• New tools for var gene assembly and analysis of genetic diversity of var2csa, the locus encoding a placental malaria vaccine candidate antigen: vaccine development implications

      Dara, Antoine; Plowe, Christopher V. (2017)
      Encoded by the var gene family, highly variable Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) proteins mediate tissue-specific cytoadherence of infected erythrocytes (iEs), resulting in immune evasion and severe malaria disease. Sequencing and assembling the 40-60 var gene complement for individual infections has been notoriously difficult, impeding molecular epidemiological studies and the assessment of var-encoded proteins as subunit vaccine candidates. VAR2CSA, a member of this family, mediates the binding of iEs to chondroitin sulfate A, a surface-associated molecule expressed in placental cells, and plays a central role in the pathogenesis of placental malaria. VAR2CSA is a target of naturally-acquired immunity and, as such, is a leading vaccine candidate against placental malaria. The gene encoding the VAR2CSA is technically challenging to sequence. Published var2csa sequences, mostly limited to specific domains, have been generated through the sequencing of cloned PCR amplicons using capillary electrophoresis, a method that is both time-consuming and costly. We developed and validated novel tools to assemble var gene sequences from clinical samples, with a focus on var2csa. These tools include Exon-Targeted Hybrid Assembly (ETHA), which is based on a combination of Pacific Biosciences (PacBio) and Illumina data, an amplicon sequencing of var2csa using PacBio, and a pipeline to reconstruct var2csa from short sequence reads. Using ETHA, we characterized the repertoire of var genes in samples from uncomplicated malaria infections in children from a single Malian village and showed them to be as genetically diverse as vars from isolates from around the globe. The amplicon sequencing allowed us to recover a segment encoding the first four domains in the 5' end of VAR2CSA (~5kb), and the full-length of the VAR2CSA's extracellular region from clinical malaria samples. The reconstruction of var2csa from short reads permitted us to generate a worldwide set of var2csa sequences. We used the VAR2CSA sequences to show that despite the extreme sequence variation, VAR2CSA from around the globe shared conserved motifs supporting the feasibility of VAR2CSA-based vaccines. The findings presented in this work will aid the understanding of malaria pathogenesis and inform the design of malaria vaccines based on PfEMP1, in particular VAR2CSA-based vaccines.