Recombinant Protein CPSIT_p8 Induced Protective Immunity Against Chlamydia Psittaci Infection In A Mouse Model

Objective: To investigate the protective immunity of recombinant protein CPSIT_p8 of Chlamydia psittaci, and provide novel and important information on the development of C. psittaci subunit vaccine.Methods: C. psittaci 6BC genome DNA was extracted as the temple and then CPSIT_p8 gene was amplified by polymerase chain reaction(PCR) and cloned into the vector to construct the prokaryotic recombinant plasmid pET-30a-CPSIT_p8, and finally transformed into E.coli BL21. The expression of recombinant protein was induced by IPTG and identifed by western blotting. The recombinant protein was purified with Ni-NTA-His affinity chromatography and concentrations were assessed by using a BCA protein assay kit. Female BALB/c mice were immunized intraperitoneally with purified recombinant protein CPSIT_p8.The titer of polyclonal antibodies was determined by indirect ELISA.Besides, using the anti-CPSIT_p8 protein antibodies to investigate and analysis the subcellular localization of CPSIT_p8 plasmid protein byconfocal laser scanning microscopy. Two weeks after the final immunization, three mice from each group were euthanized, and the spleens were assayed for IFN-γ and IL-4 production by double antibody sandwich ELISA. Meanwhile, the mice were challenged intranasally with5.0 × 105 IFUs of C. psittaci under ether anaesthesia, and the incidence,weight and survival rate of the mice were observed. The mice were euthanized 10 days after the infection. The lungs were harvested, weighed and homogenized in SPG for the detection of viable C. psittaci and cytokine levels. Intracellular inclusions were counted under a fluorescence microscopy. Lungs of immunized and the control mice were embedded in paraffin and for the histopathological evaluation lungs were sectioned and stained with haematoxylin-eosin(H&E) and S-P immunohistochemistry to evaluate protective immunity of recombinant protein CPSIT_p8. Besides, in vivo or in vitro neutralisation of C. psittaci were peformed to assess the neutralising ability of the CPSIT_p8-specific antibodies.Results: 1. The recombinant expression plasmid pET-30a-CPSIT_p8 was constructed successfully and the identification was performed by PCR and the sequencing assay. the sequencing results were in complete according to the sequences on Genbank. The recombinant proteins were expressed in supernatant with a predicted size of 17 kDa by SDS-PAGE. The identification of fusion protein CPSIT_p8was performed by western blotting. 2. Female BALB/c mice(6-8 weeks old) were immunized intraperitoneally with purified CPSIT_p8 in 3 times at 2-week intervals. The titers of IgG were 1: 1024, 000. 3. HeLa 229 cells were infected with C. psittaci 6BC and were then cultivated for 36 h before harvesting. The cells were fixed, immunolabeled with anti-CPSIT_p8, anti-C. psittaci 6BC antibodies and DAPI, and viewed under a confocal laser scanning microscopy. The endogenous CPSIT_p8protein was localized within the inclusion of C. psittaci 6BC-infected cells. 4. The cytokine levels of the spleen cells supernatants were assayed by ELISA. the level of IFN-γ in the spleen cells supernatants of CPSIT_p8-immunized mice(1, 734. 54 ± 21. 48 pg/ml) was significantly higher, compared to those of the FA(195. 35 ± 3. 22 pg/ml) and PBS(168. 91 ± 17. 74 pg/ml) control mice(P < 0. 001). The levels of IL-4were very low, in all the groups entered in the study(P > 0. 05). 5. Mice were infected with 5. 0 × 105 IFUs of C. psittaci 2 weeks after the final immunization and then euthanized 10 days later. Lung homogenates were inoculated onto HeLa 229 cell monolayers, and the chlamydial inclusions were counted by indirect immunofluorescence. Immunization of the mice with recombinant protein CPSIT_p8 elicited a significantly lower chlamydial burden, compared to the control mice(P < 0. 05). The level of IFN-γ in the lungs of immunized mice was significantly lower, compared to the control mice(P < 0. 001). The lung tissue sections observed byH&E and S-P immunohistochemistry. The results revealed that lung sections of C. psittaci infected mice immunized with CPSIT_p8 protein were lower pathological changes, compared to the control mice. The C.psittaci inclusion brown granules in the nucleus of lung tissues of mice immunized with CPSIT_p8 protein were significantly lower, compared to controls. 6. In vivo or in vitro neutralisation of C. psittaci showed that the titre of viable C. psittaci was not notably lower in the lungs of mice treated with serum of CPSIT_p8-immunized as compared to those of the control mice(P > 0. 05).Conclusions: Recombinant protein CPSIT_p8 can elicit high level of Th1 cellular immune response against C. psittaci infection in mice.