Epsilon Archive for Student Projects

Abstract

The intracellular protozoan parasite Toxoplasma gondii can infect a wide range of animal species, and is one of the main causes of infectious abortion and perinatal mortality in sheep. In humans, the parasite can cause abortion and congenital infection, and fatal disease in immunosuppressed patients. In sheep, toxoplasmosis can be controlled by vaccination with a live, attenuated vaccine. However, since such a vaccine has practical disadvantages and is not acceptable for use in humans, various strategies to develop an effective subunit vaccine have been explored. The major surface antigen of T. gondii, named SAG1, is considered as a promising vaccine candidate. Equally as important as identification of protective antigens is the choice of adjuvant. The immunostimulating complex (iscom) is an
adjuvant formulation that induces both humoral and cellular immune responses that are predominantly of type 1, and therefore is likely to be effective against intracellular
parasites.

The aim of the present study was to produce iscoms containing recombinant SAG1 (rSAG1) and to investigate their immunogenicity and protective capacity against T. gondii using a mouse model.

SAG1 expressed in E. coli as a recombinant protein with a hexahistidyl (His6) tag was coupled to preformed iscom matrix (i.e. iscom particles without any antigen) using the
affinity of the His6 tag to divalent anions. The matrix contained a chelating lipid and had been loaded with Ni2+ ions. Analytical density gradient centrifugation revealed that a substantial proportion of the SAG1 had bound to the matrix. To investigate the immunogenicity of the rSAG1 iscoms, mice were immunised twice and the cellular immune
response examined by in vitro stimulation of spleen cells. Cells from three of four immunised mice proliferated significantly when exposed to rSAG1, whereas cells from
only one of five mice were stimulated with T. gondii lysate. ELISA analysis revealed high antibody titres against rSAG1 but only low levels against T. gondii antigens. In two subsequent challenge experiments, three groups of mice were inoculated three times with
either rSAG1 iscoms, iscom matrix, or PBS. The third immunisation resulted in substantially higher antibody titres against T. gondii antigen. After inoculation with the
virulent RH strain all mice died without any significant differences in survival time between groups (p = 0.179). However, when the mice were inoculated orally with tissue
cysts of the Tg-SweF1 strain, significantly lower numbers of brain cysts were found in mice immunised with rSAG1 iscoms than in mice injected with PBS (p < 0.05).

In conclusion, although immunisation with rSAG1 iscoms did not protect mice from the lethal challenge infection, partial protection was induced as demonstrated by the reduction of brain cyst load after inoculation with an avirulent strain.