Blood donor variability is a modulatory factor for P. falciparum invasion phenotyping assays

Abstract Human erythrocytes are indispensable for Plasmodium falciparum development. Unlike other eukaryotic cells, there is no existing erythroid cell line capable of supporting long-term P. falciparum in vitro experiments. Consequently, invasion phenotyping experiments rely on erythrocytes of different backgrounds. However, the contribution of the erythrocytes variation in influencing invasion rates remains unknown, which presents a challenge for conducting large-scale comparative studies. Here, we used erythrocytes of different blood groups harboring different hemoglobin genotypes to assess the relative contribution of blood donor variability in P. falciparum invasion phenotyping assays. For each donor, we investigated the relationship between parasite invasion phenotypes and erythrocyte phenotypic characteristics, including; the expression levels of surface receptors (e.g. the human glycophorins A and C, the complement receptor 1 and decay accelerating factor), blood groups (e.g. ABO/Rh system), and hemoglobin genotypes (e.g. AA, AS and AC). Across all donors, there were significant differences in invasion efficiency following treatment with either neuraminidase, trypsin or chymotrypsin relative to the control erythrocytes. Primarily, we showed that the levels of key erythrocyte surface receptors and their sensitivity to enzyme treatment, significantly differed across donors. However, invasion efficiency correlated neither with susceptibility to enzyme treatment nor with the levels of the selected erythrocyte surface receptors. Upon further analysis, we found no relationship between P. falciparum invasion phenotype and blood group or hemoglobin genotype. Importance Assays to decipher P. falciparum invasion phenotypes are of great importance in the quest for an efficient malaria vaccine. Malaria associated mortality is mainly attributed to the blood stage of the parasite’s life cycle, a major focus of vaccine development strategies. Further, testing and validating blood stage vaccines necessitates conducting large-scale studies in endemic countries. However, comparing results from such studies is challenged by the lack of standard assays. As human erythrocytes play a pivotal role in P. falciparum invasion assays, the need to investigate the effect of blood donor variability in the outcome of such assays is apparent. The significance of our study is in reporting the absence of relationship between P. falciparum invasion efficiency and commonly shared erythrocyte features across different erythrocyte donors, therefore emphasizing the need to consider erythrocyte donor uniformity and to anticipate challenges associated to blood donor variability in early stages of large-scale study design.