Glycan Masking of Plasmodium vivax Duffy Binding Protein for Probing Protein Binding Function and Vaccine Development

Sowmya Sampath,Chetan E Chitnis,Chris Carrico,William R Schief,Ruobing Wang,Joseph D Smith,Claire Gibson,Martin Melcher,Sairam Gurumoorthy,Joel Janes

doi:10.1371/journal.ppat.1003420

Abstract

Glycan masking is an emerging vaccine design strategy to focus antibody responses to specific epitopes, but it has mostly been evaluated on the already heavily glycosylated HIV gp120 envelope glycoprotein. Here this approach was used to investigate the binding interaction of Plasmodium vivax Duffy Binding Protein (PvDBP) and the Duffy Antigen Receptor for Chemokines (DARC) and to evaluate if glycan-masked PvDBPII immunogens would focus the antibody response on key interaction surfaces. Four variants of PVDBPII were generated and probed for function and immunogenicity. Whereas two PvDBPII glycosylation variants with increased glycan surface coverage distant from predicted interaction sites had equivalent binding activity to wild-type protein, one of them elicited slightly better DARC-binding-inhibitory activity than wild-type immunogen. Conversely, the addition of an N-glycosylation site adjacent to a predicted PvDBP interaction site both abolished its interaction with DARC and resulted in weaker inhibitory antibody responses. PvDBP is composed of three subdomains and is thought to function as a dimer; a meta-analysis of published PvDBP mutants and the new DBPII glycosylation variants indicates that critical DARC binding residues are concentrated at the dimer interface and along a relatively flat surface spanning portions of two subdomains. Our findings suggest that DARC-binding-inhibitory antibody epitope(s) lie close to the predicted DARC interaction site, and that addition of N-glycan sites distant from this site may augment inhibitory antibodies. Thus, glycan resurfacing is an attractive and feasible tool to investigate protein structure-function, and glycan-masked PvDBPII immunogens might contribute to P. vivax vaccine development.

Highlights

Plasmodium vivax invasion of human reticulocytes is strongly dependent on an interaction between the P. vivax Duffy Binding Protein (PvDBP) and the Duffy Antigen Receptor for Chemokines (DARC) on the reticulocyte surface [1]
The PvDBPII dimer interface is formed between contacts across helical domains in the two monomers [13], and N-glycan acceptor sites were not added to this location
As the three native positions were located at the surface of subdomain 2 or subdomain 3 of PvDBPII distinct from the putative DARC interaction surface or dimerization surfaces (Figure 1), all three were retained in all of the hyperglycosylated variant sequences (Table 1)

Summary

Introduction

Plasmodium vivax invasion of human reticulocytes is strongly dependent on an interaction between the P. vivax Duffy Binding Protein (PvDBP) and the Duffy Antigen Receptor for Chemokines (DARC) on the reticulocyte surface [1]. An alternative pathway of P. vivax invasion has recently been described [5,6], DARC-null carriers have reduced susceptibility to P. vivax infection [4,7] and the FyA DARC allele shows reduced binding to PvDBP and is more susceptible to antibody blocking [8]. The PvDBP-DARC interaction has a critical role in P. vivax infection making it an attractive vaccine target. The extracellular region of PvDBP has been divided into six regions [10], of which DARC binding has been localized to region II (PvDBPII) [12]. PvDBPII binds to the Nterminal 65 residues of DARC with a sulfated tyrosine on DARC at position 41 having a critical role in binding [15,16]

Methods

Results

Conclusion