Expression of meningococcal epitopes in LamB of Escherichia coli and the stimulation of serosubtype-specific antibody responses.
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.
McCarvil
J
J
Division of Bacteriology, National Institute for Biological Standards and Control, South Mimms, Hertfordshire, UK.
McKenna
A J
AJ
Grief
C
C
Hoy
C S
CS
Sesardic
D
D
Maiden
M C
MC
Feavers
I M
IM
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis*; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology*; D001425:Bacterial Outer Membrane Proteins; D001428:Bacterial Vaccines; D001483:Base Sequence; D000939:Epitopes / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics* / Q000276:immunology; D022401:Meningococcal Vaccines; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000235:genetics / Q000276:immunology*; D018272:Porins / Q000235:genetics / Q000276:immunology*; D011817:Rabbits; D011991:Receptors, Virus / Q000235:genetics* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology*; D013045:Species Specificity; D014614:Vaccines, Synthetic
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001428:Bacterial Vaccines; D000939:Epitopes; D022401:Meningococcal Vaccines; D018272:Porins; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; C016532:maltoporins; C057699:porin protein, Neisseria