Worldwide genetic variability of the Duffy binding protein: insights into Plasmodium vivax vaccine development

Abstract

The dependence of Plasmodium vivax on invasion mediated by Duffy binding protein (DBP) makes this protein a prime candidate for development of a vaccine. However, the development of a DBP-based vaccine might be hampered by the high variability of the protein ligand (DBP(II)), known to bias the immune response toward a specific DBP variant. Here, the hypothesis being investigated is that the analysis of the worldwide DBP(II) sequences will allow us to determine the minimum number of haplotypes (MNH) to be included in a DBP-based vaccine of broad coverage. For that, all DBP(II) sequences available were compiled and MNH was based on the most frequent nonsynonymous single nucleotide polymorphisms, the majority mapped on B and T cell epitopes. A preliminary analysis of DBP(II) genetic diversity from eight malaria-endemic countries estimated that a number between two to six DBP haplotypes (17 in total) would target at least 50% of parasite population circulating in each endemic region. Aiming to avoid region-specific haplotypes, we next analyzed the MNH that broadly cover worldwide parasite population. The results demonstrated that seven haplotypes would be required to cover around 60% of DBP(II) sequences available. Trying to validate these selected haplotypes per country, we found that five out of the eight countries will be covered by the MNH (67% of parasite populations, range 48-84%). In addition, to identify related subgroups of DBP(II) sequences we used a Bayesian clustering algorithm. The algorithm grouped all DBP(II) sequences in six populations that were independent of geographic origin, with ancestral populations present in different proportions in each country. In conclusion, in this first attempt to undertake a global analysis about DBP(II) variability, the results suggest that the development of DBP-based vaccine should consider multi-haplotype strategies; otherwise a putative P. vivax vaccine may not target some parasite populations.

Figure 1. Frequency by country of the DBP_II haplotypes that cover at least fifty percent of local parasite population.

Among the 73 haplotypes defined using the 10 most prevalent nonsynonymous single nucleotide polymorphisms (nsSNPs), 17 haplotypes (each haplotype was represented by a color) were able to cover at least 50% of each country parasite population: 2 in South Korea (Hap 4 – dark blue and Hap 40 - orange) and in Sri Lanka (Hap 23 - red and Hap 44 – light blue); 3 in Iran (Hap 4, Hap 23 and Hap 59 – dark green), in India (Hap 12 - purple, Hap 23 and Hap 24 - yellow) and in PNG (Hap 12, Hap 55 – pink and Hap 64 – light green); 4 in Colombia (Hap 9 – light yellow, Hap 27 – dark gray, Hap 30 - green and Hap 37 - brown) and 4 in Brazil (Hap 12, Hap 23, Hap 24 and Hap 44); 6 in Thailand (Hap 8 – dark brown, Hap 15 – light pink, Hap 21 - blue, Hap 24, Hap 36 – light gray and Hap 59). The haplotype from Sal-1 reference strain was represented by purple (Hap 12), for haplotype sequences please see Table S2. The number in parentheses indicates the frequency of the selected haplotypes in the respective country (range from 0.52 to 0.63). Some F _ST values among countries are showed and represented by dashed lines.

Figure 2. Frequency of the seven nsSNP haplotypes of DBP_II that cover at least fifty percent of DBP_II sequences deposited in GenBank.

Coverage of the selected haplotypes in each country: countries that at least 50% of parasites are covered by the selected haplotypes are represented by black bars and countries that less than 50% of the parasites are covered by the selected haplotypes are represented by grey bars. The selected haplotypes were: 4, 12, 23, 24, 44, 59, 64 (for haplotype sequences see Table S2).

Figure 3. Population structure of the gene encoding DBP_II based on Bayesian cluster analysis using Structure 2.3 software.

(A) Plot of the log probability of the data [Ln P(D)] and its standard deviation (vertical bars) given values for K of 1–10. (B) Population clustering for K = 2–6. Each individual is represented by a thin vertical color. Each color represents an ancestral population (pop), and the color of individual represents their proportional membership in the different populations. Ancestral populations: red, pop1; green, pop2; blue, pop3; yellow, pop4; pink, pop5 and light blue, pop6. The figure shown for a given K is based on the highest probability run at that K.

Figure 4. Population structure for P. vivax sequences of DBP_II at K = 6.

Graphic of Structure results were produced by using the DISTRUCT program. Individual P. vivax isolate are represented by a thin vertical line from each geographic population showed above the graphic and separated by a thin black line.