STs, fHBP gene variants, and nadA presence
summarizes the distribution of STs among the isolates. Thirty-one percent of the isolates from patients in California had identical respective ST, porA, porB, and fetA genotypes, which were consistent with a specific clonal group. Isolates with this genotype were less prevalent in the other 2 collections (≤6%). With the exception of isolates from 3 patients hospitalized during a 2-week period in 1 county, the remaining California isolates with this genotype came from patients hospitalized at different times of the year and/or in different counties. Therefore, as a group the isolates were not part of a specific outbreak.
Distribution of sequence types (STs) among group B meningococcal isolates.
QPCR results showed that the fHBP gene was present in all 143 isolates. Collectively, 65% were fHBP v.1, 31% were v.2, and 4% were v.3. As shown in , the fHBP v.1 gene predominated in isolates from California (83% v.1, 13% v.2, and 4% v.3), whereas the Maryland collection had 52%, 44%, and 4% and the multicenter collection had 57%, 37% and 4%, respectively. The respective percentages in the latter 2 collections were similar to each other and were collectively different from those in the California collection (P <.01, χ2 test). In all, 71 isolates (50%) were nadA positive; 77% of the California isolates were positive for the nadA gene, compared with 32% and 37%, respectively, in the Maryland and multicenter collections (P <.001) (). All of the ST-32 isolates with identical respective porA, porB, and fetA genotypes () were positive for the fHBP v.1 and nadA genes. If these isolates are removed from the analysis of the frequencies of fHBP variants and nadA, the respective proportions in the 3 collections are still significantly different (P <.02).
Figure 1 Percentage of fHBP variant (v.) 1, 2, or 3 genes (A) and nadA genes (B), as determined by quantitative polymerase chain reaction. Error bars indicate 95% confidence intervals. Shaded bars, California isolates; hatched bars, Maryland isolates; white bars, (more ...)
Reactivity with anti-fHBP and anti-NadA antibodies
Ninety-seven percent of isolates expressed fHBP, as detected by dot immunoblotting using polyclonal antiserum prepared against fHBP v.1, v.2, and v.3. Of those with detectable expression of fHBP, there was moderate (up to 10-fold) variation in the quantity expressed by the different isolates. Of 71 isolates with the nadA gene, as detected by QPCR, 99% expressed the NadA protein, as detected by dot immunoblotting with polyclonal anti-NadA antiserum. NadA expression in cultures grown to the same optical density (0.8–0.9) was estimated to differ by as much as 100-fold. All of the isolates that were negative for the nadA gene by QPCR also were negative by dot immunoblotting.
The 6 MAbs made against fHBP v.1 or v.2 differed in their reactivity on whole-cell dot immunoblots with a panel of control strains and, therefore, appear to recognize unique epitopes. The MC58 wild-type (WT) strain (fHBP v.1) was positive by dot immunoblotting for reactivity with MAbs JAR 1, 3, and 5 (), which were prepared against recombinant fHBP v.1 expressed from the gene from MC58 [35
]. In contrast, the MC58 WT strain did not react with MAbs JAR 10, 11, or 13, which were prepared against the recombinant fHBP v.2 expressed from the gene from strain 2996 (). None of the 6 MAbs bound to the MC58 fHBP knockout (KO) strain. Group B strain NZ98/254, which expresses a v.1 fHBP that is 91.3% identical to that of MC58 [35
], was recognized by 2 of the anti–fHBP v.1 MAbs, JAR 3 and 5, as well as by the anti-v.2 MAb JAR 10. The 8047 WT strain, which expresses a v.2 fHBP, was recognized by all 3 of the anti-v.2 MAbs but by none of the anti-v.1 MAbs (). The M1239 WT strain, which expresses a v.3 fHBP, was recognized only by the anti-v.2 MAb JAR 13. The respective patterns of MAb reactivity can be used to describe different fHBP phenotypes. We established a binary system of nomenclature to indicate the pattern of reactivity of the fHBP MAbs in the order JAR 1, 3, and 5 (anti-v.1) followed by JAR 10, 11, and 13 (anti-v.2), with 1 indicating positive reactivity and 0 indicating no reactivity. Thus, the fHBP phenotype of the v.1 strain MC58 is 111-000, and that of the v.2 strain 8047 is 000–111.
Figure 2 Anti–factor H–binding protein (fHBP) monoclonal antibody (MAb) reactivity with representative Neisseria meningitidis group B strains. Heat-killed, whole meningococci were applied to a membrane, which was probed with polyclonal antibody (more ...)
Next, we examined the fHBP MAb reactivity of the 143 isolates in the 3 collections. As summarized in , among the 93 isolates expressing fHBP v.1, phenotype 111-000 was the most common (70% of v.1 isolates). Isolates with this fHBP phenotype were predominantly electrophoretic type (ET)–5 (89%), ST–32 (71%), and nadA positive (83%). Two other phenotypes, 011-000 and 011–100, constituted 9% and 10% of the v.1 isolates, respectively. These groups had heterogeneous STs, and only 11% and 38%, respectively, were nadA positive (P <.0001, compared with the proportion of nadA-positive 111-000 isolates). Other fHBP phenotypes (111-100, 001–100, and 000-000) collectively accounted for the remaining 11% of the v.1 isolates.
Prevalence of factor H–binding protein (fHBP) isolates, by fHBP phenotype.
fHBPs in the v.2 group have, on average, 85% aa identity with the v.3 group, whereas both of these groups have lower homology with fHBPs in the v.1 group (74 and 63%, respectively) [19
]. There also is considerable cross-reactivity between v.2 and v.3 proteins but not between either of these and v.1 proteins (see below). Therefore, for the purpose of analysis of the fHBP phenotypes, the data from strains expressing v.2 or v.3 proteins were combined. Among the 50 fHBP v.2 or v.3 (v.2/v.3) isolates, phenotype 000–111 occurred in 20%; this group contained both v.2 and v.3 isolates, and these were predominantly in the ST-35 complex. Phenotype 000–110 occurred in 24% of the v.2/v.3 isolates, and these isolates were all v.2 and were predominantly lineage III. Phenotype 000–001, which is typified by the fHBP v.3 strain M1239, occurred in 12% of the v.2/v.3 isolates; this group contained both v.2 and v.3 isolates, and these were primarily in the ST-162 complex. Isolates with the phenotype 000-000, which do not react with any of the MAbs, comprised 44% of the v.2/v.3 isolates. All of these isolates expressed v.2 proteins. They had diverse STs and likely represent a genetically heterogeneous collection.
Bactericidal activity of anti-fHBP antisera
We measured the bactericidal activity of anti-fHBP v.1, v.2, and v.3 antisera on 84 of the isolates (48 v.1; 36 v.2; and 3 v.3). The anti-v.1 antiserum was bactericidal (titer ≥1:8, with human complement) against 77% of the v.1 isolates, whereas the anti-v.2 and anti-v.3. antisera killed only 2% and 0%, respectively, of the v.1 isolates. v.1 isolates representative of phenotypes 111-000 and 011-000 were particularly susceptible to the bactericidal activity of the anti-v.1 antiserum (91% and 100% of isolates, respectively), whereas only 50% of isolates with phenotype 011–100 or with other v.1 phenotypes were susceptible to killing by this serum (). Together, these latter groups comprised 21% of v.1 isolates (). The respective geometric mean titers (GMTs) exhibited similar patterns. The GMTs against v.1 isolates with either the 111-000 or 011-000 phenotype were ≥1: 600, whereas those against isolates with the remaining phenotypes were between 1:30 and 1:50 ().
Figure 3 Bactericidal activity of anti–factor H–binding protein (fHBP) antisera against fHBP variant (v.) 1 isolates, by fHBP phenotype (n =8–21 isolates/group). A, Percentage of strains giving a titer ≥1:8; B, Reciprocal geometric (more ...)
The majority (74%) of the v.2/v.3 isolates were susceptible to the bactericidal activity of the anti-v.2 antiserum. Of the isolates with a 000–111, 000–110, or 000–001 phenotype, 78%–100% were susceptible, whereas only 46% of the isolates with the 000-000 phenotype were susceptible (). The GMTs of the anti-v.2 antiserum against isolates with 1 of the first 3 phenotypes were between 1:200 and 1:1500 and was 1:25 for the phenotype 000-000 (). Most of the v.2/v.3 phenotypes showed similar results with the anti-v.3 antiserum. One exception was the 000–111 phenotype; of the isolates with this phenotype, 67% were susceptible to anti-v.3 antiserum (GMT, 1:70), versus 100% being susceptible to the anti-v.2 antiserum (GMT, 1:1500).
Figure 4 Bactericidal activity of anti–factor H–binding protein (fHBP) antiserum against isolates expressing fHBP variant (v.) 2 or 3. Each fHBP phenotype had 6–13 isolates. A, Percentage of strains giving a titer ≥1:8; B, Reciprocal (more ...)
Bactericidal activity of anti-5C antiserum
Ninety-five percent of the 48 fHBP v.1 isolates tested were killed (titer ≥1: 8) by the polyclonal antiserum from mice immunized with the 5C vaccine (), which contains fHBP v.1, compared with 56% of the v.2/v.3 isolates () (P <.001). Only 2 v.1 isolates were not killed by the anti-5C antiserum, and both were in the other-phenotype category. The GMTs of the anti-5C antiserum against v.1 isolates with different fHBP phenotypes ranged from 1:150 to 1:1500. The susceptibility of the v.2/v.3 isolates with different phenotypes ranged from 33% to62% (), and no v.2/v.3 phenotype correlated strongly with susceptibility to antibodies elicited by the 5C vaccine. The respective GMTs of the v.2/v.3 isolates also were significantly lower (1:10–1:30) than those of the v.1 isolates, which may reflect the lack of bactericidal activity of anti-v.1 antibodies against v.2/v.3 isolates and the lower bactericidal activity of antibodies elicited by the other 4 antigens in the 5C vaccine.
Figure 5 Percentage of isolates giving a titer ≥1:8 to anti–5-component (5C) vaccine antiserum. Also shown are data for the respective percentages susceptible to the anti–factor H–binding protein (fHBP) variant (v.) 1 antiserum (more ...)
Contribution of GNA 2132 and NadA antigens in the 5C vaccine
To assess the contributions of antigens other than fHBP v.1 in eliciting protective antibodies, we selected strains that were killed by the anti-5C antiserum but not by the anti-v.1 antiserum and tested their susceptibility to antisera prepared against 2 of the other antigens in the 5C vaccine, GNA 2132 [16
] and NadA [18
]. Only 1 of 7 fHBP v.1 isolates tested was killed (titer ≥1:8) by the anti–GNA 2132 antiserum, compared with 8 of 10 fHBP v.2/v.3 isolates (P
<.02, Fisher’s exact test). Five of the 10 isolates expressing fHBP v.2/v.3 had the nadA
gene, and 1 of these was killed by the anti-NadA antiserum. Of the 7 v.1 isolates tested, the nadA
gene was present in 3, and none were killed by the anti-NadA antiserum.