Vaccinia Virus and Poxvirology: Methods and Protocols (Methods in Molecular Biology Vol 269)

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Less well kwn, however, are the many other firsts for vaccinia virus. Additional Product Features Place of Publication. Show More Show Less.

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Best Selling in Nonfiction. Open Borders Inc. Save on Nonfiction Trending price is based on prices over last 90 days. You may also like. Other embodiments of the invention are directed to a vaccinia virus composition produced by the methods described herein, such as those described at at paragraphs [] to [] of U. Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa.

The embodiments in the Example section are understood to be embodiments of the invention that are applicable to all aspects of the invention. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa.

Furthermore, compositions and kits of the invention can be used to achieve methods of the invention. Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

The following drawings form part of the present specification and are intended to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. Cells were infected at a multiplicity of infection of 0. Vaccinia viruses are enveloped viruses that produce four infectious forms that differ in their outer membranes: intracellular mature virion IMV , the intracellular enveloped virion IEV , the cell-associated enveloped virion CEV and the extracellular enveloped virion EEV.

The ability to evade complement inactivation and general ability to evade recognition by host immune system are important features for efficacy in systemic delivery in patients. The plasma membrane of IMV incorporates numerous host cell proteins during its assembly. Since IMV represents the majority of infectious progeny, producer cells from the same species as the target patients for the oncolytic virotherapy may be advantageous for systemic spread within the patient since the viruses will be less immunogenic.


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These host cell proteins on the IMV plasma membranes may also provide certain advantages in the infection process. Although IMV is the majority of the infectious progeny, it has not been definitively established that EEV form of the virus is not contained in the cell culture preparations. The complement activation regulating proteins from the same species as the target patients may play a major role in complement activation inhibition and therefore evading immune clearance by the host immune system.

Introduction

Thus, the inventors developed a system to produce large quantities of a vaccinia virus with a human cell component. Historically, vaccinia virus for use as a vaccine was produced from human scabs, however, subsequently it was grown on the skin of calves, sheep and water buffaloes, in the chorioallantoic membrane of chick embryos or on primary bovine embryo fibroblasts Ellner, For the planet-wide concerted effort in smallpox eradication, stocks were produced by infection through scarification of the skin of calves Fenner, The virus preparation was concentrated and lyophilized, which accounts for its stability Fenner, However, other vaccinia virus variants and strains can also be produced by the methods described herein.

For instance JX is a vaccinia virus recombinant that along with removal of the TK gene has an additional deletion of the vaccinia growth factor VGF gene constructed on a Western Reserve vaccinia backbone. This double deleted vaccinia virus vvDD has been shown to be tumor specific, and have antitumor activity in animal models McCart et al. A second vvDD variant is JX harbors and expresses the human somatostatin receptor SSTR2 in infected cells facilitating molecular imaging after systemic delivery using In-pentetreotide McCart et al.

A second generation vaccinia virus vaccine strain grown in the monkey kidney cell line Vero called ACAM has been developed. In these studies, virus was effectively purified from cellular cultures.

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In addition, cancer vaccines that employ vaccinia virus in combination with tumor specific antigens i. However, in both of these above cases, patients received local rather than systemic administration of viral preparations, and often received a fraction of the total virus dose that are required for oncolytic efficiency in vivo. In comparison with other viruses that have been put forward as oncolytic virus candidates, poxviruses such as vaccinia represent some unique challenges in large scale manufacture and purification.

HepaDNAvirus & POXvirus - NBDE & USMLE microbiology - DNA Enveloped Viruses

As some of the largest viruses in nature, poxviruses are visible by light microscopy, and measure mu in length Moss, in Fields' virology B. Fields, D. Knipe, P. Howley, D. Griffin, Eds. Due to its large size, vaccinia virus cannot be sterilized by filtration and thus any manufacturing process is typically closed.

With its entire life cycle occurring within the host cell cytoplasm, the bulk of infectious particles are not released into the cellular medium, but are retained within the infected cell thus purification requires the lysis of the infected cell and the purification of the viral particles from the cellular debris. In the morphogenesis of poxviruses, they acquire membranes from both de novo membrane synthesis early in morphogenesis, and later acquire additional membranes from the host golgi Moss, in Fields' virology B. The current methods are directed to an adherent cell process developed to produce GMP grade virus for cancer trials.

The adherent cell process can be performed in serum free conditions or in the presence of serum. One embodiment of the invention is directed to procedures for high titer JX production in cell culture. A variety of human cell lines were tested and HeLa, a human cervical cancer cell line, was found to support robust virus replication in adherent cultures.

The present methods are not limited to HeLa cells and other adherent cells may be used in the production process, but currently. HeLa cells provide the best mode cell line for production of vaccinia virus. The typical approach for assessing virus productivity is to perform a standard plaque assay, involving a dilution series followed by a day period waiting for plaques to form and then calculation of titer.


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  4. As an alternative, a quantitative PCR approach Q-PCR has been standardized that allows the determination of the number of viral genomes produced following infection. Amplification primers have been optimized that allow us to quantitate the numbers of copies of the viral E9L gene in infected cells and have correlated these results with our standard plaque assay data. The advantage of this approach is that it can be adapted to a 96 well format and the data is available within hours instead of days. Thus the standard initial screen of media and supplements, time to harvest and the affects of concentration of input virus can be rapidly performed and analyzed in 96 well plates.

    Components found in commercial serum free media were tested for their affect on virus productivity. However as mentioned above, despite excellent cell growth characteristics, the inventors observed very poor virus growth in this medium less than one virus particle per infected cell. However, dextran sulfate is also known to inhibit the replication of a wide variety of enveloped viruses including retroviruses, herpesviruses, rhabdoviruses and aerenaviruses De Clercq, A variety of concentrations of dextran sulfate were used and assessed in the rapid assay described above.

    It was found that dextran sulfate did have a dose dependent negative impact on virus productivity, a finding subsequently confirmed using the classical titer assays. While the removal of dextran sulfate does improve virus productivity it is still below the productivity routinely achieved with adherent HeLa cells in serum containing media.

    Other media components that affect virus productivity include Pluomic F, a di-functional, block, co-polymer surfactant that is generally non-toxic to cells; Tween, a commonly used detergent; and soy hydrolysate. In certain aspects of the invention the medium will lack one or more of dextran sulfate, Pluronic F, Tween and soy protein hydrolysate. Examples of some of these media formulations and serum supplements are outlined in Table 1. Since vaccinia virus prefers cell to cell contact for efficient spread, it is possible that suspension cell cultures may be limited by poor cell to cell transmission of virus.

    In certain aspects clumping of the cells can be promoted or cells can be grown on microcarriers or adherent culture can be sufficient for viral production. The cells are maintained for an additional hours at which time the cells are collected and virus harvested. During this process, the inventors discovered factors not anticipated to have had a dramatic effect on viral output. For example, the pH of media has an effect on JX replication.

    Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

    Viral production was optimized in a well buffered media at a pH of 7. Productivity from adherent cultures is significantly better than all attempts at suspension cultures. These assays will ensure that viral preparations are comparable to other confirmed lots of virus preparation and ensure the process is successful. In certain embodiments vaccinia virus can be isolated and purified. The method for purification of Vaccinia virus can include: a. In certain aspects the matrix can comprise a ligand that binds Vaccinia. The ligand can be attached to the solid-phase matrix by binding or coupling to the matrix.

    Interaction between the ligand and virus forms a reversible complex, thus, the virus is reversibly retained by the matrix. In certain aspects, glucosamine glycan GAG , in particular heparan sulfate or heparin, or a GAG-like substance is used as ligand. The ligand may also comprise or consist of sulfate.

    In a further embodiment, the ligand comprises one or more negatively charged sulfate groups. In certain aspects the ligand is a hydrophobic molecule as, for example, an aromatic phenyl group, a PPG group, a butyl group, or a hexyl group. In one aspect, the method comprises purification of Vaccinia virus with hydrophobic interaction chromatography HIC. In a further embodiment, the method comprises purification of Vaccinia virus with HIC together with affinity chromatography. The level of DNA contamination can be reduced to 0.