Transformed Toxoplasma gondii tachyzoites expressing the circumsporozoite protein of Plasmodium knowlesi elicit a specific immune response in rhesus monkeys

Abstract

Toxoplasma gondii tachyzoites were transformed with the coding sequence of the circumsporozoite (CS) protein of the primate malaria parasite Plasmodium knowlesi. A single inoculation of live transformed tachyzoites elicited an antibody response directed against the immunodominant repeat epitope (EQPAAGAGG)2 of the P. knowlesi CS protein in rhesus monkeys. Notably, these animals failed to show a positive serum conversion against T. gondii. Antibodies against Toxoplasma antigens were detected only after a second inoculation with a higher number of transformed tachyzoites. This boost induced an increased antibody response against the P. knowlesi CS protein associated with immunoglobulin class switching, thus demonstrating the establishment of immunological memory. These results indicate that the Toxoplasma-derived CS protein is efficiently recognized by the monkey immune system and represents an immunodominant antigen in transformed parasites.

FIG. 1

(A) Schematic representation of the construct pSPc-myc/PkCS. (B) Immunoblot analysis of total protein lysates of RH (left lane) and transformed Tx-PkCS7 (right lane) tachyzoites developed by using MAb 9 E10, directed against the c-Myc epitope. The migrations (in kilodaltons) of low-molecular-mass standards from Sigma are indicated on the right.

FIG. 2

Immunolocalization of the P. knowlesi CS protein in Tx-PkCS7 tachyzoites. Confocal immunofluorescence (A and C) and transmission (B and D) photomicrographs of Tx-PkCS7 (A and B) and nontransformed (C and D) tachyzoites stained by MAb 9E10 are shown. Magnification, ×630; zoom factor, 2.6 for image acquisition.

FIG. 3

Development of humoral immunity against the P. knowlesi CS protein. Monkeys in groups A (solid lines) and B (dashed lines) were inoculated (day 0, arrowhead 1) either with Tx-PkCS7 tachyzoites or with the nontransformed parental RH line, respectively. Both groups of monkeys were inoculated 4 months later (day 130, arrowhead 2) with Tx-PkCS7 tachyzoites. Specific antibodies against the P. knowlesi CS protein were revealed by comparing the reactivities of each monkey serum against the recombinant constructs PkCS 1.0 and HRPIIr. The net OD at 405 nm was calculated by subtracting the OD values in the HRPIIr ELISA from those in the P. knowlesi CS ELISA. The assays were carried out by using labelled secondary antibodies recognizing IgG(H+L) (A), IgG (B), or IgM (C) human isotypes. Each serum was tested in four replicates at a 1/200 dilution. Letter-and-number codes refer to individual monkeys. The standard error for each determination was less than 10%.

FIG. 4

Specificity analysis of anti-human immunoglobulin antibodies against human, rhesus monkey, and rat serum proteins under nonreducing (A) and reducing (B) conditions by immunoblotting with anti-IgG(H+L), anti-IgG, or anti-IgM secondary antibodies. The migrations (in kilodaltons) of high-molecular-mass standards from Sigma are indicated on the left.

FIG. 5

Fine specificity of serum reactivity against the P. knowlesi CS protein. Monkey sera collected at 5 months were assessed by ELISA (1/50 dilution) against the recombinant construct PkCS 1.0 in the presence of increasing concentrations of the synthetic peptide (EQPAAGAGG)₂ (□). The unrelated peptide Der pI (■) was employed as a specifity control and used at a concentration of 20 μg/ml. The results are the averages of four determinations normalized to control binding in the absence of synthetic peptide.

FIG. 6

Development of humoral immunity against Toxoplasma antigens in immunized rhesus monkeys. Solid and dashed lines indicate group A and B monkeys, respectively. Serum reactivity was analyzed by using a Toxoplasma ELISA kit developed for the diagnosis of human toxoplasmosis. Toxoplasma antibody levels represent the means of duplicate determinations at a 1/200 dilution.