Chlamydia Psittaci Plasmid-encoded Protein CPSIT_p7 Induced The Protective Immunity

Objective:To investigate CPSIT_p7 recombinant protein to induce specific humoral and cellular immune response,and explore the protective efficacy and protection mechanism agnist C.psittaci infection in female BALB/c mice.Methods:After the large scale expression of CPSIT_p7 protein,the recombinant protein was purified by using Ni-NTA affinity resin according to the manufacturer’s instructions and the concentrations of purified fusion proteins were determined with a BCA protein assay Kit.Pathogen-free 6-week-old female BALB/c mice were immunized three times with fortnightly interval via intraperitoneal injection（i.p.）with100μL containing 40μg purified recombinant protein CPSIT_p7 diluted in PBS or Freund’s adjuvant（FA）,FA and PBS used as controls.The indirect ELISA was used to determine the titer of polyclonal antibodies of immunized mice and western blotting was use to authenticate the CPSIT_p7 specific antibodies.Two-weeks after the last vaccination,splenic cells from each mouse were resuspended in RPMI-1640 and seeded onto culture plates,each well was added 10μg/mL of puried-CPSIT_p7 to re-stimulation,and the plate was continue to incubate for 48h.Lymphocyte proliferative capacity was estimated by using a Cell Counting Kit-8 and the spleens were also assayed for the production of IL-2,IL-4,IL-10 and IFN-γvia using ELSIA Kit.Subsequently,two-weeks after the last vaccination,each immunized and control mouse were proceed ether anesthesia and then infected intranasally（i.n.）with 5×10⁵ IFUs of C.psittaci,30μl/mouse,and the food-intake,weight and activity were observed.Ten days later,all of infection mice were sacrificed,and the lungs were dissected and homogenized in SPG buffer for determining C.psittaci titers and cytokine levels;lungs of immunized and the control mice were stained with haematoxylin-eosin（H&E）and S-P immunohistochemistry to evaluate pathological lesion.The neutralisation of in vitro or in vivo of C.psittaci were designed to evaluated the capacity of the antibodies to protect against C.psittaci infection.Meanwhile,the spleen cells collected from CPSIT_p7-immunised or control mice two weeks after the last immunisation were depleted CD4⁺or CD8⁺T cells by the selective transfer,and then infected into the tail vein of native mice.Lungs of mice were isolated and homogenised in SPG on day 7 post-infection.C.psittaci titers of the lungs were measured.Results:1.The CPSIT_p7 protein was expressed with 0.6mM IPTG,and the weight was 28 kDa.The result was verified by western blotting.2.The level of Ig G was up to 1:1,320,000 after immunized with CPSIT_p7proteins in three times.3.Lymphocyte proliferative capacity was estimated by using a Cell Counting Kit-8,and the result showed that stimulation index（SI）of group of CPSIT_p7-immunized was up to 2.8,and significantly higher than negative controls（P<0.01）;the expression of IL-4 and IL-10 were no significant differences between the CPSIT_p7-immunized and the control mice（P>0.05）,nevertheless,the expression of IFN-γ（1537.94±128.45）pg/mL of protein-immunized mice were significantly higher than PBS control group（92.79±84.86）pg/mL and FA group（81.79±34.23）pg/mL（P<0.001）.4.After the C.psittaci infection,lung homogenates were prepared,and the number of C.psittaci inclusions were detected by IFA.The mice of CPSIT_P7-immunized（3.41±0.47）×10⁵ showed to a significantly lower chlamydial load,compare to PBS（18.3±1.3）×10⁵（P<0.01）or FA-immunized mice（14.9±1.94）×10⁵（P<0.05）.Meanwhile,the concentrations of IFN-γin the lungs of CPSIT_P7-immunized mice（1041.44±187.92）pg/mL was lower than FA control mice（3892.92±236.78）pg/mL and PBS control mice（3751.3±563.37）pg/mL（P<0.001）,and there was no significant differences in the levels of IL-4,IL-6,IL-10,IL-12 and IL-17 between protein-immunized and control animal（P>0.05）.The lung tissue sections stained with H&E and S-P immunohistochemical staining.The results showed that the degree of pathological damage of protein-immunized mice was significant less than control mice,and a remarkably lower chlamydial burden（brown granule）.5.The results of neutralisation indicated that the number of C.psittaci has no different significantly between treated with protein immune sera and control sera（P>0.05）.6.The adoptive transfer of CPSIT_p7-specific spleen cells depleted of CD4⁺T cells have no significant difference in C.psittaci load（P>0.05）,while the adoptive transfer of CPSIT_p7-specific spleen cells depleted of CD8⁺T cells significantly reduced the C.psittaci burden in the lungs,compared to controls（P<0.001）.Conclusions:Vaccination with CPSIT_p7 protein was able to induce a strong Th1-type immunity response and elicited protective effect aganist C.psittaci infection,and the CPSIT_p7-specific CD4⁺T cells could reduce the chlamydial load in the lungs of native mice after the challenge.The above results provide important direction for future studies about vaccine development against C.psittaci infection.