Vaccinia virus, no longer required for immunization against smallpox, now serves as a unique vector for expressing genes within the cytoplasm of mammalian cells. As a research tool, recombinant vaccinia viruses are used to synthesize and analyze the structure-function relationships of proteins, determine the targets of humoral and cell-mediated immunity, and investigate the types of immune response needed for protection against specific infectious diseases and cancer. The vaccine potential of recombinant vaccinia virus has been realized in the form of an effective oral wild-life rabies vaccine, although no product for humans has been licensed. A genetically altered vaccinia virus that is unable to replicate in mammalian cells and produces diminished cytopathic effects retains the capacity for high-level gene expression and immunogenicity while promising exceptional safety for laboratory workers and potential vaccine recipients.

https://www.pnas.org/content/pnas/93/21/11341.full.pdf

Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety

This unit describes how to infect cells with vaccinia virus and then transfect them with a plasmid-transfer vector or PCR fragment to generate a recombinant virus. Selection and screening methods used to isolate recombinant viruses and a method for the amplification of recombinant viruses are described. Finally, a method for live immunostaining that has been used primarily for detection of recombinant modified vaccinia virus Ankara (MVA) is presented.

Generation of Recombinant Vaccinia Viruses.

Therapeutic methods and microorganisms therefor are provided. The microorganisms are designed to accumulate in immunoprivileged tissues and cells, such as in tumors and other proliferating tissue and in inflamed tissues, compared to other tissues, cells and organs, so that they exhibit relatively low toxicity to host organisms. The microorganisms also are designed or modified to result in leaky cell membranes of cells in which they accumulate, resulting in production of antibodies reactive against proteins and other cellular products and also permitting exploitation of proferating proliferating tissues, particularly tumors, to produce selected proteins and other products. Vaccines containing the microorganisms are provided. Combinations of the microorganisms and anti-cancer agents and uses thereof for treating cancer also are provided.

Microorganisms for therapy

Poxviruses as expression vectors

The present invention relates to a high efficiency method of expressing immunoglobulin molecules in eukaryotic cells. The invention is further drawn to a method of producing immunoglobulin heavy and light chain libraries, particularly using the trimolecular recombination method, for expression in eukaryotic cells. The invention further provides methods of selecting and screening for antigen-specific immunoglobulin molecules, and antigen-specific fragments thereof. The invention also provides kits for producing, screening and selecting antigen-specific immunoglobulin molecules. Finally, the invention provides immunoglobulin molecules, and antigen-specific fragments thereof, produced by the methods provided herein.

In vitro methods of producing and identifying immunoglobulin molecules in eukaryotic cells

Vaccinia virus is one of the most widely applied expression systems for use in eukaryotes in molecular biology. Expression of heterologous genes in the vaccinia virus system, however, requires integration of the foreign DNA into the vaccinia virus genome by means of homologous recombination or by direct molecular cloning. In both cases, plasmid vector constructs are required that contain the gene of interest and, usually, a marker gene, both of which are controlled by suitable promoter sequences. In order to simplify the construction of recombinants and to eliminate the need for a marker gene we have developed a modified vaccinia virus genome that allows the direct targeted insertion of DNA fragments downstream of a strong vaccinia virus promoter without any further cloning steps. The gene of interest is amplified by PCR using oligonucleotide primers that provide an SfiI site at the 5′ end and an RsrII site at the 3′ end of the PCR product. Following digestion with these restriction enzymes, the PCR product is operationally linked to a synthetic early/late promoter within the viral genomic DNA via the unique SfiI/RsrII sites of the modified vaccinia virus genome. Using this approach, intermediate plasmid constructs and bacterial hosts are not required and time consuming screening steps can be omitted, because 90% of the virus progeny carry the foreign DNA.

A novel vaccinia virus expression system allowing construction of recombinants without the need for selection markers, plasmids and bacterial hosts

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A novel vaccinia virus expression system allowing construction of recombinants without the need for selection markers, plasmids and bacterial hosts