Overexpression of cytochrome C by a recombinant rabies virus attenuates pathogenicity and enhances antiviral immunity.

The pathogenicity of individual rabies virus strains appears to correlate inversely with the extent of apoptotic cell death they induce and with the expression of rabies virus glycoprotein, a major inducer of an antiviral immune response. To determine whether the induction of apoptosis by rabies virus contributes to a decreased pathogenicity by stimulating antiviral immunity, we have analyzed these parameters in tissue cultures and in mice infected with a recombinant rabies virus construct that expresses the proapoptotic protein cytochrome c. The extent of apoptosis was strongly increased in primary neuron cultures infected with the recombinant virus carrying the active cytochrome c gene [SPBN-Cyto c(+)], compared with cells infected with the recombinant virus containing the inactive cytochrome c gene [SPBN-Cyto c(-)]. Mortality in mice infected intranasally with SPBN-Cyto c(+) was substantially lower than in SPBN-Cyto c(-)-infected mice. Furthermore, virus-neutralizing antibody (VNA) titers were significantly higher in mice immunized with SPBN-Cyto c(+) at the same dose. The VNA titers induced by these recombinant viruses paralleled their protective activities against a lethal rabies virus challenge infection, with SPBN-Cyto c(+) revealing an effective dose 20 times lower than that of SPBN-Cyto c(-). The strong increase in immunogenicity, coupled with the marked reduction in pathogenicity, identifies the SPBN-Cyto c(+) construct as a candidate for a live rabies virus vaccine.

High level expression of a human rabies virus-neutralizing monoclonal antibody by a rhabdovirus-based vector.

Humans exposed to rabies virus must be promptly treated by passive immunization with anti-rabies antibody and active immunization with rabies vaccine. Currently, antibody prepared from pooled human serum or from immunized horses is utilized. However, neither of these reagents are readily available, entirely safe, or consistent in their biological activity. An ideal reagent would consist of a panel of human monoclonal antibodies. Such antibodies are now available, their only drawback being the cost of production. Using recombinant technology, we constructed a rabies virus-based vector which expresses high levels (approximately 60 pg/cell) of rabies virus-neutralizing human monoclonal antibody. The vector is a modified vaccine strain of rabies virus in which the rabies virus glycoprotein has been replaced with a chimeric vesicular stomatitis virus glycoprotein, and both heavy and light chain genes encoding a human monoclonal antibody have been inserted. This recombinant virus can infect a variety of mammalian cell lines and is non-cytolytic, allowing the use of cell culture technology routinely employed to produce rabies vaccines.