Stable Plasmodium falciparum merozoite surface protein-1 allelic diversity despite decreasing parasitaemia in children with multiple malaria infections

Abstract

Background: Individuals experiencing recurrent malaria infections encounter a variety of alleles with each new infection. This ongoing allelic diversity influences the development of naturally acquired immunity and it can inform vaccine efficacy. To investigate the diversity and infection variability, Plasmodium falciparum merozoite surface protein 1 (PfMSP1), a crucial protein for parasite invasion and immune response, was assessed in parasites isolated from children in the Junju cohort, Kilifi County, who experienced at least 10 febrile malaria episodes over a span of 5 years.

Methods: Pfmsp1 C-terminal region (Pfmsp1₁₉) was genotyped using PCR followed by capillary sequencing in blood samples collected from the children. Sequenced reads were trimmed and aligned to the P. falciparum 3D7 reference genome. Single nucleotide polymorphisms in the Pfmsp1₁₉ region were identified from the alignment and grouped to distinct microhaplotypes whose changing frequency over time were examined across the multiple infection episodes. In addition, the variability of infections in the population was assessed using nucleotide and haplotype diversity indices.

Results: A total of eleven microhaplotypes were observed across all malaria episodes. There were 3 prevalent microhaplotypes, E-KSNG-L, Q-KSNG-L, and Q-KSNG-F in the population. Conversely, microhaplotypes such as Q-KNNG-L, E-KSSR-L, E-KNNG-L, E-KSSG-L, E-TSSR-L (3D7), Q-TSSR-L, E-TSSG-L, and E-KSNG-F were rare and maintained at low frequencies. High allelic replacements were observed, however some individuals experienced consecutive re-infections with the same microhaplotype. Notably, PfMSP1₁₉ allelic diversity as measured by haplotype diversity was stable, while nucleotide diversity decreased over time with decreasing parasitemia. Parasite PfMSP1₁₉ allelic diversity remained stable over the multiple malaria episodes, despite declining parasitaemia levels. In addition, there are reveal dynamic PfMSP1₁₉ allelic replacements across parasite infection episodes.

Conclusions: Allelic diversity was stable over time in individuals, based on this limited polymorphic region and small sample size, suggesting that there are no significant shifts in allele frequencies or replacements due to alleles being maintained under balancing selection. The dominant alleles in the population are those frequently observed in these children with multiple malaria episodes, further suggesting that early exposure to dominant alleles does not shift their frequency in the population or prevent repeat infection with the same alleles in subsequent infections. However, a blood stage merozoite vaccine is likely to require a multi-allelic formulation.

Keywords: Pfmsp1; Diversity; Malaria; Microhaplotype; Parasitaemia.

Fig. 1

Dynamics of P. falciparum msp1₁₉ microhaplotypes. A Amino- acid sequence alignment of 11 identified microhaplotypes. Polymorphic sites are shown with an asterisk (*). The nucleotide positions relative to the start position of the Pfmsp1 gene are shown below the asterisk. The dots in the alignment indicate the position corresponding to P. falciparum 3D7 with identical amino acid sequences. The epidermal growth factor (EGF)-like domains 1 and 2 are shown by arrows. The first polymorphism is located in the first EGF-like domain, whereas the second to the fifth polymorphism are located in the second EGF-like domain. B Microhaplotypes sorted by their abundance in the population. C Microhaplotypes clustered to groups based on the number of nucleotide differences between haplotypes. The dendrogram on the sides of the heatmap visually represents the relatedness of the microhaplotypes. In this context, the branches indicate distinct clusters formed through hierarchical clustering, highlighting groups of haplotypes with similar characteristic

Fig. 2

Microhaplotype patterns across the infections. The distribution of microhaplotypes in each patient across the malaria episodes. No sample—Samples not retrieved from the biobank, samples not genotyped or sequenced. No episode—No malaria episode was reported. Patient_IDs with asterisks represent cases that had 2–3 similar consecutive microhaplotypes outlined in black. Patients_IDs 4, 5 and 6 did not yield sequenced data and were excluded from the analysis

Fig. 3

Distribution of infection intervals. A Frequency histogram of the distribution of the time interval between infections in months for all infections with genotype data. B Interval in months for individuals with consecutive infections of the same microhaplotype. The size of the circles depicts the number of months between infections with the same microhaplotype depicted by the color of the circle

Fig. 4

Parasitaemia correlations with measure of genetic diversity across infection episodes. A Haplotype diversity (hap_diversity) fluctuates within a small range (between 0.6 and 1) across infection episodes as parasitaemia (parasites/µl) reduces, the correlation between haplotype diversity and parasitemia was low, 0.37. B Nucleotide diversity (nuc_diversity) reduces concurrently across infection episodes with parasitaemia with a correlation of 0.7