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1.
PLoS Pathog ; 16(8): e1008772, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866214

RESUMO

The tick-borne apicomplexan parasite, Babesia bovis, a highly persistent bovine pathogen, expresses VESA1 proteins on the infected erythrocyte surface to mediate cytoadhesion. The cytoadhesion ligand, VESA1, which protects the parasite from splenic passage, is itself protected from a host immune response by rapid antigenic variation. B. bovis relies upon segmental gene conversion (SGC) as a major mechanism to vary VESA1 structure. Gene conversion has been considered a form of homologous recombination (HR), a process for which Rad51 proteins are considered pivotal components. This could make BbRad51 a choice target for development of inhibitors that both interfere with parasite genome integrity and disrupt HR-dependent antigenic variation. Previously, we knocked out the Bbrad51 gene from the B. bovis haploid genome, resulting in a phenotype of sensitivity to methylmethane sulfonate (MMS) and apparent loss of HR-dependent integration of exogenous DNA. In a further characterization of BbRad51, we demonstrate here that ΔBbrad51 parasites are not more sensitive than wild-type to DNA damage induced by γ-irradiation, and repair their genome with similar kinetics. To assess the need for BbRad51 in SGC, RT-PCR was used to observe alterations to a highly variant region of ves1α transcripts over time. Mapping of these amplicons to the genome revealed a significant reduction of in situ transcriptional switching (isTS) among ves loci, but not cessation. By combining existing pipelines for analysis of the amplicons, we demonstrate that SGC continues unabated in ΔBbrad51 parasites, albeit at an overall reduced rate, and a reduction in SGC tract lengths was observed. By contrast, no differences were observed in the lengths of homologous sequences at which recombination occurred. These results indicate that, whereas BbRad51 is not essential to babesial antigenic variation, it influences epigenetic control of ves loci, and its absence significantly reduces successful variation. These results necessitate a reconsideration of the likely enzymatic mechanism(s) underlying SGC and suggest the existence of additional targets for development of small molecule inhibitors.


Assuntos
Antígenos de Protozoários , Babesia bovis , Conversão Gênica/imunologia , Genoma de Protozoário/imunologia , Proteínas de Protozoários , Rad51 Recombinase , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Babesia bovis/genética , Babesia bovis/imunologia , DNA de Protozoário/genética , DNA de Protozoário/imunologia , Haploidia , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Rad51 Recombinase/genética , Rad51 Recombinase/imunologia
2.
PLoS Pathog ; 16(9): e1008799, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898164

RESUMO

Professional antigen-presenting cells (APCs), like macrophages (Mϕs) and dendritic cells (DCs), are central players in the induction of natural and vaccine-induced immunity to malaria, yet very little is known about the interaction of SPZ with human APCs. Intradermal delivery of whole-sporozoite vaccines reduces their effectivity, possibly due to dermal immunoregulatory effects. Therefore, understanding these interactions could prove pivotal to malaria vaccination. We investigated human APC responses to recombinant circumsporozoite protein (recCSP), SPZ and anti-CSP opsonized SPZ both in monocyte derived MoDCs and MoMϕs. Both MoDCs and MoMϕs readily took up recCSP but did not change phenotype or function upon doing so. SPZ are preferentially phagocytosed by MoMϕs instead of DCs and phagocytosis greatly increased after opsonization. Subsequently MoMϕs show increased surface marker expression of activation markers as well as tolerogenic markers such as Programmed Death-Ligand 1 (PD-L1). Additionally they show reduced motility, produce interleukin 10 and suppressed interferon gamma (IFNγ) production by antigen specific CD8+ T cells. Importantly, we investigated phenotypic responses to SPZ in primary dermal APCs isolated from human skin explants, which respond similarly to their monocyte-derived counterparts. These findings are a first step in enhancing our understanding of pre-erythrocytic natural immunity and the pitfalls of intradermal vaccination-induced immunity.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Macrófagos/imunologia , Malária/imunologia , Plasmodium berghei/imunologia , Proteínas de Protozoários/imunologia , Pele/imunologia , Esporozoítos/imunologia , Animais , Células Cultivadas , Feminino , Humanos , Macrófagos/parasitologia , Malária/parasitologia , Camundongos , Pele/parasitologia
3.
PLoS One ; 15(9): e0238840, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32925980

RESUMO

Diagnosis of visceral leishmaniasis (VL) relies on invasive and risky aspirate procedures, and confirmation of cure after treatment is unreliable. Detection of Leishmania donovani antigens in urine has the potential to provide both a non-invasive diagnostic and a test of cure. We searched for L. donovani antigens in urine of VL patients from India and Sudan to contribute to the development of urine antigen capture immunoassays. VL urine samples were incubated with immobilised anti-L. donovani polyclonal antibodies and captured material was eluted. Sudanese eluted material and concentrated VL urine were analysed by western blot. Immunocaptured and immunoreactive material from Indian and Sudanese urine was submitted to mass spectrometry for protein identification. We identified six L. donovani proteins from VL urine. Named proteins were 40S ribosomal protein S9, kinases, and others were hypothetical. Thirty-three epitope regions were predicted with high specificity in the 6 proteins. Of these, 20 were highly specific to Leishmania spp. and are highly suitable for raising antibodies for the subsequent development of an antigen capture assay. We present all the identified proteins and analysed epitope regions in full so that they may contribute to the development of non-invasive immunoassays for this deadly disease.


Assuntos
Anticorpos Antiprotozoários/urina , Antígenos de Protozoários/urina , Leishmania donovani/imunologia , Leishmaniose Visceral/diagnóstico , Proteínas de Protozoários/urina , Adulto , Anticorpos Antiprotozoários/imunologia , Anticorpos Antiprotozoários/isolamento & purificação , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/isolamento & purificação , Estudos de Casos e Controles , Humanos , Índia/epidemiologia , Leishmania donovani/isolamento & purificação , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/urina , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/isolamento & purificação
4.
Parasitol Res ; 119(11): 3639-3648, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32930858

RESUMO

Due to its wide presence in apicomplexan parasites as well as high polymorphism and antigenic diversity, the variable merozoite surface antigen (VMSA) family in Babesia sp. has attracted increasing attention of researchers. Here, all the reported VMSA genes of Babesia spp. were obtained from GenBank, and multiple alignments were performed by using conserved regions to blast the Babesia orientalis genome database (unpublished data). Five MSA genes (named MSA-2a1, MSA-2a2, MSA-2c1, MSA-1, and MSA-2c2, respectively) were identified, sequenced, and cloned from B. orientalis, which were shown to encode proteins with open reading frames ranging in size from 266 (MSA-2c1) to 317 (MSA-1) amino acids. All the five proteins contain an MSA-2c superfamily conserved domain, with an identical signal peptide and glycosyl phosphatidyl inositol (GPI)-anchor for each of them. The five proteins were also predicted to contain B cell epitopes, with only three for BoMSA-2c1, the smallest protein in the BoVMSA family, while at least six for each of the others. Notably, BoMSA-2a1 has 2 identical copies, a specific phenomenon only present in B. orientalis. This research has determined the MSA genes of B. orientalis and provides a genetic basis for further research of functional genes in B. orientalis.


Assuntos
Antígenos de Protozoários/genética , Babesia/genética , Proteínas de Protozoários/genética , Animais , Antígenos de Protozoários/imunologia , Antígenos de Superfície/genética , Babesia/imunologia , Epitopos de Linfócito B , Glicosilfosfatidilinositóis/análise , Proteína 1 de Superfície de Merozoito/genética , Merozoítos/química , Merozoítos/imunologia , Fases de Leitura Aberta , Polimorfismo Genético , Proteínas de Protozoários/imunologia
5.
Am J Trop Med Hyg ; 103(5): 1902-1909, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32840197

RESUMO

In malaria-endemic countries, rapid diagnostic tests (RDTs) targeting Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and lactate dehydrogenase (PfLDH) have been widely used. However, little is known regarding the diagnostic performances of these RDTs in the Assosa zone of northwest Ethiopia. The objective of this study was to determine the diagnostic performances of PfHRP2 and PfLDH RDTs using microscopy and quantitative PCR (qPCR) as a reference test. A health facility-based cross-sectional study design was conducted from malaria-suspected study participants at selected health centers from November to December 2018. Finger-prick blood samples were collected for microscopy, RDTs, and qPCR method. The prevalence of P. falciparum was 26.4%, 30.3%, and 24.1% as determined by microscopy, PfHRP2 RDT, and PfLDH RDT, respectively. Compared with microscopy, the sensitivity and specificity of the PfHRP2 RDT were 96% and 93%, respectively, and those of the PfLDH RDT were 89% and 99%, respectively. Compared with qPCR, the specificity of the PfHRP2 RDT (93%) and PfLDH RDT (98%) was high, but the sensitivity of the PfHRP2 RDT (77%) and PfLDH RDT (70%) was relatively low. These malaria RDTs and reference microscopy methods showed reasonable agreement with a kappa value above 0.85 and provided accurate diagnosis of P. falciparum malaria. Thus, the current malaria RDT in the Ministry of Health program can be used in the Assosa zone of Ethiopia. However, continuous monitoring of the performance of PfHRP2 RDT is important to support control and elimination of malaria in Ethiopia.


Assuntos
Antígenos de Protozoários/imunologia , Testes Diagnósticos de Rotina/métodos , L-Lactato Desidrogenase/imunologia , Malária Falciparum/diagnóstico , Plasmodium falciparum/isolamento & purificação , Proteínas de Protozoários/imunologia , Adolescente , Adulto , Antígenos de Protozoários/genética , Criança , Pré-Escolar , Estudos Transversais , Demografia , Etiópia , Feminino , Geografia , Humanos , L-Lactato Desidrogenase/genética , Malária Falciparum/parasitologia , Masculino , Microscopia , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Reação em Cadeia da Polimerase , Proteínas de Protozoários/genética , Sensibilidade e Especificidade , Adulto Jovem
6.
PLoS Negl Trop Dis ; 14(7): e0008202, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32645098

RESUMO

Plasmodium vivax is the most widespread and difficult to treat cause of human malaria. The development of vaccines against the blood stages of P. vivax remains a key objective for the control and elimination of vivax malaria. Erythrocyte binding-like (EBL) protein family members such as Duffy binding protein (PvDBP) are of critical importance to erythrocyte invasion and have been the major target for vivax malaria vaccine development. In this study, we focus on another member of EBL protein family, P. vivax erythrocyte binding protein (PvEBP). PvEBP was first identified in Cambodian (C127) field isolates and has subsequently been showed its preferences for binding reticulocytes which is directly inhibited by antibodies. We analysed PvEBP sequence from 316 vivax clinical isolates from eight countries including China (n = 4), Ethiopia (n = 24), Malaysia (n = 53), Myanmar (n = 10), Papua New Guinea (n = 16), Republic of Korea (n = 10), Thailand (n = 174), and Vietnam (n = 25). PvEBP gene exhibited four different phenotypic clusters based on the insertion/deletion (indels) variation. PvEBP-RII (179-479 aa.) showed highest polymorphism similar to other EBL family proteins in various Plasmodium species. Whereas even though PvEBP-RIII-V (480-690 aa.) was the most conserved domain, that showed strong neutral selection pressure for gene purifying with significant population expansion. Antigenicity of both of PvEBP-RII (16.1%) and PvEBP-RIII-V (21.5%) domains were comparatively lower than other P. vivax antigen which expected antigens associated with merozoite invasion. Total IgG recognition level of PvEBP-RII was stronger than PvEBP-RIII-V domain, whereas total IgG inducing level was stronger in PvEBP-RIII-V domain. These results suggest that PvEBP-RII is mainly recognized by natural IgG for innate protection, whereas PvEBP-RIII-V stimulates IgG production activity by B-cell for acquired immunity. Overall, the low antigenicity of both regions in patients with vivax malaria likely reflects genetic polymorphism for strong positive selection in PvEBP-RII and purifying selection in PvEBP-RIII-V domain. These observations pose challenging questions to the selection of EBP and point out the importance of immune pressure and polymorphism required for inclusion of PvEBP as a vaccine candidate.


Assuntos
Variação Genética , Malária Vivax/imunologia , Plasmodium vivax/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Sequência de Aminoácidos , Anticorpos Antiprotozoários/imunologia , Ásia , Humanos , Imunidade Humoral , Malária Vivax/parasitologia , Plasmodium vivax/química , Plasmodium vivax/imunologia , Polimorfismo Genético , Proteínas de Protozoários/química , Seleção Genética , Alinhamento de Sequência
7.
BMC Infect Dis ; 20(1): 493, 2020 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-32650739

RESUMO

BACKGROUND: Toxoplasma gondii is an obligate intracellular parasite that can infect almost all warm-blooded animals, avian species and humans. Toxoplasmosis is asymptomatic in healthy individuals, whereas it may lead to death in immune suppressed or deficient patients. A vaccine against T. gondii is required to prevent consequences of the infection. The aim of this study is to generate a multivalent recombinant protein vaccine against T. gondii. METHODS: 49 previously discovered antigenic proteins of T gondii were evaluated by their expression level in E. coli and by comprehensive bioinformatics analyses to determine antigenic epitopes. Based on these analyses, six vaccine candidate proteins were selected to generate a hexavalent recombinant protein vaccine adjuvanted with Montanide ISA 50 V. Humoral and cellular immune responses were determined by flow cytometry and ELISA. Vaccinated mice were challenged with T. gondii Ankara strain tachyzoites. RESULTS: In mice vaccinated with hexavalent vaccine, strong total IgG (P < 0.0001) and IgG2a (P < 0.001) responses were induced compared to controls, the ratio of CD4+ and CD8+ T lymphocytes secreting IFN-γ increased, and significantly higher extracellular IFN-γ secretion was achieved compared to the controls (P < 0.001). The survival time of the vaccinated mice increased to 8.38 ± 2.13 days which was significantly higher than controls (P < 0.01). CONCLUSIONS: Altogether, these results show that the hexavalent vaccine which is developed for the first time against T. gondii induced strong and balanced Th1 and Th2 immune responses as well as conferred significant protection against challenge with lethal toxoplasmosis in murine model.


Assuntos
Adjuvantes Imunológicos/farmacologia , Manitol/análogos & derivados , Vacinas Protozoárias/farmacologia , Toxoplasmose/prevenção & controle , Vacinas de DNA/farmacologia , Animais , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Epitopos/imunologia , Escherichia coli/genética , Feminino , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Imunoglobulina G/sangue , Manitol/farmacologia , Camundongos , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/genética , Vacinas Protozoárias/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Toxoplasma/patogenicidade , Toxoplasmose/imunologia , Vacinas de DNA/imunologia
8.
Am J Trop Med Hyg ; 103(2): 778-784, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32602431

RESUMO

The ultrasensitive Alere Plasmodium falciparum Malaria Ag histidine-rich protein 2 rapid diagnostic test (Alere uRDT, Suwon City, South Korea) is a new diagnostic tool which is more expensive than other malaria rapid diagnostic tests (RDTs) routinely used in Ugandan clinics. The manufacturer recommends testing samples within 2 days and scoring results after 20 minutes, which may be impractical in high-volume resource-poor clinics. We compared testing by the Alere Ag rapid diagnostic test (uRDT), CareStart RDT, microscopy, and an ultrasensitive I8S rRNA quantitative reverse transcription polymerase chain reaction (qRT-PCR) using survey and clinical samples. For the Alere uRDT, we used survey blood samples stored at 4°C for 44 days and for some clinical samples deliberately scored results beyond 20 minutes. The Alere uRDT and qRT-PCR identified asymptomatic parasitemia cases in 56% and 72%, respectively, of survey samples originally scored as negative by the CareStart RDT. Using qRT-PCR as a gold standard, the Alere uRDT was superior to the CareStart RDT in estimating asymptomatic parasite prevalence in a cross-sectional survey (P = 0.007) and in detection of clinically significant malaria; both RDTs were comparable in detecting asymptomatic parasitemia in the clinic (P = 0.599). Scoring Alere uRDT results at 20 minutes produced valid results confirmed by the CareStart RDT, but there was a consistent background; scoring the Alere uRDT beyond 20 minutes produced false-positive results. The Alere uRDT outperformed the CareStart RDT (ACCESSBIO, Somerset, NJ) in a field survey in estimating malaria prevalence and in the clinic for symptomatic malarial illness. It produced reliable results using samples stored at 4°C for 44 days, but test results read beyond 20 minutes were invalid.


Assuntos
Antígenos de Protozoários/imunologia , Malária Falciparum/diagnóstico , Parasitemia/diagnóstico , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Adolescente , Adulto , Infecções Assintomáticas , Criança , Pré-Escolar , Reações Falso-Positivas , Feminino , Humanos , Lactente , Masculino , Microscopia , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , RNA Ribossômico 18S/genética , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Fatores de Tempo , Uganda
9.
Exp Parasitol ; 216: 107944, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32619431

RESUMO

Evaluation of the murine isotype antibodies is essential in subunit vaccine development because inbred mouse strains with diverse genetic backgrounds respond different to recombinant proteins. In this regard, the main goal of this study was to measuring and comparing the profile of IgG isotype responses in C57BL/6 mice. For this purpose, the extracellular region of plasmodium vivax thrombospondin-related adhesive protein (PvTRAP) gene was expressed in Escherichia coli Rosetta (DE3)-pET23a. Then, the recombinant PvTRAP alone or emulsified with Freund's complete adjuvant were applied for immunization of the C57BL/6 mice. The role of antibodies and cellular immune responses induced by recombinant PvTRAP were evaluated. The results showed the level of anti-rPvTRAP IgG2c was significantly higher than IgG2a in the groups that received rPvTRAP alone (mean OD490 = 0.798 ± 0.12 and 0.39 ± 0.1, respectively) and emulsified with CFA/IFA (mean OD490 = 1.48 ± 0.07 and 0.605 ± 0.13, respectively; P < 0.05, independent sample t-test). Additionally, the immunized mice with rPvTRAP and rPvTRAP + CFA/IFA had an intermediate-avidity IgG2a antibody but high-avidity IgG2c antibody as well as the mean of serum antibody titers results exhibited that in both rPvTRAP and rPvTRAP + CFA/IFA mouse groups, IgG2a end-point titer (1:3200 and 1:25,600, respectively) was noteworthy lower than IgG2c (1:25,600 and 1:102,400, respectively). Moreover, the results revealed the eliciting significant levels of IFN-γ (P < 0.05, independent sample t-test) and no detectable level of IL-4 in the mouse groups received rPvTRAP alone and emulsified with CFA/IFA as compared to the mouse control groups. In general, our results showed that for correctly interpreting of Th1 immune responses in C57BL/6 mouse strain it is critical to measure IgG2c instead of IgG2a along with IFN-γ.


Assuntos
Imunoglobulina G/sangue , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/imunologia , Células Th1/imunologia , Células Th2/imunologia , Animais , Afinidade de Anticorpos , Dicroísmo Circular , Feminino , Imunofluorescência , Imunoglobulina G/classificação , Interferon gama/análise , Interleucina-4/análise , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/imunologia , Vacinas de Subunidades/imunologia , Vacinas Sintéticas/imunologia
10.
Sci Rep ; 10(1): 9398, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32523082

RESUMO

Incomplete non-sterile immunity to malaria is attained in endemic regions after recurrent infections by a large percentage of the adult population, who carry the malaria parasite asymptomatically. Although blood-stage Plasmodium falciparum rapidly elicits IgG responses, the target antigens of partially protective and non-protective IgG antibodies as well as the basis for the acquisition of these antibodies remain largely unknown. We performed IgG-immunomics to screen for P. falciparum antigens and to identify epitopes associated with exposure and clinical disease. Sera from malaria cases identified five prevalent antigens recognized by all analyzed patients' IgGs. Epitope mapping of them, using adult and children sera samples from an endemic malaria region in Ghana segregated into patients with positive or negative subclinical detection of P. falciparum, revealed binding specificity for two 20-mer immunodominant antigenic regions within the START-related lipid transfer protein and the protein disulfide isomerase PDI8. These 20-mer epitopes challenged with sera samples from children under 5 years old displayed specific IgG binding in those with detectable parasitemia, even at subclinical level. These results suggest that humoral response against START and PDI8 antigens may be triggered at submicroscopic parasitemia levels in children and may eventually be used to differentially diagnose subclinical malaria in children.


Assuntos
Epitopos/imunologia , Imunoglobulina G/imunologia , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Adolescente , Adulto , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Criança , Mapeamento de Epitopos/métodos , Feminino , Gana , Humanos , Malária Falciparum/parasitologia , Masculino , Parasitemia/imunologia , Parasitemia/parasitologia , Proteínas de Protozoários/imunologia , Adulto Jovem
11.
PLoS One ; 15(6): e0233840, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32555601

RESUMO

BACKGROUND: Immunization with radiation-attenuated sporozoites (RAS) by mosquito bite provides >90% sterile protection against Plasmodium falciparum (Pf) malaria in humans. RAS invade hepatocytes but do not replicate. CD8+ T cells recognizing parasite-derived peptides on the surface of infected hepatocytes are likely the primary protective mechanism. We conducted a randomized clinical trial of RAS immunization to assess safety, to achieve 50% vaccine efficacy (VE) against controlled human malaria infection (CHMI), and to generate reagents from protected and non-protected subjects for future identification of protective immune mechanisms and antigens. METHODS: Two cohorts (Cohort 1 and Cohort 2) of healthy, malaria-naïve, non-pregnant adults age 18-50 received five monthly immunizations with infected (true-immunized, n = 21) or non-infected (mock-immunized, n = 5) mosquito bites and underwent homologous CHMI at 3 weeks. Immunization parameters were selected for 50% protection based on prior clinical data. Leukapheresis was done to collect plasma and peripheral blood mononuclear cells. RESULTS: Adverse event rates were similar in true- and mock-immunized subjects. Two true- and two mock-immunized subjects developed large local reactions likely caused by mosquito salivary gland antigens. In Cohort 1, 11 subjects received 810-1235 infected bites; 6/11 (55%) were protected against CHMI vs. 0/3 mock-immunized and 0/6 infectivity controls (VE 55%). In Cohort 2, 10 subjects received 839-1131 infected bites with a higher first dose and a reduced fifth dose; 9/10 (90%) were protected vs. 0/2 mock-immunized and 0/6 controls (VE 90%). Three/3 (100%) protected subjects administered three booster immunizations were protected against repeat CHMI vs. 0/6 controls (VE 100%). Cohort 2 uniquely showed a significant rise in IFN-γ responses after the third and fifth immunizations and higher antibody responses to CSP. CONCLUSIONS: PfRAS were generally safe and well tolerated. Cohort 2 had a higher first dose, reduced final dose, higher antibody responses to CSP and significant rise of IFN-γ responses after the third and fifth immunizations. Whether any of these factors contributed to increased protection in Cohort 2 requires further investigation. A cryobank of sera and cells from protected and non-protected individuals was generated for future immunological studies and antigen discovery. TRIAL REGISTRATION: ClinicalTrials.gov NCT01994525.


Assuntos
Mordeduras e Picadas de Insetos/imunologia , Malária/prevenção & controle , Esporozoítos/imunologia , Vacinação/métodos , Vacinas Atenuadas/efeitos adversos , Adulto , Animais , Anopheles/parasitologia , Anopheles/fisiologia , Feminino , Raios gama , Humanos , Malária/imunologia , Masculino , Pessoa de Meia-Idade , Mosquitos Vetores/parasitologia , Mosquitos Vetores/fisiologia , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/imunologia , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/imunologia , Esporozoítos/patogenicidade , Esporozoítos/efeitos da radiação , Vacinação/efeitos adversos
12.
PLoS Pathog ; 16(5): e1008572, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32413093

RESUMO

The apicomplexan Toxoplasma gondii induces strong protective immunity dependent upon recognition by Toll-like receptors (TLR)11 and 12 operating in conjunction with MyD88 in the murine host. However, TLR11 and 12 proteins are not present in humans, inspiring us to investigate MyD88-independent pathways of resistance. Using bicistronic IL-12-YFP reporter mice on MyD88+/+ and MyD88-/- genetic backgrounds, we show that CD11c+MHCII+F4/80- dendritic cells, F4/80+ macrophages, and Ly6G+ neutrophils were the dominant cellular sources of IL-12 in both wild type and MyD88 deficient mice after parasite challenge. Parasite dense granule protein GRA24 induces p38 MAPK activation and subsequent IL-12 production in host macrophages. We show that Toxoplasma triggers an early and late p38 MAPK phosphorylation response in MyD88+/+ and MyD88-/- bone marrow-derived macrophages. Using the uracil auxotrophic Type I T. gondii strain cps1-1, we demonstrate that the late response does not require active parasite proliferation, but strictly depends upon GRA24. By i. p. inoculation with cps1-1 and cps1-1:Δgra24, we identified unique subsets of chemokines and cytokines that were up and downregulated by GRA24. Finally, we demonstrate that cps1-1 triggers a strong host-protective GRA24-dependent Th1 response in the absence of MyD88. Our data identify GRA24 as a major mediator of p38 MAPK activation, IL-12 induction and protective immunity that operates independently of the TLR/MyD88 cascade.


Assuntos
Interleucina-12/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Macrófagos/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Proteínas de Protozoários/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Animais , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Interleucina-12/genética , Sistema de Sinalização das MAP Quinases/genética , Macrófagos/parasitologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Proteínas de Protozoários/genética , Toxoplasma/genética , Toxoplasmose/genética , Toxoplasmose/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética
13.
PLoS One ; 15(5): e0232786, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32379804

RESUMO

BACKGROUND: A low proportion of P. vivax-exposed individuals acquire protective strain-transcending neutralizing IgG antibodies that are able to block the interaction between the Duffy binding protein II (DBPII) and its erythrocyte-specific invasion receptor. In a recent study, a novel surface-engineered DBPII-based vaccine termed DEKnull-2, whose antibody response target conserved DBPII epitopes, was able to induce broadly binding-inhibitory IgG antibodies (BIAbs) that inhibit P. vivax reticulocyte invasion. Toward the development of DEKnull-2 as an effective P. vivax blood-stage vaccine, we investigate the relationship between naturally acquired DBPII-specific IgM response and the profile of IgG antibodies/BIAbs activity over time. METHODOLOGY/PRINCIPAL FINDINGS: A nine-year follow-up study was carried-out among long-term P. vivax-exposed Amazonian individuals and included six cross-sectional surveys at periods of high and low malaria transmission. DBPII immune responses associated with either strain-specific (Sal1, natural DBPII variant circulating in the study area) or conserved epitopes (DEKnull-2) were monitored by conventional serology (ELISA-detected IgM and IgG antibodies), with IgG BIAbs activity evaluated by functional assays (in vitro inhibition of DBPII-erythrocyte binding). The results showed a tendency of IgM antibodies toward Sal1-specific response; the profile of Sal1 over DEKnull-2 was not associated with acute malaria and sustained throughout the observation period. The low malaria incidence in two consecutive years allowed us to demonstrate that variant-specific IgG (but not IgM) antibodies waned over time, which resulted in IgG skewed to the DEKnull-2 response. A persistent DBPII-specific IgM response was not associated with the presence (or absence) of broadly neutralizing IgG antibody response. CONCLUSIONS/SIGNIFICANCE: The current study demonstrates that long-term exposure to low and unstable levels of P. vivax transmission led to a sustained DBPII-specific IgM response against variant-specific epitopes, while sustained IgG responses are skewed to conserved epitopes. Further studies should investigate on the role of a stable and persistent IgM antibody response in the immune response mediated by DBPII.


Assuntos
Antígenos de Protozoários/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Vacinas Antimaláricas/uso terapêutico , Malária Vivax/prevenção & controle , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologia , Receptores de Superfície Celular/imunologia , Adulto , Anticorpos Neutralizantes/imunologia , Formação de Anticorpos , Feminino , Humanos , Vacinas Antimaláricas/imunologia , Malária Vivax/imunologia , Masculino , Pessoa de Meia-Idade
14.
Trends Parasitol ; 36(6): 545-559, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32359873

RESUMO

Despite ongoing efforts, a highly effective vaccine against Plasmodium falciparum remains elusive. Vaccines targeting the pre-erythrocytic stages of the P. falciparum life cycle are the most advanced to date, affording moderate levels of efficacy in field trials. However, the discovery that the members of the merozoite PfRH5-PfCyRPA-PfRipr (RCR) complex are capable of inducing strain-transcendent neutralizing antibodies has renewed enthusiasm for the possibility of preventing disease by targeting the parasite during the blood stage of infection. With Phase I/II clinical trials now underway using first-generation vaccines against PfRH5, and more on the horizon for PfCyRPA and PfRipr, this review explores the rationale and future potential of the RCR complex as a P. falciparum vaccine target.


Assuntos
Vacinas Antimaláricas/imunologia , Malária/prevenção & controle , Proteínas de Protozoários/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos de Protozoários/imunologia , Proteínas de Transporte/imunologia , Ensaios Clínicos como Assunto , Malária/imunologia , Plasmodium falciparum/imunologia
15.
PLoS Pathog ; 16(5): e1008586, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32453782

RESUMO

The murine innate immune response against Toxoplasma gondii is predominated by the interaction of TLR11/12 with Toxoplasma profilin. However, mice lacking Tlr11 or humans, who do not have functional TLR11 or TLR12, still elicit a strong innate immune response upon Toxoplasma infection. The parasite factors that determine this immune response are largely unknown. Herein, we investigated two dense granule proteins (GRAs) secreted by Toxoplasma, GRA15 and GRA24, for their role in stimulating the innate immune response in Tlr11-/- mice and in human cells, which naturally lack TLR11/TLR12. Our results show that GRA15 and GRA24 synergistically shape the early immune response and parasite virulence in Tlr11-/- mice, with GRA15 as the predominant effector. Nevertheless, acute virulence in Tlr11-/- mice is still dominated by allelic combinations of ROP18 and ROP5, which are effectors that determine evasion of the immunity-related GTPases. In human macrophages, GRA15 and GRA24 play a major role in the induction of IL12, IL18 and IL1ß secretion. We further show that GRA15/GRA24-mediated IL12, IL18 and IL1ß secretion activates IFNγ secretion by peripheral blood mononuclear cells (PBMCs), which controls Toxoplasma proliferation. Taken together, our study demonstrates the important role of GRA15 and GRA24 in activating the innate immune response in hosts lacking TLR11.


Assuntos
Imunidade Inata/imunologia , Macrófagos/imunologia , Proteínas de Protozoários/imunologia , Receptores Toll-Like/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Animais , Citocinas/genética , Citocinas/imunologia , Humanos , Imunidade Inata/genética , Macrófagos/parasitologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Células RAW 264.7 , Receptores Toll-Like/genética , Toxoplasmose/genética , Toxoplasmose/patologia
16.
Nat Med ; 26(7): 1135-1145, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32451496

RESUMO

The circumsporozoite protein of the human malaria parasite Plasmodium falciparum (PfCSP) is the main target of antibodies that prevent the infection and disease, as shown in animal models. However, the limited efficacy of the PfCSP-based vaccine RTS,S calls for a better understanding of the mechanisms driving the development of the most potent human PfCSP antibodies and identification of their target epitopes. By characterizing 200 human monoclonal PfCSP antibodies induced by sporozoite immunization, we establish that the most potent antibodies bind around a conserved (N/D)PNANPN(V/A) core. High antibody affinity to the core correlates with protection from parasitemia in mice and evolves around the recognition of NANP motifs. The data suggest that the rational design of a next-generation PfCSP vaccine that elicits high-affinity antibody responses against the core epitope will promote the induction of protective humoral immune responses.


Assuntos
Anticorpos Antiprotozoários/imunologia , Vacinas Antimaláricas/imunologia , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Animais , Formação de Anticorpos/imunologia , Epitopos/imunologia , Evolução Molecular , Feminino , Humanos , Imunidade Humoral , Vacinas Antimaláricas/genética , Malária Falciparum/genética , Malária Falciparum/parasitologia , Malária Falciparum/prevenção & controle , Camundongos , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Esporozoítos/imunologia , Esporozoítos/patogenicidade
17.
Sci Rep ; 10(1): 8443, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32439948

RESUMO

Microscopy is the gold standard for malaria epidemiology, but laboratory and point-of-care (POC) tests detecting parasite antigen, DNA, and human antibodies against malaria have expanded this capacity. The island nation of Haiti is endemic for Plasmodium falciparum (Pf) malaria, though at a low national prevalence and heterogenous geospatial distribution. In 2015 and 2016, serosurveys were performed of children (ages 6-7 years) sampled in schools in Saut d'Eau commune (n = 1,230) and Grand Anse department (n = 1,664) of Haiti. Children received malaria antigen rapid diagnostic test and provided a filter paper blood sample for further laboratory analysis of the Pf histidine-rich protein 2 (HRP2) antigen, Pf DNA, and anti-Pf IgG antibodies. Prevalence of Pf infection ranged from 0.0-16.7% in 53 Saut d'Eau schools, and 0.0-23.8% in 56 Grand Anse schools. Anti-Pf antibody carriage exceeded 80% of students in some schools from both study sites. Geospatial prediction ellipses were created to indicate clustering of positive tests within the survey areas and overlay of all prediction ellipses for the different types of data revealed regions with high likelihood of active and ongoing Pf malaria transmission. The geospatial utilization of different types of Pf data can provide high confidence for spatial epidemiology of the parasite.


Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , DNA de Protozoário/genética , Testes Diagnósticos de Rotina/métodos , Malária Falciparum/diagnóstico , Plasmodium falciparum/isolamento & purificação , Proteínas de Protozoários/imunologia , Criança , DNA de Protozoário/análise , Feminino , Geografia , Haiti/epidemiologia , Humanos , Testes Imunológicos , Malária Falciparum/epidemiologia , Malária Falciparum/parasitologia , Malária Falciparum/transmissão , Masculino , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Análise Espacial
18.
Nature ; 582(7810): 104-108, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32427965

RESUMO

Malaria caused by Plasmodium falciparum remains the leading single-agent cause of mortality in children1, yet the promise of an effective vaccine has not been fulfilled. Here, using our previously described differential screening method to analyse the proteome of blood-stage P. falciparum parasites2, we identify P. falciparum glutamic-acid-rich protein (PfGARP) as a parasite antigen that is recognized by antibodies in the plasma of children who are relatively resistant-but not those who are susceptible-to malaria caused by P. falciparum. PfGARP is a parasite antigen of 80 kDa that is expressed on the exofacial surface of erythrocytes infected by early-to-late-trophozoite-stage parasites. We demonstrate that antibodies against PfGARP kill trophozoite-infected erythrocytes in culture by inducing programmed cell death in the parasites, and that vaccinating non-human primates with PfGARP partially protects against a challenge with P. falciparum. Furthermore, our longitudinal cohort studies showed that, compared to individuals who had naturally occurring anti-PfGARP antibodies, Tanzanian children without anti-PfGARP antibodies had a 2.5-fold-higher risk of severe malaria and Kenyan adolescents and adults without these antibodies had a twofold-higher parasite density. By killing trophozoite-infected erythrocytes, PfGARP could synergize with other vaccines that target parasite invasion of hepatocytes or the invasion of and egress from erythrocytes.


Assuntos
Apoptose/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Malária Falciparum/imunologia , Malária Falciparum/prevenção & controle , Parasitos/imunologia , Plasmodium falciparum/citologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Adolescente , Adulto , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Aotidae/imunologia , Aotidae/parasitologia , Caspases/metabolismo , Criança , Estudos de Coortes , DNA de Protozoário/química , DNA de Protozoário/metabolismo , Ativação Enzimática , Eritrócitos/parasitologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Quênia , Vacinas Antimaláricas/imunologia , Malária Falciparum/parasitologia , Masculino , Camundongos , Parasitos/citologia , Parasitos/crescimento & desenvolvimento , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/química , Tanzânia , Trofozoítos/citologia , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/imunologia , Vacúolos/imunologia
19.
Malar J ; 19(1): 132, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-32228596

RESUMO

BACKGROUND: During the erythrocytic cycle, Plasmodium falciparum malaria parasites express P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) that anchor the infected erythrocytes (IE) to the vascular lining of the host. The CIDRα1 domain of PfEMP1 is responsible for binding host endothelial protein C receptor (EPCR), and increasing evidence support that this interaction triggers severe malaria, accounting for the majority of malaria-related deaths. In high transmission regions, children develop immunity to severe malaria after the first few infections. This immunity is believed to be mediated by antibodies targeting and inhibiting PfEMP1, causing infected erythrocytes to circulate and be cleared in the spleen. The development of immunity to malaria coincides with acquisition of broad antibody reactivity across the CIDRα1 protein family. Altogether, this identifies CIDRα1 as an important vaccine target. However, the antigenic diversity of the CIDRα1 domain family is a challenge for vaccine development. METHODS: Immune responses in mice vaccinated with Virus-Like Particles (VLP) presenting CIDRα1 antigens were investigated. Antibody reactivity was tested to a panel of recombinant CIDRα1 domains, and the antibodies ability to inhibit EPCR binding by the recombinant CIDRα1 domains was tested in Luminex-based multiplex assays. RESULTS: VLP-presented CIDRα1.4 antigens induced a rapid and strong IgG response capable of inhibiting EPCR-binding of multiple CIDRα1 domains mainly within the group A CIDRα1.4-7 subgroups. CONCLUSIONS: The study observations mirror those from previous CIDRα1 vaccine studies using other vaccine constructs and platforms. This suggests that broad CIDRα1 antibody reactivity may be achieved through vaccination with a limited number of CIDRα1 variants. In addition, this study suggest that this may be achieved through vaccination with a human compatible VLP vaccine platform.


Assuntos
Anticorpos Antiprotozoários/imunologia , Imunização , Proteínas de Protozoários/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Domínios Proteicos
20.
Malar J ; 19(1): 126, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-32228600

RESUMO

BACKGROUND: There is an urgent need for an effective vaccine to control and eradicate malaria, one of the most serious global infectious diseases. Plasmodium merozoite surface protein 4 (MSP4) has been listed as a blood-stage subunit vaccine candidate for malaria. Infection with Plasmodium ovale species including P. ovale wallikeri and P. ovale curtisi, is also a source of malaria burden in tropical regions where it is sometimes mixed with other Plasmodium species. However, little is known about P. ovale MSP4. METHODS: The msp4 gene was amplified through polymerase chain reaction using genomic DNA extracted from blood samples of 46 patients infected with P. ovale spp. and amplified products were sequenced. Open reading frames predicted as immunogenic peptides consisting of 119 and 97 amino acids of P. ovale curtisi MSP4 (PocMSP4) and P. ovale wallikeri MSP4 (PowMSP4), respectively, were selected for protein expression. Recombinant proteins (rPoMSP4) were expressed in Escherichia coli, purified, analysed, and immunized in BALB/c mice. The specificity of anti-MSP4-immunoglobulin (Ig) G antibodies was evaluated by Western blot and enzyme-linked immunosorbent assays, and cellular immune responses were analysed via lymphocyte proliferation assays. RESULTS: Full peptide sequences of PocMSP4 and PowMSP4 were completely conserved in all clinical isolates, except in the epidermal growth factor-like domain at the carboxyl terminus where only one mutation was observed in one P. o. wallikeri isolate. Further, truncated PoMSP4 segments were successfully expressed and purified as ~ 32 kDa proteins. Importantly, high antibody responses with end-point titres ranging from 1:10,000 to 1:2,560,000 in all immunized mouse groups were observed, with high IgG avidity to PocMSP4 (80.5%) and PowMSP4 (92.3%). Furthermore, rPocMSP4 and rPowMSP4 cross-reacted with anti-PowMSP4-specific or anti-PocMSP4-specific antibodies. Additionally, anti-PoMSP4 IgG antibodies showed broad immuno-specificity in reacting against rPoMSP1 and rPoAMA1. Lastly, PocMSP4- and PowMSP4-immunized mice induced cellular immune responses with PocMSP4 (36%) and PowMSP4 cells (15.8%) during splenocyte proliferation assays. CONCLUSION: Findings from this study suggest conservation in PoMSP4 protein sequences and high immunogenicity was observed in rPoMSP4. Furthermore, induction of immune responses in PocMSP4- and PowMSP4-immunized mice informed that both humoral and cellular immune responses play crucial roles for PoMSP4 in protection.


Assuntos
Antígenos de Protozoários/imunologia , Imunogenicidade da Vacina , Imunoglobulina G/imunologia , Plasmodium ovale/imunologia , Proteínas de Protozoários/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , China , Epitopos , Feminino , Camundongos , Camundongos Endogâmicos BALB C
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