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Plasmodium falciparum-infected erythrocytes (IE) sequester in the placenta via surface protein VAR2CSA, which binds chondroitin sulfate A (CSA) expressed on the syncytiotrophoblast surface, causing placental malaria (PM) and severe adverse outcomes in mothers and their offspring. VAR2CSA belongs to the PfEMP1 variant surface antigen family; PfEMP1 proteins mediate IE adhesion and facilitate parasite immunoevasion through antigenic variation. Here we produced deglycosylated (native-like) and glycosylated versions of seven recombinant full-length VAR2CSA ectodomains and compared them for antigenicity and adhesiveness. All VAR2CSA recombinants bound CSA with nanomolar affinity, and plasma from Malian pregnant women demonstrated antigen-specific reactivity that increased with gravidity and trimester. However, allelic and glycosylation variants differed in their affinity to CSA and their serum reactivities. Deglycosylated proteins (native-like) showed higher CSA affinity than glycosylated proteins for all variants except NF54. Further, the gravidity-related increase in serum VAR2CSA reactivity (correlates with acquisition of protective immunity) was absent with the deglycosylated form of atypical M200101 VAR2CSA with an extended C-terminal region. Our findings indicate significant inter-allelic differences in adhesion and seroreactivity that may contribute to the heterogeneity of clinical presentations, which could have implications for vaccine design., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Two vaccines based on Plasmodium falciparum protein VAR2CSA are currently in clinical evaluation to prevent placental malaria (PM), but a deeper understanding of var2csa variability could impact vaccine design. Here we identified atypical extended or truncated VAR2CSA extracellular structures and confirmed one extended structure in a Malian maternal isolate, using a novel protein fragment assembly method for RNA-seq and DNA-seq data. Extended structures included one or two additional DBL domains downstream of the conventional NTS-DBL1X-6ɛ domain structure, with closest similarity to DBLɛ in var2csa and non- var2csa genes. Overall, 4/82 isolates displayed atypical VAR2CSA structures. The maternal isolate expressing an extended VAR2CSA bound to CSA, but its recombinant VAR2CSA bound less well to CSA than VAR2CSA NF54 and showed lower reactivity to naturally acquired parity-dependent antibody. Our protein fragment sequence assembly approach has revealed atypical VAR2CSA domain architectures that impact antigen reactivity and function, and should inform the design of VAR2CSA-based vaccines., Competing Interests: Competing interestsThe authors declare no competing interests., (© This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply 2019.)