Protective effect of different anti-rabies virus VHH constructs against rabies disease in mice

Abstract

Rabies virus causes lethal brain infection in about 61000 people per year. Each year, tens of thousands of people receive anti-rabies prophylaxis with plasma-derived immunoglobulins and vaccine soon after exposure. Anti-rabies immunoglobulins are however expensive and have limited availability. VHH are the smallest antigen-binding functional fragments of camelid heavy chain antibodies, also called Nanobodies. The therapeutic potential of anti-rabies VHH was examined in a mouse model using intranasal challenge with a lethal dose of rabies virus. Anti-rabies VHH were administered directly into the brain or systemically, by intraperitoneal injection, 24 hours after virus challenge. Anti-rabies VHH were able to significantly prolong survival or even completely rescue mice from disease. The therapeutic effect depended on the dose, affinity and brain and plasma half-life of the VHH construct. Increasing the affinity by combining two VHH with a glycine-serine linker into bivalent or biparatopic constructs, increased the neutralizing potency to the picomolar range. Upon direct intracerebral administration, a dose as low as 33 µg of the biparatopic Rab-E8/H7 was still able to establish an anti-rabies effect. The effect of systemic treatment was significantly improved by increasing the half-life of Rab-E8/H7 through linkage with a third VHH targeted against albumin. Intraperitoneal treatment with 1.5 mg (2505 IU, 1 ml) of anti-albumin Rab-E8/H7 prolonged the median survival time from 9 to 15 days and completely rescued 43% of mice. For comparison, intraperitoneal treatment with the highest available dose of human anti-rabies immunoglobulins (65 mg, 111 IU, 1 ml) only prolonged survival by 2 days, without rescue. Overall, the therapeutic benefit seemed well correlated with the time of brain exposure and the plasma half-life of the used VHH construct. These results, together with the ease-of-production and superior thermal stability, render anti-rabies VHH into valuable candidates for development of alternative post exposure treatment drugs against rabies.

Figure 1. Virus spread in the mouse brain following intranasal rabies virus inoculation.

The graph presents the profile of viral RNA in different parts of the brain (indicated in the left photo) upon intranasal inoculation of 10^2.5 CCID₅₀/mouse. Groups of mice (n = 7–10) were intranasally inoculated with rabies virus and sacrificed at various time points post inoculation (DPI). Viral loads were determined by qRT-PCR.

Figure 2. Co-administration of anti-rabies Rab-E8/H7 and virus directly in the brain efficiently inhibits virus infection.

Mice were inoculated intracerebrally with a mix of rabies virus and 0.12 µg (1 IU) anti-rabies Rab-E8/H7 (A) or irrelevant VHH (B) and euthanized 7 days later. The anti-rabies VHH-treated mice were protected from disease, whereas all the mock-treated mice developed severe nervous disease. The pictures represent an immunofluorescence staining for viral nucleocapsid in the brain tissue. No viral antigens were visible in the brain of anti-rabies VHH-treated mice (A), whereas green fluorescent spots indicate the abundant spread of virus in the brain of mock-treated mice (B). The graph (C) presents the viral RNA load in the brains of different groups. Viral loads were significantly different between groups treated with Rab-E8/H7 and irrelevant control VHH, between Rab-E8/H7 and uninfected controls and between irrelevant control VHH and uninfected controls (*** p<0.01).

Figure 3. Dose-dependent efficacy of anti-rabies Rab-E8/H7 upon intracerebral post-exposure treatment at one day after intranasal virus inoculation.

Two independent experiments were performed. In the first experiment (A), doses of 1, 10 and 100 µg Rab-E8/H7 were tested and in the second experiment (B), two additional doses of 33 µg and 67 µg were included. Significant protection was observed starting from a dose of 33 µg (p<0.01). One third to more than half of the mice that were treated at this or a higher dose survived the infection.

Figure 4. Viral RNA load in the brain after anti-rabies Rab-E8/H7 treatment.

(A) Mice were treated with Rab-E8/H7 (100 µg) by intracerebral injection (IC) 24 hours after intranasal virus inoculation and sacrificed at 7 DPI to assess the viral RNA loads in different brain parts. Rab-E8/H7 VHH treatment significantly reduced the spread of the virus from the front to the posterior parts of the brain (t-test, ** p<0.01, *** p<0.0001). (B) Mice were treated with Rab-E8/H7 at 24 hours before (0.12 µg) or after (100 µg) intranasal virus inoculation. Control mice were mock-treated with irrelevant VHH before virus inoculation. Viral RNA loads were measured at 35 DPI in the brain of survivor mice. Four out of five survivor mice, treated after the virus inoculation, showed residual traces of viral RNA in the brain (ΔCt 5±2.9; *** p<0.0001). These mice had however never developed signs of disease. All mock-treated mice had to be euthanized at 7–9 DPI, because of serious disease, which coincided with high viral RNA loads in their brains (ΔCt≥28).

Figure 5. Post-exposure treatment by intracerebral injection at different time points of infection.

Mice were treated with a single dose of 100 µg (463 IU) Rab-E8/H7 at increasing time points of infection. The protective effect of anti-rabies VHH diminished progressively when treatment was initiated at later stages of infection.

Figure 6. Mean brain and serum concentration of Rab-E8/H7 and HLE Rab-E8/H7.

Individual brain (circles) and serum (triangles) concentrations and mean values (lines) of HLE Rab-E8/H7 (A) and Rab-E8/H7 (B) upon intraperitoneal injection of 5 mg HLE Rab-E8/H7 or 10 mg Rab-E8/H7.

Figure 7. Mean brain/serum concentration ratio.

Mean brain/serum concentration ratio over time for HLE Rab-E8/H7 and Rab-E8/H7 upon intraperitoneal injection of 5 mg HLE Rab-E8/H7 or 10 mg Rab-E8/H7 in mice.

Figure 8. Post-exposure treatment with anti-rabies Rab-E8/H7 with or without half-life extension (HLE).

Half-life extension was accomplished by adding a third anti-albumin VHH to Rab-E8/H7. Mice were treated intraperitoneally 24 hours after intranasal virus inoculation. The clinical effect of Rab-E8/H7 was significantly improved by the half-life extension. The median survival time was prolonged by six to more than 26 days (p<0.01), depending on the dose. More than 70% of the mice were completely protected against disease upon treatment with 15 mg HLE Rab-E8/H7.

Figure 9. Post-exposure treatment with human anti-rabies immunoglobulins (Imogam).

Mice were treated intraperitoneally with 65 mg (111 IU, 1 ml) of human rabies immunoglobulins at 24 hours after intranasal virus inoculation in two independent experiments (A and B). The median survival time was prolonged by 2 days, but all mice developed serious nervous disease, requiring euthanasia.