Optimized production of HIV-1 virus-like particles by transient transfection in CAP-T cells

Sonia Gutiérrez-Granados, Laura Cervera, María de las Mercedes Segura, Jens Wölfel, Francesc Gòdia

Research output: Contribution to journalArticleResearchpeer-review

21 Citations (Scopus)

Abstract

© 2015, Springer-Verlag Berlin Heidelberg. HIV-1 virus-like particles (VLPs) have great potential as new-generation vaccines. The novel CAP-T cell line is used for the first time to produce Gag-GFP HIV-1 VLPs by means of polyethylenimine (PEI)-mediated transient transfection. CAP-T cells are adapted to grow to high cell densities in serum-free medium, and are able to express complex recombinant proteins with human post-translational modifications. Furthermore, this cell line is easily transfected with PEI, which offers the flexibility to rapidly generate and screen a number of candidates in preclinical studies. Transient transfection optimization of CAP-T cells has been performed systematically in this work. It is determined that for optimal production, cells need to be growing at mid-exponential phase, Protein Expression Medium (PEM) medium has to be added post-transfection, and cells can be transfected by independent addition of DNA and PEI with no prior complexation. A Box-Behnken experimental design is used to optimize cell density at time of transfection, DNA/cell and PEI/cell ratios. The optimal conditions determined are transfection at a density of 3.3E + 06 cells/mL with 0.5 pg of DNA/cell and 3 pg of PEI/cell. Using the optimized protocol, 6 × 1010 VLP/mL are obtained, demonstrating that CAP-T is a highly efficient cell line for the production of HIV-1 VLPs and potentially other complex viral-based biotherapeutics.
Original languageEnglish
Pages (from-to)3935-3947
JournalApplied Microbiology and Biotechnology
Volume100
Issue number9
DOIs
Publication statusPublished - 1 May 2016

Keywords

  • CAP-T
  • Design of experiments
  • HIV-1 virus-like particles
  • Transient gene expression

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