We thank Sergio G&#243;mez-Medina for excellent technical support with mouse experiments. This work was supported by funds from the Leibniz Association and the Leibniz Center of Infection. A.L. is a recipient of a pre-doctoral fellowship from the Leibniz Graduate School.

Experimentos em animais foram realizados de acordo com protocolos aprovados e seguir as orientações da lei alemã de protecção animal. Todos os funcionários que realizam experiências com animais passaram programas de treinamento de acordo com a categoria B ou C da Federação das Associações Europeias de Ciências Animais de Laboratório. 1. Geração de recombinante vivo atenuado Vacinas contra Influenza por genética reversa NOTA: O protocolo detalhado para a geração de vírus influenza recombinantes por genética reversa foi descrito em estudos anteriores 5 e está fora do âmbito do presente relatório. Resumidamente, resgate de vacinas contra a gripe adaptados ao frio (CAV) é feito em um armário de biossegurança classe II sob nível de biossegurança 2 contenção (BSL2), e envolve etapas detalhadas abaixo. O protocolo de transfecção e a infecção resulta que de Martinez-Sobrido et al. 5. <ol><li> Gerar um influenza adaptada ao frio reassortantvacina utilizando como fundo do adaptado ao frio estirpe A / Ann Arbor / 6/60 (H2N2), bem como a hemaglutinina (HA) e neuraminidase uma modificado (AN) da A / PR / 8/34 (H1N1), a linhagem A ( Figura 1A). A alteração no gene de NA consiste de uma substituição com base em PCR de uma sequência conservada no talo proteína (resíduos 65-72) por uma sequência de ADNc que codifica a curto ovalbumina de galinha (OVA) péptido -derived SIINFEKL (Figura 1B). O péptido SIINFEKL é um péptido altamente imunodominante no contexto do MHC de classe H-2b resposta de ratinhos C57BL / 6 12 I-restringida. </li><li> Placa 10 6 293T células por poço em placas de 6 poços, usando DMEM 10% de soro fetal de bovino (FBS), suplementado com 1% de penicilina / estreptomicina (P / E). </li><li> Prepare a mistura de transfecção de plasmídeo: Adicione 1 ug de cada um dos plasmídeos que codificam para os ADNc de PB2, PB1, PA, NP, M, NS de influenza A / Ann Arbor / 6/60 e uma estirpe ug dos plasmídeos que codificam para o NA -SIINFEKL eHA de influenza A / PR / 8/34. Adicionar pré-aquecido OptiMEM (15 ul, para um volume final de 100 ul / poço). </li><li> Incubar mistura de transfecção durante 30 min à TA. </li><li> Adicionar 100 ul da mistura de transfecção / cavidade e incubar durante 16 horas a 37 ° C e 5% de CO 2. </li><li> Mudar meio celular por DMEM 0,3% BSA 1% P / L a 33 ° C e 5% de CO 2 durante 5 h. </li><li> Adiciona-se 10 6 influenza células MDCK permissivas em DMSO contendo 1 ug / ml de tripsina de pâncreas de bovino tratada com L-1-tosilamida-2-feniletil-clorometilcetona (TPCK-tripsina). Manter as co-culturas 293T-MDCK para 2-3 dias a 33 ° C e 5% de CO 2. </li><li> Recolher os sobrenadantes de células MDCK-293T co-culturas e clara por centrifugação a 260 xg durante 5 min. </li><li> Inocular 9 - galinha 10 dias de idade, ovos embrionados em condições estéreis. Para fazer isso, fazer um furo na parte superior da casca do ovo, tal como indicado por Martinez-Sobrido et ai. 5. </li><li> Cubra a sagacidade casca de ovoh cera derretida usando um cotonete de algodão e incubar os ovos de galinha inoculadas durante 3 dias a 33 ° C. </li><li> Colher o fluido alantóico dos ovos embrionados infectados: Lavam-se as cascas com etanol a 70%. Abra o ovo com uma rachadura muito suavemente na parte superior e remover a membrana alantóide usando uma pinça estéril. Use uma pipeta de 10 ml para recolher tanto líquido quanto possível (tipicamente 6-10 ml). Centrifuga-se a 260 xg durante 10 min a 4 ° C e transferir o fluido alantóico foi afastada para novos tubos. </li></ol> Imunidade 2. Rastreamento vacina específica- <ol><li> Rastreamento de células dendríticas portadores de péptidos (DCs) <ol><li> Para a vacinação rato, anestesiar ratos com 5% de isoflurano em oxigênio, utilizando um vaporizador de precisão. Usar uma pipeta de 200 uL para administrar 50 uL de PBS contendo 10 3 unidades formadoras de placas (pfu) da vacina expressando o ptido SIINFEKL através de gotículas directamente para as narinas do rato. </li><li> Três dias após a vacinação, euthanize ratinhos através de anestesia de isoflurano, seguido por deslocação cervical. </li><li> Levante a pele do rato na linha média, logo abaixo da caixa torácica com uma pinça e cortar uma pequena incisão através da pele com uma tesoura. Do ponto de incisão, fazer um 3-5 cm de comprimento cortar através da pele em direção a cabeça eo rabo, em seguida, duas incisões de 2 cm da linha média lateralmente a partir de cada extremidade. Retire a pele para revelar as cavidades peritoneal e torácica. Cuidadosamente cortar através das membranas que rodeiam o peritoneu para expor o tórax. </li><li> Para aceder ao mediastino cortar o diafragma e a parte inferior da caixa torácica. Encontrar e remover dois gânglios linfáticos do mediastino (MLNS), ligeiramente dorsal e lateralmente ao lado do timo, medially adjacente à artéria braquiocefálica, perto do lado ventral da traquéia. </li><li> Para colher os MLNS usar uma pinça curva. Coloque uma pinça debaixo dos gânglios linfáticos e puxá-los para cima suavemente. Coloque gânglios linfáticos em uma placa de 6 poços contendo 1 ml de DMEM e remover quaisquer connective ou tecido gordo em torno do nó. <ol><li> Amole cada amostra cuidadosamente com duas agulhas 26 G ligados a seringas de 1 ml. Para provocar o linfonodo, segure-a com uma agulha ao quebrar aberto o linfonodo com a outra agulha. Quando isso for feito corretamente, as células concentradas estourando a partir do nó de linfa são facilmente observado na mídia. Passe todo o material de tecido através de um filtro de nylon de 70 mm para obter suspensões de células individuais. </li></ol></li><li> As suspensões de células únicas de centrífuga de 10 min a 260 xg numa centrifugadora refrigerada. Ressuspender o sedimento celular em 2 ml de glóbulos vermelhos de Lise (RBCL) Tampão para lisar as células vermelhas do sangue. Permitir que a lise continuar durante 3 minutos à temperatura ambiente. </li><li> Neutralizar RBCL tampão através de centrifugação e ressuspensão de células em 2 ml de PBS gelado. </li><li> Células da placa em forma de U-placas de 96 poços a uma concentração de 10 7 células / ml num volume final de 100 ul. </li><li> Para análise de citometria de fluxo, de células bloco Fc-receptores usando1 ug de anticorpos CD16 / CD32 anti-rato por 10 6 células durante 15 minutos em gelo. </li><li> Placas de centrífuga e descartar sobrenadantes por placa passar rapidamente. Incubar poços individuais com a combinação de anticorpo superfície de escolha. Normalmente para a detecção de células dendríticas em nódulos linfáticos, o cocktail de anticorpos deve incluir: 0,25 ug de anticorpos anti-CD11c, 0,25 ug de CD11b, 0,5 ug de MHC de classe II, 0,25 ^ g de CD103 e 0,15 ug de CD8a em um volume final de 100 ul. </li><li> Para gating negativo, adicionar 0,25 ug de anticorpos anti-CD3, CD19, ou NK1.1 uma linhagem de cocktail (Figura 2A). Estratégias de propagação alternativas, bem como os protocolos para uma compensação adequada de vários corantes fluorescentes podem ser encontradas no site do Projeto Genoma imunológica. Para além dos marcadores de superfície de escolha, adicionar 1 ug de um anticorpo contra H-2K b obrigado a SIINFEKL para o cocktail. Este anticorpo disponível comercialmente irá permitir a visualização de DCs in que o péptido SIINFEKL é ligado à superfície de MHC de classe I. </li><li> Incubar as células com anticorpos de superfície durante 30 minutos em gelo. Proteja placa da luz. Placas de centrifugar a 260 xg durante 5 minutos e lavar com 2 ml de PBS, duas vezes. Células transferir para tubos de citometria de fluxo para análise. </li></ol></li><li> Avaliação de proliferação de células T específicas de vacina <ol><li> Obtenção de células T dadoras de ratinhos disponíveis comercialmente TCR transgénica (murganhos OT-I) (C57BL / 6-Tg (TCR aTcrb) 1100Mjb / J) em que todas as células T CD8 + geradas são específicos para o péptido SIINFEKL. Eutanásia ratinhos por anestesia com isoflurano seguido por deslocação cervical. Colheita do baço através da realização de uma incisão na cavidade abdominal. Coloque baço / s em 2 ml de DMEM / baço e remover tecido conjuntivo e gordura. </li><li> Gerar suspensões de células individuais de baço a seguir o procedimento descrito no 2.1.2 e 2.1.3. </li><li> Para enriquecer ainda mais a suspensão de células individuais em células T, se aplicam os protocolos de separação de esférulas magnéticasutilizando tanto os procedimentos de selecção positiva ou negativa 12. Para diferenciar doador de células receptoras, utilize ratos congï¿½icas de modo que, por exemplo, ratinhos receptores expressar a CD45.1 marcador de leucócitos, enquanto as células T do doador expressar CD45.2 12. </li><li> Para identificar as células T, incubá-las em 1 ml de PBS contendo 5 uM de éster de succinimidilo de carboxifluoresceína (CFSE) numa densidade celular óptima de 5 x 10 6 células / ml. Incubar as células durante 20 min a 37 ° C, protegido da exposição à luz. Após a incubação, as células de centrifugação a 260 xg durante 5 min, o sobrenadante de descarte, e ressuspender as células em 3 ml de soro fetal de bovino (FBS) para neutralizar CFSE. </li><li> Células centrifugar a 260 xg durante 10 min e ressuspender em o volume adequado de PBS de modo a que 100 uL de solução contém 2 x 10 6 células. Infundir beneficiários ratos congï¿½icas com 2 x 10 6 células / rato via retro-orbital injeção sinusal 13. </li><li> Euthanize ratos receptores nos dias 3, 4 e 5 de pós-vacinação utilizando anestesia de isoflurano, seguido por deslocação cervical. Colher gânglios linfáticos do mediastino e preparar suspensões de células individuais para citometria de fluxo, tal como indicado na 2.1.2-2.1.5. </li><li> Identificar células dadoras por coloração das suspensões de células individuais em 100 uL de um cocktail contendo anticorpos: 0,25 ug de anticorpos, CD4 e CD8 anti-ratinho CD3; 0,5 ug de anti-murganho ou anticorpo CD45.1 CD45.2 (dependendo do fenótipo das células do doador). Deixar o canal FL-1 do citómetro de fluxo aberto (FITC) para determinar o perfil de diluição CFSE 12 (Figura 2B). </li></ol></li></ol> 3. ratos quiméricos para dissecar Responses vacina específica do gene NOTA: A geração de quimeras de medula óssea competitivos utilizando modelos de rato com base em DTR permite a discriminação de funções específicas de células durante a resposta imune às vacinas. Aqui nós mostramos uma estratégia adicional pelo qual combinação de DTR-expressando doador cells com células de ratos knockout licenças para estudar funções específicas de genes durante a imunidade em compartimentos celulares específicos. No exemplo que geramos camundongos com deleção específica de receptor toll-like 3 (TLR3) em Langerina + CD103 + DCs. <ol><li> Preparação de células de medula óssea dadoras <ol><li> Trabalhar sob um armário de biossegurança nível II. Evitar a contaminação por meio de instrumentos exclusivamente autoclavados. </li><li> Para gerar medula óssea ratos quiméricos, use CD45.1 congénica + camundongos C57BL / 6 do sexo feminino como destinatários, e usar TLR3 - / -, Langerina-DTR / EGFP, ou ratinhos wt expressar CD45.2 + como doadores. Utilizando murganhos dadores e receptores congï¿½icas permite a diferenciação de células dadoras e receptoras através de citometria de fluxo e isso permite que o enxerto a ser avaliada. </li><li> Para a obtenção de células de medula óssea do dador, eutanásia ratinhos dadores com anestesia isoflurano seguido por deslocação cervical. Com uma tesoura afiada realizar incisões em torno dos tornozelos, de modo que o mmusculatura ouse está exposto. </li><li> Utilizando uma pinça sem corte puxar a pele do rato e pele delicadamente do tornozelo em direção aos quadris de modo que os músculos das pernas são visíveis. </li><li> Com uma tesoura afiada cortar rato pernas na altura dos quadris e acima da cabeça do fêmur. </li><li> Coloque as pernas numa placa de Petri e em gelo. Trabalhar rápido e remover todos os músculos para expor os ossos. </li><li> Fêmures e tíbias distintas. Inserir os ossos, numa solução de etanol a 70% durante 5 minutos para remover possíveis bactérias e colocá-los imediatamente na outra placa de Petri contendo 5 ml de DMEM isento de soro. Osso cortar termina usando uma tesoura afiada e lave a medula óssea para a DMEM. Para fazê-lo, descarga 1 ml de DMEM para dentro do osso através da inserção de uma agulha de 26 G ligada a uma seringa de 5 ml. Certifique-se de que os ossos olhar branco (vazio) após a lavagem. </li><li> Recolhe todas as células da medula óssea de ratos dadores em 10 ml de DMEM. Gerar suspensões de células isoladas a partir de células de medula óssea por passagem da colheita por meio de um filtro celular de 70? M. </li><li> Clorelha suspensões de células únicas de células de medula óssea por centrifugação a 260 xg durante 10 min a 4 ° C numa centrífuga refrigerada. Ressuspender o sedimento celular em 10 ml de PBS. Destroem glóbulos vermelhos usando solução RBCL como descrito em 2.1.3 e 2.1.4. </li><li> Contagem de células de medula óssea utilizando um hemocitómetro ou um contador de células automatizado. Avaliar a viabilidade celular por determinação de células mortas através de coloração com azul de tripano 13. Idealmente, uma única produz animais cerca de 3 x 10 7 células brancas do sangue / rato, com uma viabilidade de pelo menos 80%. </li><li> No caso de quimeras totais de medula óssea, preparar TLR-3 - células doadoras ou em peso - /. A concentração óptima de células é de 2 x 10 7 células / ml desde 2 x 10 6 células em 100 ul de PBS vai ser utilizado para transplante. Se não for utilizada imediatamente, preservar células doadoras a -80 ° C, utilizando técnicas de congelação lenta e com FBS a 10% de DMSO. Para o modelo quimérico mista, misture pilhas Langerina-DTR-doadores e TLR3 - / - </sup&gt; Células do doador em uma proporção 75:25. Prepara-se uma mistura de 75: 25 Langerina-DTR: em peso de células da medula óssea para gerar ratinhos de controlo. </li></ol></li><li> Rato irradiação e transplante <ol><li> Irradiar camundongos fêmeas entre 6-8 semanas de idade em uma fonte de irradiador de césio-137. Dê camundongos duas doses de 550 rad 4 horas de intervalo. Irradiação de Split minimiza toxicidade hepática aguda e, portanto, reduz a letalidade do rato devido ao protocolo de irradiação. </li><li> Após a irradiação, manter os ratos sob tratamento com antibióticos para duas semanas. Dar antibióticos com a água potável a 2,5 - 5 mg de Baytril / kg. </li><li> 2-4 horas após a segunda irradiação, os ratinhos transplantados com células de medula óssea do dador, por via intravenosa. Realizar a injecção de células de dador através de retro-orbital injecção seio 13 e sob anestesia com isoflurano utilizando o volume máximo de 100 uL de PBS contendo 2 X 10 6 células. </li><li> Quatro semanas após o transplante, enxerto em avaliar uma amostra de 100ul de sangue periférico por citometria de fluxo utilizando uma máquina capaz de ler pelo menos duas cores. Para distinguir entre o dador e células de receptores de rádio-resistentes, disponíveis comercialmente usam CD45.1 marcado por fluorescência e anticorpos anti-ratinho CD45.2 utilizando diluições entre 1/100 e 1/200. O enxerto de células dadoras óptima deve ser de pelo menos 80% do total de células hematopoiéticas no sangue periférico. </li></ol></li><li> Depleção de vacinação e direcionados <ol><li> Vacinar os ratos com a vacina contra influenza viva atenuada recombinante via intranasal. Para a vacinação rato, anestesiar ratos com 5% de isoflurano em oxigênio, utilizando um vaporizador de precisão. Usar uma pipeta de 200 uL para administrar 50 uL de PBS contendo 10 3 unidades formadoras de placas (pfu) da vacina. </li><li> Para gerar o modelo final do rato, o tratamento de ratinhos com 50 ng de DT diluída em 50 ul de PBS, intranasalmente. Administrar DT gota a gota, directamente para as narinas de ratinhos anestesiados, tal como descrito em 3.3.1. Tratar miCE com doses diárias que iniciam o tratamento no dia -2 antes da vacinação até o dia 2 pós-vacinação (Figura 3B). </li></ol></li></ol>

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The authors have nothing to disclose.

Neste estudo, descrevemos como genética e modelos quiméricos ratinho inversa pode ser utilizada para elucidar os mecanismos fisiológicos e moleculares de imunidade induzida pela vacina. Genética reversa Influenza é estabelecida em muitos laboratórios e tem desempenhado um papel principal na compreensão patogênese da gripe, replicação e transmissão 17. Um ponto-chave em nosso protocolo é resgate de vacinas contra a gripe adaptados ao frio que expressam epitopos estrangeiros. Embora a estratégia de...

A geração de memória imunológica, na ausência de doença é a base fisiológica de vacinação eficiente 1. Recentemente, sistemas de abordagens baseadas em biologia têm revelado que as vacinas de sucesso, como a vacina contra a febre amarela, induzir uma forte indução de resposta imune inata e ativação de vários subconjuntos de células dendríticas (DCs), que por sua vez, levam a multilinhagens ativação de antígeno células T específicas 2,3. Uma vez que as DCs são a única população de células imunitárias, com a capacidade para activar células T ingénuas específicas de antigénio 4, o estudo da sua função durante a vacinação é fundamental para compreender as respostas imunitárias a vacinas e para conceber estratégias futuras contra patógenos desafiantes. Um sistema que permite o rastreio de diferentes subconjuntos DCs durante as respostas imunitárias a vacinas seria desejável, a fim de estabelecer uma cinética precisas de migração DC para tecidos linfóides, e, por conseguinte, para fornecerinsights sobre os mecanismos fisiológicos responsáveis ​​pela iniciação da imunidade adaptativa específicas de vacina. Com base genética inversa abordagens oferecem a possibilidade de gerar modificado, as vacinas vivas atenuadas que podem ser utilizadas experimentalmente com esta finalidade. Desde sua implantação, em investigação sobre a gripe, genética inversa à base de plasmídeo tem sido amplamente utilizada para gerar linhagens recombinantes de gripe, incluindo LAIVs. Os protocolos padrão para resgatar os vírus influenza recombinantes requerem multi-transfecção de linhas celulares altamente transfectáveis ​​com plasmídeos que produzem ambisense (tanto positivo como negativo de ARN senso) contendo os segmentos virais oito da gripe, bem como um sistema de amplificação no permissiva tais como Madin-Darby de rim canino ( ) células MDCK e / ou frango ovos embrionados 5. No entanto, a aplicação da genética reversa para gerar ferramentas moleculares para o estudo dos mecanismos imunológicos das vacinação permanece inexplorado. A geraçãode novos modelos de ratos, permitindo a depleção específica de subconjuntos de células imunes, incluindo CDs, abriu novas possibilidades para compreender os mecanismos imunológicos básicos subjacentes proteção provocada por vacina. A comparação entre as funções de subconjunto DC em camundongos e humanos revelou que, em grande medida, mouse e DCs humanas são funcionalmente homóloga 6,7, estes resultados sugerem fortemente que o desenvolvimento de modelos de mouse que permite a depleção específica de DCs no estado estacionário e durante condições inflamatórias, pode servir para entender a fisiologia das respostas DC em seres humanos. Nos últimos anos uma série de modelos de ratos têm sido gerado transportando transgenes que expressam o receptor da toxina da difteria de símio (DT) (DTR) sob o controlo da região do promotor de um gene de interesse 8,9. Desde tecidos de ratinho não expressam naturalmente DTR, estes modelos permitem esgotamento condicional de subconjuntos de células que carregam o gene alvo de interesse sobre inoculação em camundongos com DT. Assim, a nossa ability para esgotar as DCs específicas e outros leucócitos in vivo durante processos fisiológicos, foi grandemente melhorada pelo desenvolvimento de ro baseado no DTR. No entanto, enquanto estes modelos de ratinhos transgénicos têm sido amplamente utilizados para compreender a ontogenia do sistema imunitário, a sua aplicação para o desenvolvimento de vacinas tem sido pouco testada. Aqui, através da combinação de gripe genética reversa e quimeras de medula óssea competitivos baseados em DTR, propomos um método para estudar a cinética de imunidade da vacina, bem como a função do gene individual durante as respostas imunes às vacinas in vivo. A aplicação desta tecnologia para a avaliação pré-clínica de novas vacinas contra doenças infecciosas desafiadoras poderia ajudar a racionalizar o desenho da vacina e para testar vacinas candidatas in vivo.

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			<td>Dulbecco&#180;s Modified Eagle Medium (DMEM 1X)</td>
			<td>Gibco RL-Life Technologies</td>
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			<td>Embryonated eggs</td>
			<td>Valo biomedia Gmbh</td>
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			<td>PBS (1X)</td>
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			<td>D8537</td>
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			<td>70 &#956;M Nylon Filters</td>
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			<td>542-070</td>
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			<td>Red Blood Cell Lysing buffer (RBCL) 10X</td>
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			<td>CD16/CD32 Mouse BD Fc Block (2.4G2)</td>
			<td>BD Pharmigen</td>
			<td>553142</td>
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			<td>APC-Anti-mouse SIINFEKL-H2kb (25 D1.16)</td>
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			<td>PE-Anti-mouse CD11c (HLA3)</td>
			<td>BD Biosciences</td>
			<td>553802</td>
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			<td>eFluor 450-Anti-mouse MHCII (Md/114.15.2)</td>
			<td>eBioscience</td>
			<td>48-5321-82</td>
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			<td>PerCp/Cy5.5-Anti-mouse CD103 (2E7)</td>
			<td>Biolegend</td>
			<td>121416</td>
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			<td>PE-Anti-mouse CD45.1 (A20)</td>
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			<td>12-0453-82</td>
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			<td>V500-Anti-mouse CD45.2 (1O4)</td>
			<td>BD Bioscience</td>
			<td>562130</td>
			<td></td>
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			<td>PerCp-eFluor710 -Anti-mouse CD8a (53-6.7)</td>
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			<td>APC-Cy7-Anti-mouse CD3&#949; (145-2611)</td>
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			<td>eFluor450-Anti-mouse CD4 (GK 1.5)</td>
			<td>eBioscience</td>
			<td>48-0041-80</td>
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			<td>CFSE Proliferation dye</td>
			<td>eBioscience</td>
			<td>65-0850-85</td>
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			<td>Baytril 2.5%</td>
			<td>Bayer</td>
			<td>65-0850-85</td>
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			<td>Dymethil-Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
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			<td>Ovalbumin&#160;</td>
			<td>Molecular probes&#160;</td>
			<td>O23020</td>
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			<td>Diphteria Toxin (DT)</td>
			<td>Sigma-Aldrich</td>
			<td>D0564</td>
			<td></td>
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			<td>Trypsin-TPCK</td>
			<td>Sigma-Aldrich</td>
			<td>T1426</td>
			<td></td>
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		<tr>
			<td>BD FACsCanto II Flow cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo cell analysis software 9.5</td>
			<td>Flowjo inc.</td>
			<td></td>
			<td></td>
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			<td>Trypan Blue Stain (0.4%)&#160;</td>
			<td>Life technologies</td>
			<td>T10282</td>
			<td></td>
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			<td>Countess Automatic Cell Counter</td>
			<td>Invitrogen-Life Technologies</td>
			<td>C10227</td>
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intranasal administration of recombinant influenza vaccines in chimeric mouse models to study mucosal immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

Geração de vacinas contra a gripe vivos atenuados recombinantes pode ser conseguida por transf ecção de plasmídeos que codificam os oito segmentos de vírus influenza, sob o controlo de promotores bidireccionais 5. Uma vacina contra a gripe adaptada ao frio geralmente contém seis segmentos de uma estirpe adaptada ao frio, assim como a HA e NA da gripe de estirpe de escolha (por exemplo, H1N1) (Figura 1A). O princípio de frio adaptação baseia-se na replicação de vírus rest...

Watch this Scientific Journal Video about Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity at JoVE.com

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

A administração intranasal de recombinantes Vacinas contra Influenza em quimérico rato Modelos para estudar a imunidade mucosal

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

intranasal-administration-recombinant-influenza-vaccines-chimeric

Research

JoVE Journal

Immunology and Infection

12.4K visualizações.  Heinrich Pette Institute, Leibniz Institute for Experimental Virology. There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Vídeo: Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

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 ...

Imunologia e Infecção

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6.
Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15.
The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.

Protocolo para vacinas recombinantes RBD baseado SARS: Preparação de proteínas, Vacinação Animal e Detecção de Neutralização

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Análise quantitativa de todos Influenza Tipo A Hemaglutininas Virais e neuraminidases usando anticorpos Universal em Ensaios Blot simples slot

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system4-6. Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells4,7,8. These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages9,10. Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens7. While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life6. In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses11.
Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-&alpha; knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice12,13. As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines1,3. Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination.

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

Methods to examine contributions of the nasopharyngeal-associated lymphoreticular tissues (NALT) to nasal and systemic immune responses of mice to intranasal vaccines are described. We demonstrate a surgical procedure to establish a NALT-dependent mouse model and ex vivo cultures of extracted NALT.

Analisar o papel dos Nasopharyngeal-associado do tecido linforreticular (NALT) nas respostas do rato para Vacinas

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Um fluxo de Adesão Ensaio para o Estudo de Recrutamento de leucócitos para hepáticas humanas senoidais endotélio sob condições de estresse de cisalhamento

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Isolamento de neutrófilos e análise para determinar seu papel na linfoma sensibilidade das células a agentes terapêuticos

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

Administração intra-traqueal de<em&gt; Haemophilus influenzae</em&gt; No mouse modelos para o Estudo de inflamação das vias aéreas

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data have encouraged pulmonary tuberculosis vaccines as a promising strategy to prevent pulmonary disease, which is responsible for transmission. In this work, we describe the methodology used to demonstrate in the mouse model the benefits of intranasal BCG vaccination when compared to subcutaneous. Our data revealed greater protective efficacy following intranasal BCG administration. In addition, our results indicate that pulmonary vaccination triggers a higher immune response in lungs, including Th1 and Th17 responses, as well as an increase of immunoglobulin A (IgA) concentration in respiratory airways. Our data show correlation between protective efficacy and the presence of IL17-producing cells in lungs post-Mycobacterium tuberculosis challenge, suggesting a role for this cytokine in the protective response conferred by pulmonary vaccination. Finally, we detail the global workflow we have developed to study respiratory vaccination in the mouse model, which could be extrapolated to other tuberculosis vaccines, apart from BCG, targeting the mucosal response or other pulmonary routes of administration such as the intratracheal or aerosol.

We herein detail the methodology followed to compare protective efficacy and lung immune response induced by intranasal and subcutaneous immunization with BCG in mouse model. Our results show the benefits of pulmonary vaccination and suggest a role for IL17-mediated response in vaccine-induced protection.

A eficácia de protecção e Pulmonar resposta imune Seguindo subcutânea e intranasal BCG Administração em Ratinhos

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Medição de Inibição da Neuraminidase Influenza títulos de anticorpos por Enzyme-linked Lectina Ensaio

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

Usando modelos de Zebrafish de infecções por vírus gripe humana A para Screen antivirais e Caracterizar imune do hospedeiro respostas de células

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...