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

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

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

One of the concerns associated with the use of influenza virus-like particles (VLPs) as vaccine candidate or delivery system is their heterogeneous composition. Enveloped VLPs take up the host cell membrane at the budding site carrying out not only the viral antigenic proteins but also host proteins. In addition, the intrinsic nature of cells to produce membrane derived vesicles or extracellular vesicles (EVs), which have similar size to the VLPs, makes VLP purification process challenging. To further characterize these particles and identify proteins that are unique to each population, comparative proteomic analyses were completed to ultimately provide guidance for rational design of separation protocols. The VLPs were produced in suspension and serum free media by transient transfection of an inducible clone of a Human Embryonic Kidney (HEK-293SF) cells expressing HA and NA (H1N1/A/Puerto Rico/8/34), with a plasmid containing the gag gene of HIV-1 fused to GFP. EVs were produced independently from the non-transformed HEK-293SF cell line as a control for comparative studies. Both preparations were characterized for total nucleic acids and protein concentrations and extensively analyzed by nanoLC-MS/MS for their protein compositions. The proteomic analyses showed that aside from the recombinant VLP proteins, nucleolin was the most abundant host cell protein uniquely identified within VLPs (considering the MASCOT score value) while lactotransferrin and heat shock protein 90 were the most abundant proteins in EVs. Overall, this comparative study identifies potential target proteins as specific markers to guide VLP purification and discusses the biogenesis of enveloped particles released in HEK-293 cell suspension cultures emphasizing on the biological functions of host cell proteins identified

Rational plasmid design and bioprocess optimization to enhance recombinant adeno-associated virus (AAV) productivity in mammalian cells

Viral vectors used for gene and oncolytic therapy belong to the most promising biological products for future therapeutics. Clinical success of recombinant adeno-associated virus (rAAV) based therapies raises considerable demand for viral vectors, which cannot be met by current manufacturing strategies. Addressing existing bottlenecks, we improved a plasmid system termed rep/cap split packaging and designed a minimal plasmid encoding adenoviral helper function. Plasmid modifications led to a 12-fold increase in rAAV vector titers compared to the widely used pDG standard system. Evaluation of different production approaches revealed superiority of processes based on anchorage- and serum-dependent HEK293T cells, exhibiting about 15-fold higher specific and volumetric productivity compared to well-established suspension cells cultivated in serum-free medium. As for most other viral vectors, classical stirred-tank bioreactor production is thus still not capable of providing drug product of sufficient amount. We show that manufacturing strategies employing classical surface-providing culture systems can be successfully transferred to the new fully-controlled, single-use bioreactor system IntegrityTM iCELLisTM. In summary, we demonstrate substantial bioprocess optimizations leading to more efficient and scalable production processes suggesting a promising way for flexible large-scale rAAV manufacturing.Peer reviewed: YesNRC publication: Ye

Hemagglutinin and neuraminidase containing virus-like particles produced in HEK-293 suspension culture: An effective influenza vaccine candidate

Virus-like particles (VLPs) constitute a promising alternative as influenza vaccine. They are non-replicative particles that mimic the morphology of native viruses which make them more immunogenic than classical subunit vaccines. In this study, we propose HEK-293 cells in suspension culture in serum-free medium as an efficient platform to produce large quantities of VLPs. For this purpose, a stable cell line expressing the main influenza viral antigens hemagglutinin (HA) and neuraminidase (NA) (subtype H1N1) under the regulation of a cumate inducible promoter was developed (293HA-NA cells). The production of VLPs was evaluated by transient transfection of plasmids encoding human immunodeficiency virus (HIV) Gag or M1 influenza matrix protein. To facilitate the monitoring of VLPs production, Gag was fused to the green fluorescence protein (GFP). The transient transfection of the gag containing plasmid in 293HA-NA cells increased the release of HA and NA seven times more than its counterpart transfected with the M1 encoding plasmid. Consequently, the production of HA-NA containing VLPs using Gag as scaffold was evaluated in a 3-L controlled stirred tank bioreactor. The VLPs secreted in the culture medium were recovered by ultracentrifugation on a sucrose cushion and ultrafiltered by tangential flow filtration. Transmission electron micrographs of final sample revealed the presence of particles with the average typical size (150\u2013200 nm) and morphology of HIV-1 immature particles. The concentration of the influenza glycoproteins on the Gag-VLPs was estimated by single radial immunodiffusion and hemagglutination assay for HA and by Dot-Blot for HA and NA. More significantly, intranasal immunization of mice with influenza Gag-VLPs induced strong antigen-specific mucosal and systemic antibody responses and provided full protection against a lethal intranasal challenge with the homologous virus strain. These data suggest that, with further optimization and characterization the process could support mass production of safer and better-controlled VLPs-based influenza vaccine candidate.Peer reviewed: YesNRC publication: Ye