Study On Preparation And Application Of Humanized Anti-rabies Virus G Protein Singlechain Antibody Antibody ScFV98H

Rabies is a global human zoonotic infectious disease caused by central nervoussystem infection with rabies virus, which has100%death rate after the disease onset.The active and passive immune-therapy is recommended by the World HealthOrganization for serious exposure in order for rapid immune protection. RabiesImmune globulin (RIG) has been proved to be significant to prevent rabies infection.However, further application of RIG has been hampered by the relatively serious sideeffect of Equine Rabies Immune Globulin (ERIG), the limit of resources and potentialpathogenic contamination of Human Rabies Immune Globulin (ERIG). Thus,development of humanized antibody or humanized genetically engineered antibody isurgently required.The recent study had proved that the two types of RIG above could be replacedin rabies post-exposure prophylaxis (PEP) by the cocktail administration comprisingseveral humanized monoclonal antibodies with neutralizing capability against rabiesvirus. However, eukaryotic expression system with low expression level and highproduction-costs is required for the preparation of intact mAb. Meanwhile, productionof small molecule antibody is more flexible since it could be prepared by both theeukaryotic expression system and prokaryotic expression system, which is moreaffordable. Escherichia coli is the expression system of choice for the genetic drugsdue to the rapid process from antibody gene construction to industrial-scaleproduction and the ability to control batch-to-batch variations. Most of the proteinoften expressed as inclusion body form, the purification and refolding of inclusionbody is a challenge task. This paper is mainly focus on scFv98H production processdevelopment for diagnostic or post-exposure prophylaxis (PEP) therapeutic use. Theanalysis methods should be constructed and validated before the production process development.The investigation results:1.The validation of the analysis methodsThe rapid fluorescence focus inhibition test(RFFIT), DNA content assay(Picogreen),and protein concentration analysis method(BCA) should be constructed and validatedbefore the production process development. The validation parameters include:linearity, accuracy, precision, repeatability, precision, limit of quantitation, limit ofdetection, matrix effect. The analysis methods were tested in buffer with8M,3M,0.1M urea,10%glycerol or5%sucrose. The RFFIT results indicated that neuralizingactivity can be tested in buffer with3M,0.1M urea,10%glycerol or5%sucrose,CV<=30%, is accordance with the accept criterion; The Picogreen data indicated thatDNA content assay can be tested in buffer with3M,0.1M urea, CV<=20%, isaccordance with the accept criterion; The BCA results indicated that proteinconcentration analysis can be tested in buffer with8M,3M,0.1M urea, CV<=20%, isaccordance with the accept criterion.2. Fermentation and inclusion body washTo test the plasmid stability, the Escherichia coli seed of scFv98H was passaged10levels, the results convey that there is no loss of scFv98H plasmid,and proteinexpression. The auto-induced medium with some modification is suitable forfermentation, pH is between7.0and7.5. The net weight of Escherichia coli cell andprotein expression is with no change scaling up from3to14L. The inclusion bodyshould be centrifuged at8000rpm for10min after sonication, washed in bufferⅠfor2h, and followed by centrifuged at10000rpm for10min after wash in buffer Ⅲ.3.The development of purification and refolding process for scFv98HAfter extensive wash, the inclusion body protein of scFv98H in8M urea waschromatograph purified on His Trap HP、SP、QXL medium by bind-wash model, thedata show that scFv98H can be well separated from the impurity; when inclusionbody protein of scFv98H was chromatograph purified on DEAE、QXL、Nuvia Qmedium by flow-through model, the data indicate that scFv98H can also be wellseparated from the impurity, and with higher recovery on Nuvia Q. The process of scFv98H for diagnostic use is that IMAC first, then on QXL model by bind-washmodel, and followed by dialysis refold. The process of scFv98H for therapeutic use isthat Nuvia Q flow-through model first, then desalting, and followed by QXLon-cloumn refold.4. Preparation and diagnostic use of scFv98HFor diagnostic use, the scFv98H protein was purified on5ml His-Trap-HPImmobilized Metal Affinity Chromatography (IMAC) column. SDS-PAGE analysis,purity was about90%after one IMAC step. The pooled fractions were loaded onto theHiPrep16/10Q Sepharose XL column (HiPrep16/10QXL) again, purity wasabout95%. The antigen-specific binding characteristics, affinity(Kd=1×106M1),relative affinity was tested. The scFv98H antibody was evaluated as a diagnostic tooland compared with a commercial NP mAb for assaying the VNA level of anti-rabiesserum samples from different sources or to test the growth kinetics of RABV strainsfor vaccine manufacture in China. The results indicated that scFv98H may be used asa diagnostic tool to assay the VNA levels or virus titers. The scFv98H may be used asan alternative for the presently used diagnostic antibody for these purposes.5.Preparation and RABV PEP use of scFv98HFor RABV PEP therapeutic use, the scFv98H protein was purified on20ml Nuvia Qcolumn by flow through model, and then refolded on20ml Q Sepharose XL columnby reducing concentration of urea from8M to0.1M. The neutralization activity ofscFv98H against RABV in vitro was determined by a pseudovirus neutralization assayand rapid fluorescence focus inhibition (RFFIT) test. ScFv98H can specificallyneutralize pseudovirus and the neutralization activity was no less than1000IU/mg byRFFIT. Immunofluorescence staining and flow cytometric analysis conducted to studythe antiviral mechanism of scFv98H indicated that this novel antibody could bindwith RABV expressed on the cell surface.To test the antiviral therapeutic efficacy of scFv98H in vivo, an animal model wasconstructed. The scFv98H protein at the concentration of20IU/kg could protect80%-100%of mice from death after challenge with an LD90dose of RABV.Altogether, these findings support the potential of scFv98H as a replacement for the RIG currently used as an antiviral therapeutic agent against RABV.For human therapeutic use of the His-tag should be avioded, so the scFv98Nexpression vector was constructed. Comparision of bingding activity, neutralizingactivity in vitro and in vivo, Kd, relative affinity between scFv98N and scFv98H, dataindicate that the difference is not significant.To sum up, the scFv98H protein can be used as diagnostic or therapeutic agent.