A single immunization with recombinant rabies virus (ERAG3G) confers complete protection against rabies in mice

Abstract

Purpose: New alternative bait rabies vaccines applicable to pet dogs and wild animals are needed to eradicate rabies in Korea. In this study, recombinant rabies virus, ERAG3G strain was constructed using reverse genetic system and the safety, efficacy and immunogenicity of the ERAG3G strain was evaluated in mice and dogs.

Materials and methods: Using the full-length genome mutated amino acid at position 333 of glycoprotein of rabies virus (RABV) and helper plasmids, the ERAG3G strain was rescued in BHK/T7-9 cells successfully. Mice were inoculated with the ERAG3G strain for safety and efficacy. Safety and immunogenicity of the dog inoculated with the ERAG3G strain (1 mL, 10(8.0) FAID50/mL) via intramuscular route was evaluated for 28 days after inoculation.

Results: The ERAG3G strain rescued by reverse genetic system was propagated well in the mouse neuroblastoma cells revealing titer of 10(8.5) FAID50/mL and was not pathogenic to 4- or 6-week-old mice that received by intramuscular or intracranical route. Immunization with the ERAG3G strain conferred complete protection from lethal RABV in mice. Dogs inoculated with the vaccine candidate via intramuscular route showed high neutralizing antibody titer ranging from 2.62 to 23.9 IU/mL at 28 days postinoculation.

Conclusion: Our findings suggest that the ERAG3G strain plays an important role in inducing protective efficacy in mice and causes to arise anti-rabies neutralizing antibody in dogs.

Keywords: Animals; Rabies virus; Recombinant rabies virus; Vaccine.

Fig. 1

Vector map for the construction of rERA and ERAG3G strain. These full-length cDNA plasmids and three helper plasmids (N, P, and L plasmid) were transfected into BHK/T7-9 cells producing RNA polymerase, respectively.

Fig. 2

Cytopathic effects observed in BHK/T7-9 cell inoculated with rERA (A, ×100) and ERAG3G (B, ×200) and immunofluorescence rERA (C, ×200) and ERAG3G (D, ×100) strain by indirect fluorescent assay test using monoclonal antibodies against rabies virus (RABV) in BHK/T7-9 cells. The RABV specific fluorescent appeared in the cytoplasm of the infected cell.

Fig. 3

Rabies virus particles in cytoplasm of BHK-T7 cells inoculated with rERA (A, ×50,000) and ERAG3G (B, ×50,000) strains showing bullet shape.

Fig. 4

Change in body weight in 4-week-old (A, B) and 6-week-old mice (C, D) inoculated with ERA, rERA, and ERAG3G strains via intramuscular (IM; A, C) or intracranial (IC; B, D) route.

Fig. 5

Safety test in 4-week-old (A, B) and 6-week-old mice (C, D) inoculated with ERA, rERA, and ERAG3G strains via intramuscular (IM; A, C) or intracranial (IC; B, D) route. The 4-week-old mice inoculated with ERA and rERA strains respectively died at 12 or 13 days postinoculation. Fifty percent of the 6-week-old mice inoculated with ERA and rERA IM were alive and all of the mice inoculated with ERA and rERA IC died at 13 days postinoculation. On the contrary, both 4- and 6-week-old mice inoculated with ERAG3G IM or IC were 100% alive as control group.

Fig. 6

Change of body weight (A) and survival rate (B) in 4- and 6-week-old mice immunized with ERAG3G strain via intramuscular (IM) or intracranial (IC) route and challenged with highly pathogenic rabies virus strain (CVSN2c). The average body weight of ERAG3G group mice increased for 17 days after challenge and survival rates did not change for 17 days after challenge.

Fig. 7

Immune response in dogs inoculated with the ERAG3G strain via intramuscular route. All dogs immunized with the ERAG3G strain induced high neutralizing antibody titer >0.5 IU/mL against rabies virus. VN, virus neutralizing.