Rat and rabbit immune plasma were tested against their corresponding em P. geographically diverse panel of CSA-binding lab lines to assess antibody breadth and inhibitory activity. Results Of the five domains, DBL3, and to a lesser extent DBL5, induced antibodies that cross-reacted on five diverse CSA-binding parasite lines by circulation cytometry. By comparison, anti-DBL6 antibodies were highly strain-specific and anti-DBL1 and anti-DBL4 antibodies were poorly reactive by circulation cytometry. From this series of recombinant proteins, adhesion-blocking activity was restricted to a single rat immunized against a DBL4 recombinant protein. Conclusions Single domain name VAR2CSA recombinant proteins produced in em P. pastoris /em experienced limited efficacy in eliciting adhesion blocking antibody responses, but VAR2CSA DBL3 and DBL5 domains contain strain-transcendent epitopes that can be targeted by vaccination and may have application for vaccine development. Background Despite important advances, the burden of malaria remains very high, with more than 2.4 billion people at risk of malaria. Approximately 50 million women of child-bearing age are exposed to this risk of malaria every year [1,2]. Pregnancy associated malaria is usually a major cause of poor mother and child health and prospects to maternal anemia, prematurity, low birth excess weight and increased infant morbidity and mortality [3]. This syndrome is usually associated with em Plasmodium falciparum /em infected erythrocytes (IEs) that selectively sequester in the placenta via binding chondroitin sulfate A (CSA) [4,5]. Women become resistant to pregnancy malaria over the course of multiple pregnancies as they acquire antibodies that recognize placental isolates from geographically diverse regions [6-8], suggesting it may be feasible to develop a vaccine. Antibodies are thought to contribute to protection by blocking adhesion of IEs to CSA and by opsonizing IEs for phagocytosis [6,7,9-11]. Placental binding is usually associated with an unusually conserved em var /em gene, VAR2CSA, which is usually transcriptionally up-regulated in CSA binding parasites and expressed at Paricalcitol the surface of placental IEs [12,13]. Genetic disruption of em var2CSA /em largely abolishes CSA-binding [14-16] suggesting it is the major em var /em encoded product associated with placental sequestration. These findings support the development of a VAR2CSA-based vaccine against placental malaria. However, sequence analysis has revealed diversity among global isolates [17-19], which poses difficulties for developing a universal vaccine. The VAR2CSA extracellular region contains six Duffy binding-like (DBL) adhesion domains [13]. Several individual DBL domains (DBL2, DBL3, and DBL6) have Paricalcitol been reported to bind to CSA and a co-crystal has been solved for VAR2CSA DBL3-CSA [20,21]. However, it has been questioned whether binding interactions of single domains are physiologically relevant because many randomly expressed DBL domains from other members of the em var /em gene family also bind to CSA [22,23]. Furthermore, the full-length VAR2CSA protein binds with much greater specificity and affinity than individual domains [24,25]. Thus, it remains unclear whether VAR2CSA has one or multiple CSA-interaction sites, and binding site(s) in the full-length DBL1-6 recombinant protein remains uncharacterized. Immunization of animals with single domain name VAR2CSA recombinant proteins produced in em Baculovirus /em [26], em Escherichia coli /em [27,28] and em Pichia pastoris /em [29,30] demonstrate that it is possible to generate antibodies reactive with native VAR2CSA at the IE surface. However, there has been limited investigation into the breath of antibody reactivity and it remains hard to induce inhibitory antibodies. To date, few DBL recombinant antigens have induced anti-adhesive antibodies [28,31,32], except for an IT4-DBL4-VAR2CSA recombinant protein produced in em Baculovirus /em [31], and a refolded IT4-DBL5 recombinant protein produced in em Escherichia coli /em [33]. However, adhesion-blocking responses have been variable between different DBL4 and DBL5 antigen preparations and sensitive to construct boundaries [32,34]. The best DBL4 recombinant protein induced a broad adhesion blocking response to a range of different placental isolates [34], but not all parasites isolates were inhibited [34], and inhibitory antibodies were only observed against one of four different DBL4 alleles tested [32]. Thus, this approach has potential but more work is needed to optimize single domain name immunogens for pregnancy malaria vaccine development. In this study, the immunogenicity of single domain name VAR2CSA recombinant proteins was investigated by immunizing rats and rabbits with em P. pastoris /em DBL designed immunogens. Immune Trp53inp1 sera were examined against both the homologous parasite collection and a diverse panel of CSA binding parasite lines to identify DBL domains that elicited a broader antibody response and to define extracellular regions that contained adhesion-blocking epitopes. Methods Recombinant protein expression in em Pichia pastoris /em Cloning and production of 7G8-VAR2CSA Paricalcitol recombinant proteins was carried out in em Pichia pastoris /em as previously explained [29,35]. Construct boundaries are indicated in Physique ?Physique1.1. Recombinant proteins were analysed in 4-20% SDS-PAGE gels under non-reducing conditions. Gels were.

Rat and rabbit immune plasma were tested against their corresponding em P