Agonists of Toll-like receptors 3, 4, 7, and 9 are candidates for use as adjuvants in an outer membrane vaccine against Neisseria meningitidis serogroup B

Abstract

The bacterium Neisseria meningitidis is the causative agent of meningitis and sepsis. A generally effective vaccine against N. meningitidis serogroup B is not yet available, but outer membrane vesicle vaccines are in development. These vaccines contain lipopolysaccharide (LPS). The inclusion of N. meningitidis wild-type LPS in a vaccine is controversial because of its high toxicity. Therefore, the adjuvant activity of a panel of different Toll-like receptor (TLR) agonists in combination with LPS-deficient meningococcal outer membrane complexes was compared after immunization of mice. The results demonstrate that TLR3, TLR4, TLR7, and TLR9 agonists enhance immune responses against LPS-deficient outer membrane complexes. Their adjuvant activity was characterized by higher levels of antigen-specific immunoglobulin G (IgG), IgG2a, and IgG2b; a higher IgG2a/IgG1 ratio; lower total IgE levels; and most importantly, higher serum bactericidal antibody titers compared to LPS-deficient outer membrane complexes alone.

FIG. 1.

Antigen-specific total IgG levels. Mice were immunized with the following adjuvants: FSL-1 (TLR2/TLR6); Pam₃CSK4 (TLR2/TLR1); poly(I-C) (TLR3); N. meningitidis wild-type L3, LpxL1 LPS, B. pertussis LPS, and MPL (TLR4); B. subtilis flagellin (TLR5); Imiquimod and Loxoribine (TLR7); CpG DNA (TLR9); and MDP (NOD2). The sera of immunized mice (eight per group) were analyzed by ELISA for antigen-specific IgG. The data are expressed as means ± the SEM of log₁₀ titers. An asterisk indicates that IgG levels of a group were significantly different (P < 0.05) from pLAK33-immunized mice. The dashed line indicates the level of IgG from pLAK33-immunized mice. LPS Bp, B. pertussis LPS.

FIG. 2.

Antigen-specific antibody subclasses. The sera of immunized mice (eight per group) were analyzed by ELISA for the presence of antigen-specific IgG1 (A), IgG2a (B), and IgG2b (C). The data are expressed as means ± the SEM of log₁₀ titers. An asterisk indicates that the immunoglobulin levels of a group were significantly different (P < 0.05) from pLAK33-immunized mice. The dashed line indicates the level of immunoglobulin from pLAK33-immunized mice. LPS Bp, B. pertussis LPS.

FIG. 3.

Comparative log titers of IgG1 and IgG2a. The sera of immunized mice (eight per group) were analyzed by ELISA for the presence of antigen-specific IgG1 and IgG2a. The data are expressed as means of log₁₀ titers of IgG1 plus the means of log₁₀ titers of IgG2a.

FIG. 4.

Total IgE levels. Amounts of IgE antibodies were determined in sera of immunized mice (eight per group, except for CpG DNA 2 nmol [seven mice]) by ELISA. The data are expressed as the means ± the SEM of nanograms of IgE/milliliter. An asterisk indicates that the IgE levels of a group were significantly different (P < 0.05) from pLAK33-immunized mice. The dashed line indicates the level of IgE from pLAK33-immunized mice. LPS Bp, B. pertussis LPS.

FIG. 5.

Serum bactericidal antibodies. Serial dilutions of sera of immunized mice (eight mice per group) were incubated with N. meningitidis and rabbit complement. The highest dilution that killed ≥90% of bacteria was determined. The number of responders is indicated in the graph. A responder was defined as a mouse whose serum killed ≥90% at at least a 1:2 dilution. The data are expressed as means ± the SEM of log₁₀ titers.