OBJECTIVETo discuss the optimal immunization dose by observing the immunoprotective effects of different doses of recombinant Schistosoma japonicum (Chinese strain) signaling protein 14-3-3 (rSj14-3-3).

METHODSSj14-3-3 gene was amplified by reverse transcriptase PCR (RT-PCR), subcloned into prokaryotic expression vector pET28a, then transformed into E.coli to express by inducing. Purified rSj14-3-3 was prepared through SDS polyacrylamide gel electrophoresis (SDS-PAGE), electroelution, dialysis, then BALB/c mice were divided into 5 groups and immunized in rSj14-3-3 protein followed by challenging infection (the 1st, 2nd, and 3rd groups were immunized in 50 microg, 100 microg and 300 microg antigen, respectively. The 4th, 5th groups were immunized in Freund's adjuvant and normal saline controls). After 6 weeks of challenging infection, the mice were killed and the worm and egg reduction rates were calculated. And the mice sera in different time were taken to examine the specific anti-Sj14-3-3 IgG.

RESULTSrSj14-3-3 protein was expressed successfully. After immunizing and challenging, worm reduction was found to be 28.20% in the 1st group, 43.10% in the 2nd group, 40.00% in the 3rd group, respectively. Number of eggs in liver tissue was reduced by 41.80%, 57.50%, 55.70%, respectively. Compared the results of the tested groups to the controls, the differences were of significance by t-test (worm reduction rate: t = 6.8 in the 1st group, t = 8.7 in the 2nd group, t = 7.3 in the 3rd group, P < 0.01 in all tested groups. Egg reduction rate at the group's number above: t = 11.23, t = 11.54, t = 7.99, P < 0.01 in all tested groups). As compared the results between the tested groups by chi(2), the differences were of significance between the 1st and the 2nd groups (worm reduction rate: chi(2) = 8.96, P < 0.05; egg reduction rate: chi(2) = 15.69, P < 0.05), between the 1st and the 3rd groups, the differences were also of significance (worm reduction rate: chi(2) = 6.52, P < 0.05; egg reduction rate: chi(2) = 12.52, P < 0.05). The difference was not of significance between the 2nd and the 3rd groups (worm reduction rate: chi(2) = 1.20, P > 0.05; egg reduction rate: chi(2) = 0.93, P > 0.05). In all tested groups, total anti-Sj14-3-3 specific IgG rose markedly. IgG(1) and IgG(2a) subtypes were high, but IgG(2b) and IgG(3) were near the background in four subtypes tested.

CONCLUSIONImmunoprotection of rSj14-3-3 should have some relations with immunization dose, and the protection obtained from immunizing mice by using 100 microg antigen was the best.

14-3-3 Proteins ; administration & dosage ; immunology ; Animals ; Antibodies, Helminth ; immunology ; Antibody Formation ; Antigens, Helminth ; blood ; Female ; Helminth Proteins ; immunology ; Immunoglobulin G ; blood ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; Schistosoma japonicum ; genetics ; immunology ; Signal Transduction ; Vaccination

Schistosomiasis japonica is an endemic, zoonotic disease of major public health importance in China. Vaccination is needed as a complementary approach to the ongoing control programs. In the present study, we determined if the efficacies of DNA vaccine encoding the SjGST and Sj32 asparaginyl endopeptidase protein could be enhanced by boosting with SjGST-32 protein vaccines. Mice were inoculated with a VR1012-SjGST-32 DNA vaccine followed by boosting with rSjGST-32 at 0, 14 and 28 d. Two weeks after the final boost, mice were challenged percutaneously with cercariae. On day 45 following the challenge, all mice were sacrificed and the numbers of recovered worms and hepatic eggs were counted. Moreover, we analyzed the immune response among various vaccination groups. The results showed that DNA vaccine efficacy was enhanced when mice were boosted with protein vaccine. Adult worm and liver egg burdens were reduced 42.3% and 59.6%, respectively. We further found that DNA vaccine followed by boosting with protein significantly increased the IgG titer and T cell proliferation over those seen in mice vaccinated solely with DNA vaccines. Furthermore, the higher level of IFN-gamma expression in the splenetic CD4+ T cell showed that DNA prime-Protein boosting vaccine induced CD4+ Th1-type responses. Thus, DNA vaccine efficacy was significantly enhanced via boosting protein vaccine which might provide a basis for rational application of the Schistosoma vaccine.
Animals ; Antigens, Helminth ; immunology ; Female ; Glutathione Transferase ; administration & dosage ; immunology ; Helminth Proteins ; immunology ; Immunization, Secondary ; methods ; Mice ; Mice, Inbred C57BL ; Recombinant Fusion Proteins ; administration & dosage ; immunology ; Schistosoma japonicum ; Schistosomiasis japonica ; prevention & control ; Vaccination ; methods ; Vaccines, DNA ; administration & dosage ; immunology

Although schistosomicidal drugs and other control measures exist, the advent of an efficacious vaccine remains the most potentially powerful means for controlling this disease. In this study, native fatty acid binding protein (FABP) from Fasciola gigantica was purified from the adult worm's crude extract by saturation with ammonium sulphate followed by separation on DEAE-Sephadex A-50 anion exchange chromatography and gel filtration using Sephacryl HR-100, respectively. CD1 mice were immunized with the purified, native F. gigantica FABP in Freund's adjuvant and challenged subcutaneously with 120 Schistosoma mansoni cercariae. Immunization of CD1 mice with F. gigantica FABP has induced heterologous protection against S. mansoni, evidenced by the significant reduction in mean worm burden (72.3%), liver and intestinal egg counts (81.3% and 80.8%, respectively), and hepatic granuloma counts (42%). Also, it elicited mixed IgG1/IgG2b immune responses with predominant IgG1 isotype, suggesting that native F. gigantica FABP is mediated by a mixed Th1/Th2 response. However, it failed to induce any significant differences in the oogram pattern or in the mean granuloma diameter. This indicated that native F. gigantica FABP could be a promising vaccine candidate against S. mansoni infection.
Animals ; Antibodies, Helminth/immunology ; Fasciola/*chemistry ; Fatty Acid-Binding Proteins/*administration & dosage/immunology/isolation & purification ; Female ; Helminth Proteins/*administration & dosage/immunology/isolation & purification ; Humans ; Immunization ; Mice ; Mice, Inbred Strains ; Schistosoma mansoni/immunology/*physiology ; Schistosomiasis mansoni/immunology/parasitology/*prevention & control

OBJECTIVE: To determine the immune-protective effect of Japan Schistosoma (Chinese mainland strain) 23 kD membrane protein-heat shock protein (SjC23-Hsp70) DNA vaccine plus adjuvantinduced interleukin-12 (IL-12) plasmid DNA on Schistosoma japonicum infection in water buffalos. METHODS: Forty-five health water buffalos (8-10 months old) in non-endemic area of schistosomiasis were randomly assigned into group A (SjC23-Hsp70+IL-12, 300 μg), group B (SjC23+IL-12, 300 μg) and group C (pVAX+IL-12, 300 μg), 15 in each group. Each buffalo was immuned by shoulder intramuscular injection for 3 times, at an interval of 28 days. Twenty-eight days after the last immunization, each buffalo was infected with 1000 Japan cercariae of Schistosoma. Fecal examinations were conducted 2 days and 1 day before the perfusion, and on the day of perfusion. The number of hatching miracidia and eggs per gram feces was recorded. Fifty-six days after the infection, the buffalos were sacrificed and perfused via the descending aorta. The recovered adult worms and eggs in the liver tissue were counted. RESULTS: We compared group A and B with group C: the estrogen reduction rate was 45.7% and 26.61%; bug reduction rate was 44.51% and 25.84%; the fecal egg reduction rate was 41.1% and 31.63%; the miracidium reduction rate was 48.11% and 38.07%; and the liver egg reduction rate was 43.39% and 31.95%. The above rates in group A were higher than those in group B (P<0.05). CONCLUSION SjC23-Hsp70 DNA vaccine combined with IL-12 may have a significant immunoprotective effect on buffalos.
Animals ; Antigens, Helminth ; immunology ; Buffaloes ; Cattle ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Helminth Proteins ; immunology ; Immunization ; methods ; Interleukin-12 ; genetics ; immunology ; Membrane Proteins ; immunology ; Schistosomiasis japonica ; immunology ; prevention & control ; veterinary ; Vaccines, DNA ; administration & dosage ; immunology ; Vaccines, Synthetic ; immunology

Fascioliasis is one of the public health problems in the world. Cysteine proteinases (CP) released by Fasciola gigantica play a key role in parasite feeding, migration through host tissues, and in immune evasion. There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infections. Cysteine proteinases were purified and tested in vaccine trials of sheep infected with the liver fluke. Multiple doses (2 mg of CP in Freund's adjuvant followed by 3 booster doses 1 mg each at 4 week intervals) were injected intramuscularly into sheep 1 week prior to infect orally with 300 F. gigantica metacercariae. All the sheep were humanely slaughtered 12 weeks after the first immunization. Changes in the worm burden, ova count, and humoral and cellular responses were evaluated. Significant reduction was observed in the worm burden (56.9%), bile egg count (70.7%), and fecel egg count (75.2%). Immunization with CP was also found to be associated with increases of total IgG, IgG1, and IgG2 (P<0.05). Data showed that the serum cytokine levels of pro-inflammatory cytokines, IL-12, IFN-gamma, and TNF-alpha, revealed significant decreases (P<0.05). However, the anti-inflammatory cytokine levels, IL-10, TGF-beta, and IL-6, showed significant increases (P<0.05). In conclusion, it has been found that CP released by F. gigantica are highly important candidates for a vaccine antigen because of their role in the fluke biology and host-parasite relationships.
Animals ; Antibodies, Helminth/immunology ; Cysteine Proteases/administration & dosage/*immunology/isolation & purification ; Cytokines/immunology ; Fasciola/chemistry/*enzymology/immunology ; Fasciola hepatica/immunology/physiology ; Fascioliasis/immunology/parasitology/*prevention & control ; Female ; Helminth Proteins/administration & dosage/*immunology/isolation & purification ; Humans ; Male ; Protective Agents/*administration & dosage/isolation & purification ; Sheep ; Vaccines/immunology

We have reported that a 24 kDa protein (22U homologous; As22U) of Anisakis simplex larvae could elicit several Th2-related chemokine gene expressions in the intestinal epithelial cell line which means that As22U may play a role as an allergen. In order to determine the contribution of As22U to allergic reactions, we treated mice with 6 times intra-nasal application of recombinant As22U (rAs22U). In the group challenged with rAs22U and ovalbumin (OVA), the number of eosinophils in the bronchial alveolar lavage fluid (BALF) was significantly increased, as compared to the group receiving only OVA. In addition, mice treated with rAs22U and OVA showed significantly increased airway hyperresponsiveness. Thus, severe inflammation around the airway and immune cell recruitment was observed in mice treated with rAs22U plus OVA. The levels of IL-4, IL-5, and IL-13 cytokines in the BALF increased significantly after treatment with rAs22U and OVA. Similarly, the levels of anti-OVA specific IgE and IgG1 increased in mice treated with rAs22U and OVA, compared to those treated only with OVA. The Gro-alpha (CXCL1) gene expression in mouse lung epithelial cells increased instantly after treatment with rAs22U, and allergy-specific chemokines eotaxin (CCL11) and thymus-and-activation-regulated-chemokine (CCL17) gene expressions significantly increased at 6 hr after treatment. In conclusion, rAs22U may induce airway allergic inflammation, as the result of enhanced Th2 and Th17 responses.
Administration, Intranasal ; Animals ; Anisakiasis/*immunology/parasitology ; Anisakis/*immunology/metabolism ; Bronchoalveolar Lavage Fluid ; Chemokines/metabolism ; Cytokines/analysis/*metabolism ; Eosinophils/metabolism ; Female ; Gene Expression Regulation/*immunology ; Helminth Proteins/*immunology ; Hypersensitivity/*immunology/parasitology ; Immunoglobulin E/immunology ; Immunoglobulin G/immunology ; Larva/immunology/metabolism ; Lung/metabolism ; Mice ; Mice, Inbred C57BL ; Recombinant Proteins/immunology ; Th17 Cells/metabolism ; Th2 Cells/metabolism