• Vaccine Name: CAV-2-ROP18
• Target Pathogen: Toxoplasma gondii
• Target Disease: Toxoplasmosis
• Type: Recombinant vector vaccine
• Status: Research
• Host Species for Licensed Use: Human
• Antigen: ROP18: Rhoptry protein 18: a polymorphic serine-threonine kinase which is secreted in the host cell during the invasion process, and its catalytic activity is required for the acute virulence phenotype. It is considered one of the key virulence factors in the pathogenesis of the T. gondii infection. (Li et al., 2015)
• ROP18 gene engineering:
  • Type: Recombinant vector construction
  • Description: Recombinant plasmid pPolyII-CAV-△E3-ROP18 was constructed by in vitro ligation. The plasmid includes E3 (the E3 region of CAV-2); CMV (human cytomegalovirus (hCMV) immediate-early gene promoter), ROP18, and polyA (the SV40 early mRNA polyadenylation signal). The plasmid were digested with Asc I and Pme I to release the linear recombinant genome. The recombinant genome was used to transfect MDCK cells. (Li et al., 2015)
  • Detailed Gene Information: Click Here.
• Immunization Route: Intramuscular injection (i.m.)
• Description: Recombinant canine adenovirus expressing the ROP18 gene of the RH strain of T. gondii (Li et al., 2015)

Mouse Response

• Host Strain: specific-pathogen-free (SPF) grade inbred Kunming mice (Li et al., 2015)
• Host age: 6-8 weeks old (Li et al., 2015)
• Host gender: female (Li et al., 2015)
• Vaccination Protocol: The mice were randomly assigned into four experimental groups (33 mice per group). Group I was intramuscularly inoculated once with 0.1 ml CAV-2-ROP18 (10 8.125 p.f.u. ml−1); group II received 0.1 ml CAV-2 (108.25 p.f.u. ml−1) once as a negative control; group III was inoculated intramuscularly with 0.1 ml PBS as control at weeks 0, 2 and 4; and group IV was not injected with anything as a negative control. (Li et al., 2015)
• Immune Response: Humoral: Antibody titers significantly increased in the recombinant virus CVA-2-ROP18 group at week 2, 4 and 6 after immunization, compared to CAV-2, PBS and blank control immunized group (P < 0.05). Both IgG1 and IgG2a levels were detected. There was no significant difference in IgG1 and IgG2a levels between the groups immunized CAV-2, PBS, and nothing (P > 0.05). HI antibodies against CAV-2 were detected in all mice vaccinated with CAV-2 and the recombinant CAV-2-ROP18 at 2 weeks post-primary immunization, reaching comparable titers throughout the test period. (Li et al., 2015)
  Cellular: Splenocytes from mice immunized with CAV-2-ROP18 showed a significant proliferative response to ROP18 (P < 0.05), which was significantly higher than proliferation by splenocytes from all other controls (P < 0.05). Splenocytes from all groups proliferated to comparable levels in response to the mitogen ConA. Significant CTL activity was tested in mice immunized with CAV-2-ROP18. The percentage of CD3+/CD4+ T cells and CD3+/CD8+ T cells were significantly increased in mice immunized with CAV-2-ROP18 compared to all controls. Similarly, CAV-2-ROP18 significantly altered CD4+ or CD8+ T cell profiles in terms of IFN-γ and TNF-α expression in comparison with all controls. IL-2 and IFN-γ values in CAV-2-ROP18 group are significantly higher than in the controls (P < 0.05). Low levels of IL-4 showed a slight but significantly production from CAV-2-ROP18 groups compared to the controls (P < 0.05). (Li et al., 2015)
• Challenge Protocol: Eight weeks after the immunization, 20 mice in each group were challenged intraperitoneally (i.p.) with 1 × 103 tachyzoites of the virulent T. gondii RH strain, and 10 other mice were inoculated intragastrically with 5 cysts of the PRU strain. All mice were observed daily for mortality. Two months after the challenge, the surviving mice were euthanized and their brains were removed. Each brain was homogenized in 2 ml of PBS. The mean number of cysts per brain was determined by counting in three samples of 25 μl aliquots of each homogenized brain under an optical microscope. (Li et al., 2015)
• Efficacy: RH: Mice immunized with only a single dose of CAV-2-ROP18 showed 40% protection until 60 days after challenge, while the administration of either CAV-2 or PBS did not prevent mortality (mice died within 7 days). (Li et al., 2015)
  PRU: Mice from the CAV-2-ROP18 group developed a significantly lower (P < 0.05) number of brain cysts (8000 ± 1414 cysts per brain) compared to mice from the other three control control groups (approximately 18000 cysts). (Li et al., 2015)