Expression of an Efficient Selection Marker Out of a Duplicated Site in the ITRs of a Modified Vaccinia Virus Ankara (MVA).

: Poxviruses are large DNA viruses that replicate in the host cytoplasm without a nuclear phase. As vaccine vectors, they can package and express large recombinant cassettes from different positions of their genomic core region. We present a comparison between wildtype modified vaccinia Ankara (MVA) and isolate CR19, which has significantly expanded inverted terminal repeats (ITRs).

Computationally designed Spike antigens induce neutralising responses against the breadth of SARS-COV-2 variants.

Updates of SARS-CoV-2 vaccines are required to generate immunity in the population against constantly evolving SARS-CoV-2 variants of concerns (VOCs). Here we describe three novel in-silico designed spike-based antigens capable of inducing neutralising antibodies across a spectrum of SARS-CoV-2 VOCs. Three sets of antigens utilising pre-Delta (T2_32), and post-Gamma sequence data (T2_35 and T2_36) were designed.

Parallel Multifactorial Process Optimization and Intensification for High-Yield Production of Live YF17D-Vectored Zika Vaccine.

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.

Mucoadhesive film for oral delivery of vaccines for protection of the respiratory tract.

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.

Transgene expression knock-down in recombinant Modified Vaccinia virus Ankara vectors improves genetic stability and sustained transgene maintenance across multiple passages.

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.

A rapid, high-throughput, viral infectivity assay using automated brightfield microscopy with machine learning.

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 (AVIA), 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.

Glycan masking of a non-neutralising epitope enhances neutralising antibodies targeting the RBD of SARS-CoV-2 and its variants.

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.

Process intensification strategies toward cell culture-based high-yield production of a fusogenic oncolytic virus.

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.

Cell-line screening and process development for a fusogenic oncolytic virus in small-scale suspension cultures.

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.

Development of an efficient veterinary rabies vaccine production process in the avian suspension cell line AGE1.CR.pIX.

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.

A high cell density perfusion process for Modified Vaccinia virus Ankara production: Process integration with inline DNA digestion and cost analysis.

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.

Production of Modified Vaccinia Ankara Virus by Intensified Cell Cultures: A Comparison of Platform Technologies for Viral Vector Production.

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.

Abrogation of Marek's disease virus replication using CRISPR/Cas9.

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.

Performance of an acoustic settler versus a hollow fiber-based ATF technology for influenza virus production in perfusion.

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.

A Deleted Deletion Site in a New Vector Strain and Exceptional Genomic Stability of Plaque-Purified Modified Vaccinia Ankara (MVA).

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.

Continuous influenza virus production in a tubular bioreactor system provides stable titers and avoids the "von Magnus effect".

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".

Detection of Two Highly Diverse Peribunyaviruses in Mosquitoes from Palenque, Mexico.

The family contains the genera , , , and . 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.

High titer MVA and influenza A virus production using a hybrid fed-batch/perfusion strategy with an ATF system.

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.

mRNA as novel technology for passive immunotherapy.

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.

High-cell-density cultivations to increase MVA virus production.

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.

Efficient and stable production of Modified Vaccinia Ankara virus in two-stage semi-continuous and in continuous stirred tank cultivation systems.

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.

Innate Immune Responses of Bat and Human Cells to Filoviruses: Commonalities and Distinctions.

Marburg (MARV) and Ebola (EBOV) viruses are zoonotic pathogens that cause severe hemorrhagic fever in humans. The natural reservoir of MARV is the Egyptian rousette bat (); that of EBOV is unknown but believed to be another bat species. The Egyptian rousette develops subclinical productive infection with MARV but is refractory to EBOV.

Continuous cell lines from the Muscovy duck as potential replacement for primary cells in the production of avian vaccines.

Veterinary vaccines contribute to food security, interrupt zoonotic transmissions, and help to maintain overall health in livestock. Although vaccines are usually cost-effective, their adoption depends on a multitude of factors. Because poultry vaccines are usually given to birds with a short life span, very low production cost per dose is one important challenge.

Impaired antiviral response of adenovirus-transformed cell lines supports virus replication.

Activation of the innate immune response represents one of the most important cellular mechanisms to limit virus replication and spread in cell culture. Here, we examined the effect of adenoviral gene expression on the antiviral response in adenovirus-transformed cell lines; HEK293, HEK293SF and AGE1.HN.