The Oligosaccharide of Gonococcal Lipooligosaccharide Contains Several Epitopes That Are Recognized by Human Antibodies

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 KDOα2-4KDO 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.Lipooligosaccharide (LOS) is a major antigenic and immunogenic component of pathogenic Neisseria species such as Neisseria meningitidis and N. gonorrhoeae (34). LOS produced by these bacteria consists of an oligosaccharide (OS) moiety and lipid A, and structural variation of the OS leads to LOS heterogeneity (34). As shown in Fig. ​Fig.1A,1A, Neisseria bacteria biosynthesize a core pentasaccharide, N-acetylglucosamine (GlcNAc); two heptose (Hep) and two 2-keto-3-deoxymannooctulosonic acid (KDO) residues are sequentially α-glycosidically linked. GlcNAc is linked to the 2 position of Hep[II], which is linked to Hep[I] via its 3 position. The latter Hep[I] is linked to the 5 position of the terminal KDO of KDO(α2-4)KDO. The structural variation of neisserial OS stems from how glycoses are further biosynthesized from the inner core disaccharide, Hep[II](α1-3)Hep[I], of the pentasaccharide (Fig. ​(Fig.1A).1A). Sequential addition of glycoses to the 4 position of the 3-substituted Hep[I] produces a 3,4-branched OS structure (Fig. ​(Fig.1B),1B), and addition of both to the same Hep[I]-4 position and to the 3 position of the 2-substituted Hep[II] produces a 2,3:3,4-dibranched OS (Fig. ​(Fig.1C)1C) (4, 16, 18, 25, 41, 42). Furthermore, functional groups such as phosphate and acetyl groups are expressed in the GlcNAc(α1-2)Hep[II] portion of the core (Fig. ​(Fig.1A);1A); O-phosphorylation takes place at the 3 and 6 positions of Hep[II] (6, 35), and O-acetylation may occur at the 3 position of GlcNAc (17).Open in a separate windowFIG. 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).Recently, we reported the isolation of human IgG from normal human sera (NHS) using a 2,3:3,4-dibranched LOS (Fig. ​(Fig.1C)1C) from serum-sensitive gonococcal strain JW31R as an affinity ligand (43). The purified IgG did not bind to any 3,4-branched neisserial LOSs examined and bound only to the ligand JW31R LOS and the truncated form of LOS from serum-resistant strain 15253. Both LOSs contain a disaccharide, Galβ1-4Glc (lactose), on Hep[I], and the structural difference between the two LOSs is the length of the OS linked to Hep[II]; the same disaccharide on Hep[I] is present on Hep[II] of 15253 LOS (41), whereas a tetrasaccharide, an elongated form of lactose, resides on JW31R LOS (5). We have determined that the IgG was specific for the OS moieties of the JW31R and 15253 LOSs but not their lipid A portions.The strain 15253 LOS described above is recognized by murine monoclonal antibody (MAb) 2C7, and an OS epitope recognized by MAb 2C7 is frequently expressed in vitro and in vivo on the surface of the gonococci (10). This MAb requires the strain 15253 OS structure containing lactose on both Hep[I] and Hep[II] for binding, but it does not bind to JW31R LOS (42). Although this binding difference between MAb 2C7 and the above-mentioned anti-JW31R IgG exists, recognition of strain 15253 LOS by the latter IgG showed that a 2C7-like epitope is also recognized by human IgG. This human IgG was also found to be bactericidal against strain JW31R. These previous results provided evidence that the OS moiety of LOS is immunogenic in humans and also showed that NHS contains functional antibodies specific for the branched OS.Other investigators have also reported that NHS contains bactericidal antibodies that recognize neisserial LOS (3, 15) and that the OS moiety is immunogenic in humans. However, very little is known about the OS structures of LOS that are recognized by human antibodies. Humans are the only natural host of the pathogenic neisseriae, and the molecular basis of the recognition specificity of human antibodies to LOS is important for us to understand the immune responses of the host to these bacteria.In the present study, we aimed to isolate and characterize human IgG by using 15253 LOS, which contains the MAb 2C7 epitope (10, 42), as an affinity ligand. We anticipated identifying 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 LOSs. We found that IgG2 isolated by affinity chromatography from NHS bound to the aforementioned branched LOSs and also to Salmonella mutant Rb and Re lipopolysaccharides (LPSs). The IgG2 was also found to be functional and able to facilitate the killing of serum-resistant strain 15253, which expresses the ligand LOS. The current results demonstrated that neisserial OS contains several epitopes that are recognized by human antibodies.