Immune Strategy And Optimization Study On DNA Vaccines Of Outer Membrane Protein Of Treponema Pallidum

Treponema pallidum (Tp) is the pathogen which causes syphilis, a human sexually transmitted diseases (STD). Syphilis is the second lethal cause of STD following Acquired Immune Deficiency Syndrome (AIDS). Syphilis cannot only severely impair multiple organs to cause systemic diseases in adults, but also vertically infect the fetus though placenta to cause systemic organ system infection, leading to premature births, abortions, stillbirths, terata, or congenital syphilis, and seriously affecting the birth quality. In addition, since syphilis has the same transmission routes as AIDS, syphilis may greatly increase the risk of infection and transmission of adult AIDS and congenital AIDS. Although most Tp isolates are not drug-tolerant clinically, in recent years, syphilis has a very high incidence of disease. Approximately2million new infections are reported every year worldwide. In China, infection and incidence of syphilis are linearly increasing with a highest rate among the eight monitored STDs. Therefore, the effective control and prevention of syphilis has become a worldwide public health issue. Therefore the continued research and development of efficacious vaccines against syphilis is an urgent matter.Presently, research of Tp vaccine candidates is focused on use of Tp outer membrane proteins (OMP). Tp outer membrane proteins not only play an important role in Tp virulence, but are also the major target of the host's protective immunity.TpGpd is considered the best candidate for Tp vaccine studies due to its high homologies amongst strains and relatively strong immunogenicity and protective capacity.This study is based on the work that we have constructed the single-gene DNA vaccine and finished its study about immune activity, we construct a fusion DNA vaccine of Tp Gpd and interleukin2(IL-2) vectored by chitosan (CS) nanoparticles. It is then determined whether IL-2as a molecular adjuvant and CS nanoparticles as vectors could enhance the immune response and protective efficacy of Tp Gpd vaccine in Tp-challenged rabbits.At the same time, we verify the Vaccination strategies that the Primary immunization of DNA vaccine be carried out by intramuscular injection and the Second immunization of recominant protein vaccine with CpG ODN be complied with mucosal immunization can enhance and overall stimulate the Immune effector, especially mucosal immunity.Objectives1. the immune-modulatory and vaccine effects of using an interleukin-2(IL-2) expression plasmid as a genetic adjuvant and chitosan (CS) nanoparticles as gene transfer vector to enhance Tp Gpd DNA vaccine-induced immune responses were investigated in rabbits experimentally infected with T. pallidum.2. the Vaccination strategies that the Primary immunization of DNA vaccine be carried out by intramuscular injection and the Second immunization of recominant protein vaccine with CpG ODN be complied with mucosal immunization were verified and evaluated in a T.pallidum (Tp) rabbit challenge model.Methods1.The passage and genome extraction of Tp Nichols strain:the rabbit testis frozen at low-temperature was cut and placed in sterile saline at room temperature, Tp Nichols strain be released from testis and be recovered, and then passaged by inoculating rabbit testicular2.TpGpd gene and IL-2gene was subcloned into pcDNA3.1(+) eukaryotic expression vector, Respectively. The Gpd gene and IL-2gene was fused with a linker by recombination PCR to construct pcDNA3.1(+)/Gpd-IL-2eukaryotic expression vectors. Western-blot verify that the Gpd and Gpd-IL-2antigens could be expressed in HeLa cells.3. Gpd-IL-2fusion gene was amplified by PCR and cloned into pET28a. The recombinant expression vector pET28a/Gpd-IL-2was transformed into E.coli Rssesta and induced to express Gpd-IL-2with IPTG. The obtained fusion protein, which was purified and analyzed by SDS-PAGE and Western blot. The purified protein was used to immune rabbits.4. The experimental rabbits were divided into twelve groups, with18rabbits in each group. DNA vaccine candidates i) pcD/Gpd (100μg), ii) pcD/Gpd coated with CS (30μg), iii) pcD/Gpd and pcD/IL-2(100μg), iv) pcD/IL-2and pcD/Gpd coated with CS,v) pcD/Gpd-IL-2(100μg),vi) pcD/Gpd-IL-2coated with CS were inoculated into the left hind leg quadriceps of rabbits. pIL-2coated with CS, pIL-2,pcD coated with CS, pcD, CS and PBS were also inoculated to serve as controls. The complete regimen included three immunizations, whic were administered once in every two weeks.5. At week8after the first immunization, three rabbits from each group were used to determine Splenocyte-proliferation assay and cytokine measurements. during immunity and infection,1ml of blood was collected from immunized rabbits ear veins in each group at correspondingly different times (Weeks0,2,4,6,8,10,12,14,20,24,28, and32) after the first immunization,followed by centrifugation for serum separation. ELISA analyze the TpGpd-specific antibody responses. Spleen cells were obtained from three rabbits from each group rabbits infected (n=18,15,12,9,6and3) at correspondingly different times (Weeks8,10,12,14,30, and34). The proliferation response of spleen cells was detected by MTT assay. ELISA was used for the cytokines IFN-y and IL-2in spleen lymphocyte culture medium.6.At week10,15rabbits in each group were challenged subcutaneously with the Tp Nichols strain at8sites (105bacteria/site) in the back. Skin lesions were observed and measured in the infected sites every3days till60days. Lesion spirals were investigated by dark-field (DF) and silver staining methods at day14, day21after Tp challenge. The ratio of the number of DF-positive lesions and ulcerative lesions to the total number of the lesions was calculated for each group.7.the Vaccination strategies that the Primary immunization of pcD/Gpd-IL-2DNA vaccine(100fig) be carried out by intramuscular injection and the Second immunization of Gpd-IL-2recominant protein vaccine(50μg) with CpG ODN (10μg) be complied with mucosal immunization were verified and evaluated in a T.pallidum rabbit challenge model.Results1. The Gpd-IL-2fusion gene fragment was successfully amplified. The eukaryotic expression vectors (pcD/Gpd-IL-2, pcD/Gpd,pcD/IL-2) were successfully constructed. Transient expression of TpGpd(-41kDa), IL-2(-22kDa) and Gpd-IL-2(-60kDa) fusion gene in HeLa cells were examined by immunoblotting. The prokaryotic expression recombinant vecters pET28a/Gpd-IL-2were successfully constructed.A soluble fusion protein with molecular weight about60kDa was attained after expression and purification.2. Immunization with pcD/Gpd, pcD/Gpd-IL-2or immunization with pcD/Gpd+pcD/IL-2mixed vaccine significantly enhanced anti-Gpd specific IgG antibody levels,Cytokine levels of IL-2and IFN-y and the level of splenocyte proliferation in rabbits compared with the respective negative controls (pcD/IL-2, pcD alone and PBS groups)(P<0.05). But all of the Immunoassay indexs were further increased in the pcD/Gpd-IL-2vaccine group and pcD/Gpd+pcD/IL-2mixed vaccine group compared with pcD/Gpd vaccine group (P<0.05). there were not significantly higher levels of all the Immunoassay indexs comparing pcD/Gpd-IL-2vaccine group with pcD/Gpd+pcD/IL-2mixed vaccine group (P>0.05).3. immunization with pcD/Gpd wrapped with CS nanoparticles (pcD/Gpd+CS) or immunization with pcD/Gpd+pcD/IL-2mixed vaccine wrapped with CS nanoparticles (pcD/Gpd+pcD/IL-2+CS) or pcD/Gpd-IL-2mixed vaccine wrapped with CS nanoparticles (pcD/Gpd-IL-2+CS) could not significantly enhanced anti-Gpd specific IgG antibody levels,Cytokine levels of IL-2and IFN-y and the level of splenocyte proliferation in rabbits compared with the respective controls pcD/Gpd, pcD/Gpd+pcD/IL-2and pcD/Gpd-IL-2vaccine groups, Accordingly (P>0.05).There were significantly higher levels of all the Immunoassay indexs comparing pcD/Gpd-IL-2vaccine group or pcD/Gpd+pcD/IL-2vaccine group with pcD/Gpd+CS vaccine group (P <0.05).4. When the rabbits were challenged intradermally at eight sites on their shaved backs with105T. pallidum (Nichols) spirochetes per site, Ratios of positive skin lesions and ulcer lesions in groups immunized with all pTpGpd DNA vaccines were significantly lower than all of the control groups,respectively (P<0.001). pcD/Gpd+pcD/IL-2+CS vaccine group immunized animals exhibited the lowest rates of positive skin(8.3%)and ulcer lesions(4.2%) and the fastest recover(42d).5. Immunization with pcD/Gpd-IL-2vaccine by the intramuscular route (A2group) induced a better immune response than those obtained by the Vaccination strategie of B1group(Primary immunization of pcD/Gpd-IL-2vaccine be carried out by intramuscular injection and the Second immunization of pcD/Gpd-IL-2accine be complied with nasal immunization)(P<0.05).However, B1group exhibited significantly higher levels (P<0.05) of anti-Gpd IgA in Rabbit vaginal fluid and Rabbit nasopharyngeal mucosa fluid than A2group.6. Immunization with pcD/Gpd-IL-2vaccine by the Vaccination strategie of B2group(Primary immunization of pcD/Gpd-IL-2vaccine be carried out by intramuscular injection and the Second immunization of CpGODN with pcD/Gpd-IL-2accine be complied with nasal immunization) induced a better immune response and lower Ratios of Tp positive skin lesions and ulcer lesions than B1group.7. Immunization with pcD/Gpd-IL-2vaccine by the vaccination strategie of C2group(Primary immunization of pcD/Gpd-IL-2vaccine be carried out by intramuscular injection and the Second immunization of CpGODN with Gpd-IL-2recombinant protein accine be complied with nasal immunization) induced a best immune response and lowest Ratios of Tp positive skin lesions(0%) and ulcer lesions(3.33%) than all other Vaccination strategie group (B1,B2,C1and A2group).Conclusions1. Strong cellular immunity and humoral immunity responses could be induced by all Tp Gpd DNA vaccines in New Zealand Rabbits.2. Both pcD/Gpd-IL-2vaccine and pcD/Gpd+pcD/-IL-2vaccine could induce more powerful immune response and immune protection than pcD/Gpd vaccine.3. Immunization with Tp Gpd DNA vaccines wrapped with CS nanoparticles properly promote immune response and of the appropriate Gpd DNA vaccines.4. The vaccination strategie of primary immunization of pcD/Gpd-IL-2vaccine carried out by intramuscular injection and the Second immunization of CpGODN with Gpd-IL-2recombinant protein accine complied with nasal immunization could induce a best immune response and lowest Ratios of Tp positive skin lesions(0%) and ulcer lesions(3.33%).