The oligosaccharide of gonococcal lipooligosaccharide contains several epitopes that are recognized by human antibodies

Abstract

Recently, we isolated human IgG from normal human sera (NHS) using lipooligosaccharide (LOS) from gonococcal strain JW31R as an affinity ligand. We provided evidence that the oligosaccharide (OS) moiety of LOS was immunogenic in humans and that NHS contains functional antibodies that bind to the branched OS. The present study aimed to identify bactericidal antibodies that bind to partial core OS structures or their adjacent sites expressed in the 3,4-branched and 2,3:3,4-dibranched neisserial LOSs. Using 15253 LOS from serum-resistant gonococcal strain 15253 as an affinity ligand, we isolated IgG2 and found that this preparation contained at least three different species. (i) One IgG2 species recognized a cross-reactive epitope that is expressed on 3,4-branched and 2,3:3,4-dibranched neisserial LOSs. (ii) Another IgG2 species was specific for JW31R LOS from a pyocin-resistant gonococcal strain; this IgG-defined epitope was not shared with the aforementioned branched LOSs. (iii) The third IgG2 species bound to the "Salmonella minnesota" Rb and Re mutant lipopolysaccharides (LPSs); this IgG2 recognizes a KDOalpha2-4KDO residue at the reducing end of the carbohydrate moiety of each LPS. The IgG2 was also found to be functional and facilitated the killing of strain 15253. The current results show that neisserial LOS contains several epitopes within its OS moiety that are recognized by human antibodies.

FIG. 1.

Structure of the conserved OS (A) and two major branched OS structures (B and C) biosynthesized by N. meningitidis and N. gonorrhoeae. To distinguish the two Hep residues of the core OS, the Hep linked to KDO and the other Hep were defined as Hep[I] and Hep[II], respectively, as described previously (42).

FIG. 2.

Isolation of anti-15253 LOS IgG. (A) Purification flowchart. The IgG was purified as described previously (43). (B) OS structure of 15253 LOS. (C) Analysis of affinity purification by PAGE (silver staining). Lanes: IgG, purified human IgG (control); 1, bound to the LOS column; 2, unbound to the protein G column; 3, bound to the protein G column; 4, purified IgG after the inactivated matrix column. (D) Subclass analysis. The purified IgG was analyzed with a MAb IgG/IgA subclass typing kit. (E) ELISA analysis. Each well was coated with 15253 LOS, JW31R LOS, or S. minnesota Re 595 LPS (200 ng each). Anti-human IgG (AP conjugate) and p-nitrophosphate were used, and the absorbances (405 nm) at 70 min are shown.

FIG. 3.

PAGE/Western blot analysis of 2,3:3,4-dibranched LOSs. (A) Silver staining and immunostaining. (B) The major OS structure of JW31R LOS. (C) The OS structures of the major and minor WG LOS components. PK lysates (2 to 4 μl each, ∼100 ng of LOS) were loaded onto two linear gradient gels (Protean II cell, 14 to 18%), one for silver staining and the other for immunostaining (nitrocellulose membrane). Anti-human IgG (AP conjugate, 1:3,000 dilution) and Western Blue-stabilized substrate were used for immunostaining.

FIG. 4.

PAGE/Western blot analysis of 3,4-branched LOSs. Each PK lysate was analyzed as described in the legend to Fig. 3. (A) General structure of a 3,4-branched LOS. (B) Silver staining and immunostaining. (C) Carbohydrate residues linked to Hep[I]. 220^a: the silver-stained 220 LOS components have not yet been fully characterized. The molecular masses of the 3,4-linked LOSs were estimated by using the values used for the six PID-2 LOS components as described previously (28). The 4.8-kDa F62 LOS component is designated 4.8 Kda-F62 in panels B and C.

FIG. 5.

PAGE (16%)/Western blot analysis of truncated mutant LPSs. LPSs were analyzed as described in the legend to Fig. 3. (A) Silver staining and immunostaining. (B) The OS structure of each truncated LPS mutant. Except for E. coli LPS (800 ng), 100 ng of each truncated LPS was loaded. Ra, Salmonella Ra mutant LPS; Rb, Salmonella Rb mutant LPS (R345); Rc, Salmonella Rc mutant LPS; Rd, Salmonella Rd mutant LPS; Re, Salmonella Re 595 mutant LPS.

FIG. 6.

TLC immunostaining of the partial hydrolysate of 15253 LOS with IgG2. 15253 LOS and its partial hydrolysate (30 μg each) were developed on three TLC plates in a solvent system consisting of chloroform-methanol-water-ammonium hydroxide (50:25:4:2, vol/vol/vol/vol). (A) The plate was sprayed with methanolic H₂SO₄ and heated at 120°C for 5 min. (B) After coating with polymethyl methacrylate, the plate was blocked with 1% casein in PBS and then treated with an anti-lipid A Ab (1:3,000 dilution) and anti-mouse IgG (AP conjugate). (C) After coating with the plasticizer and blocking in the same manner as in panel B, the plate was treated as described in the legend to Fig. 3. Western Blue-stabilized substrate was used for immunostaining (B and C).

FIG. 7.

PAGE (14%, Protean III cell)/Western blot analysis of WG LOS treated with glycosidases and lgtE LOS. (A) Analysis of enzyme-treated WG LOS samples (100 ng each). Lane 1, 15253 LOS. Lane 2, WG LOS. Lane 3, treatment of WG LOS with β-galactosidase. The terminal Gal was cleaved off the major component. Lane 4, treatment of sample 3 with β-N-acetylhexosaminidase. The GlcNAc and GalNAc residues were removed, respectively, from the above Gal-depleted LOS and the minor WG LOS components. (B) Analysis of the lgtE mutant LOS (100 ng each). Lane 1, 15253 LOS; lane 2, lgtE LOS. After PAGE and subsequent electroblotting, each PVDF membrane was treated with purified IgG and then with anti-human IgG (1:25,000, peroxidase conjugate). SuperSignal West Pico was used as the substrate for detection.

FIG. 8.

IgG binding to neisserial LOS before and after absorption with MS11mkA LOS. Purified IgG was diluted to 1:20, and 10 ng of each antigen was used. (A) Before absorption. (B) After absorption of IgG with ∼19 molar equivalents of MS11mkA LOS. (C) After absorption with ∼75 molar equivalents of the same LOS. Lanes: 1, PID-2 LOS; 2, MS11mkA LOS; 3, JW31R LOS; 4, 15253 LOS. Separation by PAGE (14%, Protean III cell) and analysis by Western blotting were performed as described in the legend to Fig. 7.

FIG. 9.

Analysis of IgG binding before and after absorption with 15253 LOS or Re 595 mutant LPS. Separation by PAGE and analysis by Western blotting were performed as described in the legend to Fig. 7. IgG was preabsorbed with ∼188 molar equivalents of each antigen. (A) Silver staining. (B) Before absorption. (C) After absorption with 15253 LOS. (D) After absorption with the Re 595 mutant LPS. Lanes: 1, 15253 LOS; 2, JW31R LOS; 3, MS11mkA LOS; 4, PID-II LOS; 5, Salmonella Rb mutant LPS; 6, Re 595 mutant LPS.

FIG. 10.

Bactericidal activity of 15253 IgG. (A) JW31R strain (10⁻⁷ CFU/ml). (B) 15253 strain (10⁻⁴ CFU/ml). Lanes: 1, 15253 IgG and the complement source; 2, inactivated NHS and the complement source; 3, inactivated complement source; 4, GBSS and the complement source; 5, NHS; 6, inactivated NHS. Killing activity is expressed as follows: (1 − [CFU in the test sample with intact complement/CFU in the buffer control]) × 100 CFU. The average of duplicate plates was used in the analysis.