertactin-specific memory B-cell and serum IgG levels; these were not seen in the 1-component group, as expected. In conclusion, this study shows that a 5th consecutive dose of an acellular pertussis vaccine induces B-cell responses in vaccinated adolescents. (This study has been registered at EudraCT under registration no. 2008-008195-13 and at ClinicalTrials.gov under registration no. NCT00870350.).

B-cell responses after intranasal vaccination with the novel attenuated Bordetella pertussis vaccine strain BPZE1 in a randomized phase I clinical trial.

Despite high vaccination coverage, pertussis is still a global concern in infant morbidity and mortality, and improved pertussis vaccines are needed. A live attenuated Bordetella pertussis strain, named BPZE1, was designed as an intranasal vaccine candidate and has recently been tested in man in a phase I clinical trial. Here, we report the evaluation of the B-cell responses after vaccination with BPZE1. Forty-eight healthy males with no previous pertussis-vaccination were randomized into one of three dose-escalating groups or into a placebo group. Plasma blast- and memory B-cell responses were evaluated by ELISpot against three different pertussis antigens: pertussis toxin, filamentous haemagglutinin and pertactin. Seven out of the 36 subjects who had received the vaccine were colonized by BPZE1, and significant increases in the memory B-cell response were detected against all three tested antigens in the culture-positive subjects between days 0 and 28 post-vaccination. The culture-positive subjects also mounted a significant increase in the filamentous haemagglutinin-specific plasma blast response between days 7 and 14 post-vaccination. No response could be detected in the culture-negatives or in the placebo group post-vaccination. These data show that BPZE1 is immunogenic in humans and is therefore a promising candidate for a novel pertussis vaccine. This trial is registered at ClinicalTrials.gov (NCT01188512).

A phase I clinical study of a live attenuated Bordetella pertussis vaccine--BPZE1; a single centre, double-blind, placebo-controlled, dose-escalating study of BPZE1 given intranasally to healthy adult male volunteers.

BACKGROUND: Acellular pertussis vaccines do not control pertussis. A new approach to offer protection to infants is necessary. BPZE1, a genetically modified Bordetella pertussis strain, was developed as a live attenuated nasal pertussis vaccine by genetically eliminating or detoxifying 3 toxins. METHODS: We performed a double-blind, placebo-controlled, dose-escalating study of BPZE1 given intranasally for the first time to human volunteers, the first trial of a live attenuated bacterial vaccine specifically designed for the respiratory tract. 12 subjects per dose group received 10³, 105 or 107 colony-forming units as droplets with half of the dose in each nostril. 12 controls received the diluent. Local and systemic safety and immune responses were assessed during 6 months, and nasopharyngeal colonization with BPZE1 was determined with repeated cultures during the first 4 weeks after vaccination. RESULTS: Colonization was seen in one subject in the low dose, one in the medium dose and five in the high dose group. Significant increases in immune responses against pertussis antigens were seen in all colonized subjects. There was one serious adverse event not related to the vaccine. Other adverse events were trivial and occurred with similar frequency in the placebo and vaccine groups. CONCLUSIONS: BPZE1 is safe in healthy adults and able to transiently colonize the nasopharynx. It induces immune responses in all colonized individuals. BPZE1 can thus undergo further clinical development, including dose optimization and trials in younger age groups. TRIAL REGISTRATION: ClinicalTrials.gov NCT01188512.

Antibody responses to Bordetella pertussis Fim2 or Fim3 following immunization with a whole-cell, two-component, or five-component acellular pertussis vaccine and following pertussis disease in children in Sweden in 1997 and 2007.

Bordetella pertussis fimbriae (Fim2 and Fim3) are components of a five-component acellular pertussis vaccine (diphtheria-tetanus-acellular pertussis vaccine [DTaP5]), and antibody responses to fimbriae have been associated with protection. We analyzed the IgG responses to individual Fim2 and Fim3 in sera remaining from a Swedish placebo-controlled efficacy trial that compared a whole-cell vaccine (diphtheria-tetanus-whole-cell pertussis vaccine [DTwP]), a two-component acellular pertussis vaccine (DTaP2), and DTaP5. One month following three doses of the Fim-containing vaccines (DTwP or DTaP5), anti-Fim2 geometric mean IgG concentrations were higher than those for anti-Fim3, with a greater anti-Fim2/anti-Fim3 IgG ratio elicited by DTaP5. We also determined the responses in vaccinated children following an episode of pertussis. Those who received DTaP5 showed a large rise in anti-Fim2 IgG, reflecting the predominant Fim2 serotype at the time. In contrast, those who received DTwP showed an equal rise in anti-Fim2 and anti-Fim3 IgG concentrations, indicating that DTwP may provide a more efficient priming effect for a Fim3 response following contact with B. pertussis. Anti-Fim2 and anti-Fim3 IgG concentrations were also determined in samples from two seroprevalence studies conducted in Sweden in 1997, when no pertussis vaccine was used and Fim2 isolates predominated, and in 2007, when either DTaP2 or DTaP3 without fimbriae was used and Fim3 isolates predominated. Very similar distributions of anti-Fim2 and anti-Fim3 IgG concentrations were obtained in 1997 and 2007, except that anti-Fim3 concentrations in 1997 were lower. This observation, together with the numbers of individuals with both anti-Fim2 and anti-Fim3 IgG concentrations, strongly suggests that B. pertussis expresses both Fim2 and Fim3 during infection.

Pandemic influenza A(H1N1)pdm09 seroprevalence in Sweden before and after the pandemic and the vaccination campaign in 2009.

The immunity to pandemic influenza A(H1N1)pdm09 in Sweden before and after the outbreaks in 2009 and 2010 was investigated in a seroepidemiological study. Serum samples were collected at four time points: during 2007 (n = 1968), in October 2009 (n = 2218), in May 2010 (n = 2638) and in May 2011 (n = 2513) and were tested for hemagglutination inhibition (HI) antibodies. In 2007, 4.9% of the population had pre-existing HI titres ≥40, with the highest prevalence (20.0%) in 15-24 year-olds, followed by ≥80 year-olds (9.3%). The overall prevalence of HI titres ≥40 had not changed significantly in October 2009. In May 2010 the prevalence had increased to 48.6% with the highest percentages in 5-14 year-olds (76.2%) andlowest in 75-79 year-olds (18.3%). One year later the prevalence of HI titres ≥40 had increased further to 52.2%. Children 5-14 years had the highest incidence of infection and vaccine uptake as well as the highest post-pandemic protective antibody levels. In contrast, the elderly had high vaccine uptake and low attack rate but low levels of protective antibodies, underlining that factors other than HI antibodies are involved in protection against influenza A(H1N1)pdm09. However, for all age-groups the seroprevalence was stable or increasing between 2010 and 2011, indicating that both vaccine- and infection-induced antibodies were long-lived.

Do we need a booster of Hib vaccine after primary vaccination? A study on anti-Hib seroprevalence in Sweden 5 and 15 years after the introduction of universal Hib vaccination related to notifications of invasive disease.

The prevalence of IgG ELISA antibodies against Haemophilus influenzae polyribosyl ribitol phosphate (anti-Hib) was studied in two Swedish seroepidemiologic materials. One study was performed in 1997 5 years after the introduction of universal Hib vaccination (N=3320). Ten years later, a similar study was carried out to analyze the effect of vaccination on anti-Hib prevalence (N=2383). The median values of anti-Hib concentrations (EU/mL) were almost identical in the two materials. The antigenic pressure including vaccination, natural infections and possible cross-immunizations was thus assumed to be constant. The joint median was 0.50 EU/mL (95% confidence interval: 0.46, 0.56). However, there were also indications of reduced exposure to 'Hib-antigens' over a 10-year period. The proportion above the cut-off point for protection, 0.15 EU/mL, decreased significantly for children aged 2-19 years from 78% in 1997 to 74% in 2007 (p=0.034), and there was a significant increase in values below the minimal level of detection for adults from 17% in 1997 to 20% in 2007 (p=0.009). In the 2007 material no specific age group could be identified with a lower immune profile than other age groups older than 3 years and there was a significant downward trend of invasive infections caused by Hib according to notification data for the period 1997-2008. Therefore, the conclusion is that presently there is no need for a booster dose of Hib vaccine in Sweden after primary vaccination but the situation should be carefully monitored.

Antibody response patterns to Bordetella pertussis antigens in vaccinated (primed) and unvaccinated (unprimed) young children with pertussis.

In a previous study, it was found that the antibody response to a nonvaccine pertussis antigen in children who were vaccine failures was reduced compared with the response in nonvaccinated children who had pertussis. In two acellular pertussis vaccine efficacy trials in Sweden, we studied the convalescent-phase enzyme-linked immunosorbent assay (ELISA) geometric mean values (GMVs) in response to pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), and fimbriae (FIM 2/3) in vaccine failures and controls with pertussis. In Germany, the antibody responses to Bordetella pertussis antigens PT, FHA, PRN, and FIM-2 were analyzed by ELISA according to time of serum collection after onset of illness in children with pertussis who were vaccine failures or who were previously unvaccinated. Antibody values were also compared by severity of clinical illness. In Sweden, infants who had received a PT toxoid vaccine and who were vaccine failures had a blunted response to the nonvaccine antigen FHA compared with the response in children who had received a PT/FHA vaccine. Similarly, infants who had pertussis and who had received a PT/FHA vaccine had a blunted response to the nonvaccine antigens PRN and FIM 2/3 compared with the response in children who were vaccine failures and who had received a PT, FHA, PRN, and FIM 2/3 vaccine. In Germany, in sera collected from 0 to 15 days after pertussis illness onset, the GMVs for all 4 antigens (PT, FHA, PRN, and FIM-2) were significantly lower in an unvaccinated group than in children who were diphtheria-tetanus-acellular pertussis (DTaP) vaccine failures. In the unvaccinated group, the GMV of the PT antibody rose rapidly over time so that it was similar to that of the DTaP vaccine recipients at the 16- to 30-day period. In contrast, the antibody responses to FHA, PRN, and FIM-2 at all time periods were lower in the diphtheria-tetanus vaccine (DT) recipients than in the DTaP vaccine failures. In both Sweden and Germany, children with less severe illness had lower antibody responses than children with typical pertussis. Our findings indicate that upon exposure and infection, previous vaccinees have more-robust antibody responses to the antigens contained in the vaccine they had received than to Bordetella antigens that were not in the vaccine they had received. In addition, over time the antibody responses to FHA, PRN, and FIM-2 were greater in children with vaccine failure (primed subjects) than in unvaccinated children (unprimed subjects) whereas the responses to PT were similar in the primed and unprimed children, as determined from sera collected after 15 days of illness. Our findings lend support to the idea that DTaP vaccines should contain multiple antigens.

Kinetics and sensitivity of ELISA IgG pertussis antitoxin after infection and vaccination with Bordetella pertussis in young children.

Sera from 96 young children in a vaccine trial were analysed for kinetics of ELISA IgG anti-pertussis toxin (anti-PT) after a laboratory-verified pertussis infection. The antibody decay curves after infection were biphasic and similar in shape to those after vaccination. The change from a rapid to a slower decay after the peak occurred about 4-5 months from the first day of cough. In a group of children given a two- or a five-component acellular pertussis vaccine the proportion of sera above the tentative cut-off values for anti-PT of 20, 50 or 100 EU/ml 12 months after onset of the infection were 19%, 0% and 0% respectively. Corresponding figures for a whole-cell or placebo vaccine group of infected children were significantly higher, 73%, 39% and 30%, i.e. the antibody decay after infection in young children depends on vaccination status as well as on the pertussis vaccine given. In a large group of non-infected children vaccinated with the same five-component acellular vaccine 13%, 0% and 0% had sera above 20, 50 and 100 EU/ml at 12 months after the third vaccine dose and all were below the minimum level of detection 2 years after vaccination. In conclusion, knowledge about anti-PT kinetics is essential for the interpretation of seroepidemiological data but hardly offers the possibility to establish valid cut-off values for anti-PT in single sample serology. An option would be to identify a grey zone between the positive and negative ends of the distribution for follow-up testing by a second serum.

Should fimbriae be included in pertussis vaccines? Studies on ELISA IgG anti-Fim2/3 antibodies after vaccination and infection.

The anti-Fim response and long-term persistence after vaccination and infection may be of importance in understanding population immunity. Longitudinal serum samples (n = 1330) from 542 non-infected children related to a Swedish vaccine trial showed that the post vaccination (DTPa5) antibody decay curve for pertussis ELISA IgG anti-fimbriae2/3 (anti-Fim2/3) was bi-phasic. A slower one followed an initial rapid decay approximately 5-6 months after the third dose at 12 months of age. After 71 months, however, 60% still had concentrations above > or =5 EU/ml, a level that had been shown to correlate with decreased risk of disease. Booster responses after re-vaccination with DTPa5 at 4, 5 and 6 years of age were strong and appeared within 1 week after vaccination, indicating immune memory. Ninety-six young children with verified pertussis infection, for whom we had serum samples both before, during and after the infection, showed a high response if they had been primed with fimbriae (either DTPa5 or DTPwc). In contrast, 76% of infected children not primed with fimbriae (a DTPa2 or DT group) only had concentrations below the minimum level of detection in all samples taken during and after the infection. In two Swedish seroepidemiological surveys, one from 1997 just after reintroduction of universal childhood vaccination against pertussis and one from 2007, the proportion of children 2-3 years with anti-Fim2/3 concentrations <5 EU/ml was similar and above 90%. This reflects that the two- or three-component pertussis vaccines (DTPa2 and DTPa3) that were introduced in Sweden in 1996 do not induce anti-Fim2/3 antibodies. In previous studies it was shown in multivariate analyses that levels of IgG anti-Fim2/3 > or =5 EU/ml reduced short-term risk of pertussis in small children. As the antibody response to Fim2/3 after infection is poor in children who have not been primed earlier in life, inclusion of immunogenic Fim2/3 in future pertussis vaccines should be considered.

Seroprevalence of pertussis antitoxin (anti-PT) in Sweden before and 10 years after the introduction of a universal childhood pertussis vaccination program.

The prevalence of IgG ELISA antibodies against pertussis toxin (anti-PT) was studied in two Swedish seroepidemiological studies. One was performed in 1997 when the new pertussis vaccination program was 1 year old (n = 3420). In 2007, when Pa vaccines had been used countrywide for 10 years in the universal child vaccination program, this study was repeated to analyze the effect of vaccination on anti-PT prevalence (n = 2379). Before the statistical analysis of seroprevalence, children vaccinated within the last 2 years before the serosurveys were excluded. The results indicate a reduced exposure to Bordetella pertussis in the population. The proportion of sera without measurable anti-PT antibodies increased significantly, aggregated over all comparable age groups, from 3.8% in people sampled in 1997 to 16.3% in people sampled in 2007. For cord blood, 1% was without measurable anti-PT antibodies in 1997 compared to a significantly higher level, 12%, in 2007. With anti-PT concentrations of > or =50 and > or =100 EU/ml as cutoff points for 'recent infection' the proportion above the cutoff points for younger children was significantly higher in 1997 than in 2007 at both cutoff points. For all adults, 20 years of age and older, the difference in proportions above the lower cutoff point was close to statistically significant, comparing 1997 with 2007. This was not the case at 100 EU/ml. In the 1997 samples of children, there was a significant downward trend of 'recent infections' at both cutoff points for three sampled age groups between 5 and 15 years of age from 21% at 5.0-5.5 years of age to 7% at 14.7-15.7 years for the lowest cutoff. In the 2007 samples of children, on the contrary, there was a significant continuous upward trend of 'recent infections', at both cutoff points, for four sampled age groups between 4 and 18 years of age - from 4% at 4-5 years of age to 16% at 17-18 years at the lowest cutoff. The continuous increase, with age of children with high anti-PT concentrations, supports the recent change in the general Swedish childhood vaccination program to include a pre-school booster at 5-6 years and a school-leaving booster at 14-16 years of age.

European Sero-Epidemiology Network 2: standardisation of immunoassay results for pertussis requires homogeneity in the antigenic preparations.

A standardisation process, already developed during the earlier European Sero-Epidemiology Network (ESEN) project, was employed with a more robust algorithm to harmonise results of pertussis serological assays performed in 12 European and non-European countries. Initially, results from each country's own assay were compared with those obtained at the reference laboratory by means of an in-house pertussis toxin (PT)-based ELISA: seven countries used in-house or commercial PT-ELISAs; the other countries used assays based on Bordetella pertussis whole cell extracts (WCE) (three countries) or on combined PT-FHA (filamentous haemagglutinin) antigenic preparations (two countries). The WCE assays, although admitted for diagnostic purposes, confirmed their low correlation with the PT-ELISAs and their results could not be used for standardisation; the PT-FHA ELISAs gave results that were suitable for standardisation in one country but unsatisfactory in the other; the use of purified PT in serological assays confirmed its better reliability than other preparations and all PT-ELISAs results could be calibrated against those of the reference centre. In the standardisation process two high-titre cut-offs indicative of likelihood of recent infection (from within 4 weeks of disease onset up to 1 year after) were included for evaluations as they are suggested to be more useful, for the sero-epidemiological assays of immunity to pertussis, than the cut-off of protection, commonly employed, but still not defined for pertussis. Providing PT-ELISAs are used, standardisation of pertussis assay results is always possible and, when standardisation is performed, evaluation and comparison of the impact of different interventions can be also allowed, by measuring at the distribution of high antibody titres in the populations.

Pertussis antitoxin decay after vaccination with DTPa. Response to a first booster dose 3 1/2-6 1/2 years after the third vaccine dose.

Longitudinal serum samples were collected from 542 children that had participated in a Swedish pertussis vaccine trial 1992-1995 [Gustafsson L, Hallander HO, Olin P, Reizenstein E, Storsaeter J. A controlled trial of a two-component acellular, a five-component acellular, and a whole-cell pertussis vaccine. N Engl J Med 1996;334(6):349-355] and who did not contract pertussis. The sera were analyzed for post vaccination antibody decay and for booster response of anti-PT (IgG antibodies against pertussis toxin), as measured by ELISA. Generally, an initial rapid decay of antitoxin antibody concentration was followed by a slower decay; the change occurring when the geometric mean level of antitoxin concentration reached 8-9 ELISA Units/mL (EU/mL). The time needed to reach this level was 8-9 months after the third dose in a 2, 4, and 6 months schedule. A "best-fit" combined regression model was used to predict when 50% of the children have less than the minimum level of detection of anti-PT (1EU/mL). This occurred about 65 months after dose 3 at an age of 6 years. The anti-PT response to a booster dose was evident but the post-booster geometric mean values decreased with number of years after the third dose and the response appeared later. The results indicate that a pre-school booster might be considered at 6 years of age or earlier.