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.

Serum reactome induced by Bordetella pertussis infection and Pertussis vaccines: qualitative differences in serum antibody recognition patterns revealed by peptide microarray analysis.

BACKGROUND: Pertussis (whooping cough) remains a public health problem despite extensive vaccination strategies. Better understanding of the host-pathogen interaction and the detailed B. pertussis (Bp) target recognition pattern will help in guided vaccine design. We characterized the specific epitope antigen recognition profiles of serum antibodies ('the reactome') induced by whooping cough and B. pertussis (Bp) vaccines from a case-control study conducted in 1996 in infants enrolled in a Bp vaccine trial in Sweden (Gustafsson, NEJM, 1996, 334, 349-355). METHODS: Sera from children with whooping cough, vaccinated with Diphtheria Tetanus Pertussis (DTP) whole-cell (wc), acellular 5 (DPTa5), or with the 2 component (a2) vaccines and from infants receiving only DT (n=10 for each group) were tested with high-content peptide microarrays containing 17 Bp proteins displayed as linear (n=3175) peptide stretches. Slides were incubated with serum and peptide-IgG complexes detected with Cy5-labeled goat anti-human IgG and analyzed using a GenePix 4000B microarray scanner, followed by statistical analysis, using PAM (Prediction Analysis for Microarrays) and the identification of uniquely recognized peptide epitopes. RESULTS: 367/3,085 (11.9%) peptides were recognized in 10/10 sera from children with whooping cough, 239 (7.7%) in DTPwc, 259 (8.4%) in DTPa5, 105 (3.4%) DTPa2, 179 (5.8%) in the DT groups. Recognition of strongly recognized peptides was similar between whooping cough and DPTwc, but statistically different between whooping cough vs. DTPa5 (p<0.05), DTPa2 and DT (p<0.001 vs. both) vaccines. 6/3,085 and 2/3,085 peptides were exclusively recognized in (10/10) sera from children with whooping cough and DTPa2 vaccination, respectively. DTPwc resembles more closely the whooping cough reactome as compared to acellular vaccines. CONCLUSION: We could identify a unique recognition signature common for each vaccination group (10/10 children). Peptide microarray technology allows detection of subtle differences in epitope signature responses and may help to guide rational vaccine development by the objective description of a clinically relevant immune response that confers protection against infectious pathogens.

Modeling rates of infection with transient maternal antibodies and waning active immunity: application to Bordetella pertussis in Sweden.

Serological surveys provide reliable information from which to calculate forces (instantaneous rates) of infection, but waning immunity and clinical consequences that depend on residual immunity complicate interpretation of results. We devised a means of calculating these rates that accounts for passively acquired maternal antibodies that decay or active immunity that wanes, permitting re-infection. We applied our method to pertussis (whooping cough) in Sweden, where vaccination was discontinued from 1979 to 1995. A national cross-sectional serosurvey of antibodies to pertussis toxin, which peak soon after infection and then decay, was conducted shortly after vaccination resumed. Together with age-specific contact rates in Finland, contemporary forces of infection enable us to evaluate the recent assertion that the probability of infection upon contact is age-independent. We find elevated probabilities among children, adolescents and young adults, whose contacts may be more intimate than others. Products of contact rates and probabilities of infection permit transmission modeling and estimation of the intrinsic reproduction number. In contrast to another recent estimate, ours approximates the ratio of life expectancy and age at first infection. Our framework is sufficiently general to accommodate more realistic sojourn distributions and additional lifetime infections.

Global population structure and evolution of Bordetella pertussis and their relationship with vaccination.

Bordetella pertussis causes pertussis, a respiratory disease that is most severe for infants. Vaccination was introduced in the 1950s, and in recent years, a resurgence of disease was observed worldwide, with significant mortality in infants. Possible causes for this include the switch from whole-cell vaccines (WCVs) to less effective acellular vaccines (ACVs), waning immunity, and pathogen adaptation. Pathogen adaptation is suggested by antigenic divergence between vaccine strains and circulating strains and by the emergence of strains with increased pertussis toxin production. We applied comparative genomics to a worldwide collection of 343 B. pertussis strains isolated between 1920 and 2010. The global phylogeny showed two deep branches; the largest of these contained 98% of all strains, and its expansion correlated temporally with the first descriptions of pertussis outbreaks in Europe in the 16th century. We found little evidence of recent geographical clustering of the strains within this lineage, suggesting rapid strain flow between countries. We observed that changes in genes encoding proteins implicated in protective immunity that are included in ACVs occurred after the introduction of WCVs but before the switch to ACVs. Furthermore, our analyses consistently suggested that virulence-associated genes and genes coding for surface-exposed proteins were involved in adaptation. However, many of the putative adaptive loci identified have a physiological role, and further studies of these loci may reveal less obvious ways in which B. pertussis and the host interact. This work provides insight into ways in which pathogens may adapt to vaccination and suggests ways to improve pertussis vaccines. IMPORTANCE Whooping cough is mainly caused by Bordetella pertussis, and current vaccines are targeted against this organism. Recently, there have been increasing outbreaks of whooping cough, even where vaccine coverage is high. Analysis of the genomes of 343 B. pertussis isolates from around the world over the last 100 years suggests that the organism has emerged within the last 500 years, consistent with historical records. We show that global transmission of new strains is very rapid and that the worldwide population of B. pertussis is evolving in response to vaccine introduction, potentially enabling vaccine escape.

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.

Pulsed-field gel electrophoresis analysis of Bordetella pertussis isolates circulating in Europe from 1998 to 2009.

Between 1998 and 2009, Bordetella pertussis clinical isolates were collected during three periods, i.e., 1998 to 2001 (n = 102), 2004 to 2005 (n = 154), and 2007 to 2009 (n = 140), from nine countries with distinct vaccination programs, i.e., Denmark, Finland, France, Germany, The Netherlands, Norway, Poland, Sweden, and the United Kingdom. Pulsed-field gel electrophoresis (PFGE) analysis was performed according to standardized recommendations for epidemiological typing of B. pertussis. There were 81 different PFGE profiles, five of which (BpSR3, BpSR5, BpSR10, BpSR11, and BpSR12) were observed in 61% of the 396 isolates and shown to be predominant in almost all countries. The major profile, BpSR11, showed a decreasing trend from 25% to 30% in 1998 to 2005 to 13% in 2007 to 2009, and there were increases in BpSR3 and BpSR10 from 0% and 8% to 21% and 22%, respectively. One difference between these profiles is that BpSR11 contains isolates harboring the fim3-2 allele and BpSR3 and BpSR10 contain isolates harboring the fim3-1 allele. The total proportion of the five predominant profiles increased from 44% in 1998 to 2001 to 63% in 2004 to 2005 to 70% in 2007 to 2009. In conclusion, common PFGE profiles were identified in B. pertussis populations circulating in European countries with different vaccination programs and different vaccine coverages. These prevalent isolates contain the novel pertussis toxin promoter ptxP3 allele. However, there is evidence for diversifying selection between ptxP3 strains characterized by distinct PFGE profiles. This work shows that, even within a relatively short time span of 10 years, successful isolates which spread through Europe and cause large shifts in B. pertussis populations may emerge.

Pertussis vaccination in infancy lowers the incidence of pertussis disease and the rate of hospitalisation after one and two doses: analyses of 10 years of pertussis surveillance.

OBJECTIVES: Shortly after pertussis vaccination was reintroduced in Sweden in 1996, an intensified pertussis disease surveillance programme was set up. In this study, we report on in-depth analyses of age-dose-number-specific incidences and the rate of pertussis hospitalisation for children with no, 1 or 2 doses of an acellular pertussis vaccine before pertussis disease. Vaccine coverage, the timeliness of childhood vaccination and the effect of later than scheduled pertussis vaccination(s) are also examined. STUDY DESIGN: Children with notified laboratory-confirmed (culture or PCR) pertussis disease were evaluated among the surveillance population of about 1 million infants, born between 1996 and 2007 and followed for pertussis disease from October 1997 to December 2007, for nearly 6 million person-years. Birth and vaccination dates of the diseased children are known from the surveillance programme. To estimate denominators of the age-dose-number-specific pertussis incidences, we used birth and vaccination dates from a vaccine trial with more than 72,000 infants combined with national pertussis vaccine coverage data for children in the surveillance population. RESULTS: For infants from 3 to <5 months of age, the incidence of pertussis disease with at least 14 days of cough decreased from 264/100,000 for unvaccinated infants to 155/100,000 for infants with one dose of a pertussis vaccine prior to onset of the disease. In the age range 5 to <12 months, the age-dose specific incidences were 526, 95, and 24/100,000 for infants with no, 1 and 2 doses, respectively. The rate of hospitalisation for infants with 1 dose of a pertussis vaccine prior to onset of the disease was significantly lower than for unvaccinated infants of the same age. For many infants, there is a delay in administration of the vaccine doses according to the regular 3-5-12 month schedule (which has been the case for many years). Hypothetically, if all infants had been vaccinated exactly on schedule, we would expect about 28% fewer pertussis cases with at least 14 days of cough and 38% fewer hospitalisations due to pertussis, of cases possible to influence by vaccinations on schedule. CONCLUSION: Pertussis vaccination had a significant effect among infants already after the first dose. This is particularly important for premature infants and infants with severe respiratory and cardiac diseases. A moderate decrease in the incidence of pertussis disease in infants and rate of hospitalisation could be expected if primary vaccinations were carried out closer to the scheduled time than is currently the practice in Sweden.

Glycoconjugate vaccines and immune interactions, and implications for vaccination schedules.

Conjugate vaccines using diphtheria toxoid variant (CRM(197)), diphtheria toxoid and tetanus toxoid (TT) as carrier protein may induce immune interactions (interference or impairment as measured by lower antibody levels, or enhancement [higher antibody levels]) when coadministered with other vaccines. Immune enhancement occurs when two TT conjugates are coadministered. CRM(197) conjugate vaccines induce immune bystander interference when given with diphtheria-tetanus-acellular pertussis vaccines, which reduces responses to coadministered Haemophilus influenzae type b vaccine conjugated to TT. These bystander effects are greater as the amount of CRM(197) administered increases. When large amounts of either TT or CRM(197) are coadministered, dose-related carrier-induced epitopic suppression may occur, affecting immune responses to meningococcal or pneumococcal polysaccharides. These observations have implications for vaccine scheduling. The range of available alternative vaccines means that specific vaccine coadministrations can avoid or reduce CRM(197)-induced interference. Potential interactions arising from new CRM(197) or TT conjugates will need to be thoroughly examined.

Is adolescent pertussis vaccination preferable to natural booster infections?

Pertussis is still poorly controlled in both adolescents and adults. As a result, an adolescent pertussis booster vaccine dose has already been implemented or decided on in many countries. The reasons for this have been twofold: a worrying increase of infections in the target group of adolescents and a wish to prevent serious pertussis disease among young yet unvaccinated, and partly vaccinated, infants. Currently, it is still too early to evaluate the effect of the late booster on the circulation of Bordetella pertussis owing to the lack of relevant follow-up data. A universal adolescent booster vaccination will reduce the incidence of pertussis in the target group but the duration of immunity is uncertain. It is an open question as to what extent boosters should be offered to older age groups or if natural infections would be preferable. On the one hand, circulating B. pertussis may be hazardous to the youngest unvaccinated infants. On the other hand, subclinical natural boosters might be beneficial to population immunity. As the duration of immunity is shorter after vaccination than after natural infections, an unwanted consequence of adolescent boosters might shift the infection peak to older child-bearing adults. It is therefore recommended that recurrent serosurveys are used to follow the influence of vaccination on the antigenic pressure, as well as the duration of protective immunity. For this purpose, standardization of symptoms and laboratory criteria used for notification, as well as the methodology for seroepidemiology, must be established. Adverse reactions after adolescent vaccination and outbreaks owing to new B. pertussis variants must also be carefully monitored. In this article, we have used Swedish surveillance data and the results from Swedish seroepidemiology to illustrate these problem areas.

SNP-based typing: a useful tool to study Bordetella pertussis populations.

To monitor changes in Bordetella pertussis populations, mainly two typing methods are used; Pulsed-Field Gel Electrophoresis (PFGE) and Multiple-Locus Variable-Number Tandem Repeat Analysis (MLVA). In this study, a single nucleotide polymorphism (SNP) typing method, based on 87 SNPs, was developed and compared with PFGE and MLVA. The discriminatory indices of SNP typing, PFGE and MLVA were found to be 0.85, 0.95 and 0.83, respectively. Phylogenetic analysis, using SNP typing as Gold Standard, revealed false homoplasies in the PFGE and MLVA trees. Further, in contrast to the SNP-based tree, the PFGE- and MLVA-based trees did not reveal a positive correlation between root-to-tip distance and the isolation year of strains. Thus PFGE and MLVA do not allow an estimation of the relative age of the selected strains. In conclusion, SNP typing was found to be phylogenetically more informative than PFGE and more discriminative than MLVA. Further, in contrast to PFGE, it is readily standardized allowing interlaboratory comparisons. We applied SNP typing to study strains with a novel allele for the pertussis toxin promoter, ptxP3, which have a worldwide distribution and which have replaced the resident ptxP1 strains in the last 20 years. Previously, we showed that ptxP3 strains showed increased pertussis toxin expression and that their emergence was associated with increased notification in The Netherlands. SNP typing showed that the ptxP3 strains isolated in the Americas, Asia, Australia and Europe formed a monophyletic branch which recently diverged from ptxP1 strains. Two predominant ptxP3 SNP types were identified which spread worldwide. The widespread use of SNP typing will enhance our understanding of the evolution and global epidemiology of B. pertussis.

Appearance of Fim3 and ptxP3-Bordetella pertussis strains, in two regions of Sweden with different vaccination programs.

After introduction of a mono-component vaccine, containing only pertussis toxoid (PT), the incidence of pertussis was significantly higher in the Gothenburg area among children during the period October 1, 1997 until end of 2006 compared to the Rest of Sweden where a vaccine containing PT and two other pertussis antigens was used. To investigate a possible cause of this difference, the Bordetella pertussis populations in both regions were compared by determining the fimbrial serotype (Fim), the PFGE-type and the pertussis toxin promoter allele type (ptxP). Strains with the ptxP1 allele were successively replaced by ptxP3 strains producing more pertussis toxin. In Gothenburg compared to the Rest of Sweden, Fim3 and ptxP3 strains were observed earlier and reached higher frequencies in the studied period. Since ptxP3 strains have been shown to be more virulent, their higher prevalence may have contributed to the higher incidence of pertussis in the Gothenburg area. In addition we found a high degree of linkage between PFGE-profile and ptxP3. Our results highlight the importance of strain typing to gain insight into the mechanisms of immunity-associated selection of microbial subtypes and the causes of changes in incidences of infectious diseases.

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.