| Objective: Chlamydia trachomatis(Ct)is a strictly intracellular parasitic and highly prevalent pathogen,which seriously endangering humans healthy owing to various diseases caused by infecting human.Ct infection is often asymptomatic resulting in recurrent infections and delayed treatment.Therefore,an effective vaccine is the fundamental measure to prevent and control Ct infection.Previous studies have shown that Pgp3 protein encoded by Ct plasmid is an immunodominant peptide requiring adjuvant platform to enhance the strong immune response because of its poor immunogenicity.Development of effective adjuvant relying on antigen delivery and immune enhancement is promising.Pickering emulsion as a new type of emulsion was stabilized by solid particles adsorbed to the two-phase interface,which has great potential in targeted delivery for antigens.Chitosan(CS)particles tended to absorb on the two-phase interface to form stable CS-Pickering emulsion based on its biodegradability,biocompatibility and low toxicity.In addition,the cytokine IL-12 remains a powerful immune enhancer due to its unique biological effects.Therefore,a hypothesis that an adjuvant system based on the adjuvant properties of CS-Pickering and IL-12 is contribute to enhance the immune level of Pgp3 protein vaccine,which may provide important value and significance for the prevention and control of Ct infection.Objective: Mice were immuned with vaccine preparation by combing Pgp3 protein with CS-Pickering/IL-12 adjuvant,and the immune protection was evaluated using mice models infected by Chlamydia muridarum(Cm)to explore the value of CS-Pickering/IL-12 and Pgp3 protein in Ct vaccine and adjuvant application.Methods: CS soultion was sonicated with the oil phase to obtain the optimized CS-Pickering under different conditions including ultrasonic power,p H,CS particle concentration,water/oil volume ratio and oil phase types.In addition,the adjuvant potential of CS-Pickering was assessed in vitro by examining the ability of proteins uptake by macrophages and antigen loading.CS-Pickering was prepared and subsequently combined with recombinant mouse IL-12.CS-Pickering/IL-12 adjuvant system was used to combing with Pgp3 protein to the form vaccine preparation.4-week-old female Balb/c mice were selected and randomly divided into groups,and then were immunized three times at 0,2,and 4 weeks.All groups included PBS group,Pgp3 vaccine group,CSPickering/Pgp3,IL-12/Pgp3 and CS-Pickering/IL-12/Pgp3.Ig G,Ig G1 and Ig G2 a were detected by ELISA in mouse serum at 2th,4th,and 6th weeks after immunization;The vaginal washes liquid of mice was harvested at the 6th week after immunization to evaluate s Ig A levels;Mouse splenocytes were collected at the 6th week after immunization to evaluate the splenocytes proliferation relying on CCK8 assay and the levels of cytokines(IFN-γ,TNF-α,IL-4 and IL-2)secreted by splenocyte supernatant.A month after the final immunization,the mice were intravaginally challenged with Cm and the vaginal samples of the mice were collected with swabs at three or four days apart after the challenge.IFA was utilized to evaluate the chlamydia load in the lower genital tract in mice.Mice were sacrificed on the 60 days after challenge to separate the uterine tissue.The oviduct hydrosalpinx were observated and scored with the visual scoring rules;And then the uterine pathological histological and inflammatory were further evaluated by conventional paraffin section stained by HE.The biosafety of vaccine preparation was assessed with regard to the cytotoxic effect of CS-Pickering on macrophages detected by CCK8 and the serum biochemical indicators of immunized mice detected by biochemical analyzer including LDH,ALT,BUN,AST and ALP.Results:1.The average size,average Zeta potential and PDI of the most optimized emulsion were 789.47±44.26 nm,33.2±3 m V and 0.427±0.12,respectively(P < 0.05).2.CS-Pickering emulsions were prepared by mixing Rh B-labeled CS particles with LP,and it was observed that CS particle was mainly located at the droplets interface under a fluorescence microscope.3.FITC-BSA served as model antigen mixed with CS-Pickering emulsions on ice,the results suggesting that CS-Pickering emulsions adsorbed FITC-BSA.89.04±3.8% of Pgp3 protein was adsorbed by CSPickering emulsions within 4h(P<0.05).4.Macrophages were co-cultured with the CS-Pickering emulsions absorbed FITC-BSA,and macrophages were stained after incubation.The results showed that the CS-Pickering/FITC-BSA mixture was founded in macrophages,indicating that the macrophages could uptake CSPickering/FITC-BSA.5.As demonstrated by serum specific antibody Ig G,subtypes Ig G1,Ig G2 a and vaginal washes liquid s Ig A,compared with Pgp3 vaccine group,CS-Pickering/IL-12 adjuvant system significantly enhanced the antibody level.6.As demonstrated by proliferative capacity of splenocytes and the cytokine levels(IFN-γ,TNF-α,IL-4 and IL-2),and the results showed that proliferative capacity of splenocytes was effectively promoted by CS-Pickering/IL-12 adjuvant system than other groups(P<0.05).It was found that IFN-γ,IL-2,TNF-α and IL-4 in CS-Pickering/IL-12/Pgp3 immunized groups were higher than those in PBS group and Pgp3 vaccine group.Morever,the IFN-γ level induced by CS-Pickering/IL-12 was 5 times and 2 times higher than CS-Pickering and IL-12,respectively(P<0.05),and the TNF-α induction was 2 times and 3 times higher than CS-Pickering and IL-12,respectively(P<0.05).The level of IL-2secretion induced by CS-Pickering/IL-2 was 17% higher than the IL-12 adjuvant.7.As demonstrated by the chlamydia shedding in the lower genital tract.The results showed that notable reduction of the chlamydia shedding on day 17 and infection clearance on day 21 in CSPickering/IL-12/Pgp3 group.8.The mice were sacrificed on the 60 th day after the challenge,the uterine tissue was removed to evaluate the hydrosalpinx and histopathological damage.No obvious hydrosalpinx and inflammatory cells infiltration in the oviduct tissues of mice vaccinated with the CSPickering/IL-12/Pgp3(P<0.05).9.The results revealed limited toxicity effects on macrophages caused by CS-Pickering and there was no change in serum biochemical indexes of each group mice,suggesting acceptable biosafety of vaccines.Conclusions: Chitosan-Pickering emulsion/IL-12 adjuvant system for Pgp3 subunit vaccine elicited immune protection against-genital chlamydial infection in mice. |
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