Differential effects of C3d on the immunogenicity of gene gun vaccines encoding Plasmodium falciparum and Plasmodium berghei MSP142

The complement fragment C3d mediates B-cell activation via simultaneous engagement of the B-cell receptor and CD21 by antigen/C3d conjugates. Several studies demonstrated the potential of C3d as a molecular adjuvant for vaccination. In this work, C3d exerted differential effects on humoral immune responses after gene gun immunization of mice with plasmids encoding the malaria blood stage antigen MSP1₄₂ depending on the nature of the protein (Plasmodium falciparum vs. Plasmodium berghei MSP), the localization of the C3d moiety (C-terminal vs. N-terminal), and the presence of putative N-glycosylation sites. No improvement of protective efficacy by C3d attachment or mutation of glycosylation sites could be demonstrated by in vitro parasite growth inhibition assays or in vivo blood stage parasite challenges. Our data underscore the controversial role of C3d as molecular adjuvant.     

Deletion of the parasite-specific insertions and mutation of the catalytic triad in glutathione reductase from chloroquine-sensitive Plasmodium falciparum 3D7

The flavoenzyme glutathione reductase (GR; NADPH+glutathione disulphide+H(+)-->NADP(+)+2 glutathione-SH) of Plasmodium falciparum is a promising drug target against tropical malaria. As P. falciparum genes are assumed to be highly polymorphic we have cloned and expressed the GR cDNA of the chloroquine-sensitive strain 3D7. In comparison to the known GR of the chloroquine-resistant K1 strain there are three base exchanges all of them leading to amino acid substitutions (residues 281, 285 and 335). The catalytic efficiency k(cat)/K(m) of the 3D7 enzyme is 5-fold lower than for the K1 enzyme. In contrast, vis-à-vis the drugs carmustine, methylene blue and fluorophenyliso-alloxazine the two enzyme species exhibited identical inhibition kinetics. Two structural motifs which are specific for P. falciparum GR were studied by mutational deletion analysis of 3D7 GR. Loop 126-138 appears to be important for folding and stability of the enzyme, whereas the subdomain 318-350 was found to be involved in FAD-binding. The subdomain has no major influence on the known functions of the catalytic triad Cys-40, Cys-45 and His-485'. Flavin absorption spectroscopy of inactive point mutants showed that Cys-45 forms a thiolate charge transfer complex and Cys-40 is the interchange thiol, which reduces glutathione disulphide. The mutant His-485-->Gln had a normal K(m) for glutathione disulphide reduction but only 0.8% residual catalytic activity when compared with wild-type GR, which confirms its function as an acid/base catalyst. The parasite-specific domains in combination with the reactive catalytic residues appear to be a suitable target matrix for inhibiting GR in vivo.