A Cryptosporidium parvum vaccine candidate effect on immunohistochemical profiling of CD4, CD8, Caspase-3 and NF-κB in mice

Background: Cryptosporidium parvum is a protozoan parasite of medical and veterinary importance that causes neonatal diarrhea in many vertebrate hosts. In this study, we evaluated the efficacy of an affinity-purified antigen as a C. parvum vaccine candidate using ileal and liver tissues of experimentally infected neonatal mice by immunohistochemical profiling and immune scoring of CD4, CD8, Caspase-3, and nuclear factor kappa B (NF-κB). This vaccine was prepared from the C. parvum oocyst antigen using immunoaffinity chromatography with cyanogen bromide-activated Sepharose-4B beads. Thirty neonatal mice were divided into three groups (10 mice/group): (1) nonimmunized noninfected, (2) nonimmunized infected (using gastric tubes with a single dose of 1×105 of C. parvum oocysts in 250 µl PBS solution 1 h before a meal) and (3) immunized (twice with 40 µg/kg of purified C. parvum antigen at 2-week intervals and then infected with 1×105 C. parvum oocysts simultaneously with the second group). After euthanizing the animals on the 10th day, post-infection, their ileal and liver tissues were collected and prepared for immunohistochemistry (IHC) staining to detect CD4, CD8, Caspase-3, and NF-κB levels, which are indicators for T helper cells, cytotoxic T cells, apoptosis, and inflammation, respectively. Results: The IHC results showed that CD4, CD8, Caspase-3, and NF-κB antibodies varied significantly (P <0.001) in both organs in all the groups. We also recorded high CD4 levels and low CD8 antibodies in the nonimmunized noninfected mice tissues, while the opposite was observed in the nonimmunized infected mice tissues. In the immunized infected mice, the CD4 level was higher than CD8 in both organs. While the Caspase-3 levels were higher in the ileal tissue of nonimmunized infected than immunized infected mice ileal tissues, the reverse was seen in the liver tissues of both groups. Furthermore, NF-κB expression was higher in the liver tissues of nonimmunized infected mice than in immunized infected mice tissues. Therefore, the IHC results and immune-scoring program revealed a significant difference (P <0.001) in the CD4, CD8, Caspase-3, and NF-κB antibody levels in both ileal and liver tissues of all mice groups, which might be necessary for immunomodulation in these tissues. Conclusions: The improvement observed in the immunized infected mice suggests that this vaccine candidate might protect against cryptosporidiosis.