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1.
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
2.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
Rev Soc Bras Med Trop ; 50(6): 788-794, 2017 Nov-Dec.
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
9.
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
10.
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
11.
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
12.
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 , Glicoforina/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 , 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
13.
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
14.
Rev Bras Parasitol Vet ; 24(2): 148-54, 2015 Apr-Jun.
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
15.
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
16.
PLoS One ; 9(5): e92708, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24786587

RESUMO

PSA (Promastigote Surface Antigen) belongs to a family of membrane-bound and secreted proteins present in several Leishmania (L.) species. PSA is recognized by human Th1 cells and provides a high degree of protection in vaccinated mice. We evaluated humoral and cellular immune responses induced by a L. amazonensis PSA protein (LaPSA-38S) produced in a L. tarentolae expression system. This was done in individuals cured of cutaneous leishmaniasis due to L. major (CCLm) or L. braziliensis (CCLb) or visceral leishmaniasis due to L. donovani (CVLd) and in healthy individuals. Healthy individuals were subdivided into immune (HHR-Lm and HHR-Li: Healthy High Responders living in an endemic area for L. major or L. infantum infection) or non immune/naive individuals (HLR: Healthy Low Responders), depending on whether they produce high or low levels of IFN-γ in response to Leishmania soluble antigen. Low levels of total IgG antibodies to LaPSA-38S were detected in sera from the studied groups. Interestingly, LaPSA-38S induced specific and significant levels of IFN-γ, granzyme B and IL-10 in CCLm, HHR-Lm and HHR-Li groups, with HHR-Li group producing TNF-α in more. No significant cytokine response was observed in individuals immune to L. braziliensis or L. donovani infection. Phenotypic analysis showed a significant increase in CD4+ T cells producing IFN-γ after LaPSA-38S stimulation, in CCLm. A high positive correlation was observed between the percentage of IFN-γ-producing CD4+ T cells and the released IFN-γ. We showed that the LaPSA-38S protein was able to induce a mixed Th1 and Th2/Treg cytokine response in individuals with immunity to L. major or L. infantum infection indicating that it may be exploited as a vaccine candidate. We also showed, to our knowledge for the first time, the capacity of Leishmania PSA protein to induce granzyme B production in humans with immunity to L. major and L. infantum infection.


Assuntos
Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Leishmania/imunologia , Leishmaniose/prevenção & controle , Vacinas Protozoárias/química , Vacinas Protozoárias/imunologia , Imunidade Adaptativa , Animais , Antígenos de Protozoários/biossíntese , Antígenos de Superfície/biossíntese , Antígenos de Superfície/química , Antígenos de Superfície/imunologia , Granzimas/sangue , Humanos , Imunidade Humoral , Interferon gama/sangue , Interleucina-10/sangue , Leishmaniose/sangue , Leishmaniose/imunologia , Camundongos , Fenótipo , Vacinas Protozoárias/biossíntese , Solubilidade , Fator de Necrose Tumoral alfa/sangue
17.
Parasitol Res ; 113(7): 2559-68, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24764159

RESUMO

Among the proteins involved in the invasion by merozoite, the glycosylphosphatidylinositol-anchored proteins (GPI-APs) are suggested as potential vaccine candidates because of their localization to apical organelles and the surface; these candidates are predicted to play essential roles during invasion. As a GPI-AP, Plasmodium vivax merozoite surface protein 10 (PvMSP-10) induces high antibody titers. However, such high antibody titers have shown no protective efficacy for animals challenged with P. vivax parasites in a previous study. To adequately evaluate the immunogenicity and further characterize PvMSP-10 in order to understand its vaccine potential, we assessed its immunogenicity by immunizing BALB/c mice with cell-free expressed recombinant PvMSP-10 protein. The antigenicity of MSP-10 was analyzed, and we found 42% sensitivity and 95% specificity using serum samples from P. vivax-infected Korean patients. The IgG1 and IgG3 were the predominant immunoreactive antibodies against PvMSP-10 in vivax patient sera, and IgG1 and IgG3 and Th1-type cytokines were predominantly secreted in PvMSP-10-immunized mice. We conclude that the immunogenicity and antigenicity of MSP-10 may serve as a potential vaccine against vivax malaria.


Assuntos
Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/imunologia , Malária Vivax/prevenção & controle , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/administração & dosagem , Antígenos de Protozoários/biossíntese , Feminino , Humanos , Soros Imunes , Imunização , Imunoglobulina G/sangue , Vacinas Antimaláricas/administração & dosagem , Malária Vivax/sangue , Malária Vivax/imunologia , Merozoítos/química , Merozoítos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/administração & dosagem , Proteínas de Protozoários/biossíntese , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia
18.
Expert Rev Vaccines ; 13(1): 75-85, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24308585

RESUMO

Malaria causes about 216 million clinical cases and 0.7 million deaths annually. One promising route to address malaria is vaccination. However, so far, not even a single licensed malaria vaccine has been developed. Even the effectiveness of RTS,S, the world's most advanced malaria vaccine candidate (MVC) in clinical trials, is less than 50% efficacy against the disease. This backdrop indicates that the search for a truly effective vaccine is far from over and galvanizes us to expand the arsenal of promising MVC antigens to include in a next generation subunit vaccine. In our previous proof of principle studies, we have found that the wheat germ cell-free protein synthesis system (WGCFS) is one of the optimal tools for synthesis of quality malaria proteins and hence the identification of novel MVCs. This review summarizes the initial progresses so far made regarding the identification of novel MVCs using WGCFS.


Assuntos
Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/imunologia , Descoberta de Drogas/métodos , Vacinas Antimaláricas/imunologia , Vacinas Antimaláricas/isolamento & purificação , Biossíntese de Proteínas , Extratos Celulares , Sistema Livre de Células , Humanos , Triticum
19.
Bioengineered ; 4(6): 413-9, 2013 Nov-Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23941978

RESUMO

The production of recombinant antigens in Escherichia coli and specific polyclonal antibodies for diagnosis and therapy is still a challenge for world-wide researchers. Several different strategies have been explored to improve both antigen and antibody production, all of them depending on a successful expression and immunogenicity of the antigen. Gene fusion technology attempted to address these challenges: fusion partners have been applied to optimize recombinant antigen production in E. coli, and to increase protein immunogenicity. Taking a 12-kDa surface adhesion antigen from Cryptosporidium parvum (CP12) by example, the novel H fusion partner was presented in this work as an attractive option for the development of recombinant immunogens and its adjuvant-free immunization. The H tag (of only 1 kDa) efficiently triggered a CP12-specific immune response, and it also improved the immunization procedure without requiring co-administration of adjuvants. Moreover, polyclonal antibodies raised against the HCP12 fusion antigen detected native antigen structures displayed on the surface of C. parvum oocysts. The H tag proved to be an advanced strategy and promising technology for the diagnosis and therapy of C. parvum infections in animals and humans, allowing a rapid and simple recombinant production of the CP12 antigen.


Assuntos
Antígenos de Protozoários/biossíntese , Fusão Gênica , Proteínas Recombinantes/biossíntese , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/sangue , Sequência de Bases , Cryptosporidium parvum/genética , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Camundongos , Dados de Sequência Molecular , Oocistos/imunologia
20.
Appl Microbiol Biotechnol ; 97(5): 1987-95, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22592550

RESUMO

Malaria is a widespread and infectious disease that is a leading cause of death in many parts of the world. Eradication of malaria has been a major world health goal for decades, but one that still remains elusive. Other diseases have been eradicated using vaccination, but traditional vaccination methods have thus far been unsuccessful for malaria. Infection by Plasmodium species, the causative agent of malaria, is currently treated with drug-based therapies, but an increase in drug resistance has led to the need for new methods of treatment. A promising strategy for malaria treatment is to combine transmission blocking vaccines (TBVs) that prevent spread of disease with drug-based therapies to treat infected individuals. TBVs can be developed against surface protein antigens that are expressed during parasite reproduction in the mosquito. When the mosquito ingests blood from a vaccinated individual harboring the Plasmodium parasite, the antibodies generated by vaccination prevent completion of the parasites life-cycle. Animal studies have shown that immunization with Pfs48/45 results in the production of malaria transmission blocking antibodies; however, the development of this vaccine candidate has been hindered by poor expression in both prokaryotic and eukaryotic hosts. Recently, the chloroplast of Chlamydomonas reinhardtii has been used to express complex recombinant proteins. In this study, we show that the C-terminal antigenic region of the Pfs48/45 antigen can be expressed in the chloroplast of the green algae C. reinhardtii and that this recombinant protein has a conformation recognized by known transmission blocking antibodies. Production of this protein in algae has the potential to scale to the very large volumes required to meet the needs of millions at risk for contracting malaria.


Assuntos
Antígenos de Protozoários/biossíntese , Chlamydomonas reinhardtii/genética , Expressão Gênica , Vacinas Antimaláricas/biossíntese , Glicoproteínas de Membrana/biossíntese , Proteínas de Protozoários/biossíntese , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Biotecnologia/métodos , Vacinas Antimaláricas/genética , Vacinas Antimaláricas/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Ligação Proteica , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Tecnologia Farmacêutica/métodos , Vacinas Sintéticas/biossíntese , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
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