| Objective: The adaptive immune response level and immunoprotection of recombinant adhesin Tp0954(rTp0954)of Treponema pallidum(Tp)were determined in the New Zealand rabbit model,and the serological diagnostic value of rTp0954 was evaluated,providing experimental basis for screening novel syphilis vaccine candidate and serological diagnostic antigen.Methods:1.Expression,purification and identification of rTp0954.The prokaryotic expression vector pET-30a(+)-Tp0954 was constructed and verified,and transformed into E.coli BL21 to induce expression of rTp0954.The immunoreactivity of purified rTp0954 was identified by Western Blot.2.Evaluation of serological diagnostic value of rTp0954.An indirect ELISA method based on rTp0954 was established to detect levels of specific IgG against rTp0954 in sera from syphilis patients,potentially cross-reactive infections and uninfected controls,and to evaluate the potential value of rTp0954 as a candidate antigen of syphilis diagnostics.3.Animal grouping and immunization.Male New Zealand rabbits were randomly divided into three groups: PBS group,FA group(Fredrin’s adjuvant group)and rTp0954/FA group(experimental group).The rabbits were injected/immunized with PBS,FA and mixture of purified rTp0954 and FA at multiple points through the quadriceps muscle injection and subcutaneously,respectively.The immunization was boosted at two-week intervals for 3 times in total.4.Determination of immunogenicity of rTp0954.Before each immunization,rabbit sera were collected for detection of the level of specific IgG antibodies against rTp0954 by enzyme-linked immunosorbent assay(ELISA).Rabbit spleens were collected 2 weeks after the last immunization,and the levels of IFN-γ secretion and lymphocyte proliferation were detected by ELISA and CCK-8 kits,respectively.5.Immunoprotection of rTp0954.Rabbits were challenged with live T.pallidum(Nichols strain)at 2 weeks post last immunization,and challenge sites were monitored daily for erythema,induration and painless ulceration and were measured daily to assess lesion diameter.On day 21 post challenge(PC),the rabbits were sacrificed and real-time fluorescence quantitative PCR(RT-PCR)was performed to detect TP-DNA load in blood,skin lesions,liver and spleen.Hematoxylin-eosin(HE)staining was used to observe the inflammatory cell infiltration in the skin tissue.The popliteal lymph nodes in each group were isolated aseptically,and the extracts were injected into the testis of healthy New Zealand rabbits.The asymptomatic orchitis were observed daily and rabbit seroconversion were tested every 3 weeks.T.pallidum was observed by silver staining.Results:1.Expression,purification and identification of rTp0954.The prokaryotic expression vector PET-30a(+)-TP0954 was successfully constructed by PCR identification and sequencing.Western Blot results showed specific bands with 55 KDa size.2.Evaluation of diagnostic value of rTp0954.ELISA results showed that rTp0954 had higher sensitivity(95.2%)but lower specificity(86.7%)in detecting clinical serum samples.3.rTp0954 immunization effectively induced humoral and cellular immune responses.The level of specific IgG in rTp0954 group increased gradually with the increase of immunization times,and reached the peak at the 6th week post primary immunization,with a titer of 1:10 240 000.ELISA and CCK-8 assays showed the significantly increased IFN-γsecretion(P<0.0001)and lymphocyte stimulation index(SI)(P<0.05)in rTp0954 group compared with PBS group and FA group.4.rTp0954 immunization effectively delayed the development of lesions and inhibited T.pallidum dissemination to distal tissues.After T.pallidum challenge,animals in PBS group and FA group presented 100%lesion induration by day 9 while animals in rTp0954 group delayed by day 14.The lesion diameter in PBS group and FA group was consistently larger than that in rTp0954 group over the 21-day observation period.At day 21 PC,the ulcer formation rate of rTp0954 group was significantly lower than that of PBS and FA control group.RT-PCR results showed that Tp-DNA load in blood and tissues(skin lesions,liver and spleen)in rTp0954 group was significantly lower than that in PBS group and FA group(P<0.01).Introduction of lymph nodes from PBS or FA-immunized,T.pallidum-challenged animals to naive animals induced orsitis and serological reaction,and T.pallidum was observed in silver staining,while no reaction was observed in the rTp0954-immunized within 100 days PC.5.rTp0954 immunization promoted inflammatory cell infiltration.HE staining showed that animals in rTp0954 group had higher levels of cellular infiltrates in all cell types analysed,such as macrophages,neutrophils,lymphocytes and plasmocytes,than that in PBS group and FA group.Conclusions:1.Tp0954 may not be suitable for serological diagnosis of syphilis.2.Immunization with rTp0954 produced a strong humoral and cellular immune response and attenuated lesion development,inhibited of treponemal dissemination to distant organs,and increased cellular infiltration at the lesion sites,indicating that Tp0954 is a promising syphilis vaccine candidate. |
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