Functional and immunological analysis of the different domains of "Plasmodium falciparum" merozoite surface protein 2 (MSP2)

Malaria caused by Plasmodium falciparum is still a major health problem in many tropical
countries infecting 500 million people leading to 1 to 2 millions of deaths annually. An
effective vaccine is not available but is a major goal to reach as a measure for disease control.
The blood stages responsible for the pathology exhibit great antigenic variation and diversity
in surface antigens, an immune evasion strategy of the parasite, which hampers the rapid
acquisition of protective immunity. Antigenic variation is used for parasite proteins expressed
at the surface of infected erythrocytes while antigenic diversity manifests at the surface of
merozoites, the invasive form of blood stages. Despite their extensive polymorphism,
merozoite surface antigens are among the most promising vaccine candidates since high
antibody titres against these proteins are associated with protection from clinical disease and
specific antibodies to different merozoite antigens can inhibit parasite growth in vitro.
One antigen displayed on merozoites is the merozoite surface protein 2 (MSP2). MSP2 is
GPI-anchored and an abundant component of the merozoite surface coat. Its structure shows a
central polymorphic part containing extensive tandem amino acid repeats. These repeat
regions are flanked by semi-conserved non-repetitive domains defining two allelic families
(3D7-like and FC27-like). The repeat sequences and their organisation differ considerably
between the two families. The N- and C-terminal parts of MSP2 are conserved among all
alleles.
The 3D7 allele of MSP2 was one of three components in the blood stage subunit vaccine
Combination B recently tested in an area of Papua New Guinea endemic for Plasmodium
falciparum malaria. The Combination B trial showed promising results in reducing parasite
densities and genotyping of blood samples revealed that MSP2 was a major active
component. The vaccine exerted a selective effect on infecting parasite strains favouring those
carrying an MSP2 of the FC27-type, not represented by the vaccine.
In this thesis we showed that the antibody response against the MSP2 component of
Combination B was exclusively directed against the repetitive and semi-conserved central
domains. Together with the observed selective effect on infecting parasite genotypes in the
vaccinees, this has implications for future MSP2-based vaccines. It strongly suggests the
inclusion of representatives of both allelic families.
We also analyzed the sequences of 3D7-like MSP2 alleles found during the Combinaton B
trial and compared the sequences of alleles found in vaccinees with those found in placebo
recipients. Phylogenetic analysis showed no clustering of alleles found in breakthrough
infections in vaccinees. This suggests that vaccine-induced antibodies against the familyspecific
non-repetitive part were protective.
In the search for an improved MSP2-based vaccine we designed and evaluated two long
synthetic peptides representing the two allelic families. These peptides contain the familyspecific
part and the C-terminal conserved part. We show that both peptides are well
recognized by immune sera and that recognition matches the one of recombinant proteins
corresponding to the family-specific parts. The peptides elicited high antibody titres in mice
and monoclonal antibodies raised were shown to react with parasite-derived MSP2 in
immunofluorescence assays. We also show that antibodies purified on our peptides react with
the merozoite surface and that they have the potential to inhibit parasite growth in cooperation
with human monocytes. The results obtained from our evaluation studies encourage the
further development of long synthetic peptides as vaccine candidates.
We also asked the question of the functional role of the different domains of MSP2. We
hypothezised that the immunodominant repeat region has merely an immunological role in
distracting the antibody response to non-protective epitopes and that it is dispensable for in
vitro growth. Therefore we made transgenic parasites attempting to replace the endogenous
msp2 gene with a gene showing an internal deletion of the repeat region. Our plasmid
constructs targeted the msp2 locus, however, a gene replacement was not achieved. This
indicates that the repeat region has an essential function in addition to its proposed importance
in immune evasion.
We also addressed the question of the functional role of the conserved domains of MSP2.
MSP2 is a GPI-anchored protein and therefore the mechanisms used for its transport to the
cell surface are not known. We hypothesised that putative transport signals are likely to lie
within the conserved domains. We generated a series of transgenic parasites expressing
fluorescent reporter proteins flanked with different portions of terminal MSP2 sequences. We
found that a fluorescent reporter flanked by the conserved MSP2 domains was transported to
the surface of merozoites while the same reporter only flanked by the MSP2 signal peptide
and the GPI anchor signal remained in the ER. This suggests that trafficking signals necessary
for targeting of MSP2 to the plasma membrane are located in either of the conserved domains.