Diversity analysis of MSP1 identifies conserved epitope organization in block 2 amidst high sequence variability in Indian Plasmodium falciparum isolates

Abstract

Background: Despite its immunogenicity, the polymorphic nature of merozoite surface protein 1, an important vaccine candidate for Plasmodium falciparum malaria, remains a concern. This study analyses the impact of genetic variability and parasite population structure on epitope organization of different MSP1 segments.

Methods: Altogether 98 blood samples collected from P. falciparum infected mild and severe malaria patients of Chhattisgarh and West Bengal were used to sequence regions encoding block 2 and MSP1-19 of msp1. Sequences were analysed using MEGA7, DnaSPv5, Arlequin3.5 and BepiPred.

Results: All three major MSP1 block 2 allele families namely K1, MAD20 and RO33 were detected in the samples and they together resulted in 41 indel variants. Chhattisgarh samples displayed an average MOI of 2.07 ± 1.59 which was higher in mild malaria and in age group < 18 years. Ultra-structure of block 2 alleles revealed that mutation and repeat expansion were two major mechanisms responsible for allelic variability of K1 and MAD20. Regions flanking block 2 were highly variable in Chhattisgarh with average mismatch differences (k) ranging from 1.198 to 5.156 for three families. In contrast, region encompassing MSP1-19 exhibited limited heterogeneity (k_Chhattisgarh = 1.45, k_{West Bengal} = 1.363). Of the 50 possible B cell linear epitopes predicted from block 2 variants, 94.9% (131 of 138) of the parasites could be represented by three conserved antigens.

Conclusions: Present data indicates that natural selection and transmission intensity jointly play a role in controlling allelic diversity of MSP1 in Indian parasite isolates. Despite remarkable genetic variability, a limited number of predominant and conserved epitopes are present in Indian parasite isolates reinstating the importance of MSP1 as a promising malaria vaccine candidate.

Keywords: Epitope organization; Genetic diversity; India; MSP1; Multiclonal infection; Plasmodium falciparum.

Fig. 1

Schematic representation of P. falciparum merozoite surface protein 1 (MSP1). The regions subjected to sequence analysis were highlighted using broken lines

Fig. 2

Analysis of frequencies of msp1 block 2 alleles and multiplicity of infections in different groups. a Frequencies of msp1 alleles in Chhattisgarh and West Bengal. b Distribution of msp1 alleles in Chhattisgarh patients with mild and severe malaria. c Comparison of MOI in the mild and severe malaria patients of Chhattisgarh. d Differences of MOI in two different age groups of Chhattisgarh patients. e Frequencies of K1, MAD20, RO33 alleles associated with single and multiple infections in Chhattisgarh. Asterisk indicates p < 0.05 in Chi square test

Fig. 3

Phylogenetic relationship and prevalence of different msp1 sub-alleles. a Organization of tri-peptide motifs in the alleles belonging to K1 family in Chhattisgarh parasite population and their respective proportions. b Organization and prevalence of tri-peptide motifs in the alleles belonging to MAD20 family in Chhattisgarh sample. c Organization and prevalence of tri-peptide motifs in the alleles belonging to MAD20 in West Bengal samples. Bootstrap values were shown for each branch of the Maximum Parsimony tree. SGT, SGP, SAQ, SGA, STQ and SAR repeats were present in K1 and denoted as 1, 2, 3, 4, 3* and 3^#, respectively and SGG, SVA, SVT, SKG, SGD, PGG, PVA motifs were present in MAD20 and denoted as 5, 6, 7, 8, 5*, 5^# and 6*, respectively. Each letter in the tri-peptide motifs represents an amino acid

Fig. 4

Possible mechanisms leading to allelic variability of msp1 block 2. Repeat expansion and insertion/deletion are presumably responsible for generating K1H15 and MH17 from K1H14 to MH16, respectively

Fig. 5

Worldwide distribution of P. falciparum msp1 block 2 alleles. Frequencies of K1, MAD20 and RO33 in different geographical regions. Proportion of each allele in a certain parasite population was shown using pi diagram

Fig. 6

Comparison of average epitope scores of MSP1 block 2 and MSP1-19 peptides in India. Epitopes were predicted based on a threshold score of 1.3. Asterisk indicates p < 0.05 in two-tailed Student’s t test