Population diversity and antibody selective pressure to Plasmodium falciparum MSP1 block2 locus in an African malaria-endemic setting

Abstract

Background: Genetic evidence for diversifying selection identified the Merozoite Surface Protein1 block2 (PfMSP1 block2) as a putative target of protective immunity against Plasmodium falciparum. The locus displays three family types and one recombinant type, each with multiple allelic forms differing by single nucleotide polymorphism as well as sequence, copy number and arrangement variation of three amino acid repeats. The family-specific antibody responses observed in endemic settings support immune selection operating at the family level. However, the factors contributing to the large intra-family allelic diversity remain unclear. To address this question, population allelic polymorphism and sequence variant-specific antibody responses were studied in a single Senegalese rural community where malaria transmission is intense and perennial.

Results: Family distribution showed no significant temporal fluctuation over the 10 y period surveyed. Sequencing of 358 PCR fragments identified 126 distinct alleles, including numerous novel alleles in each family and multiple novel alleles of recombinant types. The parasite population consisted in a large number of low frequency alleles, alongside one high-frequency and three intermediate frequency alleles. Population diversity tests supported positive selection at the family level, but showed no significant departure from neutrality when considering intra-family allelic sequence diversity and all families combined. Seroprevalence, analysed using biotinylated peptides displaying numerous sequence variants, was moderate and increased with age. Reactivity profiles were individual-specific, mapped to the family-specific flanking regions and to repeat sequences shared by numerous allelic forms within a family type. Seroreactivity to K1-, Mad20- and R033 families correlated with the relative family genotype distribution within the village. Antibody specificity remained unchanged with cumulated exposure to an increasingly large number of alleles.

Conclusion: The Pfmsp1 block2 locus presents a very large population sequence diversity. The lack of stable acquisition of novel antibody specificities despite exposure to novel allelic forms is reminiscent of clonal imprinting. The locus appears under antibody-mediated diversifying selection in a variable environment that maintains a balance between the various family types without selecting for sequence variant allelic forms. There is no evidence of positive selection for intra-family sequence diversity, consistent with the observed characteristics of the antibody response.

Figure 1

Temporal distribution of the relative proportion of the three allelic families in Dielmo during 1990-99. Alleles were assigned to one of three allelic families by nested PCR. Distribution is shown by calendar year. The number of samples typed each year is shown in Table 1. Colour symbols: black: K1-types, white: Mad20-types, grey RO33 types. Note that hybrid alleles were not distinguished from the Mad20-types and are included in the Mad20 group.

Figure 2

Estimated multiplicity of infection by age group. Estimated multiplicity of infection (i.e. the mean number of Pfmsp1 block 2-alleles detected per sample) was calculated from PCR fragments generated in the nested PCR reaction. There were 51, 83, 61, 60 and 51 samples in the 0-1 y, 2-5 y, 6-9 y, 10-19 y and ≥20 y age groups, respectively. The figures shown are the mean and SD.

Figure 3

Influence of seasonality on Pfmsp1 block 2 family infection rates. Data from individual years were pooled. Three seasons were defined as February-May (yellow), June-October (green) and November-January (hatched grey).

Figure 4

Frequency distribution of the number of tri-peptide motif usage in the DK and DM alleles. A: Frequency distribution of K1-type alleles (DK alleles) by number of distinct tripeptides present. B. Frequency distribution of Mad20-types (DM alleles) by number of distinct tripeptide nucleotide sequences present (DMR, DMRK and MK hybrids excluded). C. Frequency distribution of Mad20-types DM alleles (by number of distinct tripeptide protein sequences present (DMR, DMRK and MK hybrids excluded).

Figure 5

Distribution of Pfmsp1 block2 allele frequency in Dielmo. A. Distribution by family based on sequenced alleles: K1-types (N sequenced = 144), Mad20-types grouped together with hybrid types (N sequenced = 90) and RO33-types (N sequenced = 124). Each family is depicted separately, with alleles ranked clockwise by allele number coded as shown in Table 2. B. Relative individual allele frequency in the 358 sequenced fragments (top) and adjusted to the overall population based on relative family distribution established by nested PCR on 524 PCR fragments (bottom). Identical colour codes used for A and B, ordered clockwise as follows: RD types (light blue colours), Hybrids (green and orange), DM (orange-yellow) and DK alleles (indigo-dark blue colours), with alleles ranked clockwise by allele number coded as shown in Table 2.

Figure 6

Prevalence of anti-MSP1-block 2 IgG by age group. Seroprevalence was determined using sera collected during a cross-sectional survey conducted before the 1998 rainy season (on 2-3 August 1998) when 243 villagers (i.e. 95% of the village population) donated a fingerprick blood sample. The presence of anti-MSP1 block2 specific IgG was assessed by ELISA on 16 pools of biotinylated peptides (sequence and composition of the pools described in Table 5). Plasma reacting with one or more pool was considered seropositive. Number of individuals by age group: 27, 25, 26, 40, 46 and 79 in the 0-2, 3-5, 6-8, 9-14, 15-24, ≥ 25 year group, respectively.

Figure 7

Seroprevalence and specificity of anti-MSP1-block 2 IgG in Dielmo. A) Seroprevalence to each family and family distribution within the parasite population. Seroprevalence was determined using sera collected during a cross-sectional survey conducted before the 1998 rainy season (on 2-3 August 1998) when 243 villagers (i.e. 95% of the village population) donated a fingerprick blood sample. The presence of anti-MSP1 block2 specific IgG was assessed by ELISA on 16 pools of biotinylated peptides (sequence and composition of the pools described in Table 5). Plasma reacting with one or more pool was considered seropositive, and grouped by family irrespective of the number of peptides sequences recognised within each of the three family types (i.e. MR alleles were disregarded as such, seropositivity being allocated either to Mad20 or to RO33). The relative distribution of family genotypes was established by nested PCR on 306 samples collected longitudinally during the 1990-9 time period as shown in Table 1. Colour codes K1: dark blue; Mad20: orange, RO33: light blue. B) Frequency of plasma with antibodies reacting with one, two and three allelic families. The number of families recognised is shown irrespective of the actual type recognised (i.e. individuals reacting with only K1-types, only Mad20-types or only RO33-types are placed together in the group reacting with one family). C) Frequency of reaction with each peptide pool.

Figure 8

Typical profiles of the temporal evolution of MSP1 block2- specific IgG before and after the 1998 rainy season. Antibodies were assayed from 25 individuals in August 1998 (yellow) and December 1998, i.e. after a rainy season when each inhabitant was exposed to a mean of 170 infected bites. Anti-MSP1 block2 specific IgG was assessed by ELISA on 16 pools of biotinylated peptides. The upper, central and lower panels show a representative example of a reduction of specific antibodies, an essentially unchanged profile, and a boosting of pre-existing responses, respectively.

Figure 9

Temporal fluctuation of MSP1 block2- specific IgG during the 1998 rainy season. Antibodies were assayed on 16 pools of biotinylated peptides (sequence and composition of the pools described in Table 5). Typical individual patterns are shown, with the dates of blood sampling shown on each graph. A) Transient boosting of a pre-existing response in a 14 y old subject (code 11/21), who had a clinical malaria attack on 29/10/98. B) Transient loss of a pre-existing response in a 5 y old child (code 8/15), who had a clinical malaria attack on 28/08/98. C) Transient acquisition of a novel specificity in a 9.5 y old child (code 02/04), who had a clinical malaria on 10/09/98. D) Transient changes in a 5 y old child (code 03/18), who experienced three successive clinical episodes during that time period on 17/09/98, 22/10/98 and 11/12/98. For each cinical episode, an antimalarial treatment was administered to the patient on the day of diagnosis.

Figure 10

Serological longitudinal follow up of child 01/13 from 6 months to 6 years of age. Antibodies were assayed on 16 pools of biotinylated peptides (A) and to each individual peptide from positive pool 11 (B). The peptide sequence and composition of the pools are described in Table 5. The dates of blood sampling are shown to the right of the graph. A. reactivity on the peptide pool. B. reactivity of three representative blood samples on individual peptides from pool 11.