Factor H binding protein (fHbp) is an important antigen for vaccines against meningococcal serogroup B disease. The protein binds human factor H (fH), which enables the bacteria to resist serum bactericidal activity. Little is known about the vaccine-potential of fHbp for control of meningococcal epidemics in Africa, which typically are caused by non-group B strains. We investigated genes encoding fHbp in 106 serogroup A, W-135 and X case isolates from 17 African countries. We determined complement-mediated bactericidal activity of antisera from mice immunized with recombinant fHbp vaccines, or a prototype native outer membrane vesicle (NOMV) vaccine from a serogroup B mutant strain with over-expressed fHbp. Eighty-six of the isolates (81%) had one of four prevalent fHbp sequence variants, ID 4/5 (serogroup A isolates), 9 (W-135), or 74 (X) in variant group 1, or ID 22/23 (W-135) in variant group 2. More than one-third of serogroup A isolates and two-thirds of W-135 isolates tested had low fHbp expression while all X isolates tested had intermediate or high expression. Antisera to the recombinant fHbp vaccines were generally bactericidal only against isolates with fHbp sequence variants that closely matched the respective vaccine ID. Low fHbp expression also contributed to resistance to anti-fHbp bactericidal activity. In contrast to the recombinant vaccines, the NOMV fHbp ID 1 vaccine elicited broad anti-fHbp bactericidal activity, and the antibodies had greater ability to inhibit binding of fH to fHbp than antibodies elicited by the control recombinant fHbp ID 1 vaccine. NOMV vaccines from mutants with increased fHbp expression elicit an antibody repertoire with greater bactericidal activity than recombinant fHbp vaccines. NOMV vaccines are promising for prevention of meningococcal disease in Africa and could be used to supplement coverage conferred by a serogroup A polysaccharide-protein conjugate vaccine recently introduced in some sub-Saharan countries.
For more than 100 years devastating epidemics of meningococcal disease have occurred in sub-Saharan Africa [1]. In the decade 1988 to 1997, more than 700,000 cases and over 100,000 deaths were reported. Public health responses were limited by scarce resources [2]. Further, the only vaccines available, un-conjugated (plain) polysaccharides, elicited incomplete and short duration of protection in young children [3], [4], and had a minimal effect on decreasing transmission of the organism [3], [5], [6]. Control of epidemic meningococcal disease in Africa, therefore, remains an unsolved public health challenge. Most meningococcal disease in industrialized countries is caused by strains producing capsular serogroups B, C or Y, whereas most disease in sub-Saharan Africa is caused by serogroup A strains. After more than ten years of work [7], [8], a promising serogroup A polysaccharide-protein conjugate vaccine recently was developed for sub-Saharan Africa [9], [10]. As of January 21,2011, nearly 20 million people had been immunized as part of demonstration projects in three countries (http: //www. path. org/menafrivac/index. php). While this vaccine has the potential to eliminate serogroup A epidemics, widespread vaccination may result in selective pressure for replacement of strains with other capsular serogroups such as X or W-135, which have caused epidemics in this region [11]–[14]. However, with the possible exception of Spain [15], there is little evidence of serogroup replacement after widespread use of monovalent serogroup C meningococcal conjugate vaccines in Europe [16], [17]. Pneumococcal serotype replacement, in contrast, has been a problem in many countries where pneumococcal polysaccharide-protein conjugate vaccines were introduced [18]–[22]. A number of protein antigens are being developed for prevention of meningococcal serogroup B disease (Reviewed in [23], [24]). These antigens also are prevalent in meningococcal strains with other capsular serogroups [7], [25], [26]. Therefore, the vaccines have the potential to prevent disease caused by non-group B strains. One of the most promising of the new protein vaccine candidates is factor H binding protein (fHbp, which was previously referred to as GNA1870 [27] or LP2086 [28]). Recombinant fHbp is part of two vaccines in clinical development for prevention of serogroup B disease [28]–[30]. Native outer membrane vesicle vaccines from meningococcal mutants with over-expressed fHbp also are under investigation [31]–[36]. The fHbp antigen is a surface-exposed lipoprotein that binds complement fH [37], which down-regulates complement activation and enhances the ability of the organism to escape complement-mediated bacteriolysis [37]–[39]. In immunized mice and humans, antibodies to recombinant fHbp vaccines elicited complement-mediated serum bactericidal activity [27], [28], [30], [40]–[45], which in humans is the hallmark of protection against meningococcal disease [46]–[48]. The present investigation was undertaken to determine the vaccine-potential of fHbp for control of meningococcal epidemics in Africa caused by serogroup A, W-135 and X isolates.
In a previous study, we characterized fHbp sequence variants in a small collection of serogroup A, W-135 and X isolates from patients in sub-Saharan Africa [49]. The objectives of the present study were to determine fHbp sequence variants in an expanded panel of case isolates from Africa, to measure levels of fHbp expression, which in previous studies had been reported to be important for predicting susceptibility of serogroup B strains to anti-fHbp bactericidal activity [50], and to investigate the immunogenicity in mice of recombinant fHbp vaccines representative of sequence variants prevalent among invasive African strains. The recombinant vaccine immunogenicity results were compared to that of a prototype native outer membrane vesicle vaccine (NOMV) prepared from a serogroup B mutant strain engineered to over-express fHbp. Our hypothesis was that the NOMV would elicit broader serum bactericidal antibody responses against the strains from Africa than the recombinant fHbp vaccines since in previous studies, mutant NOMV vaccines with over-expressed fHbp elicited broader bactericidal activity against serogroup B strains [33]–[36], [51]. Vaccine immunogenicity was evaluated in CD 1 mice in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Children' s Hospital & Research Center at Oakland Institutional Animal Care and Use Committee. Blood collection was performed under anesthesia, and all efforts were made to minimize suffering. The human complement source for measuring serum bactericidal activity was serum from an adult who participated in a protocol that was approved by the Children' s Hospital Oakland Institutional Review Board (IRB). Written informed consent was obtained from the subject. Antibody concentrations were transformed (Log10). For calculations of geometric mean antibody concentrations, concentrations below the limit of the assay were assigned a concentration half of the lower limit. Two-tailed Student' s t tests were used to compare the geometric mean antibody concentrations between two independent groups of mice. All statistical tests were two-tailed; probability values of less than or equal to 0. 05 were considered statistically significant.
The distributions of the prevalent clonal complexes, fHbp variant groups, and fHbp sequence variants are shown in Figure 2. The serogroup A isolates were derived from three clonal complexes: CC 1 (three isolates from 1963 to 1967 and one from 1989), CC 4 (ten from 1966 to 1990), and CC 5 (three from 1988–1999, and fourteen from 2003–2007). In contrast, the 53 serogroup W-135 isolates were predominately from CC 11 (N = 47 from 1994 to 2007). Among the 22 serogroup X isolates, four were members of a new CC (designated CC 181 (http: //pubmlst. org/neisseria/). Of the remaining 18 isolates, three from the 1970′s were sequence type (ST) 3687, and 15 from 2006–2007 were ST 5403. These two STs differed by only a single nucleotide change in one of the seven loci (fumC) and, thus, may represent an undesignated CC, which accounted for 82% of the serogroup X isolates. There also were a limited number of PorA VR types (Figure S2). Among the serogroup A isolates, P1. 20,9 was present overall in 55 percent, and in 89 percent of 18 serogroup A isolates obtained since 1990. Among the serogroup W-135 isolates, P1. 5,2 and related types such as 5-1,2-2 predominated (98%), and among the serogroup X isolates, P1. 19,26 and related type P1. 19,26-4 accounted for 68%. The PorA VR typing results are consistent with previous studies of strains from sub-Saharan Africa [58], [59]. One hundred percent of the serogroup A isolates, 95% of the X isolates, and 34% of the W-135 isolates had fHbp in the variant 1 group (Figure 2, middle column). The remaining W-135 isolates had fHbp variant 2 (58%) or 3 (8%); one serogroup X isolate had fHbp variant 3. The distribution of the major fHbp amino acid sequence variants is shown in Figure 2 (right column). All of the group A isolates had fHbp sequence variants ID 4 or 5, which differed from each other by a single amino acid. The most prevalent sequence variants in the serogroup X isolates were ID 74 (67%) and ID 73 (12%); two of the serogroup X isolates in the category “other” had fHbp ID 4, which was prevalent among serogroup A isolates. With only a few exceptions the W-135 isolates were clonal based on having a common clonal complex (CC 11) and PorA VR type (P1. 5,2, see Figure S2). This clone could be subdivided into multiple subclones based on genes encoding fHbp ID 9 (variant 1), 22/23 (variant 2, and which differed from each other by one amino acid), or 349 or 111 (variant 3; included in the category “other”, Figure 2). Collectively, four fHbp sequence variants (or related variants, each differing from the respective prevalent variant by 1 amino acid) were present in 81% of the 106 isolates. These were fHbp ID 4/5,74 or 9 (variant 1 group), or ID 22/23 (variant 2 group). The percent amino acid identities between each of these sequence variants, and between fHbp sequence variants ID 1,28 and 77, which were used as control vaccines, are summarized in Table 1. We determined fHbp expression levels in bacterial cells from 44 isolates (Figure 3) using a quantitative Western blot. Six of the 16 group A isolates tested (Panel A), and nine of the 14 W-135 isolates tested with fHbp in the variant 1 group (Panel B), had low fHbp expression (defined by ≤33% of fHbp expressed by the group B reference strain H44/76, which is a naturally high expresser of fHbp ID 1 in variant group 1). All but one of seven group W-135 isolates tested with fHbp in the variant group 2 expressed ≤33% of the group B reference strain 8047, which is a naturally high expresser of fHbp ID 77 in variant group 2 (Panel D). In contrast, all but one of the seven group X isolates tested with fHbp in variant 1 group had high fHbp expression (>100% expression compared with H44/76, Panel C). The seventh isolate had ∼80% of the fHbp expressed by H44/76, which was considered intermediate. We prepared isogenic mutants with different levels of fHbp expression from three isolates: a serogroup B reference strain, and serogroup A and W-135 African isolates. Figure 4 panels A, B and C illustrate relative expression of fHbp on the surface of live bacteria as measured by flow cytometry. Panels D, E and F show the corresponding fHbp expression measured in solubilized bacterial cells by a quantitative Western blot [50]. For each set of mutants, there was a 5- to 10-fold range between lowest and highest fHbp expression. The wildtype serogroup B reference isolate had naturally high expression of fHbp ID 1. Sera from mice immunized with each of the recombinant fHbp ID 1,4, 9, or 74 (variant 1 group) vaccines had high bactericidal titers against this strain (Figure 4, Panel G). As expected, this strain was resistant (titer <1∶10) to the serum from mice immunized with the recombinant fHbp ID 22 vaccine (variant group 2). Against the isogenic mutant of H44/76 with 58% fHbp expression by Western blot as that of the wildtype H44/76 strain, the respective anti-fHbp ID 1,4, 9, or 74 bactericidal titers were ∼10-fold lower than the corresponding titers measured against the higher fHbp expressing wildtype strain (Panel G). In contrast, none of these antisera was bactericidal against the H44/76 mutant with low fHbp expression (10% of the wildtype isolate). The H44/76 wildtype strain and two fHbp mutants were equally susceptible to bactericidal activity of a positive control mAb against PorA (Panel G, yellow horizontal hatched bars). Similar respective results were observed with the mutants of the serogroup A (A3) and serogroup W-135 isolate (W13). For example, the W-135 wildtype strain with 45% fHbp expression relative to that of the reference strain was susceptible to anti-fHbp bactericidal activity only by the antiserum prepared to the recombinant fHbp vaccine ID 9 that matched that of the target isolate (titer >1∶1000, Panel I). In contrast, the isogenic mutant with increased fHbp expression (126% relative to H44/76) was susceptible to anti-fHbp bactericidal activity by antisera to any of the recombinant fHbp vaccine sequence variants in variant group 1. While the data from the mutants do not permit a precise definition of the level of fHbp expression required for homologous and cross-reactive anti-recombinant fHbp bactericidal activity, collectively the results indicated that fHbp expression below 30% of H44/76 was associated with resistance and, with increasing fHbp expression, the isolates became more susceptible to anti-fHbp cross-reactive bactericidal activity. Figure 5 shows the anti-fHbp bactericidal titers of antisera prepared to the different recombinant fHbp sequence variants in variant group 1 when measured against wildtype serogroup A, W-135 and X isolates with fHbp in variant group 1. The isolates were generally susceptible to anti-fHbp antibodies elicited by the recombinant fHbp vaccine that matched the sequence variant of the vaccine (blue bars) but not to antibodies elicited by mismatched recombinant fHbp variants. The lack of cross-reactive bactericidal activity was most notable for the anti-fHbp ID 1 and ID 74 antisera (Panels A and D, respectively). This result was surprising since these vaccines had 92 to 96% amino acid identity with the fHbp expressed by the test strains (Table 1). In Figure 5 the order of the isolates from left to right is with increasing fHbp expression (as shown in Figure 2). While there were trends for increased susceptibility to anti-fHbp bactericidal activity with increasing fHbp expression (for example, the W-135 isolates and the respective anti-fHbp ID 9 titers), the relationship is not linear, being confounded by other variables. The lack of linearity is most evident with the serogroup A isolates where some of the lowest fHbp expressers (i. e. , A1 and A2) were killed by the anti-fHbp ID 4 antiserum that matched that of the isolates, while some of the highest expressers (i. e. , A13 and A14) were resistant. Figure 6 shows the anti-fHbp bactericidal titers of antisera prepared to different recombinant fHbp sequence variants in variant groups 2 or 3 when measured against wildtype serogroup W-135 isolates with fHbp ID22/23 in variant group 2. The anti-fHbp ID 22 antiserum was bactericidal against all of the isolates, even though nearly all of these isolates were low fHbp expressers. Although these isolates also were killed by the control mismatched anti-fHbp ID 77 (variant 2) or fHbp ID 28 (variant 3) antisera (84 to 94 percent amino acid identity with fHbp ID 22/23, Table 1), the respective titers were 10 to 100-fold lower than those of the anti-fHbp ID 22 antiserum (compare anti-ID 22 bactericidal titers in Panel B to those in Panels C and D, Figure 6). We measured susceptibility of 12 African isolates to bactericidal activity of an antiserum from mice immunized with a prototype NOMV vaccine prepared from a mutant of group B strain H44/76 with over-expressed fHbp ID 1 (Figure 7). As controls, we tested antisera from mice immunized with an NOMV vaccine from an isogenic fHbp knock-out mutant (NOMV fHbp KO) or a recombinant fHbp ID 1 vaccine. Against the wildtype serogroup B H44/76 strain, which was a high expresser of fHbp ID 1 that matched the recombinant fHbp vaccine, the bactericidal titers of the control antisera to the NOMV vaccine from the fHbp knock-out mutant, or the recombinant fHbp ID 1 vaccine, were both ∼1∶10,240. Only one of the 12 heterologous African isolates (X4) was susceptible to bactericidal activity of the antiserum to the recombinant fHbp ID 1 vaccine (white bars). In contrast, 11 of the 12 isolates were killed by the antiserum from mice immunized with the NOMV vaccine from the serogroup B mutant with over-expressed fHbp ID 1 (orange bars), which included both serogroup A isolates resistant to bactericidal activity of the antiserum to the recombinant fHbp ID 4 vaccine that matched that of the isolate (gray bars). The one W-135 strain, W1, which was not killed by any of the antisera, was the lowest expresser of fHbp (Figure 3). The remaining three serogroup W-135 tested and all four serogroup X isolates were killed by sera from the mice immunized with the NOMV vaccine from the mutant with over-expressed fHbp ID 1, but the respective bactericidal titers were lower than the corresponding titers elicited by the recombinant fHbp vaccine with a sequence variant that matched that of the strain. With one exception (X5), the bactericidal titers elicited by the control NOMV vaccine from the fHbp knockout mutant were negative (titers <1∶10, gray hatched bars). Although not shown in Figure 7, when we mixed this antiserum with the antiserum to the recombinant fHbp ID 1 vaccine, the serum bactericidal titers remained negative (<1∶10,11 isolates) or unchanged (titer 1∶50, isolate X5). Thus, there was no evidence of cooperative bactericidal activity between anti-fHbp antibodies elicited by the recombinant fHbp ID 1 vaccine and antibodies elicited to other antigens in the NOMV. These results are in contrast to a previous report of cooperative bactericidal activity observed between human antibodies to recombinant fHbp ID 1 and Neisserial Heparin binding antigen, which individually lacked bactericidal activity [60]. By ELISA, the mice immunized with the NOMV vaccine from the mutant with over-expressed fHbp ID 1 had higher serum anti-fHbp ID 1 antibody concentrations than mice immunized with the recombinant fHbp ID 1 vaccine (respective geometric means of 2203 and 746 U/ml, P<0. 02, Figure 8, Panel A). By ELISA, the sera from the mice immunized with the mutant NOMV vaccine also showed greater inhibition of binding of fH to fHbp ID 4, which was the sequence variant expressed by serogroup A strains (Panel B, P<0. 05 at each dilution tested). The increased fH inhibition was not only a result of the higher serum anti-fHbp concentrations in the mutant NOMV vaccine group, but also appeared to be from a different anti-fHbp antibody repertoire, since on average the anti-fHbp antibody concentration required for inhibition of fH in this group was nearly 4-fold lower than in the recombinant fHbp vaccine ID 1 group (respective geometric means of 1. 17 vs. 4. 04 U/ml, P<0. 05, Panel C).
There are several important limitations to the present study. First, we investigated only 106 case isolates. Although these isolates were from 17 countries, 30 percent were from Burkina Faso. Africa is a large and diverse continent with a complex ecology affecting meningococcal transmission and disease. Development of a mutant NOMV fHbp-based vaccine for sub-Saharan Africa will require ongoing surveillance of meningococcal strains to assure that the vaccine antigens match those of the prevalent strains. Second, for investigation of anti-fHbp antibody activity elicited by the recombinant fHbp vaccines, we prepared hyperimmune antiserum pools in mice immunized with the vaccines given with Freunds complete and incomplete adjuvant. This adjuvant is unsuitable for humans and the high anti-fHbp titers in the hyperimmune mouse serum pools are unlikely to be achieved in humans. The poor cross-reactive bactericidal activity of these mouse antisera is, therefore, likely to be even lower in humans immunized with recombinant vaccines given with aluminum adjuvants. The broad serum bactericidal antibody responses to the mutant fHbp NOMV vaccine, however, were in mice given the vaccine with aluminum hydroxide. A third limitation was that while the data from the isogenic mutants with different levels of fHbp showed a clear relationship between fHbp expression levels and susceptibility to anti-fHbp bactericidal activity, the relatively small number of wild type strains tested, and the presence of potential confounders such as capsular serogroup [70], LOS [71], [72] or alternative fH binding ligands [73], did not provide sufficient statistical power for a formal analysis of the relationship between fHbp expression and anti-fHbp bactericidal activity. A fourth limitation of the present study is that we used a prototype NOMV vaccine from a mutant group B strain with over-expressed fHbp ID 1. We chose this vaccine since it had worked well in previous studies of group B strains, and an NOMV vaccine from a similar mutant African strain was not yet available. Neither the PorA VR type of the group B vaccine strain nor the fHbp sequence variant was present among the African isolates. We would anticipate that even higher serum bactericidal antibody responses would be elicited by NOMV vaccines prepared from mutant African isolates where the antigens in the vaccine would be matches to the Africa strains. Finally, although the serum bactericidal titers of the control mice immunized with the NOMV from the fHbp knockout were negative, we had insufficient sera from mice immunized with the NOMV vaccine from the mutant with over-expressed fHbp to prove that the bactericidal antibodies were directed against fHbp. In several previous studies however, we demonstrated that bactericidal activity against serogroup B strains was greatly or completely diminished after depletion of anti-fHbp antibodies by solid phase adsorption [34], [36], [51].
Epidemics of meningococcal meningitis are common in sub-Saharan Africa. Most are caused by encapsulated serogroup A strains, which rarely cause disease in industrialized countries. A serogroup A polysaccharide protein conjugate vaccine recently was introduced in some countries in sub-Saharan Africa. The antibodies induced, however, may allow replacement of serogroup A strains with serogroup W-135 or X strains, which also cause epidemics in this region. Protein antigens, such as factor H binding protein (fHbp), are promising for prevention of meningococcal serogroup B disease. These proteins also are present in strains with other capsular serogroups. Here we report investigation of the potential of fHbp vaccines for prevention of disease caused by serogroup A, W-135 and X strains from Africa. Four fHbp amino acid sequence variants accounted for 81% of the 106 African isolates studied. While there was little cross-protective activity by antibodies elicited in mice by recombinant fHbp vaccines from each of the four sequence variants, a prototype native outer membrane vesicle (NOMV) vaccine from a mutant with over-expressed fHbp elicited antibodies with broad protective activity. A NOMV vaccine has the potential to supplement coverage by the group A conjugate vaccine and help prevent emergence of disease caused by non-serogroup A strains.
vaccines
medicine
vaccination
clinical immunology
immunity
vaccine development
biology
microbiology
immunology
bacterial pathogens
Meningococcal Factor H Binding Proteins in Epidemic Strains from Africa: Implications for Vaccine Development