4 research outputs found
Production and purification of Zika virus for an inactivated virus vaccine candidate
Zika arbovirus is the most recent causative agent of an unattended emerging viral disease. Previously restricted to the African continent, Zika has spread rapidly during the last five years, reaching Asia and America. The emergence of Zika in Brazil revealed that pregnant women is a particular at-risk population due to the possibility of the infection during pregnancy causing congenital Zika syndrome, which in the worst cases is evidenced by severe microcephaly in neonates. Instituto Butantan as a public vaccine producer started studies for the development of an inactivated Zika vaccine as soon as the first birth defects cases came to knowledge. The first strategy chosen for Zika production was based on the production process already established for dengue vaccine. However, in opposition of what was believed at the beginning of the Zika outbreak, this virus has some differential characteristics when compared to Dengue viruses. Mainly due to the lytic behavior of Zika infection, which is not present in Dengue infection, a new process was developed to propagate and purify Zika virions. In order to establish the best culture conditions, Vero cells were seeded in different cell concentrations and culture media, in several flask sizes and types, infected with a range of Zika virus comprising MOI from 0.01 to 0.11, in kinetic studies with or without medium exchange. These studies were responsible for reaching PFU titers above 1E+07 PFU/mL in just 72 h of process with consistent reproducibility in production levels. For purification, harvested Zika was submitted to sucrose gradient ultracentrifugation or to two chromatography steps, reaching the required level of purity regarding host cell protein (\u3c 100 ng/mg) and residual DNA (\u3c 100 pg/dose). Zika vaccine was finally established in more than one formulation, after efficient inactivation with betapropiolactone. Inactivation was carefully evaluated by performing multiple passages of the inactivated material in C636 cells followed by a plaque assay. This work focused not only on generating a proof-of-concept of the immunization with inactivated Zika, but also on the development of scalable process aiming the establishment of a technology ready to enter the next phases of the vaccine development. This project has been funded in part with Federal funds from the U.S. Department of Health and Human Services, Office of the Assistant Secretary for Preparedness and Response, Biomedical Advanced Research and Development Authority, under Grant No. IDSEP130015. Supported by WHO, Butantan Institute and BARDA.
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Semliki forest virus as a vector: pros and cons for its use in biopharmaceuticals production
Purification and immunogenicity of the rabies virus glycoprotein (RVGP) expressed by S2 cells and Semliki Forest Virus systems.
O desenvolvimento de vacinas contra a raiva tão eficientes quanto as atuais ainda é considerado importante para a profilaxia dessa doença devido ao alto número de mortes por ano no mundo. Este trabalho mostra dois sistemas para a expressão recombinante do principal antÃgeno da raiva, a glicoproteÃna viral (RVGP): células Schneider 2 de Drosophila melanogaster estavelmente transfectadas (S2 rRVGP) e vÃrus Semliki Forest (SFV) carregando o RNA da RVGP (SFVRVGP). Ensaios de purificação por cromatografia de afinidade, da rRVGP de S2 rRVGP produzida em biorreator, demonstraram resultados promissores para o isolamento de monômeros da rRVGP. Para os estudos de imunogenicidade, camundongos foram vacinados com rRVGP de S2 rRVGP e SFV-RVGP. Dosagem de anticorpos anti-glicoproteÃna do vÃrus da raiva, neutralizantes, IgG1 e IgG2a, e citocinas demonstraram que o SFV-RVGP induziu predominantemente uma resposta imune do tipo celular e que os dois vetores foram capazes de expressar uma rRVGP imunogênica, apresentando um potencial uso clÃnico (veterinário e humano).The development of new and equally efficient rabies vaccines is still considered important to the prophylaxis of the disease, which is responsible for many deaths per year worldwide. This work used two systems for recombinant expression of the major rabies antigen, the viral glycoprotein (RVGP): stably transfected Drosophila melanogaster Schneider 2 cells (S2 rRVGP) and Semliki Forest Virus (SFV) carrying the RNA of RVGP (SFV-RVGP). Purification assays of the rRVGP by metal ion affinity chromatography, from S2 rRVGP cells produced in bioreactor, showed promising results for rRVGP monomers isolation. Analysis of antibodies anti-rabies virus glycoprotein, neutralizing, IgG1 and IgG2a, and citokines showed that the SFV-RVGP induced predominantly a cellular immune response, and both vectors were capable of expressing an immunogenic rRVGP, what reinforces potential clinical applications (veterinary or human)
Semliki Forest Virus as a Vector: Pros and Cons for Its Use in Biopharmaceuticals Production
The number of biopharmaceuticals for medical and veterinarian use produced in mammalian cells is increasing year after year. All of them are obtained by stable recombinant cell lines. However, it is recognized that transient gene expression produces high level expression in a short time. In that sense, viral vectors have been extensively used for producing recombinant proteins on lab-scale. Among them, Semliki Forest virus is commonly employed for this purpose. This review discusses the main aspects related to the use of Semliki Forest virus technology as well as its advantages and drawbacks which limit currently its utilization in biopharmaceutical industry on large-scale.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento CientÃfico e Tecnológico (CNPq