Identification of Reticulocyte Binding Domain of Plasmodium ovale curtisi Duffy Binding Protein (PocDBP) Involved in Reticulocyte Invasion

The Plasmodium ovale curtisi (Poc) prevalence has increased substantially in sub-Saharan African countries as well as regions of Southeast Asia. Poc parasite biology has not been explored much to date; in particular, the invasion mechanism of this malaria parasite remains unclear. In this study, the binding domain of the Duffy binding protein of P. ovale curtisi (PocDBP) was characterized as an important ligand for reticulocyte invasion. The homologous region of the P. vivax Duffy binding protein in PocDBP, named PocDBP-RII herein, was selected, and the recombinant PocDBP-RII protein was expressed in an Escherichia coli system. This was used to analyze reticulocyte binding activity using fluorescence-activated cell sorting and immune serum production in rabbits. The binding specificity was proven by treating reticulocytes with trypsin, chymotrypsin and neuraminidase. The amino acid sequence homology in the N-terminal Cys-rich region was found to be ~ 44% between PvDBP and PocDBP. The reticulocyte binding activity of PocDBP-RII was significantly higher than the erythrocyte binding activity and was concentration dependent. Erythrocyte binding was reduced significantly by chymotrypsin treatment and inhibited by an anti-PocDBP-RII antibody. This finding suggests that PocDBP may be an important ligand in the reticulocyte invasion process of P. ovale curtisi.

Genetic Diversity and Natural Selection of Plasmodium vivax Duffy Binding Protein-II From China-Myanmar Border of Yunnan Province, China

Malaria incidence has declined dramatically over the past decade and China was certified malaria-free in 2021. However, the presence of malaria in border areas and the importation of cases of malaria parasites are major challenges for the consolidation of the achievements made by China. Plasmodium vivax Duffy binding protein (PvDBP) performs a significant role in erythrocyte invasion, and is considered a promising P. vivax vaccine. However, the highly polymorphic region of PvDBP (PvDBP-II) impedes the development of blood-stage vaccine against P. vivax. In this study, we investigated the genetic diversity and natural selection of PvDBP-II among 124 P. vivax isolates collected from the China-Myanmar border (CMB) in Yunnan Province, China, during 2009–2011. To compare genetic diversity, natural selection, and population structure with CMB isolates, 85 pvdbp-II sequences of eastern Myanmar isolates were obtained from GenBank. In addition, global sequences of pvdbp-II were retrieved from GenBank to establish genetic differentiation relationships and networks with the CMB isolates. In total, 22 single nucleotide polymorphisms reflected in 20 non-synonymous and two synonymous mutations were identified. The overall nucleotide diversity of PvDBP-II from the 124 CMB isolates was 0.0059 with 21 haplotypes identified (Hd = 0.91). The high ratio of non-synonymous to synonymous mutations suggests that PvDBP-II had evolved under positive selection. Population structure analysis of the CMB and eastern Myanmar isolates were optimally grouped into five sub-populations (K = 5). Polymorphisms of PvDBP-II display that CMB isolates were genetically diverse. Mutation, recombination, and positive selection promote polymorphism of PvDBP-II of P. vivax population. Although low-level genetic differentiation in eastern Myanmar was identified along with the more effective malaria control measures, the complexity of population structure in malaria parasites has maintained. In conclusion, findings from this study advance knowledge of the understanding of the dynamic of P. vivax population, which will contribute to guiding the rational design of a PvDBP-II based vaccine.

Multiplexed Microsphere-Based Flow Cytometric Assay to Assess Strain Transcending Antibodies to Plasmodium vivax Duffy Binding Protein II Reveals an Efficient Tool to Identify Binding-Inhibitory Antibody Responders

Malaria remains a major public health problem worldwide, and Plasmodium vivax is the most widely distributed malaria parasite. Naturally acquired binding inhibitory antibodies (BIAbs) to region II of the Duffy binding protein (DBPII), a P. vivax ligand that is critical for reticulocyte invasion, are associated with a reduced risk of clinical malaria. Owing to methodological issues in evaluating antibodies that inhibit the DBPII–DARC interaction, a limited number of studies have investigated DBPII BIAbs in P. vivax-exposed populations. Based on the assumption that individuals with a consistent BIAb response are characterized by strain-transcending immune responses, we hypothesized that detecting broadly reactive DBPII antibodies would indicate the presence of BIAb response. By taking advantage of an engineered DBPII immunogen targeting conserved DBPII neutralizing epitopes (DEKnull-2), we standardized a multiplex flow cytometry-based serological assay to detect broadly neutralizing IgG antibodies. For this study, a standard in vitro cytoadherence assay with COS-7 cells expressing DBPII was used to test for DBPII BIAb response in long-term P. vivax-exposed Amazonian individuals. Taken together, the results demonstrate that this DBPII-based multiplex assay facilitates identifying DBPII BIAb carriers. Of relevance, the ability of the multiplex assay to identify BIAb responders was highly accurate when the positivity for all antigens was considered. In conclusion, the standardized DBPII-based flow cytometric assay confirmed that DBPII-BIAb activity was associated with the breadth rather than the magnitude of anti-DBPII antibodies. Altogether, our results suggest that multiplex detection of broadly DBPII-reactive antibodies facilitates preliminary screening of BIAb responders.

Sequence analysis of Plasmodium vivax Duffy binding proteins reveals the presence of unique haplotypes and diversifying selection in Ethiopian isolates

Background Red blood cell invasion by the Plasmodium vivax merozoite requires interaction between the Duffy antigen receptor for chemokines (DARC) and the P. vivax Duffy-binding protein II (PvDBPII). Given that the disruption of this interaction prevents P. vivax blood-stage infection, a PvDBP-based vaccine development has been well recognized. However, the polymorphic nature of PvDBPII prevents a strain transcending immune response and complicates attempts to design a vaccine. Methods Twenty-three P. vivax clinical isolates collected from three areas of Ethiopia were sequenced at the pvdbpII locus. A total of 392 global pvdbpII sequences from seven P. vivax endemic countries were also retrieved from the NCBI archive for comparative analysis of genetic diversity, departure from neutrality, linkage disequilibrium, genetic differentiation, PvDBP polymorphisms, recombination and population structure of the parasite population. To establish a haplotype relationship a network was constructed using the median joining algorithm. Results A total of 110 variable sites were found, of which 44 were parsimony informative. For Ethiopian isolates there were 12 variable sites of which 10 were parsimony informative. These parsimony informative variants resulted in 10 nonsynonymous mutations. The overall haplotype diversity for global isolates was 0.9596; however, the haplotype diversity was 0.874 for Ethiopia. Fst values for genetic revealed Ethiopian isolates were closest to Indian isolates as well as to Sri Lankan and Sudanese isolates but further away from Mexican, Papua New Guinean and South Korean isolates. There was a total of 136 haplotypes from the 415 global isolates included for this study. Haplotype prevalence ranged from 36.76% to 0.7%, from this 74.2% were represented by single parasite isolates. None of the Ethiopian isolates grouped with the Sal I reference haplotype. From the total observed nonsynonymous mutations 13 mapped to experimentally verified epitope sequences. Including 10 non-synonymous mutations from Ethiopia. However, all the polymorphic regions in Ethiopian isolates were located away from DARC, responsible for junction formation. Conclusion The results of this study are concurrent with the multivalent vaccine approach to design an effective treatment. However, the presence of novel haplotypes in Ethiopian isolates that were not shared by other global sequences warrant further investigation.

Plasmodium vivax Strains Use Alternative Pathways for Invasion

Plasmodium vivax has 2 invasion ligand/host receptor pathways (P. vivax Duffy-binding protein/Duffy antigen receptor for chemokines [DARC] and P. vivax reticulocyte binding protein 2b/transferrin receptor [TfR1]) that are promising targets for therapeutic intervention. We optimized invasion assays with isogenic cultured reticulocytes. Using a receptor blockade approach with multiple P. vivax isolates, we found that all strains utilized both DARC and TfR1, but with significant variation in receptor usage. This suggests that P. vivax, like Plasmodium falciparum, uses alternative invasion pathways, with implications for pathogenesis and vaccine development.

Genetic diversity, natural selection and haplotype grouping of Plasmodium vivax Duffy-binding protein genes from eastern and western Myanmar borders

Background Merozoite proteins of the malaria parasites involved in the invasion of red blood cells are selected by host immunity and their diversity is greatly influenced by changes in malaria epidemiology. In the Greater Mekong Subregion (GMS), malaria transmission is concentrated along the international borders and there have been major changes in malaria epidemiology with Plasmodium vivax becoming the dominant species in many regions. Here, we aimed to evaluate the genetic diversity of P. vivax Duffy-binding protein gene domain II (pvdbp-II) in isolates from the eastern and western borders of Myanmar, and compared it with that from global P. vivax populations. Methods pvdbp-II sequences were obtained from 85 and 82 clinical P. vivax isolates from the eastern and western Myanmar borders, respectively. In addition, 504 pvdbp-II sequences from nine P. vivax populations of the world were retrieved from GenBank and used for comparative analysis of genetic diversity, recombination and population structure of the parasite population. Results The nucleotide diversity of the pvdbp-II sequences from the Myanmar border parasite isolates was not uniform, with the highest diversity located between nucleotides 1078 and 1332. Western Myanmar isolates had a unique R391C mutation. Evidence of positive natural selection was detected in pvdbp-II gene in P. vivax isolates from the eastern Myanmar area. P. vivax parasite populations in the GMS, including those from the eastern, western, and central Myanmar as well as Thailand showed low-level genetic differentiation (FST, 0.000–0.099). Population genetic structure analysis of the pvdbp-II sequences showed a division of the GMS populations into four genetic clusters. A total of 60 PvDBP-II haplotypes were identified in 210 sequences from the GMS populations. Among the epitopes in PvDBP-II, high genetic diversity was found in epitopes 45 (379-SIFGT(D/G)(E/K)(K/N)AQQ(R/H)(R/C)KQ-393, π = 0.029) and Ia (416-G(N/K)F(I/M)WICK(L/I)-424], Ib [482-KSYD(Q/E)WITR-490, π = 0.028) in P. vivax populations from the eastern and western borders of Myanmar. Conclusions The pvdbp-II gene is genetically diverse in the eastern and western Myanmar border P. vivax populations. Positive natural selection and recombination occurred in pvdbp-II gene. Low-level genetic differentiation was identified, suggesting extensive gene flow of the P. vivax populations in the GMS. These results can help understand the evolution of the P. vivax populations in the course of regional malaria elimination and guide the design of PvDBP-II-based vaccine.