BIOLOGY AND EPIDEMIOLOGY OF FACTOR H BINDING PROTEIN
Meningococcal Factor H binding protein (fHbp, previously referred to as GNA1870 or
Lp2086)
One of the most promising of the recombinant protein meningococcal vaccine antigens in
clinical development is fHbp, which is a surface-exposed lipoprotein that is present in all N.
meningitidis strains (Masignani et al, Journal of Experimental Medicine, 2003). In collaboration
with Sanjay Ram’s group at the University of Massachusetts, we identified fHbp as a ligand on
N. meningitidis that binds factor H (fH) (Madico et al, Journal of Immunology, 2006). Factor H is
a down-regulatory molecule in the complement cascade and binding of this molecule to the
bacterial surface contributes to the ability of the organism to avoid complement-mediated killing
by non-immune human serum or whole blood. (Figure 1). When fHbp expression was restored
in the KO mutant by transformation with a plasmid containing a gene encoding fHbp, the strain
survived in blood (Welsch et al, Journal of Infectious Diseases, 2008).
Figure 1. Survival of N. meningitidis in non-immune human blood. Solid blue line with squares,
wildtype strain; dashed orange line with circles, isogenic knockout (∆fHbp) mutant. Left panel.
Growth of wildtype N.meningitidis strain H44/76 and its isogenic fHbp knockout showing the both
grow normally at 37°C in Mueller Hinton broth. Right panel. Survival of H44/76 WT and mutant
strains in human blood (symbols same as in left panel). Also shown is survival of the H44/76∆fHbp
mutant transformed with a plasmid encoding fHbp and expressing fHbp (solid magenta line,
triangle). From Welsch et al, J. Infect. Dis. 2008.
We investigated species-specificity of fH binding to N. meningitidis and the effect of the addition
of human fH on down-regulating rat (relevant for animal models) and rabbit (relevant for vaccine
evaluation) complement activation (Granoff et al, Infection and Immunity, 2009). Binding to N.
meningitidis was specific for human fH (low for chimpanzee and not detected with fH from lower
primates) (Figure 2). Further, the addition of human fH decreased rat (Figure 3) or rabbit C3
deposition on the bacterial surface, and decreased group C bactericidal titers measured with
rabbit complement by 10- to 60-fold in heat-inactivated sera from human vaccinees (Figure 4).
Meningococcal Vaccine Research Laboratory Program Description
Figure 2. Species specificity of fH binding to N. meningitidis. Panel A. Binding
of goat polyclonal anti-human fH to fH in primate sera (1:100 dilution) as
measured by Western blot. The antibody cross-reacts with fH from each of the
four species. Panel B. Binding of fH to N. meningitidis strain H44/76 after
incubation of bacteria in different primate sera. Log-phase bacteria were
washed and lysed in sample buffer. The lysate was subjected to SDS PAGE,
and binding of fH was detected by Western blot using the antisera described in
Panel A. The cells incubated with the human serum showed the most fH
binding, whereas binding of fH in chimpanzee serum was low, and binding was
barely detectable with baboon or rhesus macaque sera. From Granoff et al,
Infect Immun 2008.
Figure 3. Regulation of rat C3 binding by human fH. Live
bacteria of group B strains H44/76 and NZ98/254 were
incubated with 20% rat serum. Rat C3 deposition was
measured by flow cytometrey. Dotted histograms, control, no
serum; Open histograms (solid line), rat serum alone with 0
µg/ml of human fH; Shaded histograms, rat serum plus human
fH, 25 µg/ml. From Granoff et al, Infect Immun 2008.
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Figure 4. Effect of addition of human fH (25 µg/ml) on group C serum
bactericidal titers measured with infant rabbit complement. The postimmunization sera were from 11 adults given a group C meningococcal
conjugate vaccine, or 19 children, aged 4- to 5- years, immunized with
quadrivalent meningococcal polysaccharide vaccine. The differences
between the respective geometric mean titers measured in human
vaccinee sera using rabbit complement in the absence or presence of
human fH were significant (P<0.01). The addition of the negative control
complement regulator, human C1 esterase, had no significant effect on the
respective titers. From Granoff et al, Infect Immun 2008.
To determine whether human fH enhanced survival of N. meningitidis in vivo, we administered
different doses of human fH to infant rats, or, as a control, a human C1 esterase inhibitor that
does not bind to N. meningitides. The animals were challenged with group B strain H44/76, and
blood cultures were obtained 8 hours later. Increasing numbers of bacteria (CFU/ml) were
isolated from the blood of animals that had been administered increasing doses of human fH
(P<0.02). Collectively, the data underscored the importance of binding of human fH on survival
of N. meningitidis in vitro and in vivo. Species-specificity of binding of human fH added another
mechanism towards our understanding of why N. meningitidis is strictly a human pathogen.
The structural basis of fH binding to fHbp. Recently, the laboratories of Susan Lea at the
University of Oxford and Christopher Tang at Imperial College in London crystallized a domain
of the fH complement regulator in complex with fHbp (Schneider et al, Nature, 2009) (Figure 5).
The site in fH that bound to fHbp was predominantly in the short consensus repeat region 6
(SCR 6). Binding was mediated by charged amino acid residues in the fHbp structure, which
mimicked portions of sugar molecules that were previously identified to bind to fH SCR 6. Small
differences in the amino acid sequences between SCR 6 in human fH and that of non-human
primate and rodent fH explained the exquisite species specificity of human fH binding to
meningococcal fHbp.
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Figure 5. Structure of complement regulator human fH (green)
complexed with Neisseria meningitidis’ surface expressed fHbp
(yellow). Based on coordinates published by Schneider, M.C.,
Prosser, B.E., Caesar, J.J.E., Kugelberg, E., Li, S., Zhang, Q., et
al. Neisseria meningtidis recruits factor H using protein mimicry of
host carbohydrates. Nature on line 18 February 2009.
Meningococcal fHbp has a modular structure and can be classified into modular groups.
Based on sequence variability of the entire protein, fHbp has been divided into three variant
groups (Masignani et al, Journal of Experimental Medicine, 2003) or two sub-families (Fletcher
et al, Infection and Immunity, 2004; Murphy et al, Journal of Infectious Disease, 2009). The
relationships between the two sub-families and three variant groups based on the entire amino
acid sequences of 70 distinct fHbp variants are shown in Figure 6. We recently presented
evidence the fHbp molecular architecture is modular (Beernink and Granoff, Microbiology,
2009). From sequences of natural chimeras we identified blocks of two to five invariant residues
that flanked five modular variable segments (Figure 7, Top panel). We mapped the variable and
invariant residues onto a molecular model based on the published coordinates from the fHbp
crystal structure described above (bottom panel). Although overall, 46% of the fHbp amino acids
were invariant, the invariant blocks that flanked the modular variable segments formed a distinct
cluster on the surface of the protein that was anchored to the cell wall (visible on the models in
the center and on the right, the lower panel, Figure 7). This location suggested that there are
structural constraints involving these invariant blocks, perhaps a requirement for a partner
protein, for anchoring and/or orienting fHbp on the bacterial cell membrane.
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Figure 6. Phylogram of fHbp based
on 70 unique amino acid sequences.
For each sequence, the peptide
identification number assigned in the
fHbp peptide database at
http://Neisseria.org is shown and, if
known, the multi-locus sequence type
(MLST) clonal complex is shown in
parentheses. The lower left branch
shows variant group 1 as defined by
Masignani et al (Masignani et al.,
2003) (sub-family B of Fletcher et al
(Fletcher et al., 2004)); Sub-family A
contained two branches, variant
groups 2 and 3. The phylogram was
constructed by multiple sequence
alignment as described elsewhere
(Beernink and Granoff, Microbiology,
2009). The scale bar shown at the
bottom indicates 5 amino acid
changes per 100 residues.
Figure 7. Panel A. Schematic representation of fHbp
showing positions of blocks of invariant residues (shown
as black vertical rectangles). The top three panels show
three representative N. meningitidis fHbp variants
(groups 1, 2 and 3; peptide ID numbers, 1, 16 and 28,
respectively). The amino acid positions of the last
residue in each variable segment are shown. All of the
meningococcal fHbp sequences, as well as the two N.
gonorrhoeae fHbp orthologs, had six identical invariant
blocks of residues that flanked segments VA through VE.
Panel B. Space-filling structural models of factor H
binding protein based on the coordinates of fHbp in a
complex with a fragment of human factor H (Schneider
et al., 2009). Light gray, variable amino acids located
within the modular variable segments; black, invariant
blocks of residues separating each of the variable
segments; yellow, invariant amino acids outside of these
blocks. The model on the left shows the surface
predicted to be anchored to the cell wall. The middle
model is the surface-exposed portion viewed from
above. The left model is viewed from the side.
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Based on the positions of the blocks of invariant amino acids, the overall architecture
could be divided into an amino-terminal repetitive element and five modular variable segments,
which we designated VA- VE. Each of the five modular variable segments segregated into one of
two types. One of the types had signature amino acid residues and sequence similarity to
peptides in the antigenic variant 1 group. The other type had signature amino acid residues and
sequence similarity to peptides in the antigenic variant 3 group. For purposes of classification,
we designated the first group of segments as α types and the second group as β types. For
each segment, we calculated the percentages of the amino acid identity of each of the α type
segments with each of the corresponding α or β type segments of the 70 peptides. We
performed a second calculation of the percentages of the amino acid identity of each of the β
type segments with the corresponding α and β types. We generated histograms showing the
respective mean frequencies of peptide variants with different percentages of amino acid
identity. Figure 8 shows a representative histogram comparing each of the 48 α VA segments to
the corresponding α and β type VA segments of the 70 peptides. For each of the modular
variable segments, there was clear separation between the percent amino acid identities of the
respective the α- and β-types.
Figure 8. A histogram for Modular Segment VA showing the mean
number of peptides (Y-axis) and percent amino acid identity (Xaxis), which was generated by comparing each of the 48 α A
segments to the corresponding α and β types A segments of all 70
peptides.
fHbp modular groups. Based on the phylogenic analysis of the five modular variable segments
described above, we could categorize each of the 70 different fHbp variants into one of six
distinct fHbp modular groups (Figure 9). Forty of the 70 fHbp peptides (57%) comprised only α
(N=33) or β (N=7) type segments, which were designated fHbp modular groups I and II,
respectively. The remaining 30 peptides (43%) could be classified into one of four chimeric
groups derived from recombination of different α or β segments (designated fHbp modular
groups III, IV, V or VI). Of the 38 proteins in the Masignani variant 1 group, 33 were in modular
group 1 and five were chimeras in modular group IV. Of the 17 proteins in the variant 3 group,
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seven were in modular group II and ten were chimeras in modular group V. All 15 variant 2
proteins were chimeras in modular groups III or VI. One of the chimeric modular groups had
96% amino acid identity with fHbp orthologs in Neisseria gonorrhoeae. Collectively the data
suggested that recombination between N. meningitidis and N. gonorrhoeae progenitors
generated a family of modular, antigenically diverse meningococcal factor H-binding proteins.
Figure 9. Schematic representation of six fHbp modular groups deduced
from phylogenic analysis. Forty of the 70 peptides contained only α type
segments or β type segments, and were designated as modular groups I
and II, respectively. The segments derived from lineages designated α
are shown in gray and the segments from lineages designated β are
shown in white. The relationship between the modular group and
Masignani variant group designation, and the number of unique
sequences observed within each fHbp modular group, are shown (Figure
from Beernink and Granoff, Microbiology 1009.
Frequency of fHbp modular groups among isolates causing disease in different countries
The analysis described above provided information on the extent of fHbp modular group
diversity but did not address the frequency of different fHbp modular groups among isolates
causing disease. In a recent publication (Pajon et al, Vaccine, 2010), we used the fHbp
sequence data reported by Murphy et al (Journal of Infectious Diseases 2009) from
systematically collected group B isolates in the U.S. and Europe to determine the frequency of
fHbp modular groups in different countries. The isolates were from cases in the United States
between 2001 and 2005 (N=432), and from the United Kingdom (N=536), France (N=244),
Norway (N=23) and the Czech Republic (N=27) for the years 2001 to 2006. We supplemented
the U.S. data with fHbp sequences we had recently determined for 143 additional isolates that
had been systematically collected at multiple sites in the U.S.
Among the total of 1405 systematically collected group B isolates, modular group I was
found in 59.7%, group II in 1.7%, group III in 8.1%, group IV in 10.6%, group V in 6.1%, and
group VI in 13.6%. In this study, we identified two new modular groups, VII, VIII and IX among
242 fHbp variants, but each of these was found in ≤0.1% of the strain collections. The
respective distributions of the fHbp modular groups in the different countries as stratified by the
variant group classification of Masignani are shown in Figure 10 (For these analyses, modular
groups VII, VIII and IX were excluded because of their rarity).
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Isolates in variant 1 group of
Masignani consisted of modular
groups I and IV. Modular group I
strains, which have entirely α-type
segments, predominated in all
three countries (54 to 64 percent of
all isolates). In the UK, however,
23% of all isolates were modular
group IV, which are natural
chimeras of α- and β-type
segments (Figure 9), as compared
with <1% in the two U.S.
collections, and 3 percent of
isolates from France (P<0.001 by
chi square).
Isolates in variant 2 group
included modular groups III and VI,
which are all natural chimeras of
α- and β-type segments. Modular
groups III or VI were present in
approximately equal proportions of
isolates in the two U.S. collections,
while modular group VI
predominated among variant 2
isolates from the UK and France.
Isolates with variant 3 group included
modular group II (entirely β-type segments)
and modular groups V, which are chimeras.
In France and the UK, modular group V
accounted for the majority of the isolates
with variant 3 fHbp, while in the two U.S.
collections there were approximately equal
numbers of modular group II or V proteins.
Figure 10. Frequency of fHbp modular groups among systematically collected N. meningitidis group B case
isolates. Data are from sequences of isolates collected in the United States (N=432), United Kingdom
(N=536) and France (N=244) reported by Murphy et al, and newly obtained sequences of 143 additional
U.S. isolates from California (2003-2004), Maryland (1995 and 2005), and pediatric hospitals in 9 states
(2001-2005) from a collection previously described by Beernink et al (J. Infect Dis 2007). Figure from Pajon
et al, Vaccine 2010.
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Strain susceptibility to anti-fHbp serum bactericidal activity in relation to modular group
and fHbp expression
The results of previous studies
suggested that antibodies to fHbp in
the variant 1 group were bactericidal
primarily against strains with fHbp
variant 1 but had little or no activity
against strains with fHbp in the
variant 2 or 3 groups, and vice versa.
These studies included relatively few
strains and didn’t consider the
possible role of modular group or
level of fHbp expression on
susceptibility of a strain to anti-fHbp
bactericidal activity.
Figure 11 depicts human
complement bactericidal titers of
serum pools from mice immunized
with recombinant fHbp vaccines
representative of modular groups IVI. The heights of the bars represent
the respective median titers of each
of the six antisera (3 to 4 pools per
modular group) when tested against
the specific test strain. For example,
the upper left panel shows the data
for strain H44/76, which is a high
expresser of fHbp in modular group I
(relative expression is designated by
+++). The median bactericidal titer of
the homologous anti-fHbp modular
group I antiserum (black bar) was
~1:6000. The respective titers of the
five heterologous anti-fHbp modular
group antisera (white bars) against
strain H44/76 were 1 to 2 log10 lower,
ranging from <1:10 (antisera to
modular group II) to ~1:200 (antisera
to modular group IV). The
corresponding median titers of the
anti-modular group II and IV antisera
when tested against control strains
with homologous fHbp modular
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Figure 11. Serum bactericidal activity of serum pools from mice immunized with fHbps from modular
groups I to VI. The black bars represent the median titers of 3 to 4 serum pools for each modular
group tested against homologous test strains. The white bars represent the median titer of the
respective heterologous serum pools. against the test strain. +, refers to relative expression of fHbp
by each of the strains; strains with +/- representing low fHbp-expressing strains (From Pajon et al,
Vaccine 2010).
groups II (strain SK104 in the Variant 3 panel) or IV (NM452 in the Variant 1 panel) were
>1:2000. Thus, these and the other heterologous antisera had high antibody activity when
measured against control strains with the respective homologous fHbp modular groups.
We also observed a trend for lower cross-reactivity of anti-fHbp bactericidal activity against
strains with low expression of fHbp from heterologous modular groups than for high expressing
strains (see for example, data for low expressing strains 03S-0408 (modular group I), M01573
(modular group IV), and RM1090 (modular group III), which were killed only by their respective
homologous modular group antisera as compared with the broader bactericidal activity against
the respective higher fHbp expressing strains (H44/76, NM452, and 03S-0673; Figure 11).