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.

Development and activities, including immunocomplex formation, of biparatopic antibodies and alternative scaffold proteins

“…For monomeric alternative scaffold proteins and fragment antibodies, generation of biparatopic proteins is an extremely efficient way of increasing avidity. Thus, a large number of these proteins have been developed as biparatopic agents [12,[16][17][18][19][20][21][22][23][24][25][26][27][28][29], which is not as important with conventional antibodies because they are already bivalent [5,7,8,10,[30][31][32][33][34][35][36][37].…”

Use of camel single-domain antibodies for the diagnosis and treatment of zoonotic diseases

“…The ease of high-level production, small size, and high stability make VHHs extremely reliable for genetic and chemical modification, such as the production of VHH-based fusion proteins to increase the persistence of VHHs in serum or confer additional functions. Construction of multivalent VHHs consisting of two or more linked VHHs targeting various epitopes leads to an increase in their ability to neutralize toxins [43,44] and viruses [68,72,76,81], suggesting the development of these approaches for VHHs targeting other viruses not yet tested.…”

“…The IC 50 s of the CVS-11 (genotype 1) strain ranged from 7 to 325 nM. These VHHs were fused to an anti-albumin VHH to extend its serum half-life and were able to neutralize the virus at picomolar doses [76]. A combined treatment based on VHH and vaccine (Rabipur, Novartis) acted synergistically to protect mice in an intranasal rabies infection model [77].…”

High Throughput Combinatorial Formatting of PcrV Nanobodies for Efficient Potency Improvement

“…Formatting represents an elegant way to improve potencies by avidity and concomitant blockage of multiple epitopes without the need for cumbersome affinity maturation. For homomultimeric targets, the combination of multiple identical (or different competing) Nanobody building blocks, appropriately spaced by linkers and each targeting a single protomer, may result in increased potencies driven by the avid binding of the multivalent Nanobody, compared with its monovalent counterparts (3)(4)(5)(6)(7)(8). For monomeric or heteromultimeric targets, similar potency increases can be observed with multivalent Nanobodies combining different building blocks binding to non-overlapping epitopes on the same protomer or each binding to a different protomer, respectively (6, 8 -10).…”

“…The pathogen's innate resistance to several antibiotics and the emergence of multi-resistant strains underscore the need for alternative treatments (12). Among its large arsenal of virulence systems, the type III secretion system (T3SS) 6 is a major determinant in the pathogenesis of acute infections (13).…”