BackgroundPlasmodium vivax accounts for the majority of human malaria infections outside Africa and is being increasingly associated in fatal outcomes with anaemia as one of the major complications. One of the causes of malarial anaemia is the augmented removal of circulating non-infected red blood cells (nRBCs), an issue not yet fully understood. High levels of auto-antibodies against RBCs have been associated with severe anaemia and reduced survival of nRBCs in patients with falciparum malaria. Since there are no substantial data about the role of those antibodies in vivax malaria, this study was designed to determine whether or not auto-antibodies against erythrocytes are involved in nRBC clearance. Moreover, the possible immune mechanisms elicited by them that may be associated to induce anaemia in P. vivax infection was investigated.MethodsConcentrations of total IgG were determined by sandwich ELISA in sera from clinically well-defined groups of P. vivax-infected patients with or without anaemia and in healthy controls never exposed to malaria, whereas the levels of specific IgG to nRBCs were determined by cell-ELISA. Erythrophagocytosis assay was used to investigate the ability of IgGs purified from each studied pooled sera in enhancing nRBC in vitro clearance by THP-1 macrophages. Defocusing microscopy was employed to measure the biomechanical modifications of individual nRBCs opsonized by IgGs purified from each group.ResultsAnaemic patients had higher levels of total and specific anti-RBC antibodies in comparison to the non-anaemic ones. Opsonization with purified IgG from anaemic patients significantly enhanced RBCs in vitro phagocytosis by THP-1 macrophages. Auto-antibodies purified from anaemic patients decreased the nRBC dynamic membrane fluctuations suggesting a possible participation of such antibodies in the perturbation of erythrocyte flexibility and morphology integrity maintenance.ConclusionsThese findings revealed that vivax-infected patients with anaemia have increased levels of IgG auto-antibodies against nRBCs and that their deposition on the surface of non-infected erythrocytes decreases their deformability, which, in turn, may enhance nRBC clearance by phagocytes, contributing to the anaemic outcome. These data provide insights into the immune mechanisms associated with vivax malaria anaemia and may be important to the development of new therapy and vaccine strategies.
Clearance of non-infected red blood cells (nRBCs) is one of the main components of anemia associated with Plasmodium vivax malaria. Recently, we have shown that anemic patients with P. vivax infection had elevated levels of anti-RBCs antibodies, which could enhance in vitro phagocytosis of nRBCs and decrease their deformability. Using immunoproteomics, here we characterized erythrocytic antigens that are differentially recognized by autoantibodies from anemic and non-anemic patients with acute vivax malaria. Protein spots exclusively recognized by anemic P. vivax-infected patients were identified by mass spectrometry revealing band 3 and spectrin as the main targets. To confirm this finding, antibody responses against these specific proteins were assessed by ELISA. In addition, an inverse association between hemoglobin and anti-band 3 or anti-spectrin antibodies levels was found. Anemic patients had higher levels of IgG against both band 3 and spectrin than the non-anemic ones. To determine if these autoantibodies were elicited because of molecular mimicry, we used in silico analysis and identified P. vivax proteins that share homology with human RBC proteins such as spectrin, suggesting that infection drives autoimmune responses. These findings suggest that band 3 and spectrin are potential targets of autoantibodies that may be relevant for P. vivax malaria-associated anemia.
Anemia is a typical manifestation of Plasmodium vivax infection and one of the leading causes of morbidity and mortality, particularly in pregnant women and children less than five years old. Despite the magnitude of the problem, there remain major unsolved questions about its etiology. In this context, we have addressed the following question: Are antibodies directed to erythrocyte membrane proteins involved in the accelerated clearance of red blood cells (RBCs) by phagocytes leading to anemia during P. vivax infection? We performed immunoproteomic assays trying to characterize proteins that are targets of antibodies. Sera from four groups of subjects were tested against a noninfected O+ erythrocyte membrane extract and are included in our study: P. vivax anemic individuals (PvAN); P. vivax non-anemic individuals (PvNA); non-infected individuals with anemia by other etiologies (NIAN); and noninfected and non-anemic individuals (NINA). Immunoblots revealed a differential pattern of RBC protein recognition related to the four studied groups. The breadth and the magnitude of antibody responses against RBC membrane antigens were higher in NAPv and ANPv, corroborating our previous results obtained by ELISA and 1-D immunoblot. Now, these proteins are being identified by peptide mass fingerprint. Next, we will confirm their identity by MALDI TOF/MS-MS, validate and categorize them in relation to location, family and function. Highly immunogenic and differentially recognized proteins will represent potential candidates for diagnostic or prognostic biomarkers of anemia in P. vivax infections. Moreover, data generated here will contribute to the better understanding of mechanisms enrolled in vivax anemia pathogenesis.
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