• Vaccine Name: T. gondii DNA vaccine pIRESneo/MIC6/PLP1
• Target Pathogen: Toxoplasma gondii
• Target Disease: Toxoplasmosis
• Type: DNA vaccine
• Status: Research
• Host Species for Licensed Use: Human
• Antigen: MIC6 (Yan et al., 2012); TgPLP1 (Yan et al., 2012)
• MIC6 gene engineering:
  • Type: DNA vaccine construction
  • Description: Amplified by PCR using a forward primer introducing Sma I recognition sites and a reverse primer introducing Xba I recognition sites. (Yan et al., 2012) The amplified MIC6 was cloned into the pGEM-T Easy vector (Promega) and was sequenced. The ROP18 fragment was cleaved by SmaI/XbaI from pGEM-MIC6, purified, and then cloned into the SmaI/XbaI sites of pIRESneo, generating recombinant plasmid pIRESneo/MIC6. The purified TgPLP1 was cloned into pIRESneo/MIC6 and formed pIRESneo/MIC6/PLP1. (Yan et al., 2012)
  • Detailed Gene Information: Click Here.
• PLP1 gene engineering:
  • Type: DNA vaccine construction
  • Description: Amplified by PCR using a forward primer introducing Cla I recognition sites and a reverse primer introducing BamH I recognition sites. (Yan et al., 2012) The amplified TgPLP1 was digested, purified, and cloned into pIRESneo/MIC6 and formed pIRESneo/MIC6/PLP1. (Yan et al., 2012)
  • Detailed Gene Information: Click Here.
• Immunization Route: Intramuscular injection (i.m.)
• Description: T. gondii DNA vaccine encoding MIC6 and TgPLP1 as antigin using pIRESneo vector. pVAX I plasmids encoding IL-18 are codelivered as adjuvant. (Yan et al., 2012)

Mouse Response

• Host Strain: Specific-pathogen-free (SPF)-grade inbred Kunming mice (Yan et al., 2012)
• Host age: 6 to 8 weeks old (Yan et al., 2012)
• Host gender: female (Yan et al., 2012)
• Vaccination Protocol: Seven groups of mice (25 mice per group) were injected with 100 μg of plasmid DNA suspended in 100 μl sterile PBS. Group I mice were injected with PBS as a blank control; group II to group VI mice were injected with the empty vector pVAX I or pIRESneo, pVAX/IL-18, pVAX/TgPLP1, or pVAX/MIC6, respectively, also as controls; and groups VII and VIII were injected with pIRESneo/MIC6/TgPLP1 or pIRESneo/MIC6/TgPLP1+pVAX/IL-18 (100 μl each) respectively. Two booster injections were administered at 2 week interval. (Yan et al., 2012)
• Immune Response: Humoral: Mice injected with pIRESneo/MIC6/TgPLP1 or pIRESneo/MIC6/TgPLP1+pVAX/IL-18 had significantly high levels of anti-TgPLP1/MIC6 IgG antibodies, especially after the third immunization. Mice injected with pVAX/PLP1 or pVAX/MIC6 alone also generated anti-TgPLP1/MIC6 antibodies but at significantly lower levels (P < 0.05). Mice injected with PBS, pVAX I, pIRESneo, or pVAX/IL-18 alone did not generate anti-TgPLP1 antibodies, significantly different from the groups mentioned above (P < 0.05). (Yan et al., 2012)
  Cellular: Splenocytes from all groups proliferated to comparable levels. The level of splenocyte proliferation increased over those of the other groups when mice were coinjected with pIRESneo/MIC6/TgPLP1 + pVAX/IL-18 (P < 0.05). Mice immunized with pIRESneo/MIC6/TgPLP1 had higher lymphocyte responses than the controls (P < 0.05). Mice immunized with pVAX/TgPLP1, pVAX/MIC6, or pVAX/IL-18 alone had significantly higher lymphocyte responses than the rest of the controls (P < 0.05). Very large amounts of specific IFN-γ, IL-2, and IL-12 were produced in pIRESneo/MIC6/TgPLP1+pVAX/IL-18, pIRESneo/MIC6/TgPLP1, pVAX/IL-18, pVAX/PLP1, or pVAX/MIC6 groups. Specific amounts of IL-4 and IL-10 were synthesized in pIRESneo/MIC6/TgPLP1 or pIRESneo/MIC6/TgPLP1+pVAX/IL-18 groups. (Yan et al., 2012)
• Challenge Protocol: Five mice were randomly chosen from every group and challenged intragastrically with 80 cysts of strain PRU, and observed daily for mortality. Eight mice of each group were orally infected with 20 cysts of strain PRU at the 14th day after the last immunization. Cysts in the brain were counted 6 weeks after challenge. (Yan et al., 2012)
• Efficacy: Acute infection: All mice immunized with PBS, pIRESneo, or pVAX I died at day 25. Immunization with pIRESneo/MIC6/TgPLP1 dramatically increased the survival time (42.8 ± 2.9 days) (P < 0.05). Coimmunization with pIRESneo/MIC6/TgPLP1 and pVAX/IL-18 enhanced the survival time even further (45.0 ± 2.9 days), although the difference was not statistically significant (P > 0.05). (Yan et al., 2012)
  Chronic infection: The lowest brain cyst burdens were observed in mice immunized with pIRESneo/MIC6/PLP1+pVAX/IL-18 (1,085.67 ± 49.32 cysts per brain) or pIRESneo/MIC6/PLP1 (1,206.00 ± 10.02 cysts per brain) and were significantly lower than those in the PBS group (3,140.33 ± 96.72 cysts per brain) and the other five groups (P < 0.05). Brain cyst burdens were significantly lower (P < 0.05) in mice immunized with pVAX/PLP1 (1,759.00 ± 60.47) or pVAX/MIC6 (1,890.00 ± 46.36) than in the control animals.(Yan et al., 2012)