On the Antigenic Determinants of the Lipopolysaccharides of Vibrio cholerae O:1, Serotypes Ogawa and Inaba*

Jin WangDagger , Sylvain Villeneuve§, Jian ZhangDagger , Ping-sheng LeiDagger , Charles E. MillerDagger , Pierre Lafaye, Farida Nato, Shousun C. Szupar , Arthur Karpaspar , Slavomír Bystrickypar , John B. Robbinspar , Pavol KovácDagger , Jean-Michel Fournier§, and Cornelis P. J. GlaudemansDagger **

From the Dagger  Laboratory of Medicinal Chemistry, NIDDK and the par  Laboratory of Developmental and Molecular Immunity, NICHD, National Institutes of Health, Bethesda, Maryland 20892 and the § Unité du Choléra et des Vibrions, Centre National de Référence des Vibrions et du Choléra and  Hybridolab, Institut Pasteur, 75724 Paris Cedex 15, France

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Monoclonal, murine IgG1s S-20-4, A-20-6, and IgA 2D6, directed against Vibrio cholerae O:1 Ogawa-lipopolysaccharide exhibited the same fine specificities and similar affinities for the synthetic methyl alpha -glycosides of the (oligo)saccharide fragments mimicking the Ogawa O-polysaccharide (O-PS). They did not react with the corresponding synthetic fragments of Inaba O-PS. IgG1s S-20-4 and A-20-6 have absolute affinity constants for synthetic Ogawa mono- to hexasaccharides of from ~105 to ~106 M-1. For IgG1s S-20-4, A-20-6, and IgA 2D6, the nonreducing terminal residue of Ogawa O-PS is the dominant determinant, accounting for ~90% of the maximal binding energy shown by these antibodies. Binding studies of derivatives of the Ogawa monosaccharide and IgGs S-20-4 and A-20-6 revealed that the C-2 O-methyl group fits into a somewhat flexible antibody cavity and that hydrogen bonds involving the oxygen and, respectively, the OH at the 2- and 3-position of the sugar moiety as well as the 2'-position in the amide side chain are required.

Monoclonal IgA ZAC-3 and IgG3 I-24-2 are specific for V. cholerae O:1 serotypes Ogawa/Inaba-LPS.1 The former did not show binding with members of either series of the synthetic ligands related to the O-antigens of the Ogawa or Inaba serotypes, in agreement with its reported specificity for the lipid/core region (1). Inhibition studies revealed that the binding of purified IgG3 I-24-2 to Ogawa-LPS might be mediated by a region in the junction of the OPS to the lipid-core region of the LPS.

cDNA cloning and analysis of the anti-Ogawa antibodies S-20-4, A-20-6, and 2D6 revealed a very high degree of homology among the heavy chains. Among the light chains, no such homology between S-20-4 and A-20-6 on the one hand, and 2D6 on the other hand, exists. For the anti-Inaba/Ogawa antibodies I-24-2 and ZAC-3, their heavy chains are completely different, with some homology among the light chains.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Vibrio cholerae O:1 is subdivided into two serological types, Inaba and Ogawa. The internal part of their O-polysaccharide chains (O-antigen) are homopolymers of (1right-arrow2)-linked linear 4,6-dideoxy-4-(3-deoxy-L-glycero-tetronamido)-alpha -D-mannopyranosyl residues. The two types apparently differ by the presence of a 2-O-methyl group in the nonreducing terminal sugar of the Ogawa O-antigen, absent in the Inaba O-antigen (2, 3).

We used synthetic mono- to hexasaccharides that mimic the fragments of the O-antigen of Ogawa and Inaba O-polysaccharides (2-4), together with certain analogs of their monosaccharides to evaluate specificity. The binding of three immunoglobulins G (two specific for Ogawa and one specific for Ogawa/Inaba) and of two immunoglobulins A (one specific for Ogawa and one specific for Inaba/Ogawa) were characterized by ligand-induced fluorescence titration or ELISA inhibition. The cDNA sequences of these antibodies are also presented in this report.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Monoclonal Antibodies-- Murine ascites fluids of A-20-6 and S-20-4 both contain vibriocidal IgG1 specific for Ogawa-LPS. I-24-2, in contrast, contains IgG3 specific for both serotypes Ogawa and Inaba-LPS, and it has low vibriocidal activity (5) (clone S-20-4 comes from the same hybridoma cells as clone S-20-3 described in this reference). Murine ascites fluid 2D6 and ZAC-3 contain IgA specific for Ogawa-LPS and IgA specific for Inaba/Ogawa-LPS, respectively. The latter two hybridomas were gifts from Drs. Marian Neutra, Harvard Medical School, and Dr. Richard Weltzin, Oravax, Cambridge, MA (1, 6) and were grown in BALB/c mice. IgGs were purified using ImmunoPure® (G) IgG purification kits (Pierce). Briefly, ascites fluid (2 ml, clarified by centrifugation) was mixed with ImmunoPure® (G) binding buffer (2 ml) and applied to a protein G column. After washing the column with 5 × 2-ml aliquots of the ImmunoPure® (G) binding buffer, the bound IgG was eluted with 6 ml of ImmunoPure® (G) elution buffer, dialyzed against PBS, pH 7.4 (2000 ml) for three changes at 0 °C, frozen, and labeled. The purified A-20-6, S-20-4, and I-24-2 showed a single arc of precipitation versus goat anti-mouse IgG1 and IgG3, respectively (heavy chain-specific), and goat anti-whole mouse serum (Sigma) by immunoelectrophoresis. IgAs were purified from ascites fluid by 40% ammonium sulfate precipitation and anion-exchange DEAE-Sephadex A-25 chromatography (7). Monomeric IgA was obtained by mild reduction with 5 mM 1,4-dithiothreitol (Sigma) and alkylation with 11 mM 2-iodoacetamide (Sigma), followed by re-adsorption of the sample on DEAE-Sephadex A-25 and elution with PBS, pH 7.4. The purity of IgAs was also verified by immuno-electrophoresis against anti-mouse IgA and serum and SDS-polyacrylamide gel electrophoresis.

LPS and Synthetic Oligosaccharides-- V. cholerae O:1 LPSs were obtained from acetone-treated cells of strain 569B, classical biotype, serotype Inaba, lot VC1219; strain 3083, classical biotype, serotype Ogawa; and V. cholerae O:139 Bengal, strain 4450. Salmonella paratyphi A LPS was a field isolate in Nepal, strain NTP-6. All LPSs were purified as described (8) and at 2 mg/ml showed negative tests (Coomassie Blue) for protein. Severely base-degraded V. cholerae O:139 LPS (9) was a gift from Dr. Andrew D. Cox, National Research Council, Ottawa, Canada. De-O-acylated Ogawa-LPS (10) was oxidized in aqueous 0.8% periodate solution for 3 days in the dark, dialyzed, and freeze-dried and then reduced in aquous sodium borohydride (11) to convert aldehyde groups to alcohol groups to give O/R-DeOAc-Ogawa-LPS. Assays, as glucose (12), for carbonyl groups in both the oxidized and oxidized and reduced samples showed the reduction to be 99.1% complete. The synthesis of methyl alpha -isomaltoside has been described (13). D-Glucose, methyl alpha -D-glucopyranoside, methyl beta -D-glucopyranoside, D-galactose, methyl alpha -D-galactopyranoside, D-mannose, methyl alpha -D-mannopyranoside, D-mannoheptose, D-glucosamine hydrochloride, 2-keto-3-deoxy-D-manno-octonate, D-fructose, and dextran 10T were purchased from Sigma. Methyl beta -D-fructofuranoside was obtained from the National Institutes of Health Carbohydrate Collection. Synthetic methyl alpha -glycosides of the mono- to hexasaccharides of the O-antigen of V. cholerae O:1, serotypes Ogawa (1O to 6O) (Fig. 1) and Inaba (1I to 6I), as well as the preparation of methyl 4-acetamido-4,6-dideoxy-2-O-methyl-alpha -D-mannopyranoside (1O-Ac), methyl 4,6-dideoxy-4-(3-deoxy-L-glycero-tetronamido)-2-O-ethyl-alpha -D-mannopyranoside (1O-Et), methyl 4,6-dideoxy-4-(3-deoxy-L-glycero-tetronamido)-2-O-n-propyl-alpha -D-mannopyranoside (1O-Pr) methyl 2,4,6-trideoxy-4-(3-deoxy-L-glycero-tetronamido)-alpha -D-mannopyranoside (1-2d), methyl 3,4,6-trideoxy-4-(3-deoxy-L-glycero-tetronamido)-alpha -D-mannopyranoside (1O-3d), methyl 4,6-dideoxy-4-(3-deoxy-D-glycero-tetronamido)-2-O-methyl-alpha -D-mannopyranoside(1O-D), methyl 4,6-dideoxy-4-(4-hydroxybutyramido)-2-O-methyl-alpha -D-mannopyranoside (1O-2'd), methyl 4,6-dideoxy-4-(3,4-dideoxy-L-glycero-tetronamido)-2-O-methyl-alpha -D-mannopyranoside (1O-4'd), and methyl 4,6-dideoxy-4-(2-deoxy-L-glycero-tetronamido)-2-O-methyl-alpha -D-mannopyranoside (1O-2'd,3'OH) have been described elsewhere (14-20). 3-Deoxy-N-isopropylidene-L-glycero-tetronic acid hydrazide (TAH) was prepared as follows: 3-deoxy-L-glycero-tetronic acid hydrazide and 3-deoxy-N-isopropylidene-L-glycero-tetronic acid hydrazide (TAH): a mixture of 3-deoxy-L-glycero-tetronolactone (16) (1 g) and hydrazine hydrate (17 ml) was stirred at room temperature overnight, when TLC (5:1 CH2Cl2-MeOH) showed that all starting lactone was consumed. The solution was concentrated to give a solid residue (1.4 g, ~100%). Crystallization from MeOH-EtOAc gave 3-deoxy-L-glycero-tetronic acid hydrazide, m.p. 108-108.5 °C, [alpha ]D -18.8° (c 0.8, H2O). 1H NMR (D2O): delta  4.28 (dd, 1 H, J2,3a 4.0, J2,3b 8.8 Hz, H-2), 3.70 (deceptively simple dd, 2 H, J 5.7 and 7.3 Hz, 2J not observed, H-4a, b), 2.06-1.95, 1.87-1.76 (2 m, 2 H, H-3a, b); 13C NMR (CDCl3): delta  174.97 (CO), 68.34 (C-2), 57.81 (C-4), 35.92 (C-3); chemical ionization mass spectroscopy: m/z 135 ([M + 1]+), 152 ([M + 18]+).
<UP>C<SUB>4</SUB>H<SUB>10</SUB>N<SUB>2</SUB>O<SUB>3</SUB></UP>
<AR><R><C><UP>Calculated:</UP></C><C><UP>C 35.82%</UP></C><C><UP>H 7.51%</UP></C><C><UP>N 20.89%</UP></C></R><R><C><UP>Found:</UP></C><C><UP>C 35.86%</UP></C><C><UP>H 7.46%</UP></C><C><UP>N 20.81%</UP></C></R></AR>


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Fig. 1.   Shown are the ligands used in binding studies.

A solution of 3-deoxy-L-glycero-tetronic acid hydrazide (1 g) was treated with 1:2 MeOH-acetone (6 ml) at 50 °C for 15 min. Crystaline, pure (TLC, NMR) N-isopropylidene derivative TAH, was obtained upon concentration to a small volume, m.p. 116~117° and [alpha ]D -18.5° (c 1, H2O). 1H NMR (CD3OD): delta  4.26 (dd, 1 H, J2,3a 3.9, J2,3b 8.6 Hz, H-2), 3.69 (deceptively simple t, 2 H, J 6.6 Hz, H-4a, b), 2.12, 1.92 (2 s, overlapped, 2 CH3), 2.12-1.90 (m, overlapped, H-3a), 1.87-1.70 (m, 1 H, H-3b); chemical ionization mass spectroscopy: m/z 175 ([M + 1]+), 192 ([M + 18]+).
<UP>C<SUB>7</SUB>H<SUB>14</SUB>N<SUB>2</SUB>O<SUB>3</SUB></UP>
<AR><R><C><UP>Calculated:</UP></C><C><UP>C 48.26%</UP></C><C><UP>H 8.10%</UP></C><C><UP>N 16.09%</UP></C></R><R><C><UP>Found:</UP></C><C><UP>C 48.37%</UP></C><C><UP>H 8.18%</UP></C><C><UP>N 16.12%</UP></C></R></AR>

All saccharides were dried at 40 °C in vacuum (1 torr) overnight and then dissolved in distilled water.

cDNA Synthesis, Amplification Cloning, and Sequencing-- Hybridomas for the monoclonal antibodies 2D6 and ZAC3, provided by Oravax, were used to prepare cDNA: poly(A)+ RNA from approximately 1.5 × 107 cells was purified by the guanadine isothiocyanate method (Quikprep Micro, Pharmacia Biotech Inc.). Synthesis of the cDNA strand was accomplished by extension of approximately 1 µg of poly(A)+ mRNA with primers specific for Ig Calpha (5'-TAC ATT GGA TCC TTA AGG AGG AGG AGG AGT AGG-3') and Ig Ckappa (5'-AGG CTT AAG CTT TTA ACA CTC ATT CCT GTT GAA-3'). Reaction conditions for the synthesis of the first strand were as recommended (Superscript first strand synthesis, Life Technologies, Inc.). Following synthesis of the cDNA by reverse transcriptase, polymerase chain reaction (PCR) was achieved with primers for the constant region used in the extension reaction in combination with one of a series of hybridizing V region primers. Positive PCR products were obtained with the VH primer (5'-GCA AGC TTG TCG ACG TGC AGC T(GT)(CA) AG(CG) AGT C(AG)GG-3' for ZAC3, 5'-GCA AGC TTG TCG ACG TGA AGC T(TG)(GC) (AT)(CG)GA (AG)TC TGG-3' for 2D6) and the VL primer (5'-AGG CTT GAA TTC ATG TTG TGA TGA CCC A-3' for both 2D6 and ZAC3). Thirty cycles of amplification were applied using 20 pmol of each flanking primer in a reaction mixture containing the Elongase enzyme mix (Life Technologies, Inc.) at a concentration of 1.5 mM KCl. Thirty cycles of denaturation (94 °C), annealing (55 °C), and extension (72 °C) were done in a DNA thermal cycler (Ericomp).

Positive PCR products were purified on 1.0% low melting agarose gels using AgarACE enzyme (Promega) to extract the DNA and ligated to pCRII vectors as described by the manufacturer (Invitrogen). White colonies were selected and screened by restriction analysis for cloned inserts. The isolated VL and VH clones were grown in YT medium to produce phage, and the single-stranded DNA was purified (21). The DNA was then subjected to single-stranded DNA sequencing according to the protocol provided by the manufacturer (Sequenase, U. S. Biochemical Corp.).

For the hybridoma cells producing A-20-6, S-20-4, and I-24-2 antibodies, the total RNA was extracted by a modification of a known method (22). The reaction mixture (100 µl) containing maximally 10 µg of total RNA, 20 mM mercaptoethanol, 80 units of RNasin (RNase inhibitor, Promega) 1 × first strand buffer (Life Technologies, Inc., Basel, Switzerland), 8 mM dithiothreitol, 0.1 µg/ml primer p(dT)15 (Boehringer Mannheim GmbH, Mannheim, Germany), 500 µM each dNTP, and 400 units of Moloney murine leukemia virus reverse transcriptase (Promega, using their protocols) was incubated for 2 h at 42 °C. The reaction mixture was then used directly for PCR amplification. For that, each cDNA mixture so obtained was divided into, respectively, eight (for the VH) and ten (for the VL) separate mixtures, each one of which was then treated with the appropriate, separate, back primers. A 50-µl reaction mixture containing 2 µl of the reaction mixture from the cDNA synthesis, 30 pmol of one of the back primers, 30 pmol of forward primer, 500 µM dNTP, 1 mmol of MgSO4, 0.05 × Thermopol buffer, and 1 unit of Vent DNA polymerase (New England Biolabs) or Taq DNA polymerase (U. S. Biochemical Corp.). The reaction mixture was subjected to 40 cycles of amplification, each one 1-min denaturation, 1-min annealing (54 °C), and 2-min extension (72 °C). Products (5 µl) were analyzed on a 2% agarose gel.

The back primer that yielded products used in the amplification of the VH of clones A-20-6 and S-20-4 was VH3-D (5'-GAA GTT AAG CTC GAG CAG TCT GGA GC-3'), and the gamma -chain primer (CH) used as forward primer was gamma 1 (5'-AGG CTT ACT AGT ACA ATC CCT GGG CAC AAT-3') for the clones A-20-6 and S-20-4. For the VH of I-24-2 the Pharmacia heavy chain primer kit was used to amplify the VH. The primers used for the amplification of each VL of three clones were the following: for I-24-2 the back primer VL5 (5'-CCA GAT GTG AGC TCG TGA TGA CCC AGA CTC CA-3') was used, while VL10 (5'-GGG AAT TCA TGG CCT GGA (C,T)T(C,T) C(A,T)CT(T,C)(A,T) T(A,C)(C,T) TCT-3') was used as the back primer for A-20-6 and S-20-4. The lambda -chain primer (CL) (5'-CCC AAG CTT AGC TC(C,T) TC(A,T) G(A,T)G GA(G,C) GG(C,T) GG(A,G) AA-3') was used as forward primer for the clones A-20-6 and S-20-4. The kappa -chain primer used for the clone I-24-2 was 5'-GCG CCG TCT AGA ATT AAC ACT CAT TCC TGT TGA A-3' (23, 24).

The back primers were designed to hybridize to the partially conserved sequences in the leader or the FR1 regions of the VH or VL. The forward primers corresponded to the N-terminal beginning of the hinge region and the C-terminal part of the CL region, respectively.

Direct sequencing of these PCR products was performed on an Applied Biosystem Apparatus ABI 373A (Genome Express, Grenoble, France) using dye terminators. Sequence data were analyzed and comparisons performed, with software from the Genetics Computer Groups, Inc. (Madison, WI) and the GeneBankTM (Los Alamos, NM) and EBI (Heidelberg, Germany)

Fluorescence Titration-- Purified immunoglobulins were diluted with PBS, pH 7.4, to solutions having an A280 of 0.04. Protein solutions (1.1 ml) were added to each of two cuvettes, one for ligand addition, the other a reference. A third cuvette filled with 1.1 ml of PBS was used as a blank. Temperature was maintained at 25 °C by a circulating thermostatic bath. Ligand solutions were verified to show no fluorescence effects by themselves by addition to a suitable nonbinding protein. To obtain a good distribution of points, ligand concentrations were adjusted so that the protein would be saturated after the addition of ~20 µl of ligand solution to the test cell. Affinity constants (Ka) for the association between ligands and antibodies were obtained by monitoring the ligand-induced tryptophanyl fluorescence of the antibody as a function of ligand concentration (25, 26). We used a Perkin-Elmer LS 50 luminescence spectrophotometer and
K<SUB>a</SUB>=&ngr;/(1−&ngr;)<UP>C<SUB>L</SUB>,</UP> (Eq. 1)
where Ka is the affinity constant, nu  is the fraction of antibody sites bound to ligand (measured as the change in antibody fluorescence due to ligand addition, divided by the maximal ligand-induced fluorescence change), 1 - nu  is the fraction of free antibody sites, and CL is the concentration of free ligand. A Scatchard plot of nu /CL versus nu  gives a line whose slope is equal to the Ka. Two representative Scatchard plots are shown in Fig. 2: one for the Ka of IgG1 A-20-6 with the Ogawa monosaccharide determinant 1O and one for the Ka of IgG1 S-20-4 with the Ogawa pentasaccharide 5O. These are expressed either in M-1 or (Table I) as the free energy (kJ) of binding (Delta G0 = -RTlnKa).


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Fig. 2.   Scatchard plots of the Ka for synthetic mono- and pentasaccharides mimicking the Ogawa O-antigen fragment with immunoglobulins G1 A-20-6 and S-20-4, respectively.

ELISA for Inhibition of Antibody Binding-- The binding constants for the antibodies that did not show a significant change in fluorescence following addition of ligand were determined by ELISA inhibition. For anti-Ogawa IgA 2D6 (6), Ogawa-LPS was the capturing reagent. In the case of anti Inaba-Ogawa IgA ZAC-3 and IgG3 I-24-2, both the Ogawa- and Inaba-LPS were used as capturing reagents in screening with 1O to 4O/1I to 4I, and 1O to 6O/1I to 6I, respectively. In screening of IgG3 I-24-2 with core-related haptens (see Table II), Ogawa-LPS was the capturing reagent.

For IgA 2D6/Ogawa-LPS, the amount of ligand required for 50% inhibition in the ELISA was determined. Since Ka = nu /(1 - nu ) CL (see "Fluorescence Titration" section), at 50% inhibition, assuming solution behavior in ELISA, we have that nu  = (1 - nu ). Thus, the reciprocal of the molar concentration causing 50% inhibition was taken to be the (relative) Ka. Microtiter plates (96-well; Maxisorb, Nunc, Denmark) were coated with 100 µl of either Ogawa- or Inaba-LPS (10 µg/ml) in 10 mM MgCl2 PBS solution at room temperature overnight. Ascites fluids (1:4000 dilution), purified 2D6, ZAC-3 (1:200 dilution), and purified I-24-2 (1:800 dilution) were incubated in quadruplicate with inhibitors in various concentrations (1-2500 µg/ml PBS) or with PBS for 1 h at 37 °C, followed by 4 °C overnight. Samples were centrifuged and supernatants (100 µl) delivered to microtiter plates. ELISA was performed as described (27). A standard curve for A405 versus antibody concentration added without inhibitor was fitted to the quadratic equation aX2 + bX + c, where X is the optical density observed. Correlation factors (gamma ) of 0.999 were routinely obtained. Based on that standard curve, residual, bound antibody in the presence of inhibitor was determined (AI), and percent inhibition was calculated as follows: (1 - AI) × 100 = % inhibition.

    RESULTS AND DISCUSSION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Antigenic Epitopes on Ogawa-LPS-- IgG1s, A-20-6 and S-20-4, specific for Ogawa-LPS (vibriocidal titer: 1280), protect mice from mortal challenge with three times the LD50 of V. cholerae O:1 serotype Ogawa strain 920139 (5). Thus, a study of their combining sites is of interest, as oligosaccharide fragments of the O-antigen that bind immunoglobulins maximally will be more likely, when linked to a carrier protein, to elicit antibodies reactive with the parent polysaccharides (28). Each synthetic mono- to hexasaccharide fragment of the Ogawa O-antigen bound the two antibodies, with binding constants of from ~105 to ~106 M-1 as measured by fluorescence titration (Table I). The free energies of association for oligosaccharide with each of the two antibodies were close, indicating that these two vibriocidal antibodies have the same fine specificity for the epitope on the Ogawa O-PS. The terminal, nonreducing Ogawa-monosaccharide (1O) contributed ~90% of the maximal binding energy shown by the entire hexasaccharide. This finding differs from those in some other (homo)polysaccharide-antibody systems (29), where monosaccharide binding accounts for only 50-60% of the maximum binding energy that is shown by the binding of four to six sugars. Methyl 4,6-dideoxy-4-(3-deoxy-L-glycero-tetronamido)-2-O-ethyl-alpha -D-mannopyranoside (1O-Et) showed an affinity constant for both these antibodies that was an order of magnitude less than that of the Ogawa monosaccharide. Neither the corresponding monosaccharide with a free OH group at C-2 (1I), nor its 2-deoxy derivative (1-2d), 3-deoxy derivative (1O-3d), 2-O-propyl derivative (1O-Pr), or the one deoxygenated at the 2'-position in the tetronic acid moiety (1O-2'd), exhibited binding. Removal of the primary hydroxyl group in the tetronic acid group gave (1O-4'd), which bound to IgGs A-20-6 and S-20-4 with higher affinity than did 1O itself (Table I). A hydrophobic pocket of defined size in the antibody might interact with the 2-O-methyl group of the terminus of the Ogawa O-PS. Alternatively, the presence of an O-methyl group at C-2 could dictate a particular conformation due to a pattern of hydrogen bonding that is absent when C-2 bears an OH group. Similarly, increased hydrophobicity, or differently routed, multiple hydrogen bond interactions might also explain why 1O-4'd could have a higher affinity than does 1O. Neither N-isopropylidene-L-tetronic acid hydrazide (TAH) nor methyl 4-acetamido-4,6-dideoxy-2-O-methyl-alpha -D-mannopyranoside (1O-Ac) showed measurable binding. The monosaccharide corresponding to 1O, but bearing a D-tetronic group instead of the natural L-isomer (1O-D), bound to both IgGs A-20-6 and S-20-4, albeit with an affinity 2 orders of magnitude less (Ka = ~103 M-1) than did 1O. None of the synthesized O-antigen saccharidic fragments of the Inaba series, from mono- to hexasaccharide, i.e. those that lack the 2-O-methyl group in the upstream terminal monosaccharide, bound to either of the two IgGs specific for the Ogawa-LPS. Therefore, the 2-O-methylated, terminal monosaccharide seems to be the dominant serotype-specific determinant for Ogawa strains. Furthermore, the Ogawa-specific antibodies do not show any appreciable affinity for internal residues, shared by the O-polysaccharides of both Inaba and Ogawa strains.

                              
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Table I
The binding constants (Ka, M-1) and free energy of association (-Delta G0, kJ/mol) for vibriocidal immunoglobulins G specific for LPS of serotype Ogawa, V. cholerae O1 with synthetic Ogawa fragments and monosaccharide derivatives

Are the fine specificities of the IgA 2D6 (6) and IgGs A-20-6 and S-20-4 for the same epitope? Since IgA 2D6 did not show ligand induced fluorescence change, this was investigated by ELISA inhibition of binding of the IgA to Ogawa-LPS. In Fig. 3 it can be seen that LPS (molecular weight ~8000 daltons), as well as 1O to 4O inhibit the binding of IgA 2D6 to Ogawa-LPS, while 1I and1I-2d show (weak) nonspecific inhibition. The inhibition curves of the Ogawa tetra- and trisaccharide are identical. There was little difference in slope between the mono-, di-, tri-, and tetrasaccharide. For 50% inhibition of the binding of the antibody to Ogawa-LPS, mono-, di-, tri-, and tetrasaccharides were required in concentrations of 34, 12.5, 5, and 5 µM, respectively. Their relative affinities were therefore 3 × 104, 8 × 104, 2 × 105, and 2 × 105 M-1, respectively. So, this antibody also shows a (relative) free energy of binding for the monosaccharide 1O that is quite dominant (some 85% of the free energy of binding shown by the tetrasaccharide 4O). Thus, our results for the binding of two vibriocidal IgGs A-20-6 and S-20-4 as well as IgA 2D6 to a series of saccharides mimicking the Ogawa O-antigen have provided strong evidence that these anti-Ogawa-LPS IgGs and the IgA are specific for the same antigenic epitope. The terminal monosaccharide, bearing the 2-O-methyl group, in the O-antigen of Ogawa-LPS is most likely the serotype-specific determinant for the Ogawa strain. It is not unusual that small determinants can dictate an immune response in mice (30).


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Fig. 3.   Inhibition curves of the binding of Ogawa-LPS to sIgA 2D6 using Ogawa-LPS, synthetic mono- to tetrasaccharides mimicking the fragment of Ogawa O-antigen and Ogawa monosaccharide derivatives.

Antigenic Epitopes on Inaba-LPS-- Synthetic oligosaccharide fragments reflecting the structural difference between Ogawa and Inaba O-antigens were used to study the specificity of monoclonal IgA ZAC-3, obtained from a lymphocyte of the mouse's Peyer's patches following immunization with V. cholerae O:1 serotype Inaba (1), and monoclonal IgG3 I-24-2, obtained following immunization of mice with a lysate from V. cholerae O:1 serotype Inaba (5). Others showed (1) that the IgA ZAC-3 dimer and polymer bound all the fragments (including the 3-4-kDa fragment) of V. cholerae LPS (Inaba), indicating that the determinant epitope for this IgA is located in the lipid A or core region, and not in the O-specific side chain. These workers also showed by microcalorimetric measurements that the Ka for IgA ZAC-3 and detergent-solubilized LPS was ~6 × 105 M-1. Their proposed specificity of ZAC-3 for the lipid/core (1) would accommodate our results described below, namely that none of our Ogawa or Inaba (oligo)saccharides show binding to that IgA.

IgG3 I-24-2 is reported to possess weak agglutination titers against Ogawa and Inaba organisms and a weak vibriocidal titer against either organism (5). No ligand-induced fluorescence change was observed with antibody IgG3 I-24-2 and either 1O to 6O or 1I to 6I or with IgA ZAC-3 and either 1O to 4O or 1I to 4I. The absence of binding of these ligands to these two (ZAC-3 and I-24-2) purified antibodies was verified by ELISA inhibition systems (antibody/Ogawa- or Inaba-LPS). Indeed, these saccharides also failed to show interaction with either antibody in the ELISA system employing either LPS-Ogawa or LPS-Inaba as the capturing agent, except for Inaba monosaccharide 1I, which could moderately inhibit (50%) only the IgG3 I-24-2/Ogawa-LPS system (see Table II), but not the ZAC-3/Inaba-LPS or ZAC-3/Ogawa-LPS ELISA system. (It is puzzling to us why only the mono- and not the higher oligosaccharides of the Inaba series is able to inhibit the IgG3 I-24-2/Ogawa-LPS system.) The binding of either IgA ZAC-3 or I-24-2 to ELISA plates, using either the LPS-Ogawa or LPS-Inaba as the capturing agent, could be inhibited by either LPS.

                              
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Table II
ELISA inhibition study using various inhibitors to inhibit the binding of the immunoglobulin G3, I-24-2 specific for Ogawa and Inaba LPSs, to Ogawa-LPS

To elucidate the inhibitory effect by various other potential inhibitors in the I-24-2/Ogawa-LPS ELISA system we examined the ligands shown in Table II. Using whole ascites fluid, Inaba- and Ogawa-LPS were the most effective inhibitors (90% inhibition). V. cholerae O:139 LPS, D-glucose, Inaba monosaccharide, and D-galactose all showed moderate inhibition. The V. cholerae O:139 LPS has a very short O-PS that is structurally unrelated to the O-PS of V. cholerae O:1 (31-35) and has its core region in common with that of V. cholerae O:1 Ogawa and Inaba (9), save for the fact that the O:139 has a branch of isomaltose instead of just glucose attached to its core. The O:139 LPS did weakly inhibit the interaction of both ascites fluid and purified IgG3 I-24-2 with Ogawa-LPS as the capturing reagent. Severely base-degraded O:139 LPS (9) consists of the core region that is flanked at the upstream end by the base-stable remnant of its short O-PS, namely two carbohydrate residues: a threo-hex-4-enuronopyranosyl residue linked to 2-amino-2,6-dideoxy glucosyl residue, while at the downstream end of the core two phosphorylated glucosaminyl residues remain. That product did not react with ascites fluid, but did weakly inhibit the interaction of purified IgG3 I-24-2 with Ogawa-LPS as the capturing reagent. O/R-DeOAc-Ogawa-LPS is a substance that has only its core destroyed by the periodate oxidation (borohydride reduction was executed to render the aldehydo groups formed chemically unreactive), since the core has vicinal hydroxyl groups (33). Its O-PS is unaffected by the periodate oxidation, as we showed by measuring its Ka values with IgGs A-20-6 and S-20-4 (Ka values of 0.5 × 106 and 1.3 × 106, respectively), showing them to be nearly the same as the Ka values these antibodies have with the hexameric O-PS saccharide 6O. This O/R-DeOAc-Ogawa-LPS was able to inhibit IgG3 I-24-2 on ELISA (see Table II) only 70%, whereas the LPS itself could do so nearly completely (90%). Since it is unlikely that the lipid plays a role, this could indicate a partial specificity of IgG3 I-24-2 for the core, as that is the only part of the molecule that would be destroyed by periodate oxidation. Dextran 10T, D-glucose, methyl alpha -glucopyranoside, methyl beta -D-glucopyranoside, D-mannose, and methyl alpha -D-mannopyranoside (the latter two are structurally related to the heptose in the core region) showed moderate to significant (50 or 70%) inhibition. The inhibition by D-galactose and methyl alpha -galactopyranoside also remains unexplained. Although from the above it does appear to recognize determinants in the core region of the LPS, an immunoblotting experiment (5), shows IgG3 I-24-2 to bind only to the high molecular weight LPS from both Inaba and Ogawa V. cholerae O:1, and not to the low molecular weight fractions, presumably made up of lipid-core region only (34).

In summary, for the immunoglobulins with specificity for Ogawa/Inaba, the behavior of the IgG3 I-24-2 indicates partial specificity for the core as well as for a single residue of 4,6-dideoxy-4-(3-deoxy-L-glycero-tetronamido)-alpha -D-mannopyranoside. Such a residue is linked, as the first of many in the O-PS, to the core in the Inaba-LPS. Nevertheless the behavior of IgG3 I-24-2 remains somewhat puzzling. Our observations on IgA ZAC3 are entirely consistent with the specificity proposed by Lüllau et al. (1) for the LPS' lipid/core region. For the binding behavior of the three anti-Ogawa monoclonal antibodies, IgG1 S-20-4, IgG1 A-20-6, and IgA 2D6, our data consistently indicate the immunodeterminant to be the upstream, terminal residue of the Ogawa O-PS.

The above results were corroborated by the cDNA-derived amino acid sequences. It is clear from the VH sequences of the anti-Ogawa immunoglobulins (Fig. 4) that all three belong to the same family, as they show a high degree of homology. It is interesting that IgGs A-20-6 and S-20-4 have identical (lambda ) VL regions, whereas IgA 2D6, possessing a kappa  L-chain, has a VL sequence that is therefore quite different. Since they show identical specificity patterns, that may be dictated more by the H- than by the L-chain. It is noteworthy that the IgGs A-20-6 and S-20-4 show significant ligand-induced tryptophanyl fluorescence change, while IgA 2D6 does not. The former two (IgGs) both possess a tryptophanyl residue at position L-91, while the latter (IgA) carries a glycine at that position. Tryptophan at that position of the L-chain has been correlated before with ligand-induced changes in the tryptophanyl fluorescence (29).


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Fig. 4.   The cDNA-derived amino acid sequences of the anti- Ogawa immunoglobulins IgG1s(lambda ) A-20-6 and S-20-4, as well as IgA(kappa ) 2D6.

In comparing the cDNA sequences of IgA ZAC-3 and IgG3 I-24-2 (Fig. 5), we observe the reverse. Here, the VH sequences differ extensively, while the VL regions show some degree of homology. It should be noted, however, that there, most of the differences occur in the hypervariable regions of the VL.


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Fig. 5.   The cDNA-derived amino acid sequences of the anti-Inaba/Ogawa immunoglobulins IgA ZAC-3 and IgG3 I-24-2.

    ACKNOWLEDGEMENT

We are grateful to Dr. Andrew D. Cox for a generous gift of severely base degraded V. cholerae O:139 LPS.

    FOOTNOTES

* This work was supported in part Grant ACC-SV6 (to J.-M. F.) from the Ministère Français de la Recherche.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed. Tel.: 301-496-1266; Fax: 301-402-0589; E-mail: glau{at}helix.nih.gov.

1 The abbreviations used are: LPS, lipopolysaccharide(s); O-PS, O-polysaccharide; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; PCR, polymerase chain reaction.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References

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