Antigenic characterization of an intrinsically unstructured protein, Plasmodium falciparum merozoite surface protein 2

Abstract

Merozoite surface protein 2 (MSP2) is an abundant glycosylphosphatidylinositol (GPI)-anchored protein of Plasmodium falciparum, which is a potential component of a malaria vaccine. As all forms of MSP2 can be categorized into two allelic families, a vaccine containing two representative forms of MSP2 may overcome the problem of diversity in this highly polymorphic protein. Monomeric recombinant MSP2 is an intrinsically unstructured protein, but its conformational properties on the merozoite surface are unknown. This question is addressed here by analyzing the 3D7 and FC27 forms of recombinant and parasite MSP2 using a panel of monoclonal antibodies raised against recombinant MSP2. The epitopes of all antibodies, mapped using both a peptide array and by nuclear magnetic resonance (NMR) spectroscopy on full-length recombinant MSP2, were shown to be linear. The antibodies revealed antigenic differences, which indicate that the conserved N- and C-terminal regions, but not the central variable region, are less accessible in the parasite antigen. This appears to be an intrinsic property of parasite MSP2 and is not dependent on interactions with other merozoite surface proteins as the loss of some conserved-region epitopes seen using the immunofluorescence assay (IFA) on parasite smears was also seen on Western blot analyses of parasite lysates. Further studies of the structural basis of these antigenic differences are required in order to optimize recombinant MSP2 constructs being evaluated as potential vaccine components.

Fig 1

Epitopes mapped on MSP2 by ELISA. (A) Epitope mapping with the anti-MSP2 MAbs on 13-mer peptides overlapping by eight residues covering the full-length 3D7 MSP2 sequence (peptides 1 to 45) and the FC27 MSP2 sequence (peptides 46 to 84) lacking the first 25 residues which are identical to those in 3D7 MSP2. OD, optical density. (B) Schematic diagram showing the location of the MAb epitopes identified by the peptide mapping in the two forms of MSP2.

Fig 2

Epitopes mapped on MSP2 by NMR. Backbone amide peak heights for ¹⁵N FC27 MSP2 (44 μM) in the presence of ∼15 μM MAb, normalized to the peak height in the absence of MAb. Data for wild-type MSP2 are shown in blue, and data for C211S MSP2 are shown in red. Vertical bars indicate uncertainties in peak heights as estimated from noise levels in ¹⁵N-¹H SOFAST-HMQC spectra. The locations of epitopes identified using the peptide array data in Fig. 1A are indicated as black bars (gray bars are for weaker, cross-reactive epitopes).

Fig 3

Western blotting on recombinant (lanes 1 and 2) and parasite (lanes 3 and 4) 3D7 (lanes 1 and 3) and FC27 (lanes 2 and 4) MSP2. (A) Analyses of the three 3D7 MSP2-specific MAbs (9D11, 11E1, and 2F2) and the FC27 MSP2-specific MAb 8G10. (B) Analyses of the six MAbs that recognize epitopes in the conserved N-terminal (6D8) or C-terminal (1F7, 6C9, 9H4, 4D11, and 9G8) regions of MSP2 as well as control blots probed with polyclonal sheep anti-3D7 and anti-FC27 MSP2 antisera. α, anti.

Fig 4

Western blots of parasite 3D7 MSP2 (lanes 1 and 3) and FC27 MSP2 (lanes 2 and 4) subjected to SDS-PAGE under reducing (lanes 1 and 2) and nonreducing (lanes 3 and 4) conditions. The 6D8 epitope was enhanced by reduction of FC27 MSP2, the 6C9 epitope was destroyed by reduction of FC27 and 3D7 MSP2, and the 9G8 epitope was unaffected by reduction of both forms of MSP2.