Novo alvo potencial para vacinas contra Plasmodium vivax: estudo da variabilidade genética de uma nova proteína de formas sanguíneas

Abstract

The invasion of erythrocytes by malaria parasites guarantees the success of human infection and, consequently, the development of clinical disease. In the case of P. vivax, the process of invasion of the parasite into reticulocytes is highly dependent on the interaction between region II of the apical protein Duffy binding protein (DBPII) and its receptor present on the surface of erythrocytes, the blood group antigen Duffy receptor for chemokines (DARC). However, recent findings that P. vivax can invade DARC-negative erythrocytes through an alternative pathway, and still unknown, reinforce the hypothesis that the invasion mechanism of this parasite is much more complex than initially thought. In this context, the present study aimed to gain insight on the natural genetic diversity of a recently described P. vivax protein, named erythrocyte binding protein 2 (EBP2), which harbor all the key features of erythrocytes binding proteins such as DBPII, suggesting a role into RBC invasion process. More specifically, we evaluated the genetic diversity of the Duffy binding like (DBL) domain (region II) of PvEBP2 in the Amazon Basin, where P. vivax is the predominant malaria parasite. For this, DNA samples from 71 patients whose P. vivax infection were acquired in different States of the Brazilian Amazon (Amapá, Amazonas, Mato Grosso, Rondônia, Roraima and Pará) were used to amplify the ebp2 genes by PCR-based protocols (nucleotides 477-1455; amino acids 159-485). To compare the degree of genetic variability between region II of EBP2 and DBPII, part of samples (n = 43) was also PCR-amplified to the dbpII gene (1245bp; nucleotides 504-1748; amino acids 168-582). For both genes, the amplification products were purified with the ExoSAP-IT enzyme as described by the manufacturer (Affymetrix® - Product Numbers 78200/01/02/05) and subjected to the Sanger sequencing reaction. Sequences were aligned and compared with reference sequences available in GenBank. To identify the worldwide genetic variability of ebp2 gene, additional analyzes were conducted using all ebp2 sequences available in GenBank from eight countries: Cambodia, Madagascar, Myanmar, Papua New Guinea, South Korea, Thailand, and Vietnam. Taken together, our results demonstrated that (i) ebp2 gene was detected in all Brazilian samples; these results suggest that the ebp2 gene deletion described in some P. vivax isolates does not appear to be common in Brazil; (ii) in the studied samples, the gene that encodes DBPII demonstrated to be much more polymorphic when compared to that of PvEBP2. Specifically, 19 SNPs and 18 haplotypes were identified for dbpII and only 9 SNPs and 12 haplotypes for ebp2. The nucleotide diversity (π) of dbpII was much higher (π = 0.00698) than ebp2 (π = 0.00145); (iii) of interest, the reference ebp2 haplotype (Cambodia, C127) was the predominant haplotype in Brazil and in all countries studied, which confirms the low genetic variability of the protein. In conclusion, this pioneering study of the genetic variability of the ebp2 gene provides evidence that region II of EBP2 is less polymorphic that of DBPII in different transmission settings of the Brazilian Amazon region. The results suggest that PvEBP2-based vaccines should consider few haplotypes strategies to target the main P. vivax parasite populations circulating around the world.