Vaccines are the most cost-effective life-saving invention of the human history and their relevance in society had become evident during the recent COVID-19 pandemic. Virus-like particles (VLPs) are a promising approach for the development of new vaccine candidates. This work is focused on the production of VLPs by the recombinant expression of the HIV-1 Gag polyprotein, and their modification to present epitopes from pathogens of interest. The work can be divided in three main sections. The first one, comprising chapters one and two, studies the functionalization of Gag VLPs with the SARS-CoV-2 Spike (S) protein. The second one, comprising chapters three, four, five and six, focuses on the generation, characterization and production enhancement of different stable gene expression (SGE) cell lines for the production of Gag-VLPs. Finally, the third one, comprising chapter seven, merges the knowledge acquired in the previous sections to produce S-functionalized VLPs at bioreactor scale. In chapter one, the methodology for the generation of Gag-VLPs incorporating the S protein (S-VLPs) by the transient co-expression of S and Gag proteins is presented. The functionalization of the VLPs is achieved, their production is enhanced using design of experiments (DoE), and a bioprocess consisting of a bioreactor followed by a scalable downstream purification process is performed. In chapter two, several S-VLP variants harboring different S protein mutations are proposed. The influence of the mutations is studied in terms of VLP expression and quality. After that, COVID-19 convalescent human sera recognition assays are performed to select the best proposed variant based on its immunogenic potential. In chapter three, a stable cell line for the production of Gag-VLPs is generated by means of illegitimate random integration. The selection of the 10H9 clone is based on growth kinetics, VLP production and expression stability. In chapter four, a SGE cell line constitutively expressing Gag-VLPs is generated by locus-specific integration at the AAVS1 genomic safe harbor using CRISPR/Cas9. The selected clone, named 13++, greatly improves the production levels of the 10H9 cell line. Moreover the success of the used integration approach postulates it as an interesting methodology for the production of new cell lines. In chapter five, a Recombinase-Mediated Cassette Exchange (RMCE)-competent cell line stably expressing Gag-VLPs is generated by lentiviral transduction. The clones are screened in terms of growth kinetics, VLP production, stability, and ability to perform RMCE targeting. The selected clone, named SH5, can be used to easily generate high producer stable cell lines for the expression of virtually any gene of interest. In chapter six, the VLP production and quality of the 10H9, 13++ and SH5 cell lines are compared with transient gene expression (TGE), while the culture media used and the length of the bioprocess is studied. 13++ cell line cultured in HyCell media and harvested at day 10 improves by 2.7-fold the VLP production levels of TGE. Finally, in chapter seven, the production of S-VLPs by 13++ cell line transfection at 1 liter bioreactor is studied. The results are evaluated and discussed to define the steps to be followed in future processes for the generation of pseudotyped VLPs.
Production of Virus-Like Particles in HEK 293 cells and functionalization with SARS-CoV-2
Boix Besora, A. (Author). 11 Jul 2022
Student thesis: Doctoral thesis
Student thesis: Doctoral thesis