A blood stage malaria vaccine ideally aims to prevent or considerably reduce blood stage parasitaemia either by inhibiting merozoite invasion into erythrocytes or by targeted destruction of parasitized erythrocytes [
19,
34]. Following this approach, several merozoite surface proteins (MSPs) have been considered promising candidate antigens for malaria vaccine development due to their accessibility by antibodies and their essential roles in erythrocyte invasion [
19]. However, the genetic diversity of the MSPs identified within and among global isolates has resulted in a major obstacle hampering the development of an effective malaria vaccine. Of the MSPs, MSP-1
42 is the most outstanding vaccine candidate antigen, which is currently at an advanced stage of clinical evaluation [
34‐
37]. But, its polymorphic nature suggests that routine changes to the vaccine and continuous surveillance of the antigen diversity in field isolates would be required.
In this study, the genetic diversity and natural selection on PvMSP-1
42 in the 149
P. vivax Korean isolates were analysed. The 149 sequences were classified into 11 distinct haplotypes with amino acid changes at 40 positions as compared to the Sal I sequence. Most of the amino acid substitutions were concentrated in the PvMSP-1
33 fragment and only a dimorphic change (N1692K) was found in PvMSP-1
19. It is known that PvMSP-1
19 is highly conserved, as observed in field isolates obtained from different geographic regions, and only one amino acid change (K1709E) has been reported thus far [
38‐
41]. The amino acid change was not observed in any of the Korean isolates, but the emergence of a new amino acid change in PvMSP-1
19 in Korean isolates suggest that PvMSP-1
19 could contribute to the diversity of PvMSP-1
42. Of the 39 amino acid changes found in PvMSP-1
33 of Korean isolates, six (N1343Y, N1427Y, L1447W, K1486R, E1603V, and L1613V) were unique and had not been reported previously. These unique changes resulted in the generation of six novel haplotypes that had not been reported so far. The sequence and phylogenetic analyses revealed that none of the Korean haplotypes were identical to either the Sal I or Belem sequences, but haplotypes 1–5 were essentially Belem types, and the others were recombinant types between Belem and Sal I, in which at least one recombination may occur at the hypervariable region of PvMSP-1
33. Recently, PvMSP-1
42 was differentiated into 12 distinctive groups (group 1–12) based on sequence differences observed in hypervariable region, but there was no evidence of geographic clustering of global isolates [
42]. Phylogenetic analysis of Korean PvMSP-1
42 haplotypes suggested they were clustered into five distinct clades with the majority belonging to the Belem type, but no clear geographic relationship was also identified. Interestingly, the isolates collected in 1999–2000 showed only limited haplotypes which were closely related to the Belem type. However, a recombinant haloptype was first observed among isolates collected in 2001. Both Belem and recombinant types of the PvMSP-1
42 haplotypes were identified thereafter, with a prevalence of the recombinant types. These results coincided with several previous studies based on the genetic diversity of several major antigens including circumsporozoite protein, MSP-1, and MSP-3α, as well as microsatellite loci, suggesting that the Korean
P. vivax isolates had been genetically homologous until 2000, but the genetic diversity was rapidly disseminated thereafter [
26,
43]. It is currently unclear why Korean
P. vivax isolates showed such diverse genetic profiles and this issue should be elucidated. PvMSP-1
19 was found to be more highly conserved than PvMSP-1
33 in the Korean isolates, but the nucleotide diversity (π) of PvMSP-1
19 was considerably higher than those found in previous studies [
28,
42,
44]. This was due to a non-synonymous substitution (N1692K) in PvMSP-1
19 of the Korean isolates. Meanwhile, the π values for PvMSP-1
42 and PvMSP-1
33 found in the Korean isolates were lower than those observed in other global isolates [
28,
42,
44]. This suggests that PvMSP-1
42 of Korean isolates showed limited genetic diversity as compared to isolates from other endemic regions including India and Sri Lanka.
The rate of non-synonymous and synonymous mutations (dN-dS) is widely used as an indicator of the action of natural selection in gene sequences. An excess of dN relative to dS is a clear signal of positive selection, whereas a lack of dN relative to dS suggests a negative or purifying selection imposed by functional constraints [
31,
45]. The positive value of dN-dS (0.0067) observed in the 149 Korean PvMSP-1
42 sequences suggested that PvMSP-1
42 in the Korean
P. vivax isolates is under the influence of positive natural selection. The observation that PvMSP-1
33 had a higher dN-dS than PvMSP-1
42 also suggested that PvMSP-1
33 is under stronger positive natural selection pressure than the entire PvMSP-1
42, and this finding was comparable to observations found in
P. vivax isolates from different areas [
28,
42]. The positive values of Tajima’s D (3.0268, P < 0.01) and Fu and Li’s D (2.0839, P < 0.02) and F (2.9904 < 0.02) statistics indicated that the PvMSP-1
42 alleles occurred at more intermediate frequencies than expected and that few alleles were rare or near fixation, which is consistent with the action of the balancing selection that maintains allelic variation in a population. These results collectively suggested that strong balancing selection, presumably by host immune pressure [
28,
45,
46], occurred at PvMSP-1
42 in the Korean isolates, and the host immune responses likely played a role in generation and maintenance of the MSP-1
42 polymorphism.
The diversity of plasmodial antigens is also likely to be generated by genetic recombination during the sexual stage of the parasites in the mosquito [
45,
46]. The results obtained in this study indicated that recombination events occurred within the PvMSP-1
42 sequences in Korean isolates. This was supported by the observation of decline of LD index R
2 with increasing nucleotide distance coupled with a high level of haplotype diversity (Hd = 0.876 ± 0.009). Indeed, all recombinant types of the Korean PvMSP-1
42 haplotypes had putative recombination sites that concentrated in PvMSP-1
33 rather than being evenly distributed across the entire PvMSP-1
42, which consistent with previous reports [
28,
41,
42]. Considering the first appearance of the recombinant haplotype PvMSP-1
42 in 2001 and the subsequent prevalence of recombinant types from 2003 to recent years, new PvMSP-1
42 haplotypes are actively being generated in Korean isolates by recombination events in recent years even though the country with low malaria transmission rate.