Evaluation of Four Adjuvant Combinations, IVAX-1, IVAX-2, CpG-1826+Montanide ISA 720 VG and CpG-1018+Montanide ISA 720 VG, for Safety and for Their Ability to Elicit Protective Immune Responses in Mice against a Respiratory Challenge with Chlamydia muridarum

Abstract

There is an urgent need to produce a vaccine for Chlamydia trachomatis infections. Here, using the Chlamydia muridarum major outer membrane protein (MOMP) as an antigen, four adjuvant combinations IVAX-1 (MPLA+CpG-1018+AddaVax), IVAX-2 (MPLA+CpG-1018+AS03), CpG-1826+Montanide ISA 720 VG (CpG-1826+Mont) and CpG-1018+Montanide ISA 720 VG (CpG-1018+Mont), were tested for their local reactogenicity and ability to elicit protection in BALB/c mice against a respiratory challenge with C. muridarum. Immunization with IVAX-1 or IVAX-2 induced no significant local reactogenicity following intramuscular immunization. In contrast, vaccines containing Montanide resulted in the formation of a local granuloma. Based on the IgG2a/IgG1 ratio in serum, the four adjuvant combinations elicited Th1-biased responses. IVAX-1 induced the highest in vitro neutralization titers while CpG-1018+Mont stimulated the lowest. As determined by the levels of IFN-γ produced by T-cells, the most robust cellular immune responses were elicited in mice immunized with CpG-1018+Mont, while the weakest responses were mounted by mice receiving IVAX-1. Following the respiratory challenge, mice immunized with CpG-1018+Mont lost the least amount of body weight and had the lowest number of C. muridarum inclusion-forming units (IFUs) in the lungs, while those receiving IVAX-2 had lost the most weight and had the highest number of IFUs in their lungs. Animals vaccinated with CpG-1826+Mont had the lightest lungs while those immunized using IVAX-2 had the heaviest. To conclude, due to their safety and adjuvanticity, IVAX formulations should be considered for inclusion in human vaccines against Chlamydia.

Keywords: Chlamydia muridarum; Chlamydia trachomatis; adjuvant combinations; major outer membrane protein; mice; vaccine.

Figure 1

Evaluation of the reactogenicity/toxicity at the site of vaccination. Before the mice were euthanized, pictures were taken of the site where the vaccines were delivered. (A) No injection control; (B) PBS control; (C) CpG-1826+Mont/MOMP; (D) IVAX-1/MOMP; (E) CpG-1018+Mont/MOMP; (F) IVAX-2/MOMP; (G) MOMP only.

Figure 2

Antibody responses following immunization. (A) IgG, IgG1 and IgG2a ELISA titers (GMT ± SE) to C. muridarum EBs. Mice were immunized and blood was collected the day before the i.n. challenge. (B) In vitro neutralizing antibody GMT ± SE using serum collected before the challenge. Pre-immunization sera were used as negative controls. * p < 0.05 by the Mann–Whitney U test.

Figure 3

Binding of serum antibodies from immunized mice to synthetic C. muridarum MOMP peptides. Serum samples from mice were collected the day before the i.n. challenge. Their reactivity to 25-aa overlapping peptides corresponding to the C. muridarum mature MOMP was analyzed by ELISA.

Figure 4

IgG and IgA antibody titers in pooled vaginal washes collected the day before the i.n. challenge. Vaginal washes were collected, two pools of five mice/group were run in triplicate, and the levels of IgG and IgA were determined using C. muridarum EBs.

Figure 5

Determination of (A) IFN-γ and (B) IL-4 levels in T-cell supernatants collected from vaccinated mice the day before the i.n. challenge. The day before the intranasal challenge, four randomly selected mice from each group were euthanized, their spleens collected, and T-cells isolated using nylon wool columns and stimulated with C. muridarum EBs, with Concanavalin A as a non-specific stimulant, or with medium as a negative control. * p < 0.05 and ** p < 0.1 by the Student’s t-test. The limit of detection is indicated with a broken horizontal line.

Figure 6

Daily changes in mean body weight following the i.n. challenge with 10⁴ C. muridarum IFUs. Percentage changes in daily mean body weight following the i.n. challenge with C. muridarum. * p < 0.05 by the repeated-measures ANOVA.

Figure 7

Disease burden at day 10 following the i.n. challenge with 10⁴ C. muridarum IFUs. (A) Percentage change in body weight at 10 days following the i.n. challenge. The mean is shown as a horizontal line. Each symbol represents an animal. * p < 0.05 and ** p < 0.1 by the Student’s t-test. (B) Lung weights (g) at 10 days after the i.n. challenge. The mean is shown as a horizontal line. Each symbol represents an animal. * p < 0.05 and ** p < 0.1 by the Student’s t-test. (C) Number of C. muridarum IFUs recovered from the lungs at day 10 after the i.n. challenge. The median is shown as a horizontal line. Each symbol represents an animal. * p < 0.05 by the Mann–Whitney U test.

Figure 8

Immune responses in the lungs of mice at 10 d.p.c. (A) IFN-γ levels in lung supernatants at 10 d.p.c. The mean is shown as a horizontal line. Each symbol represents an animal. * p < 0.05 and ** p < 0.1 by the Mann–Whitney U test. (B) C. muridarum-specific IgA levels in lung supernatants at 10 d.p.c. The mean is shown as a horizontal line. Each symbol represents an animal. * p < 0.05 and ** p < 0.1 by the Student’s t-test.