Previously, DNA sequence typing has been used for well-characterized meningococcal antigens, such as the major porin PorA (19
) as well as FetA (21
), and more recently for FHBP (6
). A conventional PCR-based approach also has been used to assign capsular groups (16
) and PorB variable region types (18
DNA sequencing of the genes encoding the new meningococcal vaccine antigen, designated GNA1870 or FHBP, is useful to identify common or representative versions of the three variant groups of this promising vaccine candidate. Since DNA sequence determination of one or more genes from many strains is resource intensive, we developed a rapid, high-throughput assay to distinguish strains from different FHBP variant groups. We considered using the same respective primers for performing conventional PCR and identifying specific products by electrophoretic mobility in agarose gels. However, compared with the typing methods for PorB and capsular group mentioned above or conventional PCR, the QPCR method has advantages of being more rapid, relatively inexpensive, and scalable to grouping a large number of isolates in a single day.
A number of monoclonal antibodies (MAbs) have been raised against FHBP variant 1 (7
). However, to date, there are no MAbs that recognize all strains within a variant group. One of the MAbs, JAR-1, binds to all strains expressing the canonical variant 1 protein (examples are strains MC58, H44/76, CU385, and others from the hypervirulent lineage associated with electrophoretic type 5) (23
). In the present study, all of the California meningococcal isolates recognized by MAb JAR-1 in a whole-cell dot blot assay (n
= 35) had FHBP variant 1 genes by QPCR. Five additional California isolates identified as having FHBP variant 1 genes by QPCR were negative for binding to MAb JAR-1. By DNA sequencing, these five isolates were confirmed as having genes encoding FHBP variant 1 (Table ). FHBP group assignments of all of the isolates that were identified as variant 2 or 3 by QPCR also were confirmed by DNA sequencing of the FHBP gene. Thus, the QPCR method provided a precise grouping designated for FHBP variant 1, 2, and 3 strains.
To develop an FHBP-based group B vaccine, it is important to know the distribution of variant 1, 2, and 3 FHBP strains in different geographic regions, since antibodies raised to the variant 1 protein are bactericidal against most FHBP variant 1 strains but show little functional activity against variant 2 or 3 strains (6
). In a previous study by Masignani et al., FHBP variant 1 genes constituted 54% of strains examined (11
). These data suggested that at least two FHBP proteins (variants 1 and 2) might be required for an FHBP-based vaccine to provide broad coverage against strains causing most group B meningococcal disease. However, the strains in the study by Masignani et al. were selected to be representative of the known genetic diversity among N. meningitidis
strains and, therefore, most likely included a disproportionate number of rare isolates chosen on the basis of genetic diversity rather than disease prevalence.
In the present study, FHBP variant 1 was present in 83% of group B isolates obtained during 2003 to 2004 from patients residing in 22 counties in California. By DNA sequencing, variant 1 also was present in 69% of recent isolates from Swedish patients (8
). Thus, the variant 1 protein is the most prevalent of the three variants, and a single FHBP protein has the potential to prevent the majority of cases of disease. In devising a multicomponent group B recombinant protein vaccine, it therefore may be preferable to include FHBP variant 1 and other unrelated, promising vaccine antigens, such as GNA2132 (15
) and/or NadA (4
). Adding FHBP variant 2 and/or variant 3 proteins may provide less of a benefit, since the immune response to several antigenically unrelated proteins may help avoid selection of escape mutants. Such a strategy, however, underscores the need to determine the prevalence of different variants of FHBP among strains from additional geographic areas and to monitor carefully for emergence of FHBP variant 2 or 3 N. meningitidis
strains after a vaccine is introduced. The QPCR method described herein is rapid and precise and can be applied to large numbers of isolates in a single day. The method also is potentially suitable for monitoring genetic variation of the other proteins targeted for vaccination.