Transient expression of rabies virus G-glycoprotein using BHK-21 cells cultured in suspension

Núñez, Eutímio Gustavo Fernández; Rezende, Alexandre Gonçalves de; Puglia, Ana L.P.; Leme, Jaci; Boldorini, Vera Lucia Lopes; Caricati, Celso Pereira; Tonso, Aldo

doi:10.1007/s10529-015-1787-3

Cited by 6 publications

(5 citation statements)

References 24 publications

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“…Growth kinetic parameters including cell viability, density, and maximum specific growth rate were evaluated in several passages, with similar results observed for the BHK/SFM and BHK/FBS cells. The values obtained were in agreement with results reported previously for BHK-21 cells cultured in DMEM supplemented with FBS (Cruz et al 1998;Fernández-Núñez et al 2015), and were higher than BHK/SFM and BHK/FBS cells were infected in parallel with VRP-RVGP-SFM or VRP-RVGP-DMEM, using a MOI of 50 for a and 100 for b, c. The experiments was performed in 6-well plates, the cells were collected 3; 6; 12 and 24 h post-infection for a and 24 h post-infection for b, c. In a mRNA-RVGP expression was evaluated by qPCR. The results are average of duplicate.…”

Section: Discussionsupporting

confidence: 92%

“…RVGP is a structural membrane glycoprotein, so it requires post-translational modifications to enable it to function as an activator of the immune response (Burger et al 1991). The BHK/SFM cells were shown to be capable of transiently expressing this protein with the correct conformation and in amounts equivalent to those found in the BHK/FBS cells and in previous studies using the SFV system (Benmaamar et al 2009;Fernández-Núñez et al 2015). SFV-RVGP are able to induced levels of antibodies in mice comparable to those values obtained with a commercial rabies vaccine (Astray et al 2014).…”

Section: Discussionmentioning

confidence: 88%

“…In comparison, BHK/SFM cells required a MOI of 100 to reach 90% of fluorescent cells, but exhibited twofold higher fluorescence intensity for all MOIs tested, indicating that the BHK/SFM cells could express more protein per cell, compared to the BHK/FBS cells. Therefore, it is important to determine the best MOI, since very high virus concentrations may impair protein expression, due to cell death caused by the virus (Rhême et al 2005;Fernández-Núñez et al 2015).…”

Section: Discussionmentioning

confidence: 99%

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Semliki Forest Virus replicon particles production in serum-free medium BHK-21 cell cultures and their use to express different proteins

et al. 2019

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The production of biopharmaceuticals as vaccines in serum-free media results in reduced risk of contamination and simpler downstream processing. The production of enveloped viruses and viral vectors such as Semliki Forest Virus (SFV) typically requires lipids that are provided by supplementation with animal serum, so production under serum-free conditions is challenging. In this work, the capacity to deliver genetic material of SFV-viral replicon particles (SFV-VRPs) produced in BHK-21 cells adapted to serum-free medium (BHK/SFM) was evaluated. Three transgenes were evaluated: GFP used as a model protein, while hepatitis C virus nonstructural protein 3 protease domain (HCV-NS3p) and rabies virus glycoprotein (RVGP) were selected based on their distinct nature (enzyme and glycoprotein, respectively). BHK/SFM cells produced a sevenfold higher number of SFV-VRPs, as determined by qRT-PCR. These particles showed similar capacities of infecting BHK/FBS or BHK/SFM cells. GFP expression was evaluated by flow cytometry, HCV-NS3p activity by enzymatic assay, and RVGP expression by ELISA and Western Blot. Expression analysis revealed higher levels of GFP and HCV-NS3p in BHK/SFM, while the levels of RVGP were similar for BHK/SFM and BHK/ FBS. In conclusion, the BHK/SFM cells showed increased SFV-VRP production yields, without affecting vector infectivity or heterologous gene expression, hence validating the use of BHK/SFM for industrial applications. Keywords BHK-21 cells Á Hepatitis C virus nonstructural protein 3 protease domain Á Rabies virus glycoprotein Á Serum-free medium Á Semliki Forest Virus Á Viral replicon particles Abbreviations SFV-VRPs Viral replicon particles of Semliki Forest Virus BHK/FBS BHK-21 cells grown in DMEM supplemented with 10% of fetal bovine serum Carlos Augusto Pereira-Died October 27th, 2017.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 88%

Section: Discussionmentioning

confidence: 99%

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Semliki Forest Virus replicon particles production in serum-free medium BHK-21 cell cultures and their use to express different proteins

et al. 2019

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“…However, a number of expression systems have been explored. Mammalian expression systems based on human embryonic kidney (HEK) 293 [37], baby hamster kidney (BHK)-21 [38], or Chinese hamster ovary (CHO) [39] cell lines have been tested with varied success; they showed distinct patterns of glycosylation depending on cell substrate and culture conditions [40]. Insect cell expression system based on recombinant baculovirus [41] or transfected Drosophila melanogaster Schneider 2 cells [42] tend to be more efficient for high yield protein expression but they typically add shorter glycans with added fucose and xylose residues.…”

Section: Protein Vaccinesmentioning

confidence: 99%

New human rabies vaccines in the pipeline

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Banyard

Ertl

2019

Vaccine

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Rabies remains endemic in more than 150 countries. In 99% of human cases, rabies virus is transmitted by dogs. The disease, which is nearly always fatal, is preventable by vaccines given either before and/or after exposure to a rabid animal. Numerous factors including the high cost of vaccines, the relative complexity of post-exposure vaccination protocols requiring multiple doses of vaccine, which in cases of severe exposure have to be combined with a rabies immune globulin, lack of access to health care, and insufficient surveillance contribute to the estimated 59,000 human deaths caused by rabies each year. New, less expensive and more immunogenic rabies vaccines are needed together with improved surveillance and dog rabies control to reduce the death toll of human rabies. Here, we discuss new rabies vaccines that are in clinical and pre-clinical testing and evaluate their potential to replace current vaccines.

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“…As células BHK-21 crescendo de forma aderente foram adaptadas com sucesso ao Foi notado que as principais diferenças morfológicas das células BHK-21 aderentes cultivadas na ausência de SFB foram a presença de aglomerados e a perda da capacidade de adesão, estes eventos são considerados comuns no processo de adaptação e já foram relatados em células BHK (CRUZ et al, 1998;HESSE;WAGNER, 2000;KALLEL et al, 2002). (CRUZ et al, 1998;FERNÁNDEZ-NÚÑEZ et al, 2015).…”

Section: Célulaunclassified

Produção de proteínas recombinantes em células BHK-21 cultivadas em meio livre de soro fetal bovino.

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AGRADECIMENTOSMeus agradecimentos são só uma ínfima parte do que realmente desejo expressar, porque toda a ajuda recebida no poderia ser calculada simplesmente com palavras… Deus: Quem se encontra presente em cada coisa que eu faço, aquele que ilumina a minha vida e os meus pensamentos, esse único ser que conhece o motivo da minha presença neste mundo. Porque meu caminho é teu, não tem nada do meu destino oculto e toda conquista só pode ser possível porque o Senhor tem conquistado primeiro.Brasil: País maravilhoso, onde tenho passado anos inesquecíveis e conhecido pessoas maravilhosas.Mis padres: Por ser ejemplo de pareja, de dedicación y de entrega total. Mis conquistas son también de ustedes, porque represento una parte de cada uno. Han sido 7 años en la distancia, pero no ha pasado un día en el que me haya sentido sola, porque su espíritu me acompaña, porque su amor ha sido tan grande que han superado el sacrificio de mi ausencia para verme feliz. Jackson: Meu companheiro e amigo incondicional. Obrigada por estes 16 anos juntos, obrigada pela paciência e amor constante, por fazer parte da minha vida e por me convidar a percorrer o mesmo rio e compartilhar os mesmos sonhos. Hoje tuas palavras são uma realidade: El camino y el tiempo compartidos son el pilar de nuestros triunfos.Minha irmã Yaneth: Porque sem dar conta faz que cada dia eu queira me esforçar mais para ser melhor para conseguir meus propósitos. Obrigada por esse sobrinho lindo, Sebastián.Minha orientadora Soraia Jorge: obrigada pela confiança depositada em mim, pelos ensinamentos, pela força e pelo exemplo como pessoa.Renato Astray: pelo exemplo, não só como cientista, mas também como pessoa e sua particular forma de viver a vida.Doutor Carlos Augusto Pereira, por acreditar em mim, pela oportunidade de ser parte do seu laboratório, pelas tantas contribuições e pelo exemplo como pesquisador.Minhas segundas Mães: Mara e Joaquina, por tantos mimos, pela companhia e por tantos ensinamentos da vida.Mi segunda família: Doña Rosita, Don Fredy, Leonardo, Paola, Alejandro, Adriana, Laura, por creer en mí, por hacerme sentir como una más de la família y porque cuando estoy con ustedes las risas y las historias nunca acaban.Pessoal do iBET laboratório dos Vírus: Ana Sofia, Hugo, Helio, Ana Isabel, Ana, Miguel, por me adotar no outro continente! Fizeram que minha experiência europeia fosse maravilhosa, com certeza lembrarei de vocês para sempre. La capacidad es de poca importancia, sin una oportunidad. Napoleon bonaparte "A grandeza de um ser humano não está no quanto ele sabe, mas no quanto ele tem consciência que não sabe". Augusto Cury Sejam quais forem os resultados com êxito ou não, o importante é que no final cada um possa dizer: 'fiz o que pude'. Louis Pasteur RESUMO SUÁREZ-PATIÑO, S. F. Produção de proteínas recombinantes em células BHK-21 cultivadas em meio livre de soro fetal bovino 2016. 125 f. Tese (Doutorado em Biotecnologia) -Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, 2016. Vetores virais e células eucariotas têm sido...

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Transient expression of rabies virus G-glycoprotein using BHK-21 cells cultured in suspension

Abstract: These results establish the basis for designing bioprocess to produce RVGP.

Cited by 6 publications

References 24 publications

Semliki Forest Virus replicon particles production in serum-free medium BHK-21 cell cultures and their use to express different proteins

Semliki Forest Virus replicon particles production in serum-free medium BHK-21 cell cultures and their use to express different proteins

New human rabies vaccines in the pipeline

Produção de proteínas recombinantes em células BHK-21 cultivadas em meio livre de soro fetal bovino.

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