Heat denatured enzymatically inactive recombinant chlamydial protease-like activity factor induces robust protective immunity against genital chlamydial challenge

Abstract

We have shown previously that vaccination with recombinant chlamydial protease-like activity factor (rCPAF) plus interleukin-12 as an adjuvant induces robust protective immunity against primary genital Chlamydia muridarum challenge in mice. Since CPAF is a protease, we compared the effects of enzymatically active and inactive (heat denatured) rCPAF to determine whether proteolytic activity is expendable for the induction of protective immunity against chlamydial challenge. Active, but not inactive, rCPAF immunization induced high levels of anti-active CPAF antibody, whereas both induced robust splenic CPAF-specific IFN-gamma production. Vaccination with active or inactive rCPAF induced enhanced vaginal chlamydial clearance as early as day 6 with complete resolution of infection by day 18, compared to day 30 in mock-vaccinated and challenged animals. Importantly, significant and comparable reductions in oviduct pathology were observed in active and inactive rCPAF-vaccinated mice compared to mock-vaccinated animals. Thus, rCPAF induced anti-chlamydial immunity is largely independent of enzymatic activity and secondary or higher order protein conformation.

Figure 1. Molecular size and enzymatic activity of rCPAF

(A) GST-rCPAF was purified using glutathione Sepharose 4B and loaded in a 10% SDS-polyacrylamide gel and stained with coomassie blue. (B) GST-rCPAF and GST alone were probed with mouse anti-CPAF c-terminus monoclonal antibody. (C) Purified active or inactive rCPAF from C. muridarum, or S100 fraction from HeLa cells infected with C. trachomatis (L2S100), or GST were incubated with cytosolic extract (CE) from the HeLa cells at 37°C for 3 hr. The full length and degraded keratin-8 fragments were detected using mouse anti-human keratin-8 primary antibody followed by goat anti-mouse secondary antibody and developed using an ECL reagent. Lanes 1: CE only, 2: CE+native CPAF, 3: CE+active rCPAF, 4: CE+denatured rCPAF, and 5: CE+GST.

Figure 2. Humoral responses after vaccination

Four to five week old female BALB/c mice (n=6) were immunized i.n. with active rCPAF, inactive rCPAF, rGST, or PBS (mock) plus IL-12 on day 0, and booster immunizations were given on days 14 and 28. Mice also were given IL-12 alone on days −1 and +1. Ten days after the final immunization, mice were bled, sera were obtained and analyzed for total antibody and various isotypes of antibodies against active rCPAF using an indirect ELISA. Results are represented as mean ± SE of reciprocal titers corresponding to the 50% maximum binding. Titers of rCPAF specific antibodies induced by immunization with active rCPAF immunization were significantly (P ≤ 0.05; Student’s t test) greater than those induced by immunization with inactive rCPAF or GST or PBS. Results are representative of two individual experiments.

Figure 3. Antigen-specific IFN-γ responses following vaccination

Four to five week old female BALB/c mice (n=6) were immunized i.n. with active rCPAF, inactive rCPAF, rGST, or PBS (mock) plus IL-12 on day 0, and booster immunizations were given on days 14 and 28. Mice were also given IL-12 alone on days −1 and +1. Twenty days after the final booster immunization, the mice (n=3) were euthanized, spleens collected and single cell suspensions made and stimulated in vitro with the indicated antigens. (A) rCPAF-specific IFN-γ response. Splenic cells from each group were stimulated with active rCPAF, unrelated antigen BSA, or cultured in media alone, and analyzed for IFN- γ production using an indirect ELISA. (B) Native CPAF-specific IFN-γ response. Cells were stimulated with L2S100 (S100 fraction of Chlamydia infected HeLa cells), BSA, or cultured in media alone, and analyzed for IFN- γ production. Results are represented as CPAF-specific responses, after subtracting the responses against GST, and reported as mean ± SE of titers of mice for individual groups. Significant difference (P ≤ 0.05; Student’s t test) in cytokine secretion between mock-vaccinated mice versus active or inactive rCPAF groups.

Figure 4. Chlamydial shedding from vagina following bacterial challenge in vaccinated mice

Four to five week old female BALB/c mice (n=6) were immunized i.n. with active rCPAF, inactive rCPAF, rGST, or PBS (mock) plus IL-12 on day 0, and booster immunizations were given on days 14 and 28. Mice were also given IL-12 alone on days −1 and +1. One month after the final immunization, mice were challenged i.vag. with 5×10⁴ IFU of C. muridarum. Vaginal swabs were obtained and analyzed for numbers of chlamydial IFU recovered. The shedding of bacteria was monitored every third day for 30 days. Results are represented as mean ± SE. Significant differences between mock-vaccinated versus active rCPAF (♣) or inactive rCPAF groups (*) (P ≤ 0.05, Mann-Whitney Rank Sum test).

Figure 5. Oviduct pathology following chlamydial challenge in vaccinated mice

Four to five week old female BALB/c mice (n=6) were immunized i.n. with active rCPAF, inactive rCPAF, rGST, or PBS (mock) plus IL-12 on day 0, and booster immunizations were given on days 14 and 28. One month after the final immunization, mice were challenged with 5×10⁴ IFU of C. muridarum and pathology was observed on day 80 post challenge. (A) Macroscopic oviduct dilatation. At 80 days post-challenge, animals were euthanized and genital tracts were collected and photographed. The cross sectional diameter for dilated oviducts was measured and the highest one was reported, with mean ± SE per group of mice. Significant reduction in oviduct dilatation with active and inactive rCPAF vaccination compared to PBS or GST (P < 0.05, Student t- test). (B) Microscopic dilatation. Genital tracts were collected, embedded in paraffin blocks, and sectioned (5μm thickness). These sections were stained with haematoxylin and eosin and the extent of dilatation reported as largest oviduct diameter/wall thickness ratio with mean ± SE. Significant difference between mock-vaccinated versus active rCPAF (♠) or inactive rCPAF (♣) vaccinated animals (P ≤ 0.05, Student t test). Results are representative of two individual experiments.