Hermann Bujard´s Laboratory
Immunogenicity of MSP-1 and development of a malaria vaccine based on full size MSP-1
R. Lutz, C. Kauth, U. Wöhlbier, S. Hein, L. Guilbride, S. Druffel-Augustin
MSP-1 from P.falciparum is a 190 kDa protein complex fixed at the merozoite surface via a GPI anchor. It consists of highly conserved and dimorphic regions and, thus, can be classified into two prototypes; in addition, it contains two small oligomorphic areas near the N-terminus (Fig. 1). The rationale for developing a subunit vaccine on the basis of MSP-1 from P.falciparum strain 3D7 can be summarized as follows:
Vaccination with MSP-1 elicits a protective humoral immune response in animal models;
Various seroepidemiological studies show associations between MSP-1 specific antibody titers and reduced risk of reinfection;
Epitopes recognized by antibodies which inhibit parasite growth in vitro are distributed throughout the molecule;
Epitopes presented via MHCI and recognized by CD8+ T cells of semi-immune individuals from malaria endemic regions are located throughout the protein as well;
The induction of MSP-1 specific CD8+ T cells suggests a cellular immune response directed against the liver stage of infection.
As MSP-1 appears to contain multiple protective T and B cell epitopes, a vaccine based on the full size molecule is expected to more likely overcome genetic restrictions of vaccinees and to be less susceptible to potentially emerging resistances.
We have developed an efficient production process for MSP-1D based on a fully synthetic gene, and are in the process of producing clinical grade material which we intend to examine in a clinical phase I study shortly. We are also studying in some detail the human humoral and cellular response towards MSP-1 and enjoy in these projects the collaborations with Drs Bocar Kouyate and Boubacar Coulibaly, CRSN, Burkina Faso, as well as with Drs Hans-Georg Rammensee and Stefan Stevanovic and coworkers, Universität Tuebingen.
Structure and function of MSP-1
C. Kauth, R. Lutz, U. Woehlbier, S. Hein, M. Kern, Z. Mekkonen
MSP-1 is presented at the surface of erythrocytes as a GPI anchored protein complex generated from a single precursor molecule by a two-step proteolytic process, which initially yields four major cleavage products (or subunits), p83,p30, p38 and p42 (Fig. 1). Before erythrocyte invasion, p42 is further cleaved into p33 and p19. The various processing products remain, however, covalently associated, but during erythrocyte invasion only p19 is carried into the newly infected cell while 90% of the protein complex is shed from the parasite surface. At least, the second processing step is essential since its prevention (e.g. by antibodies directed towards specific epitopes within p19) inhibits erythrocyte invasion. MSP-1 interacts with other merozoite surface proteins as well as with the surface of the erythrocyte. Attempts to knock out the msp-1 gene failed, giving further support to the view that MSP-1 is an essential protein as one may also conclude from its high overall conservation. Therefore, MSP-1 could be a target for interfering with the parasite life cycle not only via a respective vaccine.
After devising processes that allow us to produce in E.coli, all subunits of MSP-1D and MSP-1F (representing the two MSP-1 prototypes) and permuted fusions thereof as well as the intact precursor proteins, we are in a position to address several issues in detail. Study of the interaction of the various subunits of MSP-1D allowed us to derive the first overall model showing how the different processing products of MSP-1D are arranged (Fig. 2). The complex sketched in Fig. 2 can be reliably assembled from its four or five (p33+p19 instead of p42) subunits and used for studying MSP-1 interactions with further parasitic proteins as well as with erythrocytes.
II. Controlling gene activities in vivo via the Tet system
K. Schoenig, S. Berger
We have continued to further define a locus within the mouse genome (LC-1 locus) which upon integration of a gene controlled by Ptet (tTA/rtTA responsive promoter) can be regulated without interference from the genomic surroundings. The LC-1 locus can now be targeted via homologous recombination or utilized via a BAC (bacterial artificial chromosome) transfer system.
Further methodological developments aim at conditional mouse and rat models where the gene activities placed under Tet control can be monitored non-invasively. These projects are carried out in close collaborations with Dr. Dusan Bartsch, ZI, Mannheim, and Drs Winfried Denk and Mazahir Hasan, MPI für medizinische Forschung, Heidelberg.
Several further collaborations for which we provide our colleagues with novel (unpublished) elements of the Tet system are continuing.