Conservation and accessibility of an inner core lipopolysaccharide epitope of Neisseria meningitidis

Abstract

We investigated the conservation and antibody accessibility of inner core epitopes of Neisseria meningitidis lipopolysaccharide (LPS) because of their potential as vaccine candidates. An immunoglobulin G3 murine monoclonal antibody (MAb), designated MAb B5, was obtained by immunizing mice with a galE mutant of N. meningitidis H44/76 (B. 15.P1.7,16 immunotype L3). We have shown that MAb B5 can bind to the core LPS of wild-type encapsulated MC58 (B.15.P1.7,16 immunotype L3) organisms in vitro and ex vivo. An inner core structure recognized by MAb B5 is conserved and accessible in 26 of 34 (76%) of group B and 78 of 112 (70%) of groups A, C, W, X, Y, and Z strains. N. meningitidis strains which possess this epitope are immunotypes in which phosphoethanolamine (PEtn) is linked to the 3-position of the beta-chain heptose (HepII) of the inner core. In contrast, N. meningitidis strains lacking reactivity with MAb B5 have an alternative core structure in which PEtn is linked to an exocyclic position (i.e., position 6 or 7) of HepII (immunotypes L2, L4, and L6) or is absent (immunotype L5). We conclude that MAb B5 defines one or more of the major inner core glycoforms of N. meningitidis LPS. These findings support the possibility that immunogens capable of eliciting functional antibodies specific to inner core structures could be the basis of a vaccine against invasive infections caused by N. meningitidis.

FIG. 1

Representation of the structure of meningococcal LPS oligosaccharides of immunotypes L1 to L9. Immunotypes are indicated to the extreme left. The vertical line marks the junction between the inner core structures to the right and outer core structures to the left. The epitope recognized by MAb B5 is indicated in boldface (MAb B5 positive). Arabic numerals indicate the linkage between sugars or amino sugars. Alpha and beta indicate the carbon 1 linkage at the nonreducing end of the sugar. Genes for incorporating each of the key sugars or amino sugars into the LPS oligosaccharide in the biosynthetic pathway are indicated with arrows indicating where in the pathway the gene product is required. Abbreviations: Kdo, 2-keto-2-deoxyoctulosonic acid; Gal, galactose: GlcNAc, N-acetylglucosamine; Glu, glucose; Hep, heptose. Immunotype L5 has no PEtn on the second heptose. The gene that adds the glucose to the second heptose (lgtG) is phase variable.

FIG. 2

Cross-reactivity of MAb B5 with selected immunotypes and mutants of N. meningitidis LPS and O-deacylated (odA) LPS as determined by solid-phase ELISA. LPS glycoforms of immunotypes L2 (35E) (solid bars), L3 (H44/76) (open bars), L4 (89I) (hatched bars), L5 (M981) (open bars), L8 (M978) (horizontal-line-filled bars), wild-type, and respective mutants (galE, lgtA, or lgtB), in a native or O-deacylated form, were coated onto ELISA plates (see Materials and Methods), and the reactivity of MAb B5 was determined by standard ELISA (OD, A₄₁₀).

FIG. 3

Space-filling three-dimensional molecular models of the calculated (MMC) lowest-energy states of the core oligosaccharide from galE mutants of L3 (a), L4 (b), and L8-dephosphorylated (c). The Kdo moiety indicated in gray is substituted at the O-5 position by the heptose disaccharide inner core unit (red); HepI provides the point via a glucose residue (dark green) for extension to give α-chain epitopes, while HepII is substituted by an N-acetylglucosamine residue (lighter green) at O-2. PEtn (brown) is shown in O-3 position in the L3 immunotype and O-6 in the L4 immunotype.

FIG. 4

Cross-reactivity of MAb B5 with genetically modified L3 LPS and chemically modified L8 LPS from N. meningitidis, as determined by solid-phase ELISA. LPS glycoforms of immunotype L8 (M978) (horizontal-line-filled bars) chemically modified by O deacylation and HF treatment and immunotype L3 (H44/76) (open bars) galE, icsB, icsA, lsi, and PB4 mutants (O deacylated) were coated onto ELISA plates (see Materials and Methods), and the reactivity of MAb B5 was determined by standard ELISA (OD, A₄₁₀).

FIG. 5

(a and b) Confocal immunofluorescence microscopy of in vitro-grown N. meningitidis MC58 adherent to Huvecs. (a) Fluorescein tagging with rabbit polyclonal antibody specific for group B N. meningitidis capsule. (b) rhodamine tagging of MAb B5 specific for galE LPS. (c and d) Confocal immunofluorescence microscopy of in vivo-grown MC58 organisms stained as described for panels a and b. (c) Anticapsular antibody (green). (d) MAb B5 (red). Magnification, ×2,400 (all four panels).

FIG. 6

Silver-stained tricine gels of LPS preparations (10 μg/lane) from N. meningitidis group B strains which were not reactive with MAb B5. These LPS preparations were either not treated (−) or treated with neuraminidase (+) to show the presence of sialic acid. (a) MAb B5-negative strains: lanes 1 and 2, NGE30; lanes 3 and 4, BZ157; lanes 5 and 6, EG328; lanes 7 and 8, 1000; lanes 9 and 10, 3906. (b) MAb B5-negative strains: lanes 1 and 2, EG327; lanes 3 and 4, NGH38; lanes 5 and 6, NGH15; and MAb B5-positive strain: lanes 7 and 8, MC58. The presence of sialic acid is indicated (NeuAc). This band was seen in untreated (−) and removed in treated (+) neuraminidase preparations.