Virus-like particles (VLPs) are nanostructures that resemble the structures of viruses. They are composed of one or more structural proteins that can be arranged in several layers and can also contain a lipid outer envelope. VLPs trigger a high humoral and cellular immune response due to their repetitive structures. A key factor regarding VLP safety is the lack of viral genomic material, which enhances safety during both manufacture and administration. Contemporary VLP production may take advantage of several systems, including bacterial, yeast, insect and mammalian cells. The choice of production platform depends on several factors, including cost and the need for post-translational modifications (PTMs), which can be essential in generating an optimal immune response. Some VLP-based vaccines designed to prevent several infectious diseases are already approved and on the market, with many others at the clinical trial or research stage. Interest in this technology has recently increased due to its advantages over classical vaccines. This paper reviews the state-of-the-art of VLP production systems and the newest generation of VLP-based vaccines now available.

The manufacturing of biopharmaceuticals in mammalian cells typically relies on the use of stable producer cell lines. However, in recent years, transient gene expression has emerged as a suitable technology for rapid production of biopharmaceuticals. Transient gene expression is particularly well suited for early developmental phases, where several potential therapeutic targets need to be produced and tested in vivo. As a relatively new bioprocessing modality, a number of opportunities exist for improving cell culture productivity upon transient transfection. For instance, several compounds have shown positive effects on transient gene expression. These transfection enhancers either facilitate entry of PEI/DNA transfection complexes into the cell or nucleus or increase levels of gene expression. In this work, the potential of combining transfection enhancers to increase Gag-based virus-like particle production levels upon transfection of suspension-growing HEK 293 cells is evaluated. Using Plackett-Burman design of experiments, it is first tested the effect of eight transfection enhancers: trichostatin A, valproic acid, sodium butyrate, dimethyl sulfoxide (DMSO), lithium acetate, caffeine, hydroxyurea, and nocodazole. An optimal combination of compounds exhibiting the highest effect on gene expression levels was subsequently identified using a surface response experimental design. The optimal consisted on the addition of 20 mM lithium acetate, 3.36 mM valproic acid, and 5.04 mM caffeine which increased VLP production levels 3.8-fold, while maintaining cell culture viability at 94%.

BACKGROUND Transient gene expression has been widely used for virus‐like particles (VLP) production in both small‐ and large‐scale systems. The extended gene expression (EGE) technique is based on repeated medium exchanges and retransfections of cell culture to prolong the production phase. The aim of this study was to demonstrate scalability of the approach by operating EGE continuously in a controlled bioreactor to obtain similar results to those achieved with shake flask EGE for VLP production. A standard batch cultivation was also carried out in shake flask. RESULTS For EGE in shake flasks and in a bioreactor, cell viability was comparable; however, the bioreactor enabled much higher cell densities and specific growth rates than the shake flasks. Owing to this increased cell growth in the bioreactor, the percentage of GFP‐positive cells was considerably lower at the end than in the shake flasks. GagGFP VLP titers were similar in both shake flasks and bioreactor. Nanoparticle tracking analysis revealed that the ratio of VLPs/total particles (VLPs and cell vesicles) was higher in the shake flasks than in the bioreactor, possibly due to higher cell densities achieved in the bioreactor. CONCLUSIONS In this study, EGE methodology was carried out in a bioreactor system for the first time while maintaining GagGFP production titers. Overall, our findings call for further optimization and implementation of common downstream processing steps to improve yield and VLP quality. © 2018 Society of Chemical Industry

Transient gene expression (TGE) has been used at small and medium scale for the production of biologicals in sufficient quantities to perform pre-clinical and characterization studies. Polyethyleneimine (PEI)-mediated transfection offers a low toxicity and non-expensive method for cell transfection. DNA and PEI concentration for transient gene expression has been extensively optimized in order to increase product titers. However, the possibility to extrapolate the optimal concentrations found for a specific bioprocess when expression vectors or cell lines need to be changed has not been investigated.In this work, the combination of three different HEK293 cell lines with three different vectors was studied for the production of HIV-1 virus-like particles (VLPs). The concentration of DNA and PEI was optimized for the nine combinations. The obtained results were very similar in all cases (DNA = 2.34 ± 0.18 μg/mL and PEI = 5.81 ± 0.18 μg/mL), revealing that transfection efficiency is not dependent on the cell line or vector type, but on DNA and PEI quantities. Furthermore, two of the cell lines tested stably expressed a protein able to recognize specific origins of replication: HEK293T/SV40 and HEK293E/oriP. Origins of replication were included in the vector sequences in order to test their capacity to increase production titers. HEK293T/SV40 resulted in a decrease of cell density and productivity of 2.3-fold compared to a control plasmid. On the other hand, HEK293E/OriP platform enabled a threefold improvement in HIV-1 VLP production keeping the same cell densities and viabilities compared to a control plasmid.

Gag polyprotein from HIV-1 is able to generate virus-like particles (VLPs) when recombinantly expressed in animal cell platforms. HIV-1 VLP production in HEK293 cells can be improved by the use of different strategies for increasing product titers. One of them is the so-called extended gene expression (EGE), based on repeated medium exchanges and retransfections of the cell culture to prolong the production phase. Another approach is the media supplementation with gene expression enhancers such as valproic acid and caffeine, despite their detrimental effect on cell viability. Valproic acid is a histone deacetylase inhibitor while caffeine has a phosphodiesterase inhibition effect. Here, the combination of the EGE protocol with additive supplementation to maximize VLP production is first tested. As an alternative to the direct additive supplementation, the replacement of these chemical additives by iRNA for obtaining the same inhibition action is also tested. The combination of the EGE protocol with caffeine and valproic acid supplementation resulted in a 1.5-fold improvement in HIV-1 VLP production compared with the EGE protocol alone, representing an overall 18-fold improvement over conventional batch cultivation. shRNAs encoded in the expression vector were tested to substitute valproic acid and caffeine. This novel strategy enhanced VLP production by 2.3 fold without any detrimental effect on cell viability (91.7%) compared with the batch cultivation (92.0%). Finally, the combination of shRNA with EGE resulted in more than 15.6-fold improvement compared with the batch standard protocol traditionally used. The methodology developed enables the production of high titers of HIV-1 VLPs avoiding the toxic effects of additives.