| Part 1 Selection and evaluation of oncolytic coxsackievirusEnterovirus?EV?is a class of positive single-stranded RNA virus,including coxsackievirus,poliovirus,echovirus,etc.,and is the main pathogen of hand,foot and mouth disease?HFMD?,herpangina,poliomyelitis and so on.It can infect some tumor cell lines,and induce cytopathic effect because of its short replicative cycle.Nowadays,many kinds of oncolytic EVs have been reported like coxsackievirus A21?CV-A21?.However,most of them are devoted to treat tumors with high incidence in European and America like malignant melanoma,and there are less researches of EVs to cure the most common cancers in Asia like lung and hepatic carcinomas.In this study,oncolytic enterovirus coxsackievirus B5?CV-B5?/ Faulkner?Gen Bank: AF114383?was screened out which could be applied to lung cancer treatment,and some oncolytic mechanisms were probed.The results showed that only CV-B5/Faulkner and CV-B5/JS417 could selectively infect lung cancer cells but not normal lung cells when we used CV-B3/Nancy,CV-B3/112,CV-B5/Faulkner,CV-B5/JS417,coxsackievirus A6?CV-A6?/Gdula to infect many kinds of lung cancer cell lines,normal lung tissue cell lines,liver cancer cell lines,normal liver cell line and cervical cancer cell line.Salient dose dependent effect could be showed when using gradient dilutions of CV-B5/Faulkner to infect lung cancer cells,including A549,H1299 and NCI-H460 cell lines.There is no significance to compare the difference of viability between MRC-5,KMB-17,2BS cells infected by 100 MOI of CVB5/Faulkner and control?P>0.05?.The western blotting and flow cytometry results indicated that coxsackievirus and adenovirus receptor?CAR?,the major receptor of CV-B5,only expressed at the surface of lung cancer cells,which ensured the selective infectivity of CV-B5 to some extent.A bearing cancer BALB/c nude mice model was established by means of transplanting A549,H1299,NCI-H460 cells.The results indicated that a serial of five intratumoral injections(5 ×106TCID50 per)can completely restrain or even cure tumors of H1299?P<0.01?,restrain tumors of A549 significantly?P<0.05?,but cannot suppress tumors of NCI-H460?P>0.05?when the diameters of tumors were reaching 4-5mm.The results of using different injections to H1299 tumors showed that merely one or three injections?5×106TCID50 per?can completely restrain the tumors of 4-5mm or 8-9mm in diameters respectively,extend the survival time to 30-90 days.In order to testify the tumor targeting in distance,bilateral bearing mice were treated by 5 unilateral injections(5 ×106TCID50 per)of CV-B5/Faulkner,and the results showed that non-treated tumors shrunk and were restrained by the treatment of the lateral significantly?P<0.05?.The pathological evidences demonstrated main organs of mice after treated by CV-B5/Faulkner,including brain,lung,liver,pancreas,spleen,kidney and heart,showed no apparent histological and pathological changes,which proved the security of CV-B5/Faulkner serving as a oncolytic virus.Moreover,the results indicated that the oncolytic effect of CV-B5 relied on the cascade activation of apoptosis induced by virus infection.What's more,it is an obstacle that CV-B5 of 0.1MOI can only induce relatively weaker oncolysis of NCI-H460 cells in vitro.A great number of inhibitors of signal transduction were screened in vitro combined with CV-B5/Faulkner,and inhibitors of protein kinase B?PKB?was screened out which can significantly synergized with the oncolytic effect of NCI-H460?P<0.05?.In this study,an oncolytic enterovirus CV-B5/Faulkner had specific oncolytic effect on lung cancer cells by recognizing CAR probably in vitro and in vivo.Apoptosis pathway was activated by the infection of CV-B5 which is a part of oncolytic mechanisms.Furthermore,PKB pathway inhibitors can be used to synergized with the oncolytic effect of some refractory tumors.These results indicated that CV-B5/Faulkner has the potential to be studied and become an oncolytic vaccine.Part 2 Establishment and validation of a uniform quantitative assay for Coxsackievirus A16 antigenCoxsackievirus A16?CV-A16?,a member of EV A class,usually leads to mild and severe HFMD.A great many manufacturers are dedicated to develop CV-A16 vaccines in China nowadays.It is a key link for vaccine development,quality control?QC?and evaluation to quantify content of CV-A16 antigen.A uniform method to quantifying CV-A16 antigen is lacking universally,which is a challenge for vaccine development and evaluation.To guarantee the smooth development of CV-A16 vaccine in China,an enzyme linked immunosorbent assay?ELISA?was adopted to develop a uniform method.In this research,totally 5 monoclonal antibodies?m Abs?were compared of its neutralizing ability,binding ability and in vivo protective effect.A conformational m Ab 16E1 from mouse was screened out,and it could neutralize subgenotype B1 b and B2 b of CV-A16?titer?9600?,and bind subgenotype B1 a,B1b and B2 b of CV-A16?S/CO>30?.Moreover,10?g/g 16E1 can protect 100% neonatal mouse against virus challenge of lethal dose at day 1.By groping the reactionary condition and main parameters,we established a detective system using CV-A16 national antigen standard?CV-A16 NS?as standard substance and 16E1 as coating antibody and horseradish peroxidase conjugated polyclonal antibody?p Ab?from rabbit?neutralizing titer:1/6144,binding titer:106?as secondary antibody.The linear range was 12.5-200U/ml when using parallel line analysis to curve-fit.Methodological validation results showed that this kit did not cross react to other enteroviruses like enterovirus A71,and the accuracy of quantifying CV-A16 NS of different concentrations ranged from 80% to 115%,the CV values of repeatability and intermediate precision were less than 15%,which all satisfied the requirements of pharmacopeia.Applicability research enrolled six labs developing CV-A16 inactivation vaccines in China.These labs quantified their own vaccine bulks,harvested and purified products developed with individual strains,including subgenotype B1 a,B1b,B2 a,B2b.The results indicated that the parallelism and linearity were consistent with CV-A16 NS?P>0.05?,and the slope of different samples merely had a small fluctuation?-0.85-1.25?,which indicated that this kit could be applied to quantify the concentration of CV-A16 antigen of bulks which were developed from different crafts and strains.Additionally,the parallelism and linearity between intermediate products which included harvested products,purified virus and bulk,and CV-A16 NS were acceptable,and there was no significant difference?P>0.05?.The slopes of these three curves only fluctuated a little?-0.97-1.04?,which showed that this kit was insusceptible to the interference of other substances existing in samples and could be used to quantify the content of effective antigen in different intermediates.In this study,a m Ab was screened and selected out which have high potency and broad spectrum neutralization capability.By means of using sandwich ELISA?m Ab-p Ab?,a CV-A16 antigen detection kit was established and validated,and the specificity,repeatability and precision were all acceptable.The results of applicability research encompassing six labs and three intermediate products indicated that this kit could satisfy different subgenotypes and crafts,and be used as a uniform kit,which can guarantee the precision,repeatability and comparability of the evaluation of antigen activity and effective appraisal of CV-A16 inactivated vaccine and is a tool for CV-A16 vaccine development. |
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