fimbriae have been shown to be important components of acellular pertussis vaccines, with a significant association between clinical protection in household exposure studies and the presence of preexposure antibodies to Fim2 plus Fim3 (3
). Many studies have quantified the IgG response to fimbriae following immunization with pertussis vaccines, but copurified mixtures of Fim2 and Fim3 have been used which do not contain a defined ratio of antigens. In this study, we have purified separate Fim2 and Fim3 from Fim2- or Fim3-expressing B. pertussis
strains so that the immune response to the individual fimbrial antigens could be quantified. The purification of these antigens has been previously described (23
), but we were not able to replicate these methods successfully. Despite modification of the methods, yields of separate antigens remained low, and large fermenter cultures were required to obtain sufficient purified protein. Fim2 was purified from a serotype Fim2 strain by homogenization and repeated ammonium sulfate precipitation in the same way as Fim2 plus Fim3 from a strain expressing both antigens. Fim3 cannot be isolated using this method, and extraction with buffer containing 4 M urea was required, as previously described (23
). The proteins appeared highly pure by SDS-PAGE analysis, although Fim2 plus Fim3 and Fim2 contained large amounts of LPS. In contrast, Fim3 prepared using 4 M urea extraction contained low levels of LPS. The exquisite serotype specificity of the antigens following immunization in mice () demonstrates that this LPS contamination does not cause cross-reaction of the antibody response to heterologous antigen preparations and so is unlikely to contribute to the antibody responses seen with Fim2 plus Fim3 and Fim2 in human subjects, although this cannot be completely discounted and will be the subject of investigation in future studies. The lack of availability of separate Fim2 and Fim3 was noted by Xu et al. (32
) and prompted development of recombinant Fim2 and Fim3. These antigens had the predicted subunit molecular weight on SDS-PAGE and were recognized by specific Fim2 and Fim3 monoclonal antibodies. However, these antigens did not provide protection in a mouse intranasal infection model as described for native antigens (22
), suggesting the lack of protective epitopes in the recombinant antigens.
We developed an ELISA to determine IgG concentrations to separate Fim2 and Fim3 using the assigned concentration of NIBSC standard 89/530, which has been calibrated against U.S. reference pertussis antiserum lot 3 and has been used in many studies (16
). The NIBSC 89/530 serum was a pool that contained serum from individuals immunized with 5-component acellular pertussis vaccine or following recent pertussis disease and thus had detectable responses to both Fim2 and Fim3. The Fim units assigned were obtained in studies using copurified mixtures of Fim2 and Fim3 where the ratios of Fim2 to Fim3 were not known. The Fim2-Fim3 preparation obtained in this study was approximately 60% Fim2 and 40% Fim3 () and thus was not used to assign concentration values for Fim2 and Fim3, as this would have skewed the Fim2 and Fim3 antibody binding ratio. Thus, we used Fim2 and Fim3 at a defined 1:1 ratio of as the coating antigens, which showed that 89/530 contains a higher proportion of antibodies that recognize Fim2 than Fim3 (). We have shown that 4 M urea-treated fimbriae make suitable coating antigens. Fim2 was treated with 4 M urea before use in the ELISA to match the Fim3 preparation available. Also, sera from mice immunized with Fim2 treated with urea produced an IgG response similar to that produced by native Fim2 (), and use of urea-treated Fim2 as a coating antigen did not affect the magnitude of the response seen with sera raised against native or urea-treated Fim2. In addition, the avidity of the anti-Fim2 IgG population for its antigen is assumed to be the same as that for anti-Fim3 population and Fim3. This was shown to be the case, as the slopes of the Fim2 and Fim3 dose response curves are similar (). The higher result assigned to 89/530 for Fim2 compared to Fim3 is thus a true reflection of how much antibody is present for each serotype.
This ELISA was used to quantify Fim2 plus Fim3, Fim2, and Fim3 IgG concentrations in small panels of sera from 15-month-old children, 4- to 6-year-old children, and 11- to 18-year-olds immunized with 5-component acellular pertussis vaccine. All individuals showed rises in IgG concentration to both Fim2 and Fim3, confirming that both components of the copurified Fim2-Fim3 mixture in the vaccine are immunogenic. Higher geometric mean responses to Fim2 than Fim3 were seen in the younger two age groups, but this difference was reduced in the 11- to 18-year-old subjects. Responses to both proteins were greater in the older vaccinees, indicating a strong boost response to these antigens. The significance of the lower IgG concentrations seen in response to Fim3 following vaccination of young children is not known. Two studies performed in Sweden suggest that 5-component acellular pertussis vaccine may be less effective against Fim3 strains. Hallander et al. (8
) report a higher percentage of Fim3 breakthrough strains among children who received 5-component acellular vaccine than in an unvaccinated control group. This was also seen in the 1992 Swedish study, with a higher percentage of Fim3 breakthrough strains seen in the 5-component acellular and whole-cell vaccine groups, compared to an equal ratio of serotypes between two-component acellular vaccine and control groups (26
We have also used the Fim2 and Fim3 ELISA to determine IgG concentrations in individuals with evidence of recent pertussis by Ptx IgG ELISA. It has been observed previously that individuals with anti-Ptx IgG above the diagnostic threshold were more likely to have high levels of anti-FHA and anti-Fim2-Fim3 IgG (1
), but responses to individual Fim2 and Fim3 have not previously been analyzed. Because United Kingdom B. pertussis
isolates have been almost exclusively Fim3 serotype during the time period examined, it would be expected that individuals would show increased Fim3 IgG concentrations following disease, and this was demonstrated in panels of sera from 2009, 2010, and 2011. However, the number of individuals with moderate Fim2 IgG concentrations in 2009 and 2010 was surprising given the predominant serotypes of isolates obtained in those years (). This could mean either that Fim2 strains were circulating and not represented in the small number of isolates obtained or that B. pertussis
may express both Fim types in vivo
. This was suggested by Heikkinen et al. (11
), who used a monoclonal antibody blocking ELISA, due to the unavailability of separate Fim2 and Fim3, to determine serotype-specific antibody responses following infection. They found that patients infected by Fim3 strains had antibodies which blocked the binding of monoclonal antibodies to Fim3 but not to Fim2. However, one-third of the Fim2 strain-infected patients developed antibodies capable of blocking binding of both Fim2 and Fim3 monoclonal antibodies, indicating a difference in fimbrial expression in vivo
and in vitro
. The expression of fimbriae is known to be dependent on a run of C residues in the promoter regions of fim2
which renders the genes capable of phase variation through spontaneous variation in the poly(C) tract length (2
). As strains appear to possess both fimbrial genes, it is possible that variable expression could occur during disease. However, despite this genetic flexibility, the predominant serotypes are stable over a number of years in most countries, with periodic switching (13
), and we present preliminary evidence from 2011 that this may be occurring in the United Kingdom. An additional possibility for the high Fim2 ELISA IgG concentrations seen in some individuals may be anti-LPS antibodies reacting with the higher level of LPS in the Fim2 ELISA coating antigen, and this will be the subject of further study.
The strong anti-Fim2 and -Fim3 IgG responses seen following disease may be, in part, influenced by previous vaccination, as individuals have been shown to produce strong responses to fimbriae if they had received prior fimbria-containing pertussis vaccine (9
). In addition, blunted responses to antigens not contained in pertussis vaccines previously received have been observed (4
). United Kingdom individuals in this study are likely to have received whole-cell pertussis vaccination which primed for response to both Fim2 and Fim3.
Thus, in this study, we have purified separate Fim2 and Fim3 and characterized the immune response to these antigens following vaccination, showing that both proteins are immunogenic. In addition, the anti-Fim IgG response following recent disease was greater for Fim3, consistent with the predominant serotype of circulating B. pertussis isolates. However, a surprising number of individuals have moderate responses to Fim2, raising a number of questions about the serotype of circulating strains or in vivo expression of fimbriae. With the resurgence of pertussis seen in a number of countries with high levels of vaccine coverage, improved vaccines are required that may reduce transmission of the pathogen, and improved responses to both Fim2 and Fim3 may be important to achieve this aim.