Capsular polysaccharide-based vaccines currently exist for meningococcal serogroups A, C, Y and W. However, due to the antigenic similarity of the meningococcal serogroup B (MnB) capsular polysaccharide with sialic acid structures on human neuronal tissue,1–3 the MnB capsule is poorly immunogenic in humans. Outer membrane protein vesicle-based vaccines have been used regionally to control epidemics in Norway, Cuba, New Zealand and France,4,5 where the infecting MnB strains are genetically homogeneous and share the same porin A protein as the vaccine strain. Unfortunately, these vaccines are not effective in endemic situations where the infecting strains are genetically diverse for porin A, thus highlighting the narrow application of this approach.4,6,7 Alternative approaches are therefore needed for a broadly protective MnB vaccine that can be used globally.
A multicomponent vaccine (Bexsero, Novartis, Basel, Switzerland) containing 1 recombinant factor H–binding protein (fHBP) was recently licensed in Europe,8 and other vaccines are in clinical development.9–11 Factor H is a negative regulator of the complement pathway, and binding factor H via fHBP aids the meningococcus in evading innate immune mechanisms.12–14 Neisseria meningitidis fHBP is a lipoprotein, and studies with the lipidated protein reveal 2 immunologically distinct subfamilies, designated A and B,15,16 that also segregate genetically with ~84% or greater amino acid identity within subfamilies, but only 60–75% identity between subfamilies.17 Although additional studies suggested that the fHBP family could be further segregated genetically in up to 6 subgroups,18,19 preclinical studies have shown that monovalent vaccines composed of recombinant lipidated forms of fHBP (rLP2086) generate mainly subfamily-specific responses, whereas bivalent rLP2086 vaccines containing 1 variant from each subfamily elicit broadly protective anti-MnB serum bactericidal antibody (SBA) against strains harboring either subfamily.16,20 Phase 1 and 2 studies of an investigational bivalent rLP2086 vaccine conducted in adults, adolescents and toddlers have demonstrated SBA activity against diverse MnB strains.11,21–24
Although a clinically devastating disease, the incidence of invasive meningococcal disease and the sporadic occurrence of cases of disease do not permit the conduct of phase 3 clinical studies based on clinical disease endpoints. The meningococcal polysaccharide-conjugate vaccines for serogroups A, C, Y and W have thus been licensed based on SBA assays as a serological correlate of protection.25 Assay strains for licensure of the A/C/Y/W vaccines were corresponding capsule-expressing reference strains that are considered representative of disease-causing strains and therefore predictive of efficacy.25 For protein-based vaccines aimed at the prevention of MnB disease, selection of appropriate strains for use in SBA assays for the evaluation of phase 3 immune sera and the bridging of these strains to predict efficacy in different populations is a much more challenging task. SBA assays using human complement (hSBA) are required, and the strains used for testing need to represent the diversity of the protein antigens in strains causing disease. A comprehensive molecular survey of systematically collected isolates from national reference laboratories in the United States and several European countries was therefore initiated to evaluate the presence of and sequence conservation of the candidate fHBP vaccine antigen. An initial strain pool of 1263 systematically collected isolates from England, Wales, Northern Ireland (collectively referred to as the UK countries), Norway, France, the Czech Republic and the United States was characterized for fHBP sequence and was used for the random selection of representative SBA assay strains to monitor serum bactericidal activity.17,26 In this study, this large prevalence collection was further expanded with additional strains from Germany and Spain to confirm that there are no major differences in fHBP variant distribution or prevalence between countries. Furthermore, fHBP variant distribution relative to patient age in the total set of 1841 MnB disease isolates was evaluated to assess the potential vaccine coverage in different target age populations.
Systematically Collected MnB Strains
Previously characterized strains17 include invasive MnB isolates from the public health laboratories of the United States, UK countries (England, Wales and Northern Ireland), Norway, France and the Czech Republic. As previously described, approximately 13% of disease isolates were tested from each country. In the United States, the set consisted of all isolates collected at active bacterial core surveillance (ABCs) sites over the period of the study; the Centers for Disease Control and Prevention estimates that the surveillance system covers 13% of the US population.27 For the European countries, strains were collected in a systematic way by order of date received at the reference laboratory. Every seventh (Czech Republic) or eighth (UK countries, France and Norway) isolate from 2001 to 2006 was included, in this way approximately matching the 13% coverage in the United States. In the previous study, data were included from 2001 to 2006 for Czech Republic, UK countries, France and Norway, but in the United States strains were from the years 2000 to 2005. In this study, the collection period for the United States was aligned to the other countries, for a net increase of 27 US strains compared with the earlier study.17 New isolates from Spain and Germany were also collected systematically, with every eighth invasive MnB isolate collected by the reference laboratories in Germany and Spain during the years 2001 to 2006 evaluated. A total of 205 strains were obtained from Germany and 346 strains from Spain. The system for collecting and reporting of MnB in Germany was evaluated through a capture-recapture analysis in 2003.28 From 2002 to 2006, samples from 70% of notified invasive meningococcal disease cases were typed at the German National Reference Laboratory. Similarly, the meningococcal strains received at the Spanish National Reference Laboratory represent 70–85% of all notified invasive meningococcal disease cases within Spain. Thus, the isolates included in this set represent ~13% of invasive MnB isolates from each of the respective reference laboratories for 2001 to 2006, the years covered by the study.
Information on patient age was provided for each isolate where available, and the age distribution of the patients from whom the isolates were obtained were generally similar to the overall national age-based disease prevalence for each country.
The fhbp gene sequences were amplified as described previously,17 using the universal primers 5′-CTATTCTGCGTATGACTAGGAG-3′ and 5′-GTCCGAAC-GGTAAATTATCGTG-3′. One additional polymerase chain reaction primer (5′-GTTCTGCCAAACGATAAAGGC-3′) was used in rare instances where the initial 3′ universal fHBP primer failed. Amplicons were purified using AMPure magnetic beads (Agencourt; Beckman Coulter, Inc., Brea, CA) and sequenced using both the conserved and the subfamily-specific primers.16 All reactions were run on each strain; only those for the correct subfamily were successful. fHBP variant numbers were assigned sequentially by subfamily as new amino acid sequences were identified. Each variant differs from another by at least 1 amino acid. New fHBP sequences have been deposited in GenBank under accession numbers JN580493–JN580552 and at http://pubmlst.org/neisseria/.29
Multilocus sequence typing (MLST) was performed according to the protocols at the PubMLST website: http://pubmlst.org/neisseria/. Sequence type and clonal complex (CC) were used to further define the diversity of MnB isolates.
The fHBP subfamily distribution by patient age was modeled using binomial distributions with logit link. The P values for between age group comparisons were obtained from likelihood ratio tests of generalized linear models. The SAS PROC GENMOD (Cary, NC) was used. The Simpson Diversity Index (SDI) was used to quantitatively measure the diversity of fHBP variants within MLST groups. The lower the index obtained, the higher the number of fHBP types observed within the CC; likewise, the higher the index, the less diverse the fHBP within the CC.
Oregon is not representative of the rest of the United States because of the ongoing hyperendemic serogroup B meningococcal disease in this state.30,31 Therefore, analysis of US data includes a weighting factor of 0.1 for strains from Oregon and 0.9 for strains from other ABCs sites (weighting based on Oregon representing 10% of the ABCs population).31 All analyses reported here include this weighting factor, unless otherwise indicated.
fHBP Sequence Analysis of Strains From Germany and Spain and Comparison With Other Countries
Systematically collected invasive MnB disease isolates from Spain (n = 346) and Germany (n = 205) were evaluated and compared with a previously characterized set from the United States, Norway, France, England, Wales, Northern Ireland and the Czech Republic. US isolates were updated to match the years covered in the European set (2001 to 2006). Meta analysis of the entire set showed that all 1841 strains were positive for fhbp. Only 1 strain (included in the earlier set of Murphy et al17) was found to have a premature stop codon leading to truncation of the C-terminal domain of the fHBP protein. The fHBP variants for the isolates from Spain and Germany fell within the previously established phylogenetic tree17 and were similar to those found in the United States and other European countries (Figs, Supplemental Digital Content 1 and 2, http://links.lww.com/INF/B568 and http://links.lww.com/INF/B569).32,33 New variants identified in this study were all closely related to those previously identified (>95% amino acid identity). New variants, with their corresponding pubmlst.org/neisseria and GenBank accession numbers, are listed in Table (Supplemental Digital Content 3, http://links.lww.com/INF/B570).16,19 The fHBP subfamily distribution in Spain and Germany was 40% A/60% B and 21% A/79% B, respectively (Table 1). Combining all countries, the overall subfamily distribution for all the isolates was 31% subfamily A and 69% subfamily B.
Although prevalent variants in Spain and Germany were also common in the other countries (Fig., Supplemental Digital Content 2, http://links.lww.com/INF/B569), some differences can be observed. B64, which differs from the common fHBP variant B09 by only 2 amino acids, appeared in 9.5% of the MnB strains from Spain but was observed just twice in the rest of the collection. Conversely, B16 was relatively rare in Spain (4 of the 346 strains), while moderately prevalent in the other countries, especially in UK countries, which were also distinct with respect to a relatively high proportion of B44 and low proportion of B24-containing isolates. This was reversed for the other countries, including Spain and Germany.
Distribution of fHBP Variants Within MLST CCs
The 3 most common CCs were CC41/44 (n = 544), CC32 (n = 487) and CC269 (n = 263). As described previously, multiple fHBP variants were found in each CC and consequently CCs were not predictive of fHBP subfamily or variant despite some degree of correlation, such as A22 allele and CC41/44 isolates and B24 allele and CC32 isolates (Fig., Supplemental Digital Content 5, http://links.lww.com/INF/B572). The diversity of fHBP variants within CCs was assessed using the SDI (Table, Supplemental Digital Content 6, http://links.lww.com/INF/B573). Among these 3 most common CCs, CC41/44 (n = 544) was the most diverse (SDI 0.21) and CC32 (n = 487) was the least diverse (SDI 0.74), despite containing representatives of 46 fHBP variants. CC269 (n = 263) had a diversity index of 0.35.
fHBP Distribution Over Time
Over the 6 years evaluated, no change was detected in the overall distribution of the 2 subfamilies (Fig. A, Supplemental Digital Content 7, http://links.lww.com/INF/B574). A stable proportion between 28% and 35% for subfamily A and between 65% and 72% for subfamily B was observed. CC (Fig. B, Supplemental Digital Content 7, http://links.lww.com/INF/B574) and fHBP variants (Fig. C, Supplemental Digital Content 7, http://links.lww.com/INF/B574) also did not show major changes over time.
Patient Age Distribution
Although strains with subfamily B fHBP variants caused more cases of disease overall, significantly more disease in infants (<1 year of age) was due to subfamily A strains compared with disease isolates from adolescents and young adults (Fig. 1). This difference was significant for all countries combined (P < 0.0001 by the likelihood ratio test), as well as for individual countries contributing to large numbers of strains (P < 0.0001 for Spain, Germany and the United States; P = 0.0004 for France; P = 0.0157 for UK countries). Differences were also seen in subfamily distribution between infants and the 1- to 10-year age group (significant for all countries combined and for individual countries), and between infants and the 26- to 64-year age group (significant for all countries combined and for the United States, Spain and France; Fig. 1). Subfamily distributions were not different between infants <1 year and adults >64 years for any country. For all the countries combined, 47.7% of cases of disease in infants <1 year of age were due to subfamily A strains. Strains with fHBP variants A22, A12 and A19 were frequent causes of disease for infants <1 year of age (Tables, Supplemental Digital Content 8 and 9, http://links.lww.com/INF/B575 and http://links.lww.com/INF/B576). Likewise, 47.3% of strains causing disease in adults >64 years were subfamily A.
The distribution of MLST CC was also assessed by age; the proportions of CCs CC41/44 and CC 213 and unassigned isolates detected in the <1 year age group were higher compared with the 11- to 25-year age group (Table, Supplemental Digital Content 10, http://links.lww.com/INF/B577). These isolates have a higher proportion of subfamily A fHBPs (Fig., Supplemental Digital Content 5, http://links.lww.com/INF/B572). Conversely, more CC32 isolates (that show a higher proportion of subfamily B fHBPs) were detected in the 11- to 25-year age group. The CC32 group is dominated by the subfamily B variant B24. For CC41/44 isolates, no single fHBP variant is dominant, and this group contains similar proportions of different variants that include both subfamilies (Fig. B, Supplemental Digital Content 5, http://links.lww.com/INF/B572).
Vaccines containing fHBPs are licensed and in late-stage development for the prevention of invasive MnB disease. fHBP is 1 of 2 proteins associated with factor H binding in N. meningitidis.34 fHBP has been shown to be an important virulence factor for MnB that protects the organism from being killed by alternative complement and antimicrobial peptide (LL-37) host defense mechanisms.14 Genetically constructed MnB knockout strains lacking fHBP become sensitive to killing by complement and/or antimicrobial peptide LL-37, despite the presence of the second factor H–binding protein NspA, suggesting that fHBP mutants would be rare in clinical settings.12,14 Unlike many other meningococcal surface proteins,35,36 fHBP is not phase variable and the gene encoding fHBP appears to be maintained broadly in meningococci of all serogroups.31 These properties make fHBP an attractive vaccine candidate. No strains lacking fhbp were identified in this study of 1841 invasive MnB disease isolates, and other studies have found only an extremely small number of isolates that either lack the gene37 or harbor a premature stop codon leading to truncation of the C-terminal domain of the 2-domain fHBP protein.17,31,37,38 Analysis of strains with premature stop codons using flow cytometry and polyclonal anti-fHBP antibody demonstrated surface binding to fHBP in subpopulations of cells,38 indicating that the protein was still being expressed; however, strains were not tested in hSBA to determine whether they would be susceptible to lysis by anti-fHBP antibodies. Harris et al38 also identified 4 meningococcal serogroup C (MnC) strains that had the identical mutation but had acquired compensatory suppressor mutations that restored the full-length fHBP. This suggests strong selective pressure for the maintenance of fHBP, and combined with the rarity of identification of meningococcal disease strains with fHBP gene mutations or deletion is consistent with the important role that fHBP plays in the survival of the organism in vivo.39
The subfamily distribution of fHBP was stable over the 6 years evaluated, although some differences from country to country were observed, with 21–43% of the strains belonging to subfamily A. The overall fHBP subfamily distribution was 31% subfamily A and 69% subfamily B (Table 1). This is an important consideration for vaccine development, as previous studies have shown that inclusion of a member of each subfamily provides broad vaccine coverage for fHBP-based vaccines.16,20 Studies in Brazil40 and South Africa41 found subfamily A/B distributions of approximately 20%/80% and 59%/41%, respectively, for invasive MnB isolates.
It is particularly important to understand the distribution of vaccine antigens in isolates causing disease in different age groups when considering vaccination strategies. Our analysis of patient age found that both infants (<1 year) and adults (>64 years) had a significantly higher proportion of disease caused by strains with subfamily A variants than adolescents and young adults (47.7% for infants, 47.3% for adults >64 years versus 19.5% for adolescents and young adults for all countries combined). For vaccines that only contain fHBP antigens, the inclusion of both A and B subfamilies in the vaccine is therefore crucial if infants are the target for vaccination. For vaccines that contain additional antigens, it is equally important to understand their contribution to vaccine effectiveness in the specific age groups being considered for immunization. By understanding the epidemiology of vaccine antigens, it is possible to identify relevant strains for monitoring SBA to demonstrate the breadth of coverage of the vaccine in the applicable age group. For example, MnC conjugate vaccines have been shown to reduce the acquisition of MnC carriage in adolescents, the major carriers of meningococci, and impart herd protection by reducing transmission to susceptible individuals.42,43 The comprehensive catch-up campaign including the vaccination of adolescents was considered key to the success of MnC conjugate vaccines in the United Kingdom.44 Control of meningococcal disease in the community might also be achieved by targeting adolescents with a MnB vaccine. Although adolescents experience more fHBP subfamily B than subfamily A disease, they carry MnB subfamily A strains at higher rates than subfamily B strains,45,46 and if reduction in carriage can be achieved with fHBP vaccines, inclusion of a subfamily A and B component in a vaccine given to adolescents may also reduce subfamily A and B disease via a herd effect. Studies addressing the potential effect of this bivalent MnB vaccine on the acquisition of carriage would therefore be important.
The available data do not permit conclusions to be made with regard to the factors responsible for the age differences in fHBP variant distribution observed in this study. Isolates from infants seem to belong to CCs with higher proportions of isolates of subfamily A (CC41/44, CC213 and unassigned). These lineages are reported to be frequent among carriage isolates.47 Furthermore, they had low SDI results, suggesting the most fHBP diversity (Fig., Supplemental Digital Content 5, http://links.lww.com/INF/B572). High acquisition by infants of these isolates from their household contacts may be an explanation; the study of household contacts of infants with meningococcal disease could help to answer this question. In this study, we demonstrate the importance of assessing the distribution of vaccine antigens and the potential coverage of fHBP-based vaccines in different age groups under consideration for vaccination.
The authors thank the ABCs team for providing the MnB isolates used in this analysis: Arthur Reingold (California Emerging Infections Program); Ken Gershman (Colorado Emerging Infections Program); Matt Cartter (Connecticut Emerging Infections Program); Monica Farley (Georgia Emerging Infections Program–Emory University and the Atlanta Veterans Administration Medical Center); Lee Harrison (Maryland Emerging Infections Program); Ruth Lynfield (Minnesota Emerging Infections Program); Joan Baumbach (New Mexico Emerging Infections Program); Nancy Bennett (New York Emerging Infections Program); Ann Thomas (Oregon Emerging Infections Program); and William Schaffner (Tennessee Emerging Infections Program), and the Centers for Disease Control and Prevention Meningitis Laboratory for strain handling and technique assistance. Heike Claus (Würzburg, Germany) is gratefully acknowledged for strain selection and shipment of isolates.
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