However, antibodies that inhibited adhesion of parasites to CSA were only elicited in rabbits immunized with full-length immunogen and inhibition was restricted to the homologous CSA-binding parasite. in rabbits immunized with full-length immunogen and inhibition was restricted to the homologous CSA-binding parasite. These findings demonstrate that partial and LY500307 full-length VAR2CSA immunogens induce cross-reactive antibodies, but inhibitory antibody responses to full-length immunogen were highly allele-specific and variable between animal species. == Introduction == Pregnancy associated malaria (PAM) is usually a major cause Mouse monoclonal antibody to Pyruvate Dehydrogenase. The pyruvate dehydrogenase (PDH) complex is a nuclear-encoded mitochondrial multienzymecomplex that catalyzes the overall conversion of pyruvate to acetyl-CoA and CO(2), andprovides the primary link between glycolysis and the tricarboxylic acid (TCA) cycle. The PDHcomplex is composed of multiple copies of three enzymatic components: pyruvatedehydrogenase (E1), dihydrolipoamide acetyltransferase (E2) and lipoamide dehydrogenase(E3). The E1 enzyme is a heterotetramer of two alpha and two beta subunits. This gene encodesthe E1 alpha 1 subunit containing the E1 active site, and plays a key role in the function of thePDH complex. Mutations in this gene are associated with pyruvate dehydrogenase E1-alphadeficiency and X-linked Leigh syndrome. Alternatively spliced transcript variants encodingdifferent isoforms have been found for this gene of poor mother/child health and increases the risk for maternal LY500307 anemia, prematurity, low birth weight and infant morbidity and mortality[1]. A key feature of PAM is the selective accumulation ofPlasmodium falciparuminfected erythrocytes (IEs) in the placenta mediated by adhesion to chondroitin sulfate A (CSA)[2]. Women become resistant to pregnancy malaria as they acquire antibodies to antigens expressed on the surface of IEs that inhibit placental adhesion and mediate opsonic phagocytosis[3][7]. VAR2CSA belongs to thePlasmodium falciparumerythrocyte membrane protein 1 (PfEMP1) family encoded byvargenes[8][10]and is the leading PAM vaccine candidate[11][17]. Thevargene family encodes cytoadhesion receptors that facilitate IE binding to microvascular endothelium through a variety of different host receptor interactions[18]. Adhesion of IEs to CSA or placental syncytiotrophoblast leads to specific transcriptional upregulation ofvar2csa[14][16]and native VAR2CSA binds to CSA[19]. Furthermore, VAR2CSA appears to be a major target of the antibody mediated immune response in pregnant women[15],[20], and parasites in whichvar2csais genetically disrupted largely drop the ability to bind to CSA[12],[17]and placental syncytiotrophoblast[21], suggesting it is the primaryvargene responsible for placental sequestration. Key obstacles to the development of a pregnancy malaria vaccine are the limited understanding of the targets of inhibitory antibodies and the best way to induce these responses by vaccination. VAR2CSA is usually a large (350 kDa) and polymorphic protein with six different Duffy binding-like (DBL) domains and two other large interdomain regions[14],[22]. A significant component of the acquired antibody response to VAR2CSA appears to target polymorphic epitopes[20], many of which are shared between different VAR2CSA alleles due to extensive gene mosaicism[22]. This patchwork of polymorphic epitopes appears to contribute to cross-reactive antibody responses LY500307 between different CSA-binding parasite lines[23][29], and may facilitate parasite escape from antibody-dependent protective mechanisms, as inhibitory epitopes appear to be at least partially strain-dependent[30][32]. Although CSA-binding properties have been mapped to several VAR2CSA domains usingin vitrobinding assays[13],[33],[34], recent published data[35],[36]casts doubt around the specificity of single DBL domains for CSA. Recently, the whole extracellular region of two different VAR2CSA variants were successfully produced as recombinant LY500307 proteins and demonstrated to have significantly higher affinity and specificity for CSA than individual domains[37],[38], indicating that multiple domains may be involved in binding or come together to form a high affinity binding site(s). Furthermore, a low resolution structure of full-length VAR2CSA protein revealed a much more compact structure than predicted from x-ray crystallographic analysis of individual domains[38], consistent with three dimensional modeling that VAR2CSA surface polymorphism is usually biased with surprising amounts of invariant surface when individual domains are considered alone[22],[39]. Taken together, interactions between VAR2CSA domains are likely to occur and single domain recombinant proteins may display off target epitopes that are buried in the native protein. While it has been possible to generate surface reactive antibodies with single-domain VAR2CSA recombinant proteins, it has been difficult to generate adhesion blocking antibody responses against individual VAR2CSA domains[23],[24],[29],[40][42]. To date, the most potent inhibitory antibodies response has been generated against the highly conserved DBL4 domain name[43], but induction has not been consistently achieved against different VAR2CSA DBL4 alleles[42],[43]. Potential explanations are that single-domain immunogens may not possess the correct quaternary interactions of the native protein or reproduce the high affinity binding site(s). Thus, there is significant interest in characterizing larger, multidomain VAR2CSA immunogens that may better mimic the native protein structure. Whereas nearly all VAR2CSA vaccination studies have employed single domains, a recent study suggested that a full-length VAR2CSA recombinant protein was superior to the best single domain name immunogen for inducing inhibitory antibodies[37]. However, the breadth of antibody inhibition against different CSA-binding parasite lines was not examined, and overall there has been limited characterization of multidomain VAR2CSA immunogens. In this study, mice and rabbits were immunized with a partial length (DBL4-6) or a full-length VAR2CSA (DBL1-6) recombinant protein produced in the human embryonic kidney 293 cell line. Antibody cross-reactivity was assessed against individual VAR2CSA domains and on a diverse panel of.