In vitro and in vivo evaluation of a single chain antibody fragment generated in planta with potent rabies neutralisation activity

Abstract

Rabies causes more than 60,000 human deaths annually in areas where the virus is endemic. Importantly, rabies is one of the few pathogens for which there is no treatment following the onset of clinical disease with the outcome of infection being death in almost 100% of cases. Whilst vaccination, and the combination of vaccine and rabies immunoglobulin treatment for post-exposure administration are available, no tools have been identified that can reduce or prevent rabies virus replication once clinical disease has initiated. The search for effective antiviral molecules to treat those that have already developed clinical disease associated with rabies virus infection is considered one of the most important goals in rabies research. The current study assesses a single chain antibody molecule (ScFv) based on a monoclonal antibody that potently neutralises rabies in vitro as a potential therapeutic candidate. The recombinant ScFv was generated in Nicotiana benthamiana by transient expression, and was chemically conjugated (ScFv/RVG) to a 29 amino acid peptide, specific for nicotinic acetylcholine receptor (nAchR) binding in the CNS. This conjugated molecule was able to bind nAchR in vitro and enter neuronal cells more efficiently than ScFv. The ability of the ScFv/RVG to neutralise virus in vivo was assessed using a staggered administration where the molecule was inoculated either four hours before, two days after or four days after infection. The ScFv/RVG conjugate was evaluated in direct comparison with HRIG and a potential antiviral molecule, Favipiravir (also known as T-705) to indicate whether there was greater bioavailability of the ScFv in the brains of treated mice. The study indicated that the approach taken with the ScFv/RVG conjugate may have utility in the design and implementation of novel tools targetting rabies virus infection in the brain.

Keywords: Blood brain barrier (BBB); Clinical disease; Immunoglobulin; N-acetylcholine receptor; Nicotiana benthamiana; Rabies virus; Single-chain antibody (ScFv).

Fig. 1

Characterisation of ScFv and ScFv/RVG conjugate. The plant-produced ScFv was purified by Ni affinity chromatography. The ScFv was chemically conjugated to chemically synthesized RVG peptide to produce ScFv/RVG. ScFv and ScFv/RVG conjugate were analysed by SDS-PAGE under reducing conditions, followed by (A) staining with Coomassie blue or (B) blotting onto nitrocellulose and probing with a mouse anti-E tag antiserum. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Rabies virus (ERA stain) neutralization by ScFv and ScFv/RVG conjugate compared with 62-71-3 mAb IgG antibody as assessed by RFFIT on BSR cells. Antibody starting concentrations were 0.5 mg/ml. Assays were performed in triplicate. Error bars indicate the SD.

Fig. 3

Binding and entry of 62-71-3 ScFv to 293 cells overexpressing nAchR by flow cytometry. Binding (A) and entry (B) were detected with mouse anti-E antiserum and cy5 conjugated goat anti-mouse IgG antiserum, Solid line: ScFv, Dotted line: ScFv/RVG conjugate. A representative result from triplicate experiments is shown.

Fig. 4

Inhibition of entry of ScFv/RVG conjugate into nAchR-overexpressing 293 cells and neuroscreen cells by irradiated rabies virus and α-bungarotoxin. Flow cytometry on nAchR-overexpressing 293 cells pre-treated with irradiated rabies virus (A, B) and α-bungarotoxin (C, D) before incubation with ScFv (A and C), and ScFv/RVG conjugate (B and D). Flow cytometry on neuronal 2a cells pre-treated with irradiated rabies virus (E, F) and α-bungarotoxin (G, H) before incubation with ScFv (E and G) and ScFv/RVG conjugate (F and H). Solid line: no inhibitor, Dotted line: pre-treated with irradiated rabies virus or α-bungarotoxin; A representative result from triplicate experiments is shown.

Fig. 5

ScFv/RVG conjugate transports across in vitro BBB model. 10 μg antibodies were added to the upper chamber of hCMEC/D3 cells in the transwell. Media in the bottom well was tested for rabies virus neutralization assay after 2 and 18 h. A representative result from triplicate experiments is shown.

Fig. 6

(a) Mouse survival curves for the three treatments ScFv/RVG, HRIG and PBS only controls at three different time points following inoculation with rabies virus. (b) Mouse survival curves for the five treatments ScFv/RVG, T-705, ScFv/RVG with T-705, HRIG and PBS only controls, combining observations across three different timings, which did not significantly affect treatment effects.

Fig. 7

Logit mortality for four treatments ScFv/RVG, T-705, ScFv/RVG with T-705 and HRIG relative to PBS only controls, assuming treatment effects did not interact with timing. Bars show 95% confidence intervals estimated from a mixed effects logistic regression. Letters above the bars group treatments with similar mortality; treatments differ significantly at the 95% confidence level if they do not share any matching letters.