Rapid Identification of Malaria Vaccine Candidates Based on α-Helical Coiled Coil Protein Motif

To identify malaria antigens for vaccine development, we selected α-helical coiled coil domains of proteins predicted to be present in the parasite erythrocytic stage. The corresponding synthetic peptides are expected to mimic structurally “native” epitopes. Indeed the 95 chemically synthesized peptides were all specifically recognized by human immune sera, though at various prevalence. Peptide specific antibodies were obtained both by affinity-purification from malaria immune sera and by immunization of mice. These antibodies did not show significant cross reactions, i.e., they were specific for the original peptide, reacted with native parasite proteins in infected erythrocytes and several were active in inhibiting in vitro parasite growth. Circular dichroism studies indicated that the selected peptides assumed partial or high α-helical content. Thus, we demonstrate that the bioinformatics/chemical synthesis approach described here can lead to the rapid identification of molecules which target biologically active antibodies, thus identifying suitable vaccine candidates. This strategy can be, in principle, extended to vaccine discovery in a wide range of other pathogens

Cell biological characterization of the malaria vaccine candidate trophozoite exported protein 1.

In a genome-wide screen for alpha-helical coiled coil motifs aiming at structurally defined vaccine candidates we identified PFF0165c. This protein is exported in the trophozoite stage and was named accordingly Trophozoite exported protein 1 (Tex1). In an extensive preclinical evaluation of its coiled coil peptides Tex1 was identified as promising novel malaria vaccine candidate providing the rational for a comprehensive cell biological characterization of Tex1. Antibodies generated against an intrinsically unstructured N-terminal region of Tex1 and against a coiled coil domain were used to investigate cytological localization, solubility and expression profile. Co-localization experiments revealed that Tex1 is exported across the parasitophorous vacuole membrane and located to Maurer's clefts. Change in location is accompanied by a change in solubility: from a soluble state within the parasite to a membrane-associated state after export to Maurer's clefts. No classical export motifs such as PEXEL, signal sequence/anchor or transmembrane domain was identified for Tex1

Phosphatidylinositol 3-Kinase C2α Is Essential for ATP-dependent Priming of Neurosecretory Granule Exocytosis

Neurotransmitter release and hormonal secretion are highly regulated processes culminating in the calcium-dependent fusion of secretory vesicles with the plasma membrane. Here, we have identified a role for phosphatidylinositol 3-kinase C2α (PI3K-C2α) and its main catalytic product, PtdIns3P, in regulated exocytosis. In neuroendocrine cells, PI3K-C2α is present on a subpopulation of mature secretory granules. Impairment of PI3K-C2α function specifically inhibits the ATP-dependent priming phase of exocytosis. Overexpression of wild-type PI3K-C2α enhanced secretion, whereas transfection of PC12 cells with a catalytically inactive PI3K-C2α mutant or a 2xFYVE domain sequestering PtdIns3P abolished secretion. Based on these results, we propose that production of PtdIns3P by PI3K-C2α is required for acquisition of fusion competence in neurosecretion

The Clinical and Genotypic Spectrum of Scoliosis in Multiple Pterygium Syndrome: A Case Series on 12 Children

Background: Multiple pterygium syndrome (MPS) is a genetically heterogeneous rare form of arthrogryposis multiplex congenita characterized by joint contractures and webbing or pterygia, as well as distinctive facial features related to diminished fetal movement. It is divided into prenatally lethal (LMPS, MIM253290) and nonlethal (Escobar variant MPS, MIM 265000) types. Developmental spine deformities are common, may present early and progress rapidly, requiring regular fo llow-up and orthopedic management. Methods: Retrospective chart review and prospective data collection were conducted at three hospital centers. Molecular diagnosis was confirmed with whole exome or whole genome sequencing. Results: This case series describes the clinical features and scoliosis treatment on 12 patients from 11 unrelated families. A molecular diagnosis was confirmed in seven; two with MYH3 variants and five with CHRNG. Scoliosis was present in all but our youngest patient. The remaining 11 patients spanned the spectrum between mild (curve ≤ 25°) and malignant scoliosis (≥50° curve before 4 years of age); the two patients with MYH3 mutations presented with malignant scoliosis. Bracing and serial spine casting appear to be beneficial for a few years; non-fusion spinal instrumentation may be needed to modulate more severe curves during growth and spontaneous spine fusions may occur in those cases. Conclusions: Molecular diagnosis and careful monitoring of the spine is needed in children with MPS

Malaria vaccine candidate: design of a multivalent subunit α-helical coiled coil poly-epitope.

A new strategy for the rapid identification of new malaria antigens based on protein structural motifs was previously described. We identified and evaluated the malaria vaccine potential of fragments of several malaria antigens containing α-helical coiled coil protein motifs. By taking advantage of the relatively short size of these structural fragments, we constructed different poly-epitopes in which 3 or 4 of these segments were joined together via a non-immunogenic linker. Only peptides that are targets of human antibodies with anti-parasite in vitro biological activities were incorporated. One of the constructs, P181, was well recognized by sera and peripheral blood mononuclear cells (PBMC) of adults living in malaria-endemic areas. Affinity purified antigen-specific human antibodies and sera from P181-immunized mice recognised native proteins on malaria-infected erythrocytes in both immunofluorescence and western blot assays. In addition, specific antibodies inhibited parasite development in an antibody dependent cellular inhibition (ADCI) assay. Naturally induced antigen-specific human antibodies were at high titers and associated with clinical protection from malaria in longitudinal follow-up studies in Senegal