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

Md Atique Ahmed,Rehan Haider Zaidi,Saurav Jyoti Patgiri,Mohammed Abdulrahman Alshahrani,Fu-Shi Quan,Gauspasha Yusuf Deshmukh,Ahmed Saif,Syeda Wasfeea Wazid

doi:10.3389/fcimb.2021.810398

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.

Highlights

Malaria is a vector-borne disease which is prevalent in more than a hundred countries with 228 million malaria cases and an incidence of 405,000 deaths in 2019, the majority of which are due to Plasmodium falciparum infections (World Health Organisation, 2019)
In order to determine the relationship between PkRhopH2 sequences, phylogenetic analyses were conducted using deduced amino acid sequences using the maximum likelihood (ML) method based on the Poisson correction model as described in MEGA 5.0 with 1,000 bootstrap replicates to test the robustness of the trees
The phylogenetic analysis performed using deduced amino acid sequences in the ML method showed that PkRhopH2 is more closely related with P. coatneyi in comparison to its other orthologs in P. vivax, P. cynomolgi, and P. falciparum (Figure 1B)

Summary

Introduction

Malaria is a vector-borne disease which is prevalent in more than a hundred countries with 228 million malaria cases and an incidence of 405,000 deaths in 2019, the majority of which are due to Plasmodium falciparum infections (World Health Organisation, 2019). P. knowlesi, for example, caused 5% of malaria cases in Sabah in 2004, but 98% in 2017 (William et al, 2013; Cooper et al, 2020). This simian malaria parasite, P. knowlesi, was reported as a major cause of human malaria in Sarawak, Malaysia, in a paper published 16 years ago (Singh et al, 2004). Several Southeast Asian countries have reported zoonotic malaria cases due to P. knowlesi. The wholegenome and genetic studies on P. knowlesi identified that there are at least three subpopulations in clinical samples from Malaysia, and out of these, two of them are linked to the primary monkey hosts, Macaca nemestrina and Macaca fascicularis (Ahmed et al, 2014; Assefa et al, 2015; Pinheiro et al, 2015; Ahmed et al, 2016)

Methods

Results

Conclusion