RESUMO
Reverse genetics, the generation of influenza viruses from cDNA, presents a rapid method for creating vaccine strains. The technique necessitates the use of cultured cells. Due to technical and regulatory requirements, the choice of cell lines for production of human influenza vaccines is limited. PER.C6 cells, among the most extensively characterized and documented cells, support growth of all influenza viruses tested to date, and can be grown to high densities in large bioreactors in the absence of serum or micro carriers. Here, the suitability of these cells for the generation of influenza viruses by reverse genetics was investigated. A range of viruses reflective of vaccine strains was rescued exclusively using PER.C6 cells by various transfection methods, including an animal component-free procedure. Furthermore, a whole inactivated vaccine carrying the HA and NA segments of A/HK/156/97 (H5N1) that was both rescued from and propagated on PER.C6 cells, conferred protection in a mouse model. Thus PER.C6 cells provide an attractive platform for generation of influenza vaccine strains via reverse genetics.
Assuntos
Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H3N2/crescimento & desenvolvimento , Virus da Influenza A Subtipo H5N1/crescimento & desenvolvimento , Vacinas contra Influenza/genética , Vírus Reordenados/crescimento & desenvolvimento , Animais , Anticorpos Antivirais/sangue , Técnicas de Cultura de Células , Linhagem Celular , Feminino , Testes de Inibição da Hemaglutinação , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/imunologia , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Vírus Reordenados/genética , Vírus Reordenados/imunologia , Índice de Gravidade de Doença , Análise de Sobrevida , Transfecção/métodos , Ensaio de Placa ViralRESUMO
Ensuring complete viral inactivation is critical for the safety of vaccines based on an inactivated virus. Detection of residual infectious virus is dependent on sensitivity of the assay, sample volume analyzed and the absence of interference with viral infection. Here we describe the development and qualification of a sensitive cell-based assay for the detection of residual infectious West Nile Virus (WNV). The results of the assay are in good agreement with the assumption that at low concentrations the number of infectious units in relatively small samples follows a Poisson distribution. The assay can detect 1 infectious unit with a confidence of 99%, provides statistical controls for interference and can easily be scaled up to test large amounts of vaccine material. Furthermore, we show equivalence in sensitivity between the cell-based assay and an in vivo assay for detection of infectious WNV. Finally, the assay has been used for successful release testing of clinical lots of inactivated WNV vaccine. Given the principle and generic setup of the method we envision broad applicability to the detection of very low concentrations of infectious virus.
Assuntos
Propiolactona/farmacologia , Vacinas de Produtos Inativados , Inativação de Vírus , Vacinas contra o Vírus do Nilo Ocidental , Vírus do Nilo Ocidental/patogenicidade , Animais , Animais Lactentes , Linhagem Celular , Chlorocebus aethiops , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C3H , Células Vero , Febre do Nilo Ocidental/mortalidade , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/efeitos dos fármacos , Vírus do Nilo Ocidental/isolamento & purificação , Vírus do Nilo Ocidental/fisiologiaRESUMO
Reliable assays for accurate titration of influenza virus in infectious samples are pivotal to both influenza research and vaccine development. A titration assay adopted commonly for this purpose is the plaque assay on Madin-Darby canine kidney (MDCK) cells, despite it being time and labour consuming. A novel assay is described for titration of influenza viruses based on the detection of intracellular viral nucleoprotein (NP) by fluorescence-activated cell sorting (FACS). By using a panel of viruses of different type, subtype and origin, it is demonstrated that there is a mathematical correlation between titres measured by immunotitration and by classical plaque assay on MDCK cells. Moreover, the availability of NP antibodies specific for type A or type B influenza virus ensures the specificity of the assay. Based on speed, accuracy and specificity, it is concluded that the FACS-based immunotitration of influenza virus represents a valid and efficient alternative to the classical plaque assay.
Assuntos
Citometria de Fluxo/métodos , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza B/isolamento & purificação , Nucleoproteínas/metabolismo , Proteínas do Core Viral/metabolismo , Animais , Linhagem Celular , Cães , Humanos , Vírus da Influenza A/metabolismo , Vírus da Influenza B/metabolismo , Proteínas do Nucleocapsídeo , Fatores de Tempo , Ensaio de Placa ViralRESUMO
Influenza viruses for vaccine production are currently grown on embryonated eggs. This manufacturing system conveys many major drawbacks such as inflexibility, cumbersome down stream processing, inability of some strains to replicate on eggs to high enough yields, and selection of receptor-binding variants with reduced antigenicity. These limitations emphasize the need for a cell line-based production system that could replace eggs in the production of influenza virus vaccines in a pandemic proof fashion. Here we present the efficient propagation of influenza A and B viruses on the fully characterized and standardized human cell line PER.C6.
