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1.
J Appl Microbiol ; 135(6)2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38849307

RESUMO

AIMS: Hepatitis E virus (HEV) is responsible for ∼20 million human infections worldwide every year. The genotypes HEV-3 and HEV-4 are zoonotic and are responsible for most of the autochthonous HEV cases in high-income countries. There are several cell culture systems that allow for propagation of different HEV genotypes in vitro. One of these systems uses human lung carcinoma cells (A549), and was further optimized for propagation of HEV-3 47832c strain. In this study, we investigated the effect of different media supplements as well as microRNA-122 (miR-122) on improving the replication of HEV-3 47832c in A549 cells. METHODS AND RESULTS: We observed that supplementation of maintenance media with 5% fetal bovine serum was sufficient for efficient replication of HEV-3, and verified the positive effect of media supplementation with Amphotericin B, MgCl2, and dimethyl sulfoxide on replication of HEV-3. We have also demonstrated that adding miR-122 mimics to the culture media does not have any significant effect on the replication of HEV-3 47832c. CONCLUSIONS: Herein, we detected over a 6-fold increase in HEV-3 replication in A549/D3 cells by adding all three supplements: Amphotericin B, MgCl2, and dimethyl sulfoxide to the culture media, while demonstrating that miR-122 might not play a key role in replication of HEV-3 47832c.


Assuntos
Meios de Cultura , Genótipo , Vírus da Hepatite E , Replicação Viral , Vírus da Hepatite E/genética , Humanos , MicroRNAs/genética , Hepatite E/virologia , Células A549 , Cultura de Vírus/métodos
2.
Appl Microbiol Biotechnol ; 108(1): 383, 2024 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-38896301

RESUMO

Herpes simplex virus type 1 (HSV-1) plays an important role in the field of gene therapy and viral vaccines, especially as an oncolytic virus. However, the mass production of HSV-1 viral vectors remains a challenge in the industry. In this study, a microcarrier-mediated serum-reduced medium culture was used to improve the bioprocess of HSV-1 production and increase HSV-1 yields. The composition of the culture media, which included a basal medium, serum concentration, and glutamine additive, was optimized. The process was successfully conducted in a 1 L bioreactor, and virus production was threefold greater than that of conventional processes with a 10% serum medium. The bead-to-bead transfer process was also developed to further increase scalability. In spinner flasks, the detachment rate increased from 49.4 to 80.6% when combined agitation was performed during digestion; the overall recovery proportion increased from 37.9 to 71.1% after the operational steps were optimized. Specifically, microcarrier loss was reduced during aspiration and transfer, and microcarriers and detached cells were separated with filters. Comparable cell growth was achieved with the baseline process using 2D culture as the inoculum by exchanging the subculture medium. To increase virus production after bead-to-bead transfer, critical parameters, including shear stress during digestion, TrypLE and EDTA concentrations in the subculture, and the CCI, were identified from 47 parameters via correlation analysis and principal component analysis. The optimized bead-to-bead transfer process achieved an average of 90.4% overall recovery and comparable virus production compared to that of the baseline process. This study is the first to report the optimization of HSV-1 production in Vero cells cultured on microcarriers in serum-reduced medium after bead-to-bead transfer. KEY POINTS: • An HSV-1 production process was developed that involves culturing in serum-reduced medium, and this process achieved threefold greater virus production than that of traditional processes. • An indirect bead-to-bead transfer process was developed with over 90% recovery yield in bioreactors. • HSV-1 production after bead-to-bead transfer was optimized and was comparable to that achieved with 2D culture as inoculum.


Assuntos
Reatores Biológicos , Meios de Cultura , Herpesvirus Humano 1 , Cultura de Vírus , Herpesvirus Humano 1/crescimento & desenvolvimento , Reatores Biológicos/virologia , Meios de Cultura/química , Chlorocebus aethiops , Cultura de Vírus/métodos , Células Vero , Animais
3.
Methods Mol Biol ; 2808: 153-165, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38743369

RESUMO

Domestic cats are the natural host of feline morbilliviruses (FeMV). Although other species can also be infected (such as dogs and opossums), no laboratory animal infection model is established so far. In vitro models for studying the molecular pathogenesis are therefore needed. For this purpose, propagation and titration of FeMV are key techniques. Unlike other morbilliviruses, such as canine distemper virus (CDV) or measles virus (MV), FeMV is a slow growing virus in cell culture and is difficult to titrate using classical plaque techniques. Here we describe methods for the efficient isolation of FeMV from natural sources (e.g., urine), the propagation of viral stocks, and their titration. In addition, we establish the generation of a three-dimensional infection model mimicking the feline tubular epithelium.


Assuntos
Infecções por Morbillivirus , Morbillivirus , Animais , Gatos , Morbillivirus/patogenicidade , Morbillivirus/genética , Morbillivirus/fisiologia , Infecções por Morbillivirus/veterinária , Infecções por Morbillivirus/virologia , Rim/virologia , Rim/citologia , Doenças do Gato/virologia , Células Cultivadas , Cultura de Vírus/métodos , Modelos Animais de Doenças , Cultura Primária de Células/métodos
4.
Viruses ; 16(5)2024 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-38793654

RESUMO

Based on several clinical observations it was hypothesized that herpesviruses may influence the replication of human bocaviruses, the second known parvoviruses that have been confirmed as human pathogens. While several cell lines support the growth of HSV-1, HBoV-1 was exclusively cultivated on air-liquid interface cultures, the latter being a rather complicated, slow, and low throughput system. One of the cell lines are T84 cells, which are derived from the lung metastasis of a colorectal tumor. In this study, we provide evidence that T84 also supports HBoV replication when cultivated as monolayers, while simultaneously being permissive for HSV-1. The cell culture model thus would enable co-infection studies of both viruses and is worth being optimized for high throughput studies with HBoV-1. Additionally, the study provides evidence for a supporting effect of HSV-1 on the replication and packaging of HBoV-1 progeny DNA into DNase-resistant viral particles.


Assuntos
Coinfecção , Herpesvirus Humano 1 , Bocavirus Humano , Replicação Viral , Herpesvirus Humano 1/fisiologia , Humanos , Coinfecção/virologia , Bocavirus Humano/fisiologia , Bocavirus Humano/genética , Linhagem Celular , Linhagem Celular Tumoral , Técnicas de Cultura de Células/métodos , Herpes Simples/virologia , Infecções por Parvoviridae/virologia , Chlorocebus aethiops , Cultura de Vírus/métodos
5.
Viruses ; 16(5)2024 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-38793649

RESUMO

Influenza vaccines, which are recommended by the World Health Organization (WHO), are the most effective preventive measure against influenza virus infection. Madin-Darby canine kidney (MDCK) cell culture is an emerging technology used to produce influenza vaccines. One challenge when purifying influenza vaccines using this cell culture system is to efficiently remove impurities, especially host cell double-stranded DNA (dsDNA) and host cell proteins (HCPs), for safety assurance. In this study, we optimized ion-exchange chromatography methods to harvest influenza viruses from an MDCK cell culture broth, the first step in influenza vaccine purification. Bind/elute was chosen as the mode of operation for simplicity. The anion-exchange Q chromatography method was able to efficiently remove dsDNA and HCPs, but the recovery rate for influenza viruses was low. However, the cation-exchange SP process was able to simultaneously achieve high dsDNA and HCP removal and high influenza virus recovery. For the SP process to work, the clarified cell culture broth needed to be diluted to reduce its ionic strength, and the optimal dilution rate was determined to be 1:2 with purified water. The SP process yielded a virus recovery rate exceeding 90%, as measured using a hemagglutination units (HAUs) assay, with removal efficiencies over 97% for HCPs and over 99% for dsDNA. Furthermore, the general applicability of the SP chromatography method was demonstrated with seven strains of influenza viruses recommended for seasonal influenza vaccine production, including H1N1, H3N2, B (Victoria), and B (Yamagata) strains, indicating that the SP process could be utilized as a platform process. The SP process developed in this study showed four advantages: (1) simple operation, (2) a high recovery rate for influenza viruses, (3) a high removal rate for major impurities, and (4) general applicability.


Assuntos
Vacinas contra Influenza , Vírion , Animais , Cães , Células Madin Darby de Rim Canino , Vírion/isolamento & purificação , Cromatografia por Troca Iônica/métodos , Cultura de Vírus/métodos , Orthomyxoviridae/isolamento & purificação , Técnicas de Cultura de Células/métodos
6.
Biotechnol J ; 19(5): e2400090, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38719592

RESUMO

The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to 4.64 × 107 TU mL-1 in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38 × 107 TU mL-1 in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of >1 × 107 TU mL-1 day-1 for up to 29 consecutive days in a non-optimized 5 L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34+ cells, reducing the LV quantities required for gene and cell therapy applications.


Assuntos
Reatores Biológicos , Vetores Genéticos , Lentivirus , Lentivirus/genética , Humanos , Vetores Genéticos/genética , Meios de Cultura Livres de Soro , Linhagem Celular , Técnicas de Cultura de Células/métodos , Cultura de Vírus/métodos , Células HEK293 , Transfecção/métodos
7.
J Virol Methods ; 328: 114952, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38754768

RESUMO

Primary cell cultures derived from human embryo lung play a crucial role in virology by aiding virus propagation and vaccine development. These cultures exhibit a notable ability to undergo multiple subcultures, often reaching up to 70 passages. However, finding alternative primary cell cultures with similar longevity and usefulness is challenging. In this study, we introduce a novel primary culture cells derived from equine embryo brain (FEB), which cells exhibited remarkable long-term cultivation potential. The FEB was established and maintained using Sumitomo Nerve-Cell Culture System Comparison studies were conducted with fetal equine kidney cell line (FEK-Tc13) to assess growth rates and subculture longevity. Immunological characterization was performed using neuronal markers to confirm the neural nature of FEB cells. Viral growth assessments were conducted using equine herpesviruses (EHV-1 and EHV-4) to evaluate infectivity and cytopathic effects in FEB cells. PCR analysis and real-time PCR assays were employed to detect viral genomic DNA and transcription activity of EHVs in infected FEB cells. FEB cells demonstrated faster growth rates compared to fetal equine kidney cell line (FEK-Tc13 cells) and exhibited sustained subculture capability exceeding 50 passages. Immunostaining confirmed the glial identity of FEB cells. Both equine herpesviruses 1 and 4 EHV-1 and EHV-4 viruses efficiently replicated in FEB cells, resulting in clear cytopathic effects. PCR analysis detected genomic DNA of EHVs in infected FEB cells, indicating successful viral infection. The establishment of FEB cells with extended subculture capability highlights their potential utility as a model system for studying neural cell biology and viral infections.


Assuntos
Encéfalo , Animais , Cavalos/virologia , Encéfalo/virologia , Encéfalo/embriologia , Encéfalo/citologia , Cultura Primária de Células/métodos , Herpesvirus Equídeo 1/crescimento & desenvolvimento , Herpesvirus Equídeo 1/fisiologia , Linhagem Celular , Neurônios/virologia , Cultura de Vírus/métodos , Técnicas de Cultura de Células/métodos , Técnicas de Cultura de Células/veterinária , Células Cultivadas , Replicação Viral
8.
Diagn Microbiol Infect Dis ; 109(3): 116282, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38663335

RESUMO

The effect of freeze-thaw on SARS-CoV-2 viral viability is not well established. We isolated virus from 31 split clinical samples cultured fresh or after a 7- or 17/18-day freeze. We found that freeze-thaw did not significantly affect viral culture isolation. Therefore, frozen samples may be used to assess SARS-CoV-2 infectiousness.


Assuntos
COVID-19 , Congelamento , SARS-CoV-2 , Humanos , COVID-19/virologia , Manejo de Espécimes/métodos , Viabilidade Microbiana , Cultura de Vírus/métodos , Criopreservação
9.
Biotechnol Bioeng ; 121(7): 2175-2192, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38613199

RESUMO

In the era of Biopharma 4.0, process digitalization fundamentally requires accurate and timely monitoring of critical process parameters (CPPs) and quality attributes. Bioreactor systems are equipped with a variety of sensors to ensure process robustness and product quality. However, during the biphasic production of viral vectors or replication-competent viruses for gene and cell therapies and vaccination, current monitoring techniques relying on a single working sensor can be affected by the physiological state change of the cells due to infection/transduction/transfection step required to initiate production. To address this limitation, a multisensor (MS) monitoring system, which includes dual-wavelength fluorescence spectroscopy, dielectric signals, and a set of CPPs, such as oxygen uptake rate and pH control outputs, was employed to monitor the upstream process of adenovirus production in HEK293 cells in bioreactor. This system successfully identified characteristic responses to infection by comparing variations in these signals, and the correlation between signals and target critical variables was analyzed mechanistically and statistically. The predictive performance of several target CPPs using different multivariate data analysis (MVDA) methods on data from a single sensor/source or fused from multiple sensors were compared. An MS regression model can accurately predict viable cell density with a relative root mean squared error (rRMSE) as low as 8.3% regardless of the changes occurring over the infection phase. This is a significant improvement over the 12% rRMSE achieved with models based on a single source. The MS models also provide the best predictions for glucose, glutamine, lactate, and ammonium. These results demonstrate the potential of using MVDA on MS systems as a real-time monitoring approach for biphasic bioproduction processes. Yet, models based solely on the multiplicity and timing of infection outperformed both single-sensor and MS models, emphasizing the need for a deeper mechanistic understanding in virus production prediction.


Assuntos
Adenoviridae , Reatores Biológicos , Humanos , Células HEK293 , Reatores Biológicos/virologia , Adenoviridae/genética , Análise Multivariada , Cultura de Vírus/métodos
10.
J Virol ; 98(4): e0166323, 2024 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-38470106

RESUMO

Human norovirus (HuNoV) is a major cause of acute gastroenteritis and foodborne diseases, affecting all age groups. Despite its clinical needs, no approved antiviral therapies are available. Since the discovery of HuNoV in 1972, studies on anti-norovirals, mechanism of HuNoV infection, viral inactivation, etc., have been hampered by the lack of a robust laboratory-based cultivation system for HuNoV. A recent breakthrough in the development of HuNoV cultivation systems has opened opportunities for researchers to investigate HuNoV biology in the context of de novo HuNoV infections. A tissue stem cell-derived human intestinal organoid/enteroid (HIO) culture system is one of those that supports HuNoV replication reproducibly and, to our knowledge, is most widely distributed to laboratories worldwide to study HuNoV and develop therapeutic strategies. This review summarizes recently developed HuNoV cultivation systems, including HIO, and their use in antiviral studies.


Assuntos
Norovirus , Humanos , Antivirais/farmacologia , Infecções por Caliciviridae/tratamento farmacológico , Infecções por Caliciviridae/virologia , Gastroenterite/tratamento farmacológico , Gastroenterite/virologia , Intestinos/virologia , Norovirus/efeitos dos fármacos , Norovirus/fisiologia , Animais , Organoides/efeitos dos fármacos , Organoides/virologia , Cultura de Vírus
11.
J Fish Dis ; 47(6): e13934, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38421376

RESUMO

Carp oedema virus (CEV) has distinct molecularly identified genogroups of viral mutations, denoted as I, IIa, and IIb. Failure to propagate CEV in vitro limits studies towards understanding its interactions with host cells. Here, virus isolates belonging to genogroup I collected during natural outbreaks in the Czech Republic were employed for routine CEV cultivation in monolayers of carp-derived primary cells, common carp brain (CCB) cells, and epithelioma papulosum cyprinid (EPC) cells. Induction of cytopathic effects (CPEs) was observed and recorded in affected cells. Cell survival rate was evaluated under serial dilutions of the CEV inoculum. Virus cell entry was quantified and visualized by qPCR and transmission electron microscopy, respectively. Study findings indicate primary gills epithelia likely present the most suitable matrix for CEV growth in vitro. Cells of the head kidney and spleen facilitate virus entry with microscopically confirmed CPEs and the presence of cytoplasmic pleomorphic virus particles. Cells of the trunk kidney and gonads are unlikely to permit virus cell entry and CPEs development. Although CEV cultivation in cell lines was inconclusive, EPC cells were CEV permissible. Monolayers of carp-derived primary cells show promise for CEV cultivation that could enable elaborate study of mechanisms underlying cellular binding and responses.


Assuntos
Carpas , Doenças dos Peixes , Poxviridae , Animais , Carpas/virologia , Poxviridae/fisiologia , Poxviridae/genética , Doenças dos Peixes/virologia , Infecções por Poxviridae/veterinária , Infecções por Poxviridae/virologia , Cultura de Vírus/métodos , Linhagem Celular , República Tcheca , Células Cultivadas , Genótipo
12.
Biotechnol J ; 19(1): e2300244, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37767876

RESUMO

Oncolytic viruses (OVs) have emerged as a novel cancer treatment modality, and four OVs have been approved for cancer immunotherapy. However, high-yield and cost-effective production processes remain to be developed for most OVs. Here suspension-adapted Vero cell culture processes were developed for high titer production of an OV model, herpes simplex virus type 1 (HSV-1). Our study showed the HSV-1 productivity was significantly affected by multiplicity of infection, cell density, and nutritional supplies. Cell culture conditions were first optimized in shake flask experiments and then scaled up to 3 L bioreactors for virus production under batch and perfusion modes. A titer of 2.7 × 108 TCID50 mL-1 was obtained in 3 L batch culture infected at a cell density of 1.4 × 106 cells mL-1 , and was further improved to 1.1 × 109 TCID50 mL-1 in perfusion culture infected at 4.6 × 106 cells mL-1 . These titers are similar to or better than the previously reported best titer of 8.6 × 107 TCID50 mL-1 and 8.1 × 108 TCID50 mL-1 respectively obtained in labor-intensive adherent Vero batch and perfusion cultures. HSV-1 production in batch culture was successfully scaled up to 60 L pilot-scale bioreactor to demonstrate the scalability. The work reported here is the first study demonstrating high titer production of HSV-1 in suspension Vero cell culture under different bioreactor operating modes.


Assuntos
Herpesvirus Humano 1 , Vírus Oncolíticos , Animais , Chlorocebus aethiops , Herpesvirus Humano 1/genética , Células Vero , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Cultura de Vírus
13.
Viruses ; 15(6)2023 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-37376622

RESUMO

A Japanese rabbit hepatitis E virus (HEV) strain, JP-59, has been identified in a feral rabbit. When this virus was transmitted to a Japanese white rabbit, it caused persistent HEV infection. The JP-59 strain shares an <87.5% nucleotide sequence identity with other rabbit HEV strains. Herein, to isolate JP-59 by cell culture, we used a 10% stool suspension recovered from a JP-59-infected Japanese white rabbit and contained 1.1 × 107 copies/mL of the viral RNA and using it to infect a human hepatocarcinoma cell line, PLC/PRF/5. No sign of virus replication was observed. Although long-term virus replication was observed in PLC/PRF/5 cells inoculated with the concentrated and purified JP-59 containing a high titer of viral RNA (5.1 × 108 copies/mL), the viral RNA of JP-59c that was recovered from the cell culture supernatants was <7.1 × 104 copies/mL during the experiment. The JP-59c strain did not infect PLC/PRF/5 cells, but its intravenous inoculation caused persistent infection in rabbits. The nucleotide sequence analyses of the virus genomes demonstrated that a total of 18 nucleotide changes accompanying three amino acid mutations occurred in the strain JP-59c compared to the original strain JP-59. These results indicate that a high viral RNA titer was required for JP-59 to infect PLC/PRF/5 cells, but its replication capability was extremely low. In addition, the ability of rabbit HEVs to multiply in PLC/PRF/5 cells varied depending on the rabbit HEV strains. The investigations of cell lines that are broadly susceptible to rabbit HEV and that allow the efficient propagation of the virus are thus needed.


Assuntos
Vírus da Hepatite E , Cultura de Vírus , Replicação Viral , Animais , Humanos , Coelhos , Hepatite E/veterinária , Vírus da Hepatite E/fisiologia , RNA Viral/genética , RNA Viral/análise , Linhagem Celular Tumoral
14.
Biotechnol Bioeng ; 120(9): 2639-2657, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-36779302

RESUMO

We present a proof-of-concept study for production of a recombinant vesicular stomatitis virus (rVSV)-based fusogenic oncolytic virus (OV), rVSV-Newcastle disease virus (NDV), at high cell densities (HCD). Based on comprehensive experiments in 1 L stirred tank reactors (STRs) in batch mode, first optimization studies at HCD were carried out in semi-perfusion in small-scale cultivations using shake flasks. Further, a perfusion process was established using an acoustic settler for cell retention. Growth, production yields, and process-related impurities were evaluated for three candidate cell lines (AGE1.CR, BHK-21, HEK293SF)infected at densities ranging from 15 to 30 × 106 cells/mL. The acoustic settler allowed continuous harvesting of rVSV-NDV with high cell retention efficiencies (above 97%) and infectious virus titers (up to 2.4 × 109 TCID50 /mL), more than 4-100 times higher than for optimized batch processes. No decrease in cell-specific virus yield (CSVY) was observed at HCD, regardless of the cell substrate. Taking into account the accumulated number of virions both from the harvest and bioreactor, a 15-30 fold increased volumetric virus productivity for AGE1.CR and HEK293SF was obtained compared to batch processes performed at the same scale. In contrast to all previous findings, formation of syncytia was observed at HCD for the suspension cells BHK 21 and HEK293SF. Oncolytic potency was not affected compared to production in batch mode. Overall, our study describes promising options for the establishment of perfusion processes for efficient large-scale manufacturing of fusogenic rVSV-NDV at HCD for all three candidate cell lines.


Assuntos
Vírus Oncolíticos , Animais , Vírus Oncolíticos/genética , Técnicas de Cultura de Células , Reatores Biológicos , Linhagem Celular , Vesiculovirus/genética , Cultura de Vírus
15.
Appl Microbiol Biotechnol ; 106(13-16): 4945-4961, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35767011

RESUMO

Oncolytic viruses (OVs) represent a novel class of immunotherapeutics under development for the treatment of cancers. OVs that express a cognate or transgenic fusion protein is particularly promising as their enhanced intratumoral spread via syncytia formation can be a potent mechanism for tumor lysis and induction of antitumor immune responses. Rapid and efficient fusion of infected cells results in cell death before high titers are reached. Although this is an attractive safety feature, it also presents unique challenges for large-scale clinical-grade manufacture of OVs. Here we evaluate the use of four different suspension cell lines for the production of a novel fusogenic hybrid of vesicular stomatitis virus and Newcastle disease virus (rVSV-NDV). The candidate cell lines were screened for growth, metabolism, and virus productivity. Permissivity was evaluated based on extracellular infectious virus titers and cell-specific virus yields (CSVYs). For additional process optimizations, virus adaptation and multiplicity of infection (MOI) screenings were performed and confirmed in a 1 L bioreactor. BHK-21 and HEK293SF cells infected at concentrations of 2 × 106 cells/mL were identified as promising candidates for rVSV-NDV production, leading to infectious titers of 3.0 × 108 TCID50/mL and 7.5 × 107 TCID50/mL, and CSVYs of 153 and 9, respectively. Compared to the AGE1.CR.pIX reference produced in adherent cultures, oncolytic potency was not affected by production in suspension cultures and possibly even increased in cultures of HEK293SF and AGE1.CR.pIX. Our study describes promising suspension cell-based processes for efficient large-scale manufacturing of rVSV-NDV. KEY POINTS: • Cell contact-dependent oncolytic virus (OV) replicates in suspension cells. • Oncolytic potency is not encompassed during suspension cultivation. • Media composition, cell line, and MOI are critical process parameters for OV production. • The designed process is scalable and shows great promise for manufacturing clinical-grade material.


Assuntos
Neoplasias , Terapia Viral Oncolítica , Vírus Oncolíticos , Animais , Linhagem Celular , Vírus da Doença de Newcastle/genética , Vírus Oncolíticos/genética , Cultura de Vírus/métodos , Replicação Viral
17.
Biotechnol Prog ; 38(5): e3277, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35633106

RESUMO

The Ambr15 system is an automated, high-throughput bioreactor platform which comprises 24 individually controlled, single-use stirred-tank reactors. This system plays a critical role in process development by reducing reagent requirements and facilitating high-throughput screening of process parameters. However, until now, the system was used to simulate processes involving cells in suspension or growing on microcarriers and has never been tested for simulating cells growing on macrocarriers. Moreover, to our knowledge, a complete production process including cell growth and virus production has never been simulated. Here, we demonstrate, for the first time, the amenability of the automated Ambr15 cell culture reactor system to simulate the entire SARS-CoV-2 vaccine production process using macrocarriers. To simulate the production process, accessories were first developed to enable insertion of tens of Fibra-Cel macrocarries into the reactors. Vero cell adsorption to Fibra-Cels was then monitored and its adsorption curve was studied. After incorporating of all optimized factors, Vero cells were adsorbed to and grown on Fibra-Cels for several days. During the process, culture medium was exchanged, and the quantity and viability of the cells were followed, resulting in a typical growth curve. After successfully growing cells for 6 days, they were infected with the rVSV-ΔG-Spike vaccine virus. The present results indicate that the Ambr15 system is not only suitable for simulating a process using macrocarriers, but also to simulate an entire vaccine production process, from cell adsorption, cell growth, infection and vaccine virus production.


Assuntos
COVID-19 , Cultura de Vírus , Animais , Reatores Biológicos , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Técnicas de Cultura de Células/métodos , Chlorocebus aethiops , Humanos , SARS-CoV-2 , Células Vero , Cultura de Vírus/métodos
18.
Microbiol Immunol ; 66(7): 361-370, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35545856

RESUMO

The practical use of cell-based seasonal influenza vaccines is currently being considered in Japan. From the perspective of adventitious virus contamination, we assessed the suitability of NIID-MDCK cells (NIID-MDCK-Cs) as a safe substrate for the isolation of influenza viruses from clinical specimens. We first established a sensitive multiplex real-time PCR system to screen for 27 respiratory viruses and used it on 34 virus samples that were isolated by passaging influenza-positive clinical specimens in NIID-MDCK-Cs. Incidentally, the limit of detection (LOD) of the system was 100 or fewer genome copies per reaction. In addition to influenza viruses, human enterovirus 68 (HEV-D68) genomes were detected in two samples after two or three passages in NIID-MDCK-Cs. To further investigate the susceptibility of NIID-MDCK-Cs to adventitious viruses, eight common respiratory viruses were subjected to passages in NIID-MDCK-Cs. The genome copy numbers of seven viruses other than parainfluenza 3 decreased below the LOD by passage 4. By passaging in NIID-MDCK-Cs, the genome numbers of the input HEV-D68, 1 × 108 copies, declined to 102 at passage 3 and to under the LOD at passage 4, whereas those of the other six viruses were under the LOD by passage 3. These results implied that during the process of isolating influenza viruses with NIID-MDCK-Cs, contaminating viruses other than parainfluenza 3 can be efficiently removed by passages in NIID-MDCK-Cs. NIID-MDCK-Cs could be a safe substrate for isolating influenza viruses that can be used to develop cell-based influenza vaccine candidate viruses.


Assuntos
Vacinas contra Influenza , Influenza Humana , Orthomyxoviridae , Infecções por Paramyxoviridae , Vírus , Animais , Cães , Humanos , Vacinas contra Influenza/genética , Influenza Humana/prevenção & controle , Células Madin Darby de Rim Canino , Desenvolvimento de Vacinas , Cultura de Vírus/métodos
19.
Virol Sin ; 37(4): 547-557, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35504535

RESUMO

Virus identification is a prerequisite not only for the early diagnosis of viral infectious diseases but also for the effective prevention of epidemics. Successful cultivation is the gold standard for identifying a virus, according to the Koch postulates. However, this requires screening for a permissive cell line, which is traditionally time-, reagent- and labor-intensive. Here, a simple and easy-to-operate microfluidic chip, formed by seeding a variety of cell lines and culturing them in parallel, is reported for use in virus cultivation and virus-permissive host-cell screening. The chip was tested by infection with two known viruses, enterovirus 71 (EV71) and influenza virus H1N1. Infection with EV71 and H1N1 caused significant cytopathic effects (CPE) in RD and MDCK cells, respectively, demonstrating that virus cultivation based on this microfluidic cell chip can be used as a substitute for the traditional plate-based culture method and reproduce the typical CPE caused by virus infection. Using this microfluidic cell chip method for virus cultivation could make it possible to identify an emerging virus in a high-throughput, automatic, and unprecedentedly fast way.


Assuntos
Enterovirus , Vírus da Influenza A Subtipo H1N1 , Linhagem Celular , Microfluídica , Cultura de Vírus/métodos
20.
Vaccine ; 40(13): 2036-2043, 2022 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-35216843

RESUMO

The adenovirus vector vaccines induce humoral and cellular immune responses and have been used to develop vaccines for effective prevention of life-threating viruses, such as Ebola and Coronaviruses. High demand of vaccines worldwide requires optimization of the production process. Perfusion process increases cell concentration and volumetric productivity, so that it becomes the commonly used strategy in vaccine production In this study, we optimized and developed a perfusion process for the adenovirus-based zoster vaccine production efficiently. We first tested different perfusion strategies in shake flasks, showing semi-continuous strategies for optimal HEK 293 cell growth. We then evaluated three empirical key process parameters (cell concentration at the time of infection (VCC), multiplicity of infection (MOI), virus production pH) by the design of experiment (DoE) method, from which the robust setpoint (VCC 1.04 × 107 cells/mL, MOI 9, and virus production pH 7.17) was confirmed in both shake flask and 2 L benchtop bioreactor. In the bioreactor, we compared the performances of two perfusion systems, the commercially-available XCell ATF® system and a novel peristaltic pump-driven alternating tangential flow perfusion system (PATFP system) that we developed. During cell cultivation stage, both perfusion systems have comparable performances regarding viable cell concentration and cell viability. At 2 dpi, the PATFP system resulted in an adenovirus titer of 2.1 × 1010 IFU/mL and cell-specific virus yield of 2,062 IFU/cell, reaching 75% and 77% of values for XCell ATF® system. This study demonstrates the perfusion process to be superior strategy for adenovirus-based vaccine production compared to the batch-mode strategy (1,467 IFU/cell). Furthermore, our PATFP system shows potential to be comparable to the XCell ATF® system, and it would become an alternative perfusion strategy for the vaccine production.


Assuntos
Vacinas contra Adenovirus , Vacina contra Herpes Zoster , Adenoviridae/genética , Reatores Biológicos , Células HEK293 , Humanos , Perfusão/métodos , Cultura de Vírus/métodos
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