DNA coding for bacteriophage T7 RNA polymerase was ligated to a vaccinia virus transcriptional promoter and integrated within the vaccinia virus genome. The recombinant vaccinia virus retained infectivity and stably expressed T7 RNA polymerase in mammalian cells. Target genes were constructed by inserting DNA segments that code for beta-galactosidase or chloramphenicol acetyltransferase into a plasmid with bacteriophage T7 promoter and terminator regions. When cells were infected with the recombinant vaccinia virus and transfected with plasmids containing the target genes, the latter were expressed at high levels. Chloramphenicol acetyltransferase activity was 400-600 times greater than that observed with conventional mammalian transient-expression systems regulated either by the enhancer and promoter regions of the Rous sarcoma virus long terminal repeat or by the simian virus 40 early region. The vaccinia/T7 hybrid virus forms the basis of a simple, rapid, widely applicable, and efficient mammalian expression system.

https://www.pnas.org/content/pnas/83/21/8122.full.pdf

Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. The disease was initially described in the 16th century and was the first animal pathogen identified as a virus. Recent FMD outbreaks in developed countries and their significant economic impact have increased the concern of governments worldwide. This review describes the reemergence of FMD in developed countries that had been disease free for many years and the effect that this has had on disease control strategies. The etiologic agent, FMD virus (FMDV), a member of the Picornaviridae family, is examined in detail at the genetic, structural, and biochemical levels and in terms of its antigenic diversity. The virus replication cycle, including virus-receptor interactions as well as unique aspects of virus translation and shutoff of host macromolecular synthesis, is discussed. This information has been the basis for the development of improved protocols to rapidly identify disease outbreaks, to differentiate vaccinated from infected animals, and to begin to identify and test novel vaccine candidates. Furthermore, this knowledge, coupled with the ability to manipulate FMDV genomes at the molecular level, has provided the framework for examination of disease pathogenesis and the development of a more complete understanding of the virus and host factors involved.

Foot-and-Mouth Disease

DNA recombinant technology has radically changed hepatitis B virus (HBV) virology. The genetic organization, transcription and replication of the virus are basically understood, structures of integrated HBV sequences in hepatocellular carcinoma have been characterized, and new vaccines produced by recombinant DNA technique are being developed.

The hepatitis B virus

These revised recommendations regarding vaccinia (smallpox) vaccine update the previous Advisory Committee on Immunization Practices (ACIP) recommendations (MMWR 1991;40; No. RR-14:1-10) and include current information regarding the nonemergency use of vaccinia vaccine among laboratory and health-care workers occupationally exposed to vaccinia virus, recombinant vaccinia viruses, and other Orthopoxviruses that can infect humans. In addition, this report contains ACIP's recommendations for the use of vaccinia vaccine if smallpox (variola) virus were used as an agent of biological terrorism or if a smallpox outbreak were to occur for another unforeseen reason.

Vaccinia (smallpox) vaccine: recommendations of the Advisory Committee on Immunization Practices (ACIP), 2001.

The complete DNA sequence of vaccinia virus

Foreign DNA was inserted into two nonessential regions of the vaccinia virus genome by homologous recombination in cells infected with virus and transfected with plasmids containing the foreign DNA elements flanked by vaccinia virus DNA. Thymidine kinase-negative (TK-) recombinants were selected after inserting foreign DNA into the coding region of the TK gene of wild-type vaccinia virus; TK+ recombinants were selected after inserting the herpesvirus TK gene into TK- mutants of vaccinia virus. For TK+ expression, it was necessary to insert a 275-base-pair DNA fragment containing the initiation site and sequences upstream of an early vaccinia virus transcript next to the coding sequences of the herpesvirus gene. The unique ability of the herpesvirus TK to phosphorylate 125I-labeled deoxycytidine provided independent confirmation of gene expression. These studies demonstrate the use of vaccinia virus as a selectable cloning and expression vector, confirm the map location of the vaccinia virus TK gene, and provide initial information regarding the location of vaccinia virus transcriptional regulatory sequences.

https://www.pnas.org/content/pnas/79/23/7415.full.pdf

Vaccinia virus: a selectable eukaryotic cloning and expression vector

Potential live vaccines against hepatitis B virus have been produced. The coding sequence for hepatitis B virus surface antigen (HBsAg) has been inserted into the vaccinia virus genome under control of vaccinia virus early promoters. Cells infected with these vaccinia virus recombinants synthesize and excrete HBsAg and vaccinated rabbits rapidly produce antibodies to HBsAg.

Infectious vaccinia virus recombinants that express hepatitis B virus surface antigen

In humans, herpes simplex virus causes a primary infection and then often a latent ganglionic infection that persists for life. Because these latent infections can recur periodically, vaccines are needed that can protect against both primary and latent herpes simplex infections. Infectious vaccinia virus recombinants that contain the herpes simplex virus type 1 (HSV-1) glycoprotein D gene under control of defined early or late vaccinia virus promoters were constructed. Tissue culture cells infected with these recombinant viruses synthesized a glycosylated protein that had the same mass (60,000 daltons) as the glycoprotein D produced by HSV-1. Immunization of mice with one of these recombinant viruses by intradermal, subcutaneous, or intraperitoneal routes resulted in the production of antibodies that neutralized HSV-1 and protected the mice against subsequent lethal challenge with HSV-1 or HSV-2. Immunization with the recombinant virus also protected the majority of the mice against the development of a latent HSV-1 infection of the trigeminal ganglia. This is the first demonstration that a genetically engineered vaccine can prevent the development of latency.

https://science.sciencemag.org/content/sci/228/4700/737.full.pdf

Vaccinia virus recombinant expressing herpes simplex virus type 1 glycoprotein D prevents latent herpes in mice

A transient expression system in which chimeric genes are expressed in cells infected with vaccinia virus was developed. Recombinant plasmids containing the promoter regions of vaccinia virus genes ligated to the coding segment of the prokaryotic chloramphenicol acetyltransferase (CAT) gene were constructed. When the plasmids were introduced into vaccinia virus-infected cells by transfection, the chimeric gene was expressed and significant levels of CAT accumulated. CAT activity was not detected when the same recombinant plasmid was introduced into uninfected cells, nor was activity detected when the vaccinia virus promoter was absent from the plasmid or was replaced by simian virus 40 or Rous sarcoma virus promoters. This specificity indicated that expression is dependent on a cis-acting vaccinia virus promoter region within the recombinant plasmid and diffusible trans-acting transcription factors produced during virus infection. The lack of effect of a simian virus 40 enhancer element inserted upstream of the vaccinia virus promoter region also distinguished this system from systems dependent on RNA polymerase II. Although replication of the recombinant plasmid could not be detected in either uninfected or vaccinia virus-infected cells, an inhibitor of DNA synthesis significantly reduced CAT expression. This result, as well as the kinetics of CAT synthesis, suggests that replication of viral DNA templates can enhance transcription of chimeric genes in recombinant plasmids.

https://www.pnas.org/content/pnas/82/1/19.full.pdf

Eukaryotic transient expression system dependent on transcription factors and regulatory DNA sequences of vaccinia virus

Recombinant poxviruses, such as vaccinia, are provided that comprises a segment comprised of (A) a first DNA sequence encoding a polypeptide that is foreign to poxvirus and (B) a poxvirus transcriptional regulatory sequence, wherein (i) said transcriptional regulatory sequence is adjacent to and exerts transcriptional control over said first DNA sequence and (ii) said segment is positioned within a nonessential genomic region of said recombinant poxvirus. Vaccines, carriers, cells, and media comprising recombinant poxviruses, and methods of immunization with recombinant poxviruses also are provided.

Michael Mackett

Papers

Vaccinia virus: a selectable eukaryotic cloning and expression vector

Infectious vaccinia virus recombinants that express hepatitis B virus surface antigen

Vaccinia virus recombinant expressing herpes simplex virus type 1 glycoprotein D prevents latent herpes in mice

Eukaryotic transient expression system dependent on transcription factors and regulatory DNA sequences of vaccinia virus

Recombinant poxviruses having foreign DNA expressed under the control of poxvirus regulatory sequences