Immunogenicity And Protective Efficacy Against Treponema Pallidum Immunized With Plasmid DNA Encoding Flagellin

As a sexually transmitted infection,syphilis typically occurs as a result of infection with Treponema pallidum subsp.pallidum?T.pallidum?.T.pallidum infection commences clinically with the appearance of an ulcerative lesion,and cardiovascular and neurological complications were the most serious form for patients.Despite the existence of effective antibiotic treatment regimens,more than 12 million new syphilis cases are estimated to occur yearly throughout the world.Vaccination would be an extremely valuable and preferred approach to prevent haematogenous spread of T.pallidum within the host.This study was designed to evaluate the relative immunogenicity and inhibition of T.pallidum dissemination in New Zealand rabbits by intramuscular injection of a plasmid DNA vaccine carrying the T.pallidum flagellin flaB3 gene.In order to demanstrant that wether CpG mitifs is an effective vaccine strategy against T.pallidum dissemination in a murine model and is significantly better that vaccination with pcDNA3/FlaB3,the plasmid DNA encoding FlaB3 has been modified by the addition of two immunostimulatory CpG motifs.PART?IMMUNOGENICITY AND PROTECTIVE EFFICACY AGAINST TREPONEMA PALLIDUM IN NEW ZEALAND RABBITS IMMUNIZED WITH PLASMID DNA ENCODING FLAGELLINObjective:This study was designed to evaluate the relative immunogenicity and inhibition of T.pallidum dissemination in New Zealand rabbits by intramuscular injection of a plasmid DNA vaccine carrying the T.pallidum flagellin flaB3 gene.Methods:The mammalian expression vector pcDNA3/FlaB3 was constructed from pET28a/FlaB3 using appropriate restriction sites flanking the flaB3 gene;HeLa 229 cells were transfected with either pcDNA3 or pcDNA3/FlaB3 using the Lipofectamine^TM 2000 Reagent;The serum samples were tested for specific antibodies using the indirect ELISA method;the induction of IFN-?,CD8+T cells,IL-6 and IL-8 were evaluated using a rabbit ELISA kit according to the manufacturers'instructions;DNA was extracted on blood and tissues collected from T.pallidum-challenged rabbits using the Qiagen QIAamp DNA Mini Kit according to the manufacturer's instructions;At 21 days following the last immunization,the all animals were intradermally challenged with1×10⁷ T.pallidum per mL in 0.9%saline at each of eight sites on their shaved backs and observed and measured skin lesions in the infected sites every 3 days;Quantitative PCR?qPCR?was used to assess the T.pallidum burdens in the primary lesion sites,blood,spleen,liver,and testicles on day 21 postinoculation;H&E staining on day 21 postinfection was used to visualize the primary lesion sites and testicular tissue histology in rabbits infected with T.pallidum.Results:1.pcDNA3/FlaB3 was transfected into HeLa cells and the total proteins from these transfected cells reacted with anti-flagellin sera by Western blotting and gave a specific immunoreactive band of 34 kDa;2.A specific antibody was produced in the pcDNA3/FlaB3 group at week 2,and then the antibody titers against flagellin gradually increased;3.The upregulation of IFN-?,CD8+T cells,IL-6 and IL-8 production was significantly increased in the rabbits immunized with pcDNA3/FlaB3compared with that of the rabbits immunized with either recombinant flagellin or vaccinated with the vector DNA alone;On day 14,lesions?rash and ulcerative?were present on all animals,but the control animals presented 100%lesion induration on days 6-8,whereas the animals immunized with pcDNA3/FlaB3 or FlaB3 matched the level of induration observed in the control animals until days 11-12.Ulcerative lesions?ulceration but typically not purulent?in the control group were evident as early as day 14.However,the animals immunized with FlaB3 or pcDNA3/FlaB3 developed ulceration of the lesion on day 16,and the pcDNA3/FlaB3-immunized rabbits had fewer ulcerative lesions than the unimmunized controls.The lesion diameter measured in the control animals was larger than that in the immunized animals within 21 days postinfection.Thirty lesions were investigated by darkfield microscopy.Rabbits immunized with pcDNA3/FlaB3 had significantly fewer lesions with detectable treponemes than the rabbits vaccinated with the recombinant protein;4.The results of RT-PCR showed slightly increased T.pallidum concentrations at the skin lesions in the flagellin plasmid DNA-vaccinated rabbits compared to those of the recombinant protein-vaccinated rabbits and darkfield microscopy analysis of the primary lesion showed fewer treponemes in all immunized animals.However,the treponemal burden in blood,spleen,liver and testicles of the rabbits immunized with plasmid DNA was still significantly lower than that of the rabbits immunized with recombinant protein on day 21;5.Cellular infiltration of the skin?i.e.,the area around the hair follicle filled with inflammatory cells,including lymphocytes,macrophages,and plasma cells?was significantly higher among the rabbits immunized with pcDNA3/FlaB3 than among the rabbits immunized with either recombinant flagellin or vaccinated with vector DNA alone.However,inflammation was also typically observed in the interstitial testicular tissue?i.e.,the interstitial testicular tissue filled with inflammatory cells?in the rabbits vaccinated with vector DNA and PBS at day 21postinoculation.Conclusions:The results presented in this study showed that intramuscular immunization of New Zealand rabbits with the mammalian expression vector pcDNA3/FlaB3 elicited a high level of specific antibodies against flagellin and upregulated IFN-?secretion.Furthermore,animals immunized with pcDNA3/FlaB3 showed an altered course of lesion development and inhibited T.pallidum dissemination to distant organ sites.PART ? CPG-MODIFIED PLASMID DNA ENCODING FLAB3 CONFERS PROTECTION IN A MURINE MODEL OF INFECTION WITH TREPONEMA PALLIDUMObjective: we have explored its protective role against T.pallidum in C57BL/6 mice by using plasmid DNA vaccination.Further,the plasmid DNA encoding Fla B3 has been modified by the addition of two immunostimulatory Cp G motifs.Methods: The oligodeoxynucleotides?ODNs?are made up of DNA consisting of an unmethylated Cp G motif?5'-TCTCCCAGCGTGCGCCAT-3'?as synthesized by Sangon Biotech;He La 229 cells were transfected with either pc DNA3,pc DNA3/Fla B3,pc DNA3/Cp G-Fla B3 using the LipofectamineTM 2000 Reagent;The serum samples were tested for specific antibodies using the indirect ELISA method;Cp G-modified DNA specific splenocytes proliferation was determined at day 14 post-third boost in 3 mice per group;We used multi-parameter intracellular flow cytometry staining?ICS?analysis to identify Cp G-modified DNA specific T-cell response;Several typical cytokines of IFN-?,TNF-?,IL-4,IL-6,and IL-10 secreted by splenocytes were next assessed;The quantitative real-time PCR?q PCR?was used to evaluate T.pallidum burdens in blood,lymph nodes,testicle,brain,liver,and spleen;The differences in spirochete burden were observed by q PCR,rapid plasma regain?RPR?test,T.pallidum particle agglutination?TPPA?assay,and darkfield microscopic analysis of rabbits testicular aspirates;Spirochetes were detected in the testicle,liver,and spleen of infection mice by immunohistochemistry?IHC?.Results: 1.The two Cp G motifs were added into the plasmid?pc DNA3/Fla B3?and functional in eukaryotic cells;2.The flagellin-specific Ig G production in C57BL/6 mice immunized with pc DNA3/Cp G-Fla B3 was higher than that in the mouse group immunized with pc DNA3/Fla B3.Splenocyte proliferation from mice treated with the pc DNA3/Cp G-Fla B3 displayed higher proliferation index compared with pc DNA3/ Fla B3 or control groups;3.The multi-parameter intracellular flow cytometry staining have a significant response following immunization;CD4+ T cells secreted only IFN-? but not IL-4;4.IFN-?,TNF-?,IL-6,and IL-10 level in mice immunized with pc DNA3/Fla B3 and pc DNA3/Cp G-Fla B3 significantly increased compared with the control group;5.Mice immunized with pc DNA3/Cp G-Fla B3 contained fewer treponemal burdens in blood,lymph nodes,testicle,brain,liver,and spleen compared with the control group at 30 days after inoculation;6.Compared to the normal appearing testes in rabbits inoculated with lymph nodes from immunized mice,testes inoculated with lymph nodes from the control unimmunized animals resulted in evidence of seroconversion by day 20.The rabbits inoculated with lymph nodes from pc DNA3/Fla B3 and pc DNA3/Cp G-Fla B3 immunized mice matched the level of seroconversion until day 42 and 51,respectively;7.Strikingly,abundant spirochetes were discovered within testicle,liver,and spleen of control group by immunohistochemistry?IHC?Conclusions: In this study,the C57BL/6 mice,an experimental model,was attempted to evaluate immunogenicity and protection of pc DNA3/Cp G-Fla B3 against T.pallidum infection.pc DNA3/Cp G-Fla B3 is an effective vaccine strategy against T.pallidum dissemination in a murine model and is significantly better that vaccination with pc DNA3/Fla B3.