Expression of meningococcal epitopes in LamB of Escherichia coli and the stimulation of serosubtype‐specific antibody responses

Abstract

The class 1 outer membrane protein (OMP), a major variable surface antigen of Neisseria meningitidis, is a component of novel meningococcal vaccines currently in field trials. Serological variants of the protein are also used to serosubtype meningococci. Most of the amino acid changes that give rise to antigenic variants of the protein occur in two variable regions (VR1 and VR2) that are thought to form loops on the cell surface. The polymerase chain reaction (PCR) was used to amplify the nucleotide sequences encoding VR1 and VR2 from the chromosomal DNA of N. meningitidis strain M1080. These were cloned in frame into the lamB gene of the Escherichia coli expression vector pAJC264. Whole-cell enzyme-linked immunosorbent assays (ELISAs), using monoclonal antibodies, and SDS-PAGE confirmed that, upon induction, strains of E. coli carrying these constructs expressed hybrid LamB proteins containing the N. meningitidis surface loops. These strains were used to immunize rabbits and the resultant polyclonal antisera reacted specifically with the class 1 OMP of reference strain M1080 (P1.7). Immunogold labelling of meningococcal cells and whole-cell dot-blot analyses with these antisera showed that the variable epitopes were exposed on the cell surface and confirmed that this approach could be used to obtain serosubtype-specific antisera. The binding profiles of the antisera were determined from their reactions with overlapping synthetic peptides and their reactivity compared with that of relevant serosubtype-specific monoclonal antibodies. This approach was used successfully to raise antisera against two other class 1 OMP VR2s. A fourth antiserum raised against a VR2, including the P1.1 epitope, was not subtype specific.

Analysis of Neisseria meningitidis class 3 outer membrane protein gene variable regions and type identification using genetic techniques

“…This has been addressed primarily by inclusion of the most epidemiologically prevalent strains in vaccine preparations. Genetic engineering of the class 1 protein utilizing antigenic hybrids (22) or expression of multiple recombinant epitopes (12) has also been proposed. The patterns of identical VR sequences found among serotypes described here may predict patterns of immunologic cross-reactivity and may have important implications regarding strain selection for vaccine use.…”

The recombinant Klebsiella pneumoniae outer membrane protein OmpA has carrier properties for conjugated antigenic peptides

“…OmpA is one of the major proteins in the outer membrane topes: proteins such as LamB [17], PhoE [18], OmpC [19] and OmpA [20,21] have been demonstrated to mediate expression of gram-negative bacteria, present at about 10 5 copies/cell. It is believed to occur in a monomeric form in its native state.…”

Emergence of a new virulent clone within the electrophoretic type 5 complex of serogroup B meningococci in Norway

“…These substitutions did not affect binding of the P1.12 reference MAb, implying that these amino acids were not part of that epitope. VR1 of strain 138/91 was similar to one designated subtype P1.18 present in reference strain M990 (21,25).…”

“…In addition, porA genes from case strains with other subtypes in VR1 were also analyzed. The deduced amino acid sequences of VR1 and VR2 are given in Table 2 together with published sequences of the reference strains S3032 and M990 (21,25). Strains reacting with the P1.13 MAb had a sequence of five threonine residues at the end of VR2, whereas those reacting with MAb 202,G-12 had four threonine residues (Table 2).…”