Accelerated mass production of influenza virus seed stocks in HEK-293 suspension cell cultures by reverse genetics

Despite major advances in developing capacities and alternative technologies to egg-based production of influenza vaccines, responsiveness to an influenza pandemic threat is limited by the time it takes to generate a Candidate Viral Vaccine (CVV) as reported by the 2015 WHO Informal Consultation report titled “Influenza Vaccine Response during the Start of a Pandemic”. In previous work, we have shown that HEK-293 cell culture in suspension and serum free medium is an efficient production platform for cell culture manufacturing of influenza candidate vaccines. This report, took advantage of, recombinant DNA technology using Reverse Genetics of influenza A/Puerto Rico/8/34 H1N1 strain, and advances in the large-scale transfection of suspension cultured HEK-293 cells. Transfection in shake flasks was performed using 1ug of total plasmid and 1x106 cells/mL. The supernatant was harvested after 48 hpt and used to infect a new shake flasks at 1x106 cells/mL for virus amplification. 3-L bioreactor was inoculated and transfected at 1x106 cells/mL with 1ug of total plasmid and harvested after 48hpt and the virus generated was amplified in shake flask. Quantification by TCID50, SRID, Dot-blot and TRPS were performed as well as characterization by TEM and HA and NA sequencing. Small-scale transfection in shake flasks generated 1.5x105 IVP/mL after 48 hpt and 1x107 IVP/mL after 96 hpi. For large-scale experiment a 3-L controlled stirred tank bioreactor resulted in supernatant (P0) virus titer of 5x104 IVP/mL and 2.8x107 IVP/mL after only one amplification (P1) in HEK-293 suspension cells. We demonstrate the efficent generation of H1N1 with the PR8 backbone reassortant under controlled bioreactor conditions in two sequential steps (transfection/rescue and infection/production). This approach could deliver a CVV for influenza vaccine manufacturing within two-weeks, starting from HA and NA pandemic sequences. Thus, this innovative approach is better suited to rationally design and mass produce the CVV within timelines dictated by pandemic situations and produce effective responsiveness than previous methodolog

Evaluation of growth and recombinant protein production of human cell lines adapted to serum-free suspension cultures

Linhagens celulares humanas tem despertado interesse como plataformas de produção de proteínas terapêuticas recombinantes por sua capacidade de realizar modificações pós-traducionais complexas e de modo similar à humana, sem gerar epítopos imunogênicos como ocorre com proteínas produzidas em células de mamíferos. Para a produção de uma proteína com correta qualidade terapêutica, as agências regulatórias recomendam processos livres de componentes animais de modo a evitar contaminação com vírus e príons. Deste modo, esse trabalho visa a produção do fator VII da coagulação sanguínea recombinante (FVIIr) utilizada no tratamento de hemofílicos com inibidores em células humanas adaptadas para meios de cultura livres de soro fetal bovino. As linhagens humanas SK-Hep-1, HKB-11 e Huh-7 foram adaptadas para suspensão e meios livres de soro fetal bovino (SFB). Essas células adaptadas foram transfectadas de forma transiente com o vetor lentiviral p1054-GFP e o reagente polietilenimina. No entanto, a baixa eficiência de transfecção nas células SK-Hep-1 e Huh-7 mostraram que essas linhagens são difíceis de transfectar por esse método, e mesmo a transfecção da célula HKB-11 só foi possível após a variação de alguns parâmetros, resultando em uma transfecção de 49,5% de células HKB-11 GFP-positivas. Desta forma, a expressão estável foi avaliada e as células adaptadas foram transduzidas com um ciclo de lentivírus (MOI = 1) contendo o vetor p1054-FVII. Foram observadas porcentagens de células GFP-positivas acima de 35% nas três linhagens celulares humanas modificadas. As células transduzidas foram submetidas a dois processos de sorting por citometria de fluxo, no qual a população obtida apresentava mais de 90% de células GFP-positivas. As três células foram avaliadas com relação à expressão de FVIIr após a adição de vitamina K no cultivo, no entanto, não foi possível detectar níveis de FVIIr no sobrenadante de 48 horas do cultivo dessas células pelo teste ELISA. As células foram transduzidas com um segundo ciclo de lentivírus (MOI = 2). A quantificação por ELISA do sobrenadante de 48 horas de cultivo das três células detectou 240,96 ng/mL, 217,42 ng/mL e 78,46 ng/mL de FVII total, respectivamente, nos cultivos das células HKB-11-F7-2C, SK-Hep-1-F7-2C e Huh-7-F7-2C. A expressão relativa de RNA mensageiro por RT-PCR também foi observada nos três cultivos. Paralelamente, foi analisado o proteoma das três células adaptadas e não-adaptadas em triplicata sendo identificadas de forma abundante proteínas do citoesqueleto, do metabolismo celular, da síntese, enovelamento e degradação de proteínas, relacionadas à apoptose, ao ciclo celular e ao crescimento, proteínas contra estresse oxidativo e osmótico, com ação antioxidante, entre outras.Human cell lines have attracted great interest as a plarform for recombinant therapeutic proteins production, due their ability to perform complex posttranslational modification in a similar manner to human proteins. These proteins do not carry immunogenic epitopes as occurs with proteins produced in mammalian cells. These therapeutic proteins should be produced in a animal-free process avoiding virus and prion contamination, as recommended by regulatory agencies for quality control. Thus, this work aims the production of recombinant blood coagulation factor VII (rFVII) used in the treatment of hemophiliacs with inhibitors in human cell lines adapted to serum-free suspension cultures. Human cell lines SK-Hep-1, HKB-11 and Huh-7 were adapted to suspension and serum-free media. These adapted cells were transiently transfected with p1054-GFP lentiviral vector and the polyethyleneimine reagent. However, low transfection efficiency in SK-Hep-1 and Huh-7 cells showed that these cells are difficult to transfect by this method, and even transfection of HKB-11 cell was only possible after varying some parameters, resulting in a 49,5% HKB-11 GFP-positive cells. Stable transfection was assessed and adapted cells were transduced with lentivirus particles containing p1054-FVII vector in one cycle (MOI=1). Percentages of GFP-positive cells above 35% were observed in three modified human cell lines. Transduced cells were sorted by FACS and more than 90% of GFP-positive cells were obtained. The expression of rFVII were evaluated by ELISA test after vitamin K supplementation, however, it was not possible to detect FVII levels in the 48 hour culture supernatant. Cells were transduced again with a second lentivirus cycle (MOI = 2). ELISA quantification of the 48 hour culture supernatant detected 240,96 ng/mL, 217,42 ng/mL and 78,46 ng/mL total FVII, respectively, in the cultures of HKB-11-F7-2C, SK-Hep-1-F7-2C and Huh-7-F7-2C cells. Relative expression of mRNA by RT-PCR was also observed in the three cultures assessed. In parallel, a proteomic analysis of adapted and non-adapted cells was performed in triplicate. Proteins related to cellular metabolism, cytoskeletal structure, apoptosis, cell cycle and cell growth, against oxidative and osmotic stress, antioxidant action were found

Serum-free suspension adaptation of human cell lines

Linhagens celulares humanas têm atraído grande interesse devido a sua capacidade de glicosilar proteínas de maneira mais semelhante às proteínas nativas humanas, reduzindo o potencial de respostas imunológicas contra epítopos não humanos. No entanto, por se tratar de uma aplicação recente, essas células ainda não foram extensamente caracterizadas e cultivadas em condições reprodutíveis da escala industrial, ou seja, em suspensão e em meios de cultura livres de soro fetal bovino (SFB). Em função disso, o objetivo principal deste trabalho foi estabelecer culturas livres de SFB e em suspensão para as linhagens celulares humanas SK-Hep-1, HepG2 e HKB-11, que têm despertado grande interesse devido ao potencial de produção de proteínas recombinantes. Para isso, quatro formulações comerciais livres de SFB foram avaliadas. As células que apresentaram bons resultados na adaptação aos meios realizada em garrafas estáticas foram então adaptadas para crescimento em suspensão. Foi possível realizar a adaptação satisfatória da célula HKB-11 ao meio FreeStyle e da célula SK-Hep-1 ao meio SFMII bem como a criopreservação das mesmas também em condições livres de SFB. A caracterização cinética das células adaptadas mostrou que a célula HKB-11 apresentou concentração celular quatro vezes superior a da célula SK-Hep-1 (8,6x106 e 1,9x106 células/mL, respectivamente) e apresentou crescimento celular durante 18 dias em cultura. A velocidade específica de crescimento máxima (?max) foi semelhante nas duas células (0,0159 h-1 para a HKB-11 e 0,0186 h-1 para SK-Hep-1). A limitação do crescimento das células adaptadas não parece estar associada à exaustão de glicose e glutamina, tampouco à formação de lactato em concentrações inibitórias. Todavia, para ambos os casos, foi observada produção de amônia em concentrações consideradas inibitórias (2 - 5 mM). De maneira geral, foi possível estabelecer culturas celulares em condições compatíveis com o desenvolvimento de um bioprocesso reprodutível, seguro e em concordância com as boas práticas de fabricação.Human cell lines have attracted great interest since they are capable of producing glycosylated proteins in a more similar way to native human proteins, reducing the potential for immune responses against non-human epitopes. However, these human cell lines have not been extensively characterized and cultured in large scale and in serum-free suspension conditions. As a result, the main objective of this work was to adapt three human cell lines: SK-Hep-1, HepG2 and HKB-11 to serum-free suspension cultures, since they are promising systems of recombinant protein expression. For this task, four commercial serum-free media were tested. Adapted cell lines in T-flasks were further adapted to suspension cultures. Results showed that both HKB-11 and SK-Hep-1 were adapted to serum-free suspension cultures in FreeStyle and SFMII, respectively and were cryopreservated in serum-free formulations. Kinetic characterization showed that HKB-11 cell concentration was four times higher than SK-Hep-1 cell (8,6x106 and 1,9x106 cells/ml, respectively) and showed cell growth in culture over 18 days. The maximum specific growth rate (?max) was similar for both cell lines (0,0159 h-1 to HKB-11 and 0,0186h-1 to SK-Hep-1). Growth limitation of adapted human cell lines does not seem to be associated with depletion of glucose and glutamine, nor with the formation of lactate in inhibitory concentrations. However, in both cases, ammonia production achieved inhibitory concentrations (2 - 5 mM). In general, it was possible to establish human cell cultures that are compatible with reproducible and safe bioprocess conditions and in compliance with good manufacturing practices

SOROPREVALÊNCIA DA PNEUMONIA PROGRESSIVA OVINA (MAEDI-VISNA) NA REGIÃO DE BOTUCATU – SP PREVALENCE OF SERUM ANTIBODIES TO OVINE PROGRESSIVE PNEUMONIA (MAEDI-VISNA) IN BOTUCATU REGION

O presente estudo visou determinar a soroprevalência da pneumonia progressiva ovina, na região de Botucatu, mediante prova de imunodifusão em gel de ágar (IDGA). Foram avaliadas quatrocentas amostras de soro sanguíneo de ovinos de oito propriedades de corte, com criação em sistema semi-intensivo, de diferentes municípios da região. Nenhuma das amostras de soro foi reagente na prova de IDGA. A análise desses resultados mostra discordância com estudos realizados em outros estados brasileiros, nos quais a prevalência da doença vem aumentando progressivamente.<br /><br />PALAVRAS-CHAVES: IDGA, lentivírus, ovinos. <p>The present study aimed to verify the prevalence of the ovine progressive pneumonia in Botucatu region by agar gel immunodiffusion test (AGID).  Serum samples of 400 sheep from eight specific farms for meat, with type of semi-intensive breeding of different areas. All the samples tested were negative to Maedi-Visna. The analysis of results was discordant with studies made in others Brazilians states, where the prevalence of the disease comes increasing progressively.</p><p>KEY WORDS: AGID, lentivirus, sheep.</p&gt

Longitudinal Study of Delta-Aminolevulinate Dehydratase Activity and Oxidative Profile in Healthy Pregnant Women

Pregnancy is characterized by changes in various organs, triggering changes in the use of energy substrates and increased oxygen consumption. In addition, gestation is an oxidative event that can be assessed by the relationship between free radicals and antioxidants produced by the body. Excessive production of free radicals has detrimental effects such as damage to enzymes, carbohydrates, and DNA. Thus, the objective of this study was to evaluate the oxidative status and antioxidant responses throughout pregnancy through a longitudinal study. Reactive oxygen species were analyzed by means of thiobarbituric acid reactive substances and nitric oxide, the antioxidant system through vitamin C, sulfhydryl groups, total antioxidant capacity, and ferric reducing ability of plasma as well as enzymes such as catalase and delta-aminolevulinate-dehydratase in pregnant women in the three gestational trimesters (n = 30). According to the results, the markers of oxidative damage showed significant differences in the different gestational trimesters where they were increased in the second trimester when compared to the first trimester. The antioxidant defenses responded differently in each gestational trimester, suggesting a response pattern to try to combat the damage caused by free radicals, in order to stabilize the increase of oxidative stress caused in the second gestational trimester