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Institute for Biological Sciences, National Research Council of Canada, Ottawa, Canada
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have hypothesized that the immune system selects epitopes situated on extended helical domains of GBSPIII rather than those on the predominant random coil form (7). Because of a lack of symmetry, the random coil form is more structurally related to the short oligosaccharides associated with self Ags. Using nuclear magnetic resonance (NMR) and molecular dynamics studies on GBSPIII and its fragments, we confirmed that GBSPIII was capable of forming extended helices and that sialic acid, although not immunodominant itself, is important in the formation of these helices (7). Here we define the conformational epitope with greater precision by using a much larger repertoire of defined GBPSIII fragments to bind to GBSPIII mAbs, and by employing a number of complementary techniques to study this binding.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pn14PS was purchased from American Type Culture Collection (ATCC, Manassas, VA). GBSPIII and tetanus toxoid were gifts from Dr. F. Michon of North American Vaccine (Beltsville, MD). HSA and BSA were products of Sigma (St. Louis, MO). All chemicals were purchased from Aldrich Chemical (Milwaukee, WI) and used without further purification.
Preparation of anti-GBSPIII mAbs
Female BALB/c mice, 6–8 wk of age, were immunized intraperitoneally with GBSPIII-TT conjugate (8). Each mouse received 1 µg of polysaccharide in 0.2 ml Ribis complete adjuvant (Ribi Immunochem Research, Hamilton, MT) per injection. The mice were boosted on day 21 with an equivalent amount of conjugate vaccine and were trial bled on day 31. The two mice showing the highest Ab titer to the homologous Ag were given two final injections on day 41, an i.p. injection as given previously, and an i.v. injection with the conjugate vaccine in saline (0.1 ml). The fusion was performed 3 days following the last injections. Stimulated spleen cells from the two immunized mice were fused with SP2/0-Ag14 myeloma cells in a ratio of 10:1 in 33% (w/w) PEG 1500. Putative hybrids resulting from hypoxanthine/aminopterin/thymidine (HAT) selection were screened by ELISA against GBSPIII-HSA and Pn14PS-HSA. Those producing Ab of interest were cloned twice by limiting dilution to ensure stability and clonality. Ig subclass was determined on spent supernatant using an EIA mouse mAb isotyping kit (Amersham Canada, Oakvile, ON). Clones were expanded as ascites by i.p. injection of 106 hybridoma cells in BALB/c mice 10–14 days following i.p. treatment with 0.5 ml 2,6,10,14-tetra-methy1-pentadecane(pristane). Ascitic fluid was tapped 7–14 days postinjection.
Preparation of Fab fragment
mAb 1B1 was purified from ascites fluid using a protein A column (Pharmacia, Uppsala, Sweden). The purified mAb in PBS at pH 7.4 was concentrated by Centricon-10 (Amicon, Beverly, MA), and then lyophilized. Fab was prepared from the mAb using an "ImmunoPure Fab Preparation Kit" (Pierce, Rockford, Ill). mAb was digested into Fab by incubation overnight (15 h) in a buffer containing immumoblized papain in a water bath shaker at high speed. The resultant soluble Fab, Fc fragments, and undigested IgG were separated from the immobilized papain gel by a separator tube. Then the Fab was separated from the Fc and undigested IgG by using a protein A column. The eluate of the column was collected, dialyzed against PBS at pH 7.4 overnight, and concentrated by Centricon-10.
ELISA
Culture supernatant and ascitic fluid were assayed against the HSA conjugates (0.5–1.0 µg/100 µl PBS/well) in 96-well EIA plates (Linbro, Aurora, OH). Wells were coated at 37°C for 1 h and then washed with PBS-T (0.05% Tween 20) and blocked with 200 µl 1% BSA-PBS for 1 h at room temperature. Samples of culture supernatants or ascites, serially diluted in 1% BSA-PBS, were added and incubated for 1–3 h at room temperature. Following washing with PBS-T, alkaline phosphatase-labeled goat anti-mouse IgG (Caltag Laboratories, San Francisco, CA) diluted 1:3000 in 1% BSA-PBS was added for 1 h at room temperature. The plates were then washed and developed with p-NPP Phosphate Substrate System (Kirkegaard & Perry Laboratories, Gaithersburg, MD). After 20 min at room temperature, the plates were scanned at 410 nm in a Dynatech (Chantilly, VA) MR 5000 microplate reader.
Preparation of oligosaccharides and polysaccharides with different chain length
Oligosaccharides (1 RU to 20 RU) were derived by controlled
partial N-deacetylation, enzymatic sialylation, and nitrous
acid deamination from Pn14PS (Fig. 1
) as
previously reported (9). Pn14PS was partially
N-deacetylated by base treatment, and the resultant product
was enzymatically sialylated at the 3-O position of the
terminal galactose residues. The glucosamine residues in the partially
N-deacetylated GBSPIII, on treatment with nitrous acid, were
converted to 2,5-anhydromannose derivatives. This procedure
simultaneously fragmented the polysaccharide and introduced terminal
aldehyde group in each of the fragments. Thus, all the fragments made
using this degradative procedure contained complete RU of GBSPIII,
together with an appendage consisting of a terminal transformed RU
(Fig. 1). The fragments were separated on a Superdex 30 column eluted
with PBS to afford oligomers from 1 RU to 7 RU. Oligosaccharides
representing 1 RU to 4 RU were shown to be pure by spectroscopic
analysis. After obtaining the 7 RU fraction, the eluate of the column
containing the higher m.w. fractions was also collected. It had an
average size of 20 RU, as estimated by comparison with standard dextran
samples by HPLC (Hewlett Parkard Series 1100) using a Superose 12 10/30
column (Pharmacia).
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The oligosaccharides were conjugated to HSA by a previously
described procedure (12). In brief, a solution of
oligosaccharide (3 mg), HSA (3.5 mg), and sodium cyanoborohydride (3
mg) in 0.1 M sodium bicarbonate buffer (0.3 ml, pH 8.1) was kept at
37°C for 3–4 days. The progress of conjugation was monitored by
analyzing small aliquots from the reaction mixture by HPLC with PBS as
eluant. Conjugation was indicated by small shift of the protein peak to
a relatively lower KAV value. All the
conjugates were purified on a Biogel A 0.5 column eluted with PBS.
Fractions containing the conjugates were pooled, dialyzed, and
lyophilized and were analyzed for carbohydrate content by the
phenol-sulfuric acid method (13) using GBSPIII as a
standard (Table I).
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These glycoconjugates were prepared by procedures previously described (8). In brief, GBSPIII fragments of different chain lengths were oxidized at C8-C9 of the sialic acid residues with NaIO4 to achieve 10–15% oxidation as determined by glc-ms analysis. Conjugation to HSA and purification of the resultant glycoconjugates was performed under the same conditions as described above.
Direct binding of mAbs to oligosaccharide-HSA conjugates in ELISA
Microwell plates were coated with 1 µg per well of HSA conjugates in PBS. The plates were then blocked with 0.5% skim milk or 1% BSA in PBS. Serial dilutions of mAbs were applied, and the plates were incubated and washed twice. The Abs were detected with goat anti-mouse IgG (H+L) conjugated to HRP at 1/200 dilution and tetramethylbenzidine (TMB) substrate (Kirkegaard & Perry). Tween 20 (0.05%) in PBS was used as washing buffer between each step. OD were recorded as the reciprocal of the dilution at 450 nm.
ELISA inhibition of the binding of mAbs 1B1 and 1A6 to GBSPIII-HSA by GBSPIII fragments
Competitive inhibition assays were performed following the ELISA
procedure as described above, with the following modifications. After
the blocking step, 50 µl of oligosaccharide (concentration 2–3.5
mg/ml) in 0.5% BSA/0.02% Tween/PBS buffer was added to the wells,
which were serially diluted 2-fold with the same buffer. Then 50 µl
of mAb, which was diluted 100x in the same buffer to give an OD of
1 in the absence of inhibitors, was added, and the mixture was
incubated at room temperature for 3 h. The remainder of the
procedure was followed as described above. Inhibitory activity is
presented as percentage calculated as [OD (no inhibitor) - OD
(with inhibitor)/OD (no inhibitor)] x 100%. Inhibition vs log
concentration curves were plotted for each inhibitor (see Fig. 3), and the concentrations required for
IC50 were determined from extrapolated curves
(Table II).
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Binding kinetics and affinities were determined by SPR using a
BIACORE 1000 biosensor system (Biacore, Piscataway, NJ)
(14). Glycoconjugates of GBSPIII and its fragments were
immobilized on research grade CM5 sensor chips (Biacore) using the
amine coupling kit supplied by the manufacturer (Biacore).
Immobilizations were conducted in 10 mM sodium acetate (pH 4.5) at
conjugate concentrations of 50 µg/ml. The immobilized surface density
was 200 resonance units in each instance. Measurements were
conducted in 10 mM HEPES (pH 7.4), 150 mM NaCl, 3.4 mM EDTA at 25°C,
and at a flow rate of 20 µl/min. Following IgG or Fab binding,
conjugate surfaces were regenerated with 100 mM HCl and a contact time
of 6 s. Sensorgram data were analyzed using BIAevaluation 3
software (Biacore).
To determine the capacity of different length oligosaccharides to bind
Fab, 50 µM 1B1 Fab was injected over 2 RU, 6–7 RU, and 20 RU
conjugate surfaces. Surface densities were 200 resonance units for
each conjugate, and all three conjugates had very similar degrees of
substitution with oligosaccharide (Table I
). This Fab concentration is
at least 10x the KD in each instance
and should, therefore, result in almost complete saturation of the
surface with Fab at equilibrium. The maximum response was used to
compare binding stoichiometrics.
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Results |
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Conjugation of fragments 1 RU to 20 RU by reductive amination gave
conjugates where each saccharide chain was attached to HSA by a single
covalent bond. Because they were randomly activated, conjugation of
fragments 25 RU to 80 RU (8) resulted in the occurrence of
some cross-linking. The analysis of the conjugates is shown in Table I
and indicates that conjugates made with fragments 1 RU to 20 RU all had
similar saccharide loadings ranging from 9.8 to 16.2%, whereas those
made with larger saccharides had much higher saccharide loadings, of
the same order as that obtained with the GBSPIII–HSA conjugate.
Competitive inhibition of the binding of GBSPIII to mAbs by GBSPIII fragments
For these ELISA inhibition experiments, both mAbs 1B1 and 1A6 were
used, and the GBSPIII-HSA conjugate was used as coating Ag. The
inhibition curves are shown in Fig. 3 and indicate that, while 1 RU was
a very poor inhibitor, inhibition improved dramatically at 2 RU and
improved further at 3 RU. The inhibitory properties of 2 RU, whether
obtained by degradative methods (2 RU) or by synthesis (2
RUs) (Fig. 2), were the same, thus demonstrating
that the residual terminal oligosaccharide appendage of the former did
not add to its inhibitory properties. Further increases in saccharide
length from 3 RU to 7 RU did not increase the inhibitory properties to
any extent. However, substantial increases in inhibition were obtained
when larger fragments were employed. The inhibitory power of these
larger fragments (25 RU to 80 RU) increased sequentially with
increasing length, GBSPIII being the most potent inhibitor.
The binding affinity of GBSPIII fragments to the mAbs was also
evaluated by comparing their IC50 data. These
results are shown in Table II and again confirm the dramatic leap in
1B1 and 1A6 affinity that accompanies progression from 1 RU to 2 RU.
For 1B1, affinity maximizes at 3 RU and remains constant up to 7 RU. In
the case of mAb 1A6, inhibition maximized at 4–5 RU and also remained
constant up to 7 RU. This is consistent with the binding site of 1A6
being longer than that of 1B1. These results also indicate that the
inhibitory activity of GBSPIII is 200-fold better than that of
fragments of 2 RU to 7 RU.
Binding kinetics and affinities of GBSPIII fragments by SPR analysis
The binding of anti-GBSPIII mAb 1B1 and its Fab fragment to
HSA conjugates of GBSPIII fragments were investigated by SPR. The
binding of 1B1 Fab fragments to immobilized oligosaccharide conjugates
showed good fitting to a 1:1 interaction model. The simultaneous
fitting of the association and dissociation data of individual
sensorgrams shown in Fig. 4 for the 20 RU
conjugate is representative of that observed with all conjugates.
Association and dissociation rate constants for 1B1 Fab binding to the
complete glycoconjugate panel were derived by this fitting procedure
(Table III). For 1B1, the pattern of
affinities exhibited by the saccharide fragments with increasing size
is in good agreement with that obtained by other approaches. The
binding of 1 RU surfaces was very weak; Fab binding was not detectable
and the level of IgG binding was significant only at high surface
densities (4000 resonance units). Epitopic stabilization appears to
occur at 2 RU with the KD remaining
virtually constant from 2 RU to 7 RU. A gradual 3-fold increase in
affinity, attributable to equal contributions from a faster
Ka and slower
Kd, occurs with increasing saccharide
size from 6 RU to 20 RU. Further increases in chain length did not lead
to much improved binding. Thus, there is dramatic epitopic
stabilization at 2 RU with a further significant stabilization between
6 RU and 20 RU.
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reveal
identical steady state binding levels for 2-RU and 6- to 7-RU surfaces
of the same density (Rmax = 148 resonance units)
and that a 20-RU surface of the same density had a 3-fold greater
capacity for Fab binding (Rmax = 486 resonance
units). This is consistent with a Fab-to-Ag binding stoichiometry of
1:1 for the 2-RU and 6- to 7-RU surface and 3:1 for the 20-RU surface
and indicates that epitope multivalency occurs between 6–7 RU and
20 RU.
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). However, the differences between the
dissociation rates for IgG and Fab increased with increasing saccharide
length, indicating a higher proportion of bivalent binding at higher Ag
multivalency. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), thus confirming that both mAbs has a specificity similar to
that of the previously defined conformational epitope (6, 8). Even though each of the fragments used in our studies
contained an additional terminal oligosaccharide appendage, they were
designated on the basis of the number of complete RUs that they
contained. That this designation had relevance in addition to
convenience was established in inhibition experiments, the result of
which demonstrated that the appendage made no contribution to the
inhibitory properties of the fragments. Having established 2 RU as the minimum binding structure, we designed additional experiments to study in more detail factors behind the increasing affinity of GBSPIII with increasing length (6, 8). Using mAbs 1B1 and 1A6 and a more extensive series of well-defined chemically derived GBSPIII fragments, we attempted to answer two questions that were not addressed in previous studies (6, 8): 1) at what fragment length does the conformational epitope optimize, and 2) at what length of saccharide does epitope multivalency occur? ELISA inhibition of the binding of GBSPIII to mAbs 1B1 and 1A6 using GBSPIII fragments demonstrated that the initial formation of the conformational epitope occurs at 2 RU. A small increase in inhibition occurs from 2 RU to 3 RU, with little subsequent increase in inhibition from 3 RU to 6–7 RU. The inhibition results using mAb 1A6 were very similar. These data are consistent with the presence of only one conformational epitope from 2 RU to 7 RU, and with little further optimization of epitope in this region. The above data were confirmed by studies on 1B1 binding to GBSPIII-HSA by SPA. Further increases in inhibition and binding with fragments larger than 7 RU were initially attributed to epitope multivalency, but SPR data obtained using 1B1 Fab clearly demonstrated that optimization of epitope was still a factor even at this length of saccharide.
Because of IgG binding, both the SPR and inhibition studies could not
distinguish between the contribution of epitope multivalency and
epitope optimization to the increasing affinity with increasing length
of GBSPIII. However, valency effects were eliminated by binding 1B1 Fab
to GBSPIII-HSA, and the results up to 7 RU thus obtained were again in
excellent agreement with the inhibition data. The SPR data showed that
oligosaccharides in the 2 RU to 7 RU range were monovalent with respect
to epitope presentation and bound to Fab with a
KD of 4 µM. The
IC50 values for these monovalent Ags, which
should be equivalent to the KD of
their interactions with Ab, were 4 µM, based on an epitope size of 3
RU (Table III). However, the SPR data also demonstrated that affinity
still increased 3-fold from 7 RU to 20 RU, with little increase in
affinity with increasing length beyond this point. Thus, optimization
of the conformational epitope occurs between 7 RU and 20 RU and is a
factor, together with multivalency of epitope, in the increasing
affinity of 1B1 IgG with increasing GBSPIII length. Interestingly,
saturation-binding SPR experiments using 1B1 Fab indicate that
multivalency of epitope also occurs in the same region (7 RU to 20
RU).
The characteristics of the GBSPIII conformational epitope as defined in
the above experiments are similar to those associated with the immune
response to the capsular polysaccharides of group B Neisseria
meningitidis (15) and type 14 Streptococcus
pneumoniae (10, 16). Because they all exhibit
structural mimicry with self Ags, which usually consist of short
oligosaccharides, the immune system selects length dependent epitopes
unique to the polysaccharides for generating high affinity Abs. We have
hypothesized (7) that these epitopes are located on
extended helical domains of the polysaccharides rather than on its
random coil form. Because of lack of symmetry, the random coil form is
structurally related to short oligosaccharide self Ags. Although we
have no direct evidence for the existence of extended helical epitopes
on GBSPIII or Pn14PS, nuclear magnetic resonance and
molecular dynamics studies indicate that both the above polysaccharides
(17) and the group B meningococcal polysaccharide
(18) are capable of forming extended helices.
Additionally, there is a precedent for this type of epitope because
there is strong evidence, based on x-ray crystallographic studies
(18), that the extended epitope of the group B
meningococcal polysaccharide is helical. Interestingly, both the length
dependency of the GBSPIII epitope and its distribution are similar to
those found for Pn14PS and the group B meningococcal polysaccharide.
Studies on the immune response to Pn14PS conjugates in rabbits are
consistent with the length-dependent epitope forming at 4 RU and
persisting as a single epitope up to 20 RU (10).
Similarly, the 
2,8-polysialic acid helical epitope of group B
N. meningitidis requires 10 sialic acid residues to form,
and the helix is not duplicated until there are 40 linear sialic
acid residues present (18).
Wessels et al. (6) proposed a model of Ab binding to
GBSPIII in which the binding of the first Ab induces a second epitope
in the same chain, which by means of a process of epitope propagation
results in the accumulation of Abs on previously bound chains rather
than on naive chains. The SPR data reported here do not support this
model of Ab binding to GBSPIII. Such a model would not give good
fitting of Fab association data to a 1:1 interaction since the epitope
density would remain constant throughout the association at
nonsaturating concentrations of Fab. Our model of binding requires that
helical epitopes are intermittently located between random coil
segments of GBSPIII. This pattern of epitope distribution is not
conducive to an induction process that would require the formation of
contiguous helices. In our model, epitopes form spontaneously, and the
number of epitopes per polysaccharide chain would be more restricted.
This unusual Ag characteristic may be reflected in the association rate
constants for 1B1 Fab binding to Ag, which are, to our knowledge, the
lowest reported for Ag-Ab interactions. Values in the range of
104-105
M-1s-1
are typical for these interactions whereas those reported here for 1B1
Fab are 2 x 103
M-1s-1
for the longer oligosaccharides and slightly lower for the shorter
chain Ags. The reason for these unusually slow rates may lie in the
dynamic nature of the Ag. The extended helical conformation represents
a minor proportion of the total polysaccharide, with helices forming
intermittently and reverting to random coil. Despite the slow
association rate constant for the interaction, a slow dissociation
constant contributes to an overall affinity that is unusually high for
an anti-carbohydrate Ab. With a KD
of 0.65 µM, 1B1 Fab is at the upper end of the affinity range for
protein-carbohydrate interactions. For example, the affinities of Abs
that are specific for the Salmonella serogroup B
O-polysaccharide and the human blood group A determinant
have been determined, by SPR, to be 8 µM (19) and 290
µM (20), respectively.
In our model of binding, we would predict one helical epitope per Fab bound, which would be consistent with the SPR data and the x-ray crystallographic data obtained on the Fab binding of the conformationally related group B meningococcal polysaccharide (18). In these circumstances, bivalent Ab binding to one saccharide chain could not readily occur, especially in the shorter chains (3 RU to 6–7 RU) where only one binding site resides. Any contribution from steric hindrance in addition to the conformational length requirement in restricting binding to a single Fab at lengths as long as 7 RU (>200 Å) is unlikely, but cannot be ruled out on our evidence. The SPR data for 1B1 IgG binding to the panel of glycoconjugates showed that bivalent binding is both intersaccharide and intrasaccharide in nature. Avidity effects were observed even with conjugates presenting short chain oligosaccharides with single epitopes. This avidity could arise only through intersaccharide binding. With conjugates presenting longer chain oligosaccharides, the avidity effects were much more pronounced, indicating an additional contribution from intrasaccharide bivalent binding.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Harold J. Jennings, Institute for Biological Sciences, National Research Council of Canada, Ottawa, Canada K1A 0R6. E-mail address:
3 Abbreviations used in this paper: GBSPIII, type III group B Streptococcus capsular polysaccharide; HSA, human serum albumin; SPR, surface plasmon resonance; RU, complete repeating units in GBSPIII fragments derived by combined enzymatic and degradative procedures; RUS, synthetically derived GBSPIII repeating unit.
Received for publication January 19, 1999. Accepted for publication May 6, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(2–8)-linked sialic acid polysaccharide. Biochemistry 31:4996.[Medline]
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