Antibody immunity to Plasmodium falciparum erythrocyte membrane protein 1 and severe malaria

© 2018 Dr. Janavi Suresh RambhatlaPlasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins are expressed on the surface of the infected erythrocyte (IE). These proteins mediate the adhesion of the parasite IE to host cells which could result in severe forms of malaria. PfEMP1 is a variable protein and consists of several domains. PfEMP1s are encoded by var genes and composed of multiple duffy binding-like (DBL) domains and cysteine-rich inter-domain region (CIDR) domains. PfEMP1s can be classified into groups based on the chromosomal location, transcriptional direction and upstream promotor regions of var genes, the DBL or CIDR domains expressed or the domain cassettes (DC) expressed. DCs are sets of PfEMP1 domains that usually occur together. Transcription of specific PfEMP1 subtypes and DCs have been associated with severe malaria. These proteins are important for anti-malarial immunity. Antibody immunity to the different PfEMP1 types can develop gradually with exposure with potential to protect from severe malaria. The current study investigated if antibody responses to different subsets of PfEMP1s or selections of their constituent domains differed between individuals with severe malaria and uncomplicated malaria as well as between individuals presenting with specific severe malaria syndromes. The study also investigated how such antibodies vary between disease presentation and convalescence, 1-2 months after the malaria episode. Three aims were investigated in the present study: 1) to determine if recombinant CIDR domains are targets of antibodies in children with severe or uncomplicated malaria, 2) to determine if levels of opsonising antibodies against four parasite IE lines differed between children with severe or uncomplicated malaria and 3) to determine if the various recombinant PfEMP1 domains (transcripts of which were previously shown to be associated with severe or uncomplicated malaria in adults) are targets of antibody immunity in the same adults with severe or uncomplicated malaria as well as in children with cerebral or uncomplicated malaria. To measure antibody levels against the different PfEMP1 subtypes, unique, high-throughput multiplex assays as well as flow cytometry-based assays were employed. Antibody levels were measured in children from Papua New Guinea (PNG), adults from Papua and children from Malawi. For aim 1, total IgG levels were measured and analysed against 35 recombinant CIDR domains in plasma collected from PNG children at hospital presentation (acute) and in convalescence following severe (n = 98) or uncomplicated malaria (n = 65), and in age-matched community controls (n = 112). At hospital presentation, antibody levels did not differ across the three severity groups for any of the CIDR domain types tested. During convalescence, antibody levels against the endothelial protein C receptor (EPCR) binding CIDRα1 domain types were higher in older children with severe malaria compared to uncomplicated malaria. Antibodies to the EPCR-binding CIDRα1 domains also increased from presentation to convalescence for severe malaria cases, but not uncomplicated malaria. Such differences in antibody acquisition (by disease severity) or boosting of antibody levels (with time) against non-EPCR-binding CIDR domains, such as the CD36 binding domains or domains associated with rosetting, were not observed, suggesting that these variants may not be prominently expressed in these infections. Antibodies acquired against PfEMP1 can have varied functions such as inhibiting the IE-receptor binding, opsonic phagocytosis and inhibition of rosette formation, which in turn can potentially protect from severe malaria. Using the same PNG study population as aim 1, for aim 2, levels of one type of functional antibodies -opsonising antibodies- were measured and analysed in plasma collected at acute and convalescence in children with severe (n = 163 and 158 respectively) or uncomplicated malaria (n = 101 and 99 respectively) as well as age-matched community controls (n = 235). Opsonising antibody levels against four different parasite IE expressing the entire PfEMP1 molecule, with conformations similar to the native protein and having different binding phenotypes, were measured. Opsonising antibodies against an EPCR-binding PfEMP1 were higher in children having uncomplicated malaria compared to children with severe malaria. These antibody levels were higher in children with uncomplicated malaria compared to children with severe malaria presenting with specific severe syndromes as well. The antibody levels were also boosted from presentation to convalescence for the EPCR binding IE in children with severe, but not uncomplicated, malaria. For the intercellular adhesion molecule 1 (ICAM-1) -binding PfEMP1 (which also binds to CD36), children with uncomplicated malaria showed weak evidence for boosting of antibodies from presentation to convalescence. For the rosetting IE, opsonising antibodies acquired during convalescence were higher in children with uncomplicated malaria compared to severe malaria. Antibodies against the CD36-binding PfEMP1 did not differ by disease severity and declined from presentation to convalescence in severe and uncomplicated cases. PfEMP1 is a large multi-domain protein and for aim 3, to investigate the role of antibodies against different PfEMP1 domains, the domains (whose transcription was upregulated in severe or uncomplicated malaria cases in Papua) were first expressed using the wheat germ cell free protein synthesis systems (WGCFS). Antibody levels against 32 recombinant PfEMP1 domains thus generated were measured in acute plasma from Papuan individuals with severe (n = 28) or uncomplicated malaria (n = 35). Antibody levels against these domains were also measured in acute and convalescent plasma from Malawian children with cerebral malaria (n = 119 and 68 respectively) or uncomplicated malaria (n = 118 and 85 respectively), and in age-matched healthy individuals (n = 101). In the Papuan population, antibodies were higher in individuals with uncomplicated malaria compared to severe malaria against a range of PfEMP1 domains. The differences in antibody levels by disease severity against the different domains included PfEMP1 domains such as CIDRα1.6 and DBLβ3, which were previously associated with severe malaria, as well as several novel PfEMP1 domains such as CIDRα2.4, DBLζ4, and DBLδ1. In the Malawian population, antibody levels were higher in children with cerebral malaria compared to uncomplicated malaria at presentation but not in convalescence. Antibody levels against various PfEMP1 domains were boosted from presentation to follow-up in children with uncomplicated malaria but not severe malaria. The differences in antibody levels by disease severity against the different domains for this population also included PfEMP1 domains such as CIDRα1.1 which was previously associated with severe malaria, as well as several novel PfEMP1 domains such as CIDRα2.4, DBLε3, DBLγ3, DBLζ3, DBLβ13, DBLδ7 and DBLδ1. There were some novel overlapping targets of immunity in the two populations, while some domains did not overlap as targets of immunity in the two settings, such as DBLζ4. Thus, the current study was one of the first studies to demonstrate the EPCR-binding PfEMP1 domains or types to be important targets of antibody immunity as evidenced by differences in the antibody levels by disease severity against these domains. The present study was also one of the first to show levels of opsonising antibodies against the EPCR-binding PfEMP1 to differ by disease severity with potential to protect from severe malaria and its syndromes. Weak evidence was also found for opsonising antibodies against ICAM-1/CD36-binding PfEMP1 to be associated with uncomplicated malaria. In Papuan adults, protection from severe malaria was associated with a ‘total’ antibody response against a wide range of PfEMP1 domains whose transcripts were also upregulated in severe infections affecting the same individuals. In young Malawian children with cerebral malaria, the antibody responses against the PfEMP1 domains seem to be markers of exposure to antigens causing malaria rather than markers of immunity. The overall conclusion for the study is that antibody immunity to multiple PfEMP1 domains on the IE may be required for protection from severe malaria and the current study has helped identify some of these PfEMP1 domains