Anti-idiotypic antibody as a potential candidate vaccine for Neisseria meningitidis serogroup B

Abstract

Sepsis and meningitis caused by Neisseria meningitidis serogroup B (NMGB) are serious diseases in infants and young adults, but no effective vaccine is available. The capsular polysaccharide (PS) of NMGB has poor immunogenicity and a structural similarity to polysialic acid (PSA) on neuronal tissue that may elicit autoantibodies. Using HmenB3, a protective and nonautoreactive monoclonal antibody (MAb) to NMGB capsular PS, we produced an anti-idiotypic MAb, Naid60, which mimics the capsular PS of NMGB. We produced an anti-anti-idiotypic MAb, MoB34, by using the immunogenic site on Naid60 responsible for inducing the anti-NMGB PS antibody response. MoB34 elicited the complement-mediated killing of representative strains of serogroup B meningococci. MoB34 did not bind to CHP-134, a neuroblastoma cell line expressing alpha(2-8) PSA, or to mouse brain cryosections at a high concentration. Naid60-keyhole limpet hemocyanin immunization inhibited the growth of live NMGB in intraperitoneally challenged mice; in contrast, three of five control mice developed bacteremia. Thus, Naid60 has an immunogenic site that elicits antibodies with bactericidal activity against NMGB and no autoimmunity to PSA. We suggest that the immunogenic region of Naid60 is a candidate for the development of a new vaccine against NMGB.

FIG. 1.

Characterization of the anti-idiotype Ab of Naid60. Naid60 inhibited the binding of HmenB3 to wells coated with heat-killed NMGB in a dose-dependent manner, like E. coli K1 PS, which is structurally and antigenically identical to NMGB PS. Naid59, which bound to HmenB3 but did not inhibit the binding of HmenB3 to NMGB, was used as a negative control Ab.

FIG. 2.

Surface plasmon resonance analysis of Naid60 binding to immobilized HmenB3. The data represent five injections of Naid60 at concentrations of 50, 25, 12.5, 6.25, and 3.13 nM from top to bottom (B and D). HmenB5, which is an anti-NMGB PS MAb cross-reactive with NCAM, was used for the control antibody (D). NMGB PS was used as a positive control ligand to both HmenB3 and HmenB5, and the bindings were demonstrated at concentrations of 5, 4, 3, and 2 mg/ml of NMGB PS (A and C).

FIG. 3.

Bactericidal inhibition of HmenB3 to NMGB by Naid60. NMGB bacteria were incubated with HmenB3 and various amounts of Naid60 in the presence of baby rabbit complement. Bactericidal inhibitions were 36.9%, 92.6%, and 98.0% at 4, 40, and 400 μg/ml of Naid60, respectively, and 38.3% at 40 μg/ml of E. coli K1 PS, a positive control inhibitor.

FIG. 4.

Production and characterization of Naid60-scFv. (A) Recombinant scFv of Naid60 was produced as described in Materials and Methods. Affinity-purified Naid60-scFv was run on a 10% sodium dodecyl sulfate-polyacrylamide gel, and Western blotted using HmenB3 as a primary antibody after transfer to nitrocellulose, and identified as a ca. 30-kDa protein. (B and C) Naid60-scFv inhibited the binding of HmenB3 to wells coated with NMGB bacteria (B) and to wells coated with Naid60 (C) in a dose-dependent manner. (D) Naid60-scFv inhibited the complement-mediated bactericidal activity of HmenB3 to NMGB bacteria.

FIG. 5.

Anti-NMGB Ab levels in Naid60-KLH- or Naid60-scFv-immunized mice. (A) Groups of five BALB/c mice were injected with Naid60-KLH, which was emulsified with CFA and IFA, and then boosted at 2-week intervals. After four injections, mouse sera were collected and the anti-NMGB Ab titer at a 1:100 dilution was measured using heat-killed NMGB as a coating antigen. (B) Groups of five BALB/c mice were injected with Naid60-scFv as described for panel A. Mouse sera were collected 7 days after each immunization, and the anti-NMGB Ab titer was measured at a 1:100 dilution. Error bars indicate standard deviations.

FIG. 6.

Protective efficacy for Naid60-immune mice. Groups of five BALB/c mice were subcutaneously immunized with KLH (A) or 100 μg of Naid60-KLH (B) with alum and CpG ODN five times at 2-week intervals and treated with cyclophosphamide to render the mice susceptible to meningococcal infections. Subsequently the mice were challenged with 1 × 10⁶ CFU of NMGB (ATCC 13090). To observe bacterial clearance, blood specimens were obtained from the retro-orbital plexus at the indicated times, and 10 μl of each blood sample undiluted or diluted with HBSS at 1:10 or 1:100 was cultured on chocolate agar plates at 37°C. CFU in duplicate were determined after 18 h of incubation in a candle jar.

FIG. 7.

Binding of MoB34 to whole bacterial cells and its bactericidal activity. (A) MoB34 purified from mouse ascites fluid bound to NMGB bacteria in dose-dependent manner. (B) The complement-mediated bactericidal activity of purified MoB34 was observed, and the 50% killing concentration was determined to be 16 ng/ml. (C) The bactericidal activity of MoB34 was reduced in the presence of Naid60.

FIG. 8.

Autoreactivity tests. (A) Cells were stained with anti-CD56 (green line, left) or isotype control Ab (dotted line). The culture supernatant of HmenB1 (red line) was bound to PSA on CHP-134 cells (42). One hundred micrograms per ml of MoB34 (green line, right) was used for the staining. (B) CHP-134 cells were stained with sera from mice immunized with Naid60-scFv. Five mice sera were pooled and used as a primary Ab at 1:10 (green line, center) and 1:50 (green line, right) dilutions. Preimmune sera (green line, left), the culture supernatant of HmenB1 (red line), and irrelevant Ab (dotted line) are also shown. (C) A cryosection of neonatal (less than 10-day-old) mouse brain was stained with 50 μg/ml of irrelevant Ab (anti-LPS MAb) (panel 1) and 50 μg/ml MoB34 (panel 2). HmenB1 at a concentration of 0.1 μg/ml (panel 3) and hematoxylin-eosin (panel 4) are also shown. Bars, 50 μm.