Cross-sectional Genetic Analysis of Plasmodium Falciparum Rh5 Interacting Protein (Pfripr) and Cysteine-rich Protective Antigen (Cyrpa) Genes in Kilifi, Kenya

Abstract

The discovery of antibodies against the Reticulocyte binding-protein homologue (Rh) 5 interacting protein (Ripr) and Cysteine-rich protective antigen (CyRPA), which are crucial in the invasion process across all parasite strains has brought new hope to the vaccine development field. Determining whether the Ripr and CyRPA genes in P. falciparum isolates from malaria-endemic population in Kilifi-Kenya are polymorphic provide data that is important in vaccine development targeting the two antigens. These results aid in preventing the development of a malaria blood-stage vaccine that would not proceed beyond the clinical trial stages due to the presence of multiple antigen variants. The genomic DNA of P. falciparum extracted from blood samples collected in 2013 and 2014 from 162 children aged below 8 years were used in the study. These children suffered from uncomplicated malaria and had been admitted to Kilifi County Hospital. From the extracted genomic DNA, Exon 1 and Exon 2 of CyRPA gene were separately amplified by different primer sets whereas Ripr gene was amplified using two different primer sets. Good quality amplicons were sequenced and analysed using CLC Genomics Workbench 7, MEGA 6.0 and DnaSP 5.10.01 software. Sequence assembly was done using CLC bio and subsequent analysis conducted using DnaSp software and MEGA 6.0. A total of three mutations were detected in sequences of exon 2 of CyRPA gene at positions 193, 1005 and 1086. SNPs at position 193 and 1005 resulted in non-synonymous mutations, whereas position 1086 was a synonymous mutation. The identified SNPs were under purifying selection, suggesting a possible stabilization of the CyRPA gene. The parasites tend to ensure that mutations that may interfere with the CyRPA antigen functionality are eliminated. Such a result reaffirms CyRPA antigen as a possible candidate in developing a blood-stage malaria vaccine in the future. Similar to Exon1 of CyRPA gene, the Ripr gene lacked polymorphisms. The result was obtained from analysis of 39 samples which accounted for 24.1% of the total samples analysed. The lack of polymorphism in Ripr, Exon1 of CyRPA sequences and polymorphisms in Exon 2 of CyRPA show that both these genes appear to be highly conserved with hardly any polymorphisms making them good vaccine candidates with no possible limitation of allele-specific immunity