• Vaccine Name: rCDPK6 + rROP18 + PLG
• Target Pathogen: Toxoplasma gondii
• Target Disease: Toxoplasmosis
• Type: Subunit vaccine
• Status: Research
• Host Species for Licensed Use: None
• Host Species as Laboratory Animal Model: mouse
• Antigen: CDPK6 (Zhang et al., 2016); ROP18 (Zhang et al., 2016)
• CDPK6 gene engineering:
  • Type: Recombinant protein preparation
  • Description: The ORF of TgCDPK6 were amplified with RT–PCR using primers containing Kpn I and Not I restriction sites. The RT–PCR products were inserted into the pET-30a(+) via restriction sites, forming pET-CDPK6. E. coli BL21(DE3) was transformed with pET-CDPK6. rTgCDPK6 was expressed, purified, and encapsulated in 50:50 PLG using the water-in-oil-in-water double emulsion solvent evaporation technique. (Zhang et al., 2016)
  • Detailed Gene Information: Click Here.
• ROP18 gene engineering:
  • Type: Recombinant protein preparation
  • Description: The ORF of TgROP18 were amplified with RT–PCR using primers containing BamH I and Sal I restriction sites. The RT–PCR products were inserted into the pET-30a(+) via restriction sites, forming pET-ROP18. E. coli BL21(DE3) was transformed with pET-ROP18. rTgROP18 was expressed, purified, and encapsulated in 50:50 PLG using the water-in-oil-in-water double emulsion solvent evaporation technique. (Zhang et al., 2016)
  • Detailed Gene Information: Click Here.
• Immunization Route: subcutaneous injection
• Description: T. gondii subunit vaccine of CDPK6 and ROP18 encapsulated in PLG (Zhang et al., 2016)

Mouse Response

• Host Strain: Specific-pathogen-free Kunming mice (Zhang et al., 2016)
• Vaccination Protocol: A total of 13 groups of mice were injected subcutaneously with 10 μg of each protein. The vaccinated groups were immunized with rCDPK6, rROP18, rCDPK6 + rROP18, rCDPK6 + Montanide™ ISA 206 VG (206), rROP18 + 206, rCDPK6 + rROP18 + 206, rCDPK6 + PLG, rROP18 + PLG, or rCDPK6 + rROP18 + PLG. The control groups were immunized with PBS alone, PBS + 206, or PBS + PLG, or was not treated. (Zhang et al., 2016)
• Immune Response: Humoral: Higher levels of IgG in all protein vaccines. The levels of IgG increased sequentially with successive immunizations with the tested antigens, and reached their highest levels in week 8. Six weeks after the last vaccination, the levels of IgG antibodies in the mice immunized with rROP18 + PLG or rCDPK6 + rROP18 + PLG were significantly higher than those in the mice immunized with rROP18 or rCDPK6 + rROP18 (P < 0.01), but were not statistically different from those in the mice immunized with various proteins + 206 adjuvant (P > 0.05). (Zhang et al., 2016)
  Cellular: The splenocytes proliferated more strongly in the vaccinated mice than in the controls, and the highest proliferative response was detected in rCDPK6 + rROP18 + PLG mice (P < 0.001). Higher levels of T-cell subtypes were detected in mice immunized with protein vaccines. protein–PLG induced significantly higher levels of CD4+ (P < 0.001) and CD8+ T lymphocytes (P < 0.01) than the controls. rROP18 + 206 and rROP18 + PLG induced significantly higher levels of CD4+ than the controls (P < 0.05). Only mice from rROP18 + 206 group were showed significantly higher level of CD8+ compared to that in controls (P < 0.05). (Zhang et al., 2016)
• Challenge Protocol: Six weeks after the immunization, 10 mice in each group were challenged intraperitoneally with 10^3 tachyzoites of the T. gondii RH strain, and their survival was recorded daily until all the mice had died. Six mice in each group were inoculated orally with 10 PRU tissue cysts, and their brain cysts were determined 4 weeks after chronic infection was established. (Zhang et al., 2016)
• Efficacy: Acute: The average survival time of the mice immunized with the protein vaccines (8.56 days) was slightly longer than that in the controls (8 days). Immunization with rROP18 + PLG (10.9 days ± 2.58), rROP18 (10.1 days ± 1.52), and rCDPK6 + PLG (9.1 days ± 0.24) significantly prolong the average survival time in mice compared with that of the controls (P < 0.05). (Zhang et al., 2016)
  Chronic: The tissue cyst loadings in the brains of mice vaccinated with the proteins varied from 47.7 % to 73.6 %, and were significantly lower than those in the control groups (P < 0.001). The protein antigens plus PLG microparticles provided more effective protection to the mice than immunizations only with proteins, but the differences were not statistically significant (P > 0.05). (Zhang et al., 2016)