Toward the Development of the Next Generation of a Rapid Diagnostic Test: Synthesis of Glycophosphatidylinositol (GPI) Analogues of Plasmodium falciparum and Immunological Characterization

Abstract

A large number of proteins in malaria parasites are anchored using glycophosphatidylinositols (GPIs) with lipid tails. These GPIs are structurally distinct from human GPIs. Plasmodium falciparum GPIs have been considered as potential vaccine candidates because these molecules are involved in inducing inflammatory responses in human hosts, and natural anti-GPI antibody responses have been shown to be associated with protection against severe disease. GPIs can also be considered as targets for rapid diagnostic tests. Because isolation of native GPIs in large quantities is challenging, development of synthetic GPI molecules can facilitate further exploration of GPI molecules for diagnostics. Here, we report synthesis and immunological characterization of a panel of malaria-specific GPI analogues. A total of three GPI analogues were chemically synthesized and conjugated to a carrier protein to immunize and generate antibodies in rabbits. The rabbit immune sera showed reactivity with synthetic GPIs and native GPIs extracted from P. falciparum parasite, as determined by Luminex and ELISA methods.

Detection of Anti-Toxoplasma gondii Antibodies in Human Sera Using Synthetic Glycosylphosphatidylinositol Glycans on a Bead-Based Multiplex Assay

“…Various strategies for coupling glycan structures to carboxyl-functionalized beads have been described, e.g., via end-biotinylated ABO blood group glycans to streptavidin-coated beads, 35 by functionalization of pneumococcal polysaccharides with 4-(4,6-dimethoxy[1,3,5]triazin-2-yl)-4-methyl-morpholinium (DMTMM) before adding to beads, 36 or more classically by standard carbodiimide-based coupling protocols for glycans with a terminal amine group. 37−39 However, in the case of GPI 1 , the free amine groups of glucosamine and phosphoethanolamine are important for the specific binding of antibodies to GPIs. 16,40 Thus, we selected a conjugation method using maleimide-activated beads and reaction with a thiol linker that is attached at the phospho-myo-inositol unit of GPI 1 to retain the natural orientation of the glycan.…”

“…To evaluate the potential of synthetic GPI 1 for the determination of the T. gondii serostatus in humans using a BBMA, we first established the conjugation of the antigen to carboxylate microspheres. Various strategies for coupling glycan structures to carboxyl-functionalized beads have been described, e.g., via end-biotinylated ABO blood group glycans to streptavidin-coated beads, by functionalization of pneumococcal polysaccharides with 4-(4,6-dimethoxy­[1,3,5]­triazin-2-yl)-4-methyl-morpholinium (DMTMM) before adding to beads, or more classically by standard carbodiimide-based coupling protocols for glycans with a terminal amine group. − However, in the case of GPI 1 , the free amine groups of glucosamine and phosphoethanolamine are important for the specific binding of antibodies to GPIs. , Thus, we selected a conjugation method using maleimide-activated beads and reaction with a thiol linker that is attached at the phospho-myo-inositol unit of GPI 1 to retain the natural orientation of the glycan. Initial conjugation of the maleimide beads with a test compound (Man 3 ) showed a concentration dependency and saturation of the binding sites using 65 nmol of Man 3 per 10 5 beads.…”

Unveiling the Sugary Secrets of Plasmodium Parasites

“…In recent years, the immunogenicity of different parts of the GPI core structure was examined in more detail. Immunizing rabbits with synthetic GPI glycoconjugates yielded polyclonal sera capable of binding to native Plasmodium GPIs (Gurale et al, 2016). Vaccinating mice with glycoconjugates composed of synthetic GPI substructures conjugated to a carrier protein revealed the Man 3 -PEtN fragment to be highly immunogenic, while the presence of the fourth Man reduces immunogenicity (Malik et al, 2019).…”

Isolation and purification of glycosylphosphatidylinositols (GPIs) in the schizont stage of Theileria annulata and determination of antibody response to GPI anchors in vaccinated and infected animals

“…Focusing on the detailed structures of schizont stage of T. annulata is the prime focus of current research and, as already mentioned, the schizont stage of T. annulata is the most prominent stage in parasite invasion and pathogenicity [ 26 ]. As yet, there is no information available regarding the isolation, characterization and biological function of GPIs of T. annulata .…”