In silico study to predict promiscuous T cell and B cell-epitopes derived from the vaccine candidate antigens of Plasmodium vivax binding to MHC class-II alleles

Malaria is one of the major causes of health and disability globally, even after tremendous efforts to eradicate it. Till date no highly effective vaccine is available for its control. The primary reason for the low efficacy of vaccines is extensive polymorphism in potential vaccine candidate antigen genes and HLA polymorphisms in the human population. This problem can be resolved by developing a vaccine using promiscuous peptides to combine the number of HLA alleles. This study predicted T and B cell epitopes (promiscuous peptides) by targeting PPPK-DHPS and DHFR-TS proteins of Plasmodium vivax, using different in silico tools. Selected peptides were characterized as promiscuous peptides on the basis of their immunogenicity, antigenicity and hydrophobicity. Furthermore, to confirm their immunogenicity, these peptides were utilized for molecular modelling and docking analysis. For determining the requisite affinity with distinct HLA Class-I, and HLA Class-II alleles, only five peptides for DHFR-TS and 3 peptides for PPPK-DHPS were chosen as promiscuous peptides. The D1 peptide has the maximum binding energy with HLA alleles, according to HLA-peptide complex modelling and binding interaction analyses. These findings could lead to the development of epitope-based vaccinations with improved safety and efficacy. These epitopes could be major vaccine targets in P. vivax as they possess a higher number of promiscuous peptides. Also, the B cell epitopes possess maximum affinity towards different alleles as analyzed by docking scores. However, further investigation is warranted in vitro and in vivo.

Desenvolvimento de teste diagnóstico não invasivo capaz de detectar anticorpos anti-SARS-CoV-2 em urina / Development of a non-invasive diagnostic test capable of detecting anti-SARS-CoV-2 antibodies in urine

A plataforma de ELISA (ensaio de imunoabsorção por ligação enzimática) tem sido amplamente utilizada para detectar anticorpos anti-SARS-CoV-2 gerados após a exposição ao vírus ou à vacinação. A amostra comumente utilizada para a realização do teste é o soro. Até o momento, nenhum estudo havia investigado a urina do paciente como amostra para detectar anticorpos específicos para o vírus SARS-CoV-2. A urina é um espécime biológico que traz vantagens significativas inerentes ao tipo de amostra, que compreende coleta não invasiva, de fácil manuseio e armazenamento. Neste trabalho, propomos um ELISA indireto in house baseado no uso de urina e proteínas recombinantes do Nucleocapsídeo (N) ou da Spike (S) do vírus SARS-CoV-2. As proteínas recombinantes (r) de SARS-CoV-2, N e as subunidades da proteína S (S-Glic, S1-NGlic e RBD-NGlic), foram avaliadas usando um painel composto por aproximadamente 200 amostras de urina e de soro. A presença de anticorpos anti-SARS-CoV-2 na urina foi detectada com sensibilidade e especificidade similares ou superiores ao soro, nas quais foram obtidos valores de sensibilidade de 94,0%, 75,0%, 81,38% e 89,66%, e especificidade de 100%, 96,0%, 96,77% e 96,77%, frente às proteínas rSARS-CoV-2 N, S-Glic, S1-NGlic e RBDNGlic, respectivamente. Dessa forma, os dados apresentados sugerem que a urina poderia ser considerada como uma potencial amostra biológica para aplicação em plataformas de imunodiagnóstico para a infecção por SARS-CoV-2, trazendo benefícios tanto no contexto individual quanto populacional.

The Enzyme-linked immunosorbent assay (ELISA) method has been widely used to detect anti-SARS-CoV-2 antibodies generated after exposure to the virus or vaccination. The sample usually used to perform the test is the serum. Thus far, no study has investigated the urine of patients as biological sample to detect specific SARS-CoV-2 antibodies. Urine is a biological specimen with significant advantages inherent to the type of sample, which comprises non-invasive collection, easy handling and storage. In this work, we propose an in house urine-based indirect ELISA using recombinant proteins from Nucleocapsid (N) and Spike (S) of the SARSCoV-2 virus. SARS-CoV-2 recombinant N and S protein subunits (Gly-S, NonGly-S1 and NonGly-RBD) were evaluated in an ELISA platform with a panel composed about 200 urine and serum samples. The presence of anti-SARS-CoV-2 antibodies in urine was detected with similar or superior sensitivity and specificity to serum, in which sensitivity values of 94.0%, 75.0%, 81.38% and 89.66% were obtained, while specificity values were of 100.0%, 96.0%, 96.77% and 96.77%, respectively, against rSARS-CoV-2 N, S-Glic, S1-NGlic and RBD-NGlic proteins. In conclusion, the data presented suggest that urine could be considered as a potential biological sample for application in immunodiagnostic platforms for SARS-CoV-2 infection, with benefits to the individual and population context.

Identification of T-Cell Epitopes in Proteins of Novel Human Coronavirus, SARS-Cov-2 for Vaccine Development

Recently, a novel human coronavirus, SARS-CoV-2 led to a worldwide serious health concern, causing severe respiratory tract infections in humans. It is the third highly pathogenic and transmissible coronavirus after severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in humans. The source of origin, transmission to humans and mechanisms associated with the pathogenicity of SARS-CoV-2 are not clear yet, however, its resemblance with SARS-CoV and several other bat coronaviruses was recently confirmed through genome sequencing related studies. It has been an emergent need to develop a potent and adequate number of drugs and vaccines to control the spread of coronavirus. We have screened the specific proteins such as ORF1ab polyprotein, surface glycoprotein, membrane glycoprotein and nucleocapsid phosphoprotein of SARS-CoV-2 for identification of T-cell epitopes using immunoinformatics tools. In this study we used different bioinformatics tools for analysis of genome and proteome. We retrieved gene sequence from NCBI. The expected molecular weight and isoelectric point (pI) values were also verified using Generunner and ExPaSy. These epitopes have showed the highest binding affinity with major histocompatibility complex (MHC) class I and II molecules. These findings may be useful as an immunodiagnostic tool for the development of peptide based novel vaccines.

Meta-analyses of the validity and performance of screening tests for Hepatitis C Virus in the blood banks, 2000-2018 / Metaanálisis de la validez y el desempeño de las pruebas de tamización del virus de la hepatitis c en bancos de sangre, 2000-2018

RESUMEN Este estudio evaluó la validez y desempeño del inmunodiagnóstico del virus de la hepatitis C (VHC), con base en estudios publicados en la literatura científica mundial. Se diseñó y validó un protocolo de búsqueda y selección de investigaciones en las fases de la guía PRISMA, se analizaron los parámetros de sensibilidad, especificidad, cocientes de probabilidad, razón de odds y curva ROC, en MetaDisc. Se tamizaron 4602 estudios, de los cuales sólo 545 se realizaron en bancos de sangre y 18 evaluaron la validez diagnóstica de las pruebas para el VHC. La mayoría de los estudios fueron de Europa y Asia, con un 78 % basados en determinación de anticuerpos. Los estudios con detección de anticuerpos se realizaron en 21 483 donantes sanos y 3 145 infectados en quienes se halló una sensibilidad de 97,8 % (IC 95 % = 97,3 - 98,2), especificidad 99,0 % (IC 95 % = 98,9 - 99,2), cociente de probabilidad positivo 75,4 (IC 95 % = 27,2 - 209,2) y negativo de 0,02 (IC 95 % = 0,01 - 0,07) y área bajo la curva de 99,8 %. Se concluye que la detección de anticuerpos presenta excelente validez, desempeño y utilidad diagnóstica para la detección del VHC en donantes de sangre y población general.

ABSTRACT This study evaluated the validity and performance of the immunodiagnosis of the Hepatitis C Virus (HCV), based on studies published in the worldwide scientific literature. A search and selection research protocol was designed and validated in the phases of the PRISMA guide, the parameters of sensitivity, specificity, likelihood ratios, odds ratio, and ROC curve were analyzed in MetaDisc. 4602 studies were screened, of which only 545 were performed in blood banks and 18 evaluated the diagnostic validity of the tests for HCV. Most studies were from Europe and Asia, with 78 % based on antibody determination. Studies with antibody detection were carried out in 21483 healthy donors and 3145 infected patients in whom a sensitivity of 97.8 % (95 % CI = 97.3 - 98.2) was found, 99.0 % specificity (95 % CI = 98.9 - 99.2), positive likelihood ratio 75.4 (95 % CI = 27.2 - 209.2) and negative of 0.02 (95% CI = 0.01 - 0.07) and area under the curve 99.8 %. It is concluded that the detection of antibodies presents excellent validity, performance, and diagnostic utility for the detection of HCV in blood donors and the general population.

Evaluación y uso de un péptido sintético para la detección de anticuerpos específicos contra Plasmodium falciparum / Development and use of a synthetic peptide for the detection of specific antibodies against Plasmodium falciparum

Resumen Introducción. La malaria continúa siendo una de las enfermedades que causa mayor morbi-mortalidad a nivel mundial. Por esta razón es importante desarrollar herramientas diagnósticas eficaces que se implementen como estrategias para el control de la enfermedad. Objetivos. Estandarizar las condiciones del inmunoensayo enzimático (ELISA), para la detección de IgG específica contra Plasmodium falciparum en sueros de pacientes diagnosticados por gota gruesa con malaria no complicada por P. falciparum, empleando como antígeno un extracto proteico obtenido a partir de cultivo de P. falciparum o un péptido sintético derivado de la proteína de superficie de merozoito GLURP (del inglés: glutamate rich protein). Materiales y métodos. Para la estandarización de la técnica, se utilizaron 22 sueros de pacientes positivos para malaria por P. falciparum y 11 diagnosticados positivos para malaria por P. vivax utilizando la técnica de gota gruesa. Como controles negativos se utilizaron 44 sueros de individuos sanos. Los sueros fueron probados contra extracto de proteínas del parásito y el péptido sintético IMT 94 derivado de la proteína GLURP, para evaluar las concentraciones y las diluciones óptimas de cada componente del sistema. Para la validación de la técnica se utilizaron 251 sueros de pacientes positivos para P. falciparum y 44 sueros de individuos sanos, diagnosticados utilizando la técnica de gota gruesa. Resultados. La técnica estandarizada con el péptido sintético permitió observar diferencia significativa en el reconocimiento de sueros de pacientes, controles positivos y negativos por los antígenos (extracto de proteínas y péptidos sintéticos). Conclusiones. La metodología usada permite identificar la respuestas inmune específica contra P. falciparum.

Abstract Introduction. Malaria continues being one of the diseases causing the greatest morbi-mortality around the world. For that reason, effective diagnostic tools must thus be developed which can be used in strategies for controlling the disease. Objectives. To standardise enzyme- linked immunosorbent assay (ELISA) conditions for detecting Plasmodium falciparum specific IgG in sera from patients diagnosed by thick smear as suffering non-complicated malaria caused by P. falciparum. A protein extract obtained from P. falciparum culture or a synthetic peptide derived from glutamate rich protein (GLURP) merozoite surface protein would be used as antigen. Materials and Methods. 22 serum samples from patients diagnosed as suffering from P. falciparum malaria, 11 serum samples from patients diagnosed as suffering from P. vivax and 44 from healthy donors, diagnosed by using the thick smear tecnique were used for standarising the technique. Serum samples were tested against parasite protein extract and GLURP- derived IMT 94 synthetic peptide for standardisign optimum dilutions and concentrations for each component in the system. 251 serum samples from patients diagnosed as suffering from P. falciparum malaria and 44 from healthy donors diagnosed by using the thick smear tecnique were used to validate the technique. Results. The technique led to significant differences being observed in antigens (protein extract and synthetic peptides) recognising serum from positive and negative patients and controls. Conclusions. The methodology used led to identifying specific immune response against P. falciparum.

Evaluation of the recombinant antigens Wb14 and WbT for the capture antibody diagnosis of lymphatic filariasis

BACKGROUND Lymphatic filariasis (LF) is a parasitic disease caused mainly by the Wuchereria bancrofti worm and that affects up to 120 million people worldwide. LF is the second cause of chronic global deformity, responsible for 15 million people with lymphedema (elephantiasis) and 25 million men with scrotal hydrocele. Its diagnosis is still associated with numerous difficulties, such as the sample collection periods (microfilaria nocturnal periodicity) and limited diagnostic kits. OBJECTIVES The aim of this work was to evaluate two recombinant antigens (Wb14 and WbT) as part of an enzyme-linked immunosorbent assay (ELISA) based antibody capture tests for LF. METHODS The recombinant antigens rWb14 and rWbT were expressed in Escherichia coli BL21 and an antibody capture ELISA was performed. For this, sera were used from microfilaremic individuals with W. bancrofti (MF), chronic pathology (CP), individuals infected with Strongyloides (SP) and healthy controls from endemic (EN) and non-endemic (NE) areas. FINDINGS Both tests showed similar results, with 90% sensitivity and 96.6% specificity. In comparison with the BM14 ELISA commercial test, the Wb14 and WbT antigens performed with identical sensitivity but greater specificity. Reduced positivity with the CP suggested a potential to monitor cure. This was not confirmed, however, when sera from individuals up to seven years after treatment were assayed. MAIN CONCLUSIONS The Wb14 and WbT ELISAs were considered efficient and promising diagnostic tests. Due to the importance of antibody capture analysis to evaluate the Global Program to Eliminate Lymphatic Filariasis (GPELF), the tests proposed here appear as great alternatives to the available commercial system.

Standardization and validation of Dot-ELISA assay for Paracoccidioides brasiliensis antibody detection

Background Paracoccidioidomycosis (PCM) is a neglected systemic mycosis caused by a dimorphic fungus of the Paracoccidioides genus. The standard diagnosis is based on isolation of the fungi in culture, and by microscopic visualization of characteristic multiple budding yeast cells in biological samples. However, in some situations, access to the site of injury prevents the collection of biological material. A variety of immuno-serological techniques has proven useful for allowing inferring diagnosis with a certain degree of certainty, thus optimizing time. The aim of this study was to standardize and validate the Dot-ELISA (DE) assay, comparing it with the serological standard, double immunodiffusion (DI). Methods In order to standardize the DE assay, 143 serum samples were used. Out of those, 23 were from apparently healthy patients, 77 were from patients with confirmed PCM and 43 were from patients with other lung infections (tuberculosis, aspergillosis and histoplasmosis). To validate the DE technique, 300 serum samples from patients with PCM clinical suspicion (probable and possible cases) were employed, and these results were compared with those of DI. Results The DE assay showed sensitivity of 91%, specificity of 95.4%, positive predictive value of 96%, negative predictive value of 98.2%, accuracy of 93%, and great precision (k = 0.93). In addition, the nitrocellulose membranes have proved to be viable for using at least 90 days after P. brasiliensis B-339 antigen sensitization. Conclusion Dot-ELISA method was found to be an extremely promising tool as serologic screening technique, because of its high sensitivity. Furthermore, Dot-ELISA shows the prospect of being transferred to laboratories of mycoserology including those with fewer resources or even to be used directly in the field. It has an excellent shelf life - membranes coated with antigen can be used for testing without changes in the pattern of reactivity among laboratories - and presents reliable values of sensitivity, specificity, predictive values, accuracy and a high correlation with the serological standard methodology. Based on the present findings, it possible to state that this technique constitutes a remarkable option to be used in routine diagnosis for public health centers.(AU)

Standardization and validation of Dot-ELISA assay for Paracoccidioides brasiliensis antibody detection

Abstract Background Paracoccidioidomycosis (PCM) is a neglected systemic mycosis caused by a dimorphic fungus of the Paracoccidioides genus. The standard diagnosis is based on isolation of the fungi in culture, and by microscopic visualization of characteristic multiple budding yeast cells in biological samples. However, in some situations, access to the site of injury prevents the collection of biological material. A variety of immuno-serological techniques has proven useful for allowing inferring diagnosis with a certain degree of certainty, thus optimizing time. The aim of this study was to standardize and validate the Dot-ELISA (DE) assay, comparing it with the serological standard, double immunodiffusion (DI). Methods In order to standardize the DE assay, 143 serum samples were used. Out of those, 23 were from apparently healthy patients, 77 were from patients with confirmed PCM and 43 were from patients with other lung infections (tuberculosis, aspergillosis and histoplasmosis). To validate the DE technique, 300 serum samples from patients with PCM clinical suspicion (probable and possible cases) were employed, and these results were compared with those of DI. Results The DE assay showed sensitivity of 91%, specificity of 95.4%, positive predictive value of 96%, negative predictive value of 98.2%, accuracy of 93%, and great precision (k = 0.93). In addition, the nitrocellulose membranes have proved to be viable for using at least 90 days after P. brasiliensis B-339 antigen sensitization. Conclusion Dot-ELISA method was found to be an extremely promising tool as serologic screening technique, because of its high sensitivity. Furthermore, Dot-ELISA shows the prospect of being transferred to laboratories of mycoserology including those with fewer resources or even to be used directly in the field. It has an excellent shelf life membranes coated with antigen can be used for testing without changes in the pattern of reactivity among laboratories and presents reliable values of sensitivity, specificity, predictive values, accuracy and a high correlation with the serological standard methodology. Based on the present findings, it possible to state that this technique constitutes a remarkable option to be used in routine diagnosis for public health centers.

Development and characterization of IgY antibodies against synthetic peptides from Brucella abortus OMP25 and BP26 proteins / Desarrollo y caracterización de anticuerpos IgY contra péptidos sintéticos de las proteínas OMP25 y BP26 de Brucella abortus

Brucellosis is a zoonose produced by bacterial species from the Brucella genus. Its isolation and identification in food using classical microbiological techniques is not practical due to its slow growth rate. Therefore, it is necessary to establish fast and specific methods for the detection of the bacteria in food. The goal of this work was the production and characterization of monospecific polyclonal antibodies in chicken (IgY) against synthetic peptides from Brucella abortus OMP25 and BP26 proteins, suitable for an antigen-capture assay. Conformational as well as antigenic predictions were performed using the ANTHEPROT package. Chemical synthesis was carried out by the multiple manual synthesis using the t-boc strategy. The peptides were used as antigens for the preparation of polyclonal antibodies in chicken. Experimental animals produced specific antibodies against the OMP25 and BP26 peptides constructs determined by ELISA and MABA assays showing correspondence between the predictive study and the immunogenicity obtained in chicken. The IgY proved to be able to recognize B. abortus by MABA assays. The binding activity and specificity of antibodies was determined by Western blot with cell extract from B. abortus. In this study, we demonstrated that OMP25 and BP26 peptides constructs are good candidates for production of specific IgY antipeptide antibodies capable of recognizing proteins from sonicated B. abortus strain S19, indicating the potential usefulness of the IgY antibody for development of immunoassays for detection of Brucella abortus.

La brucelosis es una zoonosis producida por especies del género Brucella. El aislamiento e identificación de la bacteria en alimentos usando las técnicas clásicas de microbiología no es práctico debido a su lenta tasa de crecimiento. Por lo tanto, es necesario establecer métodos rápidos para la detección de la bacteria en alimentos. En el presente trabajo se desarrollaron y caracterizaron anticuerpos policlonales monoespecíficos en gallinas (IgY) contra péptidos sintéticos de las proteínas OMP25 y BP26 de Brucella abortus, que puedan ser utilizados en un ensayo de captura. Para ello, se realizaron estudios conformacionales y de predicción de epítopes en la selección de los péptidos, los cuales se utilizaron como antígenos para la producción de las IgY. Los animales desarrollaron anticuerpos específicos contra los péptidos, mostrando correspondencia entre los estudios predictivos y la inmunogenicidad obtenida. Las IgY reconocieron a B. abortus en un ensayo de MABA y la actividad de unión y especificidad fue determinada por western blot con extracto celular de B. abortus. En este estudio, demostramos que los péptidos de las proteínas OMP25 y BP26 de B. abortus son buenos candidatos para la producción de anticuerpos IgY especificos capaces de reconocer proteínas de extracto de B. abortus cepa S19, indicando el potencial uso de anticuerpos IgY para el desarrollo de inmunoensayos para la detección de Brucella abortus.

Produção da proteína recombinante Sm15 com potencial para o diagnóstico de pacientes infectados com Schistosoma mansoni

O exame parasitológico por Kato-Katz ainda é considerado o padrão ouro no diagnóstico da esquistossomose mansônica, entretanto, este apresenta baixa sensibilidade para utilização em inquéritos epidemiológicos. Além disso, as técnicas de diagnóstico imunológico, apresentam reações cruzadas com outros helmintos, protozoários e até bactérias como ocorre com a utilização dos extratos brutos do parasita. Nesse sentido, salientamos que Abath et al. identificaram um peptídeo de 15kDa denominado Sm15, que apresentou uma boa reatividade com soros de animais infectados pelo verme e, portanto, possui potencial para abordagens imunoprofiláticas e para testes diagnósticos. Neste estudo obtivemos o polipeptídio recombinante Sm15 em Escherichia coli e verificamos seu potencial para realização do diagnóstico a partir de amostras de soros de pacientes com diferentes manifestações clínicas da esquistossomose. Através de ELISA constatamos que o Sm15 apresentou maior reatividade frente a soros de pacientes esquistossomóticos, quando comparado ao extrato bruto SEA (P=0.0043). O Sm15 ainda demonstrou melhor desempenho ao apresentar maiores valores de sensibilidade, especificidade e área abaixo da curva ROC (P=0.0030). Além disso, o Sm15 foi capaz de diferenciar pacientes esquistossomóticos quanto à forma clínica, aguda ou crônica (P=0.0007). Os resultados obtidos neste estudo indicam, além de ratificar o potencial diagnóstico apresentado pelo polipeptídeo Sm15, que o mesmo poderá ser capaz de gerar uma alternativa de imunodiagnóstico de elevada acurácia, suprindo assim as lacunas existentes com relação aos testes parasitológicos e sorológicos atualmente disponíveis. Além disso, possibilitará o diagnostico precoce da esquistossomose, realização de inquéritos epidemiológicos em áreas de baixa endemicidade, impedindo assim a evolução da doença para formas clínicas de maior gravidade.

The parasitological examination by Kato-Katz still considered the gold standard in the diagnosis of schistosomiasis, however, it has low sensitivity for use in epidemiological surveys. Moreover, the techniques of immunological diagnosis, have cross-reactivity with other helminth, protozoa and even bacteria as occur with the use of crude parasite extracts. In this regard, we note that Abath and colleagues identified a 15kDa peptide termed SM15, which showed good reactivity with sera from animals infected by the worm, and therefore has potential immunoprophylactic and diagnostic testing approaches. In this study we obtained the recombinant polypeptide in Escherichia coli SM15 and check its potential for making the diagnosis from samples of patient sera with different clinical manifestations of schistosomiasis. By ELISA we found that the SM15 showed higher reactivity towards sera from schistosomiasis patients, when compared to the crude extract SEA (P = 0.0043). The SM15 also demonstrated better performance by presenting higher sensitivity, specificity, and area under the ROC curve (P = 0.0030). In addition, the SM15 was able to differentiate schistosomiasis patients about the clinical presentation, acute or chronic (P = 0.0007). The results of this study indicate not only ratifies the diagnostic potential presented by the SM15 polypeptide, that it may be able to generate an immunodiagnostic alternative high accuracy, thereby supplying the gaps in the parasitological and serological tests currently available. Also, it enables the early diagnosis of schistosomiasis, carrying out epidemiological surveys in low endemicity areas, thereby preventing disease progression to more severe clinical forms.

Expressão heteróloga da EMA-2 (equi merozoite antigen) de Theileria equi em Pichia pastoris com potencial utilização em imunobiológicos / Heterologous expression of EMA-2 (equi merozoite antigen) of Theileria equi in Pichia pastoris with potential use in immunobiologics

A piroplasmose equina causada por Theileria equi acomete os equinos de forma endêmica no Brasil e em diversos outros países tropicais e subtropicais. Considerada uma das mais importantes doenças de equinos, causa danos à saúde animal e perdas econômicas. A proteína equi merozoite antigen (EMA-2) é uma das principais proteínas de superfície, expressa nos diversos estágios do ciclo do parasita, estimula resposta imune em animais infectados, tornando-se um possível candidato para utilização em diagnóstico. O gene EMA-2 foi clonado e expresso na levedura Pichia pastoris. A proteína EMA-2 recombinante (rEMA-2) foi caracterizada antigenicamente por Western Blot e por ELISA indireto, utilizando-se soro de equino positivo para theileriose. O resultado do ELISA demonstrou uma especificidade de 90,9% e sensibilidade de 83,3%, quando comparado ao padrão, sendo superior à imunofluorescência (80,6% de especificidade e 75,0% de sensibilidade), o que sugere que a rEMA-2 expressa em P. pastoris é um promissor antígeno para ser utilizado como ferramenta no imunodiagnóstico de theileriose equina.

Theileria equi, the causative agent of equine piroplasmosis, is endemic in Brazil and many other tropical and subtropical countries. It is considered one of the most important diseases of horses causing animal health problems and significant economic loss. The Protein equi merozoite antigen-2 (EMA-2) is a major surface protein that is expressed in different parasite cycle stages and induces immune response in infected animals, being a possible candidate to be used in diagnose. EMA-2 gene was cloned and expressed in the yeast Pichia pastoris, and the recombinant protein EMA-2 (rEMA-2) was characterized by Western Blot and indirect ELISA using equine positive sera. The ELISA results demonstrated a specificity of 90.9% and a sensitivity of 83.3% compared to the standard ELISA and being superior to immunofluorescence (80.6% of specificity and 75.0% of sensitivity) suggesting that the rEMA-2 expressed in P. pastoris is a promising antigen to be used as a tool in immunodiagnostic of theileriasis.

Identification of T-Cell epitopes in structural proteins of tick borne encephalitis virus for vaccine development.

Tickborne encephalitis (TBE) is a human viral infectious disease caused by tickborne encephalitis virus (TBEV). It is transmitted by the bite of an infected tick and also initiated the swelling of brain (encephalitis) and spinal cord. There is a pressing need to develop potent and sufficient amount of drugs and vaccines for control of TBE. We have selected the structural proteins such as anchored core protein C, core protein C, premembrane, matrix and envelope proteins of TBEV for identification of T-cell epitopes using immunoinformatics tools. These epitopes were showed the highest binding affinity with major histocompatibility complex (MHC) class I and II molecules. These finding may be used as an immunodiagnostic agent and also development of peptide based novel vaccines.

Comparison of four diagnostic methods for detecting rabies viruses circulating in Korea

It is essential to rapidly and precisely diagnose rabies. In this study, we evaluated four diagnostic methods, indirect fluorescent antibody test (FAT), virus isolation (VI), reverse transcriptase polymerase chain reaction (RT-PCR), and rapid immunodiagnostic assay (RIDA), to detect rabies in animal brain homogenates. Out of the 110 animal brain samples tested, 20 (18.2%) were positive for rabies according to the FAT. Compared to the FAT, the sensitivities of VI, RT-PCR, and RIDA were 100, 100, and 95%, respectively. The specificities of VI, RT-PCR and RIDA were found to be 100, 100, and 98.9%, respectively. Rabies viruses circulating in Korea were isolated and propagated in murine neuroblastoma (NG108-15) cells with titers ranging from 101.5 to 104.5 TCID50/mL. Although the RIDA findings did not completely coincide with results obtained from FAT, VI, and RT-PCR, RIDA appears to be a fast and reliable assay that can be used to analyze brain samples. In summary, the results from our study showed that VI, RT-PCR, and RIDA can be used as supplementary diagnostic tools for detecting rabies viruses in both laboratory and field settings.

Inmunodiagnóstico en Didelphis marsupialis usando un antígeno de Paragonimus de Venezuela / Immunodiagnostic in Didelphis marsupialis using an antigen of Venezuelan Paragonimus

Los Paragonimus son trematodos que habitualmente viven en los pulmones de mamíferos carnívoros y omnívoros, entre ellos el hombre. En el oriente venezolano se encuentra el único foco de Paragonimus sp. donde Didelphis marsupialis es el único reservorio demostrado hasta ahora. Con el fin de tener herramientas de inmunodiagnóstico que detecten la presencia de Paragonimus sp. en esta especie, se elaboraron varios reactivos para realizar un ensayo inmunoenzimático ELISA. Entre ellos se obtuvo un antígeno crudo soluble de vermes adultos de Paragonimus y una inmunoglobulina de gallina anti-IgG de Didelphis marsupialis. Los mismos se capturaron en la localidad de Aguas Blancas, municipio Montes, estado Sucre, Venezuela, y se obtuvieron muestras sanguíneas; en el caso de estar infectados, los vermes adultos se extrajeron del pulmón. Los parásitos se homogenizaron y ultracentrifugaron para obtener la fracción soluble del parásito (FSPA) como antígeno para el ELISA y Western blot y detectar los anticuerpos en los Didelphis marsupialis. El análisis electroforético mostró 22 moléculas entre 6 y 82 kDa; por Western blot se presentó un reconocimiento antigénico de 8 moléculas siendo las de 112 kDa y 268 kDa las más reconocidas por los sueros positivos. Los sueros negativos no reconocieron ninguna proteína. La producción de IgY en gallinas permitió desarrollar las técnicas de inmunodiagnóstico para la búsqueda de anticuerpos específicos anti-Paragonimus sp. en Didelphis, cuya aplicación permitirá establecer la vigilancia epidemiológica de estos reservorios en áreas endémicas sin sacrificio de los mismos.

Paragonimus are trematodes that normally live in the lungs of carnivorous and omnivorous mammals such as humans. An outbreak of Paragonimus sp. in which Didelphis marsupialis was the only wild reservoir incriminated was described in eastern Venezuela. In order to have immunological tools to detect the presence of Paragonimus sp. in this reservoir, a whole antigen of the adult worm of this parasite was elaborated. Didelphis marsupialis were captured in the locality of Aguas Blancas, Montes municipality, Sucre state, Venezuela, from which blood samples were obtained and a search for worms was performed in lungs. Worms were homogenized and ultracentrifugated to obtain FSPA to perform immunoassay (ELISA) to detect antibodies in opossums. The electrophoresis analysis showed a pattern of 22 molecules between 6 and 82 kDa; by western blot, the antigenic recognition of 8 antigenic molecules appeared,112 kDa and 268 kDa molecules being the most strongly recognized by positive sera. The negative sera did not recognize any band. The production of IgY in chicken enabled the development of reagents capable of performing a standard immunodiagnosis technique to find specific anti-Paragonimus sp. in Didelphis marsupialis in order to establish epidemiological surveillance of these reservoirs in endemic areas.

Anti-Toxoplasma gondii secretory IgA in tears of patients with ocular toxoplasmosis: immunodiagnostic validation by ELISA

Toxoplasma gondii causes posterior uveitis and the specific diagnosis is based on clinical criteria. The presence of anti-T. gondii secretory IgA (sIgA) antibodies in patients' tears has been reported and an association was found between ocular toxoplasmosis and the anti-T. gondii sIgA isotype in Brazilian patients. The purpose of this study was to provide an objective validation of the published ELISA test for determining the presence of anti-T. gondii sIgA in the tears of individuals with ocular toxoplasmosis. Tears from 156 patients with active posterior uveitis were analysed; 82 of them presented characteristics of ocular toxoplasmosis (standard lesion) and 74 patients presented uveitis due to other aetiologies. Cases of active posterior uveitis were considered standard when a new inflammatory focus satellite to old retinochoroidal scars was observed. The determination of anti-T. gondii sIgA was made using an ELISA test with crude tachyzoite antigenic extracts. Tears were collected without previous stimulation. Detection of sIgA showed 65.9 percent sensitivity (95 percent CI = 54.5-74.4), 71.6 percent specificity (95 percent CI = 59.8-81.2), a positive predictive value of 72 percent (95 percent CI = 60.3-81.5) and a negative predictive value of 65.4 percent (95 percent CI = 54.0-75.4). sIgA reactivity was higher in the tears of patients with active posterior uveitis due to T. gondii (p < 0.05). The test is useful for differentiating active posterior uveitis due to toxoplasmosis from uveitis caused by other diseases.