Rabies is one of the oldest diseases know to man, but its successful control has remained elusive. Although effective vaccines of tissue culture origin against rabies do exist, such preparations are expensive. Live vaccinia virus (VV) recombinants expressing influenza or hepatitis B antigens have recently been used to immunize against these diseases. We have now used this approach to produce a novel rabies vaccine. We first altered the rabies glycoprotein cDNA by site-directed mutagenesis and removed the poly(dG) tail. We then aligned the modified cDNA with an early VV promoter sequence inserted within a cloned copy of the vaccinia thymidine kinase gene and transfected this plasmid into VV-infected cells. Recombination between the virus and the plasmid resulted in a recombinant virus harbouring the rabies glycoprotein cDNA. Inoculation of rabbits with the live recombinant virus induced high titres of rabies virus-neutralizing antibodies, and scarification with the recombinant VV protected mice against challenge with street rabies virus.

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 104 plaque-forming units of V-RGpro8 virus. fi-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. VRGpro8 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.

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Animal rabies control has been frustrated by the existence of multiple wildlife reservoirs and the lack of efficacious oral vaccines. In this investigation, raccoons fed a vaccinia-rabies glycoprotein recombinant virus in a sponge bait developed rabies virus-neutralizing antibody (0.6-54.0 units) and resisted street rabies virus infection 28 and 205 days after feeding. Additional raccoons immunized by oral infusion with attenuated antigenic variants of rabies virus strains CVS-11 and ERA failed to develop rabies virus-neutralizing antibody. This work demonstrates the feasibility of a recombinant virus vaccine containing the rabies glycoprotein gene for immunization of raccoons, and possibly other wildlife, to obtain longterm protection against rabies.Annually, 25,000-30,000 humans in the U.S. are treated for possible rabies virus infection after exposure to known or suspected rabid animals. Infected wildlife constitute a significant reservoir of rabies virus for humans and domestic animals alike (1); therefore, a major aim toward control of sylvatic rabies has been to develop an efficacious, safe, and economical oral rabies vaccine suitable for effective field distribution. Initially, the red fox (Vulpes vulpes) was the predominant target for oral immunization (2-5), with protection demonstrated both in the laboratory and in European field trials. While these studies provided encouraging results, other species (e.g., the striped skunk Mephitis mephitis and the raccoon Procyon lotor), which are major rabies virus vectors within North America (6), have been refractory to efficient oral immunization against rabies (7). Due to a major rabies epizootic among raccoons in the mid-Atlantic region of the U.S. (8) (vol/vol) fetal calf serum] by oral infusion. Twenty raccoons were given 1.0 ml of undiluted V-RG virus in a 3-cm polyurethane sponge cube coated with paraffin and containing 100-200 mg of tetracycline HCl as a calciphilic biomarker (17); this sponge is under consideration as a vaccine field deployment bait (18). Thirteen control animals received a comparable volume of cell culture medium in a bait (sham immunization). A bait was placed in each cage, and after 48 hr untouched bait was removed. Any animal not eating a bait within this period was given 1.0 ml of either 108 or 106 pfu of V-RG virus by oral infusion, as described above. Blood was collected from all animals at the time of immunization and 16-28 days later. Rabies virus-neutralizing antibody levels were determined in vitro by a modification of the standard rapid fluorescent focus inhibition test (19, 20). Titers were expressed in units/ml using the National Institutes of Health reference serum (lot R-3) as standard.Virus Challenge and Post-Mortem Evaluation. V-RG virustreated and sham-immunized animals were challenged 28 or 205 days after immunization with 0.3 ml of rabies virus MD 5951 in the right dorsolateral aspect of the cervical musculature approximately 3.0 cm from the occipital junction. Animals were observed daily and were euthanat...

Pathogenic parental rabies virus and apathogenic variant virus were shown to differ in their ability to infect neurons in vivo and neuroblastoma cells in vitro. After intracerebral inoculation, the distribution of infected neurons in the brain was similar for both viruses, but the rate of spread throughout the brain, the number of infected neurons, and the degree of cellular necrosis were much lower in the case of apathogenic virus. After adsorption to mouse neuroblastoma cells, apathogenic virus was less rapidly internalized than pathogenic virus, and cell-to-cell spread of apathogenic variant virus was completely prevented by the addition of rabies virus-neutralizing antibody, whereas the spread of pathogenic virus was not affected.