Produção e caracterização de anticorpos monoclonais contra o vírus rábico

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Programa de Pós-Graduação em Biotecnologia, Florianópolis, 2010Os primeiros anticorpos monoclonais (AcM) contra o vírus rábico foram produzidos por Wiktor e Koprowski em 1978 e, desde então, diversos outros foram produzidos e descritos. No entanto, apesar do grande número de AcM antirrábicos descritos na literatura, eles nem sempre são facilmente obtidos, principalmente porque a produção de AcM no Brasil é bastante limitada. Ademais, devido à grande diversidade de variantes de vírus rábico existentes no Brasil, torna-se importante produzir novos AcM que possam atender a interesses mais específicos. Assim, o objetivo do presente trabalho foi a produção e caracterização de AcM contra o vírus rábico. Para tanto, foram realizados cinco protocolos de imunização, nos quais camundongos Balb/c foram imunizados com os seguintes antígenos: (1) vacina antirrábica (VeroRab®, Aventis-Pasteur) para uso em humanos; (2) suspensão inativada de tecido de sistema nervoso central de camundongos infectados com vírus rábico selvagem isolado de um bovino naturalmente infectado por morcego hematófago em Passos de Torres/SC; (3) fragmento recombinante da glicoproteína do vírus rábico cepa ERA compreendendo os aminoácidos 179 a 281 (denominado rGERA179-281) seguido da inoculação de vírus rábico cepa PV na pata; (4) rGERA179-281 seguida por um reforço com a mesma proteína ou com vírus rábico cepa PV; (5) rGERA179-281 e vacina VeroRab®. Esplenócitos dos camundongos imunizados foram fusionados com células de mieloma da linhagem P3X63Ag8.653 utilizando polietilenoglicol 4000 e os hibridomas secretores de anticorpos antirrábicos foram triados por imunofluorescência indireta em células BHK-21 infectadas com vírus rábico. Diluições limitantes foram realizadas e sete clones estáveis foram obtidos, sendo denominados LIA 02 (fusão 1), 6H8 e 7B7 (fusão 2) e 3E6, 8D11, 9C7 e 8B6 (fusão 3). Nos dois últimos protocolos, não foram obtidos hibridomas secretores de anticorpos antirrábicos estáveis. Dentre os AcM, três foram caracterizados como IgG2a (6H8, 8D11 e 8B6), dois como IgM (7B7 e 9C7), um como IgG2b (LIA 02) e um como IgG3 (3E6), todos com cadeia leve kappa. Por ensaio de neutralização, foi verificado que o AcM 8D11 foi capaz de neutralizar o vírus rábico cepa PV in vitro. Além disso, o 7B7 reconheceu a rGERA179-281 e apenas o 8D11 não reconheceu o vírus presente na vacina VeroRab®, conforme demonstrado por ELISA. Os AcM também foram testados contra vírus rábico selvagem isolado de diferentes animais (bovinos, equinos, morcegos, cães e canídeos silvestres) e de humano (uma amostra) oriundas de diferentes regiões do Brasil. O AcM 6H8 reconheceu todas as amostras testadas. Os AcM LIA 02, 3E6 e 9C7 juntos também reconheceram todas elas. Assim, a partir dos resultados obtidos neste trabalho, novos estudos utilizando esses AcM como ferramentas biotecnológicas poderão ser desenvolvidos

Vector competence for West Nile virus and St. Louis encephalitis virus (flavivirus) of three tick species of the genus Amblyomma (Acari: Ixodidae)

Many species of Amblyomma ticks are commonly found infesting wild birds in South America, where birds are important hosts for several arboviruses, such as West Nile virus (WNV) and St. Louis encephalitis virus (SLEV). In this study, WNV and SLEV transmission experiments were performed to evaluate the vector competence of three South American tick species: Amblyomma ovale, Amblyomma tigrinum, and Amblyomma tonelliae. Larval and nymphal ticks of each species were allowed to feed on chicks needle inoculated with WNV or SLEV. All three Amblyomma species acquired either WNV or SLEV through larval feeding, with infection rates varying from 3.1% to 100% for WNV and from 0% to 35.7% for SLEV in engorged larvae. Transstadial perpetuation of the viruses was demonstrated in the molted nymphs, with WNV infection rates varying from 0% to 33.7% and SLEV infection rates from 13.6% to 23.8%. Although nymphal ticks also acquired either virus through feeding, transstadial perpetuation to adult ticks was lower, with virus detection in only 3.2% of A. tigrinum and 11.5% of A. tonelliae unfed adult ticks. On the other hand, vector competence for nymphs (exposed to WNV or SLEV through larval feeding) and adult ticks (exposed to WNV or SLEV through larval or nymphal feeding) was null in all cases. Although our results indicate transstadial perpetuation of WNV or SLEV in the three tick species, the ticks were not competent to transmit these agents to susceptible hosts. The role of these ixodid tick species in the epidemiology of WNV and SLEV might be insignificant, even though at least A. ovale and A. tigrinum are frequent bird ticks in Latin America, so the virus could survive winter in the fed larvae. However, future studies are required to determine the implications that this could have, as well as analyze the vector competence of other common bird tick species in South America.Fil: Flores, Fernando Sebastián. Universidad Nacional de Córdoba. Facultad de Medicina; ArgentinaFil: Zanluca, Camila. Instituto Carlos Chagas, Curitiba;Fil: Guglielmone, Alberto Alejandro. Instituto Nacional de Tecnología Agropecuaria Eea, Rafaela; ArgentinaFil: Duarte dos Santos, Claudia N.. Instituto Carlos Chagas, Curitiba;Fil: Labruna, Marcelo B.. Universidade de Sao Paulo; BrasilFil: Diaz, Luis Adrian. Universidad Nacional de Córdoba. Facultad de Medicina; Argentina. Universidad Nacional de Córdoba; Argentin

Zika Virus Infection at Different Pregnancy Stages: Anatomopathological Findings, Target Cells and Viral Persistence in Placental Tissues

Zika virus (ZIKV) infection in humans has been associated with congenital malformations and other neurological disorders, such as Guillain-Barré syndrome. The mechanism(s) of ZIKV intrauterine transmission, the cell types involved, the most vulnerable period of pregnancy for severe outcomes from infection and other physiopathological aspects are not completely elucidated. In this study, we analyzed placental samples obtained at the time of delivery from a group of 24 women diagnosed with ZIKV infection during the first, second or third trimesters of pregnancy. Villous immaturity was the main histological finding in the placental tissues, although placentas without alterations were also frequently observed. Significant enhancement of the number of syncytial sprouts was observed in the placentas of women infected during the third trimester, indicating the development of placental abnormalities after ZIKV infection. Hyperplasia of Hofbauer cells (HCs) was also observed in these third-trimester placental tissues, and remarkably, HCs were the only ZIKV-positive fetal cells found in the placentas studied that persisted until birth, as revealed by immunohistochemical (IHC) analysis. Thirty-three percent of women infected during pregnancy delivered infants with congenital abnormalities, although no pattern correlating the gestational stage at infection, the IHC positivity of HCs in placental tissues and the presence of congenital malformations at birth was observed. Placental tissue analysis enabled us to confirm maternal ZIKV infection in cases where serum from the acute infection phase was not available, which reinforces the importance of this technique in identifying possible causal factors of birth defects. The results we observed in the samples from naturally infected pregnant women may contribute to the understanding of some aspects of the pathophysiology of ZIKV

Placental Morphologic Similarities Between ZIKV-Positive and HIV-Positive Pregnant Women

Zika virus (ZIKV) caused global concern due to Brazil's unexpected epidemic, and it was associated with congenital microcephaly and other gestational intercurrences. The study aimed to analyze the placenta morphometric changes of ZIKV-infected pregnant women (ZIKV group; n = 23) compared to placentas of HIV-infected (HIV group; n = 24) and healthy pregnant women (N-control group; n = 22). It also analyzed the relationship between the morphometric results and pathological alterations on conventional microscopy, gestational trimester of infection, and presence of the congenital Zika syndrome (CZS). There was a significant increase in area (p = 0.0172), as well as a higher number of knots (p = 0.0027), sprouts (p < 0.0001), and CD163 +Hofbauer cells (HCs) (p < 0.0001) in the ZIKV group compared to the N-control group, suggesting that villous dysmaturity and HCs hyperplasia could be associated with ZIKV infections. The HIV group had a higher area (p < 0.0001), perimeter (p = 0.0001), sprouts (p < 0.0001), and CD163 + HCs (p < 0.0001) compared to the N-control group, demonstrating that the morphometric abnormalities found in the ZIKV and HIV group are probably similar. However, when ZIKV and HIV groups are compared, it was observed a higher number of sprouts (p = 0.0066) and CD163+ HCs (p < 0.0001) in the first one, suggesting that placental ZIKV congenital changes could be more pronounced

Padronização de ensaio imunoenzimático para diagnóstico de vírus dos gêneros Flavivirus e Alphavirus

Orientadora : Drª. Claudia Nunes Duarte dos SantosTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 09/02/2015Inclui referênciasÁrea de concentração : Biologia Celular e MolecularResumo: Os flavivírus e alfavírus são vírus transmitidos por artrópodes (arbovírus) e representam um problema de saúde pública em vários países. A emergência e re-emergência dos arbovírus são fenômenos naturais relacionados à evolução e adaptação desses vírus. O Brasil, por ter grandes áreas recobertas por florestas tropicais, possui condições ideais para a ocorrência e manutenção do ciclo silvestre de diversas arboviroses. A crescente urbanização, o fluxo migratório, a destruição de habitats naturais e a presença de mosquitos dos gêneros Culex e Aedes favorecem a rápida disseminação desses vírus. Além disso, os sintomas iniciais das infecções causadas por esses vírus são semelhantes a outras infecções que ocorrem em diversas regiões do Brasil e não existe tratamento específico para essas viroses, nem vacina licenciada para uso humano. O diagnóstico laboratorial diferencial, portanto, é fundamental para o correto manejo clínico dos pacientes. Assim, este trabalho teve por objetivo padronizar ensaios imunoenzimáticos para o diagnóstico sorológico do vírus Chikungunya (CHIKV), pertencente ao gênero Alphavirus, e dos vírus da dengue (DENV), da encefalite de Saint Louis (SLEV) e do oeste do Nilo (WNV), pertencentes ao gênero Flavivirus. Para tanto, as proteínas E1 e E2 do CHIKV foram expressas em células S2 de Drosophila melanogaster e purificadas por cromatografia de afinidade. A proteína E2 (denominada rE2-CHIKV) apresentou melhores resultados durante sua expressão e purificação e foi selecionada para dar prosseguimento ao trabalho. Foram então gerados três hibridomas secretores de anticorpos monoclonais (AcM) contra esta proteína, os quais foram caracterizados como sendo do isotipo IgG1k. Nenhum dos AcM reconheceu os vírus da encefalite equina venezuelana (VEEV), SLEV, WNV e DENV sorotipos 1 a 4. Foi padronizado um ensaio do tipo ELISA indireto utilizando a rE2-CHIKV como antígeno para a detecção de IgM anti-CHIKV, o qual apresentou sensibilidade de 70% e especificidade de 97% para o painel de amostras utilizado. Apesar da baixa sensibilidade, esses valores são comparáveis aos dos testes disponíveis comercialmente. O segundo vírus abordado nesse trabalho foi o vírus da dengue. Foram obtidos 22 AcM produzidos contra isolados brasileiros de DENV-1, -2 e -3. Os AcM são dos isotipos IgG2bk, IgG2ak e IgG1k, a maioria com especificidade para as proteínas de envelope ou pré-membrana do DENV. Quando testados contra os quatro sorotipos de DENV, diferentes padrões de reatividade foram identificados: grupo-específico, subcomplexo-específico (DENV-1, -3 e -4 ou DENV-2 e -3) e sorotipo-específico (DENV-2 ou -3). Além disso, alguns AcM apresentaram reatividade cruzada com vírus da febre amarela, WNV e SLEV, mas nenhum deles reconheceu o alfavírus VEEV. De acordo com nossos conhecimentos, esses são os primeiros AcM produzidos contra isolados brasileiros de DENV. O AcM denominado D3 424/8G, que apresentou reatividade com todos os flavivírus testados, foi conjugado com peroxidase e utilizado no desenvolvimento de um ensaio do tipo MAC-ELISA para detecção de IgM anti-DENV no soro de pacientes com infecção aguda por DENV. Como antígeno, foi utilizada uma mistura de DENV dos quatro sorotipos, produzidos em cultivo celular, purificados por colchão de sacarose e inativados por radiação gama. O ensaio apresentou sensibilidade de 91% e especificidade de 95% com as amostras testadas. No caso do SLEV, foram produzidas partículas-tipo vírus (VLP) em células HEK 293T. As SLE_VLPs, juntamente com o AcM D3 424/8G-HRP, foram utilizadas em um ensaio do tipo MAC-ELISA para diagnóstico de infecção aguda causada por SLEV. O teste desenvolvido apresentou sensibilidade de 95% e especificidade de 98% com o painel de amostras utilizado. Por fim, foram produzidas VLPs de WNV em células HEK 293T, as quais foram utilizadas na padronização de um ensaio do tipo MAC-ELISA para diagnóstico de infecção por WNV. Como conjugado, também foi utilizado o AcM D3 424/8G-HRP. No decorrer do trabalho, no entanto, foi obtida apenas uma amostra de soro de indivíduo com infecção aguda por WNV. Por isso, não foi possível calcular os valores de sensibilidade e especificidade do teste desenvolvido. Os insumos produzidos neste trabalho e os testes padronizados mostraram-se importantes ferramentas para serem utilizadas no diagnóstico dos vírus CHIKV, DENV, SLEV e WNV, além de poderem ser utilizados em pesquisa básica. Palavras-chave: Vírus Chikungunya. Vírus da dengue. Vírus da encefalite de Saint Louis. Vírus do oeste do Nilo. Proteínas recombinantes. Partículas-tipo vírus. Anticorpos monoclonais. Ensaio imunoenzimático. Diagnóstico.Abstract: The flavivirus and alphavirus are viruses transmitted by arthropods (arboviruses), which constitute a public health problem in many countries. The emergence and reemergence of arboviruses are natural phenomena related to species evolution and adaptation. Brazil has large areas covered by tropical forests, which provide ideal conditions for the existence and maintenance of the sylvatic cycle of many arboviruses. Increasing urbanization, migration, the destruction of natural habitats and the wide distribution of Culex and Aedes mosquitoes favor the rapid spread of these viruses. Furthermore, the initial symptoms of infections caused by these viruses are similar to other infections that occur in different regions of Brazil and there is no specific treatment for these viruses, nor licensed vaccine for human use. Laboratory differential diagnosis is therefore essential for the correct clinical management of patients. Therefore, this study aimed to develop enzyme immunoassays for the diagnosis of Chikungunya virus (CHIKV), belonging to the genus Alphavirus, and for the diagnosis of dengue virus (DENV), Saint Louis encephalitis virus (SLEV) and West Nile virus (WNV), belonging to the genus Flavivirus. For this, CHIKV E1 and E2 proteins were expressed in Drosophila melanogaster S2 cells and were purified by affinity chromatography. The E2 protein (named rE2-CHIKV) showed better results of expression and purification and was selected to continue the work. Three hybridomas secreting monoclonal antibodies (MAbs) against rE2-CHIKV were obtained. The MAbs were characterized as IgG1k isotype. None of the MAbs recognized the Venezuelan equine encephalitis virus (VEEV), SLEV, WNV and DENV serotypes 1 to 4. It was standardized an indirect ELISA for the detection of IgM anti-CHIKV using rE2-CHIKV as antigen, which had a sensitivity of 70% and specificity of 97%. Despite the low sensitivity, these values are comparable to those of commercially available tests. The second virus in this work was the dengue virus. Twenty two MAbs against Brazilian isolates of DENV-1, -2 and -3 were obtained. The MAbs include IgG2bk, IgG2ak and IgG1k isotypes, and most were raised against the envelope or the pre-membrane proteins of DENV. When the antibodies were tested against the four DENV serotypes, different reactivity patterns were identified: group-specific, subcomplex specific (DENV-1, -3 and -4 and DENV-2 and -3) and dengue serotype-specific (DENV-2 or -3). Additionally, some MAbs cross-reacted with yellow fever virus, WNV and SLEV. None of the MAbs recognized the alphavirus VEEV. To our knowledge, these are the first MAbs raised against Brazilian DENV isolates. The MAb D3 424/8G, which cross-reacted with all the flaviviruses tested, was conjugated to peroxidase and used to the development of a MAC-ELISA for detection of anti-DENV IgM in sera from patients with acute dengue. As antigen, we used a mixture of DENV particles serotypes 1 to 4, produced in cell culture, purified by sucrose cushion and inactivated by gamma radiation. The test had a sensitivity of 91% and a specificity of 95%. For SLEV, virus-like particles (VLPs) were produced in HEK 293T cells. The SLE_VLP together with the MAb D3 424/8G-HRP were used in the development of a MAC-ELISA for the diagnosis of acute infection caused by SLEV. The assay showed sensitivity of 95% and specificity of 98%. Finally, WNV VLP were produced in HEK 293T cells, and were used for standardizing a MAC-ELISA for the diagnosis of WNV infection. As conjugate it was also used MAb D3 424/8G-HRP. During the study, however, only one serum sample from patient with acute WNV infection was obtained. Therefore, it was not possible to calculate the sensitivity and the specificity of the test developed. The reagents produced and the diagnostic assays developed here have proved to be important tools to be used in the diagnosis of CHIKV, DENV, SLEV and WNV, and can be used for basic research. Keywords: Chikungunya virus. Dengue virus. Saint Louis encephalitis virus. West Nile virus. Recombinant proteins. Virus like particles. Monoclonal antibodies. Enzyme immunoassays. Diagnosis

Zika virus – an overview

Submitted by Claudete Queiroz ([email protected]) on 2016-04-26T13:35:06Z No. of bitstreams: 1 Zika virus - an overview.pdf: 312071 bytes, checksum: 0acf5bfe3b5192619976e1eb870a716b (MD5)Approved for entry into archive by Claudete Queiroz ([email protected]) on 2016-04-26T13:43:31Z (GMT) No. of bitstreams: 1 Zika virus - an overview.pdf: 312071 bytes, checksum: 0acf5bfe3b5192619976e1eb870a716b (MD5)Made available in DSpace on 2016-04-26T13:43:31Z (GMT). No. of bitstreams: 1 Zika virus - an overview.pdf: 312071 bytes, checksum: 0acf5bfe3b5192619976e1eb870a716b (MD5) Previous issue date: 2016Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, Brasil.Zika virus (ZIKV) is currently one of the most important emerging viruses in the world. Recently, it has caused outbreaks and epidemics, and has been associated with severe clinical manifestations and congenital malformations. However to date, little is known about the pathogenicity of the virus and the consequences of ZIKV infection. In this paper, we provide an overview of the current knowledge on ZIKV

Zika virus damages the human placental barrier and presents marked fetal neurotropism

Submitted by Claudete Queiroz ([email protected]) on 2016-05-10T13:36:21Z No. of bitstreams: 1 Zika virus damages the human placental barrier and presents marked fetal neurotropism.pdf: 1105746 bytes, checksum: b1d12ab552d6ba6aa4d3f65652e5dd73 (MD5)Approved for entry into archive by Claudete Queiroz ([email protected]) on 2016-05-10T13:47:39Z (GMT) No. of bitstreams: 1 Zika virus damages the human placental barrier and presents marked fetal neurotropism.pdf: 1105746 bytes, checksum: b1d12ab552d6ba6aa4d3f65652e5dd73 (MD5)Made available in DSpace on 2016-05-10T13:47:39Z (GMT). No. of bitstreams: 1 Zika virus damages the human placental barrier and presents marked fetal neurotropism.pdf: 1105746 bytes, checksum: b1d12ab552d6ba6aa4d3f65652e5dd73 (MD5) Previous issue date: 2016Pontifícia Universidade Católica do Paraná. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, Brasil.Universidade Federal do Rio Grande do Norte. Instituto de Medicina Tropical. Natal, RN, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Virologia Molecular. Curitiba, PR, Brasil.An unusually high incidence of microcephaly in newborns has recently been observed in Brazil. There is a temporal association between the increase in cases of microcephaly and the Zika virus (ZIKV) epidemic. Viral RNA has been detected in amniotic fluid samples, placental tissues and newborn and fetal brain tissues. However, much remains to be determined concerning the association between ZIKV infection and fetal malformations. In this study, we provide evidence of the transplacental transmission of ZIKV through the detection of viral proteins and viral RNA in placental tissue samples from expectant mothers infected at different stages of gestation. We observed chronic placentitis (TORCH type) with viral protein detection by immunohistochemistry in Hofbauer cells and some histiocytes in the intervillous spaces. We also demonstrated the neurotropism of the virus via the detection of viral proteins in glial cells and in some endothelial cells and the observation of scattered foci of microcalcifications in the brain tissues. Lesions were mainly located in the white matter. ZIKV RNA was also detected in these tissues by real-time-polymerase chain reaction. We believe that these findings will contribute to the body of knowledge of the mechanisms of ZIKV transmission, interactions between the virus and host cells and viral tropism

Development, Characterization and Application of Monoclonal Antibodies against Brazilian Dengue Virus Isolates

<div><p>Dengue is the most prevalent human arboviral disease. The morbidity related to dengue infection supports the need for an early, quick and effective diagnostic test. Brazil is a hotspot for dengue, but no serological diagnostic test has been produced using Brazilian dengue virus isolates. This study aims to improve the development of immunodiagnostic methods for dengue virus (DENV) detection through the production and characterization of 22 monoclonal antibodies (mAbs) against Brazilian isolates of DENV-1, -2 and -3. The mAbs include IgG2bκ, IgG2aκ and IgG1κ isotypes, and most were raised against the envelope or the pre-membrane proteins of DENV. When the antibodies were tested against the four DENV serotypes, different reactivity patterns were identified: group-specific, subcomplex specific (DENV-1, -3 and -4 and DENV-2 and -3) and dengue serotype-specific (DENV-2 or -3). Additionally, some mAbs cross-reacted with yellow fever virus (YFV), West Nile virus (WNV) and Saint Louis encephalitis virus (SLEV). None of the mAbs recognized the alphavirus Venezuelan equine encephalitis virus (VEEV). Furthermore, mAbs D3 424/8G, D1 606/A12/B9 and D1 695/12C/2H were used to develop a capture enzyme-linked immunosorbent assay (ELISA) for anti-dengue IgM detection in sera from patients with acute dengue. To our knowledge, these are the first monoclonal antibodies raised against Brazilian DENV isolates, and they may be of special interest in the development of diagnostic assays, as well as for basic research.</p></div