Custom Vaccinia Virus Vector Production Service
Overview of Vaccinia Virus Vector
The vaccinia virus (VACV or VV) is the largest and most intricate DNA virus. About 200 genes can be encoded by its linear, 190 kb double-stranded DNA genome. The virus replicates and multiplies in the cytoplasm rather than integrating into the genome of the host cell, and it has a huge capacity for introducing foreign genes. For various global gene delivery needs in gene therapy, Vaccinia is an acceptable and occasionally excellent vector for delivering genes into mammalian cells both in vitro and in vivo due to several properties.
- It has a wide host range. It infects virtually all cell types from mice to man.
- Its genome consists of about 190 kb dsDNA, with which >25kb of foreign DNA can be inserted without deletion of any viral sequence.
- It has a rapid life cycle within the cytoplasm of the host cell and can produce high levels of protein in short periods.
- It can be easily produced in high titers for applications.
Creative Biolabs offers highly attenuated, host-restricted, non- or weakly reproducing vaccinia virus strains. The Modified Vaccinia Ankara (MVA) virus was created from the vaccinia strain Ankara with a loss of around 10% of the genome. MVA is frequently employed in clinical research because of the attenuated phenotype's high level of safety.
Production
In contrast to many other viral genomes, the genomic DNA of the MVA does not produce a virus when it is transfected into receptive cells. This is because MVA needs its own transcription and replication machinery to start a productive infection, not the host's. This means that the production of recombinant MVA often involves the concurrent infection and transfection of an infectious parent virus with a shuttle vector plasmid that contains the required antigen cassette. Following selection utilizing markers, successfully recombined viruses are clonally separated. Shuttle vectors are multicopy bacterial plasmids that include both a marker cassette and an antigen cassette flanked by homology arms for recombination into the MVA genome. MVA vectors are typically made in primary CEF cells, though DF-1 and BHK-21 cells can also be utilized for virus amplification for preclinical testing.
After 2-3 days of transfection, simply harvest the cells and medium. MVA can be detected using fluorescent reporters without extra growth material. The shuttle plasmid's flanking DNA sequences are designed to make it possible to delete the fluorescent reporter cassette from the MVA genome. As a result, extra plaque purification rounds are carried out to remove the reporter gene from MVA while looking for nonfluorescent foci of virus-infected cells.
Fig.1 Summary of workflow for the production of recombinant MVA.1
Purification
Preparation of MVA requires the infection of cells, the extraction of the virus from the cell lysate, and crude purification to remove cell debris from the sample. For in vitro research, VVs are purified using the PEG concentration method. To produce ultra-purified vaccinia viruses, viral particles are further purified and concentrated using sucrose density gradient centrifugation.
Quality Control
Titer tests, Sterility tests, Mycoplasma tests, and other quality assurance procedures are used for VVs. Fluorescence transduction tests are used to determine if the VV vectors express fluorescent proteins. For ultra-purified VVs, we provide Endotoxin tests additionally. The goal of Creative Biolabs is to make the VV vectors safer while lowering the possibility of cytopathic consequences in gene therapy research. We offer one-stop services for your requirements about the development of gene delivery vectors. We welcome your contact and are eager to collaborate with you.
Reference
- Pavot, Vincent et al. "Generation and Production of Modified Vaccinia Virus Ankara (MVA) as a Vaccine Vector." Methods in molecular biology (Clifton, N.J.) vol. 1581 (2017): 97-119. doi:10.1007/978-1-4939-6869-5_6
