title: Evaluation on the immune response induced by DNA vaccine encoding MIC8 co-immunized with IL-12 genetic adjuvant against Toxoplasma gondii infection.
abstract: OBJECTIVE
  To examine the immunoprotection effect induced by MIC8 DNA vaccine co-immunized with a plasmid encoding murine IL-12 (pcIL-12) as an adjuvant in mice against the challenge of Toxoplasma gondii.
  
  
  METHODS
  The gene sequence encoding MIC8 of T. gondii RH strain was inserted into eukaryotic expression vector pcDNA3.1 to construct the pcMIC8 expression plasmid. The recombinant plasmid was transfected into HeLa cells to test its expression and the recombinant protein was then characterized by Western blotting. Eighty Kunming mice were randomly divided into 5 groups (16 per group): 3 control groups (PBS, pcDNA3.1, and pcIL-12), pcMIC8 group, and pcMIC8 plus pcIL-12 group. Mice in the pcMIC8 plus pcIL-12 group were co-injected intramuscularly at a dosage of 100 microl each of pcMIC8 and pcIL-12 suspended in 100 microg sterile PBS. Mice in other groups were inoculated with PBS, pcDNA3.1, pcIL-12, and pcMIC8 respectively following the same protocol. All the mice received three immunizations at 2-week intervals. Serum samples were collected on day 0, 13, 27, 41, and 55 before each inoculation for determining antibody IgG, IgG subclass IgG2a. Four weeks after the final immunization, IFN-gamma and IL-4 levels in splenocytes cultures from immunized mice were detected by ELISA. The mice were challenged with 10(3) tachyzoites of the virulent T. gondii RH strain three weeks after the last immunization to observe the survival time.
  
  
  RESULTS
  Western blotting showed that the protein extracts in HeLa cells upon transfection with pcMIC8 were effectively expressed in cells. The levels of IgG (0.51 +/- 0.028) and IgG2a (0.261 +/- 0.04)(on day 55) in mice immunized with pcMIC8 plus pcIL-12 were higher than pcMIC8 group (497.65 +/- 98.15) and control groups (PBS 47.18 +/- 2.73, pcDNA3.1 50.08 +/- 4.62, pcIL-12 118.15 +/- 12.73) (P < 0.05). There was no significant difference in the level of IgG 1 and IL-4 among the five groups (P > 0.05). After a lethal challenge of T. gondii RH strain, the survival time in mice immunized with pcMIC8 plus pcIL-12 (15d) was prolonged in comparison to that of pcMIC8 (10d) and control groups (PBS 5 d, pcDNA3.1 6d, pcIL-12 8 d) (P < 0.05).
  
  
  CONCLUSIONS
  The immune responses induced by the combined use of the recombinant plasmid encoding MIC8 of T. gondii with murine IL-2 gene adjuvant can be enhanced.
authors.forename: Huan-Ge
authors.initials: HG
authors.lastname: Zhao
authors.forename: Feng-Ying
authors.initials: FY
authors.lastname: Huang
authors.forename: Jun-Li
authors.initials: JL
authors.lastname: Guo
authors.forename: Guang-Hong
authors.initials: GH
authors.lastname: Tan
mesh_terms: D000276:Adjuvants, Immunologic / Q000235:genetics; D000818:Animals; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000276:immunology*; D006367:HeLa Cells; D006801:Humans; D007114:Immunization; D018664:Interleukin-12 / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008815:Mice, Inbred Strains; D010957:Plasmids; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D014122:Toxoplasma; D014123:Toxoplasmosis / Q000517:prevention & control*; D014162:Transfection; D019444:Vaccines, DNA / Q000235:genetics / Q000276:immunology* / Q000627:therapeutic use
chemical_list: D000276:Adjuvants, Immunologic; D015815:Cell Adhesion Molecules; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D019444:Vaccines, DNA; C530346:microneme protein 8, Toxoplasma gondii; D018664:Interleukin-12