Abstract: Liu Anyuan, Tao Lijian, Zhao Tie, Zhao Feijun, Zhang Xiaohong Xiangya School of Public Health, Central South University, Changsha 410078, China （Liu AY, Tao LJ）; Institute of Pathogenic Biology, University of South China, Hunan Provincial Key Laboratory for Special Pathogens Prevention and Control, Hengyang 421001, China （Zhang XH, Zhao FJ, Zhao T） Corresponding author: Zhang Xiaohong, Email: hengyangzhfj@126.com 【Abstract】 Objective To evaluate immune protective effects of Treponema pallidum （Tp） pcD/Tp92 DNA vaccine delivered through different inoculation routes against Tp?induced skin infection in New Zealand rabbits. Methods A total of 108 New Zealand rabbits were randomly and equally divided into 6 groups: A1 and A2 groups treated with intramuscular injection of empty plasmids pcD and pcD/Tp92 DNA vaccine respectively for 2 sessions, B1, B2, C1 and C2 groups firstly treated with intramuscular injection of the pcD/Tp92 DNA vaccine for 1 session for primary immunization, then receiving nasogastric feeding with pcD/Tp92 DNA vaccine, pcD/Tp92 DNA vaccine + cytosine?phosphate?guanine （CpG） oligodeoxynucleotide （ODN）, and recombinant Tp92 protein, and recombinant Tp92 protein + CpG ODN respectively for booster immunization. Enzyme?linked immunosorbent assay （ELISA） was conducted to detect the serum level of anti?Tp92 IgG antibody at week 0, 2, 4, 6, 8 after immunization, the SIgA level in the nasopharyngeal region and vaginal mucosa at week 8 after immunization, as well as levels of interleukin?2 （IL?2） and interferon?γ （IFN?γ） in the culture supernatant of rabbit spleen cells at week 8 after immunization, and methyl thiazolyl tetrazolium （MTT） assay was performed to estimate proliferative activity of rabbit splenic lymphocytes in three rabbits from each group. At week 10 after immunization, other 15 rabbits from each group were subcutaneously inoculated with Tp standard strain, and changes of skin lesions at the inoculation site during early?stage infection were observed and recorded. Results At week 8 after immunization, the C2 group showed significantly higher serum level of anti?Tp92 IgG antibody （1.825 ± 0.175）, supernatant levels of IL?2 （154.7 ± 14.6） and IFN?γ （277.4 ± 24.4）, and proliferative activity of T cells （3.57 ± 0.24） compared with the A2 （1.372 ± 0.322, 112.3 ± 13.4, 232.8 ± 25.3, 3.08 ± 0.22, respectively, all P < 0.05）, B1 （0.893 ± 0.297, 76.6 ± 21.5, 165.7 ± 22.6, 2.12 ± 0.14, respectively, all P < 0.05） and B2 （1.294 ± 0.124, 97.3 ± 18.7, 211.3 ± 24.6, 2.88 ± 0.18, respectively, all P < 0.05） groups. In addition, effective immunoprotection was achieved in the C2 group with more production of mucosa?specific SIgA antibody, as well as the lowest Tp?positive rate （6.67%） and ulcer formation rate （6.67%） in skin lesions at the inoculation sites. Conclusion The effective vaccination strategy, namely intramuscular injection of the pcD/Tp92 DNA vaccine for primary immunization followed by nasogastric feeding with mucosal adjuvant CpG ODN combined with recombinant Tp92 protein for booster immunization, can induce the strongest mucosal immune responses and immune protective effects.