Protective Immunity Induced by TgMIC5 and TgMIC16 DNA Vaccines Against Toxoplasmosis.

Toxoplasma gondii is an obligate intracellular parasite, which is responsible for a widely distributed zoonosis. Effective vaccines against toxoplasmosis are necessary to protect the public health. The aim of this study is to evaluate the immune efficacy of DNA vaccines encoding TgMIC5 and TgMIC16 genes against T. gondii infection. The recombinant plasmid pVAX-MIC5 and pVAX-MIC16 were constructed and injected intramuscularly in mice. The specific immune responses and protection against challenge with T. gondii RH tachyzoites were evaluated by measuring the cytokine levels, serum antibody concentrations, lymphocyte proliferation, lymphocyte populations, and the survival time. The protection against challenge with the T. gondii RH tchyzoites and PRU cysts was examined by evaluation of the reduction in the brain cyst burden. The results indicated that immunized mice showed significantly increased levels of IgG, IFN-γ, IL-2, IL-12p70, and IL-12p40 and percentages of CD4+ and CD8+ T cells. Additionally, vaccination prolonged the mouse survival time and reduced brain cysts compared with controls. Mouse groups immunized with a two-gene cocktail of pVAX-MIC5 + pVAX-MIC16 were more protected than mouse groups immunized with a single gene of pVAX-MIC5 or pVAX-MIC16. These results demonstrate that TgMIC5 and TgMIC16 induce effective immunity against toxoplasmosis and may serve as a good vaccine candidate against T. gondii infection.

Adjuvantic cytokine IL-33 improves the protective immunity of cocktailed DNA vaccine of ROP5 and ROP18 against toxoplasma gondii infection in mice.

Toxoplasma gondii is a threat for immunocompromized individuals, and no treatment is available for enhancing immunity against infection. Molecular adjuvants may improve the efficacy of DNA vaccine-induced T cell immunity. Here, we report that cocktailed DNA immunization with ROP5 and ROP18 boosted immune responses induced by a single DNA immunization with ROP5 or ROP18, but also that co-administration of molecular adjuvant IL-33 enhanced immune efficacy induced by this cocktailed DNA vaccination. These improved immune responses were characterized by higher Toxoplasma-specific IgG2a titers, Th1 responses associated with the production of IFN-γ, IL-2, IL-12, as well as cell-mediated activity with higher frequencies of CD8+ and CD4+ T cells. More importantly, this enhanced immunity has the ability to confer remarkable protection against a high dose lethal challenge of the T. gondii RH strain and thus against chronic infection with the T. gondii PRU strain. These data show that IL-33 is a promising immunoadjuvant to facilitate humoral as well as cellular immunity in a vaccine setting against T. gondii, and suggest that it should be evaluated in strategies against other apicomplexan parasites.

TITLE: La cytokine IL-33 utilisée comme adjuvant améliore l'immunité protectrice du vaccin à cocktail d'ADN de ROP5 et ROP18 contre l'infection à Toxoplasma gondii chez la souris. ABSTRACT: Toxoplasma gondii est une menace pour les individus immunodéprimés et aucun traitement n'est disponible pour renforcer l'immunité contre l'infection. Les adjuvants moléculaires peuvent améliorer l'efficacité de l'immunité des cellules T induite par un vaccin à ADN. Ici, nous rapportons que l'immunisation par le cocktail d'ADN de ROP5 et ROP18 a stimulé les réponses immunitaires induites par une seule immunisation par l'ADN de ROP5 ou ROP18, mais aussi que la co-administration de l'adjuvant moléculaire IL-33 a amélioré l'efficacité immunitaire induite par cette vaccination par cocktail d'ADN. Ces réponses immunitaires améliorées ont été caractérisées par des titres d'IgG2a spécifiques à Toxoplasma plus élevés, des réponses Th1 associées à la production d'IFN-γ, IL-2, IL-12 ainsi qu'une activité à médiation cellulaire où les fréquences des cellules T CD8+ et CD4+ étaient plus élevées. Plus important encore, cette immunité renforcée a la capacité de conférer une protection remarquable contre une provocation létale par haute dose de la souche RH de T. gondii et donc contre une infection chronique par la souche PRU de T. gondii. Ces données montrent qu'IL-33 est un immunoadjuvant prometteur pour faciliter l'immunité humorale et cellulaire dans un contexte de vaccination contre T. gondii et suggèrent qu'il devrait être évalué dans des stratégies contre d'autres parasites apicomplexes.

[Epidemiological investigation on the intermediate hosts of Paragonimus in Ninghai County of Zhejiang Province].

Freshwater crabs and snails were collected from Ninghai County in Zhejiang Province, and examined respectively for Paragonimus metacercariae and cercariae. Among 97 freshwater crabs found, the prevalence was 11.3% (11/97) with a mean intensity of 1 metacercariae per crab. It was 10.2% (5/49) and 20.2% (4/20) in the groups weighted 5-15 g and 15-25 g respectively, with an average intensity of 1, and no metacercariae were found in weight group of 25-35 g. Two positive crabs were found from 20 crabs with a low weight (< 5 g). Male to female crabs ratio was 2.5:1, and there was no significant difference in prevalence between males [12.7%(7/55)] and females [9.1% (2/22)]. No cercariae or metacercariae were found in 200 snails (Semisulcospira libertino).

[Fusion expression and antigenicity analysis of miracidial antigen from eggs of Schistosoma japonicum].

OBJECTIVES: To express the miracidial antigen from eggs of Schistosoma japonicum (Chinese mainland strain) (SjMP10), and investigate the role of the miracidial antigen during the hepatic granuloma formation of schistosomiasis. METHODS: A pair of specific primers was designed and synthesized according to the nucleotide sequence of the open reading frame of the miracidial antigen gene. The open reading frame of the miracidial antigen gene was amplified, digested by restrictive enzyme (BamHI, SolI), and cloned directly into the expression plasmid pGEX-4T-3 to construct the recombinant plasmid. The recombinant plasmids were transformed into E. coli BL21(DE3), and induced by IPTG to express the fusion protein of GST-SjMP10. The expressed fusion protein was purified by electric elution method, and its antigenicity was examined by Western blotting and lymphocyte proliferation test. RESULTS: The gene of miracidial antigen was cloned into the expression plasmid pGEX-4T-3. After induced by IPTG, the recombinant expressed a fusion protein of GST-SjMP10, with a molecular weight of 39 000 approximately. The purified fusion protein showed proper antigenicity that could be recognized by the sera of rabbits heavily infected by Schistosoma japonicum and could stimulate the proliferation of splenic lymphocytes of infected BALB/c mice. CONCLUSION: The miracidial antigen from eggs of Schistosoma japonicum was expressed successfully.

In vitro effect of cyclosporine A on juvenile Schistosoma mansoni labeled by AF18 (5-N-octadecanoyl aminofluorescein).

OBJECTIVE: To explore the in vitro effect of cyclosporine A on the tegument of juvenile Schistosoma mansoni labeled by AF18 and investigate the effect of cyclosporine A on schistosomula surface membrane fluidity. METHODS: Preparation of transformed schistosomula, adding cyclosporine A into tubes containing schistosomula and labeling of transformed schistosomula with AF18, then observe schistosomula under fluorescence microscope. RESULTS: Schistosomula of different groups labeled by AF18 were damaged by cyclosporine A in vitro. CONCLUSION: Cyclosporine A increases the uptake of AF18 by schistosomula in vitro which is dose-dependent, and decreases the parasite surface membrane fluidity.

[Expression and characterization of 21.7 kDa membrane protein of Schistosoma japonicum].

OBJECTIVE: To express, purify and characterize the 21.7 kDa membrane protein of Chinese strain S. japonicum (SjC21.7). METHODS: The gene of SjC21.7 was subcloned into the expression vector pGEX-4T-3 to form recombinant plasmid. The recombinant plasmids were transformed into E. coli BL21 and the GST-SjC21.7 fusion protein was expressed by IPTG induction. The recombinant SjC21.7 molecule was prepared by affinity chromatography and digested by thrombin. The Kunming strain mice were immunized with the recombinant SjC21.7 molecule to produce anti-SjC21.7 antibody. The purified SjC21.7 was recognized by the immunized mouse serum and the sera of rabbits infected by S. japonicum. RESULTS: The SjC21.7 gene was subcloned into expression vector pGEX-4T-3, then transformed into E. coli BL21 to express the GST-SjC21.7 fusion protein. The recombinant SjC21.7 molecule obtained from the fusion protein could stimulate the mice to produce a high titer of specific antibody and could be recognized by sera of both the immunized and infected rabbits. The sera of immunized mice could also recognize the 21.7 kDa protein molecule of the adult worm antigen (AWA). CONCLUSION: The recombinant and purified SjC21.7 was prepared and showed similar immunological characteristics to the natural SjC21.7 molecule, providing a basis for further investigation of the immunological protection of the recombinant SjC21.7.