Malaria has been a major global health concern of humans throughout history and is a leading cause of disease and death across many tropical and subtropical countries. In 2019, an estimated 229 million malaria cases and 409,000 malaria-caused deaths globally were reported (WHO, 2020). Among the five species of Plasmodium that infect humans, P. falciparum infection causes the highest mortality and morbidity and the most serious clinical symptoms (Buffet et al., 2011).

The resurgence and spread of antimalarial drug resistance (Ménard et al., 2015; WWARN, 2015) along with vector resistance to insecticides (Dhiman and Veer, 2014; Strode et al., 2014) have the potential to reduce the impact of existing malaria control strategies and make vaccines a public health priority. Although extensive studies have been conducted on several blood-stage antigens, few have shown the quality required for a candidate vaccine. In a systematic screen of uncharacterized P. falciparum proteins for potential blood-stage vaccine candidates, using data from transcriptome studies of P. falciparum, data-mining analysis of the genes with peak mRNA expression levels in late schizogony was performed (Bozdech et al., 2003; Le Roch et al., 2003) and another study on the prediction of PfSUB-1 protease specificity (Gilson et al., 2006). The results showed that P. falciparum surface-related antigen (PfSRA) emerged as the top hit with both signal peptide and a predicted glycosylphosphatidylinositol (GPI) attachment site. PfSRA is localized on the surfaces of both gametocytes and merozoites. The processed 32-kDa PfSRA protein fragment binds normal human erythrocytes. Immunoepidemiological studies in malaria-infected populations suggest the presence of naturally acquired protective antibodies against PfSRA. Parasite growth inhibition assays indicated that the antibodies against PfSRA could potently inhibit the invasion of merozoite on erythrocytes. Overall, the structural and functional characteristics of PfSRA indicate that it would be a promising vaccine target (Amlabu et al., 2018).

The low protective efficacy of vaccines against clinical malaria has been in part limited by extensive genetic diversity, which enables parasites to evade human immune responses and may lead to vaccine failure (Takala et al., 2002). However, the evidence for pfsra genetic diversity is limited. Therefore, it is necessary to study the characteristics of pfsra toward finding suitable vaccine candidates and understanding its population genetic structure. Accordingly, this study analyzed the full-length sequence of sra from the P. falciparum collected from infected migrant workers returning to the Jiangsu Province from Africa. We determined the nucleotide divergence and polymorphisms level of sra sequences to trace signatures of selection and to determine the extent of genetic variation in P. falciparum by characterizing the sra sequence at the nucleotide and protein levels.

Apart from the complex life cycle of the malaria parasite involving the mosquito vector and human host, the malaria parasite exhibits extensive antigenicity and genetically diverse stages that may pose an adverse obstacle to malarial control strategies. Thus, a deeper understanding of patterns and mechanisms of sequence variation and genetic recombination may contribute to the design of a vaccine that represents the global repertoire of polymorphic malaria surface antigens (Bharti et al., 2012). Systematic screens for uncharacterized P. falciparum invasion-related proteins evaluated PfSRA as one of the top hits that emerged; it contains coiled-coil domains known to be less polymorphic (Villard et al., 2007; Kulangara et al., 2009; Amlabu et al., 2018). Our investigation into the extent of sequence variation is consistent with this. In addition, coiled-coil domains form a stable structure, which elicit functional antibodies, thus blocking the related domains in many organisms and were considered to be the basis for the chemical synthesis of three PfSRA peptides designed to generate antibodies (Tripet et al., 2006; Gustchina et al., 2013; Jiang et al., 2016; Amlabu et al., 2018). Furthermore, these domains have been evaluated as potential targets for immunotherapy such as peptide-based vaccine strategies (Stoute et al., 1995; Demangel et al., 1998; Adda et al., 1999). We analyzed the full-length of pfsra (74 isolates) and found that the C-terminal fragments of the pfsra (π = 0.00198) show polymorphism probably due to selection pressure. Comparatively, the N-terminal of sra is a relatively conserved sequence (π = 0.00083).

A previous study suggested that people infected with malaria have naturally acquired antibodies against PfSRA and PfSRA N-terminal antibodies could partially inhibit merozoite invasion of erythrocytes by parasite growth inhibition assays (Amlabu et al., 2018). Now, the evidence of relatively conservative N-terminus might raise the possibility that it has the potential to be a candidate for anti-malarial vaccine. The ratio of non-synonymous (dN) to synonymous (dS) substitutions across sites was used as an index to evaluate selection pressure; dN/dS > 1 indicates diversifying positive selection. Further neutrality tests were carried out to determine the types and characteristics of natural selection on the pfsra. Statistically significant negative values of neutrality tests suggest an excess of rare polymorphisms in the population and provide evidence of purifying or directional (positive) selection (Fu and Li, 1993; Akey et al., 2004). The phylogenetic tree of 11 alleles of sra gene of 11 species showed that pfsra and other species occupied distinct bifurcating branches, supporting an ancient divergence times of the malarial parasite lineage.

The nucleotide diversity of pfsra in Southern Africa (π = 0.00186 ± SD 0.00086), Central Africa (π = 0.00208 ± SD 0.00073), and Western Africa (π = 0.00252 ± SD 0.00072) was lower than that in Eastern Africa (π = 0.00437 ± SD 0.00092), which may be related to the higher transmission rate of P. falciparum in Eastern Africa. Furthermore, more samples are needed in future research to support our findings and to control the limitations of small sample size (large confidence interval) in a single area. A previous study had also shown that P. falciparum has a spectrum of population structure: linkage “equilibrium,” low levels of differentiation and high diversity in regions with high levels of transmission (Anderson et al., 2000; Huang et al., 2020). Mutation, recombination, gene flow, and natural selection may contribute to the genetic diversity of malaria parasites (Cole-Tobian and King, 2003).

In the analysis of pfsra full-length, there were abundant polymorphisms found. Samples from the four Africa regions showed their own distinct diversity patterns. Interestingly, two larger-size parasite population (Western Africa and Central Africa) showed more polymorphisms compared to those in Eastern Africa and Southern Africa. Some mutations showed the regional differences based on the geographical isolation effect; for example, the 15th amino acid mutant (M15Y) only occurred in Central Africa; K333E was only found in Western Africa. These phenomena indicate that it is necessary to continuously monitor these regional characteristic mutations in order to explore their association with regional malaria epidemics. Overall, apart from the conserved N-terminus, the composition of PfSRA vaccine should consider the high-frequency alleles instead of the C-terminus of wild-type ones (Conway, 2007).

Epidemiological studies have indicated that the level of heterologous mating in malaria populations is positively correlated with the prevalence of mixed allele infections and transmission rates (Chenet et al., 2008). The generation of relevant genetic, immunologic, and epidemiologic data for the sra gene is necessary, especially in areas with low malaria endemicity. Even in geographical areas with low transmission, the development of vaccine strategies should include results of diversity analysis. The uneven geographical distribution of alleles may jeopardize the development and use of vaccines targeting specific variable site, as local variation may not be taken into account in vaccine design (Cole-Tobian and King, 2003). The study of different genes and their alleles is helpful for us to understand the trends of genetic variation and if alleles could render vaccine ineffective. Given the genetic diversity found in the region, an alternative to improve the vaccine effectiveness is to create a construct with the most common region-specific alleles (Cole-Tobian and King, 2003; Chenet et al., 2008).

The C-terminal fragments of the sra gene of P. falciparum showed polymorphism due to positive diversifying selection, which would hinder SRA-based vaccine development. Comparatively, in addition to the coiled-coil domains that have been evaluated as potential targets of peptide-based vaccines previously, the conserved N-terminal of pfsra is also a promising vaccine candidate against P. falciparum infection.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

This study was approved by the Ethics Committee of Jiangsu Institute of Parasitic Diseases (JIPD) (IRB00004221), Wuxi, China. The patients/participants provided their written informed consent to participate in this study.

BY and YC conceptualized the study, wrote the manuscript and contributed to the interpretation of the data. BY, HL, Q-WX, SX, HZ, J-XT, and G-DZ collected and analyzed the samples. Y-FS performed statistical and bioinformatics analysis. BY, YC, Y-BL, and JC revised the manuscript critically for important intellectual content. All the authors contributed to this article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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