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

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Abstract

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.

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Acknowledgments

The authors would like to thank Cevec Pharmaceuticals for kindly providing the CAP-T cell line as well as for valuable comments and discussions. The following reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: pGag-EGFP (Cat#11468) from Dr. Marilyn Resh. The help of Pablo Castro and Meritxell Vendrell from Servei de Microscòpia of UAB is greatly appreciated. We would also like to thank Dr. Salvador Bartolomé (Departament de Bioquímica i de Biologia Molecular, UAB), Manuela Costa (Institut de Biotecnologia i Biomedicina, UAB), and Jose Amable Bernabé (ICMAB, CSIC) for the assistance with fluorometry, cytometry, and NTA, respectively. The authors acknowledge the support with the scientific equipment and scientific and technical assistance of Dr. Camille Roesch (Izon Science Europe Ltd., Magdalen Centre, The Oxford Science Park, Oxford, UK). This work is supported by a grant of SEIDI-Ministerio de Economía y Competitividad of Spain (BIO2012-31251) and Generalitat de Catalunya (2014 SGR 1544). Sonia Gutiérrez-Granados is a recipient of a FPU grant from the Ministerio de Educación y Deportes of Spain. Laura Cervera was a recipient of a PIF scholarship from UAB.

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Correspondence to Sonia Gutiérrez-Granados.

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This article does not contain any studies with animals or human participants performed by any of the authors.

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Jens Wölfel is an employee of CEVEC Pharmaceuticals, the company that has developed the CAP-T cell line and has contributed with scientific advice to this work.

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Gutiérrez-Granados, S., Cervera, L., Segura, M. et al. Optimized production of HIV-1 virus-like particles by transient transfection in CAP-T cells. Appl Microbiol Biotechnol 100, 3935–3947 (2016). https://doi.org/10.1007/s00253-015-7213-x

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