Generation and characterization of a genetically modified live rabies vaccine strain with attenuating mutations in multiple viral proteins and evaluation of its potency in dogs.

Live rabies vaccines have advantageous features that can facilitate mass vaccination for dogs, the most important reservoirs/transmitters of rabies. However, some live vaccine strains have problems in their safety, namely, risks from the residual pathogenicity and the pathogenic reversion of live vaccine strains. The reverse genetics system of rabies virus provides a feasible option to improve the safety of a live vaccine strain by, for example, artificially introducing attenuating mutations into multiple viral proteins. It was previously demonstrated in separate studies that introduction of amino acid residues Leu at position 333 in the viral glycoprotein (G333), Ser at G194, and Leu/His at positions 273/394 in the nucleoprotein (N273/394) enhance the safety of a live vaccine strain. In this study, to test our hypothesis that combinational introduction of these residues would significantly increase the safety level of a vaccine strain, we generated a novel live vaccine candidate, ERA-NG2, that is attenuated by mutations at N273/394 and G194/333, and we examined its safety and immunogenicity in mice and dogs. ERA-NG2 did not cause any clinical signs in mice after intracerebral inoculation. After 10 passages in suckling mouse brains, ERA-NG2 retained all of the introduced mutations except the mutation at N394 and the highly attenuated phenotype. These findings indicate that the ERA-NG2 is highly and stably attenuated. After confirming that ERA-NG2 induced a virus-neutralizing antibody (VNA) response and protective immunity in mice, we immunized dogs intramuscularly with a single dose (105-7 focus-forming units) of ERA-NG2 and found that, at all of the tested doses, the strain induced a VNA response in dogs without inducing any clinical signs. These findings demonstrate that ERA-NG2 has a high level of safety and a substantial level of immunogenicity in dogs and thus is a promising live vaccine candidate that can facilitate vaccination in dogs.

Safety enhancement of a genetically modified live rabies vaccine strain by introducing an attenuating Leu residue at position 333 in the glycoprotein.

To improve the safety of genetically modified live rabies vaccine strains, most studies have utilized an attenuating Arg-to-Glu mutation at position 333 in the glycoprotein (G333), which is responsible for attenuation of the live vaccine strain SAG2. The Glu residue requires two nucleotide substitutions to revert to pathogenic Arg, thus significantly lowering the probability of pathogenic reversion caused by the Glu-to-Arg mutation at G333. However, only one nucleotide substitution is sufficient to convert the Glu residue to another pathogenic residue, Lys, and thereby to cause pathogenic reversion. This indicates a potential safety problem of SAG2 and the live vaccine candidates attenuated by Glu at G333. In this study, aiming to solve this problem, we examined the utility of a Leu residue, which requires two nucleotide substitutions to be both Arg and Lys, as an attenuating mutation at G333. Using a reverse genetics system of the live vaccine strain ERA, we generated ERA-G333Leu by introducing an Arg-to-Leu mutation at G333. Similar to ERA-G333Glu, which is attenuated by an Arg-to-Glu mutation at G333, ERA-G333Leu did not cause obvious clinical signs in 6-week-old mice after intracerebral inoculation. Importantly, after 10 passages in suckling mouse brains, ERA-G333Glu acquired a pathogenic Lys or Arg at G333 and a high level of lethality in mice, whereas ERA-G333Leu retained the attenuating Leu at G333 and only showed a modest level of virulence probably caused by a mutation at G194. In addition, ERA-G333Leu and ERA-G333Glu induced neutralizing antibody response and protective immunity in mice with similar efficiencies. The results demonstrate that, compared to ERA-G333Glu, ERA-G333Leu is more stably attenuated, also indicating the high utility of a Leu residue as an attenuating mutation at G333 in the development of live rabies vaccine strains with a high level of safety.