Exploring insect cells versatility for production of influenza virus-like particles

• Autor(a) principal: SEQUEIRA, Daniela Filipa Policarpo
• Data de Publicação: 2015
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/19341
• topic: Microbiologia médica; Virologia; Vírus da gripe; Tratamento; Vacinas; Domínio/Área Científica::Ciências Médicas

A potential strategy to produce safer and broadly protective influenza vaccines is to co-express, in the same cell host, multiple hemagglutinins (HA) with a matrix protein (M1) which self-assemble in virus-like particles (VLPs). This study demonstrates the suitability of combining stable expression and the baculovirus-expression vector system (BEVs) in insect Hi5 cells for production of such multi-HA Influenza VLPs. Stable pools of Hi5 cells expressing two HAs were generated and later infected with a M1-encoding baculovirus at two cell concentrations (CCIs; 2×106 cells/mL and 3×106 cells/mL). The HA concentration in culture supernatant was followed over time, with more productive infections observed at higher CCIs. To extend the culture time, a re-feed strategy was implemented based on the identification of key nutrients which were exhausted during cell growth. Afterwards, supplemented cultures infected at a CCI of 4×106 cells/mL allowed a 4-fold increase in HA concentration, at harvest, when compared to cultures infected at a CCI of 2×106 cells/mL. The production of multi-HA influenza VLPs using the aforementioned strategy could be successfully scaled-up to 2L bioreactor cultures with even higher volumetric (1.5-fold) HA yields. To surpass the unpredictability of gene expression promoted by the random integration strategy mentioned above, the recombinase-mediated cassette exchange (RMCE) technology was explored. The feasibility of having two cassettes flanked by distinct pairs of flippase recognition target sites (FRTs) was evaluated. Unfortunately, significant cross-interaction was observed between the selected pairs. To circumvent this bottleneck, a backup strategy consisting in the co-expression of two genes from the same locus after implementation of one cassette system, in insect Sf9 cells, was attempted. However, the isolated clones showed low expression of both M1 and HA proteins. Ongoing work focuses on the isolation of clones tagged in high expression loci by fluorescence activated cell sorter technology. This work demonstrates how the versatility of insect cell expression technology can be explored to produce Influenza VLPs as vaccine candidates.