Internalization of PEI-based complexes in transient transfection of HEK293 cells is triggered by coalescence of membrane heparan sulfate proteoglycans like Glypican-4.

Polymer-cationic mediated gene delivery is a well-stablished strategy of transient gene expression (TGE) in mammalian cell cultures. Nonetheless, its industrial implementation is hindered by the phenomenon known as cell density effect (CDE) that limits the cell density at which cultures can be efficiently transfected. The rise in personalized medicine and multiple cell and gene therapy approaches based on TGE, make more relevant to understand how to circumvent the CDE. A rational study upon DNA/PEI complex formation, stability and delivery during transfection of HEK293 cell cultures has been conducted, providing insights on the mechanisms for polyplexes uptake at low cell density and disruption at high cell density. DNA/PEI polyplexes were physiochemically characterized by coupling X-ray spectroscopy, confocal microscopy, cryo-transmission electron microscopy (TEM) and nuclear magnetic resonance (NMR). Our results showed that the ionic strength of polyplexes significantly increased upon their addition to exhausted media. This was reverted by depleting extracellular vesicles (EVs) from the media. The increase in ionic strength led to polyplex aggregation and prevented efficient cell transfection which could be counterbalanced by implementing a simple media replacement (MR) step before transfection. Inhibiting and labeling specific cell-surface proteoglycans (PGs) species revealed different roles of PGs in polyplexes uptake. Importantly, the polyplexes uptake process seemed to be triggered by a coalescence phenomenon of HSPG like glypican-4 around polyplex entry points. Ultimately, this study provides new insights into PEI-based cell transfection methodologies, enabling to enhance transient transfection and mitigate the cell density effect (CDE).

Extracellular vesicle depletion and UGCG overexpression mitigate the cell density effect in HEK293 cell culture transfection.

The hitherto unexplained reduction of cell-specific productivity in transient gene expression (TGE) at high cell density (HCD) is known as the cell density effect (CDE). It currently represents a major challenge in TGE-based bioprocess intensification. This phenomenon has been largely reported, but the molecular principles governing it are still unclear. The CDE is currently understood to be caused by the combination of an unknown inhibitory compound in the extracellular medium and an uncharacterized cellular change at HCD. This study investigates the role of extracellular vesicles (EVs) as extracellular inhibitors for transfection through the production of HIV-1 Gag virus-like particles (VLPs) via transient transfection in HEK293 cells. EV depletion from the extracellular medium restored transfection efficiency in conditions that suffer from the CDE, also enhancing VLP budding and improving production by 60%. Moreover, an alteration in endosomal formation was observed at HCD, sequestering polyplexes and preventing transfection. Overexpression of UDP-glucose ceramide glucosyltransferase (UGCG) enzyme removed intracellular polyplex sequestration, improving transfection efficiency. Combining EV depletion and UGCG overexpression improved transfection efficiency by ∼45% at 12 × 106 cells/mL. These results suggest that the interaction between polyplexes and extracellular and intracellular vesicles plays a crucial role in the CDE, providing insights for the development of strategies to mitigate its impact.

Gag Virus-like Particles Functionalized with SARS-CoV-2 Variants: Generation, Characterization and Recognition by COVID-19 Convalescent Patients' Sera.

The robustness, safety, versatility, and high immunogenicity of virus-like particles (VLPs) make them a promising approach for the generation of vaccines against a broad range of pathogens. VLPs are recombinant macromolecular structures that closely mimic the native conformation of viruses without carrying viral genetic material. Particularly, HIV-1 Gag-based VLPs are a suitable platform for the presentation of the SARS-CoV-2 Spike (S) protein on their surface. In this context, this work studies the effect of different rationally engineered mutations of the S protein to improve some of its characteristics. The studied variants harbored mutations such as proline substitutions for S stabilization, D614G from the early dominant pandemic form, the elimination of the S1/S2 furin cleavage site to improve S homogeneity, the suppression of a retention motif to favor its membrane localization, and cysteine substitutions to increase its immunogenicity and avoid potential undesired antibody-dependent enhancement (ADE) effects. The influence of the mutations on VLP expression was studied, as well as their immunogenic potential, by testing the recognition of the generated VLP variants by COVID-19 convalescent patients' sera. The results of this work are conceived to give insights on the selection of S protein candidates for their use as immunogens and to showcase the potential of VLPs as carriers for antigen presentation.

Continuous cultivation of microalgae yields high nutrient recovery from nitrified urine with limited supplementation.

Microalgae can play a key role in the bioeconomy, particularly in combination with the valorisation of waste streams as cultivation media. Urine is an example of a widely available nutrient-rich waste stream, and alkaline stabilization and subsequent full nitrification in a bioreactor yields a stable nitrate-rich solution. In this study, such nitrified urine served as a culture medium for the edible microalga Limnospira indica. In batch cultivation, nitrified urine without additional supplements yielded a lower biomass concentration, nutrient uptake and protein content compared to modified Zarrouk medium, as standard medium. To enhance the nitrogen uptake efficiency and biomass production, nitrified urine was supplemented with potentially limiting elements. Limited amounts of phosphorus (36 mg L-1), magnesium (7.9 mg L-1), calcium (12.2 mg L-1), iron (2.0 mg L-1) and EDTA (88.5 mg Na2-EDTA.2H2O L-1) rendered the nitrified urine matrix as effective as modified Zarrouk medium in terms of biomass production (OD750 of 1.2), nutrient uptake (130 mg N L-1) and protein yield (47%) in batch culture. Urine precipitates formed by alkalinisation could in principle supply enough phosphorus, calcium and magnesium, requiring only external addition of iron, EDTA and inorganic carbon. Subsequently, the suitability of supplemented nitrified urine as a culture medium was confirmed in continuous Limnospira cultivation in a CSTR photobioreactor. This qualifies nitrified urine as a valuable and sustainable microalgae growth medium, thereby creating novel nutrient loops on Earth and in Space, i.e., in regenerative life support systems for human deep-space missions.

Downstream process design for Gag HIV-1 based virus-like particles.

Virus-like particles-based vaccines have been gaining interest in recent years. The manufacturing of these particles includes their production by cell culture followed by their purification to meet the requirements of its final use. The presence of host cell extracellular vesicles represents a challenge for better virus-like particles purification, because both share similar characteristics which hinders their separation. The present study aims to compare some of the most used downstream processing technologies for capture and purification of virus-like particles. Four steps of the purification process were studied, including a clarification step by depth filtration and filtration, an intermediate step by tangential flow filtration or multimodal chromatography, a capture step by ion exchange, heparin affinity and hydrophobic interaction chromatography and finally, a polishing step by size exclusion chromatography. In each step, the yields were evaluated by percentage of recovery of the particles of interest, purity, and elimination of main contaminants. Finally, a complete purification train was implemented using the best results obtained in each step. A final concentration of 1.40 × 1010 virus-like particles (VLPs)/mL with a purity of 64% after the polishing step was achieved, with host cell DNA and protein levels complaining with regulatory standards, and an overall recovery of 38%. This work has resulted in the development of a purification process for HIV-1 Gag-eGFP virus-like particles suitable for scale-up.

An engineered HIV-1 Gag-based VLP displaying high antigen density induces strong antibody-dependent functional immune responses.

Antigen display on the surface of Virus-Like Particles (VLPs) improves immunogenicity compared to soluble proteins. We hypothesised that immune responses can be further improved by increasing the antigen density on the surface of VLPs. In this work, we report an HIV-1 Gag-based VLP platform engineered to maximise the presence of antigen on the VLP surface. An HIV-1 gp41-derived protein (Min), including the C-terminal part of gp41 and the transmembrane domain, was fused to HIV-1 Gag. This resulted in high-density MinGag-VLPs. These VLPs demonstrated to be highly immunogenic in animal models using either a homologous (VLP) or heterologous (DNA/VLP) vaccination regimen, with the latter yielding 10-fold higher anti-Gag and anti-Min antibody titres. Despite these strong humoral responses, immunisation with MinGag-VLPs did not induce neutralising antibodies. Nevertheless, antibodies were predominantly of an IgG2b/IgG2c profile and could efficiently bind CD16-2. Furthermore, we demonstrated that MinGag-VLP vaccination could mediate a functional effect and halt the progression of a Min-expressing tumour cell line in an in vivo mouse model.

Enhancing control systems of higher plant culture chambers via multilevel structural mechanistic modelling.

Modelling higher plant growth is of strategic interest for modern agriculture as well as for the development of bioregenerative life support systems for space applications, where crop growth is expected to play an essential role. The capability of constraint-based metabolic models to cope the diel dynamics of plants growth is integrated into a multilevel modelling approach including mass and energy transfer and enzyme kinetics. Lactuca sativa is used as an exemplary crop to validate, with experimental data, the approach presented as well as to design a novel model-based predictive control strategy embedding metabolic information. The proposed modelling strategy predicts with high accuracy the dynamics of gas exchange and the distribution of fluxes in the metabolic network whereas the control architecture presented can be useful to manage higher plants chambers and open new ways of merging metabolome and control algorithms.

The cell density effect in animal cell-based bioprocessing: Questions, insights and perspectives.

One of the main challenges in the development of bioprocesses based on cell transient expression is the commonly reported reduction of cell specific productivity at increasing cell densities. This is generally known as the cell density effect (CDE). Many efforts have been devoted to understanding the cell metabolic implications to this phenomenon in an attempt to design operational strategies to overcome it. A comprehensive analysis of the main studies regarding the CDE is provided in this work to better define the elements comprising its cause and impact. Then, examples of methodologies and approaches employed to achieve successful transient expression at high cell densities (HCD) are thoroughly reviewed. A critical assessment of the limitations of the reported studies in the understanding of the CDE is presented, covering the leading hypothesis of the molecular implications. The overall analysis of previous work on CDE may offer useful insights for further research into manufacturing of biologics.

Transduction of HEK293 Cells with BacMam Baculovirus Is an Efficient System for the Production of HIV-1 Virus-like Particles.

Gag virus-like particles (VLPs) are promising vaccine candidates against infectious diseases. VLPs are generally produced using the insect cell/baculovirus expression vector system (BEVS), or in mammalian cells by plasmid DNA transient gene expression (TGE). However, VLPs produced with the insect cell/BEVS are difficult to purify and might not display the appropriate post-translational modifications, whereas plasmid DNA TGE approaches are expensive and have a limited scale-up capability. In this study, the production of Gag VLPs with the BacMam expression system in a suspension culture of HEK293 cells is addressed. The optimal conditions of multiplicity of infection (MOI), viable cell density (VCD) at infection, and butyric acid (BA) concentration that maximize cell transduction and VLP production are determined. In these conditions, a maximum cell transduction efficiency of 91.5 ± 1.1%, and a VLP titer of 2.8 ± 0.1 × 109 VLPs/mL are achieved. Successful VLP generation in transduced HEK293 cells is validated using super-resolution fluorescence microscopy, with VLPs produced resembling immature HIV-1 virions and with an average size comprised in the 100-200 nm range. Additionally, evidence that BacMam transduction occurs via different pathways including dynamin-mediated endocytosis and macropinocytosis is provided. This work puts the basis for future studies aiming at scaling up the BacMam baculovirus system as an alternative strategy for VLP production.

Optimization, Production, Purification and Characterization of HIV-1 GAG-Based Virus-like Particles Functionalized with SARS-CoV-2.

Virus-like particles (VLPs) constitute a promising approach to recombinant vaccine development. They are robust, safe, versatile and highly immunogenic supra-molecular structures that closely mimic the native conformation of viruses without carrying their genetic material. HIV-1 Gag VLPs share similar characteristics with wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, making them a suitable platform for the expression of its spike membrane protein to generate a potential vaccine candidate for COVID-19. This work proposes a methodology for the generation of SARS-CoV-2 VLPs by their co-expression with HIV-1 Gag protein. We achieved VLP functionalization with coronavirus spike protein, optimized its expression using a design of experiments (DoE). We also performed the bioprocess at a bioreactor scale followed by a scalable downstream purification process consisting of two clarifications, an ion exchange and size-exclusion chromatography. The whole production process is conceived to enhance its transferability at current good manufacturing practice (cGMP) industrial scale manufacturing. Moreover, the approach proposed could be expanded to produce additional Gag-based VLPs against different diseases or COVID-19 variants.

Differential N- and O-glycosylation signatures of HIV-1 Gag virus-like particles and coproduced extracellular vesicles.

Human immunodeficiency virus 1 (HIV-1) virus-like particles (VLPs) are nanostructures derived from the self-assembly and cell budding of Gag polyprotein. Mimicking the native structure of the virus and being noninfectious, they represent promising candidates for the development of new vaccines as they elicit a strong immune response. In addition to this, the bounding membrane can be functionalized with exogenous antigens to target different diseases. Protein glycosylation depends strictly on the production platform and expression system used and the displayed glycosylation patterns may influence downstream processing as well as the immune response. One of the main challenges for the development of Gag VLP production bioprocess is the separation of VLPs and coproduced extracellular vesicles (EVs). In this study, porous graphitized carbon separation method coupled with mass spectrometry was used to characterize the N- and O- glycosylation profiles of Gag VLPs produced in HEK293 cells. We identified differential glycan signatures between VLPs and EVs that could pave the way for further separation and purification strategies to optimize downstream processing and move forward in VLP-based vaccine production technology.

Micrometric DNA/PEI polyplexes correlate with higher transient gene expression yields in HEK 293 cells.

DNA delivery with polyethylenimine (PEI) has been widely used in the last three decades for the transfection of mammalian cells. Advances in novel characterization techniques at the nanometric scale offer new opportunities to revisit the physicochemical properties of DNA/PEI polyplexes that lead to efficient transfection. In this work, these properties are tuned by studying the synergies between simple parameters such as NaCl concentration, pH and incubation time in the DNA/PEI polyplex preparation protocol by means of Design of Experiments (DoE). By doing so, a model is obtained where an optimal NaCl concentration of 125 mM and an incubation time of 11 min provided the highest transfection yields. Correlation analyses between the physicochemical properties of DNA/PEI polyplexes and the predicted model responses revealed the existence of an optimal degree of aggregation in the pre-complexing solution to attain the highest transfection efficiencies. The presence of these micrometric DNA/PEI polyplex aggregates was confirmed by several nanoparticle characterization techniques including cryo-TEM, DLS and flow virometry. The findings provide a better understanding of the role of DNA/PEI aggregates in transient gene expression approaches, in particular considering that similar complexation protocols and saline solutions are widely used for the transfection of mammalian cell cultures.

Dynamics of long-term continuous culture of Limnospira indica in an air-lift photobioreactor.

MELiSSA (Microecological Life Support System Alternative) is a developing technology for regenerative life support to enable long-term human missions in Space and has developed a demonstration Pilot Plant. One of the components of the MELiSSA Pilot Plant system is an 83L external loop air-lift photobioreactor (PBR) where Limnospira indica (previously named Arthrospira sp. PC8005) is axenically cultivated in a continuous operation mode for long-periods. Its mission is to provide O2 and consume CO2 while producing edible material. Biological and process characterization of this PBR is performed by analysing the effect of two main variables, dilution rate (D) and PFD (Photon Flux Density) illumination. A maximum oxygen productivity ( r O 2 ) of 1.35 mmol l-1 h-1 is obtained at a D of 0.025 h-1 and PFD of 930 µmol m-2 s-1 . Photoinhibition can occur when a 1 g l-1 cell density culture is exposed to PFD higher than 1700 µmol m-2 s-1 . This process is reversible if the illumination is returned to dim light (150 µmol m-2 s-1 ), proving the cell adaptability and capacity to respond at different illumination conditions. Influence of light intensity in cell composition is also described. Specific photon flux density (qPFD) has a direct effect on phycobiliproteins and chlorophyll content causing a decrease of 62.5% and 47.8%, respectively, when qPFD increases from 6.1 to 19.2 µmol g-1 s-1 . The same trend is observed for proteins and the opposite for carbohydrate content. Morphological and spiral structural features of L. indica are studied by confocal microscopy, and size distribution parameters are quantified. A direct effect between trichome width and CDW/OD ratio is observed. Changes in size distribution are not correlated with environmental factors, further confirms the adaptation capacity of the cells. The systematic analysis performed provides valuable insights to understand the key performance criteria of continuous culture in air-lift PBRs.

Corrigendum: An Alternative Perfusion Approach for the Intensification of Virus-Like Particle Production in HEK293 Cultures.

[This corrects the article DOI: 10.3389/fbioe.2020.00617.].

A Four-Step Purification Process for Gag VLPs: From Culture Supernatant to High-Purity Lyophilized Particles.

Gag-based virus-like particles (VLPs) have high potential as scaffolds for the development of chimeric vaccines and delivery strategies. The production of purified preparations that can be preserved independently from cold chains is highly desirable to facilitate distribution and access worldwide. In this work, a nimble purification has been developed, facilitating the production of Gag VLPs. Suspension-adapted HEK 293 cells cultured in chemically defined cell culture media were used to produce the VLPs. A four-step downstream process (DSP) consisting of membrane filtration, ion-exchange chromatography, polishing, and lyophilization was developed. The purification of VLPs from other contaminants such as host cell proteins (HCP), double-stranded DNA, or extracellular vesicles (EVs) was confirmed after their DSP. A concentration of 2.2 ± 0.8 × 109 VLPs/mL in the lyophilized samples was obtained after its storage at room temperature for two months. Morphology and structural integrity of purified VLPs was assessed by cryo-TEM and NTA. Likewise, the purification methodologies proposed here could be easily scaled up and applied to purify similar enveloped viruses and vesicles.

Chimeric VLPs Based on HIV-1 Gag and a Fusion Rabies Glycoprotein Induce Specific Antibodies against Rabies and Foot-and-Mouth Disease Virus.

Foot and mouth disease is a livestock acute disease, causing economic losses in affected areas. Currently, control of this disease is performed by mandatory vaccination campaigns using inactivated viral vaccines. In this work, we describe the development of a chimeric VLP-based vaccine candidate for foot-and-mouth disease virus (FMDV), based on the co-expression of the HIV-1 Gag protein and a novel fusion rabies glycoprotein (RVG), which carries in its N-term the FMDV main antigen: the G-H loop. It is demonstrated by confocal microscopy that both Gag-GFP polyprotein and the G-H loop colocalize at the cell membrane and, that the Gag polyprotein of the HIV virus acts as a scaffold for enveloped VLPs that during the budding process acquires the proteins that are being expressed in the cell membrane. The obtained VLPs were spherical particles of 130 ± 40 nm in diameter (analyzed by TEM, Cryo-TEM and NTA) carrying an envelope membrane that efficiently display the GH-RVG on its surface (analyzed by gold immunolabeling). Immunostainings with a FMDV hyperimmune serum showed that the heterologous antigenic site, genetically fused to RVG, is recognized by specific G-H loop antibodies. Additionally, the cVLPs produced expose the G-H loop to the liquid surrounding (analyzed by specific ELISA). Finally, we confirmed that these FMD cVLPs are able to induce a specific humoral immune response, based on antibodies directed to the G-H loop in experimental animals.

Characterization of HIV-1 virus-like particles and determination of Gag stoichiometry for different production platforms.

The importance of developing new vaccine technologies towards versatile platforms that can cope with global virus outbreaks has been evidenced with the most recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Virus-like particles (VLPs) are a highly immunogenic, safe, and robust approach that can be used to base several vaccine candidates on. Particularly, HIV-1 Gag VLPs is a flexible system comprising a Gag core surrounded by a lipid bilayer that can be modified to present diverse types of membrane proteins or antigens against several diseases, like influenza, dengue, West Nile virus, or human papillomavirus, where it has been proven successful. The size distribution and structural characteristics of produced VLPs vary depending on the cell line used to produce them. In this study, we established an analytical method of characterization for the Gag protein core and clarified the current variability of Gag stoichiometry in HIV-1 VLPs depending on the cell-based production platform, directly determining the number of Gag molecules per VLP in each case. Three Gag peptides have been validated to quantify the number of monomers using parallel reaction monitoring, an accurate and fast, mass-spectrometry-based method that can be used to assess the quality of the produced Gag VLPs regardless of the cell line used. An average of 3617 ± 17 monomers per VLP was obtained for HEK293, substantially varying between platforms, including mammalian and insect cells. This offers a key advantage in quantification and quality control methods to characterize VLP production at a large scale to accelerate new recombinant vaccine production technologies.

Metabolic engineering of HEK293 cells to improve transient transfection and cell budding of HIV-1 virus-like particles.

HIV-1 Gag virus-like particles (VLPs) are promising candidates for the development of future vaccines. Recent viral outbreaks have manifested the need of robust vaccine production platforms able to adapt to new challenges while achieving mass production capacity. For the rapid production of VLPs, the method of transient gene expression (TGE) have proved highly efficient. Based on a previous characterization of the HEK293 cell line upon transient transfection using multiplexed quantitative proteomics, molecular production bottlenecks and metabolic pathways likely to be optimized were identified. In this study, these molecular components and metabolic pathways have been explored and modulated via transient metabolic engineering using approaches like design of experiments to fully exploit and optimize VLP production, transfection and budding efficiency. Upon overexpression of endosomal sorting complex required for transport accessory proteins like NEDD4L and CIT, VLP production increased 3.3 and 2.9-fold, respectively. Overexpression of glycosphingolipid precursor enzyme UGCG improved transfection efficiency by 17% and knocking-down the Gag-binding protein CNP improved 2.5-fold VLP specific productivity. Combining CNP inhibition and UGCG overexpression further improved budding efficiency by 37.3%. Modulating VLP production and accessory pathways like intracellular budding, demonstrated the potential of metabolic engineering to optimize and intensify the development of robust production platforms for future vaccines.

Accelerating HIV-1 VLP production using stable High Five insect cell pools.

Stable cell pools are receiving a renewed interest as a potential alternative system to clonal cell lines. The shorter development timelines and the capacity to achieve high product yields make them an interesting approach for recombinant protein production. In this study, stable High Five cell pools are assessed for the production of a simple protein, mCherry, and the more complex HIV-1 Gag-eGFP virus-like particles (VLPs). Random integration coupled to fluorescence-activated cell sorting (FACS) in suspension conditions is applied to accelerate the stable cell pool generation process and enrich it with high producer cells. This methodology is successfully transferred to a bioreactor for VLP production, resulting in a 2-fold increase in VLP yields with respect to shake flask cultures. In these conditions, maximum viable cell concentration improves by 1.5-fold, and by-product formation is significantly reduced. Remarkably, a global increase in the uptake of amino acids in the Gag-eGFP stable cell pool is observed when compared with parental High Five cells, reflecting the additional metabolic burden associated with VLP production. These results suggest that stable High Five cell pools are a robust and powerful approach to produce VLPs and other recombinant proteins, and put the basis for future studies aiming to scale up this system.

Molecular Characterization of the Coproduced Extracellular Vesicles in HEK293 during Virus-Like Particle Production.

Vaccine therapies based on virus-like particles (VLPs) are currently in the spotlight due to their potential for generating high immunogenic responses while presenting fewer side effects than conventional vaccines. These self-assembled nanostructures resemble the native conformation of the virus but lack genetic material. They are becoming a promising platform for vaccine candidates against several diseases due to the ability of modifying their membrane with antigens from different viruses. The coproduction of extracellular vesicles (EVs) when producing VLPs is a key phenomenon currently still under study. In order to characterize this extracellular environment, a quantitative proteomics approach has been carried out. Three conditions were studied: non-transfected, transfected with an empty plasmid as control, and transfected with a plasmid coding for HIV-1 Gag polyprotein. A shift in EV biogenesis has been detected upon transfection, changing the production from large to small EVs. Another remarkable trait found was the presence of DNA being secreted within vesicles smaller than 200 nm. Studying the protein profile of these biological nanocarriers, it was observed that EVs were reflecting an overall energy homeostasis disruption via mitochondrial protein deregulation. Also, immunomodulatory proteins like ITGB1, ENO3, and PRDX5 were identified and quantified in VLP and EV fractions. These findings provide insight on the nature of the VLP extracellular environment defining the characteristics and protein profile of EVs, with potential to develop new downstream separation strategies or using them as adjuvants in viral therapies.