Enhanced Immune Response And Protection Efficacy Of A Treponema Pallidum Tp92 DNA Vaccine Induced By DNA Vaccine Combined With IL-2 Genetic Adjuvant And Chitosan Nanoparticles As Gene Transfer Vector

Objectives:In this study, 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 DNA vaccine-induced immune responses were investigated in rabbits experimentally infected with T. pallidum.Methods:The male New Zealand White rabbits were randomly assigned to eight groups with eighteen members each and immunized with plasmid constructs. Each immunization was intramuscularly administered with plasmid construct (100μg) in PBS for Experiment I (for the pcD/Tp92+pcD/IL-2 vaccine group, the pcD/Tp92 vaccine group, and the pcD and PBS control groups) or with plasmid construct (100μg) in PBS mixed with CS nanoparticles (30μg) for Experiment II (for the CS+pcD/Tp92+pcD/IL-2 vaccine group, the CS+pcD/Tp92 vaccine group, and the CS+pcD and CS+PBS control groups) three times at 2-week intervals (Weeks 0, 2, and 4). Two days before DNA inoculation, the quadricep muscles were injected with 100μl of a solution containing 0.25% bupivacaine hydrochloride to enhance subsequent DNA absorption.Two weeks before the challenge (Week 8), three rabbits from each group were used for cytokine measurements and fifteen rabbits from each group were used for challenges (Week 10). After six weeks of the last boost (Week 10), all immunized and nonimmunized control rabbits (n=15) were challenged intradermally at eight sites on their shaved backs with 105 T. pallidum (Nichols) spirochetes per site. Diameters and appearances of the lesions were recorded. Aspirates were taken from the lesions 16–20 days after the challenge and examined for the presence of treponemes using dark-field (DF) microscopy and silver staining.To evaluate anti-Tp92 humoral responses, during immunity and infection, sera were collected from immunized rabbits in each group at correspondingly different times after the first immunization; the levels of anti-Gpd total IgG antibody in serum samples were determined using ELISA.Two weeks before the challenge (Week 8), Three rabbits from each group (n=18) were sacrificed; their spleen cells were obtained as previously described. Cells were stimulated with the recombinant protein Tp92 at 10μg ml-1,Culture supernatants were harvested and kept at -80°C until detection and assay by ELISA analysis for IL-2 and IFN-γ.Spleen cells were obtained from three rabbits from each group rabbits infected at correspondingly different times, as previously described. Spleen lymphocyte proliferative response was measured using the MTT colorimetric method.Results:1. The expression of fusion protein Tp92 was examined by Western blot analysis of the cell lysates after transfection of HeLa cells with pcD/Tp92. The present study confirms that cells transfected with these plasmids expressed the corresponding proteins, based on the anticipated sizes for Tp92 (i.e., ~77 kDa) .2. Immunization with DNA encoding Tp92 mixed with chitosan nanoparticles or immunization with or DNA encoding Tp92 using pcD/IL-2 as adjuvant and mixed with chitosan nanoparticles could more significantly enhance anti-Tp92 specific IgG antibody levels and maintain in a stable and high levels throughout the immunity period in rabbits. 1) both pcD/Tp92 and pcD/Tp92+pcD/IL-2 mixed vaccines elicited significantly higher levels of anti-Tp92 IgG antibodies in rabbits during immunity (P<0.01 for both pcD/Tp92 and pcD/Tp92+pcD/IL-2 immunized rabbits compared with the respective negative controls pcD and PBS). Furthermore, pcD/Tp92+pcD/IL-2 vaccination significantly increased the levels of IgG antibodies in rabbits compared with pcD/Tp92 immunization (P<0.05 ). 2) both CS+pcD/Tp92 and CS+pcD/Tp92+ pcD/IL-2 mixed vaccines elicited significantly higher levels of anti-Tp92 IgG antibodies in rabbits during immunity and infection ( P < 0.05 for both CS+pcD/Tp92 and CS+pcD/Tp92+pcD/IL-2 immunized rabbits compared with the respective controls pcD/Tp92 and pcD/Tp92+pcD/IL-2).3.Immunization with pcD/Tp92 or immunization with pcD/Tp92+pcD/IL-2 mixed vaccine significantly enhanced Cytokine levels of IL-2 and IFN-γin rabbits. But the level of IL-2 and IFN-γwere further increased in the later( P < 0.05 ). immunization with pcD/Tp92 wrapped with chitosan nanoparticles or immunization with pcD/Tp92+ pcD/IL-2 mixed vaccine wrapped with chitosan nanoparticles could more significantly enhance levels of IL-2 and IFN-γin rabbits. But the level of IL-2 and IFN-γwere further increased in the later ( P < 0.05 ). However, there were not significantly higher levels of IL-2 and IFN-γcomparing CS+pcD/Tp92+pcD/IL-2 with pcD/Tp92+pcD/IL-2 ( P >0.05), or CS+ pcD/Tp92 with pcD/Tp92 ( P > 0.05 ). 4.Immunization with DNA encoding Tp92 or immunization with DNA encoding Tp92 using pcD/IL-2 as adjuvant significantly enhanced cell proliferation in rabbits. But the level of splenocyte proliferation was further increased in the later. immunization with DNA encoding Tp92 mixed with chitosan nanoparticles or immunization with DNA encoding Tp92 using pcD/IL-2 as adjuvant and mixed with chitosan nanoparticles could more significantly enhance cell proliferation levels and maintain in a stable and high levels throughout the infection period in rabbits. 1) Both pcD/Tp92 and pcD/Tp92+pcD/IL-2 elicited significantly higher levels lymphocyte proliferation activities in rabbits infected for 10 to 180 days with the treponema pallidum Nichols strain. (P<0.01 for both pcD/Tp92 and pcD/Tp92+pcD/IL-2 immunized rabbits compared with the respective negative controls). Furthermore, pcD/Tp92+pcD/IL-2 vaccination significantly increased the levels of lymphocyte proliferation in rabbits compared with pcD/Tp92 immunization (P<0.05).2)both CS+pcD/Tp92 and CS+pcD/Tp92+ pcD/IL-2 mixed vaccines elicited significantly higher levels lymphocyte proliferation activities in rabbits during immunity and infection(P<0.05 for both CS+pcD/Tp92 and CS+pcD/Tp92+pcD/IL-2 immunized rabbits compared with the respective controls pcD/Tp92 and pcD/Tp92+ pcD/IL-2).5. When the rabbits were challenged intradermally at eight sites on their shaved backs with 105 T. pallidum (Nichols) spirochetes per site,the co-injection of IL-2 plasmid with Tp92 DNA vaccine conferred significantly better protection than Tp92 DNA vaccine did, as characterized by lower detectable treponemes (20%)), lower ulcerative lesion scores (10%), and faster recovery. Individuals treated with co-injection of the IL-2 plasmid with the Tp92 DNA vaccine wrapped with CS nanoparticles were the least likely to have detectable treponemes (12.5%) and ulcerations (7.5%).Conclusions:These results indicate that the co-injection of an IL-2 plasmid with Tp92 DNA vaccine wrapped with CS nanoparticles can efficiently induce Th1-dominant immune responses, improve protection against T.pallidum spirochete infection, and most effectively attenuate syphilitic lesion development.