Targeting Lipooligosaccharide (LOS) for a Gonococcal Vaccine

Abstract

The increasing incidence of gonorrhea worldwide and the global spread of multidrug-resistant strains of Neisseria gonorrhoeae, constitute a public health emergency. With dwindling antibiotic treatment options, there is an urgent need to develop safe and effective vaccines. Gonococcal lipooligosaccharides (LOSs) are potential vaccine candidates because they are densely represented on the bacterial surface and are readily accessible as targets of adaptive immunity. Less well-understood is whether LOSs evoke protective immune responses. Although gonococcal LOS-derived oligosaccharides (OSs) are major immune targets, often they undergo phase variation, a feature that seemingly makes LOS less desirable as a vaccine candidate. However, the identification of a gonococcal LOS-derived OS epitope, called 2C7, that is: (i) a broadly expressed gonococcal antigenic target in human infection; (ii) a virulence determinant, that is maintained by the gonococcus and (iii) a critical requirement for gonococcal colonization in the experimental setting, circumvents its limitation as a potential vaccine candidate imposed by phase variation. Difficulties in purifying structurally intact OSs from LOSs led to "conversion" of the 2C7 epitope into a peptide mimic that elicited cross-reactive IgG anti-OS antibodies that also possess complement-dependent bactericidal activity against gonococci. Mice immunized with the 2C7 peptide mimic clear vaginal colonization more rapidly and reduce gonococcal burdens. 2C7 vaccine satisfies criteria that are desirable in a gonococcal vaccine candidate: broad representation of the antigenic target, service as a virulence determinant that is also critical for organism survival in vivo and elicitation of broadly cross-reactive IgG bactericidal antibodies when used as an immunogen.

Keywords: Lipooligosaccharide; N. gonorrhoeae; complement; peptide mimic; vaccine.

Figure 1

General structure of gonococcal lipooligosaccharide (LOS). Gonococcal LOS consists of three oligosaccharide (OS) chains. The OS chains branch from two heptose residues attached to lipid A via two 2-keto-3-deoxy-mannooctulosonic acid (KDO) molecules. One OS chain elongates from the first heptose (Hep I) outward; two chains extend from the second heptose (Hep II). Lacto-N-neotetraose structure (Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-4-) or P^k (Galα1-4Galβ1-4Glcβ1-4-) extend outward from Hep I. Phase variable genes involved in LOS biosynthesis (lgtA, C, D, and G) are shown in red; non-variable genes (lgtF, lgtE, and B) in blue. Neu5Ac (sialic acid) is shown in the orange boxes. Sialylation of LOS occurs via α2-6 or α2-3 linkage to galactose (Gal) residues. LOS branching is terminated (“capped”) either by Neu5Ac (sialic acid) or otherwise extend(s) outward by adding hexose(s). LOS epitopes are defined by mAbs 2C7, L8, 3F11, L1, and 1-1M.

Figure 2

Mechanisms of complement regulation by sialic acid on gonococcal lacto-N-neotetraose (LNnT). Gonococci can add N-acetylneuraminic acid (Neu5Ac; the form of sialic acid found in humans) to the terminal Gal of the lacto-N-neotetraose (LNnT) LOS structure (a schematic of sialylated LNnT is shown on the left side of the Figure). The presence of Neu5Ac on LNnT LOS reduces the binding of IgG to select targets. As an example, binding of mAbs to PorB, but not to Opacity protein (Opa), is inhibited (79). Sialylation of LNnT also inhibits binding of “natural” IgG in NHS to the gonococcal surface (80). Binding of mannan binding lectin (MBL) to the surface of gonococci is inhibited by LNnT LOS sialylation (81). MBL binds to gonococcal LOS that terminates in GlcNAc (82), which is elongated (“capped”) by Gal and Neu5Ac (shown on the left side of the Figure) and to Opa and PorB (83) [Opa and PorB (shown in the Classical Pathway frame to the left of the Lectin Pathway frame in the Figure)]. Neu5Ac that caps LNnT also regulates the alternative pathway of complement by enhancing binding of factor H (FH; shown as a “string of beads” in the Alternative Pathway frame) (40). Enhanced FH binding to sialylated gonococci is restricted to the LNnT structure; sialylation of the P^k-like LOS (84), or lactose on HepII (78) does not enhance FH binding. Binding of FH is also dependent on expression of PorB (85) and occurs through the C-terminal domains of FH (SCR18-20) (86). Bound FH acts as a cofactor in the factor I (FI) cleavage of C3b to iC3b (cofactor activity) and also irreversibly dissociates the C3 convertase, C3bBb (decay accelerating activity).

Figure 3

Schematic representation of the conversion of carbohydrate (OS) epitopes into peptide mimotopes. Peptide mimics of the 2C7 epitope were identified using a random FliTrx (Flagellin-Thioredoxin) peptide display library that were screened (Biopanned) using mAb 2C7 (98). The peptide–containing clones that bound to mAb 2C7 were eluted and sequenced. An optimal peptide that contained the consensus motif was synthesized as an octameric peptide (shown here as a multiantigenic peptide [MAP]) on a lysine backbone (TetraMAP), which is the currently used configuration.