Clinical and Diagnostic Laboratory Immunology, November 1999, p. 838-843, Vol. 6, No. 6
1071-412X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Comparative Analysis of Two Meningococcal
Immunotyping Monoclonal Antibodies by Resonant Mirror Biosensor and
Antibody Gene Sequencing
Bambos M.
Charalambous,1
Janet
Evans,2
Ian M.
Feavers,2 and
Martin C. J.
Maiden3,*
Department of Biochemistry and Molecular Biology, Royal Free and
University College Medical School, Royal Free Campus,
London NW3 2PF,1 Division of
Bacteriology, National Institute for Biological Standards and Control,
South Mimms, Potters Bar, Herts EN6 3QG,2
and Wellcome Trust Centre for the Epidemiology of Infectious
Diseases, Department of Zoology, University of Oxford, Oxford OX1
3PS,3 United Kingdom
Received 10 February 1999/Returned for modification 9 June
1999/Accepted 29 July 1999
 |
ABSTRACT |
Lipooligosaccharide (LOS) is a major surface component of the cell
walls of Neisseria meningitidis, which is important for its
roles in pathogenesis and antigenic variation, as a target for
immunological typing, and as a possible vaccine component. Although the
structures of many antigenic variants have been determined, routine
immunological typing of these molecules remains problematic. Resonant
mirror analysis was combined with gene sequencing to characterize two
monoclonal antibodies (MAbs) used in typing panels that were raised
against the same LOS immunotype, L3,7,9. The two MAbs (MAb 4A8-B2 and
MAb 9-2-L379) were of the same immunoglobulin subtype, but while MAb
9-2-L379 was more than a 1,000-fold more sensitive in immunotyping
assays of both whole meningococcal cells and purified LOS, MAb 4A8-B2
was more specific for immunotype L3,7,9. The differences in sensitivity
were a consequence of MAb 9-2-L379 having a 44-fold-faster association
constant than MAb 4A8-B2. Comparison of the amino acid sequences of the
variable chains of the MAbs revealed that they had very similar heavy
chains (81% amino acid sequence identity) but diverse light chains
(54% sequence identity). The differential binding kinetics and
specificities observed with these MAbs were probably due to differences
in the epitopes recognized, and these were probably a consequence of the different immunization protocols used in their production.
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INTRODUCTION |
Neisseria meningitidis,
an etiological agent of meningitis and septicemia, is a normally
commensal bacterium that nevertheless causes significant morbidity and
mortality worldwide (3). Lipooligosaccharide (LOS) is an
essential glycolipid component of the meningococcal outer membrane that
is equivalent to the longer chained LOSs of the enteric bacteria, which
is important in strain identification, vaccine development,
pathogenesis and host damage (17). Twelve LOS immunotypes
have been described in the literature (20); however, the L3,
L7, and L9 immunotypes have an identical carbohydrate structure and
have therefore been designated L3,7,9 (8). Specific and
cross-reactive epitopes are located on the oligosaccharide part of the
LOS molecule. Immunotypes L1 to L9 are associated primarily, but not
exclusively, with serogroup B and C meningococci, while immunotypes L10
to L12 are mainly associated with serogroup A isolates (18).
Immunotype L3,7,9 is often found in strains thought to be particularly
virulent (13, 18) and may contribute to the resistance of
these meningococci to complement-mediated lysis (14, 16).
This is perhaps due to the fact that the oligosaccharide structure
invariably terminates in a moiety that is structurally similar to the
terminal sequence of human glycosphingolipids (17). Meningococcal LOS of immunotypes L3,7,9, L2, and L5, in common with
that of the related gonococcus, can be further modified in vivo by
sialylation or by the addition of cytidine
5'-monophosphate-N-acetylneuraminic acid (7, 15,
17).
Although the complete oligosaccharide structures of LOS molecules
corresponding to most immunotypes have been elucidated, making it
possible to correlate the immunotype-specific epitopes with defined
oligosaccharide structures (20), immunological characterization of the variants for both routine epidemiological and
research purposes remains problematic, requiring a relatively complex
algorithm based on the reactivity of meningococcal whole cells or
purified LOS in enzyme-linked immunosorbent assays (ELISAs) with a
panel of monoclonal antibodies (MAbs) (18). For protein antigens it is frequently possible to correlate amino acid sequences of
antigenically variable proteins, deduced from gene sequences, with
immunological reactivity, and genetic techniques are consequently playing an increasing role in the characterization and study of such
molecules. In the case of carbohydrates, including meningococcal LOS,
such techniques are unlikely to provide a viable alternative to
immunological studies, despite advances in understanding of the
biosynthetic genes responsible for their production (10). Consequently, an improved understanding of LOS-antibody interactions is
necessary for epidemiological surveillance and studies of the vaccine
potential of this antigen.
In the present study, two mouse MAbs that were raised against LOSs of
immunotype L3,7,9 were compared. The hybridoma cell lines producing
these MAbs were made by immunizing animals with either
oligosaccharide-tetanus toxoid conjugate (hybridoma 4A8-B2) (21) or outer membrane complexes (hybridoma 9-2-L379)
(24). Unlike MAb 4A8-B2, MAb 9-2-L3,7,9 cross-reacted with
the L2, L5, and L8 immunotypes (18), but ascitic fluid
produced from this hybridoma appeared to be more sensitive, being
usable at a much higher dilution. The use of purified antibodies and
LOS in ELISA, together with real-time kinetic analyses with the same
reagents, established the relative sensitivities of these MAbs and
allowed us to measure their binding kinetics. These data were
correlated with the deduced primary structures of the antibodies and
known sugar structures of the relevant LOS molecules.
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MATERIALS AND METHODS |
Preparation of purified L3,7,9 LOS.
Purified immunoreactive
LOS for use in ELISA and biosensor analysis was prepared from
meningococcal isolate K454 (B:15:P1.7,16:L3,7,9) as described
previously (6). The LOS was resuspended in distilled water,
dispensed into 0.5-ml samples, vacuum dried, and stored at 
20°C
until required. Each sample contained 30 to 60 ng of LOS, as estimated
by the Limulus amoebocyte lysate (LAL) chromogenic assay
(23). The purity, number of species, and
Mr of the LOSs were determined by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and silver staining (6,
19) (data not shown).
Purification of MAbs.
The MAbs were purified from ascites
collected from pristane treated mice after intraperitoneal injection
with cells of the appropriate hybridoma cell line. The resultant
ascitic fluids were buffer exchanged on a Biogel P4 desalting column
(Pharmacia, Plc.) with 20 mM sodium phosphate buffer (pH 7.0); the MAbs
captured on a protein G column and eluted with 0.1 M glycine (pH 2.7). The peak fractions were pooled, neutralized with Tris (pH 8.8; 65 mM),
and buffer exchanged in phosphate-buffered saline (PBS; pH 7.4). The
samples were concentrated to ca. 1 µM and tested for immunoglobulin
subclass by using an isotyping kit for mouse MAbs (Serotec, Ltd.). The
ascites containing MAb 4A8-B2 gave two protein peaks after elution from
the protein G column with 0.1 M glycine (pH 2.7). The first peak
contained both immunoglobulin G2a (IgG2a) and IgM antibodies,
indicating that this sample was contaminated with serum. A second peak
contained antibodies of the IgG2a subclass alone, and only the
antibodies from this peak were used. Ascites containing MAb 9-2-L379
had a single peak, comprising antibodies of the IgG2a subclass.
PCR cloning and sequencing of the immunoglobulin variable
regions.
Hybridoma cell lines were grown to confluence in 25-ml
flasks at 37°C in a 5% CO2 atmosphere. Total RNA,
prepared with an Isoquick Kit (Orca Research, Inc.), was used to
synthesize cDNA by extension of an oligo(dT) primer by using a reverse
transcription kit (Clontech UK, Ltd.). The immunoglobulin variable (V)
regions encoding each of the MAbs were rescued by PCR from degenerate primers designed from the immunoglobulin framework regions bordering the VHC
and VL domains as described by
Kettleborough et al. (11). The PCR products encoding the
VHC domains were cloned between the AatII and
SalI sites of both the f+ and f forms of the vector pGEM5Z, while the sequences encoding the VL regions were
similarly cloned between the NcoI and XhoI sites
of both pGEM5Z vectors. The nucleotide sequences of several
independently isolated clones were determined on both strands by
"cycle sequencing" with a Taquence kit (Amersham) with
M13 forward and reverse primers radiolabelled by T4 polynucleotide
kinase with [-32P]ATP.
ELISA.
For whole-cell ELISA microtiter plate wells were
coated with N. meningitidis K454 (L3,7,9) by the method of
Abdillahi and Poolman (1). Coating of plate wells with
purified LOS was as described previously (21). To avoid
interplate variation, assays on both MAbs were carried out on the same
microtiter plate. The secondary antibody was anti-mouse IgG conjugated
to horseradish peroxidase. The absorbance was read at 450 nm 30 min
after the addition of the chromogenic substrate (0.4 mg of
1,2-phenylenediamine dihydrochloride and 0.4 mg of urea hydrogen
peroxide per ml in 0.05 M phosphate citrate buffer, pH 5.0).
Immobilization of L3,7,9 LOS to the resonant mirror biosensor
surface.
Purified LOS was biotinylated (5), lyophilized
and stored in lots of 30 to 50 ng, as estimated by the chromogenic LAL
assay, at 20°C. Immobilization of the LOS was carried out in an
IAsys resonant mirror biosensor (Affinity Sensors, Cambridge, United Kingdom), essentially according to the manufacturer's protocol. Streptavidin (Sigma) was captured onto the biotin-coated biosensor cuvette surface in PBST (10 mM sodium phosphate-138 mM NaCl-2.7 mM
KCl [pH 7.4] containing 0.05% Tween 20), and unbound streptavidin was removed by washing with PBST after 10 min. Biotinylated LOS (3 to 5 ng) was added and binding was monitored. A response of 100 arc seconds
was observed on the addition of LOS to the cuvette. Further additions
did not increase the sensitivity of the assay (data not shown). A final
bovine serum albumin (BSA) blocking step was performed by reacting the
biosensor cuvette with 0.1 mg of BSA per ml in PBST for 5 min. The
LOS-coated biosensor surface was treated with 20 mM HCl to remove any
weakly bound substances before interaction kinetics were performed and
also to regenerate the LOS surface prior to interactions with various
MAb concentrations. To obtain comparative kinetic data, the same
LOS-coated biosensor cuvette was used with both of the MAbs.
Resonant mirror biosensor analysis.
Real-time kinetic
analyses with the IAsys resonant mirror biosensor were undertaken in
PBST at 25°C, according to the methods described by the manufacturer.
The kinetic data were analyzed by curve-fitting software (FASTfit
v2.01), and the binding curves from different MAb concentrations were
overlaid and plotted by using FASTplot software (both supplied by
Affinity Sensors). Dissociation rates (KOFF)
were determined by dilution of unbound MAb in the biosensor cuvette to
zero concentration at relatively high concentrations of antibody (~10
times the dissociation equilibrium constant, KD)
and averaged to give the dissociation rate constant
Kdiss. Initially, approximate
KD values were obtained from concentrations of
MAb ranging from nano- to micromolar levels with subsequent, more-accurate KD and affinity constant
(KA) values determined from MAb concentrations
ranging from 0.01 to 10 times the approximate KD value.
The binding data at high MAb concentrations (~10 times the
KD) fitted a biphasic curve, but only the
initial rates were used in the determination of the binding kinetics.
The KON rates were calculated from the arc
second response over various periods of time and averaged. The gradient
of KON rates versus MAb concentration gave the
association rate constant Kass. The
KA was calculated for each MAb from the
Kass/Kdiss ratio, and the
KD was calculated from the
Kdiss/Kass ratio. The
binding data were also reconciled by plotting the total extent of
antibody binding in a 10-min interaction period against the MAb
concentration and then determining the KD value
from nonlinear regression analysis of these binding data (data not
shown). The y intercept of the plots of
KON versus MAb concentration give approximate
KD values, which were in agreement to within
10% of the experimentally derived KD values.
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RESULTS |
Comparison of MAbs 4A8-B2 and 9-2-L3,7,9 used for immunotyping
N. meningitidis by ELISA.
The concentration-dependent
binding of 4A8-B2 and 9-2-L379 MAbs to whole cells and to purified LOS
demonstrated that MAb 9-2-L379 exhibited approximately a
1,000-fold-greater binding to both whole cells and purified LOS than
4A8-B2 (Fig. 1). The binding observed for
both MAbs to whole cells was approximately 10-fold weaker than to the
purified LOS.
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FIG. 1.
ELISA of MAbs 4A8-B2 and 9-2-L379 against N. meningitidis cells and purified L3,7,9 LOS. The relative binding
of MAb 9-2-L379 to purified LOS ( ) and whole cells ( ) and MAb
4A8-B2 to purified LOS ( ) and whole cells ( ) of the same
meningococcal isolate are shown. Error bars represent the standard
deviation of triplicate determinations.
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Interaction kinetics of MAbs 4A8-B2 and 9-2-L379 with L3,7,9
LOS.
The real-time binding interactions of 4A8-B2 and 9-2-L379 to
immobilized biotinylated LOS, as indicated by arc second response, gave
KON rates for MAb 9-2-L379 that were more rapid
than that for MAb 4A8-B2, whereas the arc second responses were
approximately fourfold greater for MAb 4A8-B2 (Fig.
2). The Kass of
MAb 9-2-L379 was 44-fold greater than that of MAb 4A8-B2, whereas the
Kdiss were similar for both antibodies. The
KA for MAb 9-2-L379 was 31-fold greater than
that of MAb 4A8-B2 (Fig. 3; Table
1). Although real-time kinetic data
between anti-carbohydrate IgGs and their carbohydrate antigens is
limited, the binding kinetic data reported in this study are comparable
to the binding kinetics of an anti-carbohydrate IgG against
Salmonella serogroup B O-polysaccharide as
determined by using surface plasmon resonance (12).
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FIG. 2.
Real-time interaction curves for MAbs 4A8-B2 and
9-2-L379 against immobilized L3,7,9. Real-time binding interaction
curves for various concentrations of each MAb with purified LOS of
immunotype L3,7,9 are shown. (A) Overlay of the binding curves of MAb
4A8-B2 at 0.07, 0.17, 0.35, 0.52, 0.70, and 1.05 µM. (B) Overlay of
the binding curves of the MAb 9-2-L379 at 0.005, 0.01, 0.02, 0.03, 0.10, and 0.25 µM. For clarity, not all of the binding data collected
are shown, and the dissociation curves have been omitted.
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FIG. 3.
Binding kinetics for MAbs 4A8-B2 and 9-2-L379 against
immobilized L3,7,9 LOS. The association rates
(KON) of MAb 4A8-B2 () and MAb 9-2-L379 ()
with purified LOS at 25°C, plotted against MAb concentration, are
shown. The Kass constant of each MAb was derived
from the gradient of the slope.
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DNA sequence analysis and primary sequence comparison of the V
regions of MAbs 9-2-L379 and 4A8-B2.
The VHV-D-J
region of MAbs 4A8-B2 and 9-2-L379 shared 81% overall identity, both
being derived from the J558 V region family. The MAb 4A8-B2 and MAb
9-2-L379 heavy chains showed similar levels of identity to the germ
line genes VMU3.2 and 186-2 and used JH2 and
JH3, respectively. The deduced amino acid sequences of
their CDR1s were identical, their CDR2s were 88% identical, and their CDR3s had no sequence identity.
The amino acid sequences of the VL regions of the MAbs were
54% identical overall, the CDR1s, CDR2s, and CDR3s exhibiting 47, 14, and 11% identity, respectively (Fig. 4).
The VL regions of the MAbs were encoded by different V
gene families, V 8 in MAb 4A8-B2 and V ARS in MAb 9-2-L379.
Comparison of the nucleotide sequences of MAb 4A8-B2 and MAb 9-2-L379
with other murine antibody genes revealed that MAb 4A8-B2 light
chain was 82% identical to the D23 germline and used the J1 J
segment, whereas the MAb 9-2-L379 chain was 84% identical to germ
line gene 28.4.10A() and used J2. In addition to the high degree
of identity to light chains, both MAb 4A8-B2 and MAb 9-2-L379 had
the highly conserved residues phenylalanine at position 71 and
glutamine at positions 90 for MAb 9-2-L379 and 89 for MAb 4A8-B2 (Fig.
4), confirming the VL regions as light chains.
Significant differences were found between the two MAbs in the
VL CDR1, where MAb 9-2-L379 had four positively charged
residues and MAb 4A8-B2 had one, and in the VH CDR3, where
MAb 9-2-L379 had no charged residues but three hydrophilic residues and
MAb 4A8-B2 had two negatively charged residues and one positively
charged residue, with only one hydrophilic residue (Fig. 4).
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FIG. 4.
Primary sequence comparison of the variable regions
within MAbs 4A8-B2 and 9-2-L379. The aligned, deduced primary sequences
of the variable regions from MAbs 4A8-B2 and 9-2-L379 are shown. The
amino acid sequences corresponding to MAb 9-2-L379 are compared with
those of MAb 4A8-B2. Amino acid identities are represented with a
period, and differences are indicated by the appropriate letter;
hyphens represent deletions relative to the sequence of MAb 4A8-B2. The
sequences are numbered according to the Kabat database (9),
with lowercase letters representing variable-length insertion
sequences. CDRs are indicated by white text on a black background.
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DISCUSSION |
N. meningitidis presents carbohydrate structures to its
human host that mimic self-antigens and are poor immunogens. During colonization of the nasopharynx, switching between the capsulate and
acapsulate forms occurs, exposing the capsule and the outer membrane
LOS sequentially (10). Consequently, the interactions between host defences and the bacterial carbohydrate are of central importance in understanding the pathogenicity of, and in the
development of vaccines against, the meningococcus. Further, mouse MAbs
are important reagents in the immunotyping of this organism
(18). The measurement of the binding properties of
antibodies to antigens by real-time binding kinetic analysis therefore
has potential applications in both the standardization of immunotyping
reagents and assays and in the investigation of human responses to
bacterial antigens.
The two antibodies investigated in the present work, although
originally raised against the same meningococcal LOS immunotype, were
produced by distinct immunization protocols and exhibited different
apparent sensitivities and specificities in routine immunotyping ELISAs
(18). The results obtained here with purified reagents
confirmed that the less-specific MAb 9-2-L379, raised against outer
membrane complexes, was one 1,000-fold more sensitive than the
more-specific MAb 4A8-B2, which had been raised against tetanus-toxoid
conjugate. Real-time kinetic analysis by using a resonant mirror
biosensor revealed that the more sensitive reagent, MAb 9-2-L379, had a
44-fold-faster Kass and a 31-fold-higher
KA than MAb 4A8-B2, while the
Kdiss values of the two antibodies were similar.
Interestingly, the arc second response seen with MAb 4A8-B2 was
fourfold greater than that observed with MAb 9-2-L379, implying that in
the biosensor assays MAb 4A8-B2 bound in greater quantities to the
immobilized LOS. The latter observation contradicted the results of the
ELISAs, which indicated that this MAb bound relatively poorly to LOS.
As the arc second response recorded in the biosensor is dependent upon
both the quantity of antibody bound and the distance of the antibody
from the resonant surface (4), this apparent discrepancy
between assays may be the result of differences in the proximity of the
bound antibodies to the resonant mirror.
Comparisons of the deduced primary structures of the two antibodies
revealed the differences responsible for their distinct binding
activities. Notwithstanding the different protocols used for their
production in different laboratories, the two antibodies shared
practically identical heavy chains, with the exception of their CDR3s,
but possessed diverse light chains. This observation presents the
interesting prospect of using antibody engineering techniques
(2) to produce anti-L3,7,9 MAbs with different properties by
using different combinations of the various complementarity-determining regions (CDRs) reported here. An MAb with the sensitivity of MAb 9-2-L379 but the specificity of MAb 4A8-B2 would be a particularly useful reagent. Such constructs would also be potentially valuable in
improving our understanding of the immunology of LOS immunotypes.
The differences of the specificity and binding kinetics of these two
MAbs, together with the differences in their sequences and the
differences in the arc second response observed, implied that they
recognized distinct epitopes within the LOS of immunotype L3,7,9.
Previous studies of the specificity of anti-LOS immunotyping MAbs in
whole-cell ELISA demonstrated that MAb 9-2-L379 cross reacted with LOS
of immunotypes L2, L3,7,9, L5, and L8, whereas MAb 4A8-B2 was specific
for LOS of immunotype L3,7,9 (18). These observations, in
combination with the data reported here, suggest that the specificity
of MAb 4A8-B2 may be the result of interactions with both the
phosphoethanolamine (PEA) group 1-3 linked to the second heptose of the
L3,7,9 structure and the terminal disaccharide of the
lacto-N-neotetraose moiety. Conversely, MAb 9-2-L379 may interact with the galactose residue, located between the
N-acetylglucosamine and glucose residues in the
lacto-N-neotetraose moiety, in combination with the same PEA
group which are present in both the L3 and L8 immunotypes. The
relatively weak cross-reactivity of MAb 9-2-379 with LOS of immunotypes
L2 and L5 suggests that the interaction with this PEA group can be
replaced, to a limited extent, by interactions with a glucose moiety in
the same position (Fig. 5).
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FIG. 5.
Sugar structures of LOS molecules known to interact with
MAbs 9-2-L379 and 4A8-B2. The structures of LOS molecules associated
with the immunotypes L2, L3,7,9, L5, and L8 are given (7, 15, 18,
22), with the sugar moieties that are likely to be involved in
the binding interactions that distinguish the two MAbs highlighted.
Those moieties putatively contributing to the epitope recognized by MAb
9-2-L379 are shown with boldface text, while those that might play a
role in the more specific interaction of MAb 4A8-B2 with L3,7,9 LOS are
shown boxed. The PEA group that is potentially involved in binding of
both antibodies to LOS immunotype L3,7,9 is shown boxed in boldface
text.
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While much remains to be learned concerning antibody-LOS interactions,
and particularly human antibody-LOS interactions, this analysis of two
mouse MAbs by a combination of resonant mirror and antibody sequencing
technologies shows the potential of these techniques in enhancing our
understanding of antibody-carbohydrate interactions for both routine
typing and research purposes.
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ACKNOWLEDGMENTS |
We thank Jan Poolman and Wendell Zollinger for providing the
hybridomas used in this study.
This work was funded by grant number G9202857SB from the UK Medical
Research Council. M.C.J.M. is a Wellcome Trust Senior Research Fellow
in Biodiversity.
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FOOTNOTES |
*
Corresponding author. Mailing address: Wellcome Trust
Centre for the Epidemiology of Infectious Disease, Department of
Zoology, University of Oxford, South Parks Rd., Oxford OX1 3PS, United Kingdom. Phone: 44-1865-271284. Fax: 44-1865-271284. E-mail:
martin.maiden{at}zoo.ox.ac.uk.
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Clinical and Diagnostic Laboratory Immunology, November 1999, p. 838-843, Vol. 6, No. 6
1071-412X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
