Construction, purification, and evaluation of multivalent DNA vaccine against Schistosoma japonicum.

DNA vaccine encoding a multivalent antigen is a novel approach of protective immunization. Four Schistosoma japonicum candidate antigen genes, glyceraldehyde-3-phosphate dehydrogenase (SjGAPDH), 23 kDa transmembrane protein (Sj23), 14 kDa fatty-acid binding protein (SjFABP) and 26 kDa glutathione-S-transferase (Sj26), are recombined into two pieces of fusion genes SjFABP.Sj23 and Sj26.SjGAPDH, respectively. Tetravalent DNA vaccine pVIVO2-SjFABP.Sj23/Sj26.SjGAPDH is constructed by co-expressing these two fusion genes. The super-coiled DNA vaccines for large-scale clinic application were purified by sequential chromatographies including group separation chromatography and affinitive chromatographies. The purified DNA vaccines were evaluated for in vivo and in vitro transfection assay. The immunoprotective properties of the different kinds of constructed DNA vaccines were appraised by pharmacological trials. The pharmacological trials results showed that tetravalent DNA vaccine has higher protective efficiency than other tested DNA vaccines.

[Construction and expression of Schistosoma japonicum bivalent DNA vaccines and their efficiencies of protective immunity].

OBJECTIVE: To study the efficiency of protective immunity afforded by the constructed bivalent DNA vaccines of Schistosoma japonicum. METHODS: The plasmids pVIVO2-mcs-SjFABP-Sj23 and pVIVO2-mes-Sj23-SjFABP, co-expressed bivalent DNA vaccines, were constructed and identified. The presence of bivalent DNA vaccine in the mouse muscle cells was also tested by indirect immunofluorescent antibody tests (IFAT). 70 BALB/c mice were randomly divided into seven groups to be injected with normal saline, pVIVO2-mcs, pVIVO2-mcs-Sj23, pVIVO2-mcs-SjFABP, pVIVO2-mcs-Sj23-SjFABP, pVIVO2-mcs-SjFABP-Sj23 plasmid DNA, and a mixture of pVIVO2-mcs-Sj23-SjFABP plasmid DNA and amylose adjuvant, respectively. At day 45 after challenge the mice were sacrificed. The number of adult worms and hepatic eggs were counted. RESULT: Successful construction of co-expressed bivalent DNA vaccines were identified by restriction analysis and sequencing. It was confirmed by IFAT that the bivalent DNA vaccine was expressed in the plasma and on the surface of muscle cells from mouse. The worm reduction rate was 41.20%-53.85% and the egg reduction rate was 47.02%-53.83% bivalent DNA groups. Furthermore, the worm and egg reduction rates in pVIVO2-mcs-Sj23-SjFABP plasmid DNA and amylose adjuvant group were 68.89% and 84.04% respectively, significantly higher than single antigenic DNA vaccine and bivalent DNA vaccine (P < 0.05). CONCLUSION: The co-expressed bivalent DNA vaccines of Schistosoma japonicum can induce immune protection in mice. The protective immunity of amylose adjuvant group is higher than that of the univalent DNA and bivalent vaccines.

[Vaccination of mice with recombinant nucleic acid vaccine encoding the integral membrane protein Sj23 and cytokine IL-12 elicits specific immune responses against schistosoma japonica].

OBJECTIVE: To develop a Schistosoma japonica integral membrane protein Sm23 or Sj23 combined with murine IL-12 DNA-base vaccine against schistosomiasis. METHODS: Plasmids pVIVO2-Sj23 and pVIVO2-IL12-Sj23, expressing the integral membrane protein Sj23 of Schistosoma japonica and/or murine IL-12 were constructed. The plasmid pVIVO2-IL12-Sj23 was transfected into the human embryonic kidney cells of the line 293. RT-PCR was used to detect the expression of Sj23 mRNA in the 293 cells. Indirect immunofluorescence test was used to detect the expression of Sj23 protein. Fifty BALB/c mice were randomly divided into 5 equal groups to be injected with pVIVO2-IL12-Sj23 plasmid DNA, pVIVO2-Sj23 plasmid DNA, pVIVO2-IL12 plasmid DNA, pVIVO2 blank vector, and normal saline respectively into the quadriceps muscle of thigh. Four weeks after each mouse were infested with 40 +/- 2 cercariae of Schistosoma japonica. Six weeks after the infestation the mice were killed to calculate the load of schistosoma and the amount of eggs per gram (EPG) of liver so as to calculate the worm reduction rate and egg reduction rate after the vaccination. Before immunization, 4 weeks after immunization, and 6 weeks after immunization blood samples were collected from the caudal veins of mice. With soluble egg antigen (SEA) and adult worm antigen (AWA) ELISA was used to detect the serum IgG level. Western blotting was used to detect the serum specific anti-Sj23 IgG level. Six weeks after the cercaria challenge single splenocyte suspension was prepared. Splenocytes were cultured with SEA, and concanavalin A (ConA). ELISA was used to detect the levels of IL-4 and IFN-gamma in the supernatant. Flow cytometry was used to analyze the subgroups of splenocyte. RESULTS: + Forty-eight hours after the transfection, RT-PCR and indirect immunofluorescence test showed expression of Sj23 mRNA and protein in the HEK-293 cells. The worm reduction rate was 45.53% and the egg reduction was 58.35% in the pVIVO2-IL12-Sj23 group, significantly higher than those in the monovalent vaccine pVIVO2-Sj23 group (27.23% and 33.93% respectively, both P <0.05). ELISA and Western blotting analysis showed that the level of IgG specific for Sj23 significantly increased 4 weeks after vaccination in the pVIVO2-IL12-Sj23 and pVIVO2-Sj23 groups without significant difference between these 2 groups (P > 0.05). After stimulation of ConA and SEA the level of Th1 type cell factor IFN-gamma was higher and the level of the Th2 type cellular factor IL-4 was low in the supernatant of suspension of splenocytes of the pVIVO2-IL12-Sj23 group. FCM showed the percentages of CD4+ and CD8+ subgroups of the murine splenocytes of all experimental groups were all significantly lower than those of the normal mice (all P < 0.001 approximately 0.02). However, there was no significant difference in the CD4+/CD8+ ratio among the experimental groups (all CONCLUSION: pVIVO2-IL12-Sj23 is sufficient to elicit significant levels of protective immunity against Schistosoma japonica challenge infection. IL-12, a cytokine and a gene adjuvant, is able to induce Th1 responses and hence the protective immunity.

[The adjuvant effect of IL-12 on protective immunity of Schistosoma japonicum fatty acid binding protein (Sj14FABP)].

OBJECTIVE: To study the immune protection of Schistosoma japonicum fatty acid binding protein (Sj14FABP) DNA vaccine enhanced by IL-12. METHODS: The recombinant plasmids pVIVO2-Sj14FABP and pVIVO2-IL12-Sj14FABP were constructed respectively, and were prepared on a large scale after identification. 48 male BALB/c mice were divided into 4 groups randomly. In group A, B, C, and D, each mouse was injected intramuscularly with 100 microl 0.9% NaCl, 100 microg pVIVO2, 100 microg pVIVO2-Sj14FABP and 100 microg pVIVO2-IL12-Sj14FABP respectively. 30 days after immunization each mouse was challenged with 40 +/- 2 cercariae of S. japonicum. On day 45 after challenge, all mice were sacrificed to count the number of recovered adult worms and the hepatic eggs. Sera from mice were used to detect IgG antibody. The production of IL-2, IL-4 and IFN-gamma in the supernatant of spleen cells was observed by means of sandwich ABC-ELISA. RESULTS: The recombinant plasmids pVIVO2-Sj14FABP and pVIVO2-IL12-Sj14FABP were constructed. The worm reduction rate in group C and D was 24.11% and 39.4%, as well as liver egg reduction rate of 27.2% and 32.8% respectively. The level of IL-2 and IFN-gamma in group D increased significantly, while IL-4 secretion decreased (P < 0.01). 30 days after immunization, no higher titer of IgG antibody was shown in all groups. Furthermore, no significant difference on the level of IgG was found among the groups (P > 0.05). CONCLUSION: Sj14FABP DNA vaccine induces partial protective immunity in BALB/c mice. IL-12 drives the immune response toward a Th1 direction, and enhances the protective immune effect of the vaccine.

[Studies on effect enhancement of the Schistosoma japonicum DNA vaccine pVIV02-IL12-Sj23 by vegetal polysaccharides].

OBJECTIVE: To enhance the immunogenicity of the recombinant pVIVO2-IL12-Sj23 vaccine of Schistosoma japonicum by using mixed vegetal polysaccharides as adjuvant. METHODS: The plasmid pVIVO2-IL12-Sj23 was constructed. 3 groups of BALB/C mice were injected intramuscularly with normal saline (Group A), pVIVO2-IL12-Sj23 plasmid DNA (B), and pVIVO2-IL12-Sj23 plus mixed vegetal polysaccharides (C) respectively, and challenged with S. japonicum cercariae on the 4th week after immunization. Mice were killed to calculate the worm reduction rate and egg reduction rate in liver tissue on the 6th week after infection. Before and 4 weeks after immunization blood samples were collected. RESULTS: The worm reduction rate and egg reduction rate were 64.3% and 79.9%, respectively in group C, 45.5% and 58.4%, respectively in group B, showing a remarkable difference hetween them (P < 0.05). ELISA analysis showed a significantly higher level of IgG specific for Sj23 4 weeks after vaccination in groups B and C (P < 0.05). However, there was no significant difference in IgG level between groups C and B (P > 0.05). CONCLUSION: When mixed vegetal polysaccharides are used as adjuvant, the effect of the vaccine pVIVO2-IL12-Sj23 can he considerably enhanced.

[Protective immunity induced by multivalent DNA vaccine of Schistosoma japonicum Mr23 x 10(3) membrane antigen and IL-12 in mice].

OBJECTIVE: To develop multivalent DNA vaccine PV-IL12-Sj23 which co-expresses Sj23 and cytokine IL-12, and investigate its protective efficacy in BALB/c mice against challenge infection. METHODS: On the basis of the reconstructed plasmid PV-IL12-Sj23 and plasmid PV-IL12, blank plasmid PV and plasmid PV-Sj23 only expressing Sj23 were constructed. Fifty BALB/c male mice were divided into five groups, which were immunized intramuscularly with multivalent DNA vaccine PV-IL12-Sj23, plasmid PV-Sj23 expressing Sj23, plasmid PV-IL12 expressing cytokine IL-12, blank plasmid PV and saline, respectively. Each mouse was immunized with 100 microg DNA only once. All the mice were challenged with 40 cercariae at week 4, killed and perfused for collection of worms at week10. The number of recovered worms and eggs in the liver were counted. RESULTS: Blank plasmid PV and plasmid PV-Sj23 expressing Sj23 were successfully constructed. The worm reduction rate in PV-IL12-Sj23 group and PV-Sj23 group was 45.5% and 27.2% (P< 0.05) respectively. The number of eggs in liver tissue was reduced by 58.4% and 33.9% respectively. CONCLUSION: Multivalent DNA vaccine PV-IL12-Sj23 can induce protective immunity against Schistosoma japonicum in BALB/c mice significantly, with a better protective efficacy than the monovalent DNA vaccine PV-Sj23.

Congenital infection of rabbits with Schistosoma japonicum and protective immunity of offspring.

BACKGROUND: Recently congenital infection with Schistosoma japonicum (S. japonicum) has been demonstrated in pigs, rabbits, mice and dogs. We explored the rabbit as an animal model for the congenital infection of schistosomiasis japonica and assessed the effect of a congenital S. japonicum infection on the resistance of rabbit kittens to a postnatal challenge infection. METHODS: Sixteen pregnant New Zealand white rabbits were infected with a single dose of S. japonicum cercariae. The exposed animals were divided into three groups according to the gestation age at the time of infection. Diagnosis of prenatally acquired S. japonicum infection in the rabbit kittens was primarily based on serological tests in combination with parasitological and histopathological findings. Congenitally infected kittens were challenged percutaneously with 100 S. japonicum cercariae to assess the effect of a congenital S. japonicum infection on kitten resistance to a postnatal challenge infection. RESULTS: The overall prevalence of congenital infection in offspring of infected mothers was 20% (12/60). The congenital infection rate in group L (late gestation) was much higher than in group E (early gestation) and group M (mid-gestation) (P <0.05). After a postnatal challenge infection, prenatally infected kittens had a 54.66% worm reduction rate, 41.45% egg reduction rate, and 51.76% granuloma size reduction rate compared to naïve kittens. CONCLUSIONS: This study demonstrates the possibility of congenital infection of S. japonicum in rabbits and the resistance of congenitally infected kittens to a postnatal challenge infection. These results have important implications not only for epidemiological investigations, but also in designing government control programs for schistosomiasis.

Immune responses against Schistosoma japonicum after vaccinating mice with a multivalent DNA vaccine encoding integrated membrane protein Sj23 and cytokine interleukin-12.

BACKGROUND: The vaccination of mice with DNA encoding single candidate antigens has failed to induce significant protection against Schistosoma japonicum (S. japonicum) challenge infections. In this study, we evaluated the feasibility of using a multivalent DNA vaccine which co-expressed S. japonicum integral membrane protein Sj23 and murine cytokine IL-12 to induce protective immune responses. METHODS: The plasmid pVIVO2-IL12-Sj23, a eukaryotic expression vector expressing Sj23 and murine IL-12 simultaneously, was constructed, identified, and tested for expression in vitro. Its ability to protect against S. japonicum challenge infections was analyzed according to worm reduction rate and egg reduction rate after vaccination of BALB/c mice. The serum levels of specific IgG antibody were determined by enzyme-linked-immuno sorbent assay (ELISA) and Western blot analysis. Using cultured spleen cells, IFN-gamma and IL-4 post-stimulation were quantified by ELISA. The phenotypes of splenocyte populations were analyzed by flow cytometry (FCM). RESULTS: The plasmid DNA pVIVO2-IL12-Sj23 was proven to express well in vitro by transient transfection of HEK-293 cells. Immunization resulted in a worm reduction rate of 45.53% and egg reduction rate of 58.35%. ELISA and Western blot analysis indicated that immunized mice generated specific IgG against Sj23. Spleen cells showed significant increases in IFN-gamma but decreases in IL-4. No significant differences in CD4+ and CD8+ subgroup ratios were observed after the challenges. CONCLUSIONS: The multivalent DNA vaccine pVIVO2-IL12-Sj23 is sufficient to elicit moderate but highly significant levels of protective immunity against challenge infections. Cytokine IL-12, as a gene adjuvant, was able to enhance the Th1 responses and, hence, the protective immunity.

Vertical transmission of Schistosoma japonicum in the rabbit.

The aim of the present study was to confirm observations on the vertical transmission of Schistosoma japonicum in the rabbit. S. japonicum-infected pregnant rabbits were used in this study. Perfusion of mother rabbits was done 9 weeks after infection in order to obtain worm burdens in relation to their initial cercarial dose. Anti-schistosoma specific IgM antibodies in serum samples collected from rabbit kittens were detected by ELISA. Our results showed that gestation period lasted the normal 29-31 days. All the exposed mother rabbits became infected with S. japonicum. Positive IgM antibody OD values were detected in 12 out of the 60 kittens examined (20.0%). In group C and A, 40.0% and 17.9% of the kitten were congenitally infected, respectively. 18.1% of the kittens born to mothers infected with a single dose of 200 cercariae per rabbit were positives; this is not significantly different from that obtained for the 600 dose group (22.2%). Three randomly selected IgM+ kittens harbored between one and two adult worms. The livers of these kittens displayed granulomatous lesions. It is concluded that congenital S. japonicum infection does occur in the rabbit and is affected by the mother stage of pregnancy and to a lesser extent by its infection load.

[Study on CD4+ cells deletion mechanism in experimental alveolar echinococcosis].

OBJECTIVE: To study the possible mechanism of CD4+ T cells deletion in mice with alveolar echinococcosis, particularly on the relationship between Echinococcus multilocularis infection and apoptosis of T lymphocyte subsets. METHODS: BALB/c mice were infected with E. multilocularis and uninfected mice were used as control group. CD4+ T cell and CD8+ T cells were separated 12 weeks and 25 weeks after infection. Purified CD4+ and CD8+ T cell subsets were cultured in complete medium and stimulated with EmAg, anti-CD3 mAb, rIL-2, mouse rTNF alpha and PWM respectively. After 16 h of incubation, cells were collected and assessed by electron microscopy. DNA fragmentation was observed by eletrophoresis, stained by TUNEL assays and PI, analyzed by flow cytometry. RESULTS: CD4+ and CD8+ T cells in 25 weeks experiment group presented chromatin condensation, lost nuclear membrane integrity, and formed exocytoplasmic vacuolization. DNA ladder was observed by agarose gel eletrophoresis, and the appearance of DNA fragments was equivalent to approximately 200 bp. None of these appearances were observed in control group in 12 weeks post infection and CD8+ T cell in mice of 25 weeks post infection group. The apoptosis level of CD4+ and CD8+ T cells in 12 weeks post infection group was not significantly different from the control group. While the apoptosis level of CD4+ and CD8+ T cells increased significantly in 25 weeks post infection group as compared with the control (P < 0.01). Higher apoptosis in CD4+ T cells was observed than that of CD8+ T cells. Apoptosis mainly appeared during S phase of cell cycle. CONCLUSION: Apoptosis is a prominent causation of activation-induced CD4+ T cell death in later period of E. multilocularis infection. Increase of the death-promoter signals and decrease of the death suppresser signals may have been responsible, in part, for the apoptosis in CD4+ T lymphocytes in the infected mice.

[Kinetic analysis of cytokines and immunoglobulin G subclass in BALB/c mice infected with Echinococcus alveolaris].

OBJECTIVE: To observe the dynamic change of immune response in mice infected with Echinococcus alveolaris (AE) at difference period of time, and to explore hostalveo's immune regulation. METHODS: The infection lasted and was followed up for 25 weeks. The spleen cells from BALB/c mice infected with AE stimulated with EmAg and ConA or PHA in vitro. IL-2R, IFN-gamma, TNF-alpha, IL-1 and specific IgG subclasses were determined by ELISA. NO was tested by chemical assay. RESULTS: NO level sharply rose in 16 weeks after BALB/c mice were infected with AE. The levels of IgG1 and IgG3 significantly increased 8 weeks after infection, and remained elevating throughout the period of observation. IgG3 showed slight increase, IgG2a and IgG2b appeared low level following infection. The production of IL-2R and TNF alpha increased significantly 8 weeks of infection, while IL-2R sharply decreased in 12 weeks of infection. During the period of 2-12 weeks of infection there was an increase in IL-1 secreting. The level of IL-1 and TNF alpha rapidly increased since 16 weeks post infection. High level of IFN-gamma was detected during the period of observation, and showed a peak at 12 weeks. CONCLUSION: Th1 is the major response in the early stage of infection, which is replaced by Th2 response in later period of infection.

Synthesis and bioactivities of two multiple antigen peptides as potential vaccine against schistosoma.

Based on the two antigenic peptides, 26-43 (P26) and 116-131 (P116), derived from 28 kDa glutathione S-transferase of Schistosoma mansoni (Sm28GST), two multiple antigenic peptides (MAPs), (P26)4-MAP and (P116)4-MAP with the same oligomeric lysine core, were synthesized by stepwise solid-phase peptide synthesis method. The antigenicities and protective effects of these two MAPs were examined on experimental animals. As shown in the dot-ELISA result, the synthetic MAPs could be recognized and bound by immunoglobins in both patient's and infected-rabbit's sera. After Kunming mice were immunized with (P26)4-MAP, the worm burden reduction rate and the liver egg reduction rate were 59.9% and 61.1%. In (P26)4-MAP or (P116)4-MAP immunized BALB/c mice, the worm burden reduction rates were 37.5% and 62.5%, respectively, and the liver egg reduction rates were 35.1% and 54.0%, respectively.