We constructed a plasmid coexpression vector that directs the insertion of a foreign gene of interest together with the Escherichia coli beta-galactosidase (beta gal) gene into the thymidine kinase (TK) locus of the vaccinia virus genome. Tissue culture cells that had been infected with vaccinia virus were transfected with a plasmid vector containing a foreign gene. TK- recombinants could be selected by a plaque assay on TK- cells in the presence of 5-bromodeoxyuridine and distinguished from spontaneous TK- mutants by the addition of a beta-gal indicator to the agarose overlay. Plaques that expressed beta-gal stained dark blue within several hours at 37 degrees C. Alternatively, TK- selection could be eliminated, and recombinant plaques could be readily identified solely by their blue color. The reverse procedure, in which the starting virus expresses beta-gal (i.e., forms blue plaques) and the desired recombinant has deleted the entire beta-gal gene (i.e., forms white plaques), is another alternative. Each protocol was tested by constructing vaccinia virus recombinants that express hepatitis B virus surface antigen.

Vaccinia virus expression vector: coexpression of beta-galactosidase provides visual screening of recombinant virus plaques.

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

Recent advances in molecular genetics have led to the possibility of using large DNA viruses, such as vaccinia virus, as a biological delivery system for immunizing man against unrelated disease-causing agents. When live vaccinia virus recombinants expressing the hepatitis B virus surface antigen (HBsAg), the influenza A virus haemagglutinin, the herpes simplex virus (HSV) type 1 D glycoprotein, the rabies virus G glycoprotein and the vesicular stomatitis virus G glycoprotein were used for immunization, animals were protected upon challenge with the appropriate pathogenic agent. A major concern with using such vaccines, however, stems from the previously documented vaccinia virus-associated post-immunizing complications. We present here experimental evidence that thymidine kinase-negative (TK-) vaccinia virus recombinants, constructed by inserting a variety of DNA coding sequences into the vaccinia virus tk gene, are less pathogenic for mice than wild-type virus.

Decreased virulence of recombinant vaccinia virus expression vectors is associated with a thymidine kinase-negative phenotype

Vaccinia virus is no longer needed for smallpox immunization, but now serves as a useful vector for expressing genes within the cytoplasm of eukaryotic cells. As a research tool, recombinant vaccinia viruses are used to synthesize biologically active proteins and analyze structure-function relations, determine the targets of humoral- and cell-mediated immunity, and investigate the immune responses needed for protection against specific infectious diseases. When more data on safety and efficacy are available, recombinant vaccinia and related poxviruses may be candidates for live vaccines and for cancer immunotherapy.

https://science.sciencemag.org/content/sci/252/5013/1662.full.pdf

Vaccinia virus: a tool for research and vaccine development

In this report it is demonstrated for the first time that rabies-G envelope of the rabies virus is sufficient to confer retrograde axonal transport to a heterologous virus/vector. After delivery of rabies-G pseudotyped equine infectious anaemia virus (EIAV) based vectors encoding a marker gene to the rat striatum, neurons in regions distal from but projecting to the injection site, such as the dopaminergic neurons of the substantia nigra pars compacta, become transduced. This retrograde transport to appropriate distal neurons was also demonstrated after delivery to substantia nigra, hippocampus and spinal cord and did not occur when vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped vectors were delivered to these sites. In addition, peripheral administration of rabies-G pseudotyped vectors to the rat gastrocnemius muscle leads to gene transfer in motoneurons of lumbar spinal cord. In contrast the same vector pseudotyped with VSV-G transduced muscle cells surrounding the injection site, but did not result in expression in any cells in the spinal cord. Long-term expression was observed after gene transfer in the nervous system and a minimal immune response which, together with the possibility of non-invasive administration, greatly extends the utility of lentiviral vectors for gene therapy of human neurological disease.

/pdf/rabies-virus-glycoprotein-pseudotyping-of-lentiviral-vectors-12pt1f9y46.pdf

Rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral delivery

Inoculation of rabbits and mice with a vaccinia-rabies glycoprotein recombinant (V-RG) virus resulted in rapid induction of high concentrations of rabies virus-neutralizing antibodies and protection from severe intracerebral challenge with several strains of rabies virus. Protection from virus challenge also was achieved against the rabies-related Duvenhage virus but not against the Mokola virus. Effective immunization by V-RG depended on the expression of a rabies glycoprotein that registered proline rather than leucine as the eighth amino acid from its NH2 terminus (V-RGpro8). A minimum dose required for effective immunization of mice was 10(4) plaque-forming units of V-RGpro8 virus. beta-propiolactone-inactivated preparations of V-RGpro8 virus also induced high levels of rabies virus-neutralizing antibody and protected mice against intracerebral challenge with street rabies virus. V-RGpro8 virus was highly effective in priming mice to generate a secondary rabies virus-specific cytotoxic T-lymphocyte response following culture of lymphocytes with either ERA or PM strains of rabies virus.

https://www.pnas.org/content/pnas/81/22/7194.full.pdf

Protection from rabies by a vaccinia virus recombinant containing the rabies virus glycoprotein gene

Rabbit anti-idiotypic antibodies (alpha Id Ab) were prepared against five murine monoclonal antibodies (mAb) specific for the rabies virus glycoprotein. Four of the mAb were directed against three known, type-specific, neutralizing sites on the glycoprotein, and the other mAb was directed against a topographically uncharacterized, nonneutralizing epitope. An absence of significant cross-reactivity among the alpha Id Ab for heterologous mAb suggested that the alpha Id Ab were highly specific for unique variable region determinants. The binding of three of the five alpha Id Ab to their homologous mAb could be inhibited by rabies virus-soluble glycoprotein, suggesting that the alpha Id Ab possessed subpopulations similar or adjacent to the antigen-binding site of the mAb. Two of the five alpha Id Ab injected into mice elicited a specific virus-neutralizing antibody response. Mechanisms to account for the induction of the virus-neutralizing antibody by alpha Id Ab are discussed.

Anti-idiotypic antibodies induce neutralizing antibodies to rabies virus glycoprotein.

A specific, saturable receptor for rabies virus was analyzed on cultured cells of neural or non-neural origin Viral attachment kinetics were enhanced by DEAE-dextran, an effect which in turn enhanced the apparent infectivity of the virus inoculum Under optimized conditions, the attachment of metabolically labeled ERA strain rabies virus obeyed the laws of mass action, whereby the amount of virus bound to cells varied proportionally with the concentration of cells or virus Attachment was sensitive to changes of temperature and pH, did not require divalent cations such as Mg2+ or Ca2+, and occurred despite prior treatment of cells with proteolytic or sialic acid-specific enzymes Saturation of the cell surface with rabies virus could be accomplished with 3 X 10(3) to 15 X 10(3) attached virions per cell Competition for the rabies receptor occurred with rabies nonpathogenic variant virus, RV194 -2, and vesicular stomatitis virus Reovirus type 3, another neurotropic virus, failed to inhibit rabies virus binding, and West Nile virus only slightly inhibited rabies virus binding, suggesting independent cellular receptors were recognized by these viruses Isolated rabies virus glycoprotein failed to compete in an equivalent manner However, solubilization of BHK-21 cells with octylglucoside yielded a chloroform-methanol-soluble extract which blocked rabies virus attachment The binding inhibition activity of this extract was resistant to proteases but could be destroyed by phospholipases and neuraminidase, suggesting a phospholipid or glycolipid component at the receptor site These data provide evidence for a rhabdovirus-common mechanism for cellular attachment to cells in culture

K J Reagan

Papers

Protection from rabies by a vaccinia virus recombinant containing the rabies virus glycoprotein gene

Anti-idiotypic antibodies induce neutralizing antibodies to rabies virus glycoprotein.

Characterization of saturable binding sites for rabies virus.

Protection from rabies by a vaccinia virus recombinant containing the rabies virus glycoprotein gene. 1984.