Production of Rabies virus-specific monoclonal antibodies and evaluation of their neutralizing potential

Abstract

Rabies is a severe viral infection that causes acute encephalomyelitis, with a case fatality rate of nearly 100% following the onset of neurological clinical signs. Rabies irreversible clinical signs development can be effectively avoided with post-exposure prophylaxis (PEP), which includes vaccines and anti-rabies immunoglobulins (RIGs); however, there is no treatment for symptomatic rabies. The major PEP protocol faces serious access and implementation obstacles in association with a resource-limited setting, which could be successfully overcome by substituting RIGs for monoclonal antibodies (mAbs). Lower production costs, consistent supply availability, long-term storage/stability, and an improved safety profile are all advantages of mAbs. The current work focuses on the key characteristics of currently developed mAbs against rabies and highlights their potential as a novel therapeutic approach. Using immunizing Freunds adjuvanted emulsions of inactivated purified Vero cell rabies vaccine (PVRV, VERORAB) produced by Aventis Pasteur to immunize the BALB/c mice. The immunized BALB/c mice were tested for the production of anti-rabies virus-specific antibodies using Enzyme-linked immunosorbent assay (ELISA). High-responder mice were selected for the fusion process. Hybridomas recovered from the fusion process were selected and separated from the unfused cells and unfavorable fused cells by using the selective HAT medium. Twelve days post fusion the produced hybrids were screened for production of Rabies virus-specific antibodies using ELISA. Four murine hybridomas secreting rabies virus-specific monoclonal antibodies (mAbs) have been properly developed. These 4 stable hybrids were successfully cloned into 4 stable clones, namely, 1E4, 1E9, 2F3, 4E1. The rabies virus specific monoclonal antibodies produced by the 4 selected hybridomas were of IgM isotype. Using Western Blot technique, the specificity of the produced hybrids was confirmed. The neutralizing potential of the prepared mAbs was evaluated and the efficacy of mAbs cocktail prepared from the 4 hybridomas to protect mice in post exposure therapy was determined. The mAbs cocktail given to mice at 24 hours post infection was able to offer 100% protection to mice challenged with 1000 LD50 of rabies virus strain whereas all control mice developed the disease.