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

DISCUSSION

B. pertussis fimbriae have been shown to be important components of both whole-cell and acellular vaccines, and antibodies to these proteins have been associated with protection in early trials of whole-cell vaccines (by the MRC) and in household exposure studies (18, 20). IgG responses to copurified Fim2/3 have been measured in the clinical trials of acellular pertussis vaccines (13, 26) and many subsequent studies. The predominant serotype expressed by B. pertussis isolates has changed in a number of countries during different time periods (27, 28, 29), and the reasons for this are not understood. B. pertussis isolates possess fim2 and fim3 genes, and expression is dependent on a run of C residues in the promoter regions of these genes (12). Spontaneous changes in the poly(C) tract length allow or stop expression, but despite this genetic flexibility, the predominant Fim type remains stable over time until a switch in the population occurs. It is not known if this is driven by immune evasion following a rise in anti-Fim2 or anti-Fim3 IgG levels in those infected.

To understand the role of antibodies to Fim2 and Fim3, we purified individual Fim2 and Fim3 from B. pertussis and used these to quantify anti-Fim2- and anti-Fim3-specific IgG following vaccination with DTaP5 in individuals with clear evidence of on-going and recent pertussis disease (21). The ELISA IgG concentration values assigned to standard serum 89/530 in the previous study (21) were determined by aligning the ELISA dose-response curve for a 1:1 mixture of Fim2 and Fim3 with the dose-response curves obtained with this serum and separate Fim2 or Fim3 preparations coated at equal protein concentrations. Thus, the anti-Fim2 and anti-Fim3 IgG concentration values assigned to each serum sample allow comparisons of the IgG antibody responses to these antigens, although the comparisons should be treated with caution as the functional activities of the antibodies are not known. These separate Fim2 and Fim3 preparations have now been used to quantify anti-Fim2 and anti-Fim3 IgG responses in sera from Sweden trial I obtained between 1992 and 1995 (13) and from seroepidemiology samples obtained in Sweden in 1997 and 2007 (22).

A unique serum collection from a well-controlled group of small children, vaccinated with three doses of either DTaP5, DTaP2, DTwP, or placebo DT and assessed previously for anti-Fim2/3 IgG by ELISAs (13), has allowed us to determine anti-Fim2 and anti-Fim3 IgG responses. Anti-Fim2 and anti-Fim3 IgG concentrations were determined in 7-month-old children in the DTaP5 and DTwP groups only. The anti-Fim2 IgG concentrations were greater than the anti-Fim3 concentrations for both vaccines. It is interesting to note that the anti-Fim2 and anti-Fim3 IgG responses following three doses of vaccine were 7- and 14-fold lower than those previously determined in sera from 15-month-old children following a fourth dose of DTaP5 (21). This previous study showed that the ratio of the anti-Fim2 to the anti-Fim3 IgG concentration decreased with additional doses, and this study confirms that this trend continues until after dose three and that the anti-Fim2/anti-Fim3 ratio is greatest in young children. Anti-Fim2 and anti-Fim3 IgG responses following DTwP have not been reported previously and show that this particular whole-cell vaccine elicited lower anti-Fim2 IgG geometric mean titers than DTaP5 (P < 0.05) but higher anti-Fim3 IgG geometric mean titers (Table 1) with a lower ratio of anti-Fim2/anti-Fim3 values (1.6) than that of DTaP5 (6.6).

This serum collection also included children with anti-Ptx IgG-confirmed pertussis infection after vaccination with any of the vaccines used, and this allowed both the anti-Fim2 and anti-Fim3 IgG responses to be determined following disease so that the priming effect of these vaccines for anti-Fim2 and anti-Fim3 responses could be determined. The 6-month postvaccination and predisease anti-Fim2 and anti-Fim3 IgG geometric mean concentrations shown in Table 2 were low for the control (DT) and the DTaP2 vaccine without fimbriae. With both DTaP5 and DTwP, anti-Fim2 IgG values were higher than those obtained for anti-Fim3, continuing the trend observed at 7 months of age. These higher anti-Fim2 antibody levels were probably not influenced by earlier episodes of subclinical pertussis as the anti-Fim2/3 concentrations seen in the DTaP5 and DTwP subgroups in an earlier study did not differ from those seen in a larger data set of 1-year samples after vaccination (22). The values reported previously for the 15-month-old children in samples obtained before the fourth dose of DTaP5 are comparable to those reported in this study from children 6 months after three doses of DTaP5 (21).

The anti-Fim2 and anti-Fim3 IgG peak levels found in these children in a current episode of pertussis (Table 3 and Fig. 3) show the clear priming effect of prior vaccination with a Fim-containing vaccine. Children who received DTaP5 or DTwP vaccines showed much greater postdisease anti-Fim2 and anti-Fim3 IgG levels than those who previously received DT or DTaP2. The responses to Fim2 were greater than those to Fim3, corresponding with the predominant Fim2 serotype of isolates at this time. However, for interpretation of these data, it must be kept in mind that the distinction between the serotypes of isolates (2, 3 or 2/3) is based on an insensitive agglutination technique. It might be that the technique does not detect small amounts of either Fim3 or Fim2 in vitro. Also, both antigens may be expressed in vivo as suggested by Heikkinen et al. (30).

There is also the possibility that the anti-Fim2 response is more efficiently primed, particularly by DTaP5, possibly due to the greater Fim2 than Fim3 content of this vaccine. It is interesting to observe that the ratio of predisease to peak postdisease anti-Fim2 IgG levels following DTaP5 was greater than that for anti-Fim3 (Table 2 and 3), whereas the pre- to postdisease anti-Fim2 and anti-Fim3 IgG levels were similar in those who had received DTwP. Thus, it appears that DTwP is a more effective priming vaccine for an anti-Fim3 response. This is clearly shown in Fig. 2B, where the reverse cumulative distribution curve for anti-Fim3 IgG levels is clearly shifted to the right for those who had received DTwP. This result is perhaps surprising as most isolates during this time were serotyped as Fim2 but may reflect a proportion of strains expressing Fim3. However, some caution is recommended in drawing conclusions from these data as peak anti-Fim2 and anti-Fim3 IgG responses were determined with sera from only a small number of individuals. The observation that anti-Fim2 IgG responses were greater than those for anti-Fim3 following vaccination with DTaP5 may tie in with a study also performed in Sweden in 1992-1995 that suggested that DTaP5 may be less effective against Fim3 strains as described in the Introduction.

It is clear that the anti-Fim2 and anti-Fim3 responses seen following disease were greatly influenced by previous vaccination, with greater anti-Fim2 and anti-Fim3 responses seen in those who received DTaP5 or DTwP than in those vaccinated with a non-Fim vaccine. It has been shown that previous vaccination can also blunt responses to antigens not included in the vaccine (31). It can be seen in Fig. 2 that the distributions of anti-Fim2 and anti-Fim3 responses are lower in those who received DTaP2 than in those who received the control DT vaccine although this result was not significant with the sample size used. Cherry et al. (31) proposed that this was due to linked epitope suppression caused by preferential responses of memory B-cells following secondary exposure to the vaccine components. The memory B-cells thus outcompete naive B-cells for access to the Bordetella epitopes. An alternative explanation is that some protection is provided by the DTaP2 vaccine and that vaccine failures in the DTaP2 vaccine group may have milder or less prolonged disease than those in the DT group and thus have reduced responses to nonvaccine antigens.

We also had access to seroepidemiology samples collected in 1997 from children who had not received a pertussis vaccination and to samples collected in 2007 when either DTaP2 or DTaP3 had been in use for about 11 years. The reverse cumulative distribution curves in Fig. 4 show that the distributions of the IgG concentrations obtained are the same for anti-Fim2 in both years and for anti-Fim3 in 2007, while lower anti-Fim3 concentrations were obtained in 1997. The close similarity of the IgG concentration distributions was also clear from the percentages of individuals with anti-Fim2 and anti-Fim3 IgG concentrations in various ranges (Table 4). The observation that the distributions were the same shows that individuals had an equal likelihood of exposure to Fim2 or Fim3 in 2007, despite the majority of case isolates serotyped as Fim3 only. This is clear evidence that B. pertussis is able to express both Fim2 and Fim3 during infection. Expression of both fimbrial genes is affected by a poly(C) tract in the promoter region (12) with an optimal poly(C) tract length required for expression. The poly(C) tract length can be altered by slip strand mispairing during DNA replication. This may occur in vivo at a higher rate than previously thought, and this genetic flexibility may be an advantage to the organism.

Thus, in this study, we have characterized the immune responses to Fim2 and Fim3 separately using a panel of sera from Sweden trial I. This has allowed the anti-Fim2 and anti-Fim3 IgG concentrations to be determined following vaccination with either DT, DTwP, DTaP2, or DTaP5. We have also determined the responses in the same children before, during, and following an episode of pertussis disease. Thus, the priming potentials of DTaP5 and DTwP vaccines for anti-Fim2 and anti-Fim3 IgG responses were determined. As expected, due to Fim2 strains predominating at this time, there was a greater response to Fim2 than to Fim3. However, DTwP-vaccinated children produced equal boosts in responses to both Fim2 and Fim3, suggesting that either there were more circulating Fim3 strains than among those isolated and serotyped or this vaccine provides very efficient priming for response to this antigen. This also suggests that there is an opportunity for reformulating acellular vaccines to improve the priming provided by the Fim3 component. The distributions of anti-Fim2 and anti-Fim3 IgG concentrations against both antigens in seroepidemiology samples obtained in 1997 and 2007 were very similar, except that the anti-Fim3 concentrations in 1997 were lower. This observation strongly suggests that B. pertussis expresses Fim2 and Fim3 during infection irrespective of the predominant serotype of the case isolates.