Characterization Of Chlamydia Psittaci Plasmid-encoded Protein CPSIT_p7 And The Protective Immunity Induced By CPSIT_p7-based Vaccines

Background and Objective: Chlamydia psittaci is a widely distributed and strict obligate intracellular zoonotic pathogen with multiple hosts and may cause diverse diseases in humans,mammals and birds.Very recently,plasmid-deficient chlamydial strains function as live attenuated vaccines against infection.Plasmid-encoded protein Pgp3,an significant virulence factor of Chlamydia,was the only protein that was secreted into the cytosol of the infected cells,participation in host-microbe interactions,disease,and immune responses.However,former studies concerning Pgp3 were mainly focused on Chlamydia trachomatis and Chlamydia muridarum,not C.psittaci.Besides,with susceptible ability,high infectivity and subclinical or persistence infection of C.psittaci combined with low immunity of population,has highlighted a new zoonotic threat of this pathogen.Despite the existence of effective antibiotic treatment regimens,it recurs easily,therefore,arise a significant public and human health threat,underscoring the need for vaccine.The research and development of C.psittaci vaccines are of great scientific significance in improving the standard of living and animal husbandry's healthy development.In this study,we focused on the C.psittaci plasmid-encoded Pgp3 protein.The identification and characterization of C.psittaci Pgp3(CPSIT_p7)was conducted by sub-cellular localization and homology analysis.We further synthesized and developed a chimeric muti-epitope peptide vaccine and a DNA vaccine based on CPSIT_p7 and evaluate the humoral and cellular immune responses after vaccination.The protective immunity of peptide and DNA vaccines against C.psittaci pulmonary infection was evaluated in BALB/c mice.Overall,our study could provide important insights towards understanding the potential of peptide and DNA vaccines based on C.psittaci Pgp3 as new vaccine antigens for inducing protective immunity against chlamydial infection,affording potential target of great value in C.psittaci prevention and control.Methods: 1.The regions encoding C.psittaci plasmid proteins were amplified and cloned into the expression vector.The resulting plasmid was confirmed.Recombinant proteins expression were induced with IPTG and purified.After ultrafiltration and endotoxins removing of recombinant proteins,the concentrations were estimated by using BCA protein assay kit.BALB/c mice were immunized with prepared proteins.Blood samples were collected for Western Blot and antibody titers measuring.C.psittaci 6BC was propagated in He La 229 cells.Rabbit anti-chlamydial organism antibody and rabbit anti-protein antibody were stained for immunofluorescence assay to define subcellular localization of plasmid proteins in C.psittaci-infected cells.2.C.psittaci 6BC plasmid genome sequence and all the 8 plasmid gene and protein sequence were obtained from Genbank.The Pgp3 and Pgp4 gene and protein sequences of all representative strains of Chlamydia were also obtained from Gen Bank.Similarity and homology analysis of Pgp3 and Pgp4 gene and protein sequences were further conducted.CPSIT_p7 and CPSIT_p8 were predicted by bioinformatics analysis.Prediction of dominant antigen epitopes was combined with all indexes.3.The CPSIT_p7-derived multi-epitope peptides designated SP were synthesized and evaluated the protective immunity.Mice were immunized three times with SP.Blood samples were collected.Two weeks after the third immunization,half mice of each group were euthanized and the serum and spleen samples were collected for measuring antibody titers and lymphocyte response.Fourteen days after the final immunization,mice were challenged intranasally with C.psittaci to evaluate the protective efficiencies.On day 10,all mice were sacrificed and the lung tissues were isolated and partial homogenized for cytokine level and C.psittaci burden determinations.Other lung tissues were stained with hematoxylin-eosin(H&E)and streptavidin-peroxidase(S-P)immunohistochemistry.4.The region encoding CPSIT_p7 were amplified and cloned into the pc DNA3.1.The resulting eukaryotic plasmid was confirmed.Recombinant eukaryotic vector pc DNA3.1/CPSIT_p7 were extracted.He La 229 cells were transfected with either pc DNA3.1,pc DNA3.1/CPSIT_p7 and the expression in He La cells were detected by Western Blot.Mice were immunized three times.Blood samples were collected for measuring antibody titers.Fourteen days after the final immunization,mice were challenged intranasally to evaluate the protective efficiencies.On day 10,all mice were sacrificed and the lung tissues were isolated and partial homogenized for cytokine level and C.psittaci burden determinations.Other lung tissues were stained with hematoxylin-eosin(H&E)and streptavidin-peroxidase(S-P)immunohistochemistry.On day 10,all mice were sacrificed and the heart,liver,spleen,kidney and brain tissues were isolated.Parts of tissues were stained with streptavidin-peroxidase(S-P)immunohistochemistry to detect the pathology and C.psittaci burden.Quantitative PCR(q PCR)was used to assess the C.psittaci burdens.Results: 1.The target ORFs were successfully amplified and cloned into expression vector.After transformation into E.coli BL21(DE3)and induction with IPTG,the recombinant proteins CPSIT_p2,CPSIT_p6,CPSIT_p7 and CPSIT_p8 were expressed as His-tagged proteins in E.coli BL21(DE3)and purified,then ultrafiltration and endotoxins removing of recombinant proteins.With good immunogenicity,recombinant proteins CPSIT_p2,CPSIT_p6,CPSIT_p7 and CPSIT_p8 could induce high level antigen-specific antibody titers after BALB/c mice immunization and could interact with the rabbit anti-C.psittaci and-protein serums.The IFA analysis revealed that CPSIT_p2,CPSIT_p6 and CPSIT_p8 were localized mainly within the inclusion of C.psittaci-infected cells;the endogenous CPSIT_p7 protein was also most localized within the inclusion and no obvious secretary characteristics.2.Genes of CPSIT_p7 and CPSIT_p8 and their encoded proteins showed high similarities and homologies with the Pgp3 and Pgp4 gene and protein sequences of all representative strains of Chlamydia.CPSIT_p7 is the homolog of Pgp3 and CPSIT_p8 is the homolog of Pgp4 by genome annotation and above analysis.Plasmid-encoded protein CPSIT_p7 were predicted to contain abundant epitopes,which were located within or nearby its N-terminals and C-terminals.Protein CPSIT_p7 could be used as epitope-based vaccine design,but not CPSIT_p8.3.Based on the combination use of biology websites and softwares,the selected antigenic fragments were conjugated.A muti-epitope peptide antigen designed SP comprising both B-and T-cell epitopes were chemically synthesized.Immunogenicity of SP in BALB/c mice revealed that specific antibodies both Ig G and Ig A were produced in the SP group gradually increased from week 2,the SP antigen induced a significant proliferation of splenic cells compared to control mice,the upregulation of Th1-related cytokines IFN-? and IL-2 and proinflammatory cytokine IL-6 were significantly increased in the mice immunized with SP compared with that of controls,indicated that SP could induce humoral and cellular immune responses after vaccination.4.Evaluation of antigen SP against C.psittaci infection revealed that SP immunized groups showed a subclinical infection or mild signs and a decreased chlamydial load in the lungs of the SP-immunized mice.Remarkably reduced infiltration and visible pulmonary alveolus were also detected in SP-immunized mice,which shows a milder lesions.IHC analysis showed that the vaccinations with SP gave rise to an effectively lower chlamydial load.Further,the concentrations of IFN-? and IL-6 in the lungs of SP-immunized mice were significantly lower than control mice.Taken together,these results show that immunization of the mice with SP induced a protective efficacy against C.psittaci infection.5.Recombinant eukaryotic vector pcDNA3.1/CPSIT_p7 was successfully constructed.Vector pc DNA3.1/CPSIT_p7 was successfully transfected into He La 229 cells and high-efficiency expression in eukaryotic cells.Western Blot results showed that a specific reaction in 28 k Da.With good immunogenicity,pc DNA3.1/CPSIT_p7 could induce high level antigen-specific Ig G antibody titers.6.Evaluation of pc DNA3.1/CPSIT_p7 against C.psittaci infection revealed that pc DNA3.1/CPSIT_p7 immunized groups showed a decreased chlamydial load in the lungs of immunized mice.Remarkably reduced infiltration and a milder lesions were also detected in pc DNA3.1/CPSIT_p7-immunized mice.IHC analysis showed an effectively lower chlamydial load.Further,the concentrations of IFN-? and IL-6 in the lungs of pc DNA3.1/CPSIT_p7-immunized mice were significantly lower than control mice,indicating that immunization with pc DNA3.1/CPSIT_p7 induced a protective efficacy against C.psittaci infection.Vaccinations with pc DNA3.1/CPSIT_p7 gave rise to an effectively lower chlamydial load and a milder lesions of infected heart,liver,spleen,kidney and brain,inhibit C.psittaci dissemination to distant organ sites and accelerate organ sites C.psittaci clearance,especially the heart and liver,but not brain.Conclusion: 1.CPSIT_p7 and CPSIT_p8 were localized mainly within the inclusion of C.psittaci-infected cells and could induce specific immune response with good immunogenicity.Genes of CPSIT_p7 and CPSIT_p8 and their encoded proteins showed high similarities and homologies with the Pgp3 and Pgp4 gene and protein sequences of other Chlamydia.2.Plasmid-encoded protein CPSIT_p7 were predicted to contain abundant epitopes,the muti-epitope peptide antigen designed SP based on protein CPSIT_p7 could induce humoral and cellular immune responses after vaccination and immunization of mice with SP could accelerate lung C.psittaci clearance,reduce lesions and C.psittaci load,indicating that a protective efficacy was induced against C.psittaci infection.3.CPSIT_p7-based DNA vaccine pc DNA3.1/CPSIT_p7 could highly expressed in eukaryotic cells with good immunogenicity.Vaccinations with pc DNA3.1/CPSIT_p7 gave rise to an effectively lower chlamydial load and a milder lesions of infected lung,heart,liver,spleen,kidney and brain,inhibit C.psittaci dissemination to distant organ sites and accelerate C.psittaci clearance,especially the heart and liver,but not brain.