Background
Malaria is an infectious parasitic disease caused by the genus
Plasmodium which is transmitted by bites of infected
Anopheles mosquitoes.
Plasmodium falciparum and
Plasmodium vivax are the two most common malaria parasites in humans, however differing in their clinical presentation and geographic distribution.
Plasmodium falciparum causes the most severe symptoms and higher mortality, mainly among children under 5 years of age in Africa.
Plasmodium vivax generally causes milder disease, is significantly less life-threatening [
1] and is widely distributed in the Middle East, Asia, the Western Pacific, and Central and South America [
2]. Despite global efforts to control malaria transmission resulting in a significant decrease in global incidence during the last decade, it continues to challenge public health systems, particularly in tropical countries. Current global malaria control strategies will greatly benefit from the development of an effective vaccine that interrupts malaria transmission among individuals of endemic communities [
3,
4].
Proteins expressed by parasite sexual stages, namely gametocytes/gametes, could induce effective immune responses in the human host that would prevent gamete fertilization and zygote formation when ingested by the mosquito during a blood meal [
5].
Plasmodium
Ps48/45 proteins are expressed by male and female gametocytes/gametes during the parasite maturation process, and are therefore classified as pre-fertilization antigens [
6]. These proteins belong to a family common to all
Plasmodium species characterized by the presence of partially conserved domains containing six cysteine (Cys) amino acid residues that form one to three disulfide bridges, resulting in a specific tertiary structure [
7,
8]. In
P. falciparum, this protein (Pfs48/45) is expressed on the surface membrane of gametocytes [
9] and is required for male fertility [
6]. In addition, Pfs48/45 is necessary for production of high antibody titers in individuals living in endemic areas [
10,
11] that can reduce ookinete production and induce transmission-blocking activity [
12‐
14]. It is currently considered a potential target for development as a transmission-blocking vaccine. As other proteins expressed on sexual forms, Pfs48/45 is rich in Cys residues, which has made it difficult to express as a recombinant product with proper conformation [
5,
15]. Pvs48/45, the homologous protein in
P. vivax, was recently expressed in
Escherichia coli and its immunogenicity was assessed in mice and
Aotus monkeys. These studies indicated high immunogenicity in both animal models and the elicited antibodies displayed significant and reproducible transmission-blocking activity in ex vivo
P. vivax membrane-feeding assays (MFA) [
9].
Genetic diversity could generate antigenic polymorphisms, which in turn could induce changes in critical epitopes and hamper vaccine efficacy. Successful development of an effective transmission-blocking vaccine is likely dependent on an assessment of the degree of genetic diversity in
pvs48/45 among
P. vivax parasite populations in malaria-endemic locations [
16]. Although available data indicate a limited Pvs48/45 genetic polymorphism on a regional scale [
17,
18], knowledge of the sequence polymorphism on a broader scale and its potential impact on vaccine development is needed. Here, a total of 282
pvs48/45 sequences corresponding to parasites from eight countries from around the world were analysed for gene diversity to assess probable protein changes that could influence the immunogenicity and its vaccine potential.
Discussion
This study showed a limited
pvs48/45 genetic diversity in a total of 282
P. vivax isolates worldwide. Only 18 amino acid substitutions were found within the entire protein sequence of an estimated 450 amino acid residues.
Plasmodium vivax sequences corresponded to isolates from nine distant countries in three continents. Two
pvs48/45 haplotypes were found to be shared by Asian and LA parasites, with a strong geographical allele clustering. Recent studies on the diversity of
pvs48/45 among
P. vivax isolates circulating in the Korean peninsula, China and Thailand showed low levels of genetic diversity [
17,
18,
21,
37]; likewise, Brazilian isolates showed almost no variation when compared to the Sal I reference strain (PVX_083235), originally from El Salvador.
Interestingly, Korean, Colombian and Honduran isolates displayed limited sequence polymorphism. Pvs48/45 nucleotide and amino acid substitutions in isolates from Korea were found at pre-CRD (E35K), CRD-2 (H211N and K250N) and CRD-3 (D335Y, A376T, I380T, K418R). Pvs48/45 was more conserved in the Korean populations where nucleotide diversity varied from 0.00053 [
17] to 0.00147 [
17,
18], and the haplotypes V was unique for these populations.
Plasmodium vivax was recently re-introduced into the peninsula of Korea with a rapid spread pattern, suggestive of a high genetic diversity [
38]. This could explain the greater
pvs48/45 variability observed in Korean isolates as compared to isolates from Honduras and Mexico, and the almost absent variability among Brazilian isolates Despite the variability in the Korean isolates, globally, Pvs48/45 remains a highly conserved antigen compared to other transmission-blocking vaccine candidates.
Results are in agreement with previous observations that transmission-blocking vaccine candidate antigens Pvs25, Pvs28, Pvs48/45, and PvWARP showed limited sequence polymorphisms [
18]. More importantly, the limited polymorphism found here does not appear to affect the immunogenicity of predicted epitopes as only three of nine amino acid substitutions in isolates from Colombia and Honduras were located at CRD2 (H211N and K250N) and CRD3 (E353Q). No substitutions were observed in CRD1, in agreement with reports for Korean isolates and for
P. falciparum in Kenyan, Thai, Indian, and Venezuelan isolates [
39,
40]. However, further studies are required to confirm the role of these B cell epitopes and to identify T cell epitopes in defining the potential of Pvs48/45 as a transmission-blocking vaccine candidate.
Pvs48/45 Cys domains are important for proper conformation of immune epitopes [
8,
45]. In Pfs48/45, four epitopes designated epitope V (CRD1), epitope IIb (CRD2), epitope III (CRD2), and epitope I (CRD3) have been described previously [
8]. The N-terminal CRD2 and CRD3 epitopes appear to be transmission-blocking targets as specific monoclonal antibodies to these regions have demonstrated an ability to prevent parasite fertilization, and consequently mosquito infection [
12,
46].
Substitutions H211N and K250N are predicted to be located in loop regions [
18], which could serve as potential vaccine targets. Interestingly, no amino acid substitutions were found in any of the Cys residues of Pvs48/45 in the present study, which are critical for proper presentation of the transmission-blocking epitopes [
12]. However, an amino acid substitution (E353Q) was found next to a Cys residue reported to be involved in a disulfide bond. The influence of this variation on the formation of the disulfide bridge is yet to be explored.
Although parasite antigen diversity has been explained in part by immune pressure on the parasite [
41‐
43], it does not appear to apply to Pvs48/45 as the protein is highly immunogenic, and it is expressed during the entire gametocyte maturation process. For
P. vivax sexual antigens the exposure to the immune system could be longer than in
P. falciparum due to the early appearance of
P. vivax gametocytes in circulation [
44]. However it does not appear to apply to Pvs48/45 as the protein is conserved and highly immunogenic under natural conditions.
Transmission-blocking vaccines have been considered a promising strategy/tool for malaria control/elimination. In
P. vivax malaria it has been observed that gametocytogenesis occurs earlier than in
P. falciparum and remains active even in asymptomatic carriers [
44]. Consequently, malaria transmission to mosquitoes is likely to be more efficient, and thus, transmission-blocking vaccines would have a greater impact [
47].
Authors’ contributions
SH and MA conceived and designed the study; NM, AV and SH wrote the manuscript; NM and AT performed the laboratory work. AK performed structural modelling of Pvs48/45. ML and JA contributed reagents/materials/analytical tools. All authors critically revised the manuscript. All authors read and approved the final manuscript.