Intranasal immunization with chlamydial protease-like activity factor and CpG deoxynucleotides enhances protective immunity against genital Chlamydia muridarum infection

Abstract

We have reported recently that intranasal (i.n.) vaccination with chlamydial protease-like activity factor (CPAF) and interleukin-12 (IL-12) enhances protective immunity against genital chlamydial challenge. In this study, we show that i.n. or intraperitoneal (i.p.) vaccination with CPAF plus CpG deoxynucleotides (CpG), an alternative T helper 1 (Th1) adjuvant, induced robust CPAF-specific IFN-gamma responses and elevated levels of serum antibody and vaginal IgA production. CPAF+CpG vaccinated animals displayed accelerated genital chlamydial clearance, and minimal hydrosalpinx and inflammatory cellular infiltration compared to mock-immunized (PBS) challenged animals. Together, CpG dexoynucleotides are an efficacious alternative Th1 adjuvant with CPAF to induce protective anti-chlamydial immunity.

Fig. 1

Intranasal or i.p. CPAF+CpG vaccination induces robust cell-mediated immune responses. Animals (3 mice/group) were treated i.n. or i.p. with CPAF + CpG, CPAF + ODN, CpG, or PBS. On day 14, animals were euthanized and single cells prepared from spleens and analyzed for CPAF-specific recall cytokine responses by measuring IL-12, IFN-γ and IL-4 production by ELISA. Cells from each group were also stimulated with an unrelated antigen, hen egg lysozyme (HEL). * Significant differences in cytokine secretion between CPAF+CpG and CPAF+ODN immunization (P < 0.05, student's t test). Results are representative of two independent experiments.

Fig. 2

Intranasal or i.p. CPAF+CpG vaccination induces robust humoral immune responses. Animals (6 mice/group) were treated i.n. or i.p. with CPAF + CpG, CPAF + ODN, CpG, or PBS on days 0, 14, and 28. Sera or vaginal fluids were collected from animals two weeks after final immunization. (A) Systemic anti-CPAF Ab responses after immunization. Serum anti-CPAF antibody levels were analyzed by ELISA using CPAF-coated microtiter plates. Results are expressed as mean ± SD of reciprocal serum dilutions corresponding to 50% maximal binding. (B) Mucosal anti-CPAF IgA responses after immunization. Results are expressed as mean ± SD of the absorbance of undiluted vaginal fluids. * Significant differences between CPAF+CpG and CPAF+ODN immunized animals (P < 0.05, Kruskall-Wallis test). Results are representative of two independent experiments.

Fig. 3

CPAF+CpG vaccination enhances the resolution of a genital challenge. Animals (6 mice/group) were treated i.n. or i.p. with three doses of CPAF + CpG, CPAF + ODN, or PBS. One month following final vaccination, mice were challenged i.vag. with 5 × 10⁴ IFU of C. muridarum. At the indicated days following challenge, chlamydial shedding was measured. (A) & (C) Numbers of chlamydial inclusion forming units (IFUs) recovered from vaginal swabs at the indicated days after genital challenge. Results are expressed as Means ± SD. * Significant differences between PBS (mock) immunized animals and CPAF+CpG or CPAF+ODN vaccinated animals (P < 0.05, Kruskall-Wallis test). (B) & (D) Percentage of animals shedding Chlamydia after genital challenge. Significant differences were detected in the time required for resolution of infection between CPAF+CpG immunized mice and all other experimental groups (P = 0.0002, Kaplan-Meier test). Results are representative of three independent experiments.

Fig. 4

CPAF+CpG vaccination reduces the development of oviduct pathology. Animals (6 mice/group) were treated i.n. or i.p. with three doses of CPAF + CpG, CPAF + ODN, or PBS. One month following final vaccination, mice were challenged i.vag. with 5 × 10⁴ IFU of C. muridarum. At day 80 following challenge, animals were euthanized and tissues collected for further analysis. (A) Percentage of animals developing hydrosalpinx in different immunization groups after genital chlamydial challenge: B-bilateral and U-unilateral. (B) Quantitative histopathological scoring of oviduct dilatation. (C) Quantitative estimation of cellular infiltration into the genital tracts following chlamydial challenge. Means ± SD of histopathology and cellular infiltration scores are shown. * Significant differences between CPAF+CpG and mock-immunized (PBS) animals (P < 0.05, Mann-Whitney Rank-Sum test). Results are representative of two independent experiments.