Intracellular characterization of Gag VLP production by transient transfection of HEK 293 cells.

Transient transfection is a fast, flexible, and cost-effective approach to produce biological products. Despite the continued interest in transient transfection, little is known regarding the transfection process at the intracellular level, particularly for complex products, such as virus-like particles (VLPs). The kinetics of PEI-mediated transfection following an established in-house protocol is reported in this work with the aim of characterizing and understanding the complete process leading to VLP generation and identifying important events driving process improvement. For this purpose, DNA/PEI polyplexes' internalization in cells was tracked using Cy3 DNA staining. The production of a fluorescently labeled Gag polyprotein (a Gag-GFP fusion construct that forms fluorescent Gag-VLPs) was monitored by flow cytometry and confocal microscopy, and the VLP concentration in supernatants was measured by fluorometry. DNA/PEI polyplexes interact with the cell membrane immediately after polyplex addition to the cell culture. A linear increase in the number of cells expressing the protein is observed during the first 60 min of contact between the cells and polyplexes. No additional improvement in the number of cells expressing the protein (up to 60%) or VLP production (up to 1 × 1010 VLPs/mL) is observed with additional contact time between the cells and polyplexes. Polyplexes can be detected in the cytoplasm of transfected cells as early as 1.5 h post-transfection (hpt) and reach the nucleus approximately 4 hpt. GFP fluorescence is observed homogeneously in the cytoplasm of transfected cells 24 hpt, but generalized VLP budding is not observed by microscopy until 48 hpt. Although all cells have internalized a polyplex soon after transfection, only a fraction of cells (60%) express the fluorescent Gag protein. VLP production kinetics was also studied. Fluorescence in the supernatant (enveloped VLPs) is 40% less than total fluorescence, supernatant plus pellet (total Gag-GFP), indicating that there is a fraction of Gag that remains inside the cells. The maximum VLP concentration in the cell culture supernatant with cell viability >89% was observed at 72 hpt, which was determined to be the optimal harvest time. Biotechnol. Bioeng. 2017;114: 2507-2517. © 2017 Wiley Periodicals, Inc.

Extended gene expression by medium exchange and repeated transient transfection for recombinant protein production enhancement.

Production of recombinant products in mammalian cell cultures can be achieved by stable gene expression (SGE) or transient gene expression (TGE). The former is based on the integration of a plasmid DNA into the host cell genome allowing continuous gene expression. The latter is based on episomal plasmid DNA expression. Conventional TGE is limited to a short production period of usually about 96 h, therefore limiting productivity. A novel gene expression approach termed extended gene expression (EGE) is explored in this study. The aim of EGE is to prolong the production period by the combination of medium exchange and repeated transfection of cell cultures with plasmid DNA to improve overall protein production. The benefit of this methodology was evaluated for the production of three model recombinant products: intracellular GFP, secreted GFP, and a Gag-GFP virus-like particles (VLPs). Productions were carried out in HEK 293 cell suspension cultures grown in animal-derived component free media using polyethylenimine (PEI) as transfection reagent. Transfections were repeated throughout the production process using different plasmid DNA concentrations, intervals of time, and culture feeding conditions in order to identify the best approach to achieve sustained high-level gene expression. Using this novel EGE strategy, the production period was prolonged between 192 and 240 h with a 4-12-fold increase in production levels, depending on the product type considered.

Selection and optimization of transfection enhancer additives for increased virus-like particle production in HEK293 suspension cell cultures.

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%.

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.

Identification of host proteins associated with retroviral vector particles by proteomic analysis of highly purified vector preparations.

The Moloney murine leukemia virus (MMLV) belongs to the Retroviridae family of enveloped viruses, which is known to acquire minute amounts of host cellular proteins both on the surface and inside the virion. Despite the extensive use of retroviral vectors in experimental and clinical applications, the repertoire of host proteins incorporated into MMLV vector particles remains unexplored. We report here the identification of host proteins from highly purified retroviral vector preparations obtained by rate-zonal ultracentrifugation. Viral proteins were fractionated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in-gel tryptic digested, and subjected to liquid chromatography/tandem mass spectrometry analysis. Immunogold electron microscopy studies confirmed the presence of several host membrane proteins exposed at the vector surface. These studies led to the identification of 27 host proteins on MMLV vector particles derived from 293 HEK cells, including 5 proteins previously described as part of wild-type MMLV. Nineteen host proteins identified corresponded to intracellular proteins. A total of eight host membrane proteins were identified, including cell adhesion proteins integrin beta1 (fibronectin receptor subunit beta) and HMFG-E8, tetraspanins CD81 and CD9, and late endosomal markers CD63 and Lamp-2. Identification of membrane proteins on the retroviral surface is particularly attractive, since they can serve as anchoring sites for the insertion of tags for targeting or purification purposes. The implications of our findings for retrovirus-mediated gene therapy are discussed.

Predictores de la satisfacción sexual durante el confinamiento por COVID-19 en España / Predictors of sexual satisfaction during COVID-19 confinement in Spain

Esta investigación, formada por dos estudios, tiene como objetivo general analizar el impacto de la COVID-19 en la salud sexual de 347 adultos residentes en España. El estudio 1, centrado en prácticas sexuales no presenciales (sexteo [sexting] y pornografía), puso de manifiesto niveles de satisfacción sexual similares en hombres y mujeres, aunque diferentes según la edad en interacción con el consumo de pornografía y el estatus marital. El estudio 2 abordó los cambios producidos con respecto a los seis meses previos, indicando que el mantenimiento de la satisfacción sexual no parece depender del sexo, pero sí de la edad en interacción con el sexo presencial, del estatus marital y del sexo individual en interacción con un adecuado funcionamiento del interés sexual. Ante el desafío que está suponiendo esta pandemia, estos resultados resultan de utilidad para las intervenciones en salud mental y sexual que actualmente se están desarrollando a causa de la COVID-19 (AU)

This research, which consists of two studies, has the general objective of analyzing the impact of COVID-19 on the sexual health of 347 adults living in Spain. Study 1, focused on non-face-to-face sexual practices (sexting and pornography), revealed similar levels of sexual satisfaction in men and women, but with differences in age regarding interaction with the consumption of pornography and marital status. Study 2 focused on the changes produced with respect to the previous six months, indicating that the maintenance of sexual satisfaction does not depend on gender, but it does depend on age in interaction with face-to-face sex, marital status, and individual sex, in interaction with an adequate functioning of sexual interest. Given the challenge that this pandemic is posing, these results are useful for the mental and sexual health interventions that are currently being developed because of COVID-19 (AU)

Canine adenovirus downstream processing protocol.

Adenovirus vectors are efficient gene delivery tools. A major caveat with vectors derived from common human adenovirus serotypes is that most adults are likely to have been exposed to the wild-type virus and exhibit active immunity against the vectors. This preexisting immunity limits their clinical success. Strategies to circumvent this problem include the use of nonhuman adenovirus vectors. Vectors derived from canine adenovirus type 2 (CAV-2) are among the best-studied representatives. CAV-2 vectors are particularly attractive for the treatment of neurodegenerative disorders. In addition, CAV-2 vectors have shown great promise as oncolytic agents in virotherapy approaches and as vectors for recombinant vaccines. The rising interest in CAV-2 vectors calls for the development of scalable GMP compliant production and purification strategies. A detailed protocol describing a complete scalable downstream processing strategy for CAV-2 vectors is reported here. Clarification of CAV-2 particles is achieved by microfiltration. CAV-2 particles are subsequently concentrated and partially purified by ultrafiltration-diafiltration. A Benzonase(®) digestion step is carried out between ultrafiltration and diafiltration operations to eliminate contaminating nucleic acids. Chromatography purification is accomplished in two consecutive steps. CAV-2 particles are first captured and concentrated on a propyl hydrophobic interaction chromatography column followed by a polishing step using DEAE anion exchange monoliths. Using this protocol, high-quality CAV-2 vector preparations containing low levels of contamination with empty viral capsids and other inactive vector forms are typically obtained. The complete process yield was estimated to be 38-45 %.

New developments in lentiviral vector design, production and purification.

INTRODUCTION: Lentiviruses are a very potent class of viral vectors for which there is presently a rapidly growing interest for a number of gene therapy. However, their construction, production and purification need to be performed according to state-of-the-art techniques in order to obtain sufficient quantities of high purity material of any usefulness and safety. AREAS COVERED: The recent advances in the field of recombinant lentivirus vector design, production and purification will be reviewed with an eye toward its utilization for gene therapy. Such a review should be helpful for the potential user of this technology. EXPERT OPINION: The principal hurdles toward the use of recombinant lentivirus as a gene therapy vector are the low titer at which it is produced as well as the difficulty to purify it at an acceptable level without degrading it. The recent advances in the bioproduction of this vector suggest these issues are about to be resolved, making the retrovirus gene therapy a mature technology.

Generation of HIV-1 Gag VLPs by transient transfection of HEK 293 suspension cell cultures using an optimized animal-derived component free medium.

Virus-like particles (VLPs) offer great promise as candidates for new vaccine strategies. Large-scale approaches for the manufacturing of HIV-1 Gag VLPs have mainly focused on the use of the baculovirus expression system. In this work, the development and optimization of an HIV-1 Gag VLP production protocol by transient gene expression in mammalian cell suspension cultures is reported. To facilitate process optimization, a Gag-GFP fusion construct enabling the generation of fluorescent VLPs was used. The great majority of Gag-GFP present in cell culture supernatants was shown to be correctly assembled into virus-like particles of the expected size and morphology consistent with immature HIV-1 particles. Medium optimization was performed using design of experiments (DoE). Culture medium supplementation with non-animal derived components including recombinant proteins and lipids of synthetic or non-animal-derived origin resulted in improved HEK 293 cell growth and VLP production. The maximum cell density attained using the optimized Freestyle culture medium was 5.4×10(6)cells/mL in batch mode, almost double of that observed using the unsupplemented medium (2.9×10(6)cells/mL). Best production performance was attained when cells were transfected at mid-log phase (2-3×10(6)cells/mL) with medium exchange at the time of transfection using standard amounts of plasmid DNA and polyethylenimine. By using an optimized production protocol, VLP titers were increased 2.4-fold obtaining 2.8µg of Gag-GFP/mL or 2.7×10(9)VLPs/mL according to ELISA and nanoparticle tracking quantification analyses, respectively.

Development and validation of a quantitation assay for fluorescently tagged HIV-1 virus-like particles.

Upon expression, the Gag polyprotein of HIV-1 assembles spontaneously in the vicinity of the plasma membrane giving rise to enveloped virus-like particles (VLPs). These particulate immunogens offer great promise as HIV-1 vaccines. Robust VLP production and purification processes are required to generate VLPs of sufficient quality and quantity for both pre-clinical and clinical evaluation. The availability of simple, fast and reliable quantitation tools is critical to develop, optimize and monitor such processes. Traditionally, enzyme-linked immunosorbent assays (ELISA) are used to quantify p24 antigen concentrations, which reflect tightly virus particle concentrations. However, this quantitation technique is not only time-consuming, laborious and costly but it is also prone to methodological variability. As an alternative, the development and validation of a fluorescence-based quantitation assay for Gag VLPs is presented here. A Gag polyprotein fused to the enhanced green fluorescent protein was used for generation of fluorescent VLPs. A purified standard reference Gag-GFP VLP material was prepared and characterized in house. The method was validated according to ICH guidelines. The validation characteristics evaluated included accuracy, precision, specificity, linearity, range and limit of detection. The method showed to be specific for Gag-GFP. The fluorescence signal correlated well with p24 concentrations measured using a reference p24 ELISA assay. The method showed little variability compared to ELISA and was linear over a 3-log range. The limit of detection was ~10 ng of p24/mL. Finally, fluorescence-based titers were in good agreement with those obtained using transmission electron microscopy and nanoparticle tracking analysis. This simple, rapid and cost-effective quantitation assay should facilitate development and optimization of bioprocessing strategies for Gag-based VLPs.

Chromatography purification of canine adenoviral vectors.

Canine adenovirus vectors (CAV2) are currently being evaluated for gene therapy, oncolytic virotherapy, and as vectors for recombinant vaccines. Despite the need for increasing volumes of purified CAV2 preparations for preclinical and clinical testing, their purification still relies on the use of conventional, scale-limited CsCl ultracentrifugation techniques. A complete downstream processing strategy for CAV2 vectors based on membrane filtration and chromatography is reported here. Microfiltration and ultra/diafiltration are selected for clarification and concentration of crude viral stocks containing both intracellular and extracellular CAV2 particles. A DNase digestion step is introduced between ultrafiltration and diafiltration operations. At these early stages, concentration of vector stocks with good recovery of viral particles (above 80%) and removal of a substantial amount of protein and nucleic acid contaminants is achieved. The ability of various chromatography techniques to isolate CAV2 particles was evaluated. Hydrophobic interaction chromatography using a Fractogel propyl tentacle resin was selected as a first chromatography step, because it allows removal of the bulk of contaminating proteins with high CAV2 yields (88%). An anion-exchange chromatography step using monolithic supports is further introduced to remove the remaining contaminants with good recovery of CAV2 particles (58-69%). The main CAV2 viral structural components are visualized in purified preparations by electrophoresis analyses. Purified vector stocks contained intact icosahedral viral particles, low contamination with empty viral capsids (10%), and an acceptable total-to-infectious particle ratio (below 30). The downstream processing strategy that was developed allows preparation of large volumes of high-quality CAV2 stocks.

Efficient amplification of chimeric adenovirus 5/40S vectors carrying the short fiber protein of Ad40 in suspension cell cultures.

The human adenovirus 40 (Ad40) is a promising tool for gene therapy of intestinal diseases. Since the production of Ad40 in vitro is extremely inefficient, chimeric Adenovirus 5/40S vectors carrying the Ad40 short fiber on the Ad5 capsid have been developed. However, Ad5/40S productivity is low. We hypothesized that low productivity was a result of inefficient viral entry into producer cells during amplification. To this end, we have developed a production strategy based on using 211B cells (expressing Ad5 fiber) during amplification steps, while Ad5/40S infectivity is further improved by adding polybrene during infections. In addition, the optimal harvesting time was determined by evaluating the Ad5/40S viral cycle. The developed production strategy significantly reduces the number of amplification cycles and duration of the process. Finally, to further facilitate Ad5/40S production, 211B cells were adapted to suspension thus allowing to easily upscale the production process in bioreactors.

Overview of current scalable methods for purification of viral vectors.

As a result of the growing interest in the use of viruses for gene therapy and vaccines, many virus-based products are being developed. The manufacturing of viruses poses new challenges for process developers and regulating authorities that need to be addressed to ensure quality, efficacy, and safety of the final product. The design of suitable purification strategies will depend on a multitude of variables including the vector production system and the nature of the virus. In this chapter, we provide an overview of the most commonly used purification methods for viral gene therapy vectors. Current chromatography options available for large-scale purification of γ-retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes simplex virus, baculovirus, and poxvirus vectors are presented.

New protocol for lentiviral vector mass production.

Multiplasmid transient transfection is the most widely used technique for the generation of lentiviral vectors. However, traditional transient transfection protocols using 293 T adherent cells and calcium phosphate/DNA co-precipitation followed by ultracentrifugation are tedious, time-consuming, and difficult to scale up. This chapter describes a streamlined protocol for the fast mass production of lentiviral vectors and their purification by affinity chromatography. Lentiviral particles are generated by transient transfection of suspension growing HEK 293 cells in serum-free medium using polyethylenimine (PEI) as transfection reagent. Lentiviral vector production is carried out in Erlenmeyer flasks agitated on orbital shakers requiring minimum supplementary laboratory equipment. Alternatively, the method can be easily scaled up to generate larger volumes of vector stocks in bioreactors. Heparin affinity chromatography allows for selective concentration and purification of lentiviral particles in a singlestep directly from vector supernatants. The method is suitable for the production and purification of different vector pseudotypes.

A real-time PCR assay for quantification of canine adenoviral vectors.

The pre-existing humoral and cellular immunity found in the great majority of the population raises concerns about the clinical efficacy and safety of vectors derived from ubiquitous human adenovirus serotypes. To alleviate these concerns, canine adenovirus type 2 vectors (CAV-2) were developed. Owing to their extraordinary neuronal tropism and efficient axonal retrograde transport, CAV-2 vectors hold great promise for the treatment of neurodegenerative diseases. The development and validation of a SYBR Green qPCR assay for determination of CAV-2 titers is reported in the present study. This method uses specific primers designed to amplify a small genomic fragment of CAV-2 structural genes (pVI-hexon). The method was accurate and reproducible as determined by the low intra- and inter-assay variability (<15% R.S.E.). It is sensitive and useful over a 5-log range (1 x 10(3) to 1 x 10(7)genome copies/reaction). The assay can be used to quantify purified vector samples as well as crude viral lysates. The titers obtained by qPCR correlated well with both, those obtained by OD(260) and TCID(50) as indicated by the high coefficients of determination obtained by regression analysis (r(2)>0.83). The development of this simple and rapid CAV-2 quantitation method should be helpful for process development and monitoring.

Production of lentiviral vectors by large-scale transient transfection of suspension cultures and affinity chromatography purification.

The use of lentiviral vectors as gene delivery vehicles has become increasingly popular in recent years. The growing interest in these vectors has created a strong demand for large volumes of vector stocks, which entails the need for scaleable vector manufacturing procedures. In this work, we present a simple and robust process for the production of lentiviral vectors using scaleable production and purification methodologies. Lentivirus particles were produced by transient transfection of serum-free suspension-growing 293 EBNA-1 cells with four plasmids encoding the vector components using linear polyethylenimine (PEI) as transfection reagent. This process was successfully scaled-up from shake flasks to a 3-L bioreactor from which 10(10) IVP were recovered. In addition, an affinity chromatography protocol designed for purification of bioactive oncoretroviral vectors has been adapted in this work for the purification of VSV-G pseudotyped lentiviral vectors. Using heparin affinity chromatography, lentiviral particles were concentrated and purified directly from the clarified supernatants. During this step, a recovery of 53% of infective lentiviral particles was achieved while removing 94% of the impurities contained in the supernatant.