Studies On The Protective Immunity Of Schistosoma Japonicum Bivalent DNA Vaccine Encoding Sj23 And Sj14

Schistosomiasis japanica is a amphixenosis that could result in severe morbidity and mortality. Despite major advances in control with drugs against schistosoma and public health measures, schistosomiasis continues to spread to new geographic areas. In most cases schistosomiasis can be successfully treated by chemotherapy, but rapid reinfection rates for patients in endemic areas require repeated treatment. It will not only increase therapic cost but also provoke the dangers of potential resistance. Thus, safe and efficacious vaccine development is an one of important measures for long-term integrated control of schistosomiasis.DNA vaccination was introduced in 1990 reported by Wollf. It was demonstrated that foreign protein could be persistent and steady expression upon direct intramuscular injection of plasmid DNA in myocytes. Since then, vaccine against schistosomiasis went into a new stage of nucleic acid vaccine. The advantages of DNA vaccines over the traditional attenuated cercarie or subunit vaccine are the low cost of production, thermal stability and their ability to induce a wide variety of cellular and humoral immune responses. In succession several DNA vaccines encoding single antigens have been studied. The protection levels obtained by univalent DNA vaccines were far from comparable to those achieved using the attenuated vaccines, although specific antibodies and CTL responses were generated. To this day, there is still not yet a commercial DNA vaccine to the wold. These results are perhaps explained by the fact that the schistosome parasite is such an antigenically complex organism. Its genome is more complicated than that of bacteria and virus. Further more many mechanisms of immunized escape generated during long-term evolutional process together with host. Thus, the immune responses induced by single antigen vaccination may not be strong enough to combat the challenging infection. The development of multivalent vaccines is a novel strategy. There are a greater amount of protective epitopes on multivalent vaccines to obtain protection against different parasite strains and stages of parasite life cycle.In our research, Sj14 and Sj23 were cloned into plasmid pVIVO2 simultaneously to construct bivalent co-expression DNA vaccine to enhance the protection. In the present study, the protective abilities of immunization with univalent vaccine , the co-expression, gene fusion, cocktail bivalent vaccine and quadrivalent vaccine were evaluated in BALB/c mice. It was designed preliminary to explore the optimal conditions from four factors including immunized dosage, inoculated times, inoculated routes and the challenge time after last immunization.The main contents of this thesis include four parts:Part I Construction and confirmation of co-expression bivalent DNA vaccine pVIVO2-mcs-Sj14/Sj23 and pVIVO2-mcs-Sj23/Sj14 against schistosoma japonicum encoding Sj23 and Sj14The complete Sj23 and Sj14 sequences were amplified by PCR from pVIVO2-IL12-Sj23 and pVIVO2-IL12-Sj14 using two pairs primers specific for their genes. The products were cloned into one cloning site (BamHI/EcoRI) of the vector pVIVO2-mcs, generating pVIVO2-mcs-Sj23 and pVIVO2-mcs-Sj14. Similarly, Sj23 and Sj14 were obtained by using different primer pairs with different enzyme cutting sequences. After being digested, they were cloned into the other site (AvrII/BspHI) of pVIVO2-mcs-Sj23 and pVIVO2-mcs-Sj14, enabling the co-expression of two genes (pVIVO2-mcs-Sj23/Sj14 and pVIVO2-mcs-Sj14/Sj23). The two bivalent vaccines were confirmed by restriction analysis and sequencing. Result: After digestion by restriction enzyme several recombinant plasmids of pVIVO2-mcs-Sj23,pVIVO2-mcs-Sj14,pVIVO2-mcs-Sj23/Sj14 and pVIVO2-mcs-Sj14/Sj23,molecular weight of small fragments were nearly 440bp and 710bp each. Complete sequencing of the cDNA inserts confirmed the homology to the published Sj23 and Sj14 sequences. The results indicated that the recombinant plasmids were successfully constructed.At school of Life Science in Huazhong University of Science and Technology, fusion genes Sj23·Sj14 and Sj14·Sj23, which were connected by (Gly4Ser) 3, were amplified in successive PCR's instead with the method of splicing by overlap extention (SOE). Fusion genes were cloned into one cloning site (BamHI/EcoRI) of the vector pVIVO2, generating the construction of a fusion gene for expression (pVIVO2-mcs-Sj23·Sj14 and pVIVO2-mcs-Sj14·Sj23). Similarly, fusion genes Sj23·Sj14 and Sj14·Sj23 were obtained by using different primer pairs with different enzyme cutting sequences. After being digested, they were cloned into the other site (AvrII/BspHI) of pVIVO2-mcs-Sj23·Sj14 and pVIVO2-mcs-Sj14·Sj23, enabling the co-expression of two genes (pVIVO2-mcs-Sj23·Sj14/Sj14·Sj23 and pVIVO2-mcs-Sj14·Sj23/Sj23·Sj14).The cocktail vaccine was produced by mixing pVIVO2-mcs- Sj23/Sj14 and pVIVO2-mcs-Sj23·Sj14 at a molar raio of 1:1.The eukaryotic expression plasmid pVIVO2 (Invivogen, USA), a new generation multigenic vector with two transcription units, was used as a DNA vaccine vector. Since the eukaryotic expression plasmid pVIVO2 (Invivogen, USA) carries the human FerL and FerH composite promoters, the use of both promoters can eliminate the risk of transcriptional interference between the two expression cassettes. The activity of both promoters is further increased by the addition of the SV40 and CMV enhancers.Part II Eukaryotic expression of bivalent DNA vaccine pVIVO2-mcs-Sj14/Sj23 co-expression Sj 23 and Sj14It was initially important to determine that the DNA vaccine construct could direct synthesis of the immunogens in eukaryotic cells before immunization. Therefore pVIVO2-mcs-Sj14/Sj23 was selected to test the eukaryotic expression in vitro and in vivo.1. Transient expression of DNA pVIVO2-mcs-Sj14/Sj23 in vitroHEK-293 cells were transiently transfected by pVIVO2-mcs-Sj14/Sj23 and pVIVO2-mcs according to the manufacturer's instructions using LipofectamineTM 2000 Reagent. The expression of Sj23 and Sj14 were determined by testing the presence of mRNA by RT-PCR and the presence of protein using the indirect immunofluorescence assay technique 48 hours after transfection.The result of RT-PCR showed that products of the expected size (about 440bp and 710bp) were obtained using cDNA derived from cells transfected with pVIVO2–mcs- Sj14/Sj23. The presence of protein detected at the plasma and/or surface of transfected cells was confirmed by immunofluorescence microscopy. These results demonstrated that the plasmid pVIVO2-mcs-Sj14/Sj23 could be expressed in mammalian cells.2. Expression of plasmid pVIVO2-mcs-Sj14/Sj23 in muscles of BALB/c mice and persistence time.BALB/c mice (18 total) were divided into two groups. Each group was immunized by intramuscular injection with 100μg pVIVO2-mcs or pVIVO2-mcs-Sj14/Sj23. Mice were sacrificed 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, and 24 weeks separately. The quadriceps muscles of injection site were taken and made into frozen sections. The presence of proteins (Sj23 / Sj14) in the plasma and on the surface of skeletal muscle cells was confirmed by immunofluorescence microscopy. The expression of proteins was persistent at least 6 months.Part III Protection immunity induced by DNA vaccines120 BALB/c mice were divided randomly into twelve groups. Each group was immunized by intramuscular injection at a dosage of 100μg with one of the following DNA vaccine: pVIVO2-mcs, pVIVO2-mcs-Sj23, pVIVO2-mcs-Sj14, pVIVO2-mcs-Sj23/Sj14, pVIVO2-mcs-Sj14/Sj23, pVIVO2-mcs-Sj23·Sj14, pVIVO2-mcs-Sj14·Sj23, pVIVO2-mcs-Sj23·14/Sj14·23,pVIVO2-mcs-Sj14·23/Sj23·14, pVIVO2-mcs-Sj23/Sj14 (100μg)+ lentinan (100μg), the cocktail with pVIVO2-mcs-Sj23/Sj14(50μg) and pVIVO2-mcs-Sj23·Sj14(50μg), and the control group with 100μl normal saline. Four weeks after immunization, each mouse was challenged with 40±2 normal S. japonicum cercarie by abdominal skin penetration then perfused 6 weeks later. The number of adult worm recovered. The average of adult worm and eggs per gram (EPG) of liver were calculated. Granuloma of liver tissue was analyzed using a TY70 Imaging Analysis System. The diameters of all egg granulomas were measured. The protection was evaluated with worm reduction rate, egg reduction rate of the liver and granuloma diameter of single egg reduction rate.Result showed that the worm and egg burden in test groups were significantly lower than control groups (including normal saline and pVIVO2-mcs groups) (P<0.05). The levels of worm reduction of pVIVO2-mcs-Sj14/Sj23, pVIVO2-mcs-Sj23·Sj14, pVIVO2-mcs-Sj14·Sj23, cocktail group and pVIVO2-mcs-Sj23/Sj14+ lentinan group were higher than that of univalent vaccine pVIVO2-mcs-Sj23 and quadrivalent vaccines pVIVO2-mcs-Sj23·Sj14/Sj14·Sj23,pVIVO2-mcs-Sj14·Sj23/Sj23·Sj14 (P<0.05), which were higher than 50%. Worm reduction rate in pVIVO2-mcs-Sj23/Sj14+lentinan group was the highest among the test groups , and the worm/EPG reduction rates were 68.89%/84.04%. Conversely, the egg reduction rates of univalent vaccine and quadrivalent vaccine showed no significant difference when compared with bivalent vaccine (P>0.05). Furthermore, granulomas diameters of the five groups whose worm reduction rates were higher than 50% were measured. Granuloma diameter reduction rates were 18-39%. Among these seven groups (including two control groups), the average diameter of granuloma in pVIVO2-mcs- Sj23·Sj14 group was shown to be the smallest. Results showed that the protective immunity of bivalent vaccines is better than that of univalent and quadrivalent vaccine. At inducing protection of two genes co-expressed, fusion gene expression and cocktail vaccine, cocktail vaccine was shown to be the best. There appears to be no difference between two genes co-expressed and fusion gene expression in protection. Lentinan is effective adjuvant to increase potence of DNA vaccine. In this trail, univalent, bivalent and quadrivalent vaccine could decrease the worm burden, but had not anti-fecundity. The five groups whose worm reduction rates were higher than 50% could reduce granuloma diameter at some extent, and they had anti-pathology.Part IV Study on the optimal schedule with co-expression bivalent DNA vaccine pVIVO2-mcs-Sj14/Sj23In order to explore the optimal immunization conditions, bivalent vaccine pVIVO2-mcs-Sj14/Sj23 was selected to carry out second experiment (shown in Table 1). The orthogonal trial was designed by SPSS12.0 with four factors and three level of each factor. According to statistic approach, it could reduce the number of test groups chosen for incubation and help to obtain similar results with all test groups.Four factors used were immunized dosage, inoculated times, inoculated routes and the challenge time after last immunization. There were 3 different concentrations of dosage (50μg, 100μg and 200μg) inoculated, respectively. Inoculated times were once, twice, and thrice(interval time was two weeks).The three immunized routes were intramuscular, subcutaneous and intradermal injection. Challenge time was 4, 8 and 12 weeks after the last immunization. 110 BALB/c mice were divided randomly into eleven groups, including two control groups and nine test groups. Nine groups were designed with four factors three levels in orthogonal trial by using SPSS12.0 software. Two control groups were immunized by intramuscular injection with 100μg of pVIVO2 and 100μl of normal saline initially then challenged 4 weeks later. Each mouse was challenged with 40±2 normal S. japonicum cercarie by abdominal skin penetration then perfused 6 weeks later. The worm reduction rate and the egg reduction rate of the liver were calculated. Different groups were immunized at different time points, but all groups were challenged and perfused at the same time. The protection was evaluated with worm and egg reduction rate of the liver.Results showed that the worm and egg burden in test groups were significantly lower than control groups (including normal saline and pVIVO2 groups) (P<0.05). From the analysing of average value, it was concluded that the optimal schedule was 50μg, two times immunization, intradermal injection and challenged 12 weeks after the last immunization in three levels of four factors. However, the result of orthogonal design was obtained by SPSS12.0, in which F< F critical value, then P>0.05 (data not shown). There was no significant impact on immunity protection in three levels of each factor (P>0.05).Together, these data demonstrated:1. Two monovalent DNA vaccines pVIVO2-mcs-Sj23, pVIVO2-mcs-Sj14 and co-expression bivalent DNA vaccines pVIVO2-mcs-Sj14/Sj23, pVIVO2-mcs-Sj23/Sj14 were successfully constructed.2. The bivalent vaccine such as pVIVO2-mcs-Sj14/Sj23 could be expressed in vitro and vivo. The expression in vivo could be persistent at least 6 months.3. The eligible adjuvant could increase protective immunity and maintain memory effect. As one effective adjuvant to increase potence of DNA vaccine, lentinan was introduced at first time. It was worthy to reseach one step further.4. In order to enhance protective immunity, multivalence technique line was applied. The bivalent DNA vaccines were constructed by the methods of co-expressing two genes, a fusion gene expression and the combination of two kinds of plasmids (cocktail vaccine). These bivalent vaccines could induce notable protective immunity against schistosoma. There were five groups whose worm reduction rates were higher than 50%. The level protections of bivalent DNA vaccines were higher than that of univalent vaccine significantly. Undoubtedly, it provided new idea on the contrivance of multivalent vaccine. 5. In order to enhance protective immunity, we explored optimal schedule from four factors of immunized dosage, inoculated times, inoculated routes and the challenge time after last immunization and three levels each. There was no significant difference in the level of protection with four factors in three levels. While optimal schedule trended to be 50μg, two times of immunization, intracutaneous injection and attack at 12th week after last immmunization.