Informing the role of RIFINs in malaria pathogenesis, natural immunity, and design of a severe malaria vaccine

Abstract

Plasmodium falciparum is a eukaryotic parasite that causes severe malaria and contributed to 405,000 deaths worldwide in 2018. Victims of severe malaria are predominantly sub-Saharan African children, who typically present with symptoms of severe anemia or unarousable coma. The pathogenesis of severe malaria is poorly understood but mediated by the expression of adhesive variant surface antigens (VSAs) on infected red blood cells. VSAs are involved in sequestration and rosetting, unique virulence processes that allow the parasite to evade host immune responses and prevent clearance in the spleen. A relatively unstudied family of VSAs, the repetitive interspersed family (RIFIN) proteins, have recently been found to be important in rosetting and host immune suppression. RIFINs also appear to be targets for protective immunity; humoral immune responses against RIFINs have been correlated with asymptomatic infections. In this dissertation, I applied a multi-faceted approach using protein and peptide microarrays, transcriptomics, and reverse vaccinology to identify appealing RIFIN candidates for inclusion in a future severe malaria vaccine. I show that serological responses against epitopes within the semi-conserved domain of RIFINs associated with severe malaria reflected age-related malaria exposure. Sequencing and identifying specific rif genes expressed in clinical infections have not been feasible. I have addressed these challenges by adapting a novel bioinformatic pipeline and developing an HMM-based tool to process, assemble, classify, and subtype RIFIN sequences from peripheral blood samples. This takes advantage of a targeted probe capture method that I determined yields more abundant, full-length RIFIN sequences than other library enrichment approaches. Finally, I performed a comprehensive genomic survey of RIFIN gene repertoires using publicly available whole genome data of sixteen P. falciparum isolates to identify highly conserved, strain-transcendent sequences. Together, these results provide insights and powerful tools that can advance our understanding of the role RIFINs play in severe malaria pathogenesis and the development of naturally-acquired immunity to severe malaria. This work will aid efforts to determine targets for vaccines to protect children from the deadliest consequences of malaria.