Background
Although global morbidity and mortality have decreased substantially, malaria is still a great public health concern, especially in Africa [
1]. The World Malaria Report 2018 draws on data from 87 countries and areas with ongoing malaria transmission. Recent research shows that after an unprecedented period of success in global malaria control, progress has stalled. An estimated 219 million (95% confidence interval [CI]: 203–262 million) persons suffer from malaria infections worldwide, with 435,000 malaria deaths in 2017. Fifteen countries accounted for 80% of global malaria deaths in 2017, and the 10 highest burdened African countries saw an estimated 3.5 million more malaria cases in 2017 compared with the previous year [
2]. Malaria is endemic in Equatorial Guinea, a country in Central West Africa with a population of around 1 million inhabitants. In 2004, the government and private parties formed a Public–Private Partnership that has spent the last 15 years controlling malaria on Bioko Island and parts of the mainland in an effort to reduce malaria’s burden on the population. The Bioko Island Malaria Control Project (BIMCP) implemented by the U.S. NGO, Medical Care Development International (MCDI) and the Ministry of Health and Social Welfare of the Government of Equatorial Guinea, has reduced malaria prevalence from 74% (by thick blood smear) in 2003 to 11% (by rapid diagnostic test) in 2017 in children 2 to 14 years of age. Infant mortality due to malaria infection has reduced by 85% (by rapid diagnostic test). However, it has proven difficult to eliminate malaria from this region despite the increasing intensity of malaria intervention. With the emergence and geographical expansion of anti-malarial resistance worldwide, molecular markers are essential tools for the surveillance of resistant
Plasmodium parasites [
3]. The decrease of island-wide
P. falciparum prevalence was steep in the first year following the implementation of malaria intervention. However, it is still a major obstacle to public health and economic growth for countries in the tropics and subtropical regions [
4].
Vaccines are the most cost effective and efficient method of protecting against malaria. However, malaria, one of the oldest and deadliest pathogens in human history, remains without a marketed vaccine. The Equato-Guinean Malaria Vaccine Initiative (EGMVI) is engaging in conducting a series of clinical trials that will advance a
PfSPZ vaccine through to phase III clinical trials and eventually test the public health utility of the vaccine in malaria elimination projects. Therefore, the development of an effective vaccine against
P. falciparum is a necessary priority, particularly due to the increased resistance of this parasite to anti-malarial drugs [
3,
5]. Up to date, several candidate proteins, including circumsporozoite protein (CSP), Duffy-binding protein (DBP), merozoite surface protein-1 (MSP-1), apical membrane antigen-1 (AMA-1), and thrombospondin-related anonymous protein (TRAP) have been tested for their potential as candidate antigens for the development of effective vaccines [
6]. For MSP-1, previous report showed high genetic diversity and MOI values among the
P. falciparum population [
7]. A high prevalence of
Pfdhfr-
Pfdhps quadruple mutations were detected, which is associated with sulfadoxine resistance in
P. falciparum isolates on Bioko Island. This result reflects both the high endemic level of malaria and its transmission on Bioko Island [
8].
AMA-1 is an 83-kDa type I integral membrane protein localized to the apical complex [
9]. It is mainly expressed in the merozoite and sporozoite stages of malaria parasites and appears to be transported to the merozoite surface in late schizonts and free merozoites [
9‐
14]. AMA-1 consists of a signal sequence, a cysteine-rich ectodomain, a conserved cytoplasmic region and a transmembrane region [
15]. The ectodomain of AMA-1 is subdivided into three immunogenic domains, and natural immune responses against this domain have been reported in patients infected with
P. falciparum [
16,
17]. The biological function of AMA-1 is not understood thoroughly, though its stage-specific expression and localization suggest that this protein might play a crucial role in invasion [
9,
12,
18,
19]. There is also several evidence that suggest that AMA-1 forms a complex with RON2 to form the moving junction during invasion of merozoites into erythrocytes [
17‐
23]. According to a previous report, AMA-1 can induce a protective immune response that produces antibodies that can effectively inhibit
P. falciparum from invading red blood cells [
24]. However, polymorphism of protozoan antigen and immune evasion of
plasmodium have impeded the development of a malaria vaccine.
PfAMA1 is the main candidate antigen of a red-blood stage vaccine and has entered a phase II clinical trial [
25]. Due to its essential role for parasite survival and high level of immunogenicity during natural infection in humans, vaccination studies conducted in BALB/C mouse models have confirmed that AMA-1 is a potential vaccine antigen against
P. falciparum [
26].
Independent studies provide strong evidence that balancing selection acts to maintain these polymorphisms in the population, reflecting the importance of AMA-1 as a target of protective immunity [
27‐
30]. However, these antibodies can recognize either conserved or allele-specific epitopes of AMA-1, resulting in limited protection for different alleles [
31]. Several polymorphisms can be observed in DI of AMA-1, suggesting that this region seems to be the main target of anti-AMA-1 protective antibodies [
30,
32‐
35]. Nevertheless, genetic diversity of AMA-1 among
Plasmodium field isolates and the presence of variant forms in different geographic areas present hurdles in successful malaria vaccine design. Therefore, in order to design an efficient and protective malaria vaccine, it is essential to monitor genetic variations of candidate vaccine antigens in global malaria isolates circulating in endemic areas [
36].
Although the incidence of malaria has decreased significantly through BIMCP,
P. falciparum is still the most critical concern for malaria control and prevention on Bioko Island with the emergence of artemisinin resistance [
3,
5]. In this study, natural selection and genetic diversity of AMA-1 DI region in Bioko
P. falciparum isolates were analysed.
Discussion
The World Malaria Report 2018 draws on data from 87 countries and areas with ongoing malaria transmission. These reports show that after an unprecedented period of success in global malaria control, progress has stalled [
2]. This study provides useful information for the prevention and control of
P. falciparum on Bioko Island by analyzing the DI region of AMA-1 gene for a
P. falciparum vaccine, as well as characterize the genetic polymorphism and molecular evolution. Although the frequencies of these new mutation sites are not very high, four of them are distributed in B cell-3 (I158T), RBC-2 (G180C, A182V) and B cell-4 (D266N), respectively. The predicted results showed that all 8 mutations are likely to affect the structure and function of
PfAMA-1 (probably damaging). Most of these proteins are distributed in the corner area. Whether these mutations cause changes in protein structure and function and affect the binding with human host protein remains to be verified (Table
5, Fig.
2). Moreover, it was found that mutations in DI domain, especially in the C1, C1L, C2 and C3 regions, are highly correlated with the pathogenicity of the host after parasite antigen escape and infection [
49]. Nucleotide sequence analysis of these 214
PfAMA-1 sequences from Bioko populations compared to
PfAMA-1 from
P. falciparum clone 3D7 (GenBank Accession Number U65407) revealed 131 different haplotypes. Most amino acid changes identified from Bioko
PfAMA-1 isolates were clustered in C1 region, especially the C1L region, which is consistent with previous reports on the DI region mutations of
PfAMA-1 from other endemic areas [
45,
50]. Overall distribution patterns and frequencies of amino acid changes found on Bioko Island were similar to those of other
PfAMA-1 changes found globally, but several differences between Bioko Island and other global
PfAMA-1 isolates have also been identified in this study. The implication of these geographic differences is not entirely clear. Considering that only the DI region and a limited number of
PfAMA-1 isolates in each geographical area were analysed in this study, these amino acid changes and their different frequencies may not be statistically significant. In fact, many of these amino acid changes in other
PfAMA-1 genes were generally found and distributed globally in other
PfAMA-1 sequences, although their frequencies varied between and among populations. Therefore, to better understand the polymorphism of nucleotide and amino acid of
PfAMA-1 on Bioko Island, more
PfAMA-1 sequences are required.
Table 5
Function prediction scores of eight novel sites
P150T | 2.29 | 1.000 | 0.00 | 1.00 | Probably damaging |
V151I | 0.76 | 0.714 | 0.86 | 0.92 | Probably damaging |
I158S | 1.53 | 0.998 | 0.27 | 0.99 | Probably damaging |
G180C | 2.29 | 1.000 | 0.00 | 1.00 | Probably damaging |
A182V | 0.76 | 1.000 | 0.00 | 1.00 | Probably damaging |
D266N | 3.82 | 0.991 | 0.71 | 0.97 | Probably damaging |
S272N | 0.76 | 0.846 | 0.83 | 0.93 | Probably damaging |
D281H | 1.53 | 1.000 | 0.00 | 1.00 | Probably damaging |
The π value for global
PfAMA-1 varies, ranging from 0.01216 (Venezuela) to 0.02789 (Kenya). The π values of African
PfAMA-1 sequences (Bioko Island, Ghana, Tanzania, Nigeria, Gambia, Kenya and Benin) are higher than those in South American and Asian
PfAMA-1 sequences. The π values are higher on Bioko Island (0.02776) than in other African countries except Kenya (0.02789). Indeed, nucleotide diversity was not distributed throughout Bioko Island
PfAMA-1. The C2 (844–858 bp) and C3 (514–525 bp) regions show low levels of nucleotide diversity, indicating that these regions might be more conserved, with a lower frequency of polymorphisms. Moreover, much higher values of nucleotide diversity are observed in the C1L (586–621 bp) and another C2 region (724–735 bp) regions, indicating that a higher frequency of polymorphisms occurs at these regions. Indeed, DI, especially the C1 and C1L regions, are targets of the host’s immune system. The high number of DI polymorphisms in the Bioko Island population suggests that this region is under the selection of host immune pressure during evolution. Previous studies on disease-blocking vaccines using
PfAMA-1 monoclonal antibody showed that amino acid mutations in the C1 region (197/200/201/204/225) of
PfAMA-1 could block the binding of monoclonal antibody to
PfAMA-1, thereby inhibiting the effectiveness of the vaccine. In this study, the C1 region showed a high number of polymorphisms at the gene and amino acid levels, suggesting that the above region may affect the effectiveness of vaccines. Although the values are different among and between other global
PfAMA-1 isolates, highly similar distribution patterns of nucleotide diversity have been detected in global
PfAMA-1 genes analysed in this study, strongly indicating that these
PfAMA-1 genes might share highly similar nucleotide diversity. The dN − dS value for Bioko Island
PfAMA-1 DIs is positive, suggesting involvement of balancing selection. Also, the dN − dS value for all regions of DI is positive, indicating positive natural selection throughout DIs. The positive Tajima’s D values for Bioko Island
PfAMA-1 also suggested that this gene might have evolved under balancing selection. In the sliding plot analysis of Tajima’s D, general patterns of Tajima’s D values across Bioko Island
PfAMA-1 were similar to those of other
PfAMA-1 genes, although differences among and between
PfAMA-1s from different countries were also found. Regardless of the slight difference in values, C1, C2 and C3 regions all shared pretty similar distribution patterns of Tajima’s D values, strongly indicating that these regions might be major targets for the host immune response. Fu and Li’s D and F tests also provide enough evidence for balancing selection of
PfAMA-1 on Bioko Island. These values suggest that
PfAMA-1 DIs from Bioko Island populations are highly polymorphic with a strong selection force acting on it, similar to other global
PfAMA-1 genes. In addition to natural selection, recombination also contributes to the diversity of
PfAMA-1. Meiotic recombination that occurs between the adjacent polymorphic sites is responsible for the high allelic diversity in DI [
45]. These results also indicate that a high level of recombination events have occurred in
PfAMA-1 isolates on Bioko Island. High levels of recombination in
PfAMA-1 from different geographical isolates have been previously reported [
28,
35,
45,
46,
50‐
54]. High recombination events were found in Bioko Island
PfAMA-1 sequences compare to samples from other geographical areas. This might be due to special island location of Bioko region, which attribute an opportunity to
PfAMA-1 to undergo more recombination. Moreover, the incidence of recombination events in African countries is also generally higher than other areas, confirming the previous conclusions of recombination events. This is supported by the decline of the LD index R
2 with increasing nucleotide distance in
PfAMA-1 isolates on Bioko Island, consistent with previous reports [
28,
45,
46,
50‐
54]. In conclusion, recombination is the most important factor generating genetic diversity in global
PfAMA-1 populations.
The Fst value is one of the most useful methods for analyzing overall genetic differentiation (range from 0 to + 1). Fst values at each locus are considered as having no differentiation (0), low genetic differentiation (0–0.05), moderate differentiation (0.05–0.15) or high differentiation (0.15–0.25) [
54]. The Fst values for
PfAMA-1 DIs on Bioko Island show a lower level of genetic differentiation than that of
PfAMA-1 in other geographical populations except Benin, which occurs at a moderate level, as well as the values of Fst for most Asian and African countries. Also, a high level of genetic differentiation is found between Bioko Island and Venezuela. All of these indicate that Fst values between
PfAMA-1 populations belonging to the same geographical area are relatively low. The negative values in the table were considered because DI of
PfAMA-1 is too limited. However, Bioko Island shows a high Fst value for Venezuela (0.22087), providing strong evidence for geographical isolation and population segmentation in these regions. Total Fst values of DI between and among global
PfAMA-1 isolates are in the low to medium differentiation range, indicating that
PfAMA-1 has limited genetic differentiation among other parasite populations around the world.
It is important to understand the haplotype network of
PfAMA-1 on Bioko Island with other global areas in order to develop a globally effective malaria vaccine based on this gene. A total of 296 different haplotypes were identified by network analysis of 790
PfAMA-1 sequences. Haplotype network analysis shows that haplotypes on Bioko Island are scattered among other haplotypes from different countries, which is consistent with a previous report [
53]. Compared with the African continent, Bioko Island is an independent Island with a special geographical location. It is an offshore Island, with special species diversity and evolution, but very close to seriously affected areas of malaria in West Africa. As a result, the distribution of
P. falciparum on Bioko Island may to have no differences with the continent of Africa, but the distribution of the different geographic strains on the island is unclear. Therefore, the genetic diversity of
PfAMA-1 DI regions on Bioko Island were analysed to carry out a detailed investigation on the distribution of different geographical strains to help understand the situation in this region and provide a reference for drug resistance investigations and medication guidance. Many single haplotypes appear in both clusters, but no pie completely covering all haplotypes was found in all geographic areas in this study. A recent report suggested that mutations in the
PfAMA-1 sequence are not necessarily strong predictors of antigenic differences or cross-inhibitory antibody activity levels, since not all polymorphic residues contribute equally to antibody generation and escape [
47]. Due to the limited diversity of
PfAMA-1 antigens, vaccines targeting a small number of
PfAMA-1 alleles might be sufficient to cover naturally circulating populations of
P. falciparum in different endemic areas [
33]. These results also indicate that the global genetic diversity of
PfAMA-1 is relatively limited, even though substantial geographic differentiation can also be identified among populations. Nevertheless, global
PfAMA-1 is undergoing natural selection and high levels of meiotic recombination, which can produce new alleles in a gene population. Therefore, consideration should be given to the development of
PfAMA-1-based malaria vaccines using a polyallelic approach to maximize vaccine effectiveness.
To assess the association between host immune pressure and natural selection of
PfAMA-1, genetic polymorphisms in predicted B-cell epitopes and IUR regions were analysed. Based on a recent report, detailed information on potential RBC-binding regions, B-cell epitopes and IURs across the ectodomain of
PfAMA-1 were obtained [
45]. Most amino acid changes found in DI of
PfAMA-1 on Bioko Island are predicted to be localized at predicted B-cell epitopes or IUR regions in DI. In this study, B-cell epitopes 3 and 4 show high levels of nucleotide diversity in
PfAMA-1 on Bioko Island. Tajima’s D values for these predicted B-cell epitopes also suggest that these epitopes are under natural selection. The π values of RBC-2 regions in different geographical areas are close to each other, which suggests that the nucleotide diversity of DIs is similar in these regions. In Bioko Island
PfAMA-1 isolates analysed in this study, the DI region shows clustering of amino acid polymorphisms in the C1L region. The C1L region is located near the hydrophobic pocket of DIs, which affects the binding of inhibitory monoclonal antibodies and thus leads to escape from antibody targeting [
55,
56]. This shows that the important role of natural selection in generating
PfAMA-1 gene diversity is very obvious just as the effect of natural selection of
P. falciparum on Bioko Island, and also supports the idea that natural selection can promote host immune escape in this region [
57,
58]. Therefore, it may be necessary to consider the polymorphism in DI in order to obtain more efficient vaccine components.