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

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

Determination of the Molecular Basis for a Limited Dimorphism, N417K, in the Plasmodium vivax Duffy-Binding Protein

Invasion of human red blood cells by Plasmodium merozoites is vital for replication and survival of the parasite and, as such, is an attractive target for therapeutic intervention. Merozoite invasion is mediated by specific interactions between parasite ligands and host erythrocyte receptors. The P. vivax Duffy-binding protein (PvDBP) is heavily dependent on the interaction with the human Duffy blood group antigen/receptor for chemokines (DARC) for invasion. Region II of PvDBP contains many allelic polymorphisms likely to have arisen by host immune selection. Successful vaccine development necessitates a deeper understanding of the role of these polymorphisms in both parasite function and evasion of host immunity. A 3D structure of the homologous P. knowlesi DBP predicts that most variant residues are surface-exposed, including N417K, which is a dimorphic residue change that has previously been shown to be part of a linked haplotype that alters DBP sensitivity to inhibitory antibody. In natural isolates only two residues are found at this site, asparagine (N) and lysine (K). Site-directed mutagenesis of residue 417 was used to create a panel of 20 amino acid variants that were then examined for their binding phenotype and response to immune sera. Our results suggest that the observed dimorphism likely arose due to both structural requirements and immune selection pressure. To our knowledge, this is the first exhaustive examination of this kind of the role of a single amino acid residue in antigenic character and binding ability. Our results demonstrate that a single amino acid substitution can dramatically alter both the ability of the PvDBP to bind to human erythrocytes and its antigenic character

Genetic diversity and natural selection of Plasmodium vivax reticulocyte invasion genes in Ecuador

Abstract Background Knowledge of the diversity of invasion ligands in malaria parasites in endemic regions is essential to understand how natural selection influences genetic diversity of these ligands and their feasibility as possible targets for future vaccine development. In this study the diversity of four genes for merozoite invasion ligands was studied in Ecuadorian isolates of Plasmodium vivax. Methods Eighty-eight samples from P. vivax infected individuals from the Coast and Amazon region of Ecuador were obtained between 2012 and 2015. The merozoite invasion genes pvmsp-1-19, pvdbpII, pvrbp1a-2 and pvama1 were amplified, sequenced, and compared to the Sal-1 strain. Polymorphisms were mapped and genetic relationships between haplotypes were determined. Results Only one nonsynonymous polymorphism was detected in pvmsp-1-19, while 44 nonsynonymous polymorphisms were detected in pvdbpII, 56 in pvrbp1a-2 and 33 in pvama1. While haplotypes appeared to be more related within each area of study and there was less relationship between parasites of the coastal and Amazon regions of the country, diversification processes were observed in the two Amazon regions. The highest haplotypic diversity for most genes occurred in the East Amazon of the country. The high diversity observed in Ecuadorian samples is closer to Brazilian and Venezuelan isolates, but lower than reported in other endemic regions. In addition, departure from neutrality was observed in Ecuadorian pvama1. Polymorphisms for pvdbpII and pvama1 were associated to B-cell epitopes. Conclusions pvdbpII and pvama1 genetic diversity found in Ecuadorian P. vivax was very similar to that encountered in other malaria endemic countries with varying transmission levels and segregated by geographic region. The highest diversity of P. vivax invasion genes in Ecuador was found in the Amazonian region. Although selection appeared to have small effect on pvdbpII and pvrbp1a-2, pvama1 was influenced by significant balancing selection

A novel erythrocyte binding protein of Plasmodium vivax suggests an alternate invasion pathway into Duffy-positive reticulocytes

Submitted by Nuzia Santos ([email protected]) on 2017-04-17T17:15:47Z No. of bitstreams: 1 ve_Ntumngia_Francis_A Novel Erythrocyte_CPqRR_2016 .pdf: 389451 bytes, checksum: b5a5db76dc128a7a88fc1c6bb916f89b (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2017-04-17T17:19:45Z (GMT) No. of bitstreams: 1 ve_Ntumngia_Francis_A Novel Erythrocyte_CPqRR_2016 .pdf: 389451 bytes, checksum: b5a5db76dc128a7a88fc1c6bb916f89b (MD5)Made available in DSpace on 2017-04-17T17:19:45Z (GMT). No. of bitstreams: 1 ve_Ntumngia_Francis_A Novel Erythrocyte_CPqRR_2016 .pdf: 389451 bytes, checksum: b5a5db76dc128a7a88fc1c6bb916f89b (MD5) Previous issue date: 2016University of South Florida. College of Public Health. Center for Global Health and Infectious Diseases Research. Tampa, Florida, USAUniversity of South Florida. College of Public Health. Center for Global Health and Infectious Diseases Research. Tampa, Florida, USAUniversity of South Florida. College of Public Health. Center for Global Health and Infectious Diseases Research. Tampa, Florida, USA/Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrazilNational Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Malaria and Vector Research. Rockville, Maryland, USAFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrazilUniversity of South Florida. College of Public Health. Center for Global Health and Infectious Diseases Research. Tampa, Florida, USAErythrocyte invasion by malaria parasites is essential for blood-stage development and an important determinant of host range. In Plasmodium vivax, the interaction between the Duffy binding protein (DBP) and its cognate receptor, the Duffy antigen receptor for chemokines (DARC), on human erythrocytes is central to blood-stage infection. Contrary to this established pathway of invasion, there is growing evidence of P. vivax infections occurring in Duffy blood group-negative individuals, suggesting that the parasite might have gained an alternative pathway to infect this group of individuals. Supporting this concept, a second distinct erythrocyte binding protein (EBP2), representing a new member of the DBP family, was discovered in P. vivax and may be the ligand in an alternate invasion pathway. Our study characterizes this novel ligand and determines its potential role in reticulocyte invasion by P. vivax merozoites. EBP2 binds preferentially to young (CD71high) Duffy-positive (Fy+) reticulocytes and has minimal binding capacity for Duffy-negative reticulocytes. Importantly, EBP2 is antigenically distinct from DBP and cannot be functionally inhibited by anti-DBP antibodies. Consequently, our results do not support EBP2 as a ligand for invasion of Duffy-negative blood cells, but instead, EBP2 may represent a novel ligand for an alternate invasion pathway of Duffy-positive reticulocytes. IMPORTANCE For decades, P. vivax infections in humans have been defined by a unique requirement for the interaction between the Duffy binding protein ligand of the parasite and the Duffy blood group antigen receptor (DARC). Recent reports of P. vivax infections in Duffy-negative individuals challenge this paradigm and suggest an alternate pathway of infection, potentially using the recently discovered EBP2. However, we demonstrate that EBP2 host cell specificity is more restricted than DBP binding and that EBP2 binds preferentially to Duffy-positive, young reticulocytes. This finding indicates that this DBP paralog does mediate a Duffy-independent pathway of infection

DEKnull is structurally similar to Sal1 DBP-II.

<p>(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.</p

ELISA with anti-DBP conformational specific antibodies.

<p>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.</p

DiscoTope B-cell epitope prediction of Sal1 DBP-II and DEKnull.

<p>(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.</p

DEKnull mutations do not affect protein secondary structure.

<p>(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.</p

Data collection and refinement statistics for DEKnull.

<p>Data were collected from a single crystal.</p><p>*Highest resolution shell is shown in parenthesis</p><p>^†Does not include hydrogens</p><p>Data collection and refinement statistics for DEKnull.</p