Immunogenicity And Immune Protection Of Chlamydia Muridarum TC0668 Mutant Strain In Mice Models Of Reproductive Tract Infection

Background:Chlamydia is a kind of strictly intracellular parasitic prokaryotic microorganisms,which can cause diseases to both human and animals.Chlamydial infection of human conjunctiva,respiratory tract,and reproductive tract can cause conjunctivitis,respiratory and reproductive tract infection and other diseases.Chlamydia trachomatis?Ct?is one of the major pathogens of sexually transmitted infection?STI?in many countries and regions,which can not only cause trachoma and inclusion body conjunctivitis,but also cause diseases such as reproductive tract infections.Chlamydia muridarum?Cm?is often used to establish mice models of Ct infection of female reproductive tract due to its germline is closely related to that of Ct.TC0668 of Cm is a chromosomal virulence protein that is involved in inflammatory lesions of the fallopian tubes of mice.TC0668 wild-type strain(Cm TC0668^wt)and TC0668 mutant strain(Cm TC0668^mut)were inoculated in mice in parallel to establish a genital tract infection model.Compared with Cm TC0668^wtt strain,Cm TC0668^mut strain induced the decreasing pathogenicity in mice obviously,but the value of Cm TC0668^mut strain as a live attenuated vaccine in the prevention of chlamydia infective fallopian tube disease is not very clear.Objective:We tested the immunogenicity and immune protection of the Cm TC0668^mut strain as a live vaccine by immunizing mice and then use Cm Nigg?strain challenging mice,and explored the application value of Cm TC0668^mut strain in live vaccines.Methods:Cm Nigg?strain,Cm TC0668^mut strain,Cm plasmid-free?PF?strain and SPG were simultaneously immunized to BALB/c female mice,vaginal swabs of each mice were collected to detect the chlamydial load of genital tracts by IFA at 3 to 7 days after immunization.Blood was collected from the tail vein of mice at 4 and 8 weeks after immunization,and serum IgA,IgG and its subclasses IgG1 and IgG2a antibody levels were measured by ELISA.The secreted IgA?sIgA?antibody level of mice vaginal washes at 4 and 8 weeks after immunization were collected and detected by ELISA.At 60 days after immunization,mice spleen cells were collected and cytokines?IFN-?,TNF-?,IL-4 and IL-10?secreted by spleen cell supernatant were detected by ELISA,and further detected the expression level of cytokines?IFN-?and IL-4?after spleen cell stimulation by flow cytometry.The mice genital tracts were observed to evaluate the degree of hydrosalpinx of the fallopian tube,and take the pathological section of the genital tract tissue to score the degree of hydroscopic expansion and inflammation of the fallopian tube.After 60 days of immunization,Cm Nigg?strain were used to challenge each group of mice,the vaginal swabs of mice were taken every3 or 7 days after infection to detect the re-infection of Chlamydia in the lower genital tract by IFA,and the mice were sacrificed on the 60th day after the challenge.Edema was scored to evaluate the immune protection of the attenuated strain on mice infected with Cm Nigg?strain.Results:?1?Prepared the suitable concentrated?2.0?10⁵ IFUs?chlamydial strains,quantified the concentration of Chlamydia by IFA and performed PCR sequencing.The sequencing results showed that the Cm TC0668^mutut strain had mutation at specific base site,which is fully consistent with the genotype of the mutant strain.?2?The vaginal swabs of the mice were collected every 3 or 7 days to detect the chlamydial load of the genital tract after immunization.The results showed that the Chlamydia in the genital tract decreased gradually and all were cleared at 28 days after immunization.?3?Serum-specific antibody IgA,IgG and its subclasses IgG1 and IgG2a levels showed that the IgA antibodies levels produced by mice in each group were lower,and the immunized mice in Cm Nigg?strain,Cm TC0668^mut strain and Cm PF strain groups had higher levels of IgG antibodies,and their ratio of IgG1/IgG2a was<1,indicating that Cm TC0668^mut strain can induce immune response biased Th1 type.?4?The results of sIgA antibody levels in the vaginal washes showed that the sIgA antibody levels of each group of mice were lower,did not increase significantly at 8 weeks after immunization compared to 4 weeks,indicating that the immunized mice cannot induce an effective specific mucosal immune response.?5?The cytokine expression level produced by spleen cell supernatant was detected by ELISA.The results showed that the spleen cells of the Cm TC0668^mut?G13.11.1,tc0668 single gene mutant strain?,Cm TC0668^mut?G28.51.1,tc0668 and tc0237 double gene mutant strain?and Cm PF strain immunized mice produce higher levels of IFN-?and TNF-??produced by Th1 type CD4⁺T cells?and are significantly higher than the SPG negative control group.The levels of IL-4 and IL-10?produced by Th2 T cells?produced by spleen cells of mice in each group were lower,and there was no significant statistical difference between the groups.The above results indicate that the specific cellular immune response induced by the Cm TC0668^mut strain is biased toward the Th1 type.?6?Flow cytometry analysis of Th1/Th2 subtypes of spleen cells in mice after immunization showed that the CD4⁺T cells in spleen lymphocytes of Cm TC0668^mut?G13.11.1?strain,Cm TC0668^mut?G28.51.1?strain and Cm PF strain group were able to secrete higher levels of IFN-?.The level of IL-4 secreted by spleen lymphocytes of mice in each group was low,and there was no significant statistical difference between the groups.The result further indicates that the specific cellular immune response induced by the Cm TC0668^mut strain is biased towards the Th1type.?7?At 60 days after immunization,the degree of hydrosalpinx and infiltration of inflammatory cells showed that the incidences of hydrosalpinx in the Cm TC0668^mut strain and the Cm plasmid-free strain immunized mice were similar and lower than the Cm Nigg?strain,the incidence of fallopian tubes in Cm TC0668^mut?G28.51.1?strain immunized mice was lower than the Cm TC0668^mut?G13.11.1?strain.Fallopian tube dilatation and inflammatory cell infiltration showed that Cm Nigg?strain can cause fallopian tube expansion and obvious inflammatory cell infiltration.Cm TC0668^mut strain and Cm PF strain immunized mice have no obvious fallopian tube expansion and inflammation infiltration.?8?After the Cm Nigg?strain challenging the mice,the vaginal swabs of the mice are taken every 3 or 7 days to detect the chlamydial load.The results showed that the chlamydial load in the genital tract gradually decreases after the chlamydial re-infection of the mice,and all of them are fully cleared at 35 days after infection.?9?The degree of hydrosalpinx was observed at 60 days after challenge,the results showed that the degree of fallopian tube lesions caused by Cm Nigg?strain challenged with the Cm TC0668^mut strain and Cm PF strain immunized mice was slight,and no obvious fallopian tube was formed.But the degree of fallopian tube lesions caused by Cm Nigg?and SPG group was decreased.Conclusions:The pathogenicity of Cm TC0668^mut strain is lower than Cm Nigg?strain,the pathogenicity of G28.51.1 strain and Cm PF strain are similar and lower than G13.11.1 strain;TC0668^mut strain can induce specific humoral immune response;The mediated immune response of TC0668^mut strain is biased towards Th1 type;TC0668^mut strain immunized mice does not cause obvious hydrosalpinx after being challenged by Cm Nigg?strain and has the immune protection against chlamydial infection.Therefore,Cm TC0668^mut strain may be a potential attenuated live chlamydial vaccine.