Characterisation of a Live-Attenuated Rabies Virus Expressing a Secreted scFv for the Treatment of Rabies

Abstract

Rabies virus (RABV) causes possibly the oldest disease and is responsible for an estimated >59,000 human fatalities/year. Post exposure prophylaxis (PEP), the administration of vaccine and rabies immunoglobulin, is a highly effective tool which is frequently unavailable in RABV endemic areas. Furthermore, due to the constraints of the blood-brain barrier, current PEP regimes are ineffective after the onset of clinical symptoms which invariably result in death. To circumvent this barrier, a live-attenuated recombinant RABV expressing a highly RABV-neutralising scFv antibody (62-71-3) linked to the fluorescent marker mCherry was designed. Once rescued, the resulting construct (named RABV-62scFv) was grown to high titres, its growth and cellular dissemination kinetics characterised, and the functionality of the recombinant 62-71-3 scFv assessed. Encouraging scFv production and subsequent virus neutralisation results demonstrate the potential for development of a therapeutic live-attenuated virus-based post-infection treatment (PIT) for RABV infection.

Keywords: PEP; antibody; lyssavirus; rabies; rabies treatment; scFv; virus attenuation.

Figure 1

Assessing the effect of RABV-62scFv infection on mitochondrial respiration. An MTS assay was performed on N2a cells to assess the effect of RABV-62scFv and RABV-cSN infection on N2a cell viability. Output values were compared against the uninfected cell control to obtain a baseline value for mitochondrial respiration. The experiment was undertaken in triplicate to n = 3. Results were analysed using the Brown-Forsythe and Welch ANOVA Test (F* 5.299) where ns p > 0.05.

Figure 2

Growth kinetics of RABV-cSN, RABV-mCH, RABV-mCH-K226R, and RABV-62scFv. Growth kinetics were assessed by multi-step growth curves in both BHK-21 (A) and N2a cells (B). After 1 h infection of either a BHK-21 or N2a cell monolayer at 0.01 MOI, supernatant was taken at 0, 6, 12, 24, 48, 72, 96, and 120 h before FFU/mL was measured by virus titration. Experiment was performed in triplicate to n = 3.

Figure 3

Viral dissemination assay investigating the differences between the cellular dissemination of RABV-cSN, RABV-mCH, RABV-mCH-K226R, RABV-62scFv, CVS-11, and RABV-CVS-mCH over a 120 h period.

Figure 4

Representative images of foci generated in the cellular dissemination assay by either RABV-cSN, RABV-62scFv, or RABV-CVS-mCherry infection. (A,B) show images of foci generated by RABV-cSN infection ((A) at 10×, (B) at 4× magnification). (C,D) show images of foci generated by RABV-62scFv infection at similar magnifications, respectively. (E,F) show images of foci generated by RABV-CVS-mCH infection at similar magnifications, respectively. Images are displayed as brightfield, FITC fluorescence under UV, and a merged image. All cells have been stained for RABV N protein and visualised under UV. All images taken at 96 h post infection.

Figure 5

Charting r62-71-3 scFv production over time after BHK-21 cell culture monolayer infection with RABV-62scFv. Supernatant from RABV-mCH-K226R has been included as a control and results are shown as fold-increase of the negative media-only control. Where possible, error bars representing the standard error of the mean have been shown.

Figure 6

Assessment of the presence of RABV-62scFv to cross a hCMEC/D3 BBB. (A) samples taken from apical and basolateral compartments, respectively, after hCMEC/D3 monolayer infection (72 h) with RABV-62scFv (lanes 2/3) or heat-inactivated RABV-62scFv (lanes 4/5) and amplified for RABV-specific sequences. (B) Kinetics of hCMEC/D3 BBB model formation over time. At each 24 h time point after cell seeding, an apparent permeability (Papp) value was calculated. (C) Images taken of hCMEC/D3 blood-brain barrier monolayer to illustrate cellular infection with RABV-62scFv. Row (i) shows brightfield, UV illumination, and overlay images of hCMEC/D3 cells treated with live RABV-62scFv, while row (ii) shows images taken from hCMEC/D3 cells treated with heat-inactivated RABV-62scFv.

Figure 7

Survival curves of mice used to assess RABV-62scFv toxicity. Mice were given RABV-62scFv, RABV-CVS-mCH, or a media control either ic (A) or iv (B). Mice were then observed for 28 days for the development of clinical symptoms where they would be humanely sacrificed. Dosages of 10¹, 10², 10³, or 10⁴ FFU of virus (or media control) in 30 µL was delivered by needle either into the tail vein (iv) or brain (ic) of 3–4-week-old Balb/c mice.

Figure 8

Serological assessment by mFAVN of mouse sera after challenge (ic or iv) at various FFU concentrations with either RABV-62scFv or RABV-CVS-mCH or media control. (A) shows results from mice challenged ic while (B) shows results from mice challenged iv. Sera from mice given a media control were tested for neutralisation against RABV-CVS-mCH. The dotted line represents the cutoff for detection at a reciprocal antibody titre of 7.49.

Figure 9

Histopathological assessment of mouse brains after ic challenge with RABV-CVS-mCH. Images (A–E) show representative images of sections from a mouse brain. Left hand side images have been taken at 200× magnification (A–D) or 100× magnification (E), with the right-hand side images showing magnification of the areas bordered by the dotted boxes in the left-hand side images at 400× resolution. (A,B) show the hippocampus region, where brown staining for the RABV N protein is shown in (A) and staining for CD3 is shown in (B). Images (C,D) show midbrain sections where brown staining indicates the presence of RABV N protein and CD3, respectively. Lastly, (E) shows a section of the thalamus where brown staining indicates RABV N staining.