Identification, Mapping, and Genetic Diversity of Novel Conserved Cross-Species Epitopes of RhopH2 in Plasmodium knowlesi With Plasmodium vivax

Abstract

Malaria is a major public health concern, and any tangible intervention during the pre-elimination phase can result in a significant reduction in infection rates. Recent studies have reported that antigens producing cross-protective immunity can play an important role as vaccines and halt malaria transmission in different endemic regions. In this study, we studied the genetic diversity, natural selection, and discovered novel conserved epitopes of a high molecular weight rhoptry protein 2 (RhopH2) in clinical samples of Plasmodium knowlesi and Plasmodium vivax cross-protective domains, which has been proven to produce cross-protective immunity in both species. We found low levels of nucleotide diversity (P. knowlesi; π ~ 0.0093, SNPs = 49 and P. vivax π ~ 0.0014, SNPs = 23) in P. knowlesi (n = 40) and P. vivax (n = 65) samples in the PkRhopH2 cross-protective domain. Strong purifying selection was observed for both species (P. knowlesi; dS - dN = 2.41, p < 0.009, P. vivax; dS - dN = 1.58, p < 0.050). In silico epitope prediction in P. knowlesi identified 10 potential epitopes, of which 7 epitopes were 100% conserved within clinical samples. Of these epitopes, an epitope with 10 amino acids (QNSKHFKKEK) was found to be fully conserved within all P. knowlesi and P. vivax clinical samples and 80%–90% conservation within simian malaria ortholog species, i.e., P. coatneyi and P. cynomolgi. Phylogenetic analysis of the PkRhopH2 cross-protective domain showed geographical clustering, and three subpopulations of P. knowlesi were identified of which two subpopulations originated from Sarawak, Malaysian Borneo, and one comprised only the laboratory lines from Peninsular Malaysia. This study suggests that RhopH2 could be an excellent target for cross-protective vaccine development with potential for outwitting strain as well as species-specific immunity. However, more detailed studies on genetic diversity using more clinical samples from both species as well as the functional role of antibodies specific to the novel conserved epitope identified in this study can be explored for protection against infection.

In Silico Analysis Reveals High Levels of Genetic Diversity of Plasmodium knowlesi Cell Traversal Protein for Ookinetes and Sporozoites (PkCelTOS) in Clinical Samples

“…It assumes that the folded protein structure puts minimum strain on side-chain conformations, favoring low-energy positions during folding [ 48 ]. Since P. knowlesi and P. vivax are phylogenetically close and several studies have shown the importance of conserved cross-species vaccine candidates [ 11 , 49 , 50 ]. Therefore, in addition to identifying epitopes in P. knowlesi, this study examined the likelihood of epitope conservation with P. vivax .…”

“…In many countries outside of sub-Saharan Africa, P. vivax is the most prevalent species [ 1 ]. The simian malaria parasite P. knowlesi has been discovered to cause human infection, as reported from several Southeast Asian countries, particularly in Malaysian Borneo [ 4 , 9 , 10 , 11 , 12 , 13 , 14 ]. Due to its increasing incidence in recent years, P. knowlesi malaria has become a significant public health issue in the region [ 4 , 9 , 13 , 15 ].…”

“…Later on, many species of the Leucosphyrus group under the genus Anopheles were reported to transmit the parasite from macaques to humans [ 9 , 13 , 15 ]. Previous studies utilizing whole-genome and genetic analysis on clinical samples from Malaysian Borneo and Sarawak have identified the existence of at least three sub-populations [ 10 , 11 , 17 ]. Among these, two are associated with the primary monkey hosts, long-tailed macaques ( Macaca fascicularis ) and pig-tailed macaques ( Macaca nemestrina ) [ 15 , 18 ].…”

“…Thus it is important to study the antigenic diversity of potential ortholog genes of P. falciparum and P. vivax in P. knowlesi to develop an efficacious vaccine candidate for knowlesi malaria. Various studies have characterized merozoite invasion genes in P. knowlesi, analyzing their genetic diversity and population structure [ 10 , 11 , 12 , 17 , 25 , 29 ]; however, multi-stage vaccine candidates other than CSP [ 30 ] have not been genetically characterized in P. knowlesi.…”

A multi-epitope vaccine designed against blood-stage of malaria: an immunoinformatic and structural approach

“…Genetic, antigenic diversity and the ability to evade host immune responses are major obstacles hindering the development of effective anti-malaria vaccines. Many studies have focused on conserved regions or epitopes common between vast ranges of plasmodium strains 6 . P. falciparum glutamic acid-rich protein (PfGARP) is an 80 kDa antigen expressed on the surface of infected erythrocytes with P. falciparum 7 .…”