Naturally-occurring human serum antibodies to inner core lipopolysaccharide epitopes of Neisseria meningitidis protect against invasive meningococcal disease caused by isolates displaying homologous inner core structures.

Sera from healthy infants (under 1 year old), toddlers (3-4 years) and adults (18-65 years) were assayed for their ability to bind to inner core (ic) lipopolysaccharide (LPS) epitopes of Neisseria meningitidis. Antibodies (Abs) reacting to inner core structures, including different substitutions of the first heptose (HepI) and second heptose (HepII) residues of the LPS backbone, truncated and fully extended LPS glycoforms, were detected and for each structure, these inner core antibodies showed an age-related pattern of acquisition. A novel column-based methodology was used to affinity purify IgG antibodies in which purified inner core LPS (derived from a mutant MC58) was covalently linked to Sepharose 4B. Comparison of reactivity before and after affinity purification of the pooled sera showed that the purified Abs bound to the surface of N. meningitidis organisms displaying truncated and extended LPS with a homologous inner core region, promoted the deposition of C3b, were opsonophagocytic in vitro and decreased bacteraemia when used to passively protect infants rats. In addition, the purified Abs were bactericidal in vitro against the mutant strain displaying truncated LPS with a homologous inner core region. These results demonstrate that naturally occurring serum human antibodies to N. meningitidis LPS can access inner core epitopes of encapsulated organisms with a fully extended LPS.

Production of a D-glycero-D-manno-heptosyltransferase mutant of Mannheimia haemolytica displaying a veterinary pathogen specific conserved LPS structure; development and functionality of antibodies to this LPS structure.

Previous structural studies of the lipopolysaccharides from the veterinary pathogens Mannheimia haemolytica (Mh), Actinobacillus pleuropneumoniae (Ap) and Pasteurella multocida (Pm) had identified a conserved inner core oligosaccharide structure that was present in all strains investigated. In order to examine the potential of this inner core structure as a vaccine, a mutagenesis strategy was adopted to interrupt a D-glycero-D-manno-heptosyltransferase gene (losB) of Mh. This gene encodes the enzyme responsible for the addition of a D-glycero-D-manno-heptose residue, the first residue beyond the conserved inner core, and its inactivation exposed the conserved inner core structure as a terminal unit on the mutant LPS molecule. Subsequent analyses confirmed the targeted structure of the mutant LPS had been obtained, and complementation with losB in trans confirmed that the losB gene encodes an alpha-1,6-D-glycero-D-manno-heptosyltransferase. Monoclonal antibodies raised in mice to this LPS structure were found to recognise LPS and whole-cells of the truncated mutant and wild-type Mh. The antibodies were bactericidal against a wild-type Mh strain and were able to passively protect mice in a model of Mh disease. This illustrates that it is possible to raise functional antibodies against the conserved inner core LPS structure.

Development, characterization, and functional activity of a panel of specific monoclonal antibodies to inner core lipopolysaccharide epitopes in Neisseria meningitidis.

A panel of six murine monoclonal antibodies (MAbs) recognizing inner core lipopolysaccharide (LPS) epitopes of Neisseria meningitidis was prepared and characterized in order to determine the diversity of inner core LPS glycoforms among disease and carrier isolates. Two of these MAbs, L2-16 (immunoglobulin G2b [IgG2b]) and LPT3-1 (IgG2a), together with a third, previously described MAb, L3B5 (IgG3), showed reactivity, either individually or in combination, with all except 3 of 143 disease and carriage isolates (125 of 126 strains from blood, cerebrospinal fluid, or skin biopsy samples and 15 of 17 from nasopharyngeal cultures). MAbs L3B5, L2-16, and LPT3-1 were further characterized in an indirect immunofluorescence assay. All three MAbs bound to the bacterial cell surface, findings that correlated strongly with whole-cell enzyme-linked immunosorbent assay and immunodot blots. However, in contrast to our findings with L3B5, cell surface binding of L2-16 or LPT 3-1 did not correlate with functional activity as determined by bactericidal or infant rat passive protection assays against wild-type N. meningitidis strains. These findings are provocative with respect to the requirements for protective activity of antibodies and the development of inner core LPS vaccines against invasive meningococcal disease.

Highly conserved Neisseria meningitidis inner-core lipopolysaccharide epitope confers protection against experimental meningococcal bacteremia.

Inner-core lipopolysaccharide (LPS) from Neisseria meningitidis is under investigation as a vaccine for prevention of meningococcal disease caused by N. meningitidis serogroup B (NmB). We investigated the functional activity of murine monoclonal antibody (MAb) B5 that recognizes a highly conserved (galE) LPS epitope. Three patterns of MAb reactivity were observed in N. meningitidis by Western blot, depending on the relative prevalence of sialylated, nonsialylated, and/or truncated LPS glycoforms. Three representative N. meningitidis strains (8047, M986, and 2996) were investigated with MAb B5 in functional assays in vitro and in vivo. MAb B5 completely protected infant rats against bacteremia caused by 8047, partially protected against 2996, and had no protective activity against M986. Thus, an inner-core LPS epitope can be a target for protective immunity, but the affinity of MAb B5 may only be sufficient to mediate protection against NmB strains possessing at least some truncated glycoforms.