Solution structure of a Plasmodium falciparum AMA-1/MSP 1 chimeric protein vaccine candidate (PfCP-2.9) for malaria

Abstract

Background: The Plasmodium falciparum chimeric protein PfCP-2.9 is a promising asexual-stage malaria vaccine evaluated in clinical trials. This chimeric protein consists of two cysteine-rich domains: domain III of the apical membrane antigen 1 (AMA-1 [III]) and the C-terminal region of the merozoite surface protein 1 (MSP1-19). It has been reported that the fusion of these two antigens enhanced their immunogenicity and antibody-mediated inhibition of parasite growth in vitro.

Methods: The 15N-labeled and 13C/15N-labeled PfCP-2.9 was produced in Pichia pastoris for nuclear magnetic resonance (NMR) structure analysis. The chemical shift assignments of PfCP-2.9 were compared with those previously reported for the individual domains (i.e., PfAMA-1(III) or PfMSP 1-19). The two-dimensional spectra and transverse relaxation rates (R2) of the PfMSP1-19 alone were compared with that of the PfCP-2.9.

Results: Confident backbone assignments were obtained for 122 out of 241 residues of PfCP-2.9. The assigned residues in PfCP-2.9 were very similar to those previously reported for the individual domains. The conformation of the PfMSP1-19 in different constructs is essentially the same. Comparison of transverse relaxation rates (R2) strongly suggests no weak interaction between the domains.

Conclusions: These data indicate that the fusion of AMA-1(III) and MSP1-19 as chimeric protein did not change their structures, supporting the use of the chimeric protein as a potential malaria vaccine.

Figure 1

The 2D ¹⁵N-edited HSQC spectrum showing the backbone assignments of PfCP-2.9. The assigned backbone amides of PfCP-2.9 are labeled with the one-letter amino acid codes and are numbered according to the sequence of PfCP-2.9. The residue G234 (labeled in red) is folded in the spectrum.

Figure 2

Mapping of the assigned residues onto the AMA1 domain III structure. The residues with assigned backbone amides are blue. The proline residues (which have no amides) and residues without backbone assignments but with C^α and C^β atoms that could be assigned are green. The disulfide bridges are shown, and the cysteine residues are labeled. The residues are numbered according to the sequence of PfCP-2.9. The structure corresponds to the first conformer of the NMR structures of AMA1 domain III (PDB code 1HN6). The figure was generated by MOLMOL.

Figure 3

Mapping of the assigned residues onto the MSP1-19 domain structure. The residues with assigned backbone amides are blue. The proline residues (which have no amides) or residues without backbone assignments but with C^α and C^β atoms that could be assigned are green. The disulfide bridges are shown, and the cysteine residues are labeled. The residues are numbered according to the sequence of PfCP-2.9. The structure corresponds to the first conformer of the NMR structures of MSP 1-19 domain (PDB code 1CEJ). The figure was generated by MOLMOL.

Figure 4

Chemical shift comparison between PfCP-2.9 and the individual AMA1 III and MSP 1-19 domains. (A) Chemical shift differences for the H^N, N, C^α, C^β, and CO atoms between the AMA1 III domain in PfCP-2.9 and the AMA1 III domain alone (BMRB entry 4787). (B) Chemical shift differences for the H^N, N, C^α, C^β, and CO atoms between the MSP 1-19 domain in PfCP-2.9 and the MSP 1-19 domain alone (BMRB entry 4437). The residues are numbered according to the sequence of PfCP-2.9. The figure was generated by Origin 8.0.

Figure 5

Overlay of 2D ¹⁵N-edited HSQC spectra of PfCP-2.9 and MSP 1-19. The spectrum of PfCP-2.9 is shown in black and spectrum of MSP 1-19 alone is shown in red.

Figure 6

Chemical shift comparison between PfCP-2.9 and MSP 1-19. Chemical shift differences of backbone H^N atoms (upper panel) and N atoms (lower panel) between MSP 1-19 and PfCP-2.9 are shown. The chemical shift differences were calculated by subtracting the chemical shift values for each residue in the MSP 1-19 domain in PfCP-2.9 from those in MSP 1-19 alone. The residues are numbered according to the sequence of PfCP-2.9. The figure was generated by Xmgrace.

Figure 7

Comparison of transverse relaxation rates (R₂) between PfCP-2.9 and MSP 1-19. Overlay of the R₂ values of MSP 1-19 (red stars) alone and the MSP 1-19 domain in PfCP-2.9 (black circles). The scale of the R₂ values for PfCP-2.9 is on the left, and the scale for MSP 1-19 is on the right. The residues are numbered according to the sequence of PfCP-2.9. The figure was generated by Xmgrace.