Survival of rabid rabbits after intrathecal immunization

Abstract

Rabies is a fatal zoonotic disease for which no effective treatment measures are currently available. Rabies virus (RABV) has anti-apoptotic and anti-inflammatory properties that suppress nerve cell damage and inflammation in the CNS. These features imply that the elimination of RABV from the CNS by appropriate treatment could lead to complete recovery from rabies. Ten rabbits showing neuromuscular symptoms of rabies after subcutaneous (SC) immunization using commercially available vaccine containing inactivated whole RABV particles and subsequent fixed RABV (CVS strain) inoculation into hind limb muscles were allocated into three groups. Three rabbits received no further treatment (the SC group), three rabbits received three additional SC immunizations using the same vaccine, and four rabbits received three intrathecal (IT) immunizations, in which the vaccine was inoculated directly into the cerebrospinal fluid (the SC/IT group). An additional three naïve rabbits were inoculated intramuscularly with RABV and not vaccinated. The rabbits exhibited neuromuscular symptoms of rabies within 4-8 days post-inoculation (dpi) of RABV. All of the rabbits died within 8-12 dpi with the exception of one rabbit in the SC group and all four rabbits in SC/IT group, which recovered and started to respond to external stimuli at 11-18 dpi and survived until the end of the experimental period. RABV was eliminated from the CNS of the surviving rabbits. We report here a possible, although still incomplete, therapy for rabies using IT immunization. Our protocol may rescue the life of rabid patients and prompt the future development of novel therapies against rabies.

Keywords: CNS pathology; animal experiment; intrathecal immunization; rabies virus; treatment of rabies.

Figure 1

Experimental protocol. ▴, Subcutaneous (SC) immunization prior to rabies virus (RABV) inoculation; , RABV inoculation; ▪, Rabies symptoms; ♦, SC immunization; , intrathecal (IT) immunization.

Figure 2

Viral neutralizing antibody titers in the serum and CSF. Tx: treatment. , Surviving (Serum); , Surviving (CSF); , Non-surviving (Serum); , Non-surviving (CSF).

Figure 3

ELISA antibody titers in the serum and CSF. Tx: treatment. , Surviving (Serum); , Surviving (CSF); , Non-surviving (Serum); , Non-surviving (CSF).

Figure 4

The cerebellar cortex of a non-surviving rabbit from the non-treatment group showing necrosis of Purkinje cells (arrowheads). Immunohistochemistry of the cerebellum reveals abundant rabies virus (RABV) antigen in Purkinje and granular cells (inset, A). The cerebellar cortex of a surviving rabbit from the subcutaneous (SC)/intrathecal (IT) group showed the loss of Purkinje and granular cells together with gliosis of the molecular layer. RABV antigen is not found in the cerebellum (inset, B). Scale bar represents 50 μm .

Figure 5

RT-PCR of the brain. A positive signal for the rabies virus (RABV) matrix gene in the brain samples from all rabbits except one surviving rabbit in the subcutaneous (SC) group (lane 6) and three surviving rabbits in the SC/intrathecal (IT) group (lanes 9–11). HPRT, phosphoribosyltransferase.