Background
Malaria, caused by
Plasmodium spp. infections, is one of the most significant life-threatening infectious diseases to humans worldwide. According to the World Malaria Report 2019 [
1], an estimated 228 million (95% confidence interval [CI] 206–258 million) persons suffered from malaria infections worldwide, with 405,000 malaria deaths in 2018. Twenty countries accounted for 85% of global malaria cases in 2018; all these countries are in sub-Saharan Africa, except for India. Resistance to anti-malarial drugs and insecticides, coupled with the lack of availability of an effective vaccine, is the leading factors behind the parasite’s continuing burden. Apart from its complex life cycle, which alternates between the human and the mosquito host, the malaria parasite also exhibits stages characterized by extensive genetic and antigenic diversity which may present adverse obstacles to anti-malarial control measures.
Currently, there are many efforts and studies have been performed in order to develop effective vaccines, several potential vaccine candidates targeted against pre-erythrocytic, erythrocytic and sexual stages of
Plasmodium falciparum are under various stages of clinical development [
2,
3]. RTS, S/AS01 vaccine is a pre-erythrocytic stage vaccine based on the
P. falciparum circumsporozoite protein (PfCSP) [
4,
5]. In 2015, the European Medicines Agency for the immunization of children against malaria approved the RTS, S/AS01 vaccine [
6] and the phase 3 clinical trials conducted in various sites in Africa showed that the RTS, S/AS01 vaccine has a protective efficacy of 45% in children in the first twenty months after vaccination [
7,
8]. In 2018, the World Health Organization through a large-scale pilot malaria vaccine implementation program (MVIP) aimed to introduce this vaccine in three sub-Saharan countries (Ghana, Kenya, Malawi) [
6]. Besides of RTS, S/AS01, a live attenuated
Plasmodium falciparum whole sporozoite (SPZ) vaccine is also regarded as a great potential malarial vaccine. Sanaria
® PfSPZ Vaccine had conducted a clinical trial on Bioko Island where 70% vaccinees developed antibodies to
P. falciparum circumsporozoite protein, which was the first clinical trial conducted in Equatorial Guinea [
9]. It is not hard to see that
pfcsp is a very important gene for the host immune response to the
P. falciparum invasion.
PfCSP is predominantly distributed on the surface of the sporozoites with a molecular mass of about 58 kDa. PfCSP is GPI-anchored on the sporozoite surface and plays a critical role in sporozoite development, motility and hepatocyte invasion [
10,
11]. The structure of PfCSP can be divided into three distinct regions: a highly variable central repeat region flanked by a conserved N-terminal region and a C-terminal non-repeat region [
12]. The central repeat region, which has been recognized as a major target for antibody-mediated neutralization, is rich in Asn-Ala-Asn-Pro (NANP) tandem repeats and contains a small number of Asn-Val-Asp-Pro (NVDP) motifs [
12], constitutes immunodominant B cell epitopes. The C-terminal non-repeat region includes two polymorphic sub-regions, Th2R and Th3R, where T cell epitopes were identified.
The previous studies revealed higher single nucleotide polymorphisms (SNPs) of
pfcsp within the
P. falciparum population from different geographic regions [
13]. Indeed, most
P. falciparum vaccine candidate gene including
pfcsp have been found to show various genetic and antigenic polymorphisms in global parasites, which might obstruct or reduce the efficacy of vaccines [
14,
15].
Understanding the genetic nature of vaccine candidate antigens is critical for designing an effective vaccine. The aims of the present study are to investigate the polymorphism pattern of pfcsp gene and its diversifying selection of P. falciparum on Bioko Island, and to elucidate how pfcsp gene is differentiated among global P. falciparum populations. This study will fill in the blank of Bioko Island pfcsp data, as well as be helpful not only for understanding the molecular evolution of the pfcsp gene in P. falciparum, but also for designing peptide-based vaccines for the PfCSP antigen.
Discussion
Bioko Island, Equatorial Guinea, is a historically high malaria transmission region [
16,
40]. Though BIMCP had launched in Bioko Island since 2004 and achieved a remarkable result, malaria is still a major health problem in this region. The genetic diversity and natural selection were analysed in Bioko
pfcsp and global
pfcsp. In general, the polymorphism patterns between Bioko
pfcsp and African mainland
pfcsp have no obvious differentiation, although the geographic location of Bioko Island was relatively isolated. This result might be explained by the work of Guerra et al., which reported that the strong connection of human movement between Bioko and the mainland Equatorial Guinea (EG), determine a high vulnerability of Bioko to malaria importation; these studies reported that the odds of malaria infection in travellers who had been to mainland EG were more than three times the rest of the population, which confirmed that the majority malaria cases are actively imported by off-island travellers to mainland EG [
41,
42]. Furthermore, it is worth mention that the PfSPZ vaccine had been tested in Malabo and a series of clinical trials are undergoing, which might likely to affect the genetic background of the malaria parasites in this region [
9]. According to the report [
9], PfSPZ vaccine could induced the immune response to PfCSP, which might influence the genetic diversity and natural selection of
pfcsp in Malabo. The natural selection analysis revealed that Bioko
pfcsp might under a selection effect although there is no statistical significance (
p > 0.1). These findings were in line with the prior studies about
P. falciparum merozoite surface protein-1/2 (
PfMSP-1/2) and
P. falciparum apical membrane antigen-1 (
PfAMA-1) genes in Bioko Island [
43,
44].
N-terminal region of PfCSP plays an important role in the procedure of sporozoite invades to the hepatocytes [
45]. In Bioko and global
pfcsp, the genetic polymorphism of N-terminus was in a relatively low level. 19 amino acids length insertion and A98G were universally popular while several novel mutations were found with low frequency. Some scientists verified previously that the antibodies against to N-terminal region could be produced by host immune system and could evoke a partial inhibition of sporozoite invasion of hepatocytes in vitro [
46]. Now the evidences of relatively conservative N-terminus might raise the possibility that whether the N-terminus has the potential to be a component of anti-malarial vaccine.
Central repeat region is an immunodominant epitope of PfCSP, and it had been applied to the component of RTS,S malaria vaccine [
47]. Different numbers of tetrapeptide repeat was an important cause of
pfcsp polymorphism. As expected, this study revealed the diversity of the number of tetrapeptide repeat (NANP/NVNP). Through the analysis among global different geographic regions, it was found that majority of samples possessed the tetrapeptide repeat ranging from 39 to 44 times. Though some scientists hold the view that the various number of tetrapeptide repeat make no significant impacts on RTS,S vaccine efficacy [
14], it was known to correlated with the stability of CS protein structure [
48]. However, the mechanism and effect of this variation is still unclear. For the universality of this variation, deeper research towards to this region is still necessary.
In the analysis of C-terminus of pfcsp, there were abundant polymorphisms found, especially in the TSR region (including Th2R and Th3R), the proven T cell immunogenic epitopes. The C-terminus of African, Asian, American and Oceanian samples presented their own distinctive diversity patterns. Not surprisingly, more polymorphisms were performed in the two larger-size parasite population (African and Asian) compared to those of America and Oceania. Because of the geographical isolation effect, some mutations showed the regional difference, for example the mutant at 325 position (N325Y) was only occurred in Asian countries; S326A was only found in Venezuela; wild type A361 was mainly observed in Africa, and so on. These phenomena indicated us that continuous monitor to these regional characteristic mutations, and exploration on their association with regional malaria epidemic situation are necessary.
In terms of C-terminal haplotypes analysis, 29 of 34 Bioko pfcsp haplotypes were shared with African continent samples while only 5 were limited to singleton, which implied that Bioko pfcsp was not completely independent of African continent. An obvious phenomenon was found that haplotypes from Oceanian pfcsp have closer genetic relationship with Asian haplotypes. Additionally, the same phenomenon happened among the parasites from America and Africa. It reflects that worldwide genotype of pfcsp C-terminus might divide into two major groups (Africa & America and Asia & Oceania), which probably caused by the frequent communication due to geographical advantages. It provides an insight of the vaccine design based on PfCSP that the regional differentiation might be took into consideration.
The absence of 3D7-matched
pfcsp was not the uncommon finding anymore [
13,
49]. Unsurprisingly, in Bioko Island, only 2% 3D7-matched
pfcsp were found. A study about genetic diversity and protective efficacy of the RTS,S/AS01 malaria vaccine stated that the 3D7-mismatched malaria might probably weaken the efficacy of vaccine, especially the mutations at 299, 301, 317, 354, 356, 359 and 361 amino acid position [
14]. In this research, the polymorphism situation of these loci showed different degrees. It is worth mentioning that mutation rate of position 317 reached 91% and mutation rate of position 361 reached 73%. As these mutations are so common and probably affect the vaccine effect, a question raised that whether these high-frequency alleles instead of the wild-type ones could be applied in the vaccine component.
In terms of the distribution of mutations, all the 66 mutations found from global sequences were located at CD8+ T cell epitopes, while 28 of them were located at the overlap of CD8 + and CD4+ T cell epitopes. It is well known that CD8+ and CD4+ T cell are thought to play a role in natural and sporozoite vaccine induced immunity in
P. falciparum malaria [
50,
51]. This raises the question of whether these mutations affect host immunity. Mutation-effect prediction of these 28 mutations showed that more than half of them were predicted as damaging (15 of 28). Notably, when mutations located at some specific positions (including the probably harmful position 317 and 354) [
14], great changes have taken place on the free energy difference, which would result in destabilization on CS protein structure in difference extent. However, the specific mechanism of whether and how these mutations do harm to vaccine efficacy are still not clear. Therefore, continuous monitoring on these mutations and deeper exploration on the mechanism is still necessary.
According to this study, there are several new insights might be considered in the design and improvement of PfCSP-based vaccine: (1) The globally high frequency alleles instead of the wild-type ones of C-terminus might be used for composing vaccine. (2) The immunogenic and conservative N-terminus might be applied in the composition of vaccine. (3) The regional differences should be considered in the improvement of universal malaria vaccine, mainly divided as Asia-Oceania region and Africa-America region.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.