Chlamydial protease-like activity factor mediated protection against C. trachomatis in guinea pigs

Abstract

We have comprehensively demonstrated using the mouse model that intranasal immunization with recombinant chlamydial protease-like activity factor (rCPAF) leads to a significant reduction in bacterial burden, genital tract pathology and preserves fertility following intravaginal genital chlamydial challenge. In the present report, we evaluated the protective efficacy of rCPAF immunization in guinea pigs, a second animal model for genital chlamydial infection. Using a vaccination strategy similar to the mouse model, we intranasally immunized female guinea pigs with rCPAF plus CpG deoxynucleotides (CpG; as an adjuvant), and challenged intravaginally with C. trachomatis serovar D (CT-D). Immunization with rCPAF/CpG significantly reduced vaginal CT-D shedding and induced resolution of infection by day 24, compared with day 33 in CpG alone treated and challenged animals. Immunization induced robust anti-rCPAF serum IgG 2 weeks following the last immunization, and was sustained at a high-level 4 weeks post challenge. Upregulation of antigen-specific IFN-γ gene expression was observed in rCPAF/CpG-vaccinated splenocytes. Importantly, a significant reduction in inflammation in the genital tissue in rCPAF/CpG-immunized guinea pigs compared with CpG-immunized animals was observed. Taken together, this study provides evidence of the protective efficacy of rCPAF as a vaccine candidate in a second animal model of genital chlamydial infection.

Figure 1

Vaccination enhanced chlamydial clearance from the guinea pigs genital tract. Groups (n =5) of guinea pigs were immunized i.n. with rCPAF/CpG or CpG alone (mock) and boosted twice at two-week intervals. Another group (n = 5) of guinea pigs received one i.n. dose of live C. trachomatis serovar D EBs (CT-D; 1×10⁵ IFUs). One month after the final immunization, guinea pigs were challenged i.vag. with 10⁵ IFU CT-D. Chlamydial shedding was monitored every third day post challenge until day 36 and presented as mean ± SD for each group at each time point. * Significant reductions (p < 0.05; one-way ANOVA) in bacterial shedding by (a) bar graph and (b) overall area and (c) area under the curve between the indicated group and CpG-immunized (mock) guinea pigs are shown. (d) The number of guinea pigs shedding Chlamydia after genital challenge for each immunization group is summarized. (c) ** Significant reduction (p=0.002), ANOVA with Tukey B) between groups; (d) ** Significant reduction (p=0.006 and p=0.0001, respectively, Fisher’s exact test) in number of rCPAF+CpG and CT-D immunized animals, compared to CpG immunized animals, shedding chlamydia across all time-points evaluated. Results are representative of two independent experiments.

Figure 2

Vaccination induced antigen specific serum IgG responses in guinea pigs. Groups (n=5–6) of guinea pigs were immunized i.n. with CpG alone (mock), rCPAF/CpG or CT-D (1×10⁵ IFUs). Guinea pigs were rested for 4 (CpG, rCPAF/CpG) or 8 (CT-D) weeks before challenging i.vag. with 1×10⁵ IFU of CT-D. Total serum IgG reacting with rCPAF (a) and UV-inactivated CT-D (b) was determined 2 weeks prior to (pre-challenge) and 4 weeks after (post-challenge) challenge. * p < 0.05, ** p < 0.01 (One-Way ANOVA) comparison between indicated groups. Results are presented as average ± standard deviation of endpoint titer of each group. Results are representative of two independent experiments.

Figure 3

Vaccination induced antigen specific IFN-γ gene expression. Guinea pigs (n=3 per group) were euthanized one day before challenge and splenocytes were stimulated with 0.5µg of rCPAF or UV-inactivated CT-D (1×10⁵ IFUs), or unstimulated (media alone), for 24 hrs. IFN-γ gene expression was then measured by qRT-PCR analysis and normalized to respective GAPDH expression for each sample. Subsequently, within each vaccination group, IFN-γ expression by rCPAF and CT-D (UV) stimulation was calculated and presented as a relative value to medium mock stimulation. Significant increase in IFN-γ expression between stimuli was indicated * p < 0.05, ** p < 0.01 (One-Way ANOVA). Results are representative of two independent experiments.

Figure 4

Vaccination reduced histopathological lesions in indicated genital tracts following chlamydial challenge. Histopathology was assessed in naïve (uninfected/ healthy) and Chlamydia challenged CpG-, rCPAF/CpG-, CT-D-vaccinated guinea pigs. The genital tract of each guinea pig was removed at day 65 post CT-D challenge, sectioned, H&E stained, and analyzed microscopically (original magnification of the images is 100× or 200×). Histopathological injury in the genital tract of representative healthy and diseased guinea pigs was scored (a) and graphically represented for the respective group as a whole (b), for four distinct parameters (inflammatory cell infiltration, congestion, hemorrhage and edema). Obviously, the individual micrographs shown (a) represent the types of pathology observed in different regions of the genital tract of healthy and diseased animals, whereas the graph (b) summarizes these types of observations for all sections of all regions of all animals examined. The asterisk indicates significant reductions (* p <0.05; Kruskal-Wallis test with Dunns post hoc test) between CT-D or rCPAF/CpG immunized groups in comparison to CpG group for the respective parameters.