Protective efficacy of recombinant Cryptosporidium parvum CpPRP1 sushi domain against C. tyzzeri infection in mice.

Until now, there are no completely effective parasite-specific pharmaceuticals or immunotherapies for treatment against the zoonotic cryptosporidiosis. Sushi domain (CpSushi) is an important functional domain in Cryptosporidium parvum putative rhoptry protein-1 (CpPRP1), which is the only reported C. parvum rhoptry protein and may play key role in the course of invasion. Here, a 708-bp fragment encoding the CpSushi domain was amplified and expressed in E. coli. Immunofluorescence detection showed that CpSushi was located on the surface of C. parvum oocysts and the apical pole to the sporozoites that belonged to the position of rhoptry. Three-week-old female ICR mice were used for detecting the immunoreactions and immunoprotection of recombinant CpSushi (rCpSushi) to artificial C. tyzzeri infection. The results indicated that a significant increase of anti-CpSushi antibody response was induced by the recombinant protein. Compared to blank, Tris-EDTA (TE) buffer and adjuvant controls mice, rCpSushi-immunized mice produced specific spleen cell proliferation as well as enhanced IL4, IL5, IL12p70 and TNF-α production in vitro. The reduction rate of parasites shedding in stool in mice immunized with rCpSushi was 68.91% after challenging with C. tyzzeri. These results suggest that CpSushi could be a new promising cryptosporidiosis vaccine candidate antigen composition.

Huang Y ，Cao W ，Shi K ，Mi R ，Lu K ，Han X ，Chen Z ... -  《-》

Isolation and identification of sporozoite membrane protein of Cryptosporidium parvum and evaluation of calmodulin-like protein immune protection.

Until now, no completely effective parasite-specific drugs or vaccines have been approved for the treatment of cryptosporidiosis. Through the separation and identification of the sporozoite membrane protein of Cryptosporidium parvum (C. parvum), 20 related proteins were obtained. Among them, a calmodulin-like protein (CML) has a similar functional domain-exchange factor hand (EF-hand) motif as calmodulin proteins (CaMs), so it may play a similarly important role in the invasion process. A 663 bp full gene encoding the C. parvum calmodulin-like protein (CpCML) was inserted in pET28a vector and expressed in Escherichia coli. An immunofluorescence assay showed that CpCML was mainly located on the surface of the sporozoites. Three-week-old female BALB/c mice were used for modelling the immunoreactions and immunoprotection of recombinant CpCML (rCpCML) against artificial Cryptosporidium tyzzeri infections. The results indicated a significantly increased in anti-CpCML antibody response, which was induced by the immunized recombinant protein. Compared to rP23 (recombinant P23), GST6P-1 (expressed by pGEX-6P-1 transfected E. coli), GST4T-1 (expressed by pGEX-4T-1 transfected E. coli), glutathione (GSH), adjuvant and blank control groups, rCpCML-immunized mice produced specific spleen cell proliferation in addition to different production levels of IL-2, IFN-γ, TNF-α, IL-4 and IL-5. Additionally, immunization with rCpCML led to 34.08% reduction of oocyst shedding in C. tyzzeri infected mice faeces which was similar to rP23. These results suggest that CpCML may be developed as a potential vaccine candidate antigen against cryptosporidiosis.

Huang Y ，Chen Y ，Liu Y ，Mi R ，Han X ，Gong H ，Cheng L ，Chen Z ... -  《-》

Development of an immunocompetent mouse model susceptible to Cryptosporidium tyzzeri infection.

Immunocompromised mice are extensively used in the screening of vaccines and drugs for Cryptosporidium, but this study model does not reflect the real status of infection in immunocompetent animals. This study aimed to provide an optimized animal model for future studies of Cryptosporidium vaccine. Three mouse strains (ICR, BALB/c and KM) with or without immunosuppression were compared after challenge with Cryptosporidium tyzzeri (C tyzzeri). The results indicated that ICR mice shed a greater number of faecal oocysts (20 346 ± 203 oocysts/g) compared with BALB/c (2077 ± 142 oocysts/g) and KM mice (3207 ± 431 oocysts/g) after experimental infection with C tyzzeri (P < .001). However, ICR mouse model is uniquely effective for C tyzzeri, not for other Cryptosporidium spp. such as C parvum. ICR mice were then used to determine the immunoreactions and immunoprotection of P23-DNA vaccine (pVAX1-P23) to C tyzzeri experimental infection. The results showed that a significant increase in anti-P23 antibody levels was induced by the pVAX1-P23 vaccine. Compared to pVAX1, TB and blank control mice, pVAX1-P23 immunized mice produced specific spleen cell proliferation as well as enhanced IL-5, IL-12p70 and IFN-γ production in sera. After challenge with 5 × 106 C tyzzeri oocysts, the oocyst shedding of the pVAX1-P23 immunized group was reduced by 69.94% comparing to the infection control. These results provide an optimized animal model for the study of prophylactic vaccines and this model might be applied to other candidates against Cryptosporidium, not only for pVAX1-P23.

Huang Y ，Song Y ，You Y ，Mi R ，Han X ，Gong H ，Chen Z ，Liu Y ... -  《-》

Evaluation of DNA encoding acidic ribosomal protein P2 of Cryptosporidium parvum as a potential vaccine candidate for cryptosporidiosis.

The Cryptosporidium parvum acidic ribosomal protein P2 (CpP2) is an important immunodominant marker in C. parvum infection. In this study, the CpP2 antigen was evaluated as a vaccine candidate using a DNA vaccine model in adult C57BL/6 IL-12 knockout (KO) mice, which are susceptible to C. parvum infection. Our data show that subcutaneous immunization in the ear with DNA encoding CpP2 (CpP2-DNA) cloned into the pUMVC4b vector induced a significant anti-CpP2 IgG antibody response that was predominantly of the IgG1 isotype. Compared to control KO mice immunized with plasmid alone, CpP2-immunized mice demonstrated specific in vitro spleen cell proliferation as well as enhanced IFN-γ production to recombinant CpP2. Further, parasite loads in CpP2 DNA-immunized mice were compared to control mice challenged with C. parvum oocysts. Although a trend in reduction of infection was observed in the CpP2 DNA-immunized mice, differences between groups were not statistically significant. These results suggest that a DNA vaccine encoding the C. parvum P2 antigen is able to provide an effective means of eliciting humoral and cellular responses and has the potential to generate protective immunity against C. parvum infection but may require using alternative vectors or adjuvant to generate a more potent and balanced response.

Benitez A ，Priest JW ，Ehigiator HN ，McNair N ，Mead JR ... -  《-》

Hyperimmune bovine colostrum specific for recombinant Cryptosporidium parvum antigen confers partial protection against cryptosporidiosis in immunosuppressed adult mice.

Jenkins MC ，O'Brien C ，Trout J ，Guidry A ，Fayer R ... -  《VACCINE》