Rapid genetic grouping of factor h-binding protein (genome-derived neisserial antigen 1870), a promising group B meningococcal vaccine candidate

Abstract

The most important antigen component of a promising multicomponent group B meningococcal recombinant protein vaccine is based on genome-derived neisserial antigen 1870, which recently was renamed factor H-binding protein (FHBP) to reflect one of its critical functions as a complement regulatory protein. Neisseria meningitidis strains can be subdivided into three FHBP variant groups based on divergence of FHBP amino acid sequences. Within each variant group, amino acid sequences are >90% conserved. To develop an FHBP-based group B vaccine, it is important to know the distribution of FHBP variant 1, 2, and 3 strains in different geographic regions, since antibodies against FHBP are bactericidal against strains within the homologous group but show minimal activity against strains from other groups. We have devised a high-throughput, quantitative PCR-based method that allows rapid and precise assignment of FHBP genes into each of the three major variant lineages. Among 48 group B isolates from patients hospitalized in California in 2003 to 2004, 83%, 13%, and 4%, respectively, had variant 1, 2, and 3 genes. Thus, a vaccine based on the variant 1 protein has the potential to prevent the majority of cases of group B disease. The quantitative PCR-based method will be useful for determining and monitoring the prevalence of meningococcal isolates with genes encoding different FHBP variant proteins. The technique also is suitable for monitoring variation of genes encoding other protein antigens targeted for vaccination.

FIG. 1.

DNA sequence alignment of FHBP from prototype strains. FHBP from strains MC58 (variant 1; GenBank accession no. AE002098), 961-5945 (variant 2; DQ523568), and M1239 (DQ523569) were aligned using ClustalW (3). The variant 1- and 2-specific (v.1,2) primers are shown above the alignment, and the variant 2- and 3-specific (v.2,3) primers are shown below the alignment. Where the primer sequences differ from one of the FHBP sequences, an asterisk is shown adjacent to the divergent sequence. Only the regions used for primer design are shown; the numbering with respect to the full-length open reading frames is shown at the right.

FIG. 2.

Results of a conventional PCR-based assay for FHBP grouping. Three primer pairs, specific for FHBP variants 1 and 2 (v.1,2), variants 2 and 3 (v.2,3), and 16S rRNA genes, were used to amplify cognate and noncognate templates. The positions of DNA size standards in base pairs are shown at the right. (A) Variant-specific detection of plasmid templates. Lane 1, no template control; 2, pET21b-FHBP (strain MC58, variant 1); 3, pET21b-FHBP (961-5945, variant 2); 4, pET21b-FHBP (M1239, variant 3). (B) Detection of genomic DNA templates. Lane 1, MC58 wild type (WT); 2, 961-5945 WT; 3, M1239 WT; 4, MC58 FHBP knockout (KO); 5, 961-5945 FHBP KO; 6, M1239 FHBP KO.

FIG. 3.

Results of a quantitative PCR-based assay for FHBP grouping of prototype variant 1, 2, and 3 strains. The C_T is plotted versus template source. A low C_Tcorresponds with a high PCR amplification signal. Plasmid samples are pET plasmid FHBP clones from strains MC58 (variant 1), 961-5945 (variant 2), and M1239 (variant 3); WT genomic samples are wild-type (WT) genomic DNA from the same strains as the respective plasmid samples; KO genomic samples are FHBP knockout (KO) genomic DNA from the same respective strains as plasmid and WT genomic samples.

FIG. 4.

Results of a QPCR using bacterial colony suspensions as templates. A single colony from each prototype strain was suspended in buffer in five replicates, which were used as templates in QPCR with v.1,2 and v.2,3 primer pairs. Data points represent the mean C_Tvalues, and error bars correspond to the standard deviations (1σ).

FIG. 5.

QPCR grouping using as PCR template heat-killed N. meningitidis cells from strains with FHBP variant group defined by DNA sequence (Table 1). (A) QPCR data for FHBP variant 1 strains. Left, variant 1 wild-type (WT) strains; right, variant 1 knockout (KO) strains. (B) QPCR data for FHBP variant 2 and 3 strains. Descriptions correspond to those for panel A. Two disease isolates from California, 03S-0451 and 03S-0669, are shown as additional examples of variant 3 strains (confirmed by DNA sequence determination; Table 2).