Use of expanded Neisseria meningitidis serogroup B panels with the serum bactericidal antibody assay for the evaluation of meningococcal B vaccine effectiveness

ABSTRACT

Introduction

Neisseria meningitidis serogroup B (NmB) antigens are inherently diverse with variable expression among strains. Prediction of meningococcal B (MenB) vaccine effectiveness therefore requires an assay suitable for use against large panels of epidemiologically representative disease-causing NmB strains. Traditional serum bactericidal antibody assay using exogenous human complement (hSBA) is limited to the quantification of MenB vaccine immunogenicity on a small number of indicator strains.

Areas covered

Additional and complementary methods for assessing strain coverage developed previously include the Meningococcal Antigen Typing System (MATS), Meningococcal Antigen Surface Expression (MEASURE) assay, and genotyping approaches, but these do not estimate vaccine effectiveness. We provide a narrative review of these methods, highlighting a more recent approach involving the hSBA assay in conjunction with expanded NmB strain panels: hSBA assay using endogenous complement in each vaccinated person’s serum (enc-hSBA) against a 110-strain NmB panel and the traditional hSBA assay against 14 (4 + 10) NmB strains.

Expert opinion

The enc-hSBA is a highly standardized, robust method that can be used in clinical trials to measure the immunological effectiveness of MenB vaccines under conditions that mimic real-world settings as closely as possible, through the use of endogenous complement and a diverse, epidemiologically representative panel of NmB strains.

Plain Language Summary

Meningococcal disease refers to illnesses caused by the bacterium Neisseria meningitidis (meningococcus), including infections of the brain lining and spinal cord (meningitis) and bloodstream (septicemia). It is rare but often severe and can be deadly. Invasive meningococcal disease can be prevented through vaccination. Nearly all cases are caused by six serogroups (types) of meningococci, including meningococcal serogroup B. Vaccines are available against meningococcal serogroup B but, because of the uncommonness of the disease, standard clinical trials could not be performed to prove these vaccines are effective. Instead, an indirect measure, called the ‘hSBA assay' (serum bactericidal antibody assay using human complement), is used to measure the ability of vaccines to provide protection against specific N. meningitidis strains that have antigens (substances that cause the immune system to react) sharing characteristics with components of the vaccines. However, meningococcal serogroup B strains are diverse in the genetic composition and expression of vaccine antigens. Hence, a large number of N. meningitidis serogroup B strains would have to be tested to make sure that the vaccine is effective against these strains. This is not feasible using the traditional hSBA assay, which requires a human complement (a protein system, which is part of the immune system) that has not come from the vaccinated person and is difficult and time-consuming to source. Recently, an alternative hSBA assay was developed that uses the complement present in each vaccinated person’s blood (endogenous complement) and which overcomes these challenges. By allowing testing against a broad panel of N. meningitidis serogroup B strains, this new assay may enable a more accurate estimation of the effectiveness of vaccines against serogroup B meningococci.

KEYWORDS:

• 4CMenB
• complement
• invasive meningococcal disease
• MenB-FHbp
• Neisseria meningitidis
• serum bactericidal antibody assay
• vaccine
• vaccine effectiveness

1. Introduction

Invasive meningococcal disease (IMD), a life-threatening condition caused by Neisseria meningitidis (Nm), is associated with severe acute and long-term sequelae, with case fatality rates of 4.1–20.0% [1] and long-term physical or neurological effects in up to 25% of the survivors [2–4]. Twelve Nm serogroups have been identified, and six (NmA, NmB, NmC, NmW, NmX, and NmY) are responsible for most IMD worldwide [5]. While effective capsular polysaccharide conjugate vaccines have been developed for serogroups A, C, W, and Y, the NmB polysaccharide has poor immunogenicity because of similarity to polysialic acid structures on human neuronal cells [6,7]. This prompted the design of subcapsular vaccines based on meningococcal surface-exposed proteins capable of inducing bactericidal antibodies across diverse NmB strains.

Two protein-based meningococcal serogroup B (MenB) vaccines have been licensed: the four-component 4CMenB vaccine (Bexsero, GSK) [8,9] and the bivalent MenB-FHbp vaccine (Trumenba, Pfizer) [10]. 4CMenB includes three recombinant protein antigens, Neisseria adhesin A (NadA; peptide 3.8), Neisserial Heparin-Binding Antigen (NHBA; peptide 2), and factor H binding protein (fHbp; peptide 1.1, B24 variant), plus detergent-extracted outer membrane vesicles (OMV) obtained from a New Zealand outbreak strain, containing Porin A (PorA) protein as the immunodominant antigen [8,11], although OMV contain a complex mixture of antigens, several of which could assist in providing protection [12]. MenB-FHbp includes one recombinant fHbp variant from each subfamily (peptides 3.45 and 1.55, corresponding to variants A05 and B01 respectively) [10]. Pentavalent MenABCWY vaccines that combine components of approved vaccines are in phase 3 development [13,14].

While the incidence of IMD varies over time and between different geographical areas, its global incidence is low [5,15,16]. Consequently, meningococcal vaccine efficacy cannot be determined through clinical trials as this would require the enrollment of extremely large numbers of participants. The licensure of MenB vaccines has therefore relied on immunogenicity results generated by the serum bactericidal antibody (SBA) assay using an exogenous source of human complement (hSBA assay), tested against a small number of vaccine antigen-specific indicator strains or primary strains [17]. However, demonstration of SBA activity against indicator strains only does not predict vaccine effectiveness sufficiently across diverse circulating strains. N. meningitidis undergoes frequent recombination events, altering surface protein structures and expression levels through phase and antigenic variation, and is highly diverse in both the genetic features and expression of vaccine antigens [18–20]. Since protection against a particular strain is only possible if it expresses antigens cross-reactive with those contained in the MenB vaccine, evaluation of vaccine protection needs to consider this antigenic diversity and variability.

To address this issue, various methods for predicting vaccine strain coverage on genetically diverse NmB strains have been developed, including the Meningococcal Antigen Typing System (MATS) [21], Meningococcal Antigen Surface Expression (MEASURE) assay [22], Meningococcal Deduced Vaccine Antigen Reactivity (MenDeVAR) Index [23], and genetic MATS (gMATS) [24]. While these methods enable large scale, robust prediction of NmB strain coverage using protein-based MenB vaccines, each has limitations, as discussed in the current paper.

More recent approaches have assessed the use of the hSBA assay in conjunction with expanded panels of NmB strains, in addition to indicator strains or primary strains, to better predict MenB vaccine effectiveness against diverse disease-causing strains. Here, we discuss the use of the hSBA assay against expanded NmB strain panels and review the available evidence on the predicted protective effectiveness of vaccines against circulating NmB strains.

2. Assays to evaluate MenB vaccine strain coverage and effectiveness

The strengths and limitations of different assays developed to evaluate MenB vaccine strain coverage and effectiveness are outlined in Table 1. Methods that have been developed to complement the traditional hSBA assay include MATS for 4CMenB and MEASURE for MenB-FHbp, and genotyping tools, such as gMATS and MenDeVAR Index, for both licensed protein-based MenB vaccines. We provide a narrative review of these assays and of a more recent approach to improving the assessment of MenB vaccine effectiveness in clinical trial settings, involving use of the hSBA assay in conjunction with expanded NmB strain panels. Two methods are described: hSBA assay using endogenous complement present in each vaccinated subject’s serum (endogenous complement hSBA; enc-hSBA) in conjunction with a 110-strain NmB panel, and the traditional hSBA assay, which uses exogenous human complement, against 14 NmB strains (traditional hSBA 4 + 10 approach).

Table 1. Strengths and limitations of methods to evaluate MenB vaccine strain coverage and effectiveness.

	Method	Strength	Limitation
Traditional hSBA [17,18]	Measures complement-mediated killing via meningococcal vaccine-induced antibody	Established in vitro surrogate measure of meningococcal vaccine efficacy/effectiveness routinely used to support vaccine licensure Measures immunogenicity endpoints, such as antibody GMT, percentage of subjects above GMT threshold, 4-fold rise in GMT, etc.	Limited exogenous sources of seronegative human complement; process of identifying sources can be laborious and time consuming Impractical to use to evaluate coverage in large strain panels; may not sufficiently predict vaccine effectiveness across diverse circulating strains
MATS [25–27]	Evaluates in each strain the affinity of vaccine-induced antibodies against fHbp, NadA, and NHBA antigens via a sandwich ELISA combined with genotyping of PorA	Standardized, reproducible antigen typing system that assesses the level of expression and genetic diversity of antigens, allowing prediction of 4CMenB strain coverage	Specific to antigen components in 4CMenB Dedicated ELISA reaction kits are required Cannot be used on non-culture-confirmed IMD cases Underestimates predicted coverage as does not take synergistic effects of multiple antigens into account
MEASURE [18,22]	Flow cytometric method that generates phenotypic fHbp expression data via cross-reactive monoclonal antibody	Reproducible estimation of MenB-FHbp strain coverage	Specific to antigen components in MenB-FHbp Cannot be used on non-culture-confirmed cases Measures only expression level and not antigenic diversity and cross-reactivity
MenDeVAR Index [23,27,28]	Genotyping tool based on information from published MATS, MEASURE, and SBA studies	Allows prediction of vaccine strain coverage for cultured and non-cultured cases Can be used to estimate 4CMenB and MenB-FHbp strain coverage	Conservative estimate because uses published data only and because of criteria used to define antigen cross-reactivity Does not provide a real-time evaluation of coverage
gMATS [24,27]	Genotyping tool to predict 4CMenB strain coverage	Allows prediction of vaccine coverage for cultured and non-cultured cases	Predicted coverage value likely to be underestimated as does not take synergistic effects of multiple antigens into account Does not predict coverage for new alleles or alleles without available MATS data
Enc-hSBA assay against 110-strain panel [29–31]	Assessment of killing activity against 110-strain panel via hSBA assay using endogenous complement present in the serum of each vaccinated individual	Highly standardized method for measuring immunological vaccine effectiveness in clinical trial settings Ability to test large panels of epidemiologically representative NmB strains Accounts for complement activity differences among vaccinees and synergistic effects of multiple vaccine antigens Can be used for any MenB-containing vaccine and to complement data generated using traditional hSBA assay	Results reported as positive or negative only (at 1:4 dilution); not feasible to conduct quantitative measurements such as antibody GMT, etc. Large number of tests have to be performed on a panel of 110 strains Not all strains may be tested with same serum sample because of limited serum volume Persons with complement deficiencies might have impaired bactericidal activity Strain panel may need to be updated according to NmB strains causing outbreak
Traditional hSBA assay against 4 + 10 strains [32]	Quantitative assessment of killing activity of MenB-FHbp against 4 primary + 10 additional NmB strains via traditional hSBA assay, which uses exogenous complement	Relatively low operational burden Assay is established in vitro surrogate measure of meningococcal vaccine efficacy/effectiveness	Limited number (14) of test strains Focus on fHbp antigen only Limited exogenous sources of seronegative human complement; process of identifying sources can be laborious and time consuming
Real-world effectiveness data	Vaccine effectiveness data from clinical trials or the field	Demonstration of effect against disease, as opposed to immune response	Feasible only after vaccine is registered and used in large populations

4CMenB, 4-component meningococcal serogroup B vaccine; ELISA, enzyme-linked immunosorbent assay; enc-hSBA, endogenous complement hSBA; fHbp, factor H binding protein; gMATS, genetic Meningococcal Antigen Typing System; GMT, geometric mean titer; hSBA assay, serum bactericidal antibody assay using exogenous human complement; IMD, invasive meningococcal disease; MATS, Meningococcal Antigen Typing System; MEASURE, Meningococcal Antigen Surface Expression; MenB, meningococcal serogroup B; MenB-FHbp, bivalent factor H binding protein meningococcal serogroup B vaccine; MenDeVAR, Meningococcal Deduced Vaccine Antigen Reactivity; NadA, Neisseria adhesin A; NHBA, Neisserial Heparin-Binding Antigen; NmB, Neisseria meningitidis serogroup B; PorA, Porin A; SBA, serum bactericidal antibody.

2.1. Traditional hSBA assay

Immunogenicity data obtained using the hSBA assay have successfully demonstrated the MenB vaccines’ capability of inducing SBA killing against a small number of strains, each expressing one of the vaccine antigens on its surface, and possibly providing protection against other NmB strains expressing antigens similar to those in the vaccines and at similar levels [18]. However, for estimations that closely represent real-world effectiveness in the field, hSBA assay of a large number of NmB strains is required because of the multitude of clonal complexes, sequence types, and antigen genotypes that exist [18–20]. This necessitates sampling a large amount of serum from vaccinated individuals, which may be especially challenging for young children. Moreover, there is evidence that different strains can behave differently on hSBA assay depending on the source of human complement and complement donors need to be screened to ensure they have no antibodies to the tested strain [18]. Therefore, a major drawback of the traditional hSBA assay is that a suitable human complement must be identified through a laborious screening effort, i.e. complement must be collected from healthy individuals who did not acquire antibodies against Nm strains through immunization or naturally acquired immunity [33]. This is particularly problematic for MenB vaccines because the human complement is the only acceptable source for SBA assays of NmB strains [17], unlike in the assessment of MenACWY conjugate vaccines, where either human or rabbit complement can be used [33]. Antibody-depleted complement (Pel-Freez, LLC, U.S.A.) is available on the market in certain countries and various groups have developed procedures for large-scale depletion of antibodies from human serum [34–36], but these are not yet routinely used in NmB studies.

2.2. MATS

MATS is an in vitro assay developed to predict 4CMenB coverage of NmB strains in individual countries using panels of epidemiologically representative strains [25]. It combines a specialized sandwich enzyme-linked immunosorbent assay (ELISA) for protein expression and the affinity of vaccine-induced antibodies against vaccine antigens, fHbp, NadA, and NHBA, with conventional genotyping for the major component in OMV, i.e. PorA [25,26]. MATS relative potency (RP), calculated by comparing the MATS ELISA reactivity of each NmB test strain to that obtained using reference strains specific for each vaccine antigen, can predict whether a strain would be killed by SBA activity elicited by 4CMenB [26]. A strain is predicted to be covered when the MATS RP value for one of the vaccine components is equal to or higher than the positive bactericidal threshold or when it matches the PorA peptide (P1.4) present in the vaccine [26]. Coverage estimates by MATS ranged from 66% to 91% in studies conducted in Europe, North America, South America, and Australia [24,25,37–40].

The main disadvantage of MATS is that this method does not account for a potential synergistic effect of two or more vaccine components or OMV components other than PorA, with simultaneous binding of antibodies to multiple antigenic targets leading to complement activation and bactericidal killing [27]. Consequently, many strains that are not predicted to be covered by MATS are found to be killed by hSBA assay using vaccinees’ sera [18,41–43], suggesting an underestimation of strain coverage with MATS compared to assessment by hSBA assay. For example, the predicted MATS coverage for a panel of NmB isolates from England and Wales was 70% (95% confidence interval [CI]: 55–85%), while the hSBA assay estimate of 4CMenB coverage was 88% (95% CI: 72–95%) [41]. Other limitations are that MATS cannot be used for non-culture confirmed IMD cases, as it requires the growth of a viable isolate, and it is specific for 4CMenB antigens. Additionally, MATS cannot be used to assess vaccinated individuals’ responses to MenB vaccination in clinical or real-world settings.

2.3. MEASURE assay

The MEASURE assay evaluates the immunogenicity of the MenB-FHbp vaccine by generating phenotypic fHbp expression data through the use of a cross-reactive monoclonal antibody, quantifying the level of fHbp expressed on the cell surface of NmB strains with mean fluorescence intensity (MFI) as the readout [22]. This assay does not consider protein variability or distance from the vaccine component and so does not account for fHbp antigenic cross-reactivity.

The first large-scale use of MEASURE, in which IMD strains collected in the United States and Europe were evaluated, showed over 91% of 1814 NmB strains expressed sufficient levels of fHbp to be susceptible to bactericidal killing by vaccine-induced antibodies [22]. Other studies conducted on NmB strains from Greece and Canada found 63 of 66 (96%) and 93 of 102 (91%), respectively, expressed sufficient fHbp levels [44,45]. However, like MATS, this assay provides a conservative estimate because a subset of strains with fHbp expression below this level could be killed in the hSBA assay by MenB-FHbp-immune sera [22], and this method cannot be used for non-culture confirmed IMD cases or to test the human immune response. Also, the MEASURE assay only assesses antigen expression and not antigenic diversity and cross-reactivity.

2.4. Genomic approaches

Whole-genome sequencing (WGS) and antigen genotyping offer a rapid, comprehensive, and cost-effective alternative for predicting MenB vaccine coverage from large collections of strains. Genomic approaches are applicable to all IMD strains, while culture-confirmed strains are required for both MATS and MEASURE. Live meningococcal isolates are not always available, often because of rapid antibiotic treatment, and in some countries (such as Italy and the United Kingdom) many cases are confirmed by PCR alone [46,47].

The MenDeVAR Index is based on WGS data linked to information from published MATS, MEASURE, and SBA studies [23]. This can be used to estimate 4CMenB and MenB-FHbp vaccine strain coverage, categorizing NmB strains according to antigen matching with the vaccine: exact match for at least one antigen, at least one cross-reactive antigen, not cross-reactive to any of the vaccine antigens, and ‘insufficient data.’

Another genomic approach, gMATS, was developed for predicting strain coverage by 4CMenB by associating antigen genotyping and worldwide MATS results [24]. Peptides significantly associated with MATS coverage/non-coverage for each antigen are identified, and strains are defined as ‘covered’ if one or more antigen-specific gMATS prediction for the strain is covered, ‘not covered’ if all antigen-specific predictors are not covered, or ‘unpredictable’ if there are insufficient data. gMATS point estimates for vaccine coverage were between 58% and 89% for strain panels from 11 European countries, Canada, the United States, and Australia [24,28,37,48–50]. In several studies, conducted in France, Greece, and Italy, coverage predictions were performed on NmB strains using both gMATS and the MenDeVAR Index [28,44,48]. Estimates were more conservative with the MenDeVAR Index than for gMATS, probably because MenDeVAR Index uses published data, while gMATS uses all available MATS results, and because of the criteria used to define an antigen as cross-reactive or not; this mainly concerns NHBA peptide 20, for which the MenDeVAR Index did not reach the threshold considered to indicate coverage, while the coverage threshold was reached by gMATS [28]. However, like MATS, gMATS is also a conservative estimate, as indicated by the gMATS coverage estimate for invasive strains from England and Wales (72–73%), which was lower than the hSBA estimate of bacterial killing (88%) [24,41]. Moreover, genomic approaches cannot be used to evaluate the immune response to vaccination in human subjects.

2.5. Enc-hSBA assay against 110 NmB strain panel

A new approach (enc-hSBA assay) uses an endogenous complement present in each vaccinated subject’s serum, offering a binary response to killing activity elicited by vaccine-specific antibodies present in sera at a single dilution against each strain tested [29,30]. The enc-hSBA assay therefore bypasses the need to identify suitable exogenous human complement sources, which can be time-consuming, thus addressing the major technical drawback of the traditional hSBA assay, and it allows testing against large panels of NmB strains.

Hence, the enc-hSBA assay can be used in the context of a clinical trial to assess immunological vaccine effectiveness against a diverse panel of disease-causing isolates. The enc-hSBA assay is reported to be positive if at least 50% of the incubated MenB strains are killed (at 1:4 dilution) or negative if less than 50% are killed. Each serum sample is tested against multiple strains from the NmB panel, and immunological vaccine effectiveness is calculated by comparing the odds ratio of negative results in the MenB vaccinated group vs that in the control group. Unlike MATS, MEASURE, and the genomic approaches described previously, the enc-hSBA assay accounts for intersubject variability in ability to elicit complement-mediated bactericidal killing of NmB strains because of natural heterogenicity in complement concentration and activity between subjects, as well as synergistic effects of multiple MenB vaccine antigens against circulating strains with heterogeneous genetic features [29,31]. Consequently, in comparison to other assays evaluating MenB vaccine strain coverage (Table 1), the enc-hSBA assay can arguably be considered a better mimic of natural infection and hence the most relevant proxy for predicting MenB vaccine effectiveness.

The enc-hSBA assay was qualified using a panel of 110 invasive NmB strains (Table 2) and further validated using four 4CMenB vaccine antigen-specific indicator strains [29,51] (Figure 1). The 110 strains were randomly selected from 442 strains collected in 2000–2008 in 10 United States jurisdictions participating in the Centers for Disease Control and Prevention (CDC) Active Bacterial Core Surveillance (ABCs) [52,53]. The prevalence and genetic diversity of the fHbp, NHBA, and NadA antigens in the 110 NmB strains have been characterized [53] and 4CMenB coverage of the 442-strain panel was shown to be 91% by MATS and 85% by gMATS [24,52]. The genetic features of the 110-strain panel, in terms of clonal complexes distribution and MenB vaccine antigens, were similar to those of the original 442-strain panel, demonstrating that the 110-strain panel was adequately representative of the larger panel [31]. Complete genotyping information for the 110-strain panel was published previously [31].

Figure 1. Characteristics and standardization process for the endogenous complement hSBA (enc-Hsba)) assay [29,30]. The enc-Hsba assay uses active endogenous complement present in each vaccinee’s serum, i.e. serum samples are not heat-inactivated, maintaining intrinsic complement activity. Each serum sample is tested at a 1:4 dilution. After preparation of the microtiter plate with diluted sera, N. meningitidis serogroup B (NmB) bacteria from fresh log-phase liquid culture are added. Following incubation, aliquots of each well are transferred onto agar and incubated overnight, providing a positive or negative result for bactericidal killing. The assay was validated using NmB indicator strains for each of the four 4CMenB vaccine antigens, assessing robustness (assay stability), specificity, and sensitivity, and was qualified (consistency of results on triplicate testing) using the indicator strains and 110 randomly selected NmB disease-causing strains. This supported the utility of this assay for measuring the immunological effectiveness of MenB-containing vaccines against a large NmB strain panel in clinical trial settings, under conditions that are as close as possible to real-world conditions.Enc-Hsba,, serum bactericidal antibody assay using endogenous human complement; hSBA, serum bactericidal antibody assay using human complement; NmB, Neisseria meningitidis serogroup B.

Table 2. Features of the Neisseria meningitidis serogroup B (NmB) strain panel used in the endogenous complement hSBA (enc-HSBA)) assay against 110-strain panel method and the exogenous complement hSBA (traditional hSBA) assay against four primary and 10 additional (4 + 10) strains [49,57,61].

	Enc-hSBA assay against 110-strain panel	Traditional hSBA assay against 4 + 10 strains
Number of NmB strains	110	14: 4 primary plus 10 additional
NmB strain collection	110 strains randomly selected from 442 strains from US (CDC ABCs panel, 2000–2008)	4 primary: selected from 1,263 strains from US and Europe 10 additional: 9 selected from 1,263 strains, 1 (variant A07) selected from 1,814 US and Europe strains US strains from CDC ABCs panel, 2000–2005; European strains collected 2001–2006
Strain selection criteria	110 strains suitable for laboratory conditions and qualified for use in the enc-hSBA assay	4 primary: adequately reflect fHbp diversity in NmB disease strains; low to medium fHbp surface expression; low baseline hSBA seropositive rates; express fHbp variants heterologous to MenB-FHbp antigens 10 additional: fHbp variant prevalence among IMD strains in Europe or US; difference from primary test strain variants; representativeness of its variant group; technical compatibility in hSBA assay; predominant clonal complex for the variant group, if one existed
Comparison with other strain panels	4,273 strains from US, Canada, Australia, Europe: US (748 strains) Canada (407 strains) Australia (520 strains) 9 European countries (2,598 strains)	1,263 strains from US and Europe
Geographic representativeness of strain panel	Presence of at least one 4CMenB antigen in 110 strains: Overall ~89% US 95% Canada 90% Europe 87% Australia 97%	Overall ~80% (US and Europe)
Time period representativeness of strain panel	Comparison to CDC ABCs strains (2009–2014) shows stability of genetic characteristics over time	Data not available

4CMenB, 4-component meningococcal serogroup B vaccine; CDC ABCs, Centers for Disease Control and Prevention Active Bacterial Core Surveillance; fHbp, factor H binding protein; hSBA assay, serum bactericidal antibody assay using human complement; IMD, invasive meningococcal disease; MenB-FHbp, bivalent factor H binding protein meningococcal serogroup B vaccine; NmB, Neisseria meningitidis serogroup B.

Further genetic analyses showed the 110-strain panel contains a repertoire of antigen genotypes that represents approximately 89% of NmB strains circulating worldwide, ranging from 87% for the European strains to 95% for the United States and 97% for Australia [31]. Consequently, the 110-strain panel includes the most common clonal complexes and genetic variants of MenB vaccine antigens circulating in the United States and worldwide. In addition, NmB antigens from outbreak strains collected in the United States (mostly clonal complex cc232 and sequence-type ST-32) and other countries, are well represented among the 110 strains in the panel [31,54–57]. There is also evidence of stability in the genetic characteristics of the ABCs 2000–2008 panel over time, since the same predominant MenB vaccine antigen types were identified in strains collected by ABCs in 2009–2014 [58], suggesting the 110-strain panel maintains over time its representativeness of circulating strains. Some other studies that monitored circulating NmB strains over the long term also reported persistence in the most common clonal complexes and antigen variants [28,44,49,59,60].

This approach of using the enc-hSBA assay against a 110 NmB strain panel has been used to measure the immunological effectiveness of an investigational MenABCWY vaccine combining 4CMenB antigens with those from the licensed vaccine, MenACWY-CRM (Menveo, GSK) [29,30]. In this phase 2 study, United States adolescents were randomized to receive either three MenABCWY doses on a 0-2-6-month schedule or a single MenACWY-CRM dose at month 2 and placebo at 6 months as a control [30]. Immunological vaccine effectiveness was determined as the proportion of NmB strains killed in an enc-hSBA assay at 1:4 dilution by samples collected from MenABCWY-immunized individuals as opposed to the control group. The results demonstrated the MenABCWY immunological effectiveness of 67% (95% CI: 65–69%) after two doses and 71% (69–73%) after three doses against the 110-strain panel [30]. Enc-hSBA assay against the 110-strain panel will also be used to measure immunological vaccine effectiveness in a phase 3 trial (NCT04502693) of 4CMenB and the investigational MenABCWY vaccine [61].

Use of the enc-hSBA assay against the 110-strain panel has several disadvantages. The first is that results are reported as positive or negative only, and it is not feasible to conduct quantitative measurements of antibody titers, such as geometric mean titer (GMT), geometric mean ratio, and 4-fold or greater rise in GMT. Other limitations include the high operational burden from running a large number of individual assays, and it may not be possible to test all strains with the same sample because of the limited serum volume from each individual, which is particularly challenging for infant sera. However, these limitations are countered by the ability to test large panels of NmB strains and the fact that the enc-hSBA assay is a highly standardized, robust methodology, which has been successfully qualified for the panel of 110 diverse strains [29] and its utility in clinical trial settings has been demonstrated [30].

Enc-hSBA testing against the NmB 110-strain panel can be used to supplement the current standard of MenB vaccine evaluation, i.e. antibody response data generated using the traditional hSBA assay. Together, these symbolize the most exhaustive method to assess vaccine effectiveness in clinical trials at this time, which has the potential to be applied worldwide, provided epidemiological differences in NmB circulating strain genotypes are well represented. Regular monitoring of the epidemiological evolution of circulating NmB strains, including consideration of new MATS testing data of recent strain collections and evaluation of genotypes of hyperendemic and outbreak strains, will be important in the consideration of the need to adapt the NmB 110-strain panel in the future.

2.6. Traditional hSBA 4 + 10 approach

Evaluation of MenB-FHbp immunogenicity and vaccine coverage has relied on the traditional hSBA assay, which uses exogenous human complement, against four primary and 10 additional NmB strains (traditional hSBA 4 + 10 approach). These 14 strains were selected following the evaluation of fHbp sequence diversity and expression in NmB disease-causing strains via the MEASURE assay [32] (Table 3).

Table 3. Neisseria meningitidis serogroup B (NmB) strains for the hSBA assay, using an exogenous complement, against four primary and 10 additional NmB strains (traditional hSBA 4 + 10 approach), which was used in the clinical development of the MenB-FHbp vaccine [61,,63].

Strain	fHbp variant (peptide id)	fHbp expression (MFI)	Clonal complex	Country of origin	Year of isolation
Primary strains
PMB80	A22 (2.19)	3127	41/44	US	2002
PMB2001	A56 (3.187)	5002	213	France	2002
PMB2948	B24 (1.1)	6967	32	France	2001
PMB2707	B44 (1.15)	11,283	269	UK	2006
Additional strains
PMB3010	A06 (3.47)	3370	461	UK	2001
PMB3040	A07 (2.21)	1379	162	Germany	2004
PMB824	A12 (2.24)	2540	35	US	2001
PMB1672	A15 (2.25)	2995	103	France	2002
PMB1989	A19 (2.16)	1934	8	UK	2002
PMB3175	A29 (3.30)	3839	32	US	2000
PMB1256	B03 (1.14)	3976	41/44	UK	2001
PMB866	B09 (1.13)	2089	269	UK	2005
PMB431	B15 (1.252)	3785	41/44	US	2000
PMB648	B16 (1.4)	2347	41/44	UK	2003

fHbp, factor H binding protein; hSBA assay, serum bactericidal antibody assay using human complement; MenB-FHbp, bivalent factor H binding protein meningococcal serogroup B vaccine; MFI, mean fluorescence intensity on Meningococcal Antigen Typing System (MEASURE); peptide id, peptide identifier (http://pubmlst.org/neisseria/fHbp/).

As described in previous reviews [32,62], the four primary test strains harbor fHbp variants from subfamilies A (A22 and A56, corresponding to peptides 2.19 and 3.187 respectively) and B (B24 and B44, or 1.1 and 1.15). These strains were selected to adequately reflect fHbp diversity in NmB disease strains, to have low to medium fHbp surface expression to resemble the normal distribution among disease strains, and to have low baseline SBA seropositive titers, since populations at risk for IMD are characterized by non-existing or low baseline bactericidal activity to most strains [62]. The four primary test strains also had to express fHbp variants that were heterologous to the vaccine antigens to demonstrate that vaccine-induced responses provide broad coverage against NmB strains.

The 10 additional test NmB strains include prevalent fHbp variants found in collections of 1263 and 1814 disease-causing strains from the United States and Europe [32] (Table 2). Specific criteria used to select the 10 additional NmB test strains included fHbp variant prevalence among NmB strains in Europe or the United States, difference from variants expressed by primary test strains, variant group representativeness, hSBA assay technical compatibility, and belonging to a major clonal complex for the variant group, if one existed. The 10 additional test strains express the fHbp variants A06 (peptide 3.47), A07 (2.21), A12 (2.24), A15 (2.25), A19 (2.16), A29 (3.30), B03 (1.14), B09 (1.13), B15 (1.252), and B16 (1.4), differing from those in the four primary test strains and with different sequences compared to the MenB-FHbp vaccine antigens. The United States strains were from ABCs sites (2000–2005) covering approximately 13% of the population, while the European strains (2001–2006) were from the public health laboratories of Norway, France, and the Czech Republic, and the United Kingdom Health Protection Agency (now the UK Health Security Agency), representing around 13% of the invasive NmB strains. The variants expressed by the four primary test strains were present in 42% of the collection of 1263 NmB strains, and those expressed by the 10 additional test strains were present in an additional 39% of the strains. Therefore, the 14 NmB test strains corresponded to around 80% of circulating NmB IMD-causing strains in the United States and Europe at the time of collection [32].

Immune responses to two or three doses of MenB-FHbp were evaluated in two pivotal phase 3 studies of 3596 adolescents and 3304 young adults using the four primary strains; responses to the 10 additional strains were assessed in a subgroup [63]. Most participants had an SBA titer that was equal to or greater than the lower limit of quantitation (LLOQ; SBA titer 1:8 or 1:16, dependent on strain) 1-month post-dose 2 and post-dose 3 (64.0–99.1% and 87.1–99.5%, respectively) for each of the primary test strains and for the 10 additional test strains (51.6–100.0% and 71.3–99.3%, respectively) [32]. Post hoc analyses of positive predictive values (PPV) determined the association between primary and additional test strains that expressed fHbp in the same subfamily [63]. The PPV, the proportion of participants who responded (SBA titer ≥LLOQ) to the additional strain among the total number of primary strain responders, was greater than 80% for most primary/additional strain pairs within an fHbp subfamily 1-month post-dose 3. This showed that the SBA responses measured by hSBA assay using the primary test strains predicted immune responses for the additional strains within the same subfamily.

Another phase 3 trial (NCT04440163) has been completed of an investigational MenABCWY vaccine constituted from MenB-FHbp and MenACWY-TT (Nimenrix, Pfizer) [13], in which vaccine effectiveness was assessed using traditional hSBA assay against the four primary strains [64]. The primary aim of this study is to determine the immunologic noninferiority of the MenABCWY vaccine to licensed MenB-FHbp and MenACWY-CRM vaccines in more than 2400 healthy adolescents and young adults. It has been reported that the trial met all primary and secondary endpoints [65] and publication of the full results is awaited.

While the operational burden of the traditional hSBA assay is not as high as for the enc-hSBA assay against 110 strains, it is possible that testing against only four or 14 strains will not adequately mimic real-world settings to reliably predict vaccine effectiveness. This approach also has the limitation of focusing only on the fHbp antigen. Evaluations are needed to determine if differences in the distribution of fHbp variants among different age groups in various countries affect the immunogenicity, and vaccine effectiveness, of the MenB-FHbp vaccine.

3. Conclusion

Different approaches have been used to predict coverage and the clinical effectiveness of protein-based MenB vaccines against NmB disease other than the traditional hSBA assay, including MATS, MEASURE, and genomic approaches. These predict NmB strain coverage by MenB vaccines on a larger scale than the hSBA assay but have limitations that reduce the ability to obtain accurate estimations. More recent approaches include an enc-hSBA assay against a panel of 110 strains and traditional hSBA testing against a panel of four primary and 10 additional strains. These measures aim to address the diversity of circulating NmB strains in real-world settings and thus enable more accurate estimates of MenB vaccine effectiveness in a scenario where there is substantial difficulty in determining vaccine efficacy in clinical trials. The enc-hSBA assay against a panel of 110 strains is being used to assess immunological vaccine effectiveness in an ongoing phase 3 study of 4CMenB and the GSK MenABCWY candidate vaccine. Traditional hSBA assay against 14 strains has been used to assess the MenB-FHbp and the Pfizer MenABCWY candidate vaccine is being assessed in phase 3 by hSBA assay against four strains.

4. Expert opinion

It is crucial to predict the coverage of constantly evolving NmB strains both in the context of national vaccination programs and in the control of NmB disease outbreaks. In particular, wide genetic diversity and variations in antigen expression levels in NmB strains need to be taken into account when gauging the performance of MenB vaccines. While the MenB vaccine immunogenicity is measured by the hSBA assay against indicator strains, a large number of strains must be tested for an accurate assessment of MenB vaccine efficacy or effectiveness. This is not feasible because of a lack of suitable volumes of human complement for the hSBA assay, although sources of antibody-depleted complement for evaluating MenB vaccines may be used in the future.

With the introduction of two protein-based MenB vaccines, different approaches have been developed, including MATS, MEASURE, MenDeVAR Index, and gMATS, which evaluate vaccine coverage of circulating NmB strains by phenotypic or genotypic means. All these methods enable large scale, robust prediction of MenB strain coverage using protein-based MenB vaccines, but each has limitations, most notably an underestimation of vaccine coverage relative to that predicted by SBA killing. An alternative approach of using hSBA assay against expanded NmB strains to evaluate the immune response in vaccinated individuals has been explored with the traditional hSBA assay and the enc-hSBA assay that uses the endogenous complement of individual vaccinees, bypassing the need to identify suitable human complement.

An assay that measures vaccine performance under conditions as close as possible to real-world settings, through testing a large panel of strains and using (for the enc-hSBA assay) the individual vaccinee’s complement, could improve the assessment of immunological vaccine effectiveness in the context of a randomized controlled trial. There is also a possibility that this research will change the way the effectiveness of MenB-containing vaccines is considered in a broader sense, since the enc-hSBA assay against 110-strain NmB panel has the potential to provide public health regulators with a clearer picture of population-based vaccine coverage than traditional hSBA assay against a smaller number of test strains. However, the traditional hSBA assay remains a quantitative method for measuring the level of the immune response to vaccination. Together, the complementarity of the well-established traditional hSBA assay and highly standardized enc-hSBA assay would lead to a more complete assessment of vaccine effectiveness in clinical trial settings.

Practical aspects to be addressed with this new approach include the large number of tests that need to be performed to cover all strains in a qualified NmB panel. Also, the epidemiology of circulating NmB strains needs to be monitored regularly, to ensure the NmB strain panel is representative of those in different geographical regions and over long periods of time. The process of random selection of strains for the test panel may result in the omission of uncommon or emerging clonal complexes, introducing the possibility of bias. Any significant evolution of circulating NmB strains must therefore be captured in updates of the test strain panel.

In the near future, pentavalent MenABCWY vaccines or other MenB combination vaccines may become the standard of care for preventing cases of IMD in the population. The introduction of new vaccines containing NmB antigens could be facilitated by more accurate predictions of real-world vaccine effectiveness via the traditional hSBA assay supplemented by enc-hSBA or other assays against an epidemiologically representative NmB strain panel. Additionally, the demonstration of similarity in immunological vaccine effectiveness between new MenB-containing vaccines and licensed MenB vaccines could support the relevance of real-world effectiveness of licensed vaccines for new MenB-containing vaccines. A consistent approach would also enable prediction data generated for different MenB-containing vaccines to be compared.

Article highlights

• Accurate prediction of meningococcal serogroup B (MenB) vaccine coverage is challenging with the traditional human complement serum bactericidal antibody (hSBA) assay because of diversity in both the genetic features and level of expression of vaccine antigens across Neisseria meningitidis serogroup B (NmB) strains.

• Different methods exist to predict MenB vaccine strain coverage, including the Meningococcal Antigen Typing System (MATS), Meningococcal Antigen Surface Expression (MEASURE) assay, and genetic tools, such as genetic MATS (gMATS), but these do not take into account the synergistic effect of antibodies against multiple antigens and tend to provide an overly conservative estimate for multicomponent MenB vaccines.

• To complement these tools, two methods have been developed to assess MenB vaccine effectiveness in a clinical trial setting and thus comprehensively evaluate the effectiveness of MenB vaccines. These measure serum bactericidal activity against a large panel of epidemiologically representative NmB strains: hSBA assay using endogenous complement in each vaccinated person’s serum (enc-hSBA) against a 110-strain NmB panel and traditional hSBA (which uses exogenous complement) against 14 (4 + 10) NmB strains.

• The enc-hSBA assay, which provides an assessment of the killing activity of sera from recipients of MenB-containing vaccines, was qualified using 110 invasive strains representing, with their repertoire of antigen genotypes, ~89% of the strains circulating globally, 87% in Europe, 95% in the United States, 90% in Canada, and 97% in Australia.

• The traditional hSBA 4 + 10 approach is a quantitative measure of killing activity of the bivalent MenB-FHbp vaccine and was qualified against four primary and 10 additional NmB strains selected following evaluation of factor H-binding protein (fHbp) sequence diversity and expression. The four primary strains represent ~42% and the 10 additional strains represent ~38% of circulating strains in the United States and Europe (~80% combined).

• The hSBA assay against expanded NmB strain panels helps account for the diversity of circulating strains in vaccine effectiveness assessments. With a broad panel of diverse strains and the vaccinee’s own (endogenous) complement, the enc-hSBA assay aims to measure the immunological effectiveness of MenB-containing vaccines in clinical trials, under conditions that are as close as possible to real-world settings.

Declaration of interest

V Abitbol, A Andani, S Preiss, A Muzzi, L Serino, and W-Y Sohn are employed by GSK and hold shares in GSK. RB performs contract research on behalf of UKHSA for GSK, Pfizer, and Sanofi Pasteur. F Martinon-Torres reports payments to his institution from AstraZeneca, Biofabri, Seqirus, Sanofi Pasteur, MSD, Merck, Pfizer, Roche, Regeneron, Janssen, MedImmune, Novavax, Novartis, and GSK, outside the submitted work. F Martinon-Torres also reports payments made to him from Pfizer, Sanofi Pasteur, MSD, Biofabri, Seqirus, Janssen, and GSK, outside the submitted work. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or material discussed in the manuscript apart from those disclosed.

Reviewer disclosures

A reviewer on this manuscript has disclosed that they are an employee of GSK that has a commercial vaccine on MenB. Due to this conflict of interest, an additional peer reviewer was secured in order to ensure there were two further reviewers with no conflict of interest.

Author contributions

All authors substantially contributed to the conception and design of this review article and to the interpretation of the relevant literature and the development of the manuscript. All authors gave final approval before submission.

Trademark statement

Bexsero and Menveo are trademarks owned by or licensed to GSK. Trumenba and Nimenrix are trademarks of Pfizer.

Acknowledgments

The authors thank Business & Decision Life Sciences platform for editorial assistance and manuscript coordination, on behalf of GSK. Joanne Knowles (independent medical writer, on behalf of GSK) provided medical writing support. This work was presented previously at the 41st Annual Meeting of the European Society of Paediatric Infectious Diseases (ESPID), May 8–12, 2023.

Correction Statement

This article has been republished with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

GlaxoSmithKline Biologicals SA was the funding source and took in charge all costs associated with the development and publication of this manuscript.