Neisseria lactamica antigens complexed with a novel cationic adjuvant

Abstract

Colonization of the nasopharynx by non-pathogenic Neisseria species, including N. lactamica, has been suggested to lead to the acquisition of natural immunity against Neisseria meningitidis in young children. The aim of this study was to identify a model complex of antigens and adjuvant for immunological preparation against N. meningitidis B, based on cross reactivity with N. lactamica outer membrane vesicles (OMV) antigens and the (DDA-BF) adjuvant. Complexes of 25 µg of OMV in 0.1 mM of DDA-BF were colloidally stable, exhibiting a mean diameter and charge optimal for antigen presentation. Immunogenicity tests for these complexes were performed in mice. A single dose of OMV/DDA-BF was sufficient to induce a (DTH) response, while the same result was achieved only after two doses of OMV/alum. In addition, to achieve total IgG levels that are similar to a single immunization with OMV/DDA-BF, it was necessary to give the mice a second dose of OMV/alum. Moreover, the antibodies induced from a single immunization with OMV/DDA-BF had an intermediate avidity, but antibodies with a similar avidity were only induced by OMV/alum after two immunizations. The use of this novel cationic adjuvant for the first time with a N. lactamica OMV preparation revealed good potential for future vaccine design.

Keywords: Neisseria lactamica; Neisseria meningitides; bilayer fragments; dioctadecyldimethylammonium bromide; immune response; outer membrane vesicles.

Figure 1. The size distribution and zeta-potential (ζ) for different dispersions in 1 mM NaCl. One mM DDA-BF dispersion (A) and 0.1 mM DDA-BF dispersions carrying OMV of N. lactamica at concentrations of 5 (C), 25 (D) and 50 μg/mL (E). OMV alone at a concentration of 100 μg/mL is shown in (B). The interaction time for DDA-BF and protein was 1 h. In each subfigure, mean zeta-potential and diameter ± SE are quoted. The figures above the main peak correspond to the mean diameter of this specific peak.

Figure 2. IgG levels in the sera of mice immunized with OMVs from N.lactamica. The serum of 6 animals per group was collected, pooled and analyzed by ELISA: (A) 15 d after immunization; (B) 30 d after immunization; (C) 45 d after immunization; (D) Kinetics of IgG production over time. The composition of OMV-DDA-BF was statistically superior (p < 0.01) to the OMV-alum or OMV alone for the production of total IgG, both with just one dose or two doses of antigens (results represent mean ± SD of the pooled serum of six animals per group).

Figure 3. Levels of IgG1, IgG2a and IgG2b in the sera of immunized animals. The serum of 6 animals per group was collected 45 after immunization, pooled analyzed by ELISA.

Figure 4. Levels of IgG cross reactive with antigens derived from N. meningitidis The serum of 6 animals per group was collected 45 d after the first immunization, pooled and analyzed by ELISA.

Figure 5. Immunoblot analysis for cross reactivity of the antibodies produced after immunization. Twenty µg of protein of N. lactamica (A) or N. meningitides (B) whole cell lysates were submitted to electrophoresis in a polyacrylamide gel (13% SDS-PAGE). After electrophoresis, the protein fractions were transferred to nitrocellulose membranes. The membranes containing the whole cell lysates were incubated for 1 h in the presence of serum from outbred mice immunized with OMV-DDA-BF single dose (G1), OMV-alum single dose (G2), OMV single dose (G3), DDA-BF (G4), alum (G5), OMV-DDA-BF 2 doses (G6), OMV-alum 2 doses (G7), OMV 2 doses (G8) or non-immunized controls (G9), (diluted 1/500). The immunoreactions were visualized with goat anti-mouse IgG conjugated to peroxidase diluted 1/60,000. The membranes were washed with saline phosphate buffer and luminol.

Figure 6. Delayed type hypersensitivity (DTH) reaction. The percentage of swelling of the footpad of mice challenged with 10 µg/mL of outer membrane vesicles of N. meningitidis after 24 h of challenge. The percentage of swelling of the footpads of the animals vaccinated with a single dose of OMV-DDA-BF was significantly higher (p < 0.01) than the swelling of the footpads from all other animals, either vaccinated or not.