A CpG-containing oligodeoxynucleotide adjuvant for acellular pertussis vaccine improves the protective response against Bordetella pertussis

Abstract

We investigated the adjuvant effect of CpG ODN alone or in combination with aluminum hydroxide on the immune response to the three main antigens presented in current acellular pertussis vaccines: pertussis toxoid, filamentous haemagglutinin and pertactin. The development of protection in mice was investigated for the intra-peritoneal and intra-nasal immunisation routes. The results showed that CpG ODN alone, or in combination with aluminum hydroxide, gave enhancement in anti-pertussis toxin, anti- filamentous haemagglutinin and especially anti-pertactin titers after mucosal immunisation. Higher macrophage NO levels indicating activation were found when the antigens were co-formulated with CpG ODN. Vaccines containing CpG ODN gave enhanced humoral and CMI responses with a shift toward Th-1 and increased protection against challenge infection with B.pertussis in mice.

Keywords: Bordetella pertussis; CpG ODN; acellular pertussis vaccine; adjuvant.

Figure 1. CpG ODN /or CpG ODN in combination with aluminum induces strong immune response in mice. (A) Geometric mean of IgG response in sera and lung supernatant (insert) after intra-nasal immunisation, CpG ODN group; Alum group; CpG ODN + aluminum hydroxide group. Groups of five mice were immunised intra-nasally with CpC ODN (30 μg per dose), antigen mixture (PT, 6.7 μg; FHA, 6.7 μg; Pertactin, 3.35 μg per dose), antigen mixture plus aluminum hydroxide (1.3 mg), antigen mixture plus CpG ODN and antigen mixture plus CpG ODN in combination with aluminum hydroxide (0.26 mg). Levels were very low or undetectable in antigen alone group and CpC ODN negative control (data not shown). Lower and upper limits of GMs are shown in brackets. The experiment was performed three times and all showed similar trend. A representative result is shown in the figure. (B) Nitric oxide induction in murine peritoneal macrophage following intra-peritoneal immunization. Groups of five mice were immunized with CpC ODN alone (30 μg/dose); a mixture of antigens alone (PTd at 5 μg, FHA at 5 μg and PRN at 2.5 μg per dose); the antigen mixture in combination with CpG ODN (30 μg per dose) or aluminum hydroxide (1.3 mg per dose) and the antigen mixture in combination with aluminum hydroxide (0.26 mg per dose) and CpG ODN (30 μg per dose). Peritoneal macrophages were taken two weeks after boosting. Cells were re-stimulated in vitro with heat killed B. pertussis cells (HKC). After 24 h incubation NO levels in culture supernatants were determined by Griess reagent. Bars represent mean and standard deviation from three independent experiments. (C) IFN-γ production in spleen cell supernatant following intra-peritoneal immunization. Groups of five mice were immunized with CpC ODN, a mixture of antigens alone (PTd at 5 μg, FHA at 5 μg and PRN at 2.5 μg per dose); the antigen mixture in combination with CpG ODN (30 μg per dose) or aluminum hydroxide (1.3 mg per dose) and the antigen mixture in combination with aluminum hydroxide (0.26 mg per dose) and CpG ODN (30 μg per dose). Spleens were removed at two weeks after boost and cultured for 2 d. After 48 h incubation IFN-γ levels in culture supernatants were determined by ELISA. Bars represent mean and standard deviation from three independent experiments. * indicates a p-value of < 0.05.

Figure 2. CpG ODN /or CpG ODN in combination with aluminum induces stronger protection in mice against live B.pertussis challenge. (A) Protection against aerosol challenge following intra-peritoneal immunization. Groups of five mice were immunized with CpC ODN or CpG ODN alone (30 μg/dose); a mixture of antigens alone (PTd at 5 μg, FHA at 5 μg and PRN at 2.5 μg per dose); the antigen mixture in combination with CpG ODN (30 μg per dose) or aluminum hydroxide (1.3 mg per dose) and the antigen mixture in combination with aluminum hydroxide (0.26 mg per dose) and CpG ODN (30 μg per dose) followed by a booster at four weeks and aerosol challenged on day 15 after boost. Mouse lungs and trachea were removed at day 7 after the challenge. Viable counts per lung were performed on four replicates. Bars represent mean CFU per lung and standard deviations. The experiment was performed three times and all showed similar trend. A representative result is shown in the figure. (★) Viable bacteria were below detection limit. (B) Protection against aerosol challenge after intra-nasal immunisation. Groups of five mice were immunised with CpC ODN alone (30 μg/dose); a mixture of antigens alone (PTd at 6.7 μg, FHA 6.7μg and PRN 3.35 μg/dose); the antigen mixture in combination with CpG ODN (30 μg per dose) or aluminum hydroxide (1.3 mg per dose) and the antigen mixture in combination with aluminum hydroxide (0.26 mg per dose) and CpG ODN (30 μg per dose) followed by a booster at four weeks and aerosol challenged on day 15 after boost. Mouse lungs and trachea were removed at day 7 after the challenge. Viable counts per lung were performed on four replicates. Bars represent mean CFU per lung and standard deviations. The experiment was performed twice and both showed similar trend. A representative result is shown in the figure. (★) Viable bacteria were below detection limit. *Indicates a p-value of < 0.05.