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The thermostability of vaccines, particularly enveloped viral vectored vaccines, remains a challenge to their delivery wherever needed. The freeze-drying of viral vectored vaccines is a promising approach but remains challenging due to the water removal process from the outer and inner parts of the virus. In the case of enveloped viruses, freeze-drying induces increased stress on the envelope, which often leads to the inactivation of the virus. In this study, we designed a method to freeze-dry a recombinant vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike glycoprotein. Since the envelope of VSV is composed of 50% lipids and 50% protein, the formulation study focused on both the protein and lipid portions of the vector. Formulations were prepared primarily using sucrose, trehalose, and sorbitol as cryoprotectants; mannitol as a lyoprotectant; and histidine as a buffer. Initially, the infectivity of rVSV-SARS-CoV-2 and the cake stability were investigated at different final moisture content levels. High recovery of the infectious viral titer (~0.5 to 1 log loss) was found at 3-6% moisture content, with no deterioration in the freeze-dried cakes. To further minimize infectious viral titer loss, the composition and concentration of the excipients were studied. An increase from 5 to 10% in both the cryoprotectants and lyoprotectant, together with the addition of 0.5% gelatin, resulted in the improved recovery of the infectious virus titer and stable cake formation. Moreover, the secondary drying temperature of the freeze-drying process showed a significant impact on the infectivity of rVSV-SARS-CoV-2. The infectivity of the vector declined drastically when the temperature was raised above 20 °C. Throughout a long-term stability study, formulations containing 10% sugar (sucrose/trehalose), 10% mannitol, 0.5% gelatin, and 10 mM histidine showed satisfactory stability for six months at 2-8 °C. The development of this freeze-drying process and the optimized formulation minimize the need for a costly cold chain distribution system.
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Vacinas contra COVID-19 , Crioprotetores , Liofilização , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Liofilização/métodos , SARS-CoV-2/imunologia , SARS-CoV-2/química , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/química , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Crioprotetores/química , Crioprotetores/farmacologia , Trealose/química , COVID-19/prevenção & controle , COVID-19/virologia , Animais , Humanos , Manitol/química , Sacarose/química , Células Vero , Chlorocebus aethiops , Sorbitol/química , Estabilidade de Medicamentos , Histidina/química , Vírus da Estomatite Vesicular Indiana/genética , Vacinas Sintéticas/química , Vacinas Sintéticas/imunologiaRESUMO
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.
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Adenoviridae , Reatores Biológicos , Humanos , Células HEK293 , Reatores Biológicos/virologia , Adenoviridae/genética , Análise Multivariada , Cultura de Vírus/métodosRESUMO
BACKGROUND: Adeno-associated viruses (AAVs) are gaining interest in the development of cellular immunotherapy. Compared to other viral vectors, AAVs can reduce the risk of insertional oncogenesis. AAV serotype 6 (AAV6) shows the highest efficiency for transducing T cells. Nevertheless, a multiplicity of infection (MOI) of up to one million viral genomes per cell is required to transduce the target cells effectively. Cell-penetrating peptides (CPPs) are short, positively charged peptides that easily translocate the plasma membranes and can facilitate the cellular uptake of a wide variety of cargoes, including small molecules, nucleic acids, drugs, proteins and viral vectors. METHODS: The present study evaluated five CPPs (Antp, TAT-HA2, LAH4, TAT1 and TAT2) on their effects on enhancing transduction of AAV6 packaging a green fluorescent protein transgene into Jurkat T cell line. RESULTS: Vector incubation with peptides TAT-HA2 and LAH4 at a final concentration of 0.2 mm resulted in an approximately two-fold increase in transduced cells. At the lowest MOI tested (1.25 × 104 ), using LAH4 resulted in a 10-fold increase in transduction efficiency. The peptide LAH4 increased the uptake of AAV6 viral particles in both Jurkat cells and mouse primary T cells. Regardless of the large size of the AAV6-LAH4 complexes, their internalization does not appear to depend on macropinocytosis. CONCLUSIONS: Overall, the present study reports an approach to significantly improve the delivery of transgenes into T cells using AAV6 vectors. Notably, the peptides TAT-HA2 and LAH4 contribute to improving the use of AAV6 as a gene delivery vector for the engineering of T cells.
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Peptídeos Penetradores de Células , Camundongos , Animais , Peptídeos Penetradores de Células/genética , Dependovirus/genética , Transdução Genética , Sorogrupo , Linhagem Celular , Vetores Genéticos/genéticaRESUMO
Major efforts in the intensification of cell culture-based viral vaccine manufacturing focus on the development of high-cell-density (HCD) processes, often operated in perfusion. While perfusion operations allow for higher viable cell densities and volumetric productivities, the high perfusion rates (PR) normally adopted-typically between 2 and 4 vessel volumes per day (VVD)-dramatically increase media consumption, resulting in a higher burden on the cell retention device and raising challenges for the handling and disposal of high volumes of media. In this study, we explore high inoculum fed-batch (HIFB) and low-PR perfusion operations to intensify a cell culture-based process for influenza virus production while minimizing media consumption. To reduce product retention time in the bioreactor, produced viral particles were continuously harvested using a tangential flow depth filtration (TFDF) system as a cell retention device and harvest unit. The feeding strategies developed-a hybrid fed-batch with continuous harvest and a low-PR perfusion-allowed for infections in the range of 8-10 × 106 cells/mL while maintaining cell-specific productivity comparable to the batch control, resulting in a global increase in the process productivity. Overall, our work demonstrates that feeding strategies that minimize media consumption are suitable for large-scale influenza vaccine production.
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CAR-T cell therapy involves genetically engineering T cells to recognize and attack tumour cells by adding a chimeric antigen receptor (CAR) to their surface. In this study, we have used dual transduction with AAV serotype 6 (AAV6) to integrate an anti-CD19 CAR into human T cells at a known genomic location. The first viral vector expresses the Cas9 endonuclease and a guide RNA (gRNA) targeting the T cell receptor alpha constant locus, while the second vector carries the DNA template for homology-mediated CAR insertion. We evaluated three gRNA candidates and determined their efficiency in generating indels. The AAV6 successfully delivered the CRISPR/Cas9 machinery in vitro, and molecular analysis of the dual transduction showed the integration of the CAR transgene into the desired location. In contrast to the random integration methods typically used to generate CAR-T cells, targeted integration into a known genomic locus can potentially lower the risk of insertional mutagenesis and provide more stable levels of CAR expression. Critically, this method also results in the knockout of the endogenous T cell receptor, allowing target cells to be derived from allogeneic donors. This raises the exciting possibility of "off-the-shelf" universal immunotherapies that would greatly simplify the production and administration of CAR-T cells.
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Recent advances and discoveries in the structure and role of mRNA as well as novel lipid-based delivery modalities have enabled the advancement of mRNA therapeutics into the clinical trial space. The manufacturing of these products is relatively simple and eliminates many of the challenges associated with cell culture production of viral delivery systems for gene and cell therapy applications, allowing rapid production of mRNA for personalized treatments, cancer therapies, protein replacement and gene editing. The success of mRNA vaccines during the COVID-19 pandemic highlighted the immense potential of this technology as a vaccination platform, but there are still particular challenges to establish mRNA as a widespread therapeutic tool. Immunostimulatory byproducts can pose a barrier for chronic treatments and different production scales may need to be considered for these applications. Moreover, long-term storage of mRNA products is notoriously difficult. This review provides a detailed overview of the manufacturing steps for mRNA therapeutics, including sequence design, DNA template preparation, mRNA production and formulation, while identifying the challenges remaining in the dose requirements, long-term storage and immunotolerance of the product.
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COVID-19 , Pandemias , Humanos , COVID-19/terapia , Técnicas de Cultura de Células , Terapia Baseada em Transplante de Células e Tecidos , RNA Mensageiro/genéticaRESUMO
The Vero cell line is the most used continuous cell line for viral vaccine manufacturing. Its anchorage-dependent use renders scaling up challenging and operations very labor-intensive which affects cost effectiveness. Thus, efforts to adapt Vero cells to suspension cultures have been invested, but hurdles such as the long doubling time and low cell viability remain to be addressed. In this study, building on the recently published Vero cell line annotated genome, a functional genomics analysis of the Vero cells adapted to suspension is performed to better understand the genetic and phenotypic switches at play during the adaptation of Vero cells from anchorage-dependent to suspension cultures. Results show downregulation of the epithelial-to-mesenchymal transition (EMT) pathway, highlighting the dissociation between the adaptation to suspension process and EMT. Surprisingly, an upregulation of cell adhesion components is observed, notably the CDH18 gene, the cytoskeleton pathway, and the extracellular pathway. Moreover, a downregulation of the glycolytic pathway is balanced by an upregulation of the asparagine metabolism pathway, promoting cell adaptation to nutrient deprivation. A downregulation of the adherens junctions and the folate pathways alongside with the FYN gene are possible explanations behind the currently observed low-cell viability and long doubling time.
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In recent years, the use of adeno-associated viruses (AAVs) as vectors for gene and cell therapy has increased, leading to a rise in the amount of AAV vectors required during pre-clinical and clinical trials. AAV serotype 6 (AAV6) has been found to be efficient in transducing different cell types and has been successfully used in gene and cell therapy protocols. However, the number of vectors required to effectively deliver the transgene to one single cell has been estimated at 106 viral genomes (VG), making large-scale production of AAV6 necessary. Suspension cell-based platforms are currently limited to low cell density productions due to the widely reported cell density effect (CDE), which results in diminished production at high cell densities and decreased cell-specific productivity. This limitation hinders the potential of the suspension cell-based production process to increase yields. In this study, we investigated the improvement of the production of AAV6 at higher cell densities by transiently transfecting HEK293SF cells. The results showed that when the plasmid DNA was provided on a cell basis, the production could be carried out at medium cell density (MCD, 4 × 106 cells mL-1 ) resulting in titers above 1010 VG mL-1 . No detrimental effects on cell-specific virus yield or cell-specific functional titer were observed at MCD production. Furthermore, while medium supplementation alleviated the CDE in terms of VG/cell at high cell density (HCD, 10 × 106 cells mL-1 ) productions, the cell-specific functional titer was not maintained, and further studies are necessary to understand the observed limitations for AAV production in HCD processes. The MCD production method reported here lays the foundation for large-scale process operations, potentially solving the current vector shortage in AAV manufacturing.
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Dependovirus , Vetores Genéticos , Humanos , Vetores Genéticos/genética , Células HEK293 , Sorogrupo , Transfecção , Dependovirus/genética , Contagem de CélulasRESUMO
The human cell line HEK293 is one of the preferred choices for manufacturing therapeutic proteins and viral vectors for human applications. Despite its increased use, it is still considered in disadvantage in production aspects compared to cell lines such as the CHO cell line. We provide here a simple workflow for the rapid generation of stably transfected HEK293 cells expressing an engineered variant of the SARS-CoV-2 Receptor Binding Domain (RBD) carrying a coupling domain for linkage to VLPs through a bacterial transpeptidase-sortase (SrtA). To generate stable suspension cells expressing the RBD-SrtA, a single two plasmids transfection was performed, with hygromycin selection. The suspension HEK293 were grown in adherent conditions, with 20% FBS supplementation. These transfection conditions increased cell survival, allowing the selection of stable cell pools, which was otherwise not possible with standard procedures in suspension. Six pools were isolated, expanded and successfully re-adapted to suspension with a gradual increase of serum-free media and agitation. The complete process lasted four weeks. Stable expression with viability over 98% was verified for over two months in culture, with cell passages every 4-5 days. With process intensification, RBD-SrtA yields reached 6.4 µg/mL and 13.4 µg/mL in fed-batch and perfusion-like cultures, respectively. RBD-SrtA was further produced in fed-batch stirred tank 1L-bioreactors, reaching 10-fold higher yields than perfusion flasks. The trimeric antigen displayed the conformational structure and functionality expected. This work provides a series of steps for stable cell pool development using suspension HEK293 cells aimed at the scalable production of recombinant proteins.
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COVID-19 , Humanos , Células HEK293 , SARS-CoV-2 , Reatores Biológicos , Proteínas Recombinantes/genéticaRESUMO
The administration of viral vectored vaccines remains one of the most effective ways to respond to the ongoing novel coronavirus disease 2019 (COVID-19) pandemic. However, pre-existing immunity to the viral vector hinders its potency, resulting in a limited choice of viral vectors. Moreover, the basic batch mode of manufacturing vectored vaccines does not allow one to cost-effectively meet the global demand for billions of doses per year. To date, the exposure of humans to VSV infection has been limited. Therefore, a recombinant vesicular stomatitis virus (rVSV), which expresses the spike protein of SARS-CoV-2, was selected as the vector. To determine the operating upstream process conditions for the most effective production of an rVSV-SARS-CoV-2 candidate vaccine, a set of critical process parameters was evaluated in an Ambr 250 modular system, whereas in the downstream process, a streamlined process that included DNase treatment, clarification, and a membrane-based anion exchange chromatography was developed. The design of the experiment was performed with the aim to obtain the optimal conditions for the chromatography step. Additionally, a continuous mode manufacturing process integrating upstream and downstream steps was evaluated. rVSV-SARS-CoV-2 was continuously harvested from the perfusion bioreactor and purified by membrane chromatography in three columns that were operated sequentially under a counter-current mode. Compared with the batch mode, the continuous mode of operation had a 2.55-fold increase in space-time yield and a reduction in the processing time by half. The integrated continuous manufacturing process provides a reference for the efficient production of other viral vectored vaccines.
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Newcastle disease (ND) remains a critical disease affecting poultry in sub-Saharan Africa. In some countries, repeated outbreaks have a major impact on local economies and food security. Recently, we developed an adenovirus-vectored vaccine encoding the Fusion protein from an Ethiopian isolate of Newcastle disease virus (NDV). The adenoviral vector was designed, and a manufacturing process was developed in the context of the Livestock Vaccine Innovation Fund initiative funded by the International Development Research Centre (IDRC) of Canada. The industrially relevant recombinant vaccine technology platform is being transferred to the National Veterinary Institute (Ethiopia) for veterinary applications. Here, a manufacturing process using HEK293SF suspension cells cultured in stirred-tank bioreactors for the vaccine production is proposed. Taking into consideration supply chain limitations, options for serum-free media selection were evaluated. A streamlined downstream process including a filtration, an ultrafiltration, and a concentration step was developed. With high volumetric yields (infectious titers up to 5 × 109 TCID50/mL) in the culture supernatant, the final formulations were prepared at 1010 TCID50/mL, either in liquid or lyophilized forms. The liquid formulation was suitable and safe for mucosal vaccination and was stable for 1 week at 37 °C. Both the liquid and lyophilized formulations were stable after 6 months of storage at 4 °C. We demonstrate that the instillation of the adenoviral vector through the nasal cavity can confer protection to chickens against a lethal challenge with NDV. Overall, a manufacturing process for the adenovirus-vectored vaccine was developed, and protective doses were determined using a convenient route of delivery. Formulation and storage conditions were established, and quality control protocols were implemented.
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The field of lentiviral vector (LV) production continues to face challenges in large-scale manufacturing, specifically regarding producing enough vectors to meet the demand for treating patients as well as producing high and consistent quality of vectors for efficient dosing. Two areas of interest are the use of stable producer cell lines, which facilitates the scalability of LV production processes as well as making the process more reproducible and robust for clinical applications, and the search of a cell retention device scalable to industrial-size bioreactors. This manuscript investigates a stable producer cell line for producing LVs with GFP as the transgene at shake flask scale and demonstrates LV production at 3L bioreactor scale using the Tangential Flow Depth Filtration (TFDF) as a cell retention device in perfusion mode. Cumulative functional yields of 3.3 x 1011 and 3.9 x 1011 transducing units were achieved; the former over 6 days of LV production with 16.3 L of perfused media and the latter over 4 days with 16 L. In comparing to a previously published value that was achieved using the same stable producer cell line and the acoustic filter as the perfusion device at the same bioreactor scale, the TFDF perfusion run produced 1.5-fold higher cumulative functional yield. Given its scale-up potential, the TFDF is an excellent candidate to be further evaluated to determine optimized conditions that can ultimately support continuous manufacturing of LVs at large scale.
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Despite their wide use in the vaccine manufacturing field for over 40 years, one of the main limitations to recent efforts to develop Vero cells as high-throughput vaccine manufacturing platforms is the lack of understanding of virus-host interactions during infection and cell-based virus production in Vero cells. To overcome this limitation, this manuscript uses the recently generated reference genome for the Vero cell line to identify the factors at play during influenza A virus (IAV) and recombinant vesicular stomatitis virus (rVSV) infection and replication in Vero host cells. The best antiviral gene candidate for gene editing was selected using Differential Gene Expression analysis, Gene Set Enrichment Analysis and Network Topology-based Analysis. After selection of the ISG15 gene for targeted CRISPR genomic deletion, the ISG15 genomic sequence was isolated for CRISPR guide RNAs design and the guide RNAs with the highest knockout efficiency score were selected. The CRISPR experiment was then validated by confirmation of genomic deletion via PCR and further assessed via quantification of ISG15 protein levels by western blot. The gene deletion effect was assessed thereafter via quantification of virus production yield in the edited Vero cell line. A 70-fold and an 87-fold increase of total viral particles productions in ISG15-/- Vero cells was achieved for, respectively, IAV and rVSV while the ratio of infectious viral particles/total viral particles also significantly increased from 0.0316 to 0.653 for IAV and from 0.0542 to 0.679 for rVSV-GFP.
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Antivirais , Replicação Viral , Animais , Chlorocebus aethiops , Genômica , RNA Guia de Cinetoplastídeos , Células Vero , Replicação Viral/genéticaRESUMO
The COVID-19 pandemic has highlighted the need for efficient vaccine platforms that can rapidly be developed and manufactured on a large scale to immunize the population against emerging viruses. Viral-vectored vaccines are prominent vaccine platforms that have been approved for use against the Ebola virus and SARS-CoV-2. The Newcastle Disease Virus is a promising viral vector, as an avian paramyxovirus that infects poultry but is safe for use in humans and other animals. NDV has been extensively studied not only as an oncolytic virus but also a vector for human and veterinary vaccines, with currently ongoing clinical trials for use against SARS-CoV-2. However, there is a gap in NDV research when it comes to process development and scalable manufacturing, which are critical for future approved vaccines. In this review, we summarize the advantages of NDV as a viral vector, describe the steps and limitations to generating recombinant NDV constructs, review the advances in human and veterinary vaccine candidates in pre-clinical and clinical tests, and elaborate on production in embryonated chicken eggs and cell culture. Mainly, we discuss the existing data on NDV propagation from a process development perspective and provide prospects for the next steps necessary to potentially achieve large-scale NDV-vectored vaccine manufacturing.
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COVID-19 , Vacinas Virais , Animais , Humanos , Vírus da Doença de Newcastle/genética , Pandemias , SARS-CoV-2 , Vacinas Virais/genéticaRESUMO
Plasmid transfection of mammalian cells is the dominant platform used to produce adeno-associated virus (AAV) vectors for clinical and research applications. Low yields from this platform currently make it difficult to supply these activities with adequate material. In an effort to better understand the current limitations of transfection-based manufacturing, this study examines what proportion of cells in a model transfection produce appreciable amounts of assembled AAV capsid. Using conformation-specific antibody staining and flow cytometry, we report the surprising result that despite obtaining high transfection efficiencies and nominal vector yields in our model system, only 5%-10% of cells appear to produce measurable levels of assembled AAV capsids. This finding implies that considerable increases in vector titer could be realized through increasing the proportion of productive cells. Furthermore, we suggest that the flow cytometry assay used here to quantify productive cells may be a useful metric for future optimization of transfection-based AAV vector manufacturing platforms.
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Capsídeo , Dependovirus , Animais , Proteínas do Capsídeo/genética , Dependovirus/genética , Vetores Genéticos/genética , Mamíferos , TransfecçãoRESUMO
OBJECTIVE: In a multicenter, international cohort, we aimed to validate a modified Sequential Organ Failure Assessment (mSOFA) using the Richmond Agitation-Sedation Scale, hypothesized as comparable to the Glasgow Coma Scale (GCS)-based Sequential Organ Failure Assessment (SOFA). SUMMARY BACKGROUND DATA: The SOFA score, whose neurologic component is based on the GCS, can predict intensive care unit (ICU) mortality. But, GCS is often missing in lieu of other assessments, such as the also reliable and validated Richmond Agitation Sedation Scale (RASS). Single-center data suggested an RASS-based SOFA (mSOFA) predicted ICU mortality. METHODS: Our nested cohort within the prospective 2016 Fourth International Study of Mechanical Ventilation contains 4120 ventilated patients with daily RASS and GCS assessments (20,023 patient-days, 32 countries). We estimated GCS from RASS via a proportional odds model without adjustment. ICU mortality logistic regression models and c-statistics were constructed using SOFA (measured GCS) and mSOFA (measured RASS-estimated GCS), adjusted for age, sex, body-mass index, region (Europe, USA-Canada, Latin America, Africa, Asia, Australia-New Zealand), and postoperative status (medical/surgical). RESULTS: Cohort-wide, the mean SOFA=9.4+/-2.8 and mean mSOFA = 10.0+/-2.3, with ICU mortality = 31%. Mean SOFA and mSOFA similarly predicted ICU mortality (SOFA: AUC = 0.784, 95% CI = 0.769-0.799; mSOFA: AUC = 0.778, 95% CI = 0.763-0.793, P = 0.139). Across models, other predictors of mortality included higher age, female sex, medical patient, and African region (all P < 0.001). CONCLUSIONS: We present the first SOFA modification with RASS in a "real-world" international cohort. Estimating GCS from RASS preserves predictive validity of SOFA to predict ICU mortality. Alternative neurologic measurements like RASS can be viably integrated into severity of illness scoring systems like SOFA.
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Unidades de Terapia Intensiva , Escores de Disfunção Orgânica , Estudos de Coortes , Feminino , Escala de Coma de Glasgow , Humanos , Masculino , Estudos ProspectivosRESUMO
The ongoing COVID-19 pandemic drew global attention to infectious diseases, attracting numerous resources for development of pandemic preparedness plans and vaccine platforms-technologies with robust manufacturing processes that can quickly be pivoted to target emerging diseases. Newcastle Disease Virus (NDV) has been studied as a viral vector for human and veterinary vaccines, but its production relies heavily on embryonated chicken eggs, with very few studies producing NDV in cell culture. Here, NDV is produced in suspension Vero cells, and analytical assays (TCID50 and ddPCR) are developed to quantify infectious and total viral titer. NDV-GFP and NDV-FLS (SARS-CoV-2 full-length spike protein) constructs were adapted to replicate in Vero and HEK293 suspension cultures using serum-free media, while fine-tuning parameters such as MOI, temperature, and trypsin concentration. Shake flask productions with Vero cells resulted in infectious titers of 1.07 × 108 TCID50/mL for NDV-GFP and 1.33 × 108 TCID50/mL for NDV-FLS. Production in 1 L batch bioreactors also resulted in high titers in culture supernatants, reaching 2.37 × 108 TCID50/mL for NDV-GFP and 3.16 × 107 TCID50/mL for NDV-FLS. This shows effective NDV production in cell culture, building the basis for a scalable vectored-vaccine manufacturing process that can be applied to different targets.
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OBJECTIVES: To characterize the epidemiologic profiles of prediabetes mellitus (preDM), diabetes mellitus (DM), and hypertension (HTN) in Qataris using the nationally representative 2012 Qatar STEPwise Survey. METHODS: A secondary data analysis of a cross-sectional survey that included 2,497 Qatari nationals aged 18-64 years. Descriptive and analytical statistical analyses were conducted. RESULTS: Prevalence of preDM, DM, and HTN in Qataris aged 18-64 years was 11.9% (95% confidence interval [CI] 9.6%-14.7%), 10.4% (95% CI 8.4%-12.9%), and 32.9% (95% CI 30.4%-35.6%), respectively. Age was the common factor associated with the three conditions. Adjusted analyses showed that unhealthy diet (adjusted odds ratio (aOR) = 1.84, 95% CI 1.01-3.36) was significantly associated with preDM; that physical inactivity (aOR = 1.66, 95% CI 1.12-2.46), central obesity (aOR = 2.08, 95% CI 1.02-4.26), and HTN (aOR = 2.18, 95% CI 1.40-3.38) were significantly associated with DM; and that DM (aOR = 2.07, 95% CI 1.34-3.22) was significantly associated with HTN. Population attributable fraction of preDM associated with unhealthy diet was 7.7%; of DM associated with physical inactivity, central obesity, and HTN, respectively, was 14.9%, 39.8%, and 17.5%; and of HTN associated with DM was 3.0%. CONCLUSIONS: One in five Qataris is living with either preDM or DM, and one in three is living with HTN, conditions that were found to be primarily driven by lifestyle factors. Prevention, control, and management of these conditions should be a national priority to reduce their disease burden and associated disease sequelae.
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Hipertensão/epidemiologia , Estado Pré-Diabético/epidemiologia , Adolescente , Adulto , Peso Corporal , Dieta Saudável/estatística & dados numéricos , Exercício Físico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , CatarRESUMO
Viral vectors and viral vaccines are invaluable tools in prevention and treatment of diseases. Many infectious diseases are controlled using vaccines designed from subunits or whole viral structures, whereas other genetic diseases and cancers are being treated by viruses used as vehicles for delivering genetic material in gene therapy or as therapeutic agents in virotherapy protocols. Viral vectors and vaccines are produced in different platforms, from traditional embryonated chicken eggs to more advanced cell cultures. All these expression systems, like most cells and cellular tissues, are known to spontaneously release extracellular vesicles (EVs). EVs share similar sizes, biophysical characteristics and even biogenesis pathways with enveloped viruses, which are currently used as key ingredients in a number of viral vectors and licensed vaccine products. Herein, we review distinctive features and similarities between EVs and enveloped viruses as we revisit the downstream processing steps and analytical technologies currently implemented to produce and document viral vector and vaccine products. Within a context of well-established viral vector and vaccine safety profiles, this review provides insights on the likely presence of EVs in the final formulation of enveloped virus products and discusses the potential to further resolve and document these components.
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The Vero cell line is the most used continuous cell line for viral vaccine manufacturing with more than 40 years of accumulated experience in the vaccine industry. Additionally, the Vero cell line has shown a high affinity for infection by MERS-CoV, SARS-CoV, and recently SARS-CoV-2, emerging as an important discovery and screening tool to support the global research and development efforts in this COVID-19 pandemic. However, the lack of a reference genome for the Vero cell line has limited our understanding of host-virus interactions underlying such affinity of the Vero cell towards key emerging pathogens, and more importantly our ability to redesign high-yield vaccine production processes using Vero genome editing. In this paper, we present an annotated highly contiguous 2.9 Gb assembly of the Vero cell genome. In addition, several viral genome insertions, including Adeno-associated virus serotypes 3, 4, 7, and 8, have been identified, giving valuable insights into quality control considerations for cell-based vaccine production systems. Variant calling revealed that, in addition to interferon, chemokines, and caspases-related genes lost their functions. Surprisingly, the ACE2 gene, which was previously identified as the host cell entry receptor for SARS-CoV and SARS-CoV-2, also lost function in the Vero genome due to structural variations.