Comparison of immune response in sheep immunized with DNA vaccine encoding Toxoplasma gondii GRA7 antigen in different adjuvant formulations.

Immunization with plasmid DNA, a relatively novel technique, is a promising vaccination technique. To improve the immune response by DNA vaccination various methods have been used, such as chemical adjuvants or immunomodulatory molecules formulated into microparticles or liposomes. The aim of this research is to evaluate the immune responses of sheep immunized with DNA plasmids encoding Toxoplasma gondii dense granule antigen GRA7 formulated into three different adjuvant formulations. Sixty sheep were injected intramuscularly with the DNA plasmids. Twelve received the liposome-formulated plasmid pVAXIgGRA7, 12 Emulsigen P formulated plasmid pVAXIgGRA7 and 12 Emulsigen D formulated plasmid pVAXIgGRA7. Twelve animals were used as a control and received the vector alone. All the animals were inoculated at week 0, and week 4. Immunization of the sheep with plasmids encoding GRA7, with the different adjuvant formulations, effectively primed the immune response. After the first inoculation, moderate to high antibody responses were observed with the three different adjuvant formulations. A significantly elevated specific IgG2 response was observed in the sheep immunized with liposomes and Emulsigen D as adjuvants. In the group immunized with Emulsigen P as an adjuvant, lower IgG1 and IgG2 antibody levels were developed compared to the other treatment groups. In all the immunized groups, DNA immunization stimulated a IFN-gamma response. No antibody or IFN-gamma responses were detected in the control group immunized with an empty plasmid or not immunized. These results indicate that intramuscular immunization of sheep with a DNA vaccine with the adjuvants liposomes and Emulsigen D induce a significant immune response against T. gondii.

Leptin concentrations and the immune-mediated reduction of feed intake in sheep infected with the nematode Trichostrongylus colubriformis.

The hypothesis that increases in the concentration of the anorectic peptide leptin may be responsible for the immune-mediated reduction in feed intake (FI) during gastrointestinal parasitism in sheep was investigated. In a 2 x 2 x 2 factorial design, the first factor was age at the start of infection (5 months old v. 17 months old). The second factor was parasite infection (no infection v. administration of eighty L3 infective Trichostrongylus colubriformis larvae/kg live weight (LW) per d three times per week for 77 d). The third factor was immunosuppressive therapy with a corticosteroid (no therapy or weekly intramuscular injection of 40 mg methylprednisolone acetate/30 kg LW). Relative to their uninfected counterparts, a 20 % reduction in FI per unit LW (FI/LW; g DM/kg LW) was observed in infected non-suppressed 5-month-old lambs from 21 to 63 d post-infection (P < 0.001) but not in comparable17-month-old ewes or in corticosteroid-treated lambs or ewes (P>0.05 for all), allowing the suggestion that the anorexia was a consequence of the developing immune response. The reduction in FI/LW in 5-month-old lambs was not associated with an increase in plasma leptin concentration. Furthermore, plasma leptin concentrations were greater in corticosteroid-treated animals (P < 0.001) and in 17-month-old animals (P < 0.001), none of which displayed an infection-induced reduction in FI/LW. Plasma leptin was positively correlated with carcass fat percentage in both 5-month-old (P = 0.016) and 17-month-old (P < 0.001) animals and did not appear to provide a direct feedback mechanism that restricted energy intake. The results do not support the hypothesis that an increase in circulating leptin is directly responsible for the immune-mediated anorexia in lambs during T. colubriformis infection.

Cloning, sequence analysis and expression of ovine CD154 (CD40 ligand).

The CD154 (CD40 ligand) molecule is a member of the tumor necrosis factor (TNF) family and plays an important role in the interaction between antigen-specific lymphocytes and antigen-presenting cells. In this study, reverse transcription-PCR cloning was used to derive the sequence encoding ovine CD154. Sequence analysis of the cloned CD154 gene showed a similarity of 97%, 89%, and 88% with the bovine, porcine and human sequences, respectively, at the nucleic acid level. The deduced amino acid sequence for the ovine CD154 shared 97%, 91%, and 87% similarity with the CD154 protein of bovine, porcine and human. The cysteine residues characteristic of the TNF family and N-linked glycosylation sites are conserved although one of the cysteine residues (Cys9) appeared only in ovine CD154. The isolated CD154 sequence was expressed as a mature protein in Chinese hamster ovary (CHO) cells. The analysis of expression of ovine CD154 in mammalian cells by Western blot confirmed the cross reactivity with anti-CD154 antibody.

Evaluation of immune responses in sheep induced by DNA immunization with genes encoding GRA1, GRA4, GRA6 and GRA7 antigens of Toxoplasma gondii.

The dense granule proteins of Toxoplasma gondii are investigated as possible vaccine candidates against the parasite. The aim of this research was to evaluate the immune responses of sheep injected twice, intramuscularly, with DNA plasmids encoding T. gondii dense granule antigens GRA1, GRA4, GRA6 and GRA7 formulated into liposomes. Control sheep were injected with an empty vector or received no injections. The injection of sheep with DNA plasmids encoding for GRA1, GRA4, GRA6 or GRA7 elicited an immune response after the first and the second injections as indicated by the moderate to high antibody responses. The injection of pGRA7 induced a significant level of anti-GRA7 IgG2 antibody and IFN-γ responses indicating a Th1-like immune response whereas injection with pGRA1, pGRA4 and pGRA6 stimulated a IgG1 type antibody response with a limited, if any, IFN-γ response. The results demonstrate that the intramuscular injection of sheep with a DNA liposome formulated plasmid coding for GRA proteins is an effective system that induces a significant immune response against T. gondii.

Modulation of immune response to Toxoplasma gondii in sheep by immunization with a DNA vaccine encoding ROP1 antigen as a fusion protein with ovine CD154.

CD154 is a cell surface molecule expressed by activated T cells. CD40 and CD154 interaction is critically important in regulating humoral and cell-mediated immune responses. In this study we have investigated whether a DNA vaccine encoding rhoptry protein 1 (ROP1) of Toxoplasma gondii, and encoding ovine CD154 induces an enhanced ROP1-specific immune response in sheep. Two groups of twelve animals received two intramuscular injections, of a DNA plasmid encoding T. gondii ROP1 antigen (group 1) or an ROP1 antigen fused to ovine CD154 (group 2). There were two control groups of sheep. One was injected with an empty vector (group 3) and the other received no injections at all (group 4). The injection of the plasmid containing ROP1 (group 1) at weeks 0 and 4 induced a significant IgG2 response at week 2 which was amplified at week 4 after the booster injection and persisted to week 8 compared to the control animals in groups 3 and 4. For IgG1, significant differences from the control animals were only observed from week 5 onwards. The fusion of CD154 and ROP1 elicited significant IgG1 and IgG2 responses from week 1 which were amplified from weeks 5 to 8 compared to the control animals in groups 3 and 4. The IgG1 response was significantly higher in group 2 animals receiving pROP1-CD154 compared to group 1 receiving pROP1 only. There was no significant difference in IgG2 responses between groups 1 and 2. Significant differences in IFN-γ levels were only observed in treatment group 1 at week 2 and treatment group 2 at weeks 1 and 2 compared to the control animals. The results demonstrated that an intramuscular injection of pROP1-CD154 gene to sheep significantly enhanced their immune response and induced a mixed Th1/Th2 response while the intramuscular injection of pROP1 only induced a Th1-specific immune response.

The immune responses of sheep after DNA immunization with, Toxoplasma gondii MAG1 antigen-with and without co-expression of ovine interleukin 6.

The aim of this study is to compare the immune responses of sheep stimulated by the intramuscular injection of a liposome formulated-DNA plasmid encoding the Toxoplasma gondii MAG1 antigen only or co-expressed with ovine IL-6. Forty-five, 2-year-old sheep were divided into four groups. Group 1 received an empty pVAXIg plasmid, group 2 no treatment, group 3 liposome formulated plasmid pVAXIgMAG1 and group 4, pVAXIgMAG1 plus pVAXovIL-6 plasmids. All the animals were inoculated at weeks 0 and 4. The injection of sheep with a plasmid encoding for MAG1 only or a MAG1 plasmid co-expressed with a plasmid encoding for ovine IL-6 produced humoral immune responses. The plasmids containing MAG1 elevated significantly serum IgG1 and IgG2 levels 2 weeks and onwards after the first injection of the plasmids. Co-expression of IL-6 with MAG1 had no effect on IG1 or IG2 levels illustrating that IL-6 in the formulation used had no modulating effect on any measured immune response.