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BACKGROUND: There is a paucity of knowledge about the healthcare attitudes and practices of French-speaking immigrants originating from Sub-Saharan Africa (FISSA) living in minority settings. The purpose of this study was to characterize FISSA healthcare experiences and confidence in the malaria-related knowledge of health professionals in Edmonton. METHODS: A structured survey was used to examine a cohort of 382 FISSA (48% female; 52% male) living in Edmonton. FISSA general healthcare attitudes, experiences and satisfaction with the Canadian healthcare system were studied. Healthcare Competency Perception (HCP) was characterized by using an index score. Statistical analyses were performed to evaluate the impact of healthcare experiences and other outcomes. RESULTS: Intriguingly, while only 42% of FISSA had a French-speaking family physician, 83% (197/238) of those who had received health care services in Alberta found that access to medical treatment was easy, and 77% (188/243) were satisfied with received care. Although 70% (171/243) of FISSA did not receive services in French, 82% (199/243) surprisingly reported having good levels of comprehension during their visits. Satisfaction with care was associated with having a family physician (p = 0.018) and having health insurance (p = 0.041). Nevertheless, confidence in the healthcare system's ability to treat malaria effectively was significantly lower, with only 39% (148/382) receiving a positive score on the HCP index. CONCLUSION: This study provides an important insight into FISSA experience with and perception of the Alberta's healthcare system.
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Emigrantes e Imigrantes , Malária , Humanos , Masculino , Feminino , Canadá , Acessibilidade aos Serviços de Saúde , Atitude , Alberta , Malária/terapiaRESUMO
BACKGROUND: Important knowledge gaps exist in the understanding of the management of the risks of imported malaria in Canada among Francophone immigrants from sub-Saharan Africa (FISSA). The aim of this cross-sectional study was to investigate the malaria related-knowledge, attitude and practices (KAP) of FISSA in Edmonton, where these immigrants are in an official minority language situation and the impact of language barriers on these factors. METHODS: A structured survey was used to examine the KAP of 382 FISSA in the Edmonton area from 2018 to 2019. Fisher's Exact Test was applied to determine if there were associations between knowledge of malaria and different risk factors. RESULTS: Almost all FISSA (97%) had an accurate knowledge of fever as the key symptom of malaria. Interestingly, 60% of participants identified bed nets as a preventive method and only 19% of participants had accurate knowledge of malaria transmission. An accurate knowledge of symptoms was significantly associated with a high perceived risk of contracting malaria [odds ratio (OR) 4.33, 95% confidence interval (CI) 1.07-20.62]. Furthermore, even though 70% of FISSA had a high perceived risk of contracting malaria in endemic regions, only 52% of travellers had a pre-travel medical encounter. Importantly, language was not the predominant reason for not seeking pre-travel medical advice, although 84% of respondents chose French as their official language of preference when seeking medical advice. Having a French-speaking physician was correlated with satisfactory prevention knowledge (OR 1.96, 95% CI 1.16-3.35). With respect to health-seeking behaviour, 88% of respondents with a child < 5 years of age would seek medical care for fever in the child after travel to sub-Saharan Africa (SSA). CONCLUSION: This study highlights that factors other than knowledge, risk assessment, and language might determine the lack of compliance with pre-travel medical encounters. It underscores the need for effective strategies to improve this adherence in minority settings.
Assuntos
Emigrantes e Imigrantes , Malária , Criança , Estudos Transversais , Febre , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Malária/epidemiologia , ViagemRESUMO
Background: In pregnancy, Plasmodium falciparum parasites express the surface antigen VAR2CSA, which mediates adherence of red blood cells to chondroitin sulfate A (CSA) in the placenta. VAR2CSA antibodies are generally acquired during infection in pregnancy and are associated with protection from placental malaria. We observed previously that men and children in Colombia also had antibodies to VAR2CSA, but the origin of these antibodies was unknown. Here, we tested whether infection with Plasmodium vivax is an alternative mechanism of acquisition of VAR2CSA antibodies. Methods: We analyzed sera from nonpregnant Colombians and Brazilians exposed to P. vivax and monoclonal antibodies raised against P. vivax Duffy binding protein (PvDBP). Cross-reactivity to VAR2CSA was characterized by enzyme-linked immunosorbent assay, immunofluorescence assay, and flow cytometry, and antibodies were tested for inhibition of parasite binding to CSA. Results: Over 50% of individuals had antibodies that recognized VAR2CSA. Affinity-purified PvDBP human antibodies and a PvDBP monoclonal antibody recognized VAR2CSA, showing that PvDBP can give rise to cross-reactive antibodies. Importantly, the monoclonal antibody inhibited parasite binding to CSA, which is the primary in vitro correlate of protection from placental malaria. Conclusions: These data suggest that PvDBP induces antibodies that functionally recognize VAR2CSA, revealing a novel mechanism of cross-species immune recognition to falciparum malaria.
Assuntos
Antígenos de Protozoários/imunologia , Antígenos de Superfície/imunologia , Reações Cruzadas/imunologia , Malária Falciparum/imunologia , Malária Vivax/imunologia , Plasmodium falciparum/imunologia , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologia , Receptores de Superfície Celular/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antiprotozoários/sangue , Criança , Sulfatos de Condroitina , Colômbia , Eritrócitos/parasitologia , Eutérios/imunologia , Feminino , Humanos , Imunidade , GravidezRESUMO
Malaria in pregnancy can cause serious adverse outcomes for the mother and the fetus. However, little is known about the effects of submicroscopic infections (SMIs) in pregnancy, particularly in areas where Plasmodium falciparum and Plasmodium vivax cocirculate. A cohort of 187 pregnant women living in Puerto Libertador in northwest Colombia was followed longitudinally from recruitment to delivery. Malaria was diagnosed by microscopy, reverse transcription-quantitative PCR (RT-qPCR), and placental histopathology. Gestational age, hemoglobin concentration, VAR2CSA-specific IgG levels, and adhesion-blocking antibodies were measured during pregnancy. Statistical analyses were performed to evaluate the impact of SMIs on birth weight and other delivery outcomes. Twenty-five percent of women (45/180) were positive for SMIs during pregnancy. Forty-seven percent of infections (21/45) were caused by P. falciparum, 33% were caused by P. vivax, and 20% were caused by mixed Plasmodium spp. Mixed infections of P. falciparum and P. vivax were associated with lower gestational age at delivery (P = 0.0033), while other outcomes were normal. Over 60% of women had antibodies to VAR2CSA, and there was no difference in antibody levels between those with and without SMIs. The anti-adhesion function of these antibodies was associated with protection from SMI-related anemia at delivery (P = 0.0086). SMIs occur frequently during pregnancy, and while mixed infections of both P. falciparum and P. vivax were not associated with a decrease in birth weight, they were associated with significant risk of preterm birth. We propose that the lack of adverse delivery outcomes is due to functional VAR2CSA antibodies that can protect pregnant women from SMI-related anemia.
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In pregnancy, parity-dependent immunity is observed in response to placental infection with Plasmodium falciparum. Antibodies recognize the surface antigen, VAR2CSA, expressed on infected red blood cells and inhibit cytoadherence to the placental tissue. In most settings of malaria endemicity, antibodies against VAR2CSA are predominantly observed in multigravid women and infrequently in men, children, and nulligravid women. However, in Colombia, we detected antibodies against multiple constructs of VAR2CSA among men and children with acute P. falciparum and Plasmodium vivax infection. The majority of men and children (>60%) had high levels of IgGs against three recombinant domains of VAR2CSA: DBL5ε, DBL3X, and ID1-ID2. Surprisingly, these antibodies were observed only in pregnant women, men, and children exposed either to P. falciparum or to P. vivax. Moreover, the anti-VAR2CSA antibodies are of high avidity and efficiently inhibit adherence of infected red blood cells to chondroitin sulfate A in vitro, suggesting that they are specific and functional. These unexpected results suggest that there may be genotypic or phenotypic differences in the parasites of this region or in the host response to either P. falciparum or P. vivax infection outside pregnancy. These findings may hold significant clinical relevance to the pathophysiology and outcome of malaria infections in this region.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Malária Falciparum/imunologia , Malária Vivax/imunologia , Plasmodium falciparum/imunologia , Plasmodium vivax/imunologia , Adolescente , Adulto , Idoso , Afinidade de Anticorpos , Criança , Pré-Escolar , Colômbia/epidemiologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/imunologia , Malária Falciparum/epidemiologia , Malária Vivax/epidemiologia , Masculino , Pessoa de Meia-Idade , Gravidez , Adulto JovemRESUMO
BACKGROUND: Binding to chondroitin sulfate A by VAR2CSA, a parasite protein expressed on infected erythrocytes, allows placental sequestration of Plasmodium falciparum-infected erythrocytes. This leads to severe consequences such as maternal anemia, stillbirths, and intrauterine growth retardation. The latter has been clearly associated to increased morbidity and mortality of the infants. Acquired anti-VAR2CSA antibodies have been associated with improved pregnancy outcomes, suggesting a vaccine could prevent the syndrome. However, identifying functionally important regions in the large VAR2CSA protein is difficult. METHODS: Using genetic immunization, we raised polyclonal antisera against overlapping segments of VAR2CSA in mice and rabbits. The adhesion-inhibition capacities of induced antisera and of specific antibodies purified from plasma of malaria-exposed pregnant women were assessed on laboratory-adapted parasite lines and field isolates expressing VAR2CSA. Competition enzyme-linked immunosorbent assay (ELISA) was employed to analyze functional resemblance between antibodies induced in animals and those naturally acquired by immune multigravidae. RESULTS: Antibodies targeting the N-terminal sequence (NTS) up to DBL2X (NTS-DBL2X) efficiently blocked parasite adhesion to chondroitin sulfate A in a manner similar to that of antibodies raised against the entire VAR2CSA extracellular domain. Interestingly, naturally acquired antibodies and those induced by vaccination against NTS-DBL2X target overlapping strain-transcendent anti-adhesion epitopes. CONCLUSIONS: This study highlights an important step achieved toward development of a protective vaccine against placental malaria.
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Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/metabolismo , Adesão Celular/imunologia , Sulfatos de Condroitina/metabolismo , Eritrócitos/metabolismo , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Ensaio de Imunoadsorção Enzimática , Eritrócitos/parasitologia , Feminino , Expressão Gênica , Humanos , Imunidade Ativa/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Camundongos , Plasmídeos , Plasmodium falciparum/metabolismo , Gravidez , Ligação Proteica , Coelhos , VacinaçãoRESUMO
We have recently shown, using a well-defined in vitro model, that connexin 43 (Cx43) is directly involved in human cytotrophoblastic cell fusion into a multinucleated syncytiotrophoblast. Cx43 appears to interact with partner proteins within a fusogenic complex, in a multi factorial and dynamic process. This fusogenic complex remains to be characterized and constituent proteins need to be identified. In order to identify proteins interacting with the entire Cx43 molecule (extracellular, transmembrane and intracellular domains), we produced and purified full-length recombinant Cx43 fused to glutathione S-transferase (GST-Cx43) and used it as "bait" in GST pull-down experiments. Cx43 cDNA was first cloned into the pDEST15 vector in order to construct a GST-fusion protein, using the Gateway system. The fusion protein GST-Cx43 was then expressed in Escherichia coli strain BL21-AI™ and purified by glutathione-affinity chromatography. The purified fusion protein exhibited the expected size of 70 kDa on SDS-PAGE, western blot and GST activity. A GST pull-down assay was used to show the capacity of the full-length recombinant protein to interact with known partners. Our results suggest that this method has the capacity to produce sufficient full-length recombinant protein for investigations aimed at identifying Cx43 partner proteins.
Assuntos
Conexina 43/isolamento & purificação , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Análise de Variância , Cromatografia de Afinidade , Clonagem Molecular , Conexina 43/biossíntese , Conexina 43/química , Conexina 43/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Glutationa Transferase/biossíntese , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Immunoblotting , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genéticaRESUMO
The Plasmodium falciparum protein VAR2CSA is a critical mediator of placental malaria, and VAR2CSA antibodies (IgGs) are important to protect pregnant women. Although infrequently detected outside pregnancy, VAR2CSA IgGs were reported in men and children from Colombia and Brazil and in select African populations. These findings raise questions about the specificity of VAR2CSA IgGs and the mechanisms by which they are acquired outside pregnancy. Here we review the data on VAR2CSA IgGs in men and children from different malaria-endemic regions. We discuss experimental factors that may affect interpretation of the serological data and consider the biological relevance of VAR2CSA IgGs in non-pregnant populations. We propose potential mechanisms for the acquisition of VARCSA IgGs outside of pregnancy. We identify knowledge gaps and research priorities.
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Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Humanos , Malária Falciparum/sangue , Malária Falciparum/imunologia , Pesquisa/tendênciasRESUMO
Trophoblastic cell-cell fusion is an essential event required during human placental development. Several membrane proteins have been described to be directly involved in this process, including connexin 43 (Cx43), syncytin 1 (Herv-W env), and syncytin 2 (Herv-FRD env glycoprotein). Recently, zona occludens (ZO) proteins (peripheral membrane proteins associated with tight junctions, adherens junctions, and gap junctions) were shown to be involved in mouse placental development. Moreover, zona occludens 1 (ZO-1) was localized mainly at the intercellular boundaries between human trophoblastic cells. Therefore the role of ZO-1 in the dynamic process of human trophoblastic cell-cell fusion was investigated using primary trophoblastic cells in culture. In vitro as in situ, ZO-1 was localized mainly at the intercellular boundaries between trophoblastic cells where its expression substantially decreased during differentiation and during fusion. At the same time, Cx43 was localized at the interface of trophoblastic cells and its expression increased during differentiation. To determine a functional role for ZO-1 during trophoblast differentiation, small interfering RNA (siRNA) was used to knock down ZO-1 expression. Cytotrophoblasts treated with ZO-1 siRNA fused poorly, but interestingly, decreased Cx43 expression without altering the functionality of trophoblastic cell-cell communication as measured by relative permeability time constant determined using gap-FRAP experiments. Because kinetics of Cx43 and ZO-1 proteins show a mirror image, a potential association of these two proteins was investigated. By using coimmunoprecipitation experiments, a physical interaction between ZO-1 and Cx43 was demonstrated. These results demonstrate that a decrease in ZO-1 expression reduces human trophoblast cell-cell fusion and differentiation.
Assuntos
Diferenciação Celular , Fusão Celular , Junções Intercelulares/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Placenta/metabolismo , Trofoblastos/metabolismo , Forma Celular , Células Cultivadas , Gonadotropina Coriônica/metabolismo , Conexina 43/metabolismo , Desmoplaquinas/metabolismo , Feminino , Recuperação de Fluorescência Após Fotodegradação , Idade Gestacional , Humanos , Imuno-Histoquímica , Imunoprecipitação , Cinética , Proteínas de Membrana/genética , Fosfoproteínas/genética , Placenta/citologia , Gravidez , Ligação Proteica , Interferência de RNA , Fatores de Tempo , Proteína da Zônula de Oclusão-1RESUMO
BACKGROUND: Pregnant women acquire protective antibodies that cross-react with geographically diverse placental Plasmodium falciparum isolates, suggesting that surface molecules expressed on infected erythrocytes by pregnancy-associated malaria (PAM) parasites have conserved epitopes and, that designing a PAM vaccine may be envisaged. VAR2CSA is the main candidate for a pregnancy malaria vaccine, but vaccine development may be complicated by its sequence polymorphism. METHODS: The dynamics of P. falciparum genotypes during pregnancy in 32 women in relation to VAR2CSA polymorphism and immunity was determined. The polymorphism of the msp2 gene and five microsatellites was analysed in consecutive parasite isolates, and the DBL5epsilon + Interdomain 5 (Id5) part of the var2csa gene of the corresponding samples was cloned and sequenced to measure variation. RESULTS: In primigravidae, the multiplicity of infection in the placenta was associated with occurrence of low birth weight babies. Some parasite genotypes were able to persist over several weeks and, still be present in the placenta at delivery particularly when the host anti-VAR2CSA antibody level was low. Comparison of diversity among genotyping markers confirmed that some PAM parasites may harbour more than one var2csa gene copy in their genome. CONCLUSIONS: Host immunity to VAR2CSA influences the parasite dynamics during pregnancy, suggesting that the acquisition of protective immunity requires pre-exposure to a limited number of parasite variants. Presence of highly conserved residues in surface-exposed areas of the VAR2CSA immunodominant DBL5epsilon domain, suggest its potential in inducing antibodies with broad reactivity.
Assuntos
Antígenos de Protozoários/genética , Malária Falciparum/imunologia , Placenta/parasitologia , Plasmodium falciparum/genética , Complicações Parasitárias na Gravidez/genética , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Antígenos de Protozoários/metabolismo , Sequência de Bases , Estudos de Coortes , Feminino , Variação Genética , Genótipo , Idade Gestacional , Humanos , Recém-Nascido , Malária Falciparum/parasitologia , Repetições de Microssatélites/genética , Dados de Sequência Molecular , Plasmodium falciparum/imunologia , Plasmodium falciparum/isolamento & purificação , Polimorfismo Genético/genética , Gravidez , Complicações Parasitárias na Gravidez/sangue , Complicações Parasitárias na Gravidez/imunologia , Proteínas de Protozoários/metabolismo , Senegal/epidemiologiaRESUMO
Many pathogens evolve extensive genetic variation in virulence proteins as a strategy to evade host immunity. This poses a significant challenge for the host to develop broadly neutralizing antibodies. In Plasmodium falciparum, we show that a mechanism to circumvent this challenge is to elicit antibodies to cryptic epitopes that are not under immune pressure. We previously discovered that antibodies to the Plasmodium vivax invasion protein, PvDBP, cross-react with P. falciparum VAR2CSA, a distantly related virulence factor that mediates placental malaria. Here, we describe the molecular mechanism underlying this cross-species immunity. We identified an epitope in subdomain 1 (SD1) within the Duffy binding-like (DBL) domain of PvDBP that gives rise to cross-reactive antibodies to VAR2CSA and show that human antibodies affinity purified against a synthetic SD1 peptide block parasite adhesion to chondroitin sulfate A (CSA) in vitro The epitope in SD1 is subdominant and highly conserved in PvDBP, and in turn, SD1 antibodies target cryptic epitopes in P. falciparum VAR2CSA. The epitopes in VAR2CSA recognized by vivax-derived SD1 antibodies (of human and mouse origin) are distinct from those recognized by VAR2CSA immune serum. We mapped two peptides in the DBL5ε domain of VAR2CSA that are recognized by SD1 antibodies. Both peptides map to regions outside the immunodominant sites, and antibodies to these peptides are not elicited following immunization with VAR2CSA or natural infection with P. falciparum in pregnancy, consistent with the cryptic nature of these target epitopes.IMPORTANCE In this work, we describe a molecular mechanism of heterologous immunity between two distant species of Plasmodium Our results suggest a mechanism that subverts the classic parasite strategy of presenting highly polymorphic epitopes in surface antigens to evade immunity to that parasite. This alternative immune pathway can be exploited to protect pregnant women from falciparum placental malaria by designing vaccines to cryptic epitopes that elicit broadly inhibitory antibodies against variant parasite strains.
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Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Epitopos/imunologia , Imunidade Heteróloga , Plasmodium falciparum/imunologia , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologia , Receptores de Superfície Celular/imunologia , Animais , Brasil , Adesão Celular , Sulfatos de Condroitina/metabolismo , Colômbia , Reações Cruzadas , Mapeamento de Epitopos , Humanos , Malária Falciparum/imunologia , Malária Vivax/imunologia , Camundongos , Uganda , Fatores de Virulência/imunologiaRESUMO
Objectives: Blood stage malaria parasites attenuated with seco-cyclopropyl pyrrolo indole (CPI) analogues induce robust immunity in mice to homologous and heterologous malaria parasites and are being considered for the development of a human vaccine. However, it is not understood how attenuated parasites induce immunity. We showed that following vaccination, parasite DNA persisted in blood for several months, raising the possibility that ongoing immune stimulation may be critical. However, parasites were not seen microscopically beyond 24 h postvaccination. We aimed to provide a mechanistic understanding of immune induction. Methods: Mice were vaccinated with chemically attenuated Plasmodium chabaudi parasites. PCR and adoptive transfer studies were used to determine the presence of parasites and antigen in vivo. In other experiments, Plasmodium falciparum parasitised red blood cells were attenuated in vitro and RNA and antigen expression studied. Results: We show that blood transferred from vaccinated mice into naïve mice activates T cells and induces complete protective immunity in the recipient mice strongly suggesting that there is persistence of parasite antigen postvaccination. This is supported by the presence of parasite RNA in vaccinated mice and both RNA and antigen expression in P. falciparum cultures treated with CPI drugs in vitro. In addition, drugs that block parasite growth also prevent the induction of immunity in vaccinated mice, indicating that some growth of attenuated parasites is required for immune induction. Conclusions: Attenuated parasites persist at submicroscopic levels in the blood of mice postvaccination with the ability to activate T cells and induce ongoing protective immune responses.
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In Latin America, four million pregnancies are at risk of malaria annually, but malaria in pregnancy is largely overlooked. As countries progress toward malaria elimination, targeting reservoirs of transmission is a priority. Pregnant women are an important risk group because they harbor asymptomatic infections and dormant liver stages of Plasmodium vivax that cause relapses. Of significant concern is the discovery that most infections in pregnant women fail to be detected by routine diagnostics. We review here recent findings on malaria in pregnancy within Latin America. We focus on the Amazon basin and Northwest Colombia, areas that harbor the greatest burden of malaria, and propose that more sensitive diagnostics and active surveillance at antenatal clinics will be necessary to eliminate malaria from these final frontiers.
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Malária/prevenção & controle , Complicações Parasitárias na Gravidez/prevenção & controle , Erradicação de Doenças , Feminino , Humanos , América Latina , Gravidez , Complicações Parasitárias na Gravidez/diagnósticoRESUMO
In the thyroid, iodotyrosine dehalogenase acts on the mono and diiodotyrosines released during the hydrolysis of thyroglobulin to liberate iodide, which can then reenter the hormone-producing pathways. It has been reported that the deiodination of iodotyrosines occurs predominantly in the microsomes and is mediated by NADPH. Recently, two cDNAs, 7401- and 7513-base pairs long that encode proteins with a conserved nitroreductase domain were published in GenBank as iodotyrosine dehalogenase 1 (DEHAL1) and iodotyrosine dehalogenase 1B (DEHAL1B), respectively. We report here our investigation of the localization and activity of one of these isoforms, DEHAL1. DEHAL1 mRNA is highly expressed in the thyroid, is up-regulated by cAMP, and encodes a transmembrane protein that efficiently catalyzes the NADPH-dependent deiodination of mono (L-MIT) and diiodotyrosine (L-DIT), with greater activity vs. L-MIT. Iodotyrosine deiodinase was active in HEK293 cells transfected by DEHAL1 cDNA, but not in CHO cells. A fraction of DEHAL1 protein is exposed to the cell surface, as indicated by biotinylation experiments. Immunohistochemistry studies showed that DEHAL1 proteins accumulate at the apical pole of thyrocytes. Taken together, these findings indicate that the deiodination reaction occurs at the apical pole of the thyrocyte and is involved in a rapid iodide recycling process at and/or close to the organification site.
Assuntos
Hidrolases/metabolismo , Iodo/metabolismo , Proteínas de Membrana/metabolismo , Tireoglobulina/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Membrana Celular/metabolismo , Polaridade Celular , Clonagem Molecular , Vesículas Citoplasmáticas/metabolismo , Humanos , Hidrolases/genética , Membranas Intracelulares/metabolismo , Iodeto Peroxidase/metabolismo , Proteínas de Membrana/genética , NADP/metabolismo , Nitrorredutases/genética , Nitrorredutases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Glândula Tireoide/citologia , Glândula Tireoide/metabolismoRESUMO
In placental malaria (PM), sequestration of infected erythrocytes in the placenta is mediated by an interaction between VAR2CSA, a Plasmodium falciparum protein expressed on erythrocytes, and chondroitin sulfate A (CSA) on syncytiotrophoblasts. Recent works have identified ID1-DBL2Xb as the minimal CSA-binding region within VAR2CSA able to induce strong protective immunity, making it the leading candidate for the development of a vaccine against PM. Assessing the existence of population differences in the distribution of ID1-DBL2Xb polymorphisms is of paramount importance to determine whether geographic diversity must be considered when designing a candidate vaccine based on this fragment. In this study, we examined patterns of sequence variation of ID1-DBL2Xb in a large collection of P. falciparum field isolates (n=247) from different malaria-endemic areas, including Africa (Benin, Senegal, Cameroon and Madagascar), Asia (Cambodia), Oceania (Papua New Guinea), and Latin America (Peru). Detection of variants and estimation of their allele frequencies were performed using next-generation sequencing of DNA pools. A considerable amount of variation was detected along the whole gene segment, suggesting that several allelic variants may need to be included in a candidate vaccine to achieve broad population coverage. However, most sequence variants were common and extensively shared among worldwide parasite populations, demonstrating long term persistence of those polymorphisms, probably maintained through balancing selection. Therefore, a vaccine mixture including such stable antigen variants will be putatively applicable and efficacious in all world regions where malaria occurs. Despite similarity in ID1-DBL2Xb allele repertoire across geographic areas, several peaks of strong population differentiation were observed at specific polymorphic loci, pointing out putative targets of humoral immunity subject to positive immune selection.
Assuntos
Antígenos de Protozoários/genética , Malária Falciparum/parasitologia , Placenta/parasitologia , Plasmodium falciparum/isolamento & purificação , Plasmodium falciparum/metabolismo , Complicações Parasitárias na Gravidez/parasitologia , Adolescente , Adulto , Variação Antigênica/imunologia , Antígenos de Protozoários/imunologia , DNA de Protozoário/genética , Feminino , Frequência do Gene , Variação Genética , Humanos , Malária Falciparum/epidemiologia , Malária Falciparum/imunologia , Filogeografia , Placenta/imunologia , Plasmodium falciparum/classificação , Plasmodium falciparum/imunologia , Gravidez , Complicações Parasitárias na Gravidez/imunologia , Análise de Sequência de DNA , Vacinas Virais/genética , Vacinas Virais/imunologia , Adulto JovemRESUMO
BACKGROUND: VAR2CSA is a large polymorphic Plasmodium falciparum protein expressed on infected erythrocytes (IE) that allows their binding in the placenta, thus precipitating placental malaria (PM). The N-terminal part of VAR2CSA that contains the binding site to placental chondroitin sulfate A (CSA) is currently recognized as the most attractive region for vaccine development. An ultimate challenge is to define epitopes in this region that induce a broad cross-reactive adhesion inhibitory antibody response. METHODS: Based on phylogenetic data that identified a dimorphic sequence motif in the VAR2CSA DBL2X, we raised antibodies against the NTS-DBL2X constructs containing one sequence or the other (3D7 and FCR3) and tested their functional properties on P. falciparum isolates from pregnant women and on laboratory-adapted strains. RESULTS: The CSA binding inhibitory capacity of the antibodies induced varied from one parasite isolate to another (range, 10%100%), but the combined analysis of individual activity highlighted a broader functionality that increased the total number of isolates inhibited. Interestingly, the differential inhibitory effect of the antibodies observed on field isolates resulted in significant inhibition of all field isolates tested, suggesting that optimal inhibitory spectrum on field isolates from pregnant women might be achieved with antibodies targeting limited variants of the N-terminal VAR2CSA. CONCLUSIONS: Our findings indicate that the NTS-DBL2X region of VAR2CSA can elicit strain-transcending anti-adhesion antibodies and suggest that the combination of the two major variants used here could represent the basis for an effective bivalent VAR2CSA-based vaccine.
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Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Adesão Celular/efeitos dos fármacos , Eritrócitos/fisiologia , Eritrócitos/parasitologia , Plasmodium falciparum/imunologia , Plasmodium falciparum/fisiologia , Adulto , Animais , Feminino , Humanos , Gravidez , Coelhos , Adulto JovemRESUMO
Genetic vaccination, consisting in delivering a genetically engineered plasmid DNA by a non-viral vector or technique into a tissue, is currently of great interest. New delivery technique including DNA transfer by electroporation recently greatly improved the potency of this concept. Because it avoids the step of producing a recombinant protein, it is particularly of use in studying the immunogenic properties of large proteins. Here we describe the use of electroporation mediated DNA immunization to identify important protective epitopes from the large VAR2CSA protein from Plasmodium falciparum implicated in the pathology of placental malaria. Immunizing mice and rabbit with DNA plasmids encoding different fragments of VAR2CSA leads to high titer antisera. Moreover an N-terminal region of the protein was found to induce protective functional antibodies.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/imunologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Complicações Parasitárias na Gravidez/prevenção & controle , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/genética , Antígenos de Protozoários/genética , Eletroporação , Feminino , Técnicas de Transferência de Genes , Imunização , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/genética , Malária Falciparum/complicações , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Camundongos , Placenta/efeitos dos fármacos , Placenta/imunologia , Placenta/parasitologia , Plasmídeos/genética , Plasmídeos/imunologia , Plasmodium falciparum/efeitos dos fármacos , Gravidez , Complicações Parasitárias na Gravidez/imunologia , Coelhos , Vacinas de DNARESUMO
BACKGROUND: Protection against pregnancy associated malaria (PAM) is associated with high levels of anti-VAR2CSA antibodies. This protection is obtained by the parity dependent acquisition of anti-VAR2CSA antibodies. Distinct parity-associated molecular signatures have been identified in VAR2CSA domains. These two observations combined point to the importance of identifying VAR2CSA sequence variation, which facilitate parasitic evasion or subversion of host immune response. Highly conserved domains of VAR2CSA such as DBL5ε are likely to contain conserved epitopes, and therefore do constitute attractive targets for vaccine development. METHODOLOGY/PRINCIPAL FINDINGS: VAR2CSA DBL5ε-domain sequences obtained from cDNA of 40 placental isolates were analysed by a combination of experimental and in silico methods. Competition ELISA assays on two DBL5ε variants, using plasma samples from women from two different areas and specific mice hyperimmune plasma, indicated that DBL5ε possess conserved and cross-reactive B cell epitopes. Peptide ELISA identified conserved areas that are recognised by naturally acquired antibodies. Specific antibodies against these peptides labelled the native proteins on the surface of placental parasites. Despite high DBL5ε sequence homology among parasite isolates, sequence analyses identified motifs in DBL5ε that discriminate parasites according to donor's parity. Moreover, recombinant proteins of two VAR2CSA DBL5ε variants displayed diverse recognition patterns by plasma from malaria-exposed women, and diverse proteoglycan binding abilities. CONCLUSIONS/SIGNIFICANCE: This study provides insights into conserved and exposed B cell epitopes in DBL5ε that might be a focus for cross reactivity. The importance of sequence variation in VAR2CSA as a critical challenge for vaccine development is highlighted. VAR2CSA conformation seems to be essential to its functionality. Therefore, identification of sequence variation sites in distinct locations within VAR2CSA, affecting antigenicity and/or binding properties, is critical to the effort of developing an efficient VAR2CSA-based vaccine. Motifs associated with parasite segregation according to parity constitute one such site.