Summary
1. Background
The merozoite surface protein 2 (MSP2) of the malaria parasite Plasmodium falciparum has been prominent in two respects. Firstly, being a highly immunogenic surface antigen, it is considered as a potential vaccine candidate. The 3D7 variant of MSP2 constitutes one of three subunits of the malaria vaccine Combination B, which has been tested recently in a successful pilot efficacy trial in Papua New Guinea (PNG). Secondly, due to its extensive polymorphism, msp2 is now widely used as a marker gene for genotyping P. falciparum infections. However, the function and importance of the molecule is yet completely unknown.
2. Working Hypotheses
a) Humoral immunity to MSP2 is to a limited and variable degree cross reactive but short lasting.
b) Vaccination elicits an antibody response specifically directed against certain domains and Combination B elicited a 3D7-allele-specific response with little effect on the constant region of the MSP2 molecule.
c) The constant but not the variable part of MSP2 is essential for the parasite's survival.
3. Specific aims
a) To measure the immunological cross reactivity between different MSP2 alleles and the persistence of the humoral response in first time infected individuals.
b) To examine sera of vaccinated children for presence of antibodies directed against the family-specific and constant domains of MSP2.
c) To examine consequences of msp2 gene replacement in a P. falciparum in vitro culture with transfection constructs containing different and partially deleted forms of msp2.
d) To improve transfection technology for P. falciparum.
4. Experimental Design and Methods
a) A study including 80 patients attending the STI clinical services with a first malaria episode. All samples will be genotyped, sequenced, and selected msp2 alleles will be cloned and expressed. Sera will be tested against these various recombinant proteins in a checker board design. Responses will be correlated to allelic family, number of tandem repeats or similarities between different repeats.
b) Sera from the PNG vaccine trial (baseline and weeks 8,12, and 18) will be tested against recombinant proteins corresponding to family specific domains (3D7, FC27) and to the constant domain (N- and C-termini).
c) Stable transfection of P. falciparum strain 3D7 via homologous recombination (insertion/duplication) to generate expressed deleted forms of MSP2. Allelic replacement of msp2 against alleles of the alternative family. Measurements of growth rates, invasion success, and immunological recognition.
5. Expected Value of the Proposed Project
a) Improved understanding of cross-reactivity and concomitant immunity.
b) Elucidation of the functional importance of the variable or repetitive parts of MSP2.
c) Evaluation and improvement of the current formulation of MSP2 in the vaccine Combination B.
d) Improvement of transfection technology.
