Frequency of factor H-binding protein modular groups and susceptibility to cross-reactive bactericidal activity in invasive meningococcal isolates

Abstract

Meningococcal factor H-binding protein (fHbp) is a promising vaccine candidate that elicits serum bactericidal antibodies in humans. Based on sequence variability of the entire protein, fHbp has been divided into three variant groups or two sub-families. We recently reported that the fHbp architecture was modular, consisting of five variable segments, each encoded by genes from one of two lineages. Based on combinations of segments from different lineages, all 70 known fHbp sequence variants could be classified into one of six modular groups. In this study, we analyzed sequences of 172 new fHbp variants that were available from public databases. All but three variants could be classified into one of the six previously described modular groups. Among systematically collected invasive group B isolates from the U.S. and Europe, modular group I overall was most common (60%) but group IV (natural chimeras) accounted for 23% of UK isolates and <1% of U.S. isolates (P<0.0001). Mouse antisera to recombinant fHbp from each of the modular groups showed modular group-specific bactericidal activity against strains with low fHbp expression but had broader activity against strains with higher fHbp expression. Thus both modular group and relative expression of fHbp affected strain susceptibility to anti-fHbp bactericidal activity. The results confirmed the modular architecture of fHbp and underscored its importance for the design of broadly protective group B vaccines in different regions.

Figure. 1

fHbp expression measured by immunoblotting with infrared detection. Panel A, Recombinant proteins in modular groups I (ID 1) or VI (ID 77), or heat-killed bacterial cells from strains expressing fHbp in the corresponding modular groups. The modular group I proteins were detected with murine anti-fHbp mAb JAR 5, which recognizes nearly all fHbps in modular groups I and IV. The modular group VI proteins were detected with anti-fHbp mAb JAR 31, which recognizes nearly all proteins in modular groups II, III, V and VI (See Supplemental Table 3). Panel B, Standard curves from the corresponding binding of the recombinant proteins shown in Panel A.

Figure 2

Network analysis of the relatedness of the amino acid sequences of 242 unique fHbp sequence variants as generated by the program, SplitsTree [22]. The previously described classifications into sub-families A and B [16], or variant groups 1, 2 and 3 [14], are shown along with the respective nine modular groups (I to IX, see Figure 3). The most extreme modular groups (II on the left and I on the right) represent proteins for which each of the respective five segments was derived from one lineage or the other. The proteins in modular groups III to IX apparently represent recombination events that resulted in natural chimeras.

Figure 3

Schematic representations of fHbp modular structure. Panel A, Positions of blocks of invariant residues (shown as vertical rectangles), which flank five variable segments, designated V_A to V_E. The amino acid positions of the last residue in each variable segment are shown. The numbering is based on sequences of mature proteins of three N. meningitidis fHbp variants (IDs 1, 77 and 26) in variant groups 1, 2 and 3, respectively, as defined by Masignani et al [14]. Panel B. Schematic of nine fHbp modular groups deduced from phylogenic analysis of 242 unique amino acid variants. The respective Masignani variant group designations, and the number of unique sequences observed within each fHbp modular group, are shown. Adapted from Beernink and Granoff [18].

Figure 4

Summary networks of peptide sequences of each of the variant sequences in variable segments A and E as computed by the program SplitsTree [22] (See legend to Figure 2).

Figure 5

Frequency of fHbp modular groups among systematically collected N. meningitidis group B case isolates. Data are from sequences of isolates collected in the United States (N=432), United Kingdom (N=536) and France (N=244) reported by Murphy et al [16], and newly obtained sequences of 143 additional U.S. isolates from California (2003-2004), Maryland (1995 and 2005), and pediatric hospitals in 9 states (2001-2005) from a collection previously described by Beernink et al [27].

Figure 6

Serum bactericidal activity of serum pools from mice immunized with fHbps from modular groups I to VI. The black bars represent the median titers of 3 to 4 serum pools for each modular group tested against heterologous test strains. The white bars represent the median titer of the respective homologous serum pools against the test strain. +, refers to relative expression of fHbp by each of the strains; strains with +/- representing low fHbp-expressing strains (see values in Table 1).