Daniel Bout

Mic1-3 Knockout Toxoplasma gondii is a good candidate for a vaccine against T. gondii-induced abortion in sheep

This study assessed the effectiveness of a mutant strain of Toxoplasma gondii (RH strain) lacking the mic1 and mic3 genes (Mic1-3KO) against Toxoplasma abortion in sheep. Ewes were inoculated subcutaneously with 105 Mic1-3KO tachyzoïtes in three independent experiments. Following vaccination, Mic1-3KO induced a mild febrile response and serum IgG antibodies, which persisted throughout the experiments. Tissue cysts formed in the sheep, but were not, under our experimental conditions, infectious when given orally. Ewes were mated two months after vaccination and were orally challenged with the PRU strain of T. gondii at mid-gestation (400 oocysts in Experiments 1 and 2; 100 oocysts in Experiment 3). Challenge of vaccinated pregnant ewes resulted in a slight febrile response, whereas unvaccinated ewes developed a more severe, characteristic febrile response of longer duration. After challenge, all unvaccinated ewes aborted whereas 62%, 91% and 64% (Experiments 1, 2 and 3 respectively) of the lambs from vaccinated ewes were viable, with no clinical signs of infection. Mic1-3KO was as effective as S48, the strain used as a live vaccine for sheep (Toxovax®). A dose of 105 Mic1-3KO tachyzoites was sufficient to induce protection (versus a dose of 2 × 106). Both subcutaneous and intraperitoneal injections were effective. Moreover, preliminary results showed the potential of Mic1-3KO to reduce the development of tissue cysts in lambs born to vaccinated ewes. This study demonstrates that Mic1-3KO is a potent vaccine candidate.

A novel CD4–CD8α+CD205+CD11b– murine spleen dendritic cell line: establishment, characterization and functional analysis in a model of vaccination to toxoplasmosis

Dendritic cells (DCs) play an essential role in the induction of immune responses to pathogen infections. Native DCs are difficult to obtain in large numbers and consequently the vast majority of DCs employed in all experiments are derived from bone marrow progenitors. In an attempt to solve this problem, we have established a novel CD8α+ DC line (H‐2k) from spleen, which we have named SRDC line, and which is easy to culture in vitro. These cells display similar morphology, phenotype and activity to CD4–CD8α+CD205+CD11b– DCs purified ex vivo. Toxoplasma gondii antigen was shown to be taken up by these cells and to increase class I and class II major histocompatibility complex (MHC), CD40, CD80 and CD86 surface expression. We report that vaccination with T. gondii antigen‐pulsed SRDCs, which synthesize large amounts of interleukin‐12, induced protective immune responses against this intracellular pathogen in syngeneic CBA/J mice. This protection was associated with strong cellular and humoral immune responses at systemic and intestinal levels. Spleen and mesenteric lymph node cell proliferations were correlated with a Th1/Th2‐type response and a specific SRDC homing to spleen and intestine was observed. The SRDC or CD4–CD8α+CD205+CD11b– DC line can be expected to be a very useful tool for immunobiology studies of DC.

Immunogenicity of synthetic peptides from the Sm31 antigen (cathepsin B) of the Schistosoma mansoni adult worms.

The chemical synthesis of peptides may simplify the production of molecules for diagnosis of Schistosoma mansoni. Seventeen polymeric, 20‐amino acids long, peptides comprising the entire Sm31 molecule of the adult worm, were synthesized under the t‐boc strategy and their immunogenicity was evaluated. Of these, 10 peptides were immunogenic in rabbits. The peptides containing the sequence Gly74‐Ser93 (peptide IMT‐172) and the sequence Val154–Ala173 (peptide IMT‐180) were responsible for the recognition of the Sm31 molecule by Western blot. This was confirmed by the specific inhibition of recognition of each molecule with the homologous peptide. Additionally, antibodies against these peptides strongly fixed to the adult worm gut. The present results, together with the strong immunogenicity shown for the adult worm 31 kDa antigen, establish the basis for the development of an immunodiagnostic method using synthetic peptides.

Salbutamol as an adjuvant for nasal vaccination.

Salbutamol, a beta-adrenergic agonist, which transiently increases cAMP levels, was tested for its potential adjuvant activity in nasal vaccination. SAG1, the major surface protein of Toxoplasma gondii was used as vaccinating protein to protect CBA mice from a challenge with the parasite. Mice, vaccinated with SAG1 in the presence of salbutamol, showed a highly significant decrease in cerebral cysts, compared to non-vaccinated mice or mice receiving the vaccinating protein alone. Lymph node cells from BALB/c mice, receiving in the footpads a dendritic cell line pulsed with the antigen in the presence of salbutamol, showed a proliferative response in the presence of SAG1. The adjuvant properties of salbutamol are thus partially mediated by its effect on antigen presenting cells.