Molecular Characterization and Immuno-Reactivity Patterns of a Novel Plasmodium falciparum Armadillo-Type Repeat Protein, PfATRP.

Nearly half of the genes in the Plasmodium falciparum genome have not yet been functionally investigated. We used homology-based structural modeling to identify multiple copies of Armadillo repeats within one uncharacterized gene expressed during the intraerythrocytic stages, PF3D7_0410600, subsequently referred to as P. falciparum Armadillo-Type Repeat Protein (PfATRP). Soluble recombinant PfATRP was expressed in a bacterial expression system, purified to apparent homogeneity and the identity of the recombinant PfATRP was confirmed by mass spectrometry. Affinity-purified α-PfATRP rabbit antibodies specifically recognized the recombinant protein. Immunofluorescence assays revealed that α-PfATRP rabbit antibodies reacted with P. falciparum schizonts. Anti-PfATRP antibody exhibited peripheral staining patterns around the merozoites. Given the localization of PfATRP in merozoites, we tested for an egress phenotype during schizont arrest assays and demonstrated that native PfATRP is inaccessible on the surface of merozoites in intact schizonts. Dual immunofluorescence assays with markers for the inner membrane complex (IMC) and microtubules suggest partial colocalization in both asexual and sexual stage parasites. Using the soluble recombinant PfATRP in a screen of plasma samples revealed that malaria-infected children have naturally acquired PfATRP-specific antibodies

Vaccine-induced human monoclonal antibodies to PfRH5 show broadly neutralizing activity against P. falciparum clinical isolates

Vaccines to the Plasmodium falciparum reticulocyte binding-like protein homologue 5 (PfRH5) target the blood-stage of the parasite life cycle. PfRH5 has the potential to trigger the production of strain-transcendent antibodies and has proven its efficacy both in pre-clinical and early clinical studies. Vaccine-induced monoclonal antibodies (mAbs) to PfRH5 showed promising outcomes against cultured P. falciparum laboratory strains from distinct geographic areas. Here, we assessed the functional impact of vaccine-induced anti-PfRH5 mAbs on more genetically diverse P. falciparum clinical isolates. We used mAbs previously isolated from single B cells of UK adult PfRH5 vaccinees and used ex-vivo growth inhibition activity (GIA) assays to assess their efficacy against P. falciparum clinical isolates. Next-generation sequencing (NGS) was used to assess the breadth of genetic diversity in P. falciparum clinical isolates and to infer the genotype/phenotype relationship involved in antibody susceptibility. We showed a dose-dependent inhibition of clinical isolates with three main GIA groups: high, medium and low. Except for one isolate, our data show no significant differences in the mAb GIA profile between P. falciparum clinical isolates and the 3D7 reference strain, which harbors the vaccine allele. We also observed an additive relationship for mAb combinations, whereby the combination of GIA-low and GIA-medium antibodies resulted in increased GIA, having important implications for the contribution of specific clones within polyclonal IgG responses. While our NGS analysis showed the occurrence of novel mutations in the pfrh5 gene, these mutations were predicted to have little or no functional impact on the antigen structure or recognition by known mAbs. Our present findings complement earlier reports on the strain transcendent potential of anti-PfRH5 mAbs and constitute, to our knowledge, the first report on the susceptibility of P. falciparum clinical isolates from natural infections to vaccine-induced human mAbs to PfRH5

Molecular Characterization and Immuno-Reactivity Patterns of a Novel Plasmodium falciparum Armadillo-Type Repeat Protein, PfATRP.

Nearly half of the genes in the Plasmodium falciparum genome have not yet been functionally investigated. We used homology-based structural modeling to identify multiple copies of Armadillo repeats within one uncharacterized gene expressed during the intraerythrocytic stages, PF3D7_0410600, subsequently referred to as P. falciparum Armadillo-Type Repeat Protein (PfATRP). Soluble recombinant PfATRP was expressed in a bacterial expression system, purified to apparent homogeneity and the identity of the recombinant PfATRP was confirmed by mass spectrometry. Affinity-purified α-PfATRP rabbit antibodies specifically recognized the recombinant protein. Immunofluorescence assays revealed that α-PfATRP rabbit antibodies reacted with P. falciparum schizonts. Anti-PfATRP antibody exhibited peripheral staining patterns around the merozoites. Given the localization of PfATRP in merozoites, we tested for an egress phenotype during schizont arrest assays and demonstrated that native PfATRP is inaccessible on the surface of merozoites in intact schizonts. Dual immunofluorescence assays with markers for the inner membrane complex (IMC) and microtubules suggest partial colocalization in both asexual and sexual stage parasites. Using the soluble recombinant PfATRP in a screen of plasma samples revealed that malaria-infected children have naturally acquired PfATRP-specific antibodies

Image_1_Phenotypic characterization of Ghanaian P. falciparum clinical isolates reveals a homogenous parasite population.jpeg

BackgroundErythrocyte invasion by P. falciparum involves functionally overlapping interactions between the parasite’s ligands and the erythrocyte surface receptors. While some P. falciparum isolates necessarily engage the sialic acid (SA) moieties of the erythrocytes during the invasion, others use ligands whose binding is independent of SA for successful invasion. Deciphering the major pathway used by P. falciparum clinical isolates represent a key step toward developing an efficient blood stage malaria vaccine.MethodsWe collected a total of 156 malaria-infected samples from Ghanaian children aged 2 to 14 years and used a two-color flow cytometry-based invasion assay to assess the invasion phenotype diversity of Ghanaian P. falciparum clinical isolates. Anti-human CR1 antibodies were used to determine the relative contribution of the PfRh4-CR1 interaction in the parasites invasion phenotype and RT-qPCR was used to assess the expression levels of key invasion-related ligands.ResultsOur findings show no clear association between demographic or clinical data and existing reports on the malaria transmission intensity. The complete invasion data obtained for 156 isolates, showed the predominance of SA-independent pathways in Ghanaian clinical isolates. Isolates from Hohoe and Navrongo had the highest diversity in invasion profile. Our data also confirmed that the PfRh4-CR1 mediated alternative pathway is important in Ghanaian clinical isolates. Furthermore, the transcript levels of ten invasion-related genes obtained in the study showed little variations in gene expression profiles within and between parasite populations across sites.ConclusionOur data suggest a low level of phenotypic diversity in Ghanaian clinical isolates across areas of varying endemicity and further highlight its importance in the quest for new intervention strategies, such as the investigation of blood-stage vaccine targets, particularly those targeting specific pathways and able to trigger the stimulation of broadly neutralizing invasion antibodies.</p