A TLR2 agonist is a more effective adjuvant for a Chlamydia major outer membrane protein vaccine than ligands to other TLR and NOD receptors

Abstract

Chlamydia trachomatis (Ct) is the most common sexually transmitted bacterial pathogen in the World and there is an urgent need for a vaccine to prevent these infections. To determine what type of adjuvant can better enhance the immunogenicity of a Chlamydia vaccine, we formulated the recombinant major outer membrane protein (Ct-rMOMP) with several ligands for Toll-like receptors (TLR) and the nucleotide-binding oligomerization domain (NOD) including Pam(2)CSK(4) (TLR2/TLR6), Poly (I:C) (TLR3), monophosphoryl lipid A (TLR4), flagellin (TLR5), imiquimod R837 (TLR7), imidazoquinoline R848 (TRL7/8), CpG-1826 (TLR9), M-Tri-(DAP) (NOD1/NOD2) and muramyldipeptide (NOD2). Groups of female BALB/c mice were immunized intramuscularly (i.m.) three times with the Ct-rMOMP and each one of those adjuvants. Four weeks after the last immunization the mice were challenged intranasally (i.n.) with 10(4)C. trachomatis mouse pneumonitis (MoPn) inclusion forming units (IFU). As negative antigen control, mice were immunized with the Neisseria gonorrhoeae recombinant porin B (Ng-rPorB) and the same adjuvants. As a positive vaccine control, mice were inoculated i.n. with 10(4)IFU of MoPn. The humoral and cell mediated immune responses were determined the day before the challenge. Following the challenge the mice were weighed daily and, at 10 days post-challenge (p.c.), they were euthanized, their lungs weighted and the number of IFU in the lungs counted. As determined by the IgG2a/IgG1 ratio in the sera, mice immunized with Ct-rMOMP+Pam(2)CSK(4) showed a strong Th2 biased humoral immune response. Furthermore, these mice developed a robust cellular immune response with high Chlamydia-specific T cell proliferation and levels of IFN-γ production. In addition, based on changes in body weight, weight of the lungs and number of IFU recovered from the lungs, the mice immunized with Ct-rMOMP+Pam(2)CSK(4), were better protected against the i.n. challenge than any group of mice immunized with Ct-rMOMP and the other adjuvants. In conclusion, Pam(2)CSK(4) should be evaluated as a candidate adjuvant for a C. trachomatis vaccine.

Figure 1. Western blot analysis of serum samples from the day before the i.n. challenge with C. trachomatis

C. trachomatis MoPn EB were used as the antigen. Lane 1, molecular weight standards. Lanes 2 to 7, sera from mice immunized with: Ct-rMOMP + Pam₂CSK₄; Ct-rMOMP + Poly (I:C); Ct-rMOMP + CpG-1826; Ng-rPorB + Pam₂CSK₄; Ng-rPorB + Poly (I:C); Ng-rPorB + CpG-1826, respectively. Lane 8) C. trachomatis MoPn EB. Lane 9) MEM-0. Lane 10) Pre-immunization sera.

Figure 2. Changes in body weight following the i.n. challenge with C. trachomatis

A. Daily percentage change in mean body weight following the i.n. C. trachomatis challenge. *, p < 0.001 as determined by the Repeated Measures ANOVA. B. Percentage change in mean body weight of each group at day 10 after the i.n. C. trachomatis challenge. The mean is indicated as a short line. Each symbol represents a single animal.

Figure 3. Disease burden and Chlamydia yields from the lungs at day 10 following the i.n. C. trachomatis challenge

A. Lungs weight (g) at day 10 after the i.n. C. trachomatis challenge. The mean is shown as a short line. Each symbol represents a single animal. B. Number of Chlamydia IFU recovered from the lungs at day 10 after the i.n. challenge. The median is shown as a short line. Each symbol represents a single animal.