Journal of Infection 81 (2020) 862–872

Contents lists available at ScienceDirect

Journal of Infection
journal homepage: www.elsevier.com/locate/jinf

Review

Methods to evaluate serogroup B meningococcal vaccines: From

predictions to real-world evidence
Ray Borrow a,1, Muhamed-Kheir Taha b,1, Marzia Monica Giuliani c, Mariagrazia Pizza c,
Angelika Banzhoff d, Rafik Bekkat-Berkani e,∗
a
Meningococcal Reference Unit, Public Health England, Manchester, United Kingdom
b
Institut Pasteur, Invasive Bacterial Infection and National Reference Center for Meningococci and Haemophilus influenzae, Paris, France
c
GSK, Siena, Italy
d
GSK, Marburg, Germany
e
GSK, Rockville, MD, United States

a r t i c l e i n f o s u m m a r y

Article history: Serogroup B meningococci (MenB) remain a prominent cause of invasive meningococcal disease (IMD).
Accepted 28 July 2020 The protein-based multicomponent 4CMenB and the bivalent MenB-FHbp are the only currently avail-
Available online 31 July 2020
able vaccines against MenB-caused IMD. Efficacy studies are not possible, due to the low incidence of
Keywords: IMD. Therefore, the vaccines’ immunogenicity has been evaluated against several target strains chosen
Meningococcal serogroup B to quantify complement-mediated killing induced by each vaccine component in the serum bactericidal
Strain coverage antibody assay. However, due to the wide genetic diversity and different expression levels of vaccine anti-
4CMenB gens across MenB strains, vaccine performance may differ from one strain to another. Here, we review
MenB-FHbp the methods used to predict MenB strain coverage for 4CMenB and MenB-FHbp. Phenotypic assays such
hSBA as the meningococcal antigen typing system (MATS, 4CMenB-specific) and the flow cytometric meningo-
Typing coccal antigen surface expression assay (MEASURE; MenB-FHbp-specific) were developed. Genomic ap-
MATS
proaches are also available, such as genetic MATS (gMATS) and the Bexsero antigen sequence type (BAST)
MEASURE
gMATS
scheme, both 4CMenB-specific. All methods allow tentative predictions of coverage across MenB strains,
BAST including that afforded by each vaccine antigen, and are rapid and reproducible. Real-world data on vac-
cine effectiveness are needed to confirm predictions obtained by these methods.
A Video Abstract linked to this article can be found on Figshare: https://doi.org/10.6084/m9.figshare.
13234340.
© 2020 GlaxoSmithKline Biologicals SA. Published by Elsevier Ltd on behalf of The British Infection
Association.
This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)

Background qualae, whether physical or neurological;5 for instance, amputa-

Invasive meningococcal disease (IMD) is a serious condition
caused by Neisseria meningitidis. The incidence of IMD fluctuates
considerably from one region to another and over time, while re-
maining relatively low worldwide. In the last two decades, 0.01
to 3.6 cases/10 0,0 0 0 persons were estimated globally.1 IMD inci-
dence is the highest in infants, and a second, lower peak is ob-
served in adolescents,2 which is also the age group with the high-
est meningococcal carriage rates.3 IMD can be a deadly disease if
untreated and is associated with high case-fatality rates of 4.1–
20.0%, even with prompt treatment.4 Survivors can suffer afteref-
fects with long-lasting consequences and serious permanent se-
∗
Corresponding author.
E-mail address: rafik.x.bekkat-berkani@gsk.com (R. Bekkat-Berkani).
1
Contributed as first author.
https://doi.org/10.1016/j.jinf.2020.07.034
0163-4453/© 2020 GlaxoSmithKline Biologicals SA. Published by Elsevier Ltd on behalf of The British Infection Association. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
tions are reported in up to 8% and hearing loss in up to 19% of
IMD survivors who experienced the disease in infancy or child-
hood.6 While effective antibiotics against N. meningitidis are cur-
rently available, there is growing evidence of an increase in an-
timicrobial resistance worldwide.1 Considering all these elements,
IMD continues to be an important health concern.
Twelve meningococcal serogroups (Men) are identified, with
six of them (MenA, MenB, MenC, MenX, MenY, and MenW) re-
sponsible for nearly all IMD worldwide.7 The prevalence of these
serogroups varies with geographical and socio-economic status and
over time. Implementation of MenACWY vaccination has drastically
decreased the occurrence of IMD caused by MenA, MenC, MenW
and MenY in the Americas, Australia, Africa, and European coun-
tries. MenB is now one of the major causes of IMD in these regions,
albeit with an evidenced trend of decreasing incidence.1,8,9 .
 Methods to predict strain coverage of MenB vaccines / Journal of Infection 81 (2020) 862–872
Vaccines against MenB
Mono- or polyvalent meningococcal vaccines against MenA,
MenC, MenW and MenY have been available for many years, with
polysaccharide-protein conjugate vaccines preferred over polysac-
charide due to their ability to induce better booster response, long-
term immunological memory and herd protection.10 The develop-
ment of vaccines against MenB has been more challenging due
to the poor immunogenicity of the MenB capsular polysaccharide.
This prompted the design of protein vaccines, based on meningo-
coccal surface-exposed proteins able to induce bactericidal anti-
bodies across the diversified MenB strains.
Two MenB vaccines are now licensed for use in several coun-
tries worldwide. The multicomponent vaccine 4CMenB (Bexsero,
GSK) contains four antigenic constituents: factor H binding protein
(fHbp) variant 1 presented as a fusion protein with GNA2091 ac-
cessory protein, neisserial heparin binding antigen (NHBA) fused
with accessory protein GNA1030, Neisseria adhesin A (NadA), and
outer membrane vesicles (OMV) obtained from the N. meningitidis
epidemic strain NZ98/254, expressing porin A (PorA) serosubtype
P1.4. The second vaccine, MenB-FHbp (rLP2086; Trumenba, Pfizer)
contains lipidated fHbp of the subfamilies B (corresponding to vari-
ant 1) and A (variants 2 and 3). In most countries, 4CMenB vaccine
is approved for use in individuals from 2 months of age and older;
the MenB-FHbp vaccine is approved in individuals 10 years of age
and older. In the United States (US), both MenB vaccines are in-
dicated for individuals 10–25 years old. Both vaccines have been
found to be immunogenic and well tolerated in the age groups for
which their use has been approved.11,12
Introduction of MenB vaccination in national immunization
programs
4CMenB has already been introduced in the national immu-
nization program of several countries, such as the United King-
dom (UK, September 2015)13 and, more recently, Ireland (Decem-
ber 2016),14 Andorra (October 2016),15 Italy (January 2017),16 San
Marino (2017),17 Lithuania (July 2018),18 and Czechia (May 2020).19
Portugal and Malta plan to introduce 4CMenB in 2020, while re-
gional introduction is underway in other countries such as Spain
(e.g., the Castile y Leon region20 ), or already implemented in Aus-
tralia (South Australia).21 In Australia, government-funded MenB
vaccination is also offered to Aboriginal and Torres Strait Islander
children and for people of all ages with high-risk medical condi-
tions, starting with July 2020.22
In the UK, the first country to introduce a vaccine against MenB
in the national infant vaccination program, a 2 + 1 schedule is
used, with administrations at 2, 4, and 12 months of age. At three
years since introduction, a 75% reduction in MenB IMD cases in
infants was reported, corresponding to an incidence rate ratio be-
tween observed and expected cases of 0.25 (95% confidence inter-
val: 0.19–0.36). Protection is believed to be afforded at least until
the end of the third year of life.23
Assessing protective efficacy against meningococci through
strain coverage
Vaccine efficacy is defined as the percentage reduction in dis-
ease incidence in a vaccinated versus a non-vaccinated population,
under optimal conditions, such as a randomized clinical trial. How-
ever, this is not feasible for IMD, for which the incidence is very
low and evaluation of vaccine efficacy would require the enrolment
of very large, impractical numbers of individuals. Consequently, the
licensure of meningococcal vaccines was not based on vaccine effi-
cacy assessments and relied on immunogenicity results. Immuno-
genicity data were generated by assays which detect antibody-
863
dependent lysis of reference meningococcal isolates in the pres-
ence of external sources of complement. Since the structure of the
capsular polysaccharide is highly conserved within the meningo-
coccal serogroups, polysaccharide-based vaccines which show ade-
quate immune responses against reference strains are very likely
to provide protection against all strains belonging to the same
serogroups. Indeed, a titre of ≥1:4 in serum bactericidal antibody
(SBA) assays using human complement (hSBA) was found to in-
dicate protection against MenC-caused IMD;24 this threshold has
been extended and used for the licensure of polysaccharide-protein
conjugate vaccines against other meningococcal serogroups. The
availability of this established correlate of protection allows predic-
tion of efficacy against MenACWY strains based on the immune re-
sponses to reference strains. However, evaluation of protein-based
vaccines remains more challenging because MenB vaccines target
proteins that show high variation in their sequence and/or levels of
expression. Since protection against a certain meningococcal strain
is only possible if that strain expresses antigens cross-reactive with
those contained in the vaccine, evaluation of the protection af-
forded must therefore account for the wide genetic diversity of
MenB strains, which impacts the level of expression of (surface)
antigens through gene regulation, phase variation and sequence di-
versity.25 Addressing this issue lead to novel, specific methods pre-
dicting SBA activity against genetically diverse strains, accounting
for each and all vaccine components. These methods allow conser-
vative estimates of protection, based on prediction of strain cov-
erage, defined as the proportion of disease-causing meningococ-
cal isolates killed by post-vaccination immune sera in hSBA, in
a specific region and period of time. These methods are in gen-
eral accepted by regulatory authorities and constitute an innova-
tive solution for the evaluation of MenB vaccines. However, the
best practice to demonstrate vaccine effectiveness remains eval-
uation in real-life settings, which can only be performed follow-
ing implementation of MenB vaccination in national immunization
programs.
Here we review the methods used to evaluate the protective
coverage of current MenB vaccines across circulating IMD-causing
MenB strains. A plain language summary on our findings is pro-
vided in Fig. 1.
hSBA: the current correlate of protection for meningococcal
vaccines
Bactericidal antibodies were first associated with protection
against meningococcal disease almost a century ago, by Heist
et al..26 However, Goldschneider et al. were the first to propose a
correlate of protection in 1969, by testing SBA activity in sera col-
lected from infants and adults using a hSBA against MenA, MenB
and MenC strains.24 An increase in SBA activity against MenC
strains was found to be associated in most cases with the appear-
ance of immunoglobulin (Ig) G and IgM antibodies against the in-
fecting strain. Of note, while the mounting of hSBA titres ≥1:4 was
considered as protective, titres <1:4 were not necessarily indica-
tive of susceptibility to disease.24
This complement-mediated bactericidal killing assay became
the gold-standard for evaluating successful immunization and pro-
tection against meningococci. Since variations in the assay proto-
col can severely impact the results, the assay conditions must be
defined and validated, in alignment with guidelines developed by
a panel of experts from the World Health Organization (WHO) in
1976.27
The limited availability of human complement to assess SBA
was recognized very early in the development of meningococcal
vaccines and alternative sources were sought. In the assessment of
polysaccharide vaccines against MenA and MenC, baby-rabbit com-
plement28 was considered an acceptable alternative. After assess-
864 Methods to predict strain coverage of MenB vaccines / Journal of Infection 81 (2020) 862–872

Plain Language Summary

What is the context?

• Invasive meningococcal disease (IMD) is an uncommon and life-threatening disease that can
progress to death in only 24 hours. There are currently two licensed protein-based vaccines
against the bacterium Neisseria meningidis serogroup B (MenB), a leading cause of IMD:
4CMenB (Bexsero, GSK) and MenB-FHbp (Trumenba, Pfizer).
• Due to low disease incidence, it is not possible to assess vaccine efficacy in clinical trial
sengs prior to licensure. Instead:
- vaccine performance is assessed with immunogenicity assays that measure how vaccine-
induced anbodies react against a limited number of MenB strains, used as reference
strains;
- in addion, other assays can esmate possible vaccine protecon against large numbers
of circulang MenB strains;
- vaccine effecveness can be determined following implementaon of MenB vaccinaon
into naonal immunizaon programs.

Review highlights & take-home message

• We compared four methods used to predict possible vaccine protecon against MenB
strains (MATS, MEASURE, gMATS and BAST).
• While not without limitaons, these methods can complement the currently established
bactericidal anbody assay by providing large-scale, rapid and reproducible predicons of
possible vaccine protecon.
• Infant immunizaon programs with 4CMenB show the true vaccine effect against invasive
meningococcal disease observed in real-world sengs. This will allow for correlaon of
predicve laboratory methods with actual protecon against IMD.
•

Fig. 1. Plain language summary.

ment of the SBA assay using baby-rabbit serum as complement
(rSBA), the WHO recommended the following immunogenicity-
related criterion for licensure of meningococcal polysaccharide vac-
cines: ≥4-fold rise (increase of ≥2 dilutions) in SBA titres in ≥90%
of the immunized adult individuals, when tested against target
strains.27 Later on, it was noted that higher rSBA than hSBA titres
against MenC strains were observed for the majority of sera, and
rSBA titres of ≥1:8 or ≥1:128 were proposed as threshold of pro-
tection.29,30 Nevertheless, no clear correlation between hSBA and
rSBA titres has been shown to date.31
The rSBA was subsequently used in the licensure process of
meningococcal vaccines in the US and the UK. The WHO later rec-
ommended that hSBA/rSBA titres of ≥1:4/≥1:8 be used as thresh-
olds of protection against disease in the clinical development and
licensure of MenA vaccines as well.32 However, the impact of com-
plement source on the assay results is recognized and still dis-
cussed in depth.33,34 While for MenACWY vaccines both sources
are used, human complement is considered the only acceptable
source of complement for evaluation of MenB vaccines.35 Indeed,
the choice of complement profoundly impacts the assay results for
MenB strains; for instance, it has been shown that IgM antibodies
against the MenB capsular polysaccharide are bactericidal in the
presence of rabbit, but not human complement.36
hSBA for the assessment of MenB vaccines immunogenicity
Given the large number of serotypes, serosubtypes and se-
quence types of circulating MenB disease-causing strains,37-39
analysing the immune response of vaccination against each sep-
arate MenB strain by hSBA is not feasible. Therefore, during the
clinical development of MenB vaccines, immune responses were
evaluated against antigen-specific reference strains. For 4CMenB,
four strains, chosen to allow testing of SBA directed against each of
the four major vaccine antigens, were used most often, especially
in phase 3 trials: 44/76-SL for fHbp, 5/99 for NadA, NZ98/254 for
PorA, and M10713 for NHBA. The latter only became available in
the later stages of the clinical development program and up un-
til that point, an enzyme-linked immunosorbent assay (ELISA) was
used for the assessment of immune response.11 For MenB-FHbp,
 Methods to predict strain coverage of MenB vaccines / Journal of Infection 81 (2020) 862–872
four primary test strains were used, harbouring fHbp subfamilies
A (PMB80 and PMB2001) and B (PMB2948 and PMB2707). These
strains, heterologous to the fHbp variants included in the vaccine,
were selected based on several criteria, such as their representa-
tiveness of fHbp diversity in US and European IMD-causing MenB
strains and a low-to-medium surface expression of fHbp.40 The im-
munogenicity data obtained throughout the clinical development
of 4CMenB41-44 and MenB-FHbp45 demonstrate the vaccines’ abil-
ity to induce SBA killing against reference strains and to potentially
provide protection against other MenB strains expressing antigens
similar to the vaccine components. However, in view of the great
diversity and the temporal changes observed worldwide for MenB
strains, the use of a limited set of reference strains is insufficient
to derive protection against other strains. However, testing against
a large number of strains by hSBA would require a large amount
of sera from vaccinated individuals, which is especially challenging
in the case of infants and children. Moreover, several sources of
complement are needed as distinct strains may behave differently
with complement sources. Thus, some strains can be complement-
sensitive to multiple human complement sources, although the
reason behind this is not fully understood. Lipooligosaccharide ex-
pression (immunotype and degree of sylilation) and lack of PorA
expression have previously been found to impact SBA activity.35,46
In addition, the measured SBA may be influenced by the serendip-
itous presence of naturally-acquired antibodies to the tested strain
in the human sera used as complement source that even if non-
bactericidal alone can synergize with those present in the tested
samples and contribute to complement activation. The polymor-
phism of human factor H (a key regulator of the alternative com-
plement pathway),47 the level of expression and the polymorphism
of proteins other than those included in the vaccine which can
contribute to complement activation,48 or the complement protein
composition, which may vary from one individual to another49 are
several other factors impacting the behaviour of strains with dif-
ferent complement sources.
Another drawback for the use of hSBA in evaluating the strain
coverage of MenB vaccines is the limited availability of suit-
able human complement. The complement should be collected
from healthy individuals who did not acquire antibodies against
meningococcal strains through immunization or naturally-acquired
immunity. To address these limitations, the use of endogenous
complement as source was recently proposed and used to estimate
strain coverage of a vaccine containing 4CMenB components across
a panel of 110 IMD-causing MenB isolates circulating in the US.50
Methods for prediction of MenB vaccines strain coverage
MATS
The meningococcal antigen typing system (MATS) was devel-
oped in 2010 to estimate coverage of MenB strains by 4CMenB. The
method is 4CMenB-specific and combines three antigen-specific
sandwich ELISAs that measure both immunological cross-reactivity
and quantity of the antigens for fHbp, NadA and NHBA, with geno-
typing and phenotyping information for PorA (Fig. 2, Text Box 1).
MATS has been previously described in detail.51,52 Briefly, the
overall vaccine coverage is estimated by assessing ELISA reactiv-
ity for fHbp, NHBA and NadA present in the lysate of MenB strains
and genotype of PorA for the presence of the variable region 2 P1.4
that matches the PorA serosubtype of the vaccine’s OMV compo-
nent. A MATS-positive strain is a strain predicted to be covered by
antibodies directed against at least one of the four antigens con-
tained in 4CMenB. In MATS, ELISA reactivity of each strain is com-
pared to that of three reference MenB strains, specific to each of
the fHbp, NadA and NHBA antigens. The difference in reactivity is
calculated as the relative potency (RP) of each strain. A strain is
865
considered as covered if the RP is higher than a positive bacteri-
cidal threshold (PBT),52 corresponding to a previously-established
minimum RP value predictive of killing in the hSBA for each anti-
gen.53 PBT values correspond to a 80% probability that the strain
is killed in hSBA by pooled sera from 4CMenB-immunized infants
when the strain is covered by only one antigen and a 96% probabil-
ity of killing for MenB isolates positive for two or more antigens.52
The assay has been used to predict 4CMenB coverage for more
than 30 0 0 strains in several countries. Coverage estimates varied
worldwide from 66% to 91%54,55 (Fig. 3).
It is important to note that the MATS ELISAs use polyclonal an-
tibodies for both the capture and detection of fHbp, NHBA, and
NadA, providing an enhanced determination of the immunological
and cross-reactive properties of a given antigen. However, MATS
does not account for a potential synergistic effect for two or more
vaccine components, nor for OMV components other than PorA,
which leads to an underestimation of strain coverage compared to
assessment by hSBA. This was shown by comparing MATS predic-
tions with hSBA results in a panel of 40 MenB strains, selected
from a set of 535 isolates collected from England and Wales from
July 2007 to June 2008. The selection method ensured that the 40-
strain panel was representative of the large set of circulating iso-
lates in terms of MATS phenotypes and strain genotyping profile.56
MATS was found to be a conservative predictor of strain coverage
by 4CMenB in infants and adolescents, with a predicted strain cov-
erage of 70%, whereas hSBA results showed a coverage of 88% for
the same strain panel. Therefore, MATS has an 78% accuracy and
96% positive predictive value when compared with hSBA.56 It was
suggested that MATS might also underestimate the contribution of
NadA to 4CMenB strain coverage, which was found to be very low
(≤2.5%) across MenB strains collected worldwide.57-63 . This might
be due to differences between in vitro and in vivo NadA expres-
sion. A study evaluating transcription regulation of the nadA gene
in MenB strains showed that while some strains exhibit low lev-
els of NadA expression (undetectable by MATS) in vitro, infection
in vivo might upregulate NadA expression and therefore results in
efficient killing by 4CMenB-induced anti-NadA antibodies in the in-
fant rat model.64
In addition, due to the constant evolution of MenB strains,
the sequence and level of expression of 4CMenB components in
disease-causing isolates is likely to vary over time, thus MATS pre-
diction must be re-evaluated periodically to ensure it reflects the
current epidemiological trend. For instance, a phase variation in
the nadA gene was found to affect expression of NadA in meningo-
coccal strains circulating in the UK between 2010 and 2016. This
impacted MATS estimates since strains with low expression levels
are not predicted as covered by this method.65
MATS is specific to antigen components in the 4CMenB, and
therefore cannot be used for the assessments of other vaccines.
Furthermore, MATS is a phenotypic method that requires cultured
isolates and cannot be used for non-culture polymerase chain re-
action (PCR)-confirmed IMD cases.
MEASURE
The flow cytometric meningococcal antigen surface expression
(MEASURE) assay was developed in 2010 for assessment of strain
coverage by MenB-FHbp.66 The assay has been previously de-
scribed in detail.67 Briefly, a monoclonal antibody MN86-994-11
was identified as highly specific for fHbp across diverse MenB iso-
lates expressing fHbp subfamily A or B. MN86-994-11 was used for
the detection of surface-expressed fHbp by flow cytometry, and
a mean fluorescence intensity of 10 0 0 was found to correspond
to approximately 30 pg of surface-expressed fHbp/μg of total cell
protein. This value was correlated with 91.2% probability of the iso-
late to be killed in hSBA by MenB-FHbp immune sera, thus pro-
866 Methods to predict strain coverage of MenB vaccines / Journal of Infection 81 (2020) 862–872

Fig. 2. Schematic representations of methods currently used to assess immunogenicity and predict MenB strain coverage for protein-based MenB vaccine.
Note: ∗ A titre of ≥ 1 : 4, in serum bactericidal antibody (SBA) assays using human complement (hSBA) was found to indicate protection against MenC-caused invasive
meningococcal disease24 and this threshold has been extended and used for the licensure of vaccines against other meningococcal serogroups.
∗∗
Identify antigen-encoding genes predicted to be positive in MATS.

viding a cut-off for predicting susceptibility to bactericidal killing
(Fig. 2, Text Box 1).67
While providing a rapid and reproducible estimation of the
MenB-FHbp strain coverage, MEASURE also has several limitations.
First, strain susceptibility to bactericidal killing was determined
for MenB-FHbp only, and the method is not applicable to other
vaccines. Moreover, in contrast to MATS, MEASURE was devel-
oped to measure surface expression of fHbp variants in meningo-
coccal strains, but does not assess antigenic diversity, and so
it does not account for antigenic cross-reactivity between fHbp
(sub)families/variants. The use of this specific monoclonal antibody
allows for detection of a single conformational epitope conserved
across different variants. Similar to MATS, MEASURE cannot pre-
dict the percentage of individuals with SBA activity following vac-
cination, and cannot be used for non-culture PCR-confirmed IMD
cases.
gMATS
Culture-confirmed IMD isolates are not always available, mainly
due to early antibiotic treatment, and in some countries (e.g. Italy
or the UK) more than 50% of cases are confirmed by PCR only.68,69
Therefore, an alternative method was developed, using antigen
genotyping to predict vaccine coverage and thus enabling the
testing of the complete range of epidemiologically-representative
MenB strain panels. Genetic MATS (gMATS) was defined by assess-
ing the level of correlation between MATS coverage estimates and
antigen genotyping for each of the four antigenic components of
4CMenB.55 Thus, gMATS complements MATS predictions with data
generated by genotyping (Text Box 1).
In gMATS, genes encoding fHbp and NHBA are PCR-amplified
and sequenced, or their sequences are extracted from the whole
genome sequence (WGS) when available. Antigen-specific pre-
dicted strain coverage by gMATS is defined by identifying peptide
IDs significantly associated with MATS coverage/non-coverage for
that antigen. For fHbp and NHBA, only peptide IDs present in ≥5
isolates were considered for the analysis. Peptide IDs for which
the percentage of MATS-covered strains is >60% or <40% are con-
sidered predictors of coverage or non-coverage, respectively, while
peptide IDs not meeting either of the two criteria are designated as
“unpredictable”. In total, 16 fHbp peptides and nine NHBA peptides
 Methods to predict strain coverage of MenB vaccines / Journal of Infection 81 (2020) 862–872 867

Fig. 3. MATS and gMATS predictions for 4CMenB coverage across MenB strains collected worldwide. MATS, meningococcal antigen typing system; gMATS, genetic MATS;
MenB, meningococcal serogroup B.
Note: Error bars represent 95% confidence intervals for MATS and lower/upper limit estimates for gMATS.

Text Box 1. Comparison of different methods for prediction of MenB vaccines strain coverage.

were identified as predictors of coverage. The presence/absence of
the nadA gene can be scored, but this does not allow to predict
coverage as NadA is believed to be artificially under-expressed in
strains when grown for MATS analysis. Therefore, NadA is always
considered as not contributing to coverage in gMATS. Coverage for
PorA is defined as in MATS (PorA variable region 2 match with
peptide 4) (Table 1). A strain is considered as gMATS-covered if
the sequence of the antigen-encoding gene corresponds to pep-
tides predicted as “covered” on the basis of MATS, gMATS-not cov-
ered if the sequence of the antigen-encoding gene corresponds to
peptides predicted as “not-covered” on the basis of MATS, and un-
predictable in the remaining cases. Finally, when predicting strain
coverages, half of the unpredictable gMATS strains were assumed
to be covered, as 49% of them were found to be MATS-positive.55
Strain coverage for 4CMenB by gMATS was evaluated in a panel
of 3481 invasive MenB isolates from 13 countries and was con-
cordant with MATS results; 84.5%, 81.5%, 100% and 100% of strains
could be predicted as fHbp, NHBA, NadA and PorA-MATS covered
868 Methods to predict strain coverage of MenB vaccines / Journal of Infection 81 (2020) 862–872

by gMATS, leading to an overall estimate of 81.3%. gMATS vaccine

gMATS, genetic meningococcal antigen typing system; BAST, Bexsero Antigen Sequence Type; fHbp, factor H-binding protein; NS, not specified; NHBA, neisserial heparin binding antigen; NadA, Neisseria adhesin A; NA, not
strain coverage predictions ranged from 57% to 87% across 13 coun-

BAST
tries and were similar with those predicted by MATS (Fig. 3).55

NS

NS

NS
NS
Similarly to MATS, gMATS underestimates strain coverage as as-
sessed by killing in hSBA and does not account for cooperative ef-
fects between the antigens, nor for the contribution of the NadA
antigen or minor OMV components of 4CMenB. Another limitation
Unpredictable

of gMATS is that coverage cannot be predicted for new alleles of

the fhbp and nhba genes, for which MATS data are not available.
variant 1
All other

All other
peptides

peptides
gMATS

However, the assay presents the major advantage of not requiring

NHBA
fHbp

NA
NA
bacterial isolates and is therefore not limited to culture-positive
cases55 (Text Box 1).

BAST

The Bexsero Antigen Sequence Type (BAST) scheme is a WGS-

BAST

based scheme established and implemented in the Neisseria

NS

NS

NS
NS

PubMLST database (http://pubmlst.org/neisseria).70 The method

evaluates association of genetic lineage (sequence type [ST], clonal
complex [cc]) with 4CMenB antigen components and MATS70,71
(Text Box 1). Coverage is estimated by examining antigen pep-
tide sequences present in both the vaccine and an epidemiological
Peptides 6, 13, 17, 18, 19, 24, 25, 30,

dataset and genotype-phenotype modelling. However, the method

Peptide 213 and all variant 2 and 3

31, 43, 47, 58, 112, 114, 120, 122,

presents some inherent limitation in predicting coverage, mainly

due to the fact that the dataset of MATS data on fHbp subvari-
ants and NHBA peptides was quite limited when the assay was de-
veloped (2011) and therefore all fHbp subvariants and NHBA pep-
tides described later are considered as “unknown” in BAST. This is
particularly true for NHBA, as strains are predicted to be covered
160, 187, 253

by NHBA only when the encoding gene is an exact match for the
PorA VR2=4
Not covered

peptide contained in the vaccine, resulting in a significant under-

peptides

Always
gMATS

estimation of coverage prediction by BAST. Exact match for PorA

VR1 7.2 is also assessed, despite the fact that the epitope is non-
immunogenic. Peptides/variants considered as covered in BAST are
reported in Table 1.
BAST strain coverage predictions are available for isolates col-
lected from the UK and Ireland during 2010–2014, for which cov-
erages of 22.8–30.8% and 66.1% were predicted by BAST exact
Peptides 1, 4, 13,

matches and by genotype-phenotype modelling, respectively.71

14, 15, 37, 232

In certain regions in Australia, annual coverages of 44–91% were

PorA VR2=4
Any variant

predicted by BAST and MATS, from 20 0 0 to 2014.70,72 In addi-

Peptide 2
Antigen peptides/variants considered in strain coverage prediction by gMATS and BAST.

tion, the European meningococcal strain collection genome library

BAST

(EMSC-GL) for the epidemiological year 2011–2012 was character-

ized with the BAST scheme. The EMSC-GL comprised 799 meningo-
coccal strains causing IMD, submitted by 16 European countries, of
which 65.7% were MenB. Predicted BAST strain coverages varied
from 0 to 97.1% across countries, for an overall estimate of 52.4%.73
Peptides 1, 2, 3, 5, 10, 20, 21, 113, 243
110, 144, 224, 232, 245, 249, 252, 510

In a recent study in Poland, across 662 typed isolates, 292 BAST

profiles were identified and strain coverage due to exact match
Peptides 1, 2, 4, 14, 15, 37, 89, 90,

was estimated at 39.7%, while the gMATS prediction was 86.6%.74

Of note, the gMATS coverage was similar to the 83.3% coverage
predicted by extrapolation of MATS data for a subset of strains col-
lected during 2010–2011.55
As for MATS and gMATS predictions, the difference in reported
estimates of strain coverage obtained with the BAST scheme (that
PorA VR2=4

scores exact matching with the four major vaccine antigens) re-
flects changes over time and across geographical areas in the epi-
Covered
gMATS

Never

demiology of MenB-caused IMD. This method enables constant

applicable; PorA, porin A.

surveillance of 4CMenB antigens in the circulating strains, with-

out requiring a live isolate, therefore covering non-culture cases.
Moreover, it allows comparisons (albeit limited) with strain cov-
erages for other vaccines. For instance, in the EMSC-GL study, a
strain coverage of 3.4% was predicted for the bivalent MenB-FHbp,
4CMenB
antigen

based on exact peptide matches for the vaccine’s fHbp variants in

NHBA
Table 1

NadA
fHbp

PorA

the tested isolates, for which SBA data were available; no overlap
of coverage was observed between the two vaccines within the Eu-
 Methods to predict strain coverage of MenB vaccines / Journal of Infection 81 (2020) 862–872
Table 2
Predicted strain coverage and observed impact in real-world settings for 4CMenB.
Clinical trials/prediction methods,% (95% CI)
Years hSBA MATS
UK 2007–2008 88 (72–95)56 73.1 (57–87)55
2014–2015 NA 66.9 (52–81)
2015–2016 NA 73.0 (56–83)
Canada 2006–2009 NA 66.2 (46–78)
UK, United Kingdom; hSBA, serum bactericidal assay using human complement; CI, confidence interval; MATS; meningococcal antigen
typing system; gMATS, genetic MATS; NA, not available. Note: ∗ Impact was defined as percentage reduction in MenB-caused invasive
meningococcal disease incidence from pre-vaccine introduction (UK)/ pre-vaccination campaign (Canada).
ropean genomic library.73 However, as is the case for gMATS, BAST
is limited by the continuous evolution of MenB isolates, generat-
ing new STs, new alleles or combination of alleles encoding the
four vaccine antigens. In addition, cross-protection or phenotypic
expression data on which BAST relies are very limited, it is im-
portant to correlate this analysis with other functional assays. For
these reasons, the accuracy of BAST is lower than for gMATS. How-
ever, the BAST scheme can also be enhanced in the future to in-
clude new STs expressing vaccine antigens or to allow predictions
adjusted for cross-protection or synergistic effects between vaccine
components. For instance, a novel OMV typing (OMVT) scheme
was recently developed. Based on WGS, unique OMVTs were iden-
tified and grouped in a relatively small number of OMVT clusters,
for which a non-overlapping associations with BASTs was shown.75
This association further supports the presumption that immune
and/or metabolic selection is the driving force behind the presence
of a stable, structured population of IMD-causing strains, and al-
lows for the improvement of current WGS-based methods to eval-
uate strain coverage.
From prediction to observation: remaining challenges in
assessing true vaccine effectiveness
MenB vaccines are used either in national vaccination programs
(in the UK, Ireland, Italy, Lithuania, Andorra and San Marino) or
as outbreak control (in Canada, the US, and France). Prediction of
coverage of the diverse and continuously-evolving MenB strains is
crucial for both vaccination strategies. Currently, phenotypic assays
such as hSBA, MATS and MEASURE, or genomic approaches such as
gMATS and BAST are available for prediction of strain coverage.
All these methods predict relatively high strain coverages for
both licensed MenB vaccines (4CMenB and MenB-FHbp), for differ-
ent regions worldwide. However, their results need to be validated
against long-term, real-life vaccine effectiveness data. These data
continue to accumulate for 4CMenB, following its implementation
in the UK national immunization program and from a large vacci-
nation campaign in a Canadian region. Immunization in individu-
als ≤20 years of age in the Saguenay-Lac-Saint-Jean region of Que-
bec, was estimated to reduce the MenB-IMD risk with 86% (95%
confidence interval:−2–98) (unadjusted value: 96%) and to result
in a vaccine effectiveness of 79% (95% confidence interval: −231–
99) over a period of four years.76 These estimates in age groups
directly targeted for vaccination are supportive of the 75% reduc-
tion in MenB IMD cases in infants observed in the UK, over three
years from vaccine introduction.23 A study estimating the effect
of meningococcal vaccines on herd protection against N. menin-
gitidis in UK university students showed that 4CMenB and Men-
ACWY vaccines induced carriage reduction for a subset of Neisse-
ria strains, at 4–12 months after vaccination.77 Recently-concluded
studies conducted in Australia indicated that vaccination of ado-
lescents with 4CMenB did not impact (oro)pharyngeal carriage of
MenB.78,79 Additional studies are ongoing to evaluate the herd ef-
fect induced following mass immunizations.
869
Real-world settings,% (95% CI)
gMATS Years Impact∗ Vaccine effectiveness
73.2 (64–82)55 2015– 75% 59.1%
72.3 (60–85)55 2018 (−31.1–
73.3 (59–88) 87.2)23
72.3 (64–80) 2014–2018 86% 79% (−231–99%)76
MATS vaccine strain coverage predictions were 66% and 66–
73% for Canada and the UK, respectively.55,58,59,80 However, vac-
cine effectiveness estimates from these countries seem to indi-
cate a greater coverage for 4CMenB than that predicted by ei-
ther method (Table 2). Moreover, MATS and similar/complementing
methods have limitations which do not allow them to fully pre-
dict vaccine effectiveness, nor indeed to accurately predict strain
coverage. Inherently, MATS is a conservative predictor of 4CMenB’s
breadth of coverage, but can be complemented with gMATS to pro-
vide more accurate estimates, even for non-cultivable strains. In
addition, phenotypic data on the expression of vaccine antigens
may still be needed to allow the updating of genomic methods
such as gMATS and BAST.
While all these methods have been developed to predict cover-
age against MenB strains, they can potentially be extended to other
serogroups as well. Non-MenB strains also express antigens con-
tained in the protein vaccines and there is growing evidence that
4CMenB may provide cross-protection against other meningococ-
cal serogroups. For instance, pooled sera from infants and adoles-
cents immunized with 4CMenB exhibited SBA activity against non-
MenB strains. In a panel of 147 MenC, MenW, and MenY clinical
isolates collected from three European countries and Brazil, 74.1%
of strains were killed in hSBA by sera from 4CMenB-immunized in-
fants.81 Sera derived from adolescents vaccinated with two doses
of 4CMenB demonstrated killing in hSBA against four strains,
which were representative of the current MenA epidemiology, se-
lected from 1046 MenA isolates collected from different countries
between 20 0 0 and 2016.82 Sera from adolescents and infants re-
ceiving one and three doses of 4CMenB, respectively, displayed
bactericidal activity against all six tested invasive MenW isolates,
causing meningitis or septicaemia in patients from England and
Wales during 2011–2012.83 MATS data were also consistent with
the hSBA results indicating coverage of nine MenX strains collected
from Africa, but not two European ones.84 MenB-FHbp-immune
adolescent sera also demonstrated SBA activity against six non-
MenB strains (one MenA, MenC, MenY and MenX and two MenW),
although the conclusion is limited by the fact that the strains
were selected for testing based on fHbp prevalence.85 So far, MATS
has not been successfully applied to other serogroups, since PBTs
have only been established for MenB strains. However, non-MenB
meningococci seem to be less genetically diverse, therefore poten-
tially requiring a lower number of strains to evaluate the contribu-
tion of each 4CMenB antigen to coverage.86 Similarly, gMATS and
BAST may be adaptable enough to be applied to other Neisseria
serogroups or species.
While not without limitations, current methods afford large-
scale, robust and rapid prediction of MenB strain coverage by
protein-based MenB vaccines. However, prediction of true vaccine
performance remains challenging and the gold standard is repre-
sented by real-world effectiveness studies in a population once the
vaccines are broadly implemented. In addition, continuous mon-
itoring of MenB strain diversity remains paramount to anticipate
temporal and regional variations in strain coverage and adjust the
composition of current vaccines to match them, if necessary.
870 Methods to predict strain coverage of MenB vaccines / Journal of Infection 81 (2020) 862–872
Trademark statement
Bexsero is a trademark owned by or licensed to the GSK group
of companies. Trumenba is a trademark of Pfizer Inc.
Author contribution
All authors participated in the development and the review of
the manuscript and approved the final submitted version.
Declaration of Competing Interest
AB, MMG, MP and RBB are employees of the GSK group of com-
panies and hold shares as part of their employee remuneration.
MKT reports grants from the GSK group of companies and Pfizer
during the conduct of the work; grants from Sanofi, outside the
submitted work. In addition, MKT has a patent (NZ630133A) is-
sued with the GSK group of companies. RB has performed contract
research on behalf of Public Health England for the GSK group of
companies, Pfizer and Sanofi.
Acknowledgments
The authors would like to thank Eva Hong and Ala-
Eddine Deghmane (Institut Pasteur, National Reference center for
Meningococci and Haemophilus influenzae) for their helpful discus-
sion on MATS assay. The authors also thank the Modis platform
for writing support, editorial assistance and manuscript coordina-
tion, on behalf of GSK. Petronela M. Petrar provided medical writ-
ing support and Divya Kesters coordinated the manuscript devel-
opment and provided editorial support. This work was sponsored
by GlaxoSmithKline Biologicals SA, which funded all costs asso-
ciated with the development and the publishing of the present
manuscript.
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