Abstract
Over 50 million women are exposed to the risk of malaria during pregnancy every year. Malaria during pregnancy is a leading global cause of maternal morbidity and adverse pregnancy outcomes. Adhesion of Plasmodium falciparum-infected erythrocytes to placental chondroitin-4-sulfate (CSA) has been linked to the severe disease outcome of placental malaria. Accumulated evidence strongly supports VAR2CSA as the leading placental malaria vaccine candidate. Recombinant proteins encompassing the VAR2CSA high affinity CSA binding site have been generated, and their activity as immunogens that elicit functional (inhibitory) and cross-reactive antibodies against CSA-binding parasites assessed. The expression of His-tagged proteins was compared in four different expression systems and their capacity to bind specifically to CSA was analyzed. CHO cells and E. coli SHuffle cells were the two expression systems able to express some of the recombinant proteins in reasonable amounts. Larger analytical scale production of DBL1x-2× (3D7) and DBL3x-4ε (FCR3) best expressed in CHO and E. coli SHuffle cells were performed. Purified proteins were administered to rats either alone or adjuvanted with human approved adjuvants. Analysis of the functionality and cross-reactivity of the induced antibodies allowed us to down-select the DBL1x-2(3D7) expressed in E. coli SHuffle cells as the best antigen to be transitioned to further clinical development in order to protect future pregnant women living in malaria endemic areas against the severe clinical outcomes of placental malaria.
Highlights
Malaria caused by Plasmodium constitutes a major health problem and is still one of the most common deadly infectious diseases in the world
This study describes the down-selection of a VAR2CSA-based production and purity by capillary electrophoresis revealed vaccine candidate, which would be able to generate antibodies relatively modest expression yields (≈5 mg/L) and low purity for capable of (i) cross-reacting with native VAR2CSA expressed at the DBL1x-6ε (3D7), DBL1x-3× (3D7) and ID1-DBL3x (3D7) in the crude surface of erythrocytes infected with different parasite strains from supernatant (Table S1)
Flow cytometry assessment of membrane-bound IgG revealed that, as compare to preimmune sera, rats immunized with DBL1x-2× (3D7) expressed in either Chinese Hamster Ovary (CHO) cells or SHuffle bacteria were able to generate antibodies able to strongly recognize native VAR2CSA from the autologous parasite strain NF54-CSA and to cross-react with cell-surfaced exposed VAR2CSA from FCR3-CSA parasites and to a lower extent from 7G8-CSA parasites (Fig. 5)
Summary
Down-selection of the VAR2CSA DBL1-2 expressed in E. coli as a lead antigen for placental malaria vaccine development. Accumulated evidence strongly supports VAR2CSA as the leading placental malaria vaccine candidate. CHO cells and E. coli SHuffle cells were the two expression systems able to express some of the recombinant proteins in reasonable amounts. Larger analytical scale production of DBL1x-2× (3D7) and DBL3x-4ε (FCR3) best expressed in CHO and E. coli SHuffle cells were performed. Analysis of the functionality and cross-reactivity of the induced antibodies allowed us to down-select the DBL1x-2(3D7) expressed in E. coli SHuffle cells as the best antigen to be transitioned to further clinical development in order to protect future pregnant women living in malaria endemic areas against the severe clinical outcomes of placental malaria
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