Structural And Functional Research Of Anti-Rabies Virus G-protein ScFv FV57and Its Molecular Modification

Rabies is a centried infectious desease which lacks of efficient control measuresand causes55000deaths per year. Rabies is caused by the Rabies Virus’ infection tothe exposurers’ central nervous systems, and its subsequent induction of the lethalencephalomyelitis. Infection with rabies results in a mortality rate of100%oncesymptoms are evident. Based on this property of rabies, the WHO recommends to usePost-exposure Prophylaxis （PEP）, including a combined administration of rabiesvaccine and rabies immunoglobulin （RIG） to prevent the rabies. However, the RIGcurrently in use has some disadvantages has limited the extensive application of RIG,such as the high cost for production, the batch-to-batch variation, and the potentdanger to spread blood-bourne diseases. Monoclonal antibody （mAb） is a preeminentsubstitution of the RIG, due to its potential merits such as continuous production andthe absence of blood contamination. Meanwhile, single-chain variable antibodyfragment （scFv）, which has been studied and applied most among all the smallmolecular antibodies, also has good applying prospect in rabies PEP due to itspossibility of prokaryotically expression, high penetrability and low immunogenicity.In the former researches, two strains of anti-rabies virus G-protein scFv, FV4098andFV57, were prepared and evaluated based on the sequences of two strains ofanti-rabies mAbs that had been taken into clinical trial. However, FV57could notmatch FV4098in the in vivo neutralizing potency, since it only provides as high as60%protection against lethal rabies virus challenge in a mice PEP model, while theprotection rate of FV4098is100%.According to the amino acid sequences comparison the intact mAb and scFv,FV57has altogether5cysteines, two of them were located in the framework region （FR） of the heavy chain and linked in an intra-chain disulfide-bond, two were locatedin the FR region in the light chain and formed an intra-chain disulfide-bond （VL16,VL84）, while one is located in the complementary determining region （CDR） and hasa free thiol （VL85）. Thus, since the cysteine in VL85is adjacent to the cysteine inVL84, it may probably mis-matched with the cysteine in VL16, and this mis-matcheddisulfide-bond might bring about steric hindrance in the CDR region andconsequently restrain the binding of FV57and the target rabies virus. Meanwhile, therelative high structural looseness of FV57might also lead to its low neutralizingpotency against rabies virus.Thus, issuing the problems above, in this paper the cysteines in VL85and VL84of FV57were mutated to serine respectively, and two strains of mutants FV57^VL85Sand FV57^VL84S, which conrresponds to the correct and incorrect disulfide-bondfolding of FV57, were expressed from E.coli.and prepared. Then, the three scFvs’intra-chain disulfide bonds in the light chain were deleted by mutating the cysteine inVL16to serine, and the newly constructed mutants were called FV57^VL16S，FV57^VL16-85Sand FV57^VL16-84S. Then the biological activity, including the bindingactivity as well as the neutralzing potency of those6strains of scFvs against rabiesvirus were measured by ELISA and RFFIT. The results show that FV57^VL85Shashigher biological activity than its original form FV57, while activity of FV57^VL84Smuch lower than the original form. Thus, the mis-matched disulfide-bond VL16-VL85might play a negative effects on FV57’s activity.Furthermore, in the study of the three mutants without VLintra-chaindisulfide-bonds, FV57^VL16Sand FV57VL16-85both have lower activity than theirrespective original form FV57and FV57^VL85Ssince the absence of intra-chaindisulfide-bond might change the formation of CDR region, however, FV57^VL16-84Shasbetter activity than the original FV57^VL84Ssince even the loss of intra-chaindisulfide-bond might have negative effect on the CDR region’s function, the bindingbetween CDR region and antigen was improved since the absence of steric hindrancemay compensate it, and consequently the binding was finally improved. Thus, thereason for the VL16-VL85mis-matched disulfide-bond’s effects is identified to be the steric hindrance.Moreover, in order to enhance the structural domain’s intensity for better stability,in this paper an additional inter-chain disulfide-bond was added to FV57betweenVH44and VL100, and a disulfide-stabled scFv （ds-scFv） ds-FV57was obtained.Additionally, in order to avoid the cysteine in VL85’s participation in a mis-matcheddisulfide-bond, the cysteine in VL85was mutated to serine, and the new protein wasnamed ds-FV57^VL85S. Then the binding activity to rabies virus, antibody affinity,molecular stability, neutralizing potency of the three scFv were measured. The resultsshows that ds-FV57did not show significant improvement than FV57due to thedisulfide-bond mis-match. However, ds-FV57^VL85Shas much better advantage overFV57in stability and neutralizing potency, and could provide better protection ratetoward rabies virus challenge in a mice PEP model. Those results could providevaluable background and prospect for the later study of FV57and its furtherapplication in the rabies PEP.