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

<p>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.</p

Mean brain/serum concentration ratio.

<p>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.</p

Overview of average pharmacokinetic parameter values.

<p>Overview of average pharmacokinetic parameter values.</p

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

<p>(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).</p

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

<p>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).</p

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

<p>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.</p

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

<p>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.</p

Comparison of the neutralizing potency of different anti-rabies VHH constructs <i>in vitro</i> and <i>in vivo</i> following pre-incubation with rabies virus.

<p>Comparison of the neutralizing potency of different anti-rabies VHH constructs <i>in vitro</i> and <i>in vivo</i> following pre-incubation with rabies virus.</p

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

<p>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.</p