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1.
Front Immunol ; 12: 704653, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34675915

RESUMO

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.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antiprotozoários , Antígenos de Protozoários/imunologia , Citometria de Fluxo , Malária Vivax/imunologia , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologia , Receptores de Superfície Celular/imunologia , Anticorpos Antiprotozoários/imunologia , Humanos , Malária Vivax/diagnóstico
2.
Malar J ; 17(1): 76, 2018 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-29422046

RESUMO

BACKGROUND: The Plasmodium vivax Duffy binding protein (PvDBP) has been the most studied ligand binding human reticulocytes to date. This molecule has a cysteine-rich domain in region II (RII) which has been used as control for evaluating the target cell binding activity of several parasite molecules. However, obtaining rPvDBP-RII in a soluble form using the Escherichia coli expression system usually requires laborious and time-consuming steps for recovering the molecule's structure and function, considering it is extracted from inclusion bodies. The present study describes an easy and fast method for expressing and obtaining several PvDBP fragments which should prove ideal for use in protein-cell interaction assays. RESULTS: Two PvDBP encoding regions (rii and riii/v) were cloned in pEXP5-CT vector and expressed in E. coli and extracted from the soluble fraction (rPvDBP-RIIS and rPvDBP-RIII/VS) using a simple freezing/thawing protocol. After the purification, dichroism analysis enabled verifying high rPvDBP-RIIS and rPvDBP-RIII/VS secondary structure α-helix content, which was lowered when molecules were extracted from inclusion bodies (rPvDBP-RIIIB and rPvDBP-RIII/VIB) using a denaturing step. Interestingly, rPvDBP-RIIS, but not rPvDBP-RIIIB, bound to human reticulocytes, while rPvDBP-RIII/VS and rPvDBP-RIII/VIB bound to such cells in a similar way to negative control (cells incubated without recombinant proteins). CONCLUSIONS: This research has shown for the first time how rPvDBP-RII can be expressed and obtained in soluble form using the E. coli system and avoiding the denaturation and refolding steps commonly used. The results highlight the usefulness of the rPvDBP-RIII/VS fragment as a non-binding control for protein-cell target interaction assays. The soluble extraction protocol described is a good alternative to obtain fully functional P. vivax proteins in a fast and easy way, which will surely prove useful to laboratories working in studying this parasite's biology.


Assuntos
Antígenos de Protozoários/genética , Antígenos de Protozoários/isolamento & purificação , Perfilação da Expressão Gênica/métodos , Parasitologia/métodos , Plasmodium vivax/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/isolamento & purificação , Reticulócitos/metabolismo
3.
J Infect Dis ; 214(10): 1539-1546, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27578850

RESUMO

BACKGROUND: Antibodies to the cysteine-rich domain II of Plasmodium vivax Duffy binding protein (PvDBP) can inhibit binding of this parasite ligand to its receptor on red blood cells, the Duffy antigen/receptor for chemokines. These binding-inhibitory antibodies (BIAbs) also inhibit P. vivax invasion of reticulocytes in vitro. METHODS: To investigate whether naturally acquired anti-PvDBP antibodies are associated with reduced risk of clinical malaria in a population exposed to low levels of P. vivax transmission, we measured total levels of immunoglobulin G antibodies to 5 PvDBP variants and used a functional in vitro assay to quantify their binding-inhibitory activity in a cohort of 466 rural Amazonians followed up for up to 37 months. RESULTS: No association between total immunoglobulin G antibody responses to any PvDBP variant and risk of symptomatic, laboratory-confirmed vivax malaria was observed in this cohort. However, a Cox proportional hazards model, adjusted for age, sex, and genotype for the Duffy antigen/receptor for chemokines, showed a >40% decrease in the prospective risk of clinical vivax malaria in subjects with the strongest BIAb responses (upper and middle terciles). High BIAb responses were mostly PvDBP variant transcending and stable over time. CONCLUSIONS: Strong naturally acquired BIAb responses are associated with a reduced risk of clinical P. vivax malaria in rural Amazonians.


Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Malária Vivax/imunologia , Malária Vivax/prevenção & controle , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologia , Receptores de Superfície Celular/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Brasil , Criança , Pré-Escolar , Estudos Transversais , Feminino , Humanos , Imunoglobulina G/sangue , Lactente , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , População Rural , Adulto Jovem
4.
Mem. Inst. Oswaldo Cruz ; 109(5): 608-617, 19/08/2014. tab, graf
Artigo em Inglês | LILACS | ID: lil-720427

RESUMO

Plasmodium vivax infects human erythrocytes through a major pathway that requires interaction between an apical parasite protein, the Duffy binding protein (PvDBP) and its receptor on reticulocytes, the Duffy antigen/receptor for chemokines (DARC). The importance of the interaction between PvDBP (region II, DBPII) and DARC to P. vivax infection has motivated our malaria research group at Oswaldo Cruz Foundation (state of Minas Gerais, Brazil) to conduct a number of immunoepidemiological studies to characterise the naturally acquired immunity to PvDBP in populations living in the Amazon rainforest. In this review, we provide an update on the immunology and molecular epidemiology of PvDBP in the Brazilian Amazon - an area of markedly unstable malaria transmission - and compare it with data from other parts of Latin America, as well as Asia and Oceania.


Assuntos
Humanos , Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/imunologia , Malária Vivax/prevenção & controle , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologia , Receptores de Superfície Celular/imunologia , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/química , Brasil , Ensaio de Imunoadsorção Enzimática , Geografia Médica , Proteínas de Protozoários/química , Receptores de Superfície Celular/química
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