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.
Highlights
Rabies virus (Familia Rhabdoviridae, Genus Lyssavirus) is a model neurotropic RNA virus, which causes an aggressive and lethal infection in the brain of humans and mammals [1]
Characterisation of the mouse rabies model In order to test the in vivo efficacy of different anti-rabies VHH, a mouse model reflecting the neurological late stage of rabies disease was set-up and characterised
In a first series of experiments, disease symptoms and viral kinetics in the brain were assessed after intranasal inoculation of rabies virus
Summary
Rabies virus (Familia Rhabdoviridae, Genus Lyssavirus) is a model neurotropic RNA virus, which causes an aggressive and lethal infection in the brain of humans and mammals [1]. Nanobodies (a trade-name by Ablynx) or VHH are the smallest functional portions (15 kDa) of heavy chain-only antibodies naturally occurring in Camelidae, and represent the antigenbinding variable domain. The single domain nature and the small size of VHH allow easy formatting by genetic fusion into multimeric constructs with multiple specificities [4,5,6]. We developed a number of rabies virus-specific VHH directed against the rabies virus spike glycoprotein G [7]. In vitro, these VHH were fully able to neutralize the rabies virus infectivity in neuroblastoma and baby hamster kidney cells-21 (BHK-21) and could neutralize a wide spectrum of Lyssavirus species. The neutralizing potency increased massively when two VHH were combined with a glycine-serine linker into bivalent or biparatopic constructs [7]
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