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. 2015 Mar 20;9(3):e0003644.
doi: 10.1371/journal.pntd.0003644. eCollection 2015 Mar.

Structural analysis of the synthetic Duffy Binding Protein (DBP) antigen DEKnull relevant for Plasmodium vivax malaria vaccine design

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Structural analysis of the synthetic Duffy Binding Protein (DBP) antigen DEKnull relevant for Plasmodium vivax malaria vaccine design

Edwin Chen et al. PLoS Negl Trop Dis. .

Abstract

The Plasmodium vivax vaccine candidate Duffy Binding Protein (DBP) is a protein necessary for P. vivax invasion of reticulocytes. The polymorphic nature of DBP induces strain-specific immune responses that pose unique challenges for vaccine development. DEKnull is a synthetic DBP based antigen that has been engineered through mutation to enhance induction of blocking inhibitory antibodies. We determined the x-ray crystal structure of DEKnull to identify if any conformational changes had occurred upon mutation. Computational and experimental analyses assessed immunogenicity differences between DBP and DEKnull epitopes. Functional binding assays with monoclonal antibodies were used to interrogate the available epitopes in DEKnull. We demonstrate that DEKnull is structurally similar to the parental Sal1 DBP. The DEKnull mutations do not cause peptide backbone shifts within the polymorphic loop, or at either the DBP dimerization interface or DARC receptor binding pockets, two important structurally conserved protective epitope motifs. All B-cell epitopes, except for the mutated DEK motif, are conserved between DEKnull and DBP. The DEKnull protein retains binding to conformationally dependent inhibitory antibodies. DEKnull is an iterative improvement of DBP as a vaccine candidate. DEKnull has reduced immunogenicity to polymorphic regions responsible for strain-specific immunity while retaining conserved protein folds necessary for induction of strain-transcending blocking inhibitory antibodies.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Location of DEK polymorphisms on Sal1 DBP-II.
DEK polymorphisms (DEKAQQRRKQ) mapped onto the Sal1 DBP-II and DARC heterotetramer. DEK residues are shown in brown and side chains are displayed as sticks. The two DBP-II molecules are in green and yellow. The two DARC peptides are in purple and blue.
Fig 2
Fig 2. DEKnull is structurally similar to Sal1 DBP-II.
(A) DEKnull separated into three sub-domains, sub-domain 1 (S1—red), sub-domain 2 (S2—blue), and sub-domain 3 (S3—green). (B) S1 (red) contains a β-hairpin, S2 (blue) is a helix bundle, and S3 (green) is a helix bundle. Domain boundaries and disulfide bonding cysteines are labeled. (C) Structural alignment of DEKnull (solid colors) with Sal1 DBP-II (light tinted colors) with r.m.s. deviation of 0.435Å. (D) Structural alignment of individual DEKnull sub-domains (solid colors) with Sal1 DBP-II sub-domains (light tinted colors). S1 alignment has a r.m.s. deviation of 0.308 Å. S2 alignment has a r.m.s. deviation of 0.288 Å. S3 alignment has a r.m.s. deviation of 0.310 Å.
Fig 3
Fig 3. DEKnull mutations do not affect protein secondary structure.
(A) and (B) Two views of a structural alignment of Sal1 DBP-II DEKAQQRRKQ polymorphic region (white) and DEKnull ASTAATSRTS mutant region (blue). Mutated residues are labeled and shown as sticks. (C) Structural alignment of DARC binding sites on Sal1 DBP-II (white) and DEKnull (blue). DARC binding residues are labeled and shown as sticks. (D) Structural alignment of DBL dimerization interfaces on Sal1 DBP-II (white) and DEKnull (blue). Dimerization residues are labeled and shown as sticks.
Fig 4
Fig 4. DiscoTope B-cell epitope prediction of Sal1 DBP-II and DEKnull.
(A) Graphical representation of DiscoTope B-cell epitope scores for Sal1 DBP-II (blue line) and DEKnull (red line). Prediction threshold is shown in green. DEK residues are located at amino acids 339–348 and shown above the corresponding location in the graph. (B) Two views of Sal1 DBP-II predicted epitopes mapped onto crystal structure. (C) Two views of DEKnull predicted epitopes mapped onto crystal structure.
Fig 5
Fig 5. ELISA with anti-DBP conformational specific antibodies.
ELISA assays for conformational anti-DBP antibodies with BSA as a negative control, Sal1 DBP-II as a positive control, and DEKnull protein. Four inhibitory (3C9, 2H2, 2C6, 2D10) and two non-inhibitory (3D10, 2F12) antibodies were tested.

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