Efficient production of influenza virus-like particles in HEK-293SF cells

Influenza is considered a major threat to human health. The current influenza vaccines production system in embryonated eggs does not satisfy the market needs in cases of pandemic or high seasonal demand. Furthermore, even though the existent seasonal vaccines are effective to induce protective immunity in healthy adults, poor immunogenicity in the elderly and early childhood is elicited (Wu et al., 2010). Therefore, there is a need to develop new generation influenza vaccines produced in robust and flexible production platforms capable of providing complete immune protection and support the vaccines demand. Virus-like particles (VLP) constitute a promising alternative to safely elicit a potent immune response against influenza. These particles do not contain viral genome and their structure mimic influenza virus (Thompson et al., 2015). In this study, a HEK-293SF inducible stable cell line expressing hemagglutinin (HA) and neuraminidase (NA) of the strain A/PR/8/34 (H1N1) has been developed. Once the stable cell line, named 293HA/NA, was established, the production of VLPs was mediated by transient transfection with the plasmid pAdCMV5-GAGGFP that encodes the HIV GAG structural protein fused with GFP. The transient expression of GAG was efficient to abundantly release the particles in the culture medium. The VLPs production in 293HA/NA cells using GAGGFP protein as scaffold was studied in 250 mL shake flasks by following the increase of fluorescence in the supernatant (Spectrophotometry) and the accumulation of HA (Dot-Blot) (Figure 1). The process was successfully operated in a 3L-Bioreactor and at 72h post-transfection the cell supernatant was ultracentrifuged on a 25 % sucrose cushion. High levels of well-structured influenza GAG-VLPs were obtained by this production process and their concentration was estimated by electron microscopy at 5.9*109 VLPs/mL. The influenza GAG-VLPs were purified by Tangential Flow Filtration and the concentration of HA was estimated by three different techniques: Single Radial Immunodiffusion, Hemagglutination assay and Dot-Blot. With further optimization, the production system proposed in this work could be a promising alternative to support the influenza vaccines manufacturing in the future

High-Yield Production Process of Influenza Virus-Like Particles in Human Cells Toward Large-Scale Vaccine Manufacturing

Le virus influenza a été la cause d’épidémies et de pandémies parmi les plus anciennes et meurtrières rapportées dans l’histoire de l’humanité. La vaccination est le moyen le plus efficace de prévenir les infections. Par contre, la fabrication actuelle des vaccins contre l’influenza se fait dans les oeufs, avec des embryons de poulet, un procédé lent et laborieux qui limite la capacité de répondre efficacement en cas de pandémie ou suite à une forte demande pour la grippe saisonnière. De plus, ces vaccins induisent principalement une réponse humorale contre les antigènes dominants hémagglutinine (HA) et neuraminidase (NA), deux protéines du virus, causant un manque de protection croisée contre certaines des nouvelles souches. Leur efficacité est également réduite chez certains groupes plus vulnérables (par ex. personnes âgées et jeunes enfants). Par conséquent, l’industrie se tourne vers le développement d’une nouvelle gamme de vaccins plus immunogéniques et produits à partir de plateformes plus efficaces. Les particules pseudo-virales (Virus-like particles en anglais, VLPs) constituent une alternative intéressante comme vaccin. Les VLPs présentent une structure qui s’apparente à celle des virus de type sauvage, en permettant la présentation à leur surface des antigènes principaux, dans leur conformation native. De plus, les particules pseudo-virales sont non-infectieuses et incapables de se répliquer. Les cellules de mammifères offrent plusieurs avantages comme plateforme d’expression pour la synthèse de nombreux produits biopharmaceutiques; elles sont capables d’effectuer des modifications post-traductionnelles complexes, de croître à haute densité et de produire des VLPs en suspension et en bioréacteur. Jusqu’à maintenant, les études traitant des VLPs influenza (produites avec des cellules de mammifères) se sont concentrées principalement sur l’assemblage du virion et sur le mécanisme de bourgeonnement cellulaire, alors que seulement un nombre limité d’études porte sur leur production à grande échelle et leur emploi potentiel comme vaccin. Dans le cadre de cette thèse, un bioprocédé transposable à grande échelle pour produire des quantités importantes de VLPs chimériques Gag-influenza à partir de cellules HEK-293 (cellules de reins issues d’un embryon humain) a été développé. En premier lieu, nous avons généré une lignée cellulaire HEK-293 exprimant de façon stable les protéines HA et NA (souche H1N1 du virus Influenza) sous le contrôle d’un système inductible au cumate. Ensuite, la formation des VLPs chimériques a été induite et dirigée par la transfection de plasmides codant la protéine Gag du virus de l’immunodéficience humaine ou la protéine M1, vii une composante de la matrice du virus de l’influenza. La protéine Gag a été fusionnée à la protéine fluorescente verte pour faciliter le suivi de la production des VLPs. Les protéines antigéniques ont été produites 7 fois plus efficacement en présence de la protéine Gag, ce qui indique qu’il s’agit d’une meilleure protéine structurale que M1 dans ce contexte. Par conséquent, la production de VLPs contenant HA-NA et Gag (par transfection) a donc été transférée à l’échelle d’un bioréacteur de 3L avec agitation. Les VLPs ont été recueillies par ultracentrifugation sur un coussin de sucrose, puis concentrées par filtration à flux tangentiel en employant une membrane ayant des pores d’une taille limite de 1000 kDa. Plusieurs techniques ont été employées pour caractériser les VLPs produites: immunodiffusion radiale simple, essai d’hémagglutination, immunobuvardage de type Western et dot blot, ainsi que la microscopie électronique à transmission. Par ailleurs, lors d’essais sur des animaux, l’immunisation intranasale à partir de VLPs a permis d’induire une réponse immunitaire spécifique en plus de conférer une protection totale à toutes les souris soumises à l’épreuve (100% d’efficacité) avec une souche homologue de l’influenza. Après avoir démontré l’efficacité des VLPs comme vaccin in vivo (démonstration de faisabilité), une nouvelle lignée cellulaire inductible a été développée, exprimant cette fois les trois protéines HA, NA et Gag fusionnée à la GFP. Le but de générer une telle lignée était de simplifier le procédé de production en éliminant l’étape de transfection transitoire, qui peut être laborieuse à conduire à grande échelle. Sans sacrifier la production spécifique, le procédé a été optimisé pour obtenir une plus grande production volumétrique en augmentant notamment la densité cellulaire au moment de l’induction et en employant un mode de perfusion. L’opération a été réalisée à l’aide d’un bioréacteur de 3L et d’une filtration à flux tangentiel subséquente permettant d’augmenter les rendements de VLPs de 60 fois (3x1011 Gag-GFP événements fluorescents/L de culture mesurés par cytométrie de flux) en comparaison à une production sans perfusion (5x109 Gag-GFP événements fluorescents/L). Le procédé a été caractérisé en déterminant la cinétique de production des protéines d’intérêts présentes dans les VLPs (le procédé en amont) ainsi que le taux de récupération pour chaque opération unitaire de la purification (le procédé en aval). L’opération du bioréacteur, en mode de perfusion et avec la lignée stable 293-HA/NA/Gag-GFP, a permis d’obtenir 5 fois plus de protéines antigéniques HA dans les VLPs que le bioréacteur opéré sans perfusion où la transfection transitoire a été employée. Le nouveau procédé développé viii a permis de générer des rendements supérieurs à ceux publiés jusqu’à ce jour pour des VLPs influenza produits à partir de cellules de mammifères. Finalement, dans le but de répondre à certaines préoccupations de biosécurité associées à un usage potentiel de VLPs comme vaccins (parce que ce sont des particules enveloppées qui bourgeonnent d’une cellule hôte et qui renferment des protéines cellulaires, de l’ADN et de l’ARN), nous avons effectué une caractérisation du protéome des VLPs Gag-influenza produites par transfection transitoire et des vésicules extracellulaires produites par des cellules HEK-293 de type sauvage. Les fonctions des protéines identifiées dans les VLPs et dans les vésicules extracellulaires ont été discutées. Le procédé développé dans le cadre de cette thèse devrait être efficace pour produire des VLPs exposant les protéines HA et NA issues de différentes souches d’influenza. Les VLPs produites pourraient être évaluées dans le cadre d’essais cliniques dans le but de conduire au développement d’un vaccin efficace et sécuritaire pour remplacer ou complémenter le procédé actuel de production dans les embryons poulets. ---------- Of the fatal infections noted in human history, influenza epidemics and pandemics are among the most ancient. Vaccination remains the most effective tool to prevent infection. However, the current production of influenza vaccines in embryonated chicken eggs has limited capacity during pandemics or high demand seasons, and is both labor-intensive and time-consuming. Furthermore, the seasonal egg-produced vaccines mainly induce humoral response to the Hemagglutinin (HA)/Neuraminidase (NA) dominant antigens, which leads to a lack of cross-protection against other non-matching novel strains. In addition, the vaccines provide low protection in high risk groups (e.g., elderly and young children). Consequently, the industry is moving toward the development of novel, more immunogenic influenza vaccines as well as more efficient production platforms. Virus-like particles (VLPs) constitute a promising alternative as influenza vaccine. They mimic the particulate structure of wild-type viruses while they are non-infectious, non-replicative particles, and the main antigens repetitively displayed on their surface maintain the native conformation. Mammalian cell culture offers several advantages for the production of biopharmaceuticals such as their ability to perform complex post-translational modifications and the high cell densities and productivities reached in suspension culture bioreactors. Up to now, the production of influenza VLPs from mammalian cells has been mostly addressed to study influenza assembly and budding mechanisms but little attention has been paid to its potential use for large-scale manufacturing of VLPs as influenza vaccine candidate. The aim of this thesis was to develop a scalable process to produce large quantities of chimeric influenza Gag-VLPs from stable human embryonic kidney HEK-293 cells in suspension culture. First, a HEK-293 cell line stably expressing HA and NA proteins of influenza (subtype H1N1) under the regulation of the inducible cumate system was established. Then, the formation of VLPs was mediated by transient transfection of plasmids encoding human immunodeficiency virus (HIV) Gag or M1 influenza matrix protein. Gag protein was fused to the green fluorescent protein (GFP) to facilitate the monitoring of VLPs production. VLP antigenic proteins were produced seven times more efficiently in the presence of Gag, indicating that Gag is a better scaffolding protein than M1 in this context. Subsequently, the production of HA-NA containing VLPs after transient transfection of Gag as scaffold protein was successfully implemented in a 3-L controlled stirred tank bioreactor. VLPs were recovered by ultracentrifugation on a sucrose x cushion followed by concentration through tangential flow filtration (TFF) using a 1000 KDa cut-off membrane. Different techniques were employed to characterize the produced VLPs: Single radial immunodiffusion (SRID), hemagglutination assay, dot-blot, western-blot, and transmission electron microscopy (TEM). Of great significance, intranasal immunization of VLPs induced specific immunogenic response and provided complete protection in mice challenged with the homologous influenza strain. Once the proof of concept of VLPs as an efficacious influenza vaccine was demonstrated in vivo, we developed a new inducible cell line expressing the three proteins HA, NA and the Gag fused to GFP. This was performed in an effort to streamline the production process by eliminating the transient transfection step that can be cumbersome at large scale. The process was optimized to reach a high volumetric yield of VLPs by increasing the cell density at the time of induction without sacrificing the cell specific productivity. By operating a 3L-bioreactor in perfusion mode followed by TFF, the yields of VLPs were improved by 60-fold (3x1011 Gag-GFP fluorescent events/L of culture measured by flow cytometry) compared to a standard batch culture (5x109 Gag-GFP fluorescent events/L). The process was characterized for the upstream kinetics of production of VLP proteins and recovery rates for each downstream step. The production of a single bioreactor, operated in perfusion mode, with the stable cell line 293-HA/NA/Gag-GFP yielded 5-fold more total VLP antigenic HA proteins than what was produced with the 3L-batch bioreactor using transient transfection. Our process provided unprecedented yields of influenza VLPs produced from mammalian cells. Finally, because VLPs are enveloped particles that bud from a host cell potentially enclosing host cell proteins, DNA and RNA, which could pose a safety concern, we performed a proteomic characterization of the influenza Gag-VLPs produced by transient transfection and also extracellular vesicles (EVs) produced from wild-type HEK-293 cells. The functions of all proteins identified within VLPs and EVs were critically discussed. The process developed in this thesis could support the production of VLPs harboring HA and NA of different strains for clinical trials and could potentially result in a better vaccine candidate with higher efficacy and safety to replace the current labor-intensive egg-produced influenza vaccines

Proteomic characterization of influenza H1N1 Gag virus-like particles and extracellular vesicles produced in HEK-293SF

One of the major concerns associated with the use of influenza virus-like particles (VLPs) produced in cell culture as vaccine candidates is their heterogeneous composition. Enveloped VLPs take up the host cell membrane at the budding site carrying out with them not only the viral antigenic proteins but also host cell proteins. In addition, the intrinsic nature of the cells to produce membrane derived vesicles which have similar size to the VLPs and can also contain the antigenic proteins, makes the VLP purification process challenging. Certainly, the expression system and the viral recombinant proteins employed will determine the nature of the proteins within the VLPs. To further characterize cell culture produced-influenza VLPs and contribute to enable their approval as vaccine candidates, the composition and biogenesis of VLPs need to be better understood. In this study we have characterized, by nanoscale liquid chromatography tandem mass spectrometry (n-LC-MS/MS), influenza H1N1 Gag-VLPs produced in human embryonic kidney cells adapted to serum-free medium (HEK-293SF). The cells stably express HA and NA, and the VLPs production occurs following transient transfection with a plasmid containing the gag gene of HIV-1 fused to GFP. Extracellular vesicles (EVs) produced by the unmodified HEK-293SF were also characterized by n-LC-MS/MS. A total of 73 host cell proteins were identified in the VLPs, whereas 98 were detected in the extracellular vesicles. From that, 32 host cell proteins were unique to VLPs while 41 proteins were found in both. Importantly, nucleolin was the most abundant host cell differential protein identified in VLPs while lactotransferrin and heat shock protein 90 were the most present in EVs. This study provides a detailed proteomic description of the VLPs and EVs produced in HEK-293SF as well as a critical discussion of the function of each protein incorporated in both nanoparticles species. The outcome of this research also sheds light on unique target proteins differentially identified either in VLPs and EVs that could potentially be exploited for the development of novel purification protocols to separate EVs from VLPs

Advancements in molecular design and bioprocessing of recombinant adeno-associated virus gene delivery vectors using the insect-cell baculovirus expression platform.

AbstractDespite rapid progress in the field, scalable high‐yield production of adeno‐associated virus (AAV) is still one of the critical bottlenecks the manufacturing sector is facing. The insect cell‐baculovirus expression vector system (IC‐BEVS) has emerged as a mainstream platform for the scalable production of recombinant proteins with clinically approved products for human use. In this review, we provide a detailed overview of the advancements in IC‐BEVS for rAAV production. Since the first report of baculovirus‐induced production of rAAV vector in insect cells in 2002, this platform has undergone significant improvements, including enhanced stability of Bac‐vector expression and a reduced number of baculovirus‐coinfections. The latter streamlining strategy led to the eventual development of the Two‐Bac, One‐Bac, and Mono‐Bac systems. The one baculovirus system consisting of an inducible packaging insect cell line was further improved to enhance the AAV vector quality and potency. In parallel, the implementation of advanced manufacturing approaches and control of critical processing parameters have demonstrated promising results with process validation in large‐scale bioreactor runs. Moreover, optimization of the molecular design of vectors to enable higher cell‐specific yields of functional AAV particles combined with bioprocess intensification strategies may also contribute to addressing current and future manufacturing challenges

Characterization of HA and NA-containing VLPs produced in suspension cultures of HEK 293 cells

Virus like particles (VLPs) can be formulated into promising vaccines to prevent influenza infection. In addition of having a structure and composition that mimic the wild type virus, VLPs are safe since they are devoid of viral genes and consequently are not infectious. One approach to scale up the manufacturing of VLPs is to produce them in a serum-free suspension culture using a stable mammalian cell line. Importantly, with VLPs synthetized by mammalian cells, the post-translational modifications of the surface antigens should be similar to the wild type virus, and therefore should trigger a potent and specific immune response for the pathogen. As a proof of concept, we first established a cell line that was stably expressing hemagglutinin (HA) and neuraminidase (NA) proteins of influenza (subtype H1N1) using our patented cGMP human embryonic kidney (HEK293) cell line (clone 293SF-3F6). Transcription of the genes for these two glycoproteins was regulated by the inducible cumate transcription gene-switch. Next, to establish our capability to produce VLPs, we compared the formation of VLPs using these cells after forced expression of two scaffold proteins: Gag from the human immunodeficiency virus and M1 protein from influenza A (H1N1). In addition, monitoring of the VLPs was facilitated by fusing the Gag protein to the green fluorescent protein (GFP). VLP production was therefore initiated by transient transfection of plasmid encoding Gag or M1 and by addition of cumate to the culture medium. The VLPs secreted in the culture medium were recovered by ultracentrifugation on a sucrose cushion. The presence of HA an NA within the VLP fraction was demonstrated by western blot and quantified by dot blot. Interestingly, VLPs were produced more efficiently in the presence of Gag, indicating that Gag is a better scaffolding protein than M1 in this context. Under the electron microscope, the Gag-VLPs appeared as vesicles of 100 to 150 nm of diameter, containing a denser internal proteinous ring, which is a typical morphology for VLPs produced through Gag expression. The production of Gag-VLPs was also validated in a 3-L stirred tank bioreactor in serum-free medium. The immunogenicity of the VLPs is currently under investigation in a murine model for influenza. In conclusion, VLPs containing HA and NA can be manufactured in serum free suspension culture of HEK293 cells through forced expression of Gag. The efficacy of these VLPs for vaccination remains to be demonstrated

Multivalent Influenza vaccine production in HEK-293 cells in response to pandemic threats

Influenza virus infects millions of people every year worldwide, with elderly and very young people among the most critically affected. Strains that constitute a pandemic threat are characterized by the severity of the clinical manifestations and mortality rates and tend to require the urgent production of hundreds of millions of vaccine doses in very short periods of time. There is an evident need to develop new generations of influenza vaccines based on robust production systems such as mammalian or insect cell cultures. These systems may allow, in contrast to production in embryonated chicken eggs, a faster response capacity, a superior manufacturing process control and a more reliable and better characterized product. Please click Download on the upper right corner to see the full abstract

Development of scalable downstream processing platform for HEK293SF cell-based influenza vaccine production

Background: Research efforts during recent decades have demonstrated the suitability of mammalian cell culture platform for influenza vaccine production. Certainly, the potential of this system for a large-scale continuous vaccine manufacturing will enable a faster response to pandemic comparing with traditional egg-based production. Even though great advances have been achieved on the upstream processing of mammalian cell culture produced influenza vaccines, the downstream processing and quality of final product have still room for improvement or is still in development. Please click Download on the upper right corner to see the full abstract

Accelerated mass production of influenza virus seed stocks in HEK-293 suspension cell cultures by reverse genetics

Despite major advances in developing capacities and alternative technologies to egg-based production of influenza vaccines, responsiveness to an influenza pandemic threat is limited by the time it takes to generate a Candidate Viral Vaccine (CVV) as reported by the 2015 WHO Informal Consultation report titled “Influenza Vaccine Response during the Start of a Pandemic”. In previous work, we have shown that HEK-293 cell culture in suspension and serum free medium is an efficient production platform for cell culture manufacturing of influenza candidate vaccines. This report, took advantage of, recombinant DNA technology using Reverse Genetics of influenza A/Puerto Rico/8/34 H1N1 strain, and advances in the large-scale transfection of suspension cultured HEK-293 cells. Transfection in shake flasks was performed using 1ug of total plasmid and 1x106 cells/mL. The supernatant was harvested after 48 hpt and used to infect a new shake flasks at 1x106 cells/mL for virus amplification. 3-L bioreactor was inoculated and transfected at 1x106 cells/mL with 1ug of total plasmid and harvested after 48hpt and the virus generated was amplified in shake flask. Quantification by TCID50, SRID, Dot-blot and TRPS were performed as well as characterization by TEM and HA and NA sequencing. Small-scale transfection in shake flasks generated 1.5x105 IVP/mL after 48 hpt and 1x107 IVP/mL after 96 hpi. For large-scale experiment a 3-L controlled stirred tank bioreactor resulted in supernatant (P0) virus titer of 5x104 IVP/mL and 2.8x107 IVP/mL after only one amplification (P1) in HEK-293 suspension cells. We demonstrate the efficent generation of H1N1 with the PR8 backbone reassortant under controlled bioreactor conditions in two sequential steps (transfection/rescue and infection/production). This approach could deliver a CVV for influenza vaccine manufacturing within two-weeks, starting from HA and NA pandemic sequences. Thus, this innovative approach is better suited to rationally design and mass produce the CVV within timelines dictated by pandemic situations and produce effective responsiveness than previous methodolog

Generation and efficacy assessment of a chimeric antigen E2-CD154 as a marker Classical Swine Fever Virus subunit vaccine produced in HEK 293 and CHO K1 mammalian cells

The E2 glycoprotein is the major antigen that induces neutralizing and protective antibodies in CSFV infected pigs, thus a marker vaccine based on this antigen appears to be the most promising alternative to induce a protective immune response against CSFV. However, the structural characteristics of this protein state the necessity to produce glycoprotein E2 in more complex expression systems such as mammalian cells. In this study, we use a lentivirus-based gene delivery system to establish a stable recombinant HEK 293 and CHO K1 cell line for the expression of E2 fused to porcine CD154 as immunostimulatory molecule. In a first experiment, E2his and E2-CD154 were compared in an immunization trial. The average antibody titers in E2his immunized pigs was in the range of 30-40% of blocking and the average antibody titers for E2-CD154 are above 40% at day 14, meaning that the chimeric antigen is able to raise antibodies at positive levels in a shorter time. Additionally, the blocking rate of E2his vaccinated group in ELISA ranged between 66-88% and in the E2-CD154- vaccinated groups ranged between 86-92%, one week after booster immunization. The NPLA antibody titers also increased greatly. Later on, the protective capacity of purified E2-CD154 glycoprotein was demonstrated in a challenge experiment in pigs using a biphasic immunization schedule with 25 and 50 μg. The immunized animals developed neutralizing antibodies that were protective when the animals were faced to a challenge with 105 LD50 of ‘‘Margarita’’ CSFV highly pathogenic strain. No clinical signs of the disease were detected in the vaccinated pigs. Unvaccinated pigs in the control group exhibited symptoms of CSF at 3–4 days after challenge and were euthanized from 7–9 days when the pigs became moribund. These results indicate that E2-CD154 produced in recombinant HEK 293 and CHOK1cell line is a high quality candidate for the development of a safe and effective CSFV subunit vaccine. In the next steps, pilot and production scale, E2-CD154 expression levels should be increased in 10 to 50 fold, arriving to a very attractive productive platform for an implementation of a commercial subunit vaccine against CSF

Process intensification for high yield production of influenza H1N1 Gag virus-like particles using an inducible HEK-293 stable cell line

Influenza virus dominant antigens presentation using virus like particle (VLP) approach is attractive for the development of new generation of influenza vaccines. Mammalian cell platform offers many advantages for VLP production. However, limited attention has been paid to the processing of mammalian cell produced VLPs. Better understanding of the production system could contribute to increasing the yields and making large-scale VLP vaccine manufacturing feasible. In a previous study, we have generated a human embryonic kidney HEK-293 inducible cell line expressing Hemagglutinin (HA) and Neuraminidase (NA), which was used to produce VLPs upon transient transfection with a plasmid containing HIV-1 Gag. In this work, to streamline the production process, we have developed a new HEK-293 inducible cell line adapted to suspension growth expressing the three proteins HA, NA (H1N1 A/PR/8/1934) and the Gag fused to GFP for monitoring the VLP production. The process was optimized to reach higher volumetric yield of VLPs by increasing the cell density at the time of induction without sacrificing the cell specific productivity. A 5-fold improvement was achieved by doing media evaluation at small scale. Furthermore, a 3-L perfusion bioreactor mirrored the performance of small-scale shake flask cultures with sequential medium replacement. The cell density was increased to 14 7 106 cells/ml at the time of induction which augmented by 60-fold the volumetric yield to 1.54 7 1010 Gag-GFP fluorescent events/ml, as measured by flow cytometry. The 9.5-L harvest from the perfusion bioreactor was concentrated by tangential flow filtration at low shear rate. The electron micrographs revealed the presence of VLPs of 100\u2013150 nm with the characteristic dense core of HIV-1 particles. The developed process shows the feasibility of producing high quantity of influenza VLPs from an inducible mammalian stable cell line aiming at large scale vaccine manufacturing.Peer reviewed: YesNRC publication: Ye