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

Abstract

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 METHODSMonoclonal Antibodies-Murine ascites fluids of A-20-6 and S-20-4 both contain vibriocidal IgG 1 specific for Ogawa-LPS. I-24-2, in contrast, contains IgG 3 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 ϫ 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 IgG 1 and IgG 3 , respectively (heavy chainspecific), 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 DEAESephadex 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 freezedried a...

Crystal structure of an anti-carbohydrate antibody directed againstVibrio choleraeO1 in complex with antigen: Molecular basis for serotype specificity

“…The Ogawa serotype-specific epitope is probably associated with the upstream terminal perosamine residue of the O-SP, because Inaba strains are rfbT mutants of wild-type Ogawa strains in which methylation of the terminal perosamine is inactivated (2,3,7). Also, binding studies of anti-Ogawa Abs IgG1 S-20-6 and IgG1 S-20-4 with synthetic methyl ␣-glycosides of fragments, up to the hexasaccharide, of the Ogawa O-SP, as well as with analogs of the terminal monosaccharide, revealed that the terminal residue accounts for approximately 90% of the maximal binding energy (8). This hypothesis raises the question of how such a small antigenic determinant-a single methyl group at the 2-OH position of the terminal perosamine-can dictate a highly specific immune response.…”

Characterization of a novel protective monoclonal antibody that recognizes an epitope common to Vibrio cholerae Ogawa and Inaba serotypes

“…(Wang et al, 1998). The heavy chain sequences of mAb 72.1 and mAb ZAC-3 are very similar but that of mAb I-24-2 is very different.…”

“…However, the variable heavy chain of mAb I-24-2 differs extensively from those of 72.1 and ZAC-3. Previous studies have suggested that, while mAb ZAC-3 recognizes an epitope at the lipid A or core region, mAb I-24-2 recognizes an epitope at the junction of the core and O-SP region (Lullau et al, 1996;Villeneuve et al, 1999;Wang et al, 1998 In an attempt to determine if the protective epitope that is common to both Ogawa and Inaba serotype LPS can be utilized in a subunit vaccine against cholera, peptide mimics of this epitope were identified by screening phage display libraries with mAb 72.1. Eleven cyclic peptide mimics constrained by two cysteines were identified from three different phage display libraries.…”

“…For V H amplification, primer for conserved framework one region, prMND85-72.1.V H .FR CTT CCG GAA TTC SAR GTN MAG CTG SAG SAG TCW GG, and IgG3 heavy chain primer, prMND84-72.1.V H .Ig3 GGA AGA TCT AGG GAC CAA GGG ATA GAC AGA TGG (mixed base code: R5A, G; Y5C, T; M5A, C; K5G, T; S5C, G; W5A, T; V5A, C, G; N5A, C, G, T) (Wang et al, 2000), were used. For V L amplification, primer specific for conserved framework one region, prMND82-72.1.V L .FR CCA GAT GTG AGC TCG TGA TGA CCC AGA CTC CA, and the k chain-specific primer, prMND83-72.1.V L .k GCC CGT CTA GAA TTA ACA CTC ATT CCT GTT GAA (Wang et al, 1998), were used. The reaction mixture contained 20 ml of the reaction mixture from the cDNA synthesis, 50 pmol forward and reverse primers, 8 ml 106 PCR buffer, 1 ml 25 mM MgCl 2 , 0.5 ml AmpliTaq DNA polymerase (5 units ml 21 ) and deionized water to bring the volume to 100 ml.…”

“…The protective efficacy of ZAC-3 has not been reported. Inhibition studies revealed that the binding of I-24-2 to Ogawa and Inaba LPS may be mediated by a region located at the junction of the O-SP and core region of the LPS (Villeneuve et al, 1999;Wang et al, 1998). The ZAC-3 mAb binds to an epitope in the lipid A or core region but not to the O-SP (Lullau et al, 1996).…”