Safety and efficacy of C. muridarum vaccines adjuvanted with CpG-1826 and four concentrations of Montanide-ISA-720-VG

Abstract

It is recommended that the adjuvant Montanide ISA 720 VG be used at a concentration of 70% v/v. At this concentration, Montanide causes at the site of immunization a local granuloma that can last for several weeks. To determine the safety and protective efficacy of a Chlamydia muridarum MOMP vaccine, formulated with CpG-1826 and four different concentrations of Montanide (70%, 50%, 30% and 10%), BALB/c (H-2^d) female mice were immunized twice intramuscularly. Local reactogenicity was significant for vaccines formulated with 70% or 50% Montanide but not for those inoculated with 30% or 10% Montanide. Robust humoral and cell mediated memory immune responses were elicited by the 70%, 50% and 30% Montanide formulations. Mice were challenged intranasally with 10⁴ C. muridarum inclusion forming units (IFU). Based on changes in body weight, lungs's weight and number of IFU recovered, mice vaccinated with the 70%, 50% and 30% Montanide formulations were significantly protected, but not mice receiving 10% Montanide. To conclude, we recommend the 30% Montanide concentration to be tested in humans and animal models to determine its safety and efficacy, in comparison to the 70% Montanide concentration currently used. The 30% Montanide formulation could significantly facilitate licensing of this adjuvant for human use.

Fig. 1. Evaluation of injection site reactogenicity.

At euthanasia, pictures from representative mice were taken of the vaccination site. Mice immunized with Montanide ISA 720 VG: (a) 70%, (b) 50%, (c) 30% and (d) 10%; (e) MOMP without adjuvants and (f) PBS.

Fig. 2. Antibody responses in serum and vaginal washes to C. muridarum EB following immunization.

Mice were vaccinated and blood and vaginal washes from each animal were collected the day before the i.n. challenge. a IgG1 and IgG2a ELISA GMT in serum. b In vitro neutralizing antibody GMT in serum and (c) IgG and IgA antibody GMT in vaginal washes. LOD Limit of detection. *P < 0.05 by Student’s t-test.

Fig. 3. Binding of serum antibodies from immunized mice to synthetic C. muridarum MOMP peptides.

Serum samples from vaccinated mice were collected the day before the i.n. challenge. Pooled serum samples from each group of mice were tested by an ELISA for their reactivity to 25-aa overlapping synthetic peptides corresponding to the C. muridarum mature MOMP. C. muridarum EB and MOMP were used as positive controls. C, a synthetic 25 mer non MOMP peptide, was used as negative control. Peptide 25, overlaps the N- and C- terminus of C. muridarum MOMP.

Fig. 4. Determination of cytokines levels in T-cells supernatants collected from vaccinated mice the day before the i.n. challenge.

Mice were immunized and the day before the intranasal challenge they were euthanized, their spleens collected, T-cells isolated using nylon wool columns and stimulated with C. muridarum EB, or with Concanavalin A as a non-specific stimulant, or with medium as a negative control. a IFN-γ levels in T-cell supernatants and (b) Levels of IL-4 in T-cell supernatants. Mice per group four. LOD Limit of detection. *P < 0.05 by Student’s t-test.

Fig. 5. Daily changes in mean body weight following the i.n. challenge with 10⁴ C. muridarum IFU and changes in body weight at D10 p.c.

Immunized mice were challenge i.n. with 10⁴ IFU of C. muridarum 4 weeks after the boost and changes in body weight recorded daily. At 10 days post-challenge mice were euthanized. Each group included 10 mice. a Percentage changes in daily mean body weight following the i.n. challenge with C. muridarum. *P < 0.05 by the Repeated Measures ANOVA. b % Changes in body weight at D10 p.c. *P < 0.05 by Student’s t-test. **P < 0.10 by Student’s t-test.

Fig. 6. Determination of lungs’ weights and chlamydia burden at D10 following the i.n. challenge with 10⁴ C. muridarum IFU.

Vaccinated mice were challenged i.n. with 10⁴ IFU of C. muridarum. At D10 p.c mice were euthanized, their lungs weighed, homogenized in 5 ml of SPG and cultured in HeLa-229 cell monolayers. Following staining with a monoclonal antibody to MOMP, the number of chlamydial inclusions was counted using light microscopy. Each group included 10 mice. a Lungs’ 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 by Student’s t-test. **P < 0.10 by Student’s t-test. b Number of C. muridarum IFU recovered from the lungs at D10 following the i.n. challenge. The median is shown as a horizontal line. Each symbol represents an animal. LOD Limit of detection. *P < 0.05 by the Mann–Whitney U-test.

Fig. 7. Immune responses in the lungs of mice at D10 p.c.

Vaccinated mice were challenged i.n. 4 weeks after the boost with 10⁴ IFU of C. muridarum. At D10 p.c. the mice were euthanized, their lungs weighed and homogenized in 5 ml of SPG. Following homogenization, the lungs were centrifuged, the supernatants collected and used to determine the levels of IFN-γ and C. muridarum specific IgA. Each group included 10 mice. a IFN-γ levels in lungs’ supernatants at 10 d.p.c. The mean is shown as a horizontal line. Each symbol represents an animal. *P < 0.05 by Student’s t-test. **P < 0.1 by Student’s t-test. b C. muridarum-specific IgA levels in lungs’ supernatants at 10 d.p.c. The mean is shown as a horizontal line. Each symbol represents an animal. *P < 0.05 by Student’s t-test. **P < 0.1 by Student’s t-test.