A recombinant rabies virus expressing vesicular
To investigate the importance of rabies virus (RV) glycoprotein (G) in protection against rabies, we constructed a recombinant RV (rRV) in which the ecto- and transmembrane domains of RV G were replaced by the corresponding regions of the vesicular stomatitis virus (VSV) glycoprotein (rRV-VSV-G). We were able to recover rRV-VSV-G and found that the particle production was equal to rRV.
However, budding of the chimeric virus was delayed and infectious titers were reduced 10-fold compared to the parental strain of rRV containing RV G. Biochemical analysis showed equal replication rates of both viruses, and amounts Similar wild type and chimeric G were present.
in the respective viral particles. Additional studies were performed to determine if the immune response against rRV-VSV-G was sufficient to protect against rabies. Mice were primed with rRV or rRV-VSV-G and challenged with a pathogenic strain of RV 12 days later. Similar immune responses against internal viral proteins of both viruses indicated successful infection. All mice receiving the rRV vaccine survived the challenge, while immunization with rRV-VSV-G did not induce protection. The results confirm the crucial role of RV G in an RV vaccine.
Rabies virus (RV), a negative-strand RNA virus, belongs to the genus Lyssavirus within the family Rhabdoviridae. Five structural proteins, including nucleoprotein (N), phosphoprotein, matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase (L), are encoded by the 12 kb viral genome.
G, the only protein exposed on the surface of the virus particle, mediates both binding to cell receptors and entry into host cells. Being a highly immunogenic protein, virus-neutralizing antibodies that are induced against this protein (1, 2) protect against RV infection (3). In addition, RV G induces cytotoxic T lymphocytes (4) and helper T cells (5).
Although RV G appears to be the primary determinant of protection against RV infection, some researchers have reported that it is not essential in a rabies vaccine. Immunization of mice and raccoons with ribonucleoprotein RV (RNP) in complete Freund's adjuvant resulted in protection (6). Moreover, administration of N alone or via an RV N-expressing viral vector resulted in protection against lethal challenge with RV (7–9).
On the other hand, others have reported only partial protection after immunization with RV N and suggest that immunization reduces the incubation period and clinical symptoms of rabies (10). Therefore, the question of the importance of RV G alone or in combination with N in protection remains. Further clarification of this debate will help to develop more effective vaccines.
The development of an efficient salvage system for the rescue of infectious RV from cDNA has enabled direct manipulations of the RV genome and the production of genetically engineered recombinant viruses. Using this technology, RVs expressing stable heterologous proteins were rescued. Foreign genes such as CAT (11), CD4 and CXCR4 (12) or HIV-1 gp160 (13) have been inserted into the RV genome, in addition to the five standard genes.
An advantage of rhabdovirus-based vectors is the ability to incorporate foreign glycoproteins into viral particles. For RV, this process requires replacement of the cytoplasmic domain of the foreign glycoprotein with the corresponding fragment of RV G (12, 14). This process appears to be different for vesicular stomatitis virus (VSV), another rhabdovirus.
The incorporation of foreign glycoproteins into VSV particles depends only on the expression level of the glycoproteins and does not require any modification (15-17). Moreover, VSV even incorporates its own glycoprotein containing a foreign cytoplasmic domain as efficiently as the G protein of wild-type VSV.