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 ... -  《-》

Induction of immune responses in mice by a DNA vaccine encoding Cryptosporidium parvum Cp12 and Cp21 and its effect against homologous oocyst challenge.

Cp12 and Cp21 surface proteins on the sporozoite of Cryptosporidium parvum have been identified as the immunodominant antigens involved in the immune response to C. parvum infection. In the present study, the efficacy of Cp12 and Cp21 antigens as vaccine candidates was investigated in BALB/c mice that were susceptible to C. parvum infection. DNA sequences of Cp12, Cp21, Cp12-Cp21, and C (CpG oligodeoxynucleotide (ODN))-Cp12-Cp21 were amplified and then cloned into pVAX1 vector to form the four recombinant plasmids pVAX1-Cp12, pVAX1-Cp21, pVAX1-Cp12-Cp21, and pVAX1-C-Cp12-Cp21. Recombinant protein expression from these four plasmids in HeLa cells were confirmed by indirect immunofluorescence staining and Western blot analysis. The in vivo efficacies of the four DNA vaccines were tested in BALB/c mice. The results indicated that the four DNA vaccines elicited significant antibody responses and specific cellular responses when compared to control mice that received vector only or PBS. Among those four plasmids, pVAX1-C-Cp12-Cp21 elicited significantly higher levels of IgG. Also, the percentages of CD4(+) and CD8(+) T cells were significantly higher in the group with pVAX1-C-Cp12-Cp21 nasal sprays. Their efficacy in immunoprotection against homologous challenge was also detected after administration of the four DNA vaccines. The results showed that mice in the pVAX1-C-Cp12-Cp21 nasal group had a 77.5% reduction in the level of oocyst shedding and a significant difference was detected when this group was compared with the pVAX1, PBS, pVAX1-Cp12, and pVAX1-Cp21 groups. The reduction in the level of oocysts shedding from the group of pVAX1-C-Cp12-Cp21 nasal spray was also higher than that of pVAX1-Cp12-Cp21 group. These results suggested that C-Cp12-Cp21-DNA may provide an effective means of eliciting humoral and cellular responses and generating protective immunity against C. parvum infections in BALB/c mice.

Yu Q ，Li J ，Zhang X ，Gong P ，Zhang G ，Li S ，Wang H ... -  《-》

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 ... -  《-》

Application of recombinant Cryptosporidium parvum P23 for isolation and prevention.

Omidian Z ，Ebrahimzadeh E ，Shahbazi P ，Asghari Z ，Shayan P ... -  《-》

Multivalent DNA vaccine induces protective immune responses and enhanced resistance against Cryptosporidium parvum infection.

The aim of this work was to evaluate efficiency as well as the type of immune response, Th1 or Th2, induced by multivalent DNA vaccinations in C57BL/6 interleukin-12p40 (IL-12p40) knockout (KO) mice. A recombinant pVAX-15-23 plasmid DNA was constructed by inserting surface glycoprotein (cp15- and p23)-encoding DNA into the pVAX1 expression vector. Various parameters including antibody and cytokine responses, proliferation assay and oocyst shedding were used to evaluate the type of immune response and the level of protection against challenge infection. Obtained results indicated that plasmid pVAX-15-23 induced strong protective immune response against C. parvum characterized by dominance of IgG2a, high level of INF-γ and lower level of the oocysts shedding after challenge infection. Moreover, co-immunization with the multivalent DNA and pMEM12R plasmid encoding IL-12 can further enhance these responses compared with the multivalent DNA alone. The obtained results suggest that multivalent pVAX-15-23 DNA vaccine may be a candidate as a generic approach to C. parvum immunization applicable to clinical practice.

Wang C ，Luo J ，Amer S ，Guo Y ，Hu Y ，Lu Y ，Wang H ，Duan M ，He H ... -  《-》