Abstract
Rabies is an ancient and neglected zoonotic disease caused by the rabies virus, a neurotropic RNA virus that belongs to the Rhabdoviridae family, genus Lyssavirus. It remains an important public health problem as there are cost and health concerns imposed by the current human post exposure prophylaxis therapy. The use of monoclonal antibodies (mAbs) is therefore an attractive alternative. Rabies mostly affects people that reside in resource-limited areas where there are occasional failures in the cold-chain. These environmental changes may upset the stability of the mAbs. This study focused on mAbs 62-71-3 and E559; their structures, responses to freeze/thaw (F/T) and exposure to reactive oxygen species were therefore studied with the aid of a wide range of biophysical and in silico techniques in order to elucidate their stability and identify aggregation prone regions. E559 was found to be less stable than 62-71-3. The complementarity determining regions (CDR) contributed the most to its instability, more specifically: peptides 99EIWD102 and 92ATSPYT97 found in CDR3, Trp33 found in CDR1 and the oxidised Met34. The constant region “158SWNSGALTGHTFPAVL175” was also flagged by the special aggregation propensity (SAP) tool and F/T experiments to be highly prone to aggregation. The E559 peptides “4LQESGSVL11 from the heavy chain and 4LTQSPSSL11 from the light chain, were also highly affected by F/T. These residues may serve as good candidates for mutation, in the aim to bring forward more stable therapeutic antibodies, thus paving a way to a more safe and efficacious antibody-based cocktail treatment against rabies.
Highlights
Developing countries of Africa and Asia remain highly affected by rabies which is one of the oldest recorded infections of mankind
The complementarity determining regions (CDR) regions of E559 are mostly responsible for its instability, residues 99EIWD102 found in HC CDR3 and 92ATSPYT97 found on LC CDR3
The aggregation prone Trp101 from CDR3 was in spatial proximity to Trp33 which formed part of peptides that became more exposed to the solvent with an increase in F/T cycles
Summary
Developing countries of Africa and Asia remain highly affected by rabies which is one of the oldest recorded infections of mankind. There are various challenges with the current human PEP such as availability, affordability and safety This is mainly because RIG is prepared from pooled sera from hyper immunised humans (HRIG) or horses (ERIG) [5]. In previous work [9], we discussed the use of E559 and 62-71-3 in a cocktail as each mAb targets a different site of the rabies virus glycoprotein and as such prevents viral escape [4,6]. These mAbs were expressed in ΔxT/FT plants, a Nicotiana benthamiana mutant that supports production of fructose-free glycans, and were reported to be more efficacious RIG. The E559 and 62-71-3 mAb structures were studied in combination with their responses to freeze/thaw (F/T) and exposure to reactive oxygen species, to understand the mechanisms behind their degradation and to suggest ways to improve their stability
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