Agradecemos membros passados ​​e presentes em JCT e laboratórios LM-S para o desenvolvimento e melhoria das técnicas de genética reversa arenavírus e plasmídeos. Agradecemos também Snezhana Dimitrova para suporte técnico. O anticorpo monoclonal dirigido para JUNV NP (SA02-BG12) foi obtido a partir de BEI Recursos (NIAID Biodefesa e Infecções Repositório de Recursos de Investigação). BYHC foi apoiado por Grant Número GM068411 Institucional de Ruth L. Prêmio por Serviço Nacional de Investigação Kirschstein. EO-R é um Fullbright-Conicyt (BIO 2008) e uma bolsa de Rochester Vaccine (2012) destinatário. Suporte atual EO-R é fornecida por uma bolsa de pós-doutorado para a Diversidade e Excelência Acadêmica do Gabinete para o Desenvolvimento Professores e Diversidade na Universidade de Rochester. Research in LM-S laboratório é financiado pelo NIH concede RO1 AI077719, R21NS075611-01, R03AI099681-01A1, os Centros de Excelência NIAID para Influenza Pesquisa e Vigilância (HHSN266200700008C) eA Universidade de Rochester Center for Biodefense Modeling Imune (HHSN272201000055C). Pesquisa da JCT laboratório foi suportado por concessões RO1 AI047140, RO1 AI077719 e SR1 AI079665 do NIH.

1. Arenavirus Resgate transfecção O nosso sistema de resgate foi baseado na utilização de ambas as proteínas II plasmídeos de expressão que codificam a polimerase de nucleoproteína (NP) e dependente de ARN-ARN-polimerase (L), os factores que actuam em trans necessárias para a replicação viral de ARN e a expressão do gene do genoma arenavírus 7, 19, e os plasmídeos, capazes de dirigir a síntese intracelular, via celular espécies de ARN antigenoma G RNA polimerase I (pol-I), de S e 7. Nos nossos estudos que utilizam a expressão de proteínas do plasmídeo pCAGGS, que utiliza o promotor de frango β-actina e as sequências de sinal polyadenylanation (PA), e o pol-I humana do plasmídeo, o qual utiliza o promotor da polimerase I humana e sequências de terminação de murino (Figura 2) . Os segmentos de ARN L S e são clonados no plasmídeo HPOL-I numa orientação antigenomic para permitir a geração de segmentos de RNA genómicas após a transcrição por HPOL-I. Para o resgate de wild-tyep recombinante LCMV (rLCMV) e Espontânea # 1 (rCandid # 1) o pCAGGS NP e L a partir de cada um dos vírus, foram co-transfectados juntamente com o seu respectivo segmentos de ARN L (Figura 3) Pol-I humana e S. Geração de LCMV recombinante trisegmented (r3LCMV) e Espontânea # 1 (r3Candid # 1) seguido de um protocolo similar, mas o segmento S foi dividido em dois plasmídeos diferentes de pol I-S, cada um codificando um gene repórter diferente: em um, a leitura aberta NP armação (ORF) foi substituído com o (GFP), gene repórter Proteína Fluorescente Verde (HPOL-GFP I S / GP) e, no outro, o precursor da glicoproteína virai (GPC) ORF é substituído com o Gaussia luciferase (Glic) gene repórter (HPOL-I S NP / Gluc). Uma representação esquemática do protocolo é ilustrada na Figura 4. Foi estabelecido o seguinte protocolo de transfecção e infecção por 6-well-placas. <ol><li> OptiMEM-Lipofectamine 2000 (LPF2000) mistura: Preparar 250 mL de mídia OptiMEM e, dependendosobre o vírus de resgate, 10 - 12 ug de LPF2000 (1 ug / ul) por transfecção (Tabela 1). Recomenda-se uma razão de 2,5 ug LPF2000 por ug de ADN. Incubar durante 5 - 10 min à temperatura ambiente. Entretanto, prepara-se a mistura de transfecção do plasmídeo num tubo de microcentrífuga separados (passo 1.2.) </li><li> Mistura de transfecção de ADN de plasmídeo: Preparar o cocktail de transfecção do plasmídeo num tubo de microcentrífuga utilizando as quantidades recomendadas para cada vírus de resgate (ver Tabela 1). Levar o volume final de 50 ul com OptiMEM. </li><li> OptiMEM-LPF2000-mistura de plasmídeo de DNA: Adicionar 250 ul da mistura-LPF2000 OptiMEM (passo 1.1) para a mistura de transfecção de ADN do plasmídeo (passo 1.2.) Incubar a mistura por mais 20 - 30 min a RT. Enquanto isso, prepare e conte 1 x 10 6 células Vero por reação transfecção. Células Vero serão transfectados em suspensão. </li><li> Preparação de células Vero: Vero cells são cultivadas em placas de 100 mm. Antes de começar, trazer o PBS 1x, DMEM FBS a 10% 1% Meios PS e mistura de tripsina-EDTA a 37 ° C. <ol><li> Lavar as células, duas vezes, com 5 ml de PBS 1x. </li><li> Tripsinizar as células com 1 ml de tripsina-EDTA e esperar até que as células de separar (aproximadamente 5 min). Suave escutas talvez necessária para separar completamente as células dos pratos. </li><li> Cuidadosamente, ressuspender as células em 10 ml de meio DMEM 10% FBS a 1%, PS, em um tubo de centrífuga de 15 ml. </li><li> Centrifugar as células durante 5 min a 1000 rpm numa centrífuga. </li><li> Ressuspender as células em 10 ml de meio DMEM FBS a 10% 1% de PS e contar as células utilizando um hemocitómetro e ajuste a concentração de 1 x 10 6 células / ml. Foi observado que a 0,5-1 x 10 6 por transfecção de células Vero produziram os melhores resultados. </li></ol></li><li> Misturar LPF2000/DNA e células: após 20 - 30 minutos de incubação a temperatura ambiente (passo 1.3), adicionar 1 ml de células Vero (1 x 10 6) (passo 1.4) paraa mistura do plasmídeo OptiMEM-LPF2000-DNA e incubar durante 5 min à temperatura ambiente. </li><li> Semente LPF2000/DNA-cell mistura em 6 poços placas: Adicionar a mistura de ADN-LPF2000-Vero (passo 1.5) para os poços da placa de 6 poços. Agitar suavemente o-bem-placa 6 e deixar a incubar durante a noite a transfecção na incubadora a 37 ° C e 5% de CO 2. </li><li> Alterar meio de transfecção: Aproximadamente 16 - 24 horas após a transfecção, remova a mídia de transfecção, adicionar 2 ml de mídia infecção e incubar células transfectadas para um adicional de 48 horas. </li><li> Passagem celular: Após 48 horas de incubação em meio de infecção, remover o tecido de cultura sobrenadante (TCS) e passar as células transf ectadas em um prato de 100 mm. O TCS raramente contém o vírus infeccioso, porque as células transfectadas precisa de incubação adicional para gerar partículas de virus. Para a passagem de células: <ol><li> Lavar as células, duas vezes, com 2 ml de PBS 1x. </li><li> Trypsinize células w<html> om 0,5 ml de tripsina-EDTA e esperar até que as células separar. </li><li> Ressuspender as células em 1 ml de meio DMEM 10% FBS a 1%, PS, em um tubo de microcentrífuga de 1,5 ml. </li><li> Centrifugar as células durante 5 min a 5000 rpm, 4 ° C, numa microcentrifugadora. </li><li> Ressuspender cuidadosamente as células em 1 ml de meio de infecção e transferência das células para um prato de 100 mm. Levar o volume total da placa, com meios infecciosas, a 8 ml, um volume suficiente para evitar a secagem das células, bem como para a concentração do vírus. </li></ol></li><li> Agitar suavemente o prato de 100 mm e continuar a incubação de células Vero na incubadora a 37 ° C e 5% de CO 2 durante 72 horas. </li><li> Colecção de SCT: Após 72 horas de incubação, recolher o TCS em um tubo de centrífuga de 15 ml. Remover os detritos celulares por centrifugação durante 5 min a 2500 rpm numa centrífuga. Arenavírus infecciosa é encontrado na TCS, para remover detritos da célula sedimento e guardar o TCS a -80 ° C. </li></ol>le &quot;&gt; 2. Confirmação de Resgate Arenavirus recombinante A detecção de um resgate bem sucedido de rLCMV e rCandid º 1 é obtida através de um formando unidade de foco (UFF) imunofluorescência indireta (IFA). Para o tipo selvagem de vírus de resgate que utilize anticorpos específicos para o NP viral. O r3LCMV e r3Candid # 1 codificar dois genes repórter (GFP e Glic), permitindo assim a detecção de vírus e sem a necessidade de titulação de anticorpos por meio de microscopia de fluorescência (BPA) e / ou luminescência (Glic). <ol><li> Confirmar de tipo selvagem salvamento arenavírus recombinante: Um dia antes da titulação, as células Vero lavar duas vezes com PBS, e trypsinize preparar placas de 96 poços para atingir 80 - 90% de confluência no dia seguinte (4 x 4 10 células / poço). Agite as placas com as mãos para obter uma distribuição uniforme das células. Cultura das células, durante a noite, no 37 ° C incubadora com 5% de CO 2. Verifique as células ao microscópio para confirmar a monocamada, antes de prosseguir com a infecção: <ol><li> Diluir em série (10 diluições duplas), contendo o vírus TCS recuperado a partir das células transfectadas no prato de 100 mm (passo 1.10) em OptiMEM. </li><li> Remover os suportes das células Vero semeadas, lavar duas vezes com 50 ul de PBS 1x, e infectar as células com 50 ul de vírus diluído em série (passo 2.1.) Infect células durante 1,5 horas a 37 ° C e 5% de CO 2. </li><li> Após a infecção, remover o inoculo de vírus, adicionar 100 mL de meio de infecção / poço e incubar as células durante 16 - 18 h. Viral re-infecção pode ocorrer após 18 horas de incubação, o que pode resultar em excesso de estimativa dos títulos virais. </li><li> Em 16-18 horas pós-infecção, remover a TCS a partir de cada poço e fixar as células com formaldeído a 4% diluído em PBS 1x, durante 15 min à TA. </li><li> Em seguida, remover a solução de fixação e permeabilizar as células com 0,1% de triton X-100 diluído em PBS 1x, durante 10 min à temperatura ambiente. </li><li> Aspirar a solução de permeabilização, em seguida, lave as células três vezes wipo PBS 1x. </li><li> Bloquear as células com soro de albumina bovina 2,5% (BSA) em 1x PBS (solução de bloqueio) durante 1 hora à temperatura ambiente. Alternativamente, as células podem ser bloqueadas durante a noite a 4 ° C e continuou a incubação com o anticorpo, no dia seguinte. </li><li> Enquanto isso preparar o anticorpo primário. Dilui-se os anticorpos primários como 1 específicos do LCMV-Espontânea e em solução de bloqueio, e em seguida, centrifuga o anticorpo / solução de bloqueio durante 15 minutos a 3500 rpm. O anti-NP LCMV clone de anticorpo monoclonal 1.1.3 (diluição 1:30) 16 NP e o anticorpo monoclonal anti-JUNV SA02-BG12 de BEI Recursos (diluição 1:500) podem ser utilizados para a detecção de rLCMV e rCandid # 1 , respectivamente. </li><li> Após 1 hora de incubação, aspirar a solução de bloqueio e adicionar 50 ul do anticorpo primário às células e incubar durante 1 hora a 37 ° C. </li><li> Neste momento, dilui-se o anticorpo secundário em solução de bloqueio, e depois centrifugar a / solução de bloqueio de anticorpos durante 15 min a 3500 rpm. O polide coelho anti-IgG de ratinho FITC anticorpo secundário clonal para a detecção de anticorpos monoclonais primários foram utilizados para obter as imagens observadas na Figura 5. </li><li> Após 1 hora de incubação, aspirar o anticorpo primário, lavar 3 vezes com PBS 1x, e adicionar o segundo anticorpo, durante 30 min a 37 ° C. </li><li> Após 30 min de incubação, aspirar o anticorpo secundário e lavar 3 vezes com PBS 1x. As células são agora pronto para ser observada usando microscopia de fluorescência para determinar tanto o sucesso do salvamento a titulação viral e por contagem do foco formando unidades por ml (UFF / mL). </li></ol></li><li> r3LCMV e r3Candid # 1 resgate viral: Uma vez que estes vírus expressam dois genes repórter, a sua salva pode ser monitorizada por microscopia de fluorescência para detectar a expressão da GFP ou expressão Gluc na TCS. Resgate também pode ser confirmado pela expressão de Gluc na TCS. Para a titulação de vírus trisegmented, seguimos os passos 2.1 a 2.1.4, no entanto as células não precisam de be corrigido para determinar um resgate viral bem sucedida por causa da expressão GFP. A titulação do vírus vai ser determinada pela contagem do UFF / mL. Para determinar o resgate viral bem sucedida por expressão Gluc, a expressão a partir de Glic TCS pode ser medida utilizando um kit de ensaio Gluc (Biolux kit de ensaio de luciferase Gaussia, New England Biolabs). Para esse efeito: <ol><li> Pipetar 100 l de TCS em uma placa de 96 poços branca (placas de microtitulação de fundo plano, Fisher Scientific). </li><li> Configure o luminometer (LumiCount, Packard Biosciences). </li><li> Adicionar 50-100 ul de solução de ensaio Gluc (como recomendado pelo fabricante) a cada amostra e medir a expressão do gene repórter com o Glic luminómetro. Como controlo negativo, foi medida a expressão de Glic TCS a partir dos vírus do tipo selvagem, células infectadas. </li></ol></li></ol> 3. A passagem de sobrenadantes de cultura de tecidos Resgate viral depende da eficiência de transfecção. As células Vero foram mostradoster eficiências de transfecção mais baixos do que outras linhas de células 14. Se os títulos de vírus no TCS são baixos, infectar as células frescas Vero a uma multiplicidade de infecção (MOI) de 0,01 (LCMV) ou 0,1 (Espontânea # 1) durante 72 horas para amplificar o vírus.

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Geração de arenavírus recombinantes utilizando técnicas de genética reversa baseados plasmídeo tornou-se uma abordagem amplamente utilizado para a investigação de muitas facetas diferentes de biologia arenavírus. Aqui nós documentar uma melhoria crucial para o atual sistema através da realização de arenavírus resgata em células Vero, permitindo o potencial de geração de vacinas candidatas aprovadas pela FDA contra arenavírus e vetores de vacinas contra outras doenças infecciosas. <p class="jove_co...

Arenavírus são vírus com envelope com um bisegmented, genoma de RNA de cadeia negativa 3, que pertencem à família Arenaviridae. O genoma arenavírus codifica quatro proteínas de maneira AMBISENSE a partir de dois segmentos virais independentes 3. O segmento de grande (L) codifica a RNA polimerase dependente de RNA (L) eo pequeno anel (Really Interesting New Gene) proteína dedo (Z), que serve como a principal força motriz do vírus da brotação. O segmento pequeno (S) codifica a nucleoproteína viral (NP) e da glicoproteína de superfície (GP) (Figura 1). Vários membros da família Arenaviridae são responsáveis ​​pela febre hemorrágica letal (HF) 3 em seres humanos. Das preocupações principais são vírus de Lassa (LASV) e vírus Junín (JUNV), que são conhecidos por causar alta mortalidade em pacientes hospitalizados, 8, 9. Embora esses vírus são endêmicos para a África Ocidental e rural Argentina, respectivamente,há preocupações crescentes de importação de LASV e JUNV para áreas não endêmicas, devido ao aumento de viagem para 10. Além disso, embora não seja normalmente associada com doença grave em humanos, o arenavírus protótipo vírus choriomeningitis linfocítica (LCMV) é considerado um patógeno negligenciado como tem havido casos de infecção letal em pacientes imunocomprometidos, 6, 15 e é responsável por defeitos congênitos e abortos espontâneos em mulheres grávidas, 2, 13. Actualmente não há EUA aprovados pela FDA vacinas contra arenavírus e tratamento é limitada ao nucleósido análogo ribavirina, que é apenas parcialmente eficaz e, muitas vezes associada com efeitos colaterais significativos. A introdução de reverse-base plasmídeo genética 4 e geração de arenavírus recombinantes 7, 19 têm avançado muito no campo da pesquisa arenavírus. Actualmente, as células de roedores (tais como BHK-21) são utilizados para o Generção de arenavírus recombinantes devido à específica da espécie murina RNA polimerase I (pol-I) promotora dirigir a transcrição inicial dos segmentos L e S. No entanto salvamento vírus em células BHK-21 não são um método aprovado para a geração de arenavírus recombinantes como vacinas candidatas potenciais de sementes. Aqui vamos documentar o uso do ser humano pol-I promotor de resgate eficiente do Velho Mundo protótipo (OW) LCMV eo Novo Mundo (NW) JUNV Candid º 1 tensão em células Vero. Utilizando uma metodologia similar, geramos LCMV recombinante trisegmented (r3LCMV) e Candid # 1 (r3Candid # 1) arenavírus que contêm dois genes estranhos adicionais codificados em dois segmentos de RNA é diferente 5. Este novo sistema não só segue um protocolo altamente reprodutível e simples, mas pode ser imediatamente implementado na geração de arenavírus recombinantes como vacinas potenciais ou sementes vector de vacina.

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 Cell lines 
Vero E6 (African green monkey kidney epithelial cells) are maintained in a 37 &deg;C incubator with 5 % CO2 in DMEM 10 % FBS 1 % PS. Cells are available from the American Type Culture Collection (ATCC, catalogue number CRL-1586). 
 
Plasmids 
All plasmids, with the exception of hpol-I L, can be grown at 37 &deg;C, for 16-18 hr. We recommend that cultures of the hpol-I L be grown at 30 &deg;C for 24 hr. Plasmids are prepared using a plasmid maxi kit (EZNA Fastfilter Plasmid Maxi Kit, Omega Bio-tek) following the manufacturer's recommendations and stored at -20 &deg;C. The concentration of the purified DNA plasmid is determined by spectrophotometry at 260 nm, with purity being estimated using the 260:280 nm ratio. Preparations with 1.8-2.0 260:280 nm ratios are considered appropriate for virus rescue purposes. Additionally, plasmid concentration and purity should be confirmed with agarose gel electrophoresis. 
 
Viruses 
The described protocol for rescuing rLCMV (Armstrong 53b) and rCandid#1 can be executed under biosafety level (BSL) 2 conditions. Contaminated material, including TCS and cells should be sterilized before disposal. Rescue of other arenavirus may require higher BSL facilities so proper safety/security measures must be followed. 
 
Tissue culture media and solutions 
DMEM 10 %FBS 1 %PS: 445 ml Dulbecco's modified Eagle's medium (DMEM), 50 ml of Fetal Bovine Serum (FBS), and 5 ml of 100X Penicillin/Streptomycin (PS). Store at 4 &deg;C. This media will be used for maintenance of Vero cells. 
 
Infectious Media: 2-to-1 mixture of OptiMEM and DMEM 10 %FBS 1 %PS. Store at 4 &deg;C. This media will be used during viral infections. 
 
10X Phosphate buffered saline (PBS): 80 g of NaCl, 2 g of KCl, 11.5 g of Na2HPO4.7H2O, 2 g of KH2PO4. Add ddH2O up to 1 liter. Adjust pH to 7.3. Sterilize by autoclave. Store at room temperature. 
1X PBS: Dilute 10X PBS 1:10 with ddH2O. Sterilize by autoclave and store at room temperature. 
2.5 % BSA: 2.5 g of BSA in 97.5 ml of 1X PBS. Store at 4 &deg;C. This is used as a blocking solution for IFA.
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generation of recombinant arenavirus for vaccine development in fda-approved vero cells

O desenvolvimento e implementação de arenavírus reversa genética representa um avanço significativo no campo 4 arenavírus. A utilização de sistemas de arenavírus minigenome baseados em células, juntamente com a capacidade de gerar arenavírus infecciosos recombinantes com mutações predeterminadas nos seus genomas facilitou a investigação da contribuição dos determinantes virais para as diferentes etapas do ciclo de vida arenavírus, bem como vírus-hospedeiro interacções e mecanismos de patogénese arenavírus 1, 3, 11. Além disso, o desenvolvimento de arenavírus trisegmented tem permitido a utilização do genoma arenavírus para expressar genes estranhos adicionais de interesse, criando assim a possibilidade de uma aplicação vector de vacina à base de arenavírus 5. Do mesmo modo, o desenvolvimento de arenavírus infecciosas único ciclo capaz de expressar genes repórter proporciona uma nova ferramenta experimental para melhorar a segurança de pesquisa envolvendo highly patogênicos arenavírus humanos 16. A geração de arenavírus recombinantes utilizando técnicas de genética reversa baseados em plasmídeo, até agora contou com a utilização de linhas de células de roedores 7,19, o que coloca algumas barreiras para o desenvolvimento da Food and Drug Administration (FDA) licenciado vacina ou vetores vacinais. Para superar esse obstáculo, descrevemos aqui a geração eficiente de arenavírus recombinantes em células Vero FDA-aprovados.

Salvamento bem sucedido de um arenavírus do tipo selvagem recombinante será confirmada pela presença de antigénios do vírus utilizando IFA (Figura 5). No caso de vírus recombinantes trisegmented, resgate viral bem sucedida pode ser avaliada através da observação se expressão da GFP por microscopia de fluorescência (Figura 6A). Salvamento bem sucedido irá ser ainda confirmada por avaliar a expressão Gluc (Figura 6B). Os resultados representativos que ilustra...

Watch this Scientific Journal Video about Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells at JoVE.com

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

Resgate de arenavírus recombinantes a partir de ADNc clonados, uma abordagem designada por genética reversa, permite aos investigadores para investigar o papel dos produtos de genes virais específicos, bem como a contribuição dos seus vários domínios e resíduos específicos, de diferentes aspectos da biologia do arenavírus . Da mesma forma, inverter técnicas de genética em linhas de células aprovadas pela FDA (Vero) para o desenvolvimento da vacina proporciona novas possibilidades para a produção de vacinas eficazes e seguras para combater arenavírus patogénicos humanos.

Geração de Arenavirus recombinante de Desenvolvimento de Vacinas no FDA-approved células Vero

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

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Immunology and Infection

16.8K visualizações.  University of Rochester School of Medicine and Dentistry. Resgate de arenavírus recombinantes a partir de ADNc clonados, uma abordagem designada por genética reversa, permite aos investigadores para investigar o papel dos produtos de genes virais específicos, bem como a contribuição dos seus vários domínios e resíduos específicos, de diferentes aspectos da biologia do arenavírus . Da mesma forma, inverter técnicas de genética em linhas de células aprovadas pela FDA (Vero) para o desenvolvimento da vacina proporciona...

Vídeo: Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

Isolamento T de linfócitos humanos, Cultura e Análise da Migração In Vitro</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Imunologia e Infecção

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Geração de recombinante do vírus Influenza de DNA plasmidial

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDHhigh CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c.&#160;tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFN&#947; and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.

Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor cells.

Geração de uma nova vacina de células dendríticas usando melanoma e células-tronco de câncer escântico

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Usando Click Chemistry medir o efeito da infecção viral sobre a síntese de RNA do Host-Cell

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

In Vitro Análise do mediada por Myd88 Cellular resposta imune a vírus do Nilo Ocidental Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these traditionally modified Ad5 vectors in clinical trials could be primarily correlated with Ad5 pre-existing immunity (PEI) among the majority of the population. To promote Ad5-vectored vaccine development by solving the concern of Ad5 PEI, the innovative Antigen Capsid-Incorporation strategy has been employed. By merit of this strategy, Ad5-vectored we first constructed the hexon shuttle plasmid HVR1-KWAS-HVR5-His6/pH5S by subcloning the hypervariable region (HVR) 1 of hexon into a previously constructed shuttle plasmid HVR5-His6/pH5S, which had His6 tag incorporated into the HVR5. This HVR1 DNA fragment containing a HIV epitope ELDKWAS was synthesized. HVR1-KWAS-HVR5-His6/pH5S was then linearized and co-transformed with linearized backbone plasmid pAd5/&#8710;H5 (GL) , for homologous recombination. This recombined plasmid pAd5/H5-HVR1-KWAS-HVR5-His6 was transfected into cells to generate the viral vector Ad5/H5-HVR1-KWAS-HVR5-His6. This vector was validated to have qualitative fitness indicated by viral physical titer (VP/ml), infectious titer (IP/ml) and corresponding VP/IP ratio. Both the HIV epitope and His6 tag were surface-exposed on the Ad5 capsid, and retained epitope-specific antigenicity of their own. A neutralization assay indicated the ability of this divalent vector to circumvent neutralization by Ad5-positive sera in vitro. Mice immunization demonstrated the generation of robust humoral immunity specific to the HIV epitope and His6. This proof-of-principle study suggested that the protocol associated with the Antigen Capsid-Incorporation strategy could be feasibly utilized for the generation of Ad5-vectored vaccines by modifying different capsid proteins. This protocol could even be further modified for the generation of rare-serotype adenovirus-vectored vaccines.

Here, we present a protocol to generate a proof-of-principle divalent adenovirus type 5 (Ad5) vector Ad5/H5-HVR1-KWAS-HVR5-His6 by utilizing the Antigen Capsid-Incorporation strategy. This vector was demonstrated to exhibit qualitative fitness, the capability to escape Ad5-positive sera in vitro, and the antigenicity as well as immunogenicity to the incorporated antigens.

Utilizando a estratégia Capsid-Incorporação Antigen para o desenvolvimento de abordagens adenovírus subtipo 5-Vectored de vacinas

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Geração de IgG Humana Recombinante de Anticorpos Monoclonais As células seleccionadas a partir de B imortalizadas

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

Ensaio altamente sensível para a medição do Anexo de células Arenavirus

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

Geração, amplificação e titulação do vírus sincicial respiratórios recombinantes

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

Subunit vaccines offer advantages over more traditional inactivated or attenuated whole-cell-derived vaccines in safety, stability, and standard manufacturing. To achieve an effective protein-based subunit vaccine, the protein antigen often needs to adopt a native-like conformation. This is particularly important for pathogen-surface antigens that are membrane-bound proteins. Cell-free methods have been successfully used to produce correctly folded functional membrane protein through the co-translation of nanolipoprotein particles (NLPs), commonly known as nanodiscs. 
This strategy can be used to produce subunit vaccines consisting of membrane proteins in a lipid-bound environment. However, cell-free protein production is often limited to small scale (&lt;1 mL). The amount of protein produced in small-scale production runs is usually sufficient for biochemical and biophysical studies. However, the cell-free process needs to be scaled up, optimized, and carefully tested to obtain enough protein for vaccine studies in animal models. Other processes involved in vaccine production, such as purification, adjuvant addition, and lyophilization, need to be optimized in parallel. This paper reports the development of a scaled-up protocol to express, purify, and formulate a membrane-bound protein subunit vaccine. 
Scaled-up cell-free reactions require optimization of plasmid concentrations and ratios when using multiple plasmid expression vectors, lipid selection, and adjuvant addition for high-level production of formulated nanolipoprotein particles. The method is demonstrated here with the expression of a chlamydial major outer membrane protein (MOMP) but may be widely applied to other membrane protein antigens. Antigen effectiveness can be evaluated in vivo through immunization studies to measure antibody production, as demonstrated here.

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

This protocol describes using commercial, cell-free protein expression kits to produce membrane proteins supported in nanodisc that can be used as antigens in subunit vaccines.

Produção em Escala Livre de Células e Adição de Adjuvante a uma Proteína Recombinante da Membrana Externa Principal de Chlamydia muridarum para o Desenvolvimento de Vacinas