Frequent expansion of Plasmodium vivax Duffy Binding Protein in Ethiopia and its epidemiological significance

Abstract

28 Plasmodium vivax invasion of human erythrocytes depends on the Duffy Binding 29 Protein (PvDBP) which interacts with the Duffy antigen. PvDBP copy number varies 30 between P. vivax isolates, but the prevalence of PvDBP multiplications in Sub-Saharan 31 Africa and its impact are unknown. We determined the prevalence and type of PvDBP 32 duplications, as well as PvDBP copy number variation among 178 Ethiopian P. vivax 33 isolates using a PCR-based diagnostic method, a novel quantitative real-time PCR 34 assay and whole genome sequencing. For the 145 symptomatic samples, PvDBP 35 duplications were detected in 95 isolates, of which 81 had the Cambodian and 14 36 Malagasy-type PvDBP duplications. PvDBP varied from 1 to >4 copies. Isolates with 37 multiple PvDBP copies were found to be higher in symptomatic than asymptomatic 38 infections. For the 33 asymptomatic samples, PvDBP was detected with two copies in 39 two of the isolates, and both were the Cambodian-type PvDBP duplication. PvDBP copy 40 number in Duffy-negative heterozygotes was not significantly different from that in 41 Duffy-positives, providing no support for the hypothesis that increased copy number is a 42 specific association with Duffy-negativity, although the number of Duffy-negatives was 43 small and further sampling is required to test this association thoroughly. 44 45 46 3 47 Author summary 48 Plasmodium vivax invasion of human erythrocytes relies on interaction between the 49 Duffy antigen and P. vivax Duffy Binding Protein (PvDBP). Whole genome sequences 50 from P. vivax field isolates in Madagascar identified a duplication of the PvDBP gene 51 and PvDBP duplication has also been detected in non-African P. vivax-endemic 52 countries. Two types of PvDBP duplications have been reported, termed Cambodian 53 and Malagasy-type duplications. Our study used a combination of PCR-based 54 diagnostic method, a novel quantitative real-time PCR assay, and whole genome 55 sequencing to determine the prevalence and type of PvDBP duplications, as well as 56 PvDBP copy number on a broad number of P. vivax samples in Ethiopia. We found that 57 over 65% of P. vivax isolated from the symptomatic infections were detected with 58 PvDBP duplications and PvDBP varied from 1 to >4 copies. The majority of PvDBP 59 duplications belongs to the Cambodian-type while the Malagasy-type duplications was 60 also detected. For the asymptomatic infections, despite a small sample size, the 61 majority of P. vivax were detected with a single-copy based on both PCR and qPCR 62 assays. There was no significant difference in PvDBP copy number between Duffy-null 63 heterozygote and Duffy-positive homozygote/heterozygote. Further investigation is 64 needed with expanded Duffy-null homozygotes to examine the functional significance of 65 PvDBP expansion.66 67 68 69 4 70 Introduction 71 Plasmodium vivax and P. falciparum are the two major parasite species that cause 72 clinical malaria worldwide. While P. falciparum causes most malaria mortality, P. vivax 73 can also cause severe dis...

UsingPlasmodium knowlesias a model for screeningPlasmodium vivaxblood-stage malaria vaccine targets reveals new candidates

“…However, it has now clearly been demonstrated that P. vivax is also able to infect individuals who are Duffy negative, lacking DARC expression on their red blood cell surface (21,22). This could indicate that P. vivax is able to utilize other ligands for invasion such as PvRBP2b (28) and erythrocyte binding protein 2 (ebp2) (30), although it is also possible that PvDBP is still involved in the invasion of Duffy negative cells (26). It is also worth noting that the genetic essentiality of PvDBP for parasite growth has never been able to be directly tested, as P. vivax cannot be cultured and therefore cannot be genetically manipulated.…”

“…However, it has recently been shown that P. vivax is also able to infect individuals who are Duffy negative, so express little or no DARC on the surface of their erythrocytes (21)(22)(23)(24). While the invasion of Duffy negative erythrocytes could still rely on PvDBP (25,26), the sole focus on PvDBP as a vaccine candidate clearly needs to be reassessed and additional targets evaluated, either as potential substitutes for PvDBP, or to be used in combination with it. As noted above, erythrocyte invasion is a very complex process, and while the process is much less well-understood in P. vivax than it is in P.…”

The biology of unconventional invasion of Duffy-negative reticulocytes by Plasmodium vivax and its implication in malaria epidemiology and public health

“…A molecular epidemiology study conducted in Ethiopia observed that the proportion of parasites with PvDBP amplification was higher in individuals carrying the FY*A allele compared to individuals carrying the FY*B [ 51 ]. On the other hand, in Madagascar where both Duffy-negative and Duffy-positive individuals coexist, PvDBP amplification is not selected in response to the Duffy null homozygotes [ 52 ].…”

Global distribution of single amino acid polymorphisms in Plasmodium vivax Duffy-binding-like domain and implications for vaccine development efforts

“…Complicating these scenarios further are additional facets of Pv biology—several cases of Pv infection have now been observed in Duffy-negative individuals, suggesting that the parasite might have evolved an alternative pathway that is independent of DARC–DBL interaction for invasion [ 44 – 52 ]. Studies from Malagasy, Colombia and Ethiopia have also suggested that Pv DBP gene copy variations in the field are evident and indicative of Pv evolution [ 53 – 55 ]. Therefore, the development of Pv DBL-based vaccine seems to be fraught with several challenges—and here we highlight further reasons for scepticism in this direction.…”