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1.
Parasitol Res ; 119(4): 1371-1380, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31970471

RESUMO

Phosphoinositide-dependent phospholipase-C (PI-PLC) triggers the calcium signaling pathway which plays an important role in dense granule and microneme secretion and pathogenesis of Toxoplasma gondii (T. gondii). There are limited data about the effects of phospholipid analogues against T. gondii. The current study assessed the effect of edelfosine, as a phospholipid analogue, on GRA1 and MIC3 expressions using in vitro and in vivo models of acute toxoplasmosis. Infected Vero cells were treated by edelfosine in two subgroups: 24 h following the cell infection and treatment at the same time of cell infection. Animal study was performed on forty mice in four groups including non-infected, infected untreated, infected edelfosine-treated, and infected pyrimethamine-treated. Gene and protein expression analyses were done using quantitative real-time PCR and western blot, respectively. Edelfosine significantly reduced the GRA1 (P < 0.01) and MIC3 (P < 0.01) mRNA and protein expressions in 24 h following the cell infection and at the same time of cell infection groups. In vivo study showed that the edelfosine significantly reduced the GRA1 expression in eye, and MIC3 expression in brain and liver. Moreover, the edelfosine-treated infected mice had significant higher survival rate compared with uninfected mice. The reducing effect of edelfosine on GRA1 and MIC3 mRNA and protein levels 24 h following the cell infection was more than treatment at the same time of cell infection group. Moreover, the effect of edelfosine on GRA1 and MIC3 expression in animal tissues was variable. These data showed that the edelfosine may decrease the T. gondii excretory/secretory antigens through inhibition of PI-PLC.


Assuntos
Antígenos de Protozoários/biossíntese , Antiparasitários/farmacologia , Éteres Fosfolipídicos/farmacologia , Proteínas de Protozoários/biossíntese , Toxoplasma/efeitos dos fármacos , Toxoplasmose Animal/tratamento farmacológico , Animais , Antígenos de Protozoários/genética , Western Blotting , Encéfalo/metabolismo , Linhagem Celular , Chlorocebus aethiops , Olho/metabolismo , Feminino , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/genética , Toxoplasma/genética , Células Vero
2.
Exp Parasitol ; 206: 107757, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31494216

RESUMO

Equine theileriosis is a severe equine disease caused by the protozoan Theileria equi, which is prevalent in tropical and subtropical areas. In this study, a recombinant equi merozoite antigen-2 (rEMA-2) of T. equi was used as an immunogen. Two groups of 10 mice each were divided into control and vaccinated groups. Sixty mares seronegative for theileriosis were divided in two groups, one vaccinated and another group as a control animal. Mice and mares of the vaccinated groups were inoculated with 150 µL of the vaccine containing 50 µg of rEMA-2 and 2 mL of the vaccine containing 200 µg of rEMA-2, respectively, at days 0 and 21. The immunogenicity of rEMA-2 was evaluated by ELISA and fluorescent antibody test (IFAT) using serum from vaccinated mice, mares and antigenicity in naturally infected horse. At every point throughout the ELISA study, there were significant differences between the vaccinated and control groups (p < 0.05). The vaccine induced 3- and 4-fold IgG increases in mice at the 14th and 28th day, respectively, compared to the control group. The horses' IgG dynamics showed a significant (p < 0.05) increase in the total IgG titer as early as day 7, which increased until day 28 at which time a more significant (p < 0.001) IgG titer was observed. In evaluating the isotypes, we observed a trend similar to that of total IgG, where IgG(T) (IgG3-5) were significantly (p < 0.05) more elevated than the other isotypes analyzed, followed by IgGb (IgG4-7) and IgGa (IgG1). Positive fluorescence was detected by IFAT, suggesting that the protein is immunogenic and conserves some epitopes identical to the native T. equi antigens present in the equine blood smear. Thus, our results suggest that rEMA-2 can be a promising vaccinal antigen.


Assuntos
Antígenos de Protozoários/imunologia , Pichia/imunologia , Theileria/imunologia , Análise de Variância , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Cavalos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Merozoítos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Distribuição Aleatória , Proteínas Recombinantes/imunologia
3.
Appl Microbiol Biotechnol ; 103(16): 6495-6504, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31218376

RESUMO

The present study aimed to evaluate the influence of induction conditions (IPTG concentration, temperature, and induction time) on the plasmid pQE-30 stability and 503 antigen expression of Leishmania i. chagasi in Escherichia coli M15. Batch cultures were performed at 37 °C and induced by the addition of different IPTG concentrations (0.01 to 1.5 mM). Subsequently, experiments were carried out at different temperatures (27 to 42 °C), evaluating the influence of induction time (0.5 to 6 h after the start of the culture). The results showed that IPTG toxicity caused a metabolic stress in the cells and, consequently, the microorganism growth reduced. The induction with IPTG may also be associated with the plasmid pQE-30 instability, due to metabolic burden imposed by the recombinant protein expression. The optimal conditions for 503 antigen expression of Leishmania i. chagasi in Escherichia coli M15 were an IPTG concentration of 1.0 mM, temperature of 37 °C, and induction time of 2 h. The maximum antigen concentration obtained was 0.119 ± 0.009 g/L, about seven times higher than the lowest concentration. Therefore, the results showed that 503 antigen can be produced in laboratory; however, it requires more studies to minimize the plasmid instability and improve to industrial scale.


Assuntos
Antígenos de Protozoários/biossíntese , Escherichia coli/metabolismo , Expressão Gênica , Leishmania/genética , Proteínas Recombinantes/biossíntese , Ativação Transcricional , Antígenos de Protozoários/genética , Escherichia coli/genética , Instabilidade Genômica/efeitos dos fármacos , Isopropiltiogalactosídeo/metabolismo , Plasmídeos , Proteínas Recombinantes/genética , Temperatura
4.
Malar J ; 18(1): 197, 2019 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-31196098

RESUMO

BACKGROUND: Vivax malaria is the predominant form of malaria outside Africa, affecting about 14 million people worldwide, with about 2.5 billion people exposed. Development of a Plasmodium vivax vaccine is a priority, and merozoite surface protein 7 (MSP-7) has been proposed as a plausible candidate. The P. vivax genome contains 12 MSP-7 genes, which contribute to erythrocyte invasion during blood-stage infection. Previous analysis of MSP-7 sequence diversity suggested that not all paralogs are functionally equivalent. To explore MSP-7 functional diversity, and to identify the best vaccine candidate within the family, MSP-7 expression and antigenicity during bloodstream infections were examined directly from clinical isolates. METHODS: Merozoite surface protein 7 gene expression was profiled using RNA-seq data from blood samples isolated from ten human patients with vivax malaria. Differential expression analysis and co-expression cluster analysis were used to relate PvMSP-7 expression to genetic markers of life cycle stage. Plasma from vivax malaria patients was also assayed using a custom peptide microarray to measure antibody responses against the coding regions of 12 MSP-7 paralogs. RESULTS: Ten patients presented diverse transcriptional profiles that comprised four patient groups. Two MSP-7 paralogs, 7A and 7F, were expressed abundantly in all patients, while other MSP-7 genes were uniformly rare (e.g. 7J). MSP-7H and 7I were significantly more abundant in patient group 4 only, (two patients having experienced longer patency), and were co-expressed with a schizont-stage marker, while negatively associated with liver-stage and gametocyte-stage markers. Screening infections with a PvMSP-7 peptide array identified 13 linear B-cell epitopes in five MSP-7 paralogs that were recognized by plasma from all patients. CONCLUSIONS: These results show that MSP-7 family members vary in expression profile during blood infections; MSP-7A and 7F are expressed throughout the intraerythrocytic development cycle, while expression of other paralogs is focused on the schizont. This may reflect developmental regulation, and potentially functional differentiation, within the gene family. The frequency of B-cell epitopes among paralogs also varies, with MSP-7A and 7L consistently the most immunogenic. Thus, MSP-7 paralogs cannot be assumed to have equal potential as vaccines. This analysis of clinical infections indicates that the most abundant and immunogenic paralog is MSP-7A.


Assuntos
Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/imunologia , Malária Vivax/imunologia , Malária Vivax/prevenção & controle , Proteínas de Membrana/biossíntese , Proteínas de Membrana/imunologia , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/imunologia , Adolescente , Adulto , África , Idoso , Idoso de 80 Anos ou mais , Alelos , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Criança , Pré-Escolar , Feminino , Perfilação da Expressão Gênica , Humanos , Lactente , Recém-Nascido , Masculino , Proteínas de Membrana/genética , Análise em Microsséries , Pessoa de Meia-Idade , Plasmodium vivax/imunologia , Proteínas de Protozoários/genética , Análise de Sequência de RNA , Adulto Jovem
5.
Prep Biochem Biotechnol ; 48(10): 968-976, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30489218

RESUMO

Leishmaniosis is a complex of diseases that can be fatal, if not given proper attention. Despite its relevance in the public health system, there is no vaccine capable of preventing the disease in humans so far and its treatment is expensive and aggressive to human health. The present study aims to optimize the induction parameters of the 503 Leishmania i. chagasi antigen expressed in recombinant Escherichia coli M15. The induction at different cell densities was evaluated in order to analyze the influence of the induction time on the yield of the protein of interest. In this segment, lactose and isopropyl-ß-d-thiogalactopyranoside (IPTG) were used as inducer molecules, using various concentrations: 0.1 g/L, 1.0 g/L, and 10 g/L for lactose and 20 µM, 100 µM, 500 µM, and 1000 µM for IPTG. The results presented that the concentration of IPTG that obtained the higher antigen levels was that of 100 µM (0.087 g/L), a 10-fold lower concentration than was being previously used in this type of system and for lactose, it was 1 g/L (0.016 g/L). Thus, the induction with 100 µM allowed obtaining the antigen with a concentration 5.6 times higher than the lactose induction maximum concentration.


Assuntos
Antígenos de Protozoários , Escherichia coli/metabolismo , Expressão Gênica , Leishmania infantum , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/isolamento & purificação , Escherichia coli/genética , Humanos , Leishmania infantum/genética , Leishmania infantum/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação
6.
Mol Biochem Parasitol ; 224: 44-49, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30053393

RESUMO

The transmission-blocking vaccine candidate Pfs48/45 from the human malaria parasite Plasmodium falciparum is known to be difficult to express in heterologous systems, either as full-length protein or as correctly folded protein fragments that retain conformational epitopes. In this study we express full-length Pfs48/45 in the rodent parasite P. berghei. Pfs48/45 is expressed as a transgene under control of the strong P. berghei schizont-specific msp1 gene promoter (Pfs48/45@PbMSP1). Pfs48/45@PbMSP1 schizont-infected red blood cells produced full-length Pfs48/45 and the structural integrity of Pfs48/45 was confirmed using a panel of conformation-specific monoclonal antibodies that bind to different Pfs48/45 epitopes. Sera from mice immunized with transgenic Pfs48/45@PbMSP1 schizonts showed strong transmission-reducing activity in mosquitoes infected with P. falciparum using standard membrane feeding. These results demonstrate that transgenic rodent malaria parasites expressing human malaria antigens may be used as means to evaluate immunogenicity and functionality of difficult to express malaria vaccine candidate antigens.


Assuntos
Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/imunologia , Proteínas de Membrana/imunologia , Plasmodium berghei/genética , Plasmodium falciparum/genética , Proteínas Recombinantes/imunologia , Animais , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/genética , Modelos Animais de Doenças , Transmissão de Doença Infecciosa/prevenção & controle , Expressão Gênica , Malária/prevenção & controle , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Transgenes
7.
Parasitol Res ; 117(7): 2255-2263, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29779048

RESUMO

Interleukin 27 (IL-27) is a member of the IL-6/IL-12 family, and IL-27 receptor (IL-27R) consists of WSX-1 (the IL-27Rα subunit) and the signal-transducing subunit gp130. Human and mouse mast cells (MCs) express the IL-27R. To explore the expressions of IL-27/IL-27R subunits (WSX-1 and gp130) during acute ocular toxoplasmosis (OT), we established mouse model by intraocular injection of 500 Toxoplasma gondii RH strain tachyzoites. Histopathological changes were analyzed, MCs were counted by toluidine blue staining, and tryptase+/IL-27+ MCs were examined by immunofluorescence double-staining in the eyes and cervical lymph nodes (CLNs) of T. gondii-infected mice. The mRNA expressions of IL-27p28, WSX-1, gp130, and tachyzoite specific surface antigen 1 (SAG1) in the eyes and CLNs of T. gondii-infected mice, and the expressions of WSX-1 and gp130 in the murine mastocytoma cell line P815 infected with T. gondii tachyzoites in vitro were examined by using quantitative real-time reverse transcription-polymerase chain reaction. Our results showed that, after T. gondii infection, severe histopathological changes, increased numbers of total MCs and degranulated MCs, elevated expressions of IL-27p28, WSX-1, and gp130 were found in the eyes and CLNs, and significant correlations between the levels of IL-27 and SAG1 existed in the eyes and CLNs of T. gondii-infected mice. In addition, increased levels of WSX-1 and gp130 were examined in T. gondii-infected P815 cells. Our data suggested that IL-27/IL-27R expression induced by T. gondii infection may regulate MC-mediated immune response during acute OT in mouse model.


Assuntos
Receptor gp130 de Citocina/metabolismo , Interleucinas/metabolismo , Mastócitos/metabolismo , Receptores de Citocinas/metabolismo , Toxoplasmose Ocular/patologia , Animais , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/genética , Degranulação Celular/imunologia , Linhagem Celular Tumoral , Receptor gp130 de Citocina/genética , Modelos Animais de Doenças , Feminino , Humanos , Interleucinas/genética , Mastócitos/imunologia , Mastocitoma/metabolismo , Camundongos , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/genética , RNA Mensageiro/biossíntese , Receptores de Citocinas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Linfócitos T/metabolismo , Toxoplasma/genética , Toxoplasma/patogenicidade , Toxoplasmose Ocular/imunologia , Toxoplasmose Ocular/parasitologia
8.
J Biotechnol ; 266: 111-117, 2018 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-29269249

RESUMO

Malaria is an infectious disease having a large negative impact on economic growth. Vaccines are considered as a novel strategy to reduce the burden of malaria. Malaria parasite has a complex life cycle and attempts are being made to develop vaccines that target each stage of the life cycle. Oral vaccines seem to be more feasible to implement in poor countries, since they are relatively inexpensive, needle-free administrated, mostly stable at non-refrigerated conditions and painless. By using recombinant technology, suitable oral hosts could serve as antigen delivering vehicles in developing oral vaccines. Chlamydomonas reinhardtii offers beneficial attributes as oral recombinant protein expression platform. Moreover, C. reinhardtii chloroplast is an attractive platform for expressing malaria antigens because it is capable of folding complex proteins, including those requiring disulfide bond formation, while lacking the ability to glycosylate proteins; a valuable quality of any malaria protein expression system, since the Plasmodium parasite lacks N-linked glycosylation machinery. As a first step towards developing an oral vaccine candidate against malaria, here, we expressed a fusion protein consisting of PfCelTOS, a candidate for pre-erythrocytic and transmission-blocking vaccines, fused to human interleukin-2 (IL-2) as vaccine adjuvant in the chloroplast of C. reinhardtii. The effect of light and media on recombinant protein production and cell growth was then studied. Results demonstrated that expressed recombinant proteins accumulate as a soluble, properly folded and functional protein within algal chloroplasts. Moreover, results showed that the highest cell density can be achieved using mixotrophy mode. However, protein accumulation appears to be favored by cultivating in TAP medium in low light.


Assuntos
Antígenos de Protozoários , Chlamydomonas reinhardtii , Cloroplastos , Vacinas Antimaláricas , Plasmodium falciparum/genética , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/genética , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Vacinas Antimaláricas/biossíntese , Vacinas Antimaláricas/genética
9.
PLoS Negl Trop Dis ; 11(8): e0005803, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28800590

RESUMO

Delivery of various forms of recombinant Theileria parva sporozoite antigen (p67) has been shown to elicit antibody responses in cattle capable of providing protection against East Coast fever, the clinical disease caused by T. parva. Previous formulations of full-length and shorter recombinant versions of p67 derived from bacteria, insect, and mammalian cell systems are expressed in non-native and highly unstable forms. The stable expression of full-length recombinant p67 in mammalian cells has never been described and has remained especially elusive. In this study, p67 was expressed in human-derived cells as a full-length, membrane-linked protein and as a secreted form by omission of the putative transmembrane domain. The recombinant protein expressed in this system yielded primarily two products based on Western immunoblot analysis, including one at the expected size of 67 kDa, and one with a higher than expected molecular weight. Through treatment with PNGase F, our data indicate that the larger product of this mammalian cell-expressed recombinant p67 cannot be attributed to glycosylation. By increasing the denaturing conditions, we determined that the larger sized mammalian cell-expressed recombinant p67 product is likely a dimeric aggregate of the protein. Both forms of this recombinant p67 reacted with a monoclonal antibody to the p67 molecule, which reacts with the native sporozoite. Additionally, through this work we developed multiple mammalian cell lines, including both human and bovine-derived cell lines, transduced by a lentiviral vector, that are constitutively able to express a stable, secreted form of p67 for use in immunization, diagnostics, or in vitro assays. The recombinant p67 developed in this system is immunogenic in goats and cattle based on ELISA and flow cytometric analysis. The development of a mammalian cell system that expresses full-length p67 in a stable form as described here is expected to optimize p67-based immunization.


Assuntos
Antígenos de Protozoários/biossíntese , Proteínas de Protozoários/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Protozoários/imunologia , Western Blotting , Bovinos , Ensaio de Imunoadsorção Enzimática , Cabras , Células HEK293 , Humanos , Proteínas de Protozoários/imunologia , Proteínas Recombinantes de Fusão/imunologia , Theileria parva
10.
Cell Microbiol ; 19(9)2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28409866

RESUMO

The successful invasion of Plasmodium is an essential step in their life cycle. The parasite reticulocyte-binding protein homologues (RHs) and erythrocyte-binding like proteins are two families involved in the invasion leading to merozoite-red blood cell (RBC) junction formation. Ca2+ signaling has been shown to play a critical role in the invasion. RHs have been linked to Ca2+ signaling, which triggers the erythrocyte-binding like proteins release ahead of junction formation, consistent with RHs performing an initial sensing function in identifying suitable RBCs. RH5, the only essential RHs, is a highly promising vaccine candidate. RH5-basigin interaction is essential for merozoite invasion and also important in determining host tropism. Here, we show that RH5 has a distinct function from the other RHs. We show that RH5-Basigin interaction on its own triggers a Ca2+ signal in the RBC resulting in changes in RBC cytoskeletal proteins phosphorylation and overall alterations in RBC cytoskeleton architecture. Antibodies targeting RH5 that block the signal prevent invasion before junction formation consistent with the Ca2+ signal in the RBC leading to rearrangement of the cytoskeleton required for invasion. This work provides the first time a functional context for the essential role of RH5 and will now open up new avenues to target merozoite invasion.


Assuntos
Basigina/metabolismo , Sinalização do Cálcio/fisiologia , Proteínas de Transporte/metabolismo , Eritrócitos/fisiologia , Merozoítos/patogenicidade , Plasmodium falciparum/patogenicidade , Anticorpos Monoclonais/imunologia , Antígenos de Protozoários/biossíntese , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/imunologia , Linhagem Celular , Citoesqueleto/parasitologia , Citoesqueleto/patologia , Eritrócitos/parasitologia , Interações Hospedeiro-Parasita/fisiologia , Humanos , Malária Falciparum/parasitologia , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/biossíntese
11.
Protein Expr Purif ; 133: 66-74, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28263855

RESUMO

SAG1-related sequence 3 (SRS3) is one of the major Toxoplasma gondii tachyzoite surface antigens and has been shown to be potentially useful for the detection of toxoplasmosis. This protein is highly conformational due to the presence of six disulfide bonds. To achieve solubility and antigenicity, SRS3 depends on proper disulfide bond formation. The aim of this study was to over-express the SRS3 protein with correct folding for use in serodiagnosis of the disease. To achieve this, a truncated SRS3 fusion protein (rtSRS3) was produced, containing six histidyl residues at both terminals and purified by immobilized metal affinity chromatography. The refolding process was performed through three methods, namely dialysis in the presence of chemical additives along with reduced/oxidized glutathione and drop-wise dilution methods with reduced/oxidized glutathione or reduced DTT/oxidized glutathione. Ellman's assay and ELISA showed that the protein folding obtained by the dialysis method was the most favorable, probably due to the correct folding. Subsequently, serum samples from individuals with chronic infection (n = 76), probable acute infection (n = 14), and healthy controls (n = 81) were used to determine the usefulness of the refolded rtSRS3 for Toxoplasma serodiagnosis. The results of the developed IgG-ELISA showed a diagnostic specificity of 91% and a sensitivity of 82.89% and 100% for chronic and acute serum samples, respectively. In conclusion, correctly folded rtSRS3 has the potential to be used as a soluble antigen for the detection of human toxoplasmosis.


Assuntos
Anticorpos Antiprotozoários , Antígenos de Protozoários , Imunoglobulina G , Redobramento de Proteína , Toxoplasma/genética , Toxoplasma/imunologia , Toxoplasmose , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Toxoplasma/química , Toxoplasma/metabolismo , Toxoplasmose/sangue , Toxoplasmose/diagnóstico , Toxoplasmose/imunologia
12.
Protein Expr Purif ; 136: 52-57, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26578115

RESUMO

Plasmodium vivax is dependent on interaction with the Duffy antigen receptor for chemokines (DARC) for invasion of human erythrocytes. The P. vivax Duffy binding protein (PvDBP) mediates interaction of P. vivax merozoites with DARC. The DARC receptor-binding domain lies in a conserved N-terminal cysteine-rich region of PvDBP referred to as region II (PvDBPII). PvDBPII is an attractive vaccine candidate since antibodies raised against PvDBPII block erythrocyte invasion by P. vivax. Here, we describe methods to produce recombinant PvDBPII in its correctly folded conformation. A synthetic gene optimized for expression of PvDBPII in Escherichia coli and fed batch fermentation process based on exponential feeding strategy was used to achieve high levels of expression of recombinant PvDBPII. Recombinant PvDBPII was isolated from inclusion bodies, refolded by rapid dilution and purified by ion exchange chromatography. Purified recombinant PvDBPII was characterized for identity, purity and functional activity using standardized release assays. Recombinant PvDBPII formulated with various human compatible adjuvants including glycosylpyranosyl lipid A-stable emulsion (GLA-SE) and alhydrogel was used for immunogenicity studies in small animals to downselect a suitable formulation for clinical development. Sera collected from immunized animals were tested for recognition of PvDBPII and inhibition of PvDBPII-DARC binding. GLA-SE formulations of PvDBPII yielded higher ELISA and binding inhibition titres compared to PvDBPII formulated with alhydrogel. These data support further development of a recombinant vaccine for P. vivax based on PvDBPII formulated with GLA-SE.


Assuntos
Antígenos de Protozoários , Imunogenicidade da Vacina , Vacinas Antimaláricas , Plasmodium vivax/genética , Proteínas de Protozoários , Receptores de Superfície Celular , Animais , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/isolamento & purificação , Humanos , Vacinas Antimaláricas/biossíntese , Vacinas Antimaláricas/genética , Vacinas Antimaláricas/imunologia , Vacinas Antimaláricas/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium vivax/imunologia , Domínios Proteicos , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/isolamento & purificação , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação
13.
Rev Soc Bras Med Trop ; 50(6): 788-794, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29340456

RESUMO

INTRODUCTION: The production of the Montenegro antigen for skin test poses difficulties regarding quality control. Here, we propose that certain animal models reproducing a similar immune response to humans may be used in the quality control of Montenegro antigen production. METHODS: Fifteen Cavia porcellus (guinea pigs) were immunized with Leishmania amazonensis or Leishmania braziliensis , and, after 30 days, they were skin tested with standard Montenegro antigen. To validate C. porcellus as an animal model for skin tests, eighteen Mesocricetus auratus (hamsters) were infected with L. amazonensis or L. braziliensis , and, after 45 days, they were skin tested with standard Montenegro antigen. RESULTS: Cavia porcellus immunized with L. amazonensis or L. braziliensis , and hamsters infected with the same species presented induration reactions when skin tested with standard Montenegro antigen 48-72h after the test. CONCLUSIONS: The comparison between immunization methods and immune response from the two animal species validated C. porcellus as a good model for Montenegro skin test, and the model showed strong potential as an in vivo model in the quality control of the production of Montenegro antigen.


Assuntos
Antígenos de Protozoários/biossíntese , Cobaias/imunologia , Testes Intradérmicos/normas , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/diagnóstico , Modelos Animais , Animais , Leishmania/imunologia , Masculino , Valor Preditivo dos Testes , Controle de Qualidade , Sensibilidade e Especificidade
14.
Cell Microbiol ; 19(6)2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28030753

RESUMO

The extensive modification of Plasmodium falciparum-infected erythrocytes by variant surface antigens plays a major role in immune evasion and malaria-induced pathology. Here, using high-resolution microscopy, we visualize the spatio-temporal expression dynamics of STEVOR, an important variant surface antigens family, in a stage-dependent manner. We demonstrate that it is exported to the cell surface where protein molecules cluster and preferentially localize in proximity to knobs. Quantitative evidence from our force measurements and microfluidic assays reveal that STEVOR can effectively mediate the formation of stable, robust rosettes under static and physiologically relevant flow conditions. Our results extend previously published studies in P. falciparum and emphasize the role of STEVOR in rosetting, an important contributor to disease pathology.


Assuntos
Antígenos de Protozoários/genética , Antígenos de Superfície/genética , Adesão Celular/genética , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/genética , Antígenos de Protozoários/biossíntese , Antígenos de Superfície/biossíntese , Adesão Celular/fisiologia , Linhagem Celular , Eritrócitos/parasitologia , Humanos , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/biossíntese , Formação de Roseta
15.
PLoS One ; 11(10): e0164053, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27695087

RESUMO

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading asexual blood stage vaccine candidate for malaria. In preparation for clinical trials, three Diversity Covering (DiCo) PfAMA1 ectodomain proteins, designed to overcome the intrinsic polymorphism that is present in PfAMA1, were produced under Good Manufacturing Practice (GMP) in Pichia pastoris. Using identical methodology, the 3 strains were cultivated in 70-L scale fed-batch fermentations and PfAMA1-DiCos were purified by two chromatography steps, an ultrafiltration/diafiltration procedure and size exclusion chromatography, resulting in highly pure (>95%) PfAMA1-DiCo1, PfAMA1 DiCo2 and PfAMA1 DiCo3, with final yields of 1.8, 1.9 and 1.3 gram, respectively. N-terminal determinations showed that approximately 50% of each of the proteins lost 12 residues from their N-terminus, in accordance with SDS-PAGE (2 main bands) and MS-data. Under reducing conditions a site of limited proteolytic cleavage within a disulphide bonded region became evident. The three proteins quantitatively bound to the mAb 4G2 that recognizes a conformational epitope, suggesting proper folding of the proteins. The lyophilized Drug Product (1:1:1 mixture of PfAMA1-DiCo1, DiCo2, DiCo3) fulfilled all pre-set release criteria (appearance, dissolution rate, identity, purity, protein content, moisture content, sub-visible particles, immuno-potency (after reconstitution with adjuvant), abnormal toxicity, sterility and endotoxin), was stable in accelerated and real-time stability studies at -20°C for over 24 months. When formulated with adjuvants selected for clinical phase I evaluation, the Drug Product did not show adverse effect in a repeated-dose toxicity study in rabbits. The Drug Product has entered a phase Ia/Ib clinical trial.


Assuntos
Variação Antigênica , Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/biossíntese , Vacinas Antimaláricas/imunologia , Proteínas de Membrana/imunologia , Proteínas de Protozoários/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Feminino , Fermentação , Humanos , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/efeitos adversos , Malária Falciparum/imunologia , Malária Falciparum/prevenção & controle , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Estabilidade Proteica , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Controle de Qualidade , Coelhos , Proteínas Recombinantes
16.
Protein Expr Purif ; 127: 88-97, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27450536

RESUMO

An immunogenic protein, enolase 2, was identified among the secreted excretory/secretory antigens (ESAs) from Toxoplasma gondii strain RH using immunoproteomics based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Enolase 2 was cloned, sequenced, and heterologously expressed. BLAST analysis revealed 75-96% similarity with enolases from other parasites. Immunoblotting demonstrated good immunoreactivity of recombinant T. gondii enolase (Tg-enolase 2) to T. gondii-infected animal serum. Purified Tg-enolase 2 was found to catalyze dehydration of 2-phospho-d-glycerate to phosphoenolpyruvate. In vitro studies revealed maximal activity at pH 7.5 and 37 °C, and activity was inhibited by K(+), Ni(2+), Al(3+), Na(+), Cu(2+) and Cr(3+). A monoclonal antibody against Tg-enolase 2 was prepared, 1D6, with the isotype IgG2a/κ. Western blotting revealed that 1D6 reacts with Tg-enolase 2 and native enolase 2, present among T. gondii ESAs. The indirect immunofluorescence assays showed that enolase 2 could be specifically detected on the growing T. gondii tachyzoites. Immunoelectron microscopy revealed the surface and intracellular locations of enolase 2 on T. gondii cells. In conclusion, our results clearly show that the enzymatic activity of T. gondii enolase 2 is ion dependent and that it could be influenced by environmental factors. We also provide evidence that enolase 2 is an important immunogenic protein of ESAs from T. gondii and that it is a surface-exposed protein with strong antigenicity and immunogenicity. Our findings indicate that enolase 2 could play important roles in metabolism, immunogenicity and pathogenicity and that it may serve as a novel drug target and candidate vaccine against T. gondii infection.


Assuntos
Anticorpos Monoclonais Murinos/imunologia , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários , Fosfopiruvato Hidratase , Proteínas de Protozoários , Toxoplasma , Animais , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/isolamento & purificação , Antígenos de Protozoários/farmacologia , Escherichia coli/metabolismo , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Fosfopiruvato Hidratase/biossíntese , Fosfopiruvato Hidratase/imunologia , Fosfopiruvato Hidratase/isolamento & purificação , Fosfopiruvato Hidratase/farmacologia , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/farmacologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Toxoplasma/enzimologia , Toxoplasma/imunologia
17.
Parasitol Int ; 65(6 Pt A): 708-714, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27443851

RESUMO

Plasmodium reichenowi, an ape malaria parasite is morphologically identical and genetically similar to Plasmodium falciparum, infects chimpanzees but not humans. Genomic studies revealed that all primate malaria parasites belong to Laverania subgenus. Laverania parasites exhibit strict host specificity, but the molecular mechanisms underlying these host restrictions remain unexplained. Plasmodium merozoites express multiple binding ligands that recognize specific receptors on erythrocytes, including micronemal proteins belonging to P. falciparum EBL family. It was shown that erythrocyte binding antigen-175 (EBA-175), erythrocyte binding ligand-1 (EBL-1), erythrocyte binding antigen-140 (EBA-140) recognize erythrocyte surface sialoglycoproteins - glycophorins A, B, C, respectively. EBA-140 merozoite ligand hijacks glycophorin C (GPC), a minor erythrocyte sialoglycoprotein, to invade the erythrocyte through an alternative invasion pathway. A homolog of P. falciparum EBA-140 protein was identified in P. reichenowi. The amino acid sequences of both EBA-140 ligands are very similar, especially in the conservative erythrocyte binding region (Region II). It has been suggested that evolutionary changes in the sequence of EBL proteins may be associated with Plasmodium host restriction. In this study we obtained, for the first time, the recombinant P. reichenowi EBA-140 ligand Region II using baculovirus expression vector system. We show that the ape EBA-140 Region II is host specific and binds to chimpanzee erythrocytes in the dose and sialic acid dependent manner. Further identification of the erythrocyte receptor for this ape ligand is of great interests, since it may reveal the molecular basis of host restriction of both P. reichenowi and its deadliest human counterpart, P. falciparum.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Eritrócitos/metabolismo , Glicoforinas/metabolismo , Especificidade de Hospedeiro/genética , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/genética , Antígenos de Protozoários/metabolismo , Sítios de Ligação , Proteínas de Transporte/biossíntese , Linhagem Celular , Dicroísmo Circular , Proteínas de Membrana , Ácido N-Acetilneuramínico/metabolismo , Pan troglodytes , Plasmodium falciparum/genética , Ligação Proteica , Proteínas de Protozoários/biossíntese , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Células Sf9 , Ressonância de Plasmônio de Superfície
18.
Rev Bras Parasitol Vet ; 24(2): 148-54, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26083692

RESUMO

Neosporosis is a disease caused by the protozoon Neospora caninum that leads to significant economic losses in many countries. In the present study, we report on use of the recombinant protein NcSRS2 of N. caninum expressed in Pichia pastoris in an indirect immunoenzymatic assay (ELISA) for diagnosing neosporosis infection in sheep and dogs. We observed that the ELISA test yielded specificity of 94.5% and sensitivity of 100% for sheep and specificity of 93.3% and sensitivity of 100% for dogs. We observed that the sensitivity was higher than shown by the indirect fluorescent antibody test, and this was confirmed by means of Western blot. The results from this study suggest that the recombinant protein expressed in P. pastoris is a suitable antigen for use in immunodiagnosis to detect N. caninum in two important species exposed to this parasitosis.


Assuntos
Antígenos de Protozoários/sangue , Antígenos de Superfície/sangue , Doenças do Cão/sangue , Doenças do Cão/parasitologia , Ensaio de Imunoadsorção Enzimática , Neospora , Infecções Protozoárias em Animais/sangue , Proteínas de Protozoários/sangue , Doenças dos Ovinos/sangue , Doenças dos Ovinos/parasitologia , Animais , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/imunologia , Antígenos de Superfície/biossíntese , Antígenos de Superfície/imunologia , Doenças do Cão/diagnóstico , Cães , Neospora/imunologia , Pichia/metabolismo , Infecções Protozoárias em Animais/diagnóstico , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/imunologia , Ovinos , Doenças dos Ovinos/diagnóstico
19.
Rev. bras. parasitol. vet ; 24(2): 148-154, Apr-Jun/2015. graf
Artigo em Inglês | LILACS | ID: lil-750757

RESUMO

Neosporosis is a disease caused by the protozoon Neospora caninum that leads to significant economic losses in many countries. In the present study, we report on use of the recombinant protein NcSRS2 of N. caninum expressed in Pichia pastoris in an indirect immunoenzymatic assay (ELISA) for diagnosing neosporosis infection in sheep and dogs. We observed that the ELISA test yielded specificity of 94.5% and sensitivity of 100% for sheep and specificity of 93.3% and sensitivity of 100% for dogs. We observed that the sensitivity was higher than shown by the indirect fluorescent antibody test, and this was confirmed by means of Western blot. The results from this study suggest that the recombinant protein expressed in P. pastoris is a suitable antigen for use in immunodiagnosis to detect N. caninum in two important species exposed to this parasitosis.


A neosporose é uma doença causada pelo protozoário Neospora caninum que leva a perdas econômicas importantes em muitos países. No presente estudo, é descrita a utilização da proteína recombinante NcSRS2 de N. caninum expressa em Pichia pastoris em um ensaio imunoenzimático indireto (ELISA) para o diagnóstico de infecção por Neospora em ovelhas e cães. Observou-se, que utilizando-se um ELISA, o teste produziu uma especificidade de 94,5% e uma sensibilidade de 100% para ovinos; e uma especificidade de 93,3% e sensibilidade de 100% para cães. Uma maior sensibilidade foi observada em relação à IFI que foi confirmada por Western blot. Os resultados deste estudo sugerem que a proteína recombinante expressa em P. pastoris é bom antígeno para ser utilizado no diagnóstico imunológico para detectar N. caninum em duas espécies importantes expostas a esta parasitose.


Assuntos
Animais , Cães , Infecções Protozoárias em Animais/sangue , Doenças dos Ovinos/sangue , Ensaio de Imunoadsorção Enzimática , Proteínas de Protozoários/sangue , Neospora/imunologia , Doenças do Cão/parasitologia , Doenças do Cão/sangue , Antígenos de Protozoários/sangue , Antígenos de Superfície/sangue , Pichia/metabolismo , Infecções Protozoárias em Animais/diagnóstico , Doenças dos Ovinos/diagnóstico , Doenças dos Ovinos/parasitologia , Ovinos , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/imunologia , Doenças do Cão/diagnóstico , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/imunologia , Antígenos de Superfície/biossíntese , Antígenos de Superfície/imunologia
20.
J. bras. nefrol ; 36(4): 430-436, Oct-Dec/2014. tab, graf
Artigo em Português | LILACS | ID: lil-731139

RESUMO

Introdução: Atualmente, é descrita elevada prevalência de hipovitaminose D no Lúpus Eritematoso Sistêmico (LES), a qual se associa a algumas manifestações clínicas e maior atividade inflamatória. Objetivo: Avaliar a associação entre insuficiência de vitamina D com LES e marcadores inflamatórios. Métodos: Estudo transversal, tendo sido avaliados 45 pacientes com LES e 24 controles sem a doença. Níveis de 25-hidroxivitamina D [25(OH)D] menores que 30 ng/mL foram considerados insuficientes. A atividade da doença foi avaliada pelo Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). Foram avaliados, ainda, proteína C reativa ultrassensível (PCRus) e interleucina-6 (IL-6) para verificação do status inflamatório. Para avaliação do envolvimento renal, foram realizados análise de elementos anormais e sedimentoscopia urinárias (EAS), hematúria e piúria quantitativas, proteinúria e depuração de creatinina em urina de 24 horas e anti-DNA de dupla hélice sérico. Resultados: A prevalência de insuficiência de 25(OH)D foi de 55% nos pacientes lúpicos e 8% nos participantes controles (p = 0,001). A mediana da 25(OH)D foi menor nos pacientes do que no grupo controle. Os pacientes com insuficiência de 25(OH)D apresentaram níveis mais elevados de IL-6 e maior prevalência de hematúria ao EAS. Não houve correlação entre vitamina D, nefrite lúpica e SLEDAI. Conclusão: Em nosso estudo, a insuficiência de vitamina D foi mais prevalente em pacientes com LES e se associou com níveis mais elevados de IL-6 e presença de hematúria. .


Introduction: Nowadays it is described a high prevalence of hypovitaminosis D in Systemic Lupus Erythematosus (SLE), which is associated with some clinical manifestations and increased inflammatory activity. Objective: To evaluate the association between vitamin D insufficiency with SLE and inflammatory markers. Methods: Cross-sectional study, in which have been evaluated 45 SLE patients and 24 controls without the disease. Levels of 25-hydroxyvitamin D [25(OH) D] less than 30 ng/mL were considered inadequate. Disease activity was assessed by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). High sensitivity C reactive protein (hsCRP) and interleukin-6 (IL-6) were evaluated for verification of the inflammatory status. For assessment of renal involvement, analysis of abnormal elements and urinay sediment (AES), quantitative hematuria and pyuria, proteinuria and creatinine clearance in 24-hour urine and serum anti-double stranded DNA were performed. Results: The prevalence of 25(OH)D insufficiency was 55% in SLE patients and 8% in the controls participants (p = 0.001). The median of 25(OH)D was lower in patients than in controls. Patients with insufficient 25(OH)D had higher levels of IL-6 and higher prevalence of hematuria in the AES. There was no correlation between vitamin D and SLEDAI or lupus nephritis. Conclusion: In our study, vitamin D deficiency was more prevalent in patients with SLE and was associated with higher levels of IL-6 and hematuria. .


Assuntos
Animais , Coelhos , Antígenos de Protozoários/imunologia , Proteínas de Membrana/imunologia , Dobramento de Proteína , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Sarcosina/análogos & derivados , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/genética , Antígenos de Protozoários/isolamento & purificação , Cisteína , Cromatografia de Afinidade/métodos , Cromatografia por Troca Iônica/métodos , Ácido Edético , Endotoxinas , Escherichia coli , Fermentação , Expressão Gênica , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Níquel , Estrutura Terciária de Proteína , Plasmodium falciparum/genética , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Sacarose
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