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The live-attenuated yellow fever 17D strain is a potent vaccine and viral vector. Its manufacture is based on embryonated chicken eggs or adherent Vero cells. Both processes are unsuitable for rapid and scalable supply. Here, we introduce a high-throughput workflow to identify suspension cells that are fit for the high-yield production of live YF17D-based vaccines in an intensified upstream process. The use of an automated parallel ambr15 microbioreactor system for screening and process optimization has led to the identification of two promising cell lines (AGE1.CR.pIX and HEKDyn) and the establishment of optimized production conditions, which have resulted in a >100-fold increase in virus titers compared to the current state of the art using adherent Vero cells. The process can readily be scaled up from the microbioreactor scale (15 mL) to 1 L stirred tank bioreactors. The viruses produced are genetically stable and maintain their favorable safety and immunogenicity profile, as demonstrated by the absence of neurovirulence in suckling BALB/c mice and consistent seroprotection in AG129 mice. In conclusion, the presented workflow allows for the rapid establishment of a robust, scalable, and high-yield process for the production of live-attenuated orthoflavivirus vaccines, which outperforms current standards. The approach described here can serve as a model for the development of scalable processes and the optimization of yields for other virus-based vaccines that face challenges in meeting growing demands.
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The delivery of vaccines plays a pivotal role in influencing the strength and longevity of the immune response and controlling reactogenicity. Mucosal immunization, as compared to parenteral vaccination, could offer greater protection against respiratory infections while being less invasive. While oral vaccination has been presumed less effective and believed to target mainly the gastrointestinal tract, trans-buccal delivery using mucoadhesive films (MAF) may allow targeted delivery to the mucosa. Here we present an effective strategy for mucosal delivery of several vaccine platforms incorporated in MAF, including DNA plasmids, viral vectors, and lipid nanoparticles incorporating mRNA (mRNA/LNP). The mRNA/LNP vaccine formulation targeting SARS-CoV-2 as a proof of concept remained stable within MAF consisting of slowly releasing water-soluble polymers and an impermeable backing layer, facilitating enhanced penetration into the oral mucosa. This formulation elicited antibody and cellular responses comparable to the intramuscular injection, but also induced the production of mucosal IgAs, highlighting its efficacy, particularly for use as a booster vaccine and the potential advantage for protection against respiratory infections. The MAF vaccine preparation demonstrates significant advantages, such as efficient delivery, stability, and simple noninvasive administration with the potential to alleviate vaccine hesitancy.
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Vacinas contra COVID-19 , Nanopartículas , Animais , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/imunologia , Administração Oral , Nanopartículas/administração & dosagem , Mucosa Bucal/imunologia , COVID-19/prevenção & controle , Feminino , Camundongos Endogâmicos BALB C , SARS-CoV-2/imunologia , Camundongos , Sistemas de Liberação de Medicamentos/métodos , Humanos , Lipídeos/química , Lipídeos/administração & dosagem , RNA Mensageiro/administração & dosagem , LipossomosRESUMO
Modified vaccinia virus Ankara is a versatile vaccine vector, well suited for transgene delivery, with an excellent safety profile. However, certain transgenes render recombinant MVA (rMVA) genetically unstable, leading to the accumulation of mutated rMVA with impaired transgene expression. This represents a major challenge for upscaling and manufacturing of rMVA vaccines. To prevent transgene-mediated negative selection, the continuous avian cell line AGE1.CR pIX (CR pIX) was modified to suppress transgene expression during rMVA generation and amplification. This was achieved by constitutively expressing a tetracycline repressor (TetR) together with a rat-derived shRNA in engineered CR pIX PRO suppressor cells targeting an operator element (tetO) and 3' untranslated sequence motif on a chimeric poxviral promoter and the transgene mRNA, respectively. This cell line was instrumental in generating two rMVA (isolate CR19) expressing a Macaca fascicularis papillomavirus type 3 (MfPV3) E1E2E6E7 artificially-fused polyprotein following recombination-mediated integration of the coding sequences into the DelIII (CR19 M-DelIII) or TK locus (CR19 M-TK), respectively. Characterization of rMVA on parental CR pIX or engineered CR pIX PRO suppressor cells revealed enhanced replication kinetics, higher virus titers and a focus morphology equaling wild-type MVA, when transgene expression was suppressed. Serially passaging both rMVA ten times on parental CR pIX cells and tracking E1E2E6E7 expression by flow cytometry revealed a rapid loss of transgene product after only few passages. PCR analysis and next-generation sequencing demonstrated that rMVA accumulated mutations within the E1E2E6E7 open reading frame (CR19 M-TK) or deletions of the whole transgene cassette (CR19 M-DelIII). In contrast, CR pIX PRO suppressor cells preserved robust transgene expression for up to 10 passages, however, rMVAs were more stable when E1E2E6E7 was integrated into the TK as compared to the DelIII locus. In conclusion, sustained knock-down of transgene expression in CR pIX PRO suppressor cells facilitates the generation, propagation and large-scale manufacturing of rMVA with transgenes hampering viral replication.
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Vacinas Sintéticas , Vaccinia virus , Ratos , Animais , Vaccinia virus/genética , Linfócitos T CD8-Positivos , TransgenesRESUMO
The threat of spillovers of coronaviruses associated with the severe acute respiratory syndrome (SARS) from animals to humans necessitates vaccines that offer broader protection from sarbecoviruses. By leveraging a viral-genome-informed computational method for selecting immune-optimized and structurally engineered antigens, here we show that a single antigen based on the receptor binding domain of the spike protein of sarbecoviruses elicits broad humoral responses against SARS-CoV-1, SARS-CoV-2, WIV16 and RaTG13 in mice, rabbits and guinea pigs. When administered as a DNA immunogen or by a vector based on a modified vaccinia virus Ankara, the optimized antigen induced vaccine protection from the Delta variant of SARS-CoV-2 in mice genetically engineered to express angiotensin-converting enzyme 2 and primed by a viral-vector vaccine (AZD1222) against SARS-CoV-2. A vaccine formulation incorporating mRNA coding for the optimized antigen further validated its broad immunogenicity. Vaccines that elicit broad immune responses across subgroups of coronaviruses may counteract the threat of zoonotic spillovers of betacoronaviruses.
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Infectivity assays are essential for the development of viral vaccines, antiviral therapies, and the manufacture of biologicals. Traditionally, these assays take 2-7 days and require several manual processing steps after infection. We describe an automated viral infectivity assay (AVIATM), using convolutional neural networks (CNNs) and high-throughput brightfield microscopy on 96-well plates that can quantify infection phenotypes within hours, before they are manually visible, and without sample preparation. CNN models were trained on HIV, influenza A virus, coronavirus 229E, vaccinia viruses, poliovirus, and adenoviruses, which together span the four major categories of virus (DNA, RNA, enveloped, and non-enveloped). A sigmoidal function, fit between virus dilution curves and CNN predictions, results in sensitivity ranges comparable to or better than conventional plaque or TCID50 assays, and a precision of â¼10%, which is considerably better than conventional infectivity assays. Because this technology is based on sensitizing CNNs to specific phenotypes of infection, it has potential as a rapid, broad-spectrum tool for virus characterization, and potentially identification.
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The accelerated development of the first generation COVID-19 vaccines has saved millions of lives, and potentially more from the long-term sequelae of SARS-CoV-2 infection. The most successful vaccine candidates have used the full-length SARS-CoV-2 spike protein as an immunogen. As expected of RNA viruses, new variants have evolved and quickly replaced the original wild-type SARS-CoV-2, leading to escape from natural infection or vaccine induced immunity provided by the original SARS-CoV-2 spike sequence. Next generation vaccines that confer specific and targeted immunity to broadly neutralising epitopes on the SARS-CoV-2 spike protein against different variants of concern (VOC) offer an advance on current booster shots of previously used vaccines. Here, we present a targeted approach to elicit antibodies that neutralise both the ancestral SARS-CoV-2, and the VOCs, by introducing a specific glycosylation site on a non-neutralising epitope of the RBD. The addition of a specific glycosylation site in the RBD based vaccine candidate focused the immune response towards other broadly neutralising epitopes on the RBD. We further observed enhanced cross-neutralisation and cross-binding using a DNA-MVA CR19 prime-boost regime, thus demonstrating the superiority of the glycan engineered RBD vaccine candidate across two platforms and a promising candidate as a broad variant booster vaccine.
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COVID-19 , SARS-CoV-2 , Humanos , Epitopos , Vacinas contra COVID-19 , Polissacarídeos , Anticorpos NeutralizantesRESUMO
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.
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Vírus Oncolíticos , Animais , Vírus Oncolíticos/genética , Técnicas de Cultura de Células , Reatores Biológicos , Linhagem Celular , Vesiculovirus/genética , Cultura de VírusRESUMO
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.
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Neoplasias , Terapia Viral Oncolítica , Vírus Oncolíticos , Animais , Linhagem Celular , Vírus da Doença de Newcastle/genética , Vírus Oncolíticos/genética , Cultura de Vírus/métodos , Replicação ViralRESUMO
BACKGROUND: Mass vaccination of dogs as important rabies reservoir is proposed to most effectively reduce and eliminate rabies also in humans. However, a minimum coverage of 70% needs to be achieved for control of the disease in zoonotic regions. In numerous developing countries, dog vaccination rate is still dangerously low because of economic constraints and due to a high turnover in dog populations. Improved vaccine production processes may help to alleviate cost and supply limitations. In this work, we studied and optimized the replication and vaccine potency of PV rabies virus strain in the muscovy-duck derived AGE1.CR and AGE1.CR.pIX suspension cell lines. RESULTS: The BHK-21-adapted PV rabies virus strain replicated efficiently in the avian cell lines without requirement for prior passaging. CR.pIX was previously shown to augment heat shock responses and supported slightly higher infectious titers compared to the parental CR cell line. Both cell lines allowed replication of rabies virus also in absence of recombinant IGF, the only complex component of the chemically defined medium that was developed for the two cell lines. After scale-up from optimization experiments in shake flask to production in 7-l bioreactors peak virus titers of 2.4 × 108 FFU/ml were obtained. The potency of inactivated rabies virus harvest according to the NIH test was 3.5 IU/ml. Perfusion with the chemically defined medium during the virus replication phase improved the potency of the vaccine twofold, and increased the number of doses 9.6 fold. CONCLUSION: This study demonstrates that a rabies vaccine for animal vaccination can be produced efficiently in the AGE1.CR.pIX suspension cell line in a scalable process in chemically defined medium.
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Vacina Antirrábica , Raiva , Animais , Reatores Biológicos , Linhagem Celular , Cães , Patos , Raiva/prevenção & controle , Raiva/veterináriaRESUMO
By integrating continuous cell cultures with continuous purification methods, process yields and product quality attributes have been improved over the last 10 years for recombinant protein production. However, for the production of viral vectors such as Modified Vaccinia virus Ankara (MVA), no such studies have been reported although there is an increasing need to meet the requirements for a rising number of clinical trials against infectious or neoplastic diseases. Here, we present for the first time a scalable suspension cell (AGE1.CR.pIX cells) culture-based perfusion process in bioreactors integrating continuous virus harvesting through an acoustic settler with semi-continuous chromatographic purification. This allowed obtaining purified MVA particles with a space-time yield more than 600% higher for the integrated perfusion process (1.05 × 1011 TCID50 /Lbioreactor /day) compared to the integrated batch process. Without further optimization, purification by membrane-based steric exclusion chromatography resulted in an overall product recovery of 50.5%. To decrease the level of host cell DNA before chromatography, a novel inline continuous DNA digestion step was integrated into the process train. A detailed cost analysis comparing integrated production in batch versus production in perfusion mode showed that the cost per dose for MVA was reduced by nearly one-third using this intensified small-scale process.
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Reatores Biológicos/virologia , DNA Viral/metabolismo , Vaccinia virus , Cultura de Vírus , Animais , Técnicas de Cultura Celular por Lotes/instrumentação , Técnicas de Cultura Celular por Lotes/métodos , Contagem de Células , Linhagem Celular , Cromatografia em Gel , Custos e Análise de Custo , Patos , Desenho de Equipamento , Vaccinia virus/isolamento & purificação , Vaccinia virus/metabolismo , Cultura de Vírus/instrumentação , Cultura de Vírus/métodosRESUMO
Modified Vaccinia Ankara (MVA) virus is a promising vector for vaccination against various challenging pathogens or the treatment of some types of cancers, requiring a high amount of virions per dose for vaccination and gene therapy. Upstream process intensification combining perfusion technologies, the avian suspension cell line AGE1.CR.pIX and the virus strain MVA-CR19 is an option to obtain very high MVA yields. Here the authors compare different options for cell retention in perfusion mode using conventional stirred-tank bioreactors. Furthermore, the authors study hollow-fiber bioreactors and an orbital-shaken bioreactor in perfusion mode, both available for single-use. Productivity for the virus strain MVA-CR19 is compared to results from batch and continuous production reported in literature. The results demonstrate that cell retention devices are only required to maximize cell concentration but not for continuous harvesting. Using a stirred-tank bioreactor, a perfusion strategy with working volume expansion after virus infection results in the highest yields. Overall, infectious MVA virus titers of 2.1-16.5 × 109 virions/mL are achieved in these intensified processes. Taken together, the study shows a novel perspective on high-yield MVA virus production in conventional bioreactor systems linked to various cell retention devices and addresses options for process intensification including fully single-use perfusion platforms.
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Vaccinia virus , Cultura de Vírus , Animais , Técnicas de Cultura de Células , Linhagem Celular , Replicação ViralRESUMO
Marek's disease virus (MDV) is a highly cell-associated alphaherpesvirus that causes deadly lymphomas in chickens. While vaccination protects against clinical symptoms, MDV field strains can still circulate in vaccinated flocks and continuously evolve towards greater virulence. MDV vaccines do not provide sterilizing immunity, allowing the virus to overcome vaccine protection, and has increased the need for more potent vaccines or alternative interventions. In this study, we addressed if the CRISPR/Cas9 system can protect cells from MDV replication. We first screened a number of guide RNAs (gRNAs) targeting essential MDV genes for their ability to prevent virus replication. Single gRNAs significantly inhibited virus replication, but could result in the emergence of escape mutants. Strikingly, combining two or more gRNAs completely abrogated virus replication and no escape mutants were observed upon serial passaging. Our study provides the first proof-of-concept, demonstrating that the CRISPR/Cas9 system can be efficiently used to block MDV replication. The presented findings lay the foundation for future research to completely protect chickens from this deadly pathogen.
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Sistemas CRISPR-Cas , Mardivirus/efeitos dos fármacos , RNA Guia de Cinetoplastídeos/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Embrião de Galinha , Galinhas , Patos , Genes Virais , Células HEK293 , Humanos , Mardivirus/genética , Mardivirus/fisiologia , Doença de Marek/prevenção & controle , Vacinas contra Doença de Marek , Mutação , Estudo de Prova de Conceito , RNA Guia de Cinetoplastídeos/genética , Organismos Livres de Patógenos Específicos , Replicação Viral/genéticaRESUMO
Process intensification and integration is crucial regarding an ever increasing pressure on manufacturing costs and capacities in biologics manufacturing. For virus production in perfusion mode, membrane-based alternating tangential flow filtration (ATF) and acoustic settler are the commonly described cell retention technologies. While acoustic settlers allow for continuous influenza virus harvesting, the use of commercially available membranes for ATF systems typically results in the accumulation of virus particles in the bioreactor vessel. Accordingly, with one single harvest at the end of a cultivation, this increases the risk of lowering the product quality. To assess which cell retention device would be most suitable for influenza A virus production, we compared various key performance figures using AGE1.CR.pIX cells at concentrations between 25 and 50 × 106 cells/mL at similar infection conditions using either an ATF system or an acoustic settler. Production yields, process-related impurities, and aggregation of viruses and other large molecules were evaluated. Taking into account the total number of virions from both the bioreactor and the harvest vessel, a 1.5-3.0-fold higher volumetric virus yield was obtained for the acoustic settler. In addition, fewer large-sized aggregates (virus particles and other molecules) were observed in the harvest taken directly from the bioreactor. In contrast, similar levels of process-related impurities (host cell dsDNA, total protein) were obtained in the harvest for both retention systems. Overall, a clear advantage was observed for continuous virus harvesting after the acoustic settler operation mode was optimized. This development may also allow direct integration of subsequent downstream processing steps. KEY POINTS: ⢠High suspension cell density, immortalized avian cell line, influenza vaccine.
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Filtração/métodos , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Perfusão/instrumentação , Cultura de Vírus/métodos , Replicação Viral , Animais , Reatores Biológicos/virologia , Aves , Linhagem Celular Transformada , Cães , Filtração/classificação , Vírus da Influenza A Subtipo H1N1/fisiologia , Células Madin Darby de Rim Canino , Perfusão/métodos , Vírion/isolamento & purificação , Cultura de Vírus/instrumentaçãoRESUMO
Vectored vaccines based on highly attenuated modified vaccinia Ankara (MVA) are reported to be immunogenic, tolerant to pre-existing immunity, and able to accommodate and stably maintain very large transgenes. MVA is usually produced on primary chicken embryo fibroblasts, but production processes based on continuous cell lines emerge as increasingly robust and cost-effective alternatives. An isolate of a hitherto undescribed genotype was recovered by passage of a non-plaque-purified preparation of MVA in a continuous anatine suspension cell line (CR.pIX) in chemically defined medium. The novel isolate (MVA-CR19) replicated to higher infectious titers in the extracellular volume of suspension cultures and induced fewer syncytia in adherent cultures. We now extend previous studies with the investigation of the point mutations in structural genes of MVA-CR19 and describe an additional point mutation in a regulatory gene. We furthermore map and discuss an extensive rearrangement of the left telomer of MVA-CR19 that appears to have occurred by duplication of the right telomer. This event caused deletions and duplications of genes that may modulate immunologic properties of MVA-CR19 as a vaccine vector. Our characterizations also highlight the exceptional genetic stability of plaque-purified MVA: although the phenotype of MVA-CR19 appears to be advantageous for replication, we found that all genetic markers that differentiate wildtype and MVA-CR19 are stably maintained in passages of recombinant viruses based on either wildtype or MVA-CR.
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Vetores Genéticos , Instabilidade Genômica , Deleção de Sequência , Vaccinia virus/genética , Vaccinia virus/isolamento & purificação , Replicação Viral , Animais , Linhagem Celular , Patos , Genótipo , Mutação Puntual , Recombinação Genética , Ensaio de Placa ViralRESUMO
Continuous cell culture-based influenza vaccine production could significantly reduce footprint and manufacturing costs compared to current batch processing. However, yields of influenza virus in continuous mode can be affected by oscillations in virus titers caused by periodic accumulation of defective interfering particles. The generation of such particles has also been observed previously in cascades of continuous stirred tank reactors (CSTRs) and is known as the "von Magnus effect". To improve virus yields and to avoid these oscillations, we have developed a novel continuous tubular bioreactor system for influenza A virus production. It was built using a 500 mL CSTR for cell growth linked to a 105 m long tubular plug-flow bioreactor (PFBR). Virus propagation took place only in the PFBR with a nominal residence time of 20 h and a production capacity of 0.2 mL/min. The bioreactor was first tested with suspension MDCK cells at different multiplicities of infection (MOI), and then with suspension avian AGE1.CR.pIX cells at a fixed nominal MOI of 0.02. Maximum hemagglutinin (HA) titers of 2.4 and 1.6 log10(HA units/100 µL) for suspension MDCK cells and AGE1.CR.pIX cells, respectively, were obtained. Flow cytometric analysis demonstrated that 100% infected cells with batch-like HA titers can be obtained at a MOI of at least 0.1. Stable HA and TCID50 titers over 18 days of production were confirmed using the AGE1.CR.pIX cell line, and PCR analysis demonstrated stable production of full-length genome. The contamination level of segments with deletions (potentially defective interfering particles), already present in the virus seed, was low and did not increase. Control experiments using batch and semi-continuous cultures confirmed these findings. A comparison showed that influenza virus production can be achieved with the tubular bioreactor system in about half the time with a space-time-yield up to two times higher than for typical batch cultures. In summary, a novel continuous tubular bioreactor system for cell culture-based influenza virus production was developed. One main advantage, an essentially single-passage amplification of viruses, should enable efficient production of vaccines as well as vectors for gene and cancer therapy.
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Técnicas de Cultura Celular por Lotes/métodos , Reatores Biológicos , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vacinas contra Influenza/isolamento & purificação , Cultura de Vírus/métodos , Animais , Técnicas de Cultura Celular por Lotes/instrumentação , Aves , Cães , Vírus da Influenza A Subtipo H1N1/imunologia , Células Madin Darby de Rim Canino , Cultura de Vírus/instrumentação , Replicação ViralRESUMO
The Peribunyaviridae family contains the genera Orthobunyavirus, Herbevirus, Pacuvirus, and Shangavirus. Orthobunyaviruses and pacuviruses are mainly transmitted by blood-feeding insects and infect a variety of vertebrates whereas herbeviruses and shangaviruses have a host range restricted to insects. Here, we tested mosquitoes from a tropical rainforest in Mexico for infections with peribunyaviruses. We identified and characterized two previously unknown viruses, designated Baakal virus (BKAV) and Lakamha virus (LAKV). Sequencing and de novo assembly of the entire BKAV and LAKV genomes revealed that BKAV is an orthobunyavirus and LAKV is likely to belong to a new genus. LAKV was almost equidistant to the established peribunyavirus genera and branched as a deep rooting solitary lineage basal to herbeviruses. Virus isolation attempts of LAKV failed. BKAV is most closely related to the bird-associated orthobunyaviruses Koongol virus and Gamboa virus. BKAV was successfully isolated in mosquito cells but did not replicate in common mammalian cells from various species and organs. Also cells derived from chicken were not susceptible. Interestingly, BKAV can infect cells derived from a duck species that is endemic in the region where the BKAV-positive mosquito was collected. These results suggest a narrow host specificity and maintenance in a mosquito-bird transmission cycle.
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Infecções por Bunyaviridae/transmissão , Culicidae/virologia , Mosquitos Vetores/virologia , Orthobunyavirus/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Infecções por Bunyaviridae/virologia , Culicidae/fisiologia , Feminino , Genoma Viral , Humanos , México , Mosquitos Vetores/fisiologia , Orthobunyavirus/classificação , Orthobunyavirus/isolamento & purificação , Filogenia , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
A cultivation strategy to increase the productivity of Modified Vaccinia Ankara (MVA) virus in high-cell density processes is presented. Based on an approach developed in shake flask cultures, this strategy was established in benchtop bioreactors, comprising the growth of suspension AGE1.CR.pIX cells to high cell densities in a chemically defined medium before infection with the MVA-CR19 virus strain. First, a perfusion regime was established to optimize the cell growth phase. Second, a fed-batch regime was chosen for the initial infection phase to facilitate virus uptake and cell-to-cell spreading. Afterwards, a switch to perfusion enabled the continuous supply of nutrients for the late stages of virus propagation. With maximum infectious titers of 1.0 × 1010 IU/mL, this hybrid fed-batch/perfusion strategy increased product titers by almost one order of magnitude compared to conventional batch cultivations. Finally, this strategy was also applied to the production of influenza A/PR/8/34 (H1N1) virus considered for manufacturing of inactivated vaccines. Using the same culture system, a total number of 3.8 × 1010 virions/mL was achieved. Overall, comparable or even higher cell-specific virus yields and volumetric productivities were obtained using the same cultivation systems as for the conventional batch cultivations. In addition, most viral particles were found in the culture supernatant, which can simplify further downstream operations, in particular for MVA viruses. Considering the current availability of well-described perfusion/cell retention technologies, the present strategy may contribute to the development of new approaches for viral vaccine production.
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Técnicas de Cultura Celular por Lotes , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vaccinia virus/crescimento & desenvolvimento , Cultura de Vírus/métodos , Animais , Reatores Biológicos , Linhagem Celular , Patos , Vírus da Influenza A Subtipo H1N1/fisiologia , Vaccinia virus/fisiologia , Vírion/crescimento & desenvolvimento , Vírion/fisiologia , Replicação ViralRESUMO
While active immunization elicits a lasting immune response by the body, passive immunotherapy transiently equips the body with exogenously generated immunological effectors in the form of either target-specific antibodies or lymphocytes functionalized with target-specific receptors. In either case, administration or expression of recombinant proteins plays a fundamental role. mRNA prepared by in vitro transcription (IVT) is increasingly appreciated as a drug substance for delivery of recombinant proteins. With its biological role as transient carrier of genetic information translated into protein in the cytoplasm, therapeutic application of mRNA combines several advantages. For example, compared to transfected DNA, mRNA harbors inherent safety features. It is not associated with the risk of inducing genomic changes and potential adverse effects are only temporary due to its transient nature. Compared to the administration of recombinant proteins produced in bioreactors, mRNA allows supplying proteins that are difficult to manufacture and offers extended pharmacokinetics for short-lived proteins. Based on great progress in understanding and manipulating mRNA properties, efficacy data in various models have now demonstrated that IVT mRNA constitutes a potent and flexible platform technology. Starting with an introduction into passive immunotherapy, this review summarizes the current status of IVT mRNA technology and its application to such immunological interventions.
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Imunização Passiva , RNA Mensageiro/metabolismo , Animais , Anticorpos/genética , Anticorpos/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Imunoterapia Adotiva , Capuzes de RNA/química , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Mensageiro/genéticaRESUMO
Increasing the yield and the productivity in cell culture-based vaccine manufacturing using high-cell-density (HCD) cultivations faces a number of challenges. For example, medium consumption should be low to obtain a very high concentration of viable host cells in an economical way but must be balanced against the requirement that accumulation of toxic metabolites and limitation of nutrients have to be avoided. HCD cultivations should also be optimized to avoid unwanted induction of apoptosis or autophagy during the early phase of virus infection. To realize the full potential of HCD cultivations, a rational analysis of the cultivation conditions of the appropriate host cell line together with the optimal infection conditions for the chosen viral vaccine strain needs to be performed for each particular manufacturing process. We here illustrate our strategy for production of the modified vaccinia Ankara (MVA) virus isolate MVA-CR19 in the avian suspension cell line AGE1.CR.pIX at HCD. As a first step we demonstrate that the adjustment of the perfusion rate strictly based on the measured cell concentration and the glucose consumption rate of cells enables optimal growth in a 0.8â¯L bioreactor equipped with an ATF2 system. Concentrations up to 57â¯×â¯106â¯cells/mL (before infection) were obtained with a viability exceeding 95%, and a maximum specific cell growth rate of 0.019â¯h-1 (doubling timeâ¯=â¯36.5â¯h). However, not only the cell-specific MVA-CR19 virus yield but also the volumetric productivity was reduced compared to infections at conventional-cell-density (CCD). To facilitate optimization of the virus propagation phase at HCD, a larger set of feeding strategies was analyzed in small-scale cultivations using shake flasks. Densities up to 63â¯×â¯106â¯cells/mL were obtained at the end of the cell growth phase applying a discontinuous perfusion mode (semi-perfusion) with the same cell-specific perfusion rate as in the bioreactor (0.060â¯nL/(cellâ¯d)). At this cell concentration, a medium exchange at time of infection was required to obtain expected virus yields during the first 24â¯h after infection. Applying an additional fed-batch feeding strategy during the whole virus replication phase resulted in a faster virus titer increase during the first 36â¯h after infection. In contrast, a semi-continuous virus harvest scheme improved virus accumulation and recovery at a rather later stage of infection. Overall, a combination of both fed-batch and medium exchange strategies resulted in similar cell-specific virus yields as those obtained for CCD processes but 10-fold higher MVA-CR19 titers, and four times higher volumetric productivity.
Assuntos
Técnicas de Cultura de Células/métodos , Vaccinia virus/crescimento & desenvolvimento , Cultura de Vírus/métodos , Animais , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Aves , Contagem de Células , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Glucose/química , Vaccinia virus/fisiologia , Vacinas Virais , Replicação ViralRESUMO
One important aim in cell culture-based viral vaccine and vector production is the implementation of continuous processes. Such a development has the potential to reduce costs of vaccine manufacturing as volumetric productivity is increased and the manufacturing footprint is reduced. In this work, continuous production of Modified Vaccinia Ankara (MVA) virus was investigated. First, a semi-continuous two-stage cultivation system consisting of two shaker flasks in series was established as a small-scale approach. Cultures of the avian AGE1.CR.pIX cell line were expanded in the first shaker, and MVA virus was propagated and harvested in the second shaker over a period of 8-15 days. A total of nine small-scale cultivations were performed to investigate the impact of process parameters on virus yields. Harvest volumes of 0.7-1 L with maximum TCID50 titers of up to 1.0×109 virions/mL were obtained. Genetic analysis of control experiments using a recombinant MVA virus containing green-fluorescent-protein suggested that the virus was stable over at least 16 d of cultivation. In addition, a decrease or fluctuation of infectious units that may indicate an excessive accumulation of defective interfering particles was not observed. The process was automated in a two-stage continuous system comprising two connected 1 L stirred tank bioreactors. Stable MVA virus titers, and a total production volume of 7.1 L with an average TCID50 titer of 9×107 virions/mL was achieved. Because titers were at the lower range of the shake flask cultivations potential for further process optimization at large scale will be discussed. Overall, MVA virus was efficiently produced in continuous and semi-continuous cultivations making two-stage stirred tank bioreactor systems a promising platform for industrial production of MVA-derived recombinant vaccines and viral vectors.