Functional activities and epitope specificity of human and murine antibodies against the class 4 outer membrane protein (Rmp) of Neisseria meningitidis

Abstract

Antibodies against the class 4 outer membrane protein (OMP) from Neisseria meningitidis have been purified from sera from vaccinees immunized with the Norwegian meningococcal group B outer membrane vesicle vaccine. The human sera and purified antibodies reacted strongly with the class 4 OMP in immunoblots, whereas experiments with whole bacteria showed only weak reactions, indicating that the antibodies mainly reacted with parts of the class 4 molecule that were not exposed. The purified human anti-class 4 OMP antibodies and the monoclonal antibodies (MAbs) were neither bactericidal nor opsonic against live meningococci. Three new MAbs against the class 4 OMP were generated and compared with other, previously described MAbs. Three linear epitopes in different regions of the class 4 OMP were identified by the reaction of MAbs with synthetic peptides. The MAbs showed no blocking effect on bactericidal activity of MAbs against other OMPs. However, one of the eight purified human anti-class 4 OMP antibody preparations, selected from immunoblot reactions among sera from 27 vaccinees, inhibited at high concentrations the bactericidal effect of a MAb against the class 1 OMP. However, these antibodies were not vaccine induced, as they were present also before vaccination. Therefore, this study gave no evidence that vaccination with a meningococcal outer membrane vesicle vaccine containing the class 4 OMP induces blocking antibodies. Our data indicated that the structure of class 4 OMP does not correspond to standard beta-barrel structures of integral OMPs and that no substantial portion of the OmpA-like C-terminal region of this protein is located at the surface of the outer membrane.

FIG. 1

Immunoblot with sera from eight selected vaccinees immunized with the Norwegian meningococcal OMV vaccine and their corresponding purified anti-class 4 OMP human Igs against strain 44/76. Lanes a, postvaccination serum; lanes b, purified human anti-class 4 OMP Ig from the same serum. Lanes 1 to 8 show sera from vaccinees 1347, 1429, 1436, 1479, 1480, 1508, 1607, and 1610, respectively. Arrows indicate positions of class 1, class 3, and class 4 OMPs (top to bottom, respectively).

FIG. 2

Electron micrographs of meningococcal strains 44/76 (a, c, and d) and 44/76 Rmp⁻ (b) immunogold labelled with 10-nm-colloidal-gold-labelled goat anti-mouse Ig after incubation with anti-class 1 OMP MAb (151,F-9) (a) or anti-class 4 OMP MAb (185,H-8) (b to d). For panel d, the bacteria had been heated to 100°C before labelling. Magnification, ×40,000.

FIG. 3

Flow cytometry histograms showing binding of different MAbs to viable (A) and ethanol-killed (B) cells of meningococcal strain 44/76. The ethanol-killed bacteria had been stored for 1 month at room temperature. FITC, fluorescein isothiocyanate.

FIG. 4

SBA of postvaccination sera from vaccinees immunized with the Norwegian OMV vaccine against strains 44/76 and 44/76 Rmp⁻. Observations along the diagonal show sera with the same titer against both strains; broken lines indicate ±1 titer step difference. Data for SBA against strains 24/88 and 24/88 Rmp⁻ were similar (not shown).

FIG. 5

Blocking effect of anti-class 4 OMP antibodies from vaccinee 1607 on the bactericidal activity of P1.16 MAb and postvaccination sera. (a) Inhibition of the bactericidal activity of anti-class 1 OMP MAb (151,F-9) by purified Ig against class 4 OMP. The concentration of the anti-class 1 OMP MAb (effector antibody) was kept constant at the last dilution, giving 100% killing of strain 44/76, and the anti-class 4 OMP Ig (blocking antibody) was titrated. (b) Inhibition of the bactericidal activity of postvaccination serum from vaccinee 1428 by anti-class 4 OMP Ig from vaccinee 1607. ■, numbers of colonies surviving after incubation with different dilutions of serum (vaccinee 1428) without purified Ig against class 4 OMP; ▴, numbers of colonies surviving after incubation with different dilutions of the 1:4 (vol/vol) mixture of serum from vaccinee 1428 and purified Ig against class 4 OMP from vaccinee 1607. There was no indication of blocking effect with purified anti-class 4 OMP Igs from the other seven vaccinees.

FIG. 6

ELISA reactivities of nine anti-class 4 OMP MAbs with synthetic 14-mer peptides from class 4 OMP on pins. The reaction of MAb V414 was similar to the reaction of MAb AE3. Amino acid sequences corresponding to peptide numbers are given in Table 2.

FIG. 7

ELISA reactivities with synthetic 14-mer peptides from class 4 OMP on pins with purified anti-class 4 OMP antibodies (purified Igs) from eight vaccinees immunized with the Norwegian OMV vaccine. Amino acid sequences corresponding to peptide numbers are given in Table 2. O.D., optical density. The arrow in panel Ig 1607, corresponding to peptide no. 8, indicates the most-marked detected difference between this vaccinee and the others.

FIG. 8

Alignment of class 4 OMP, OprF, and OmpA proteins. From OprF and OmpA proteins, only those segments that can be aligned with the class 4 OMP are shown. Epitopes identified in class 4 OMP and in OprF are boxed (21, 39). Exposed parts of OprF, according to epitope insertion experiments (59), are shown as reversed-type amino acids. The hinge region and the putative peptidoglycan-associating region are shaded.