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Objective:To establish VSV-G qPCR assay for detection of replication competent lentivirus(RCL) and verify its application.Methods:A real-time fluorescent quantitative PCR for VSV-G envelope gene was developed. Several parameters including specificity, linear, amplification efficiency, precision, trueness, dynamic range, limit of detection, limit of quantification and robustness were verified. Preliminary application on CAR-T cells, end of production cells and the harvest of lentivirus vector was performed by using the method developed.Results:The real-time fluorescent quantitative PCR assay for VSV-G was specific for the detection VSV-G without specific amplification on 293T, PBMC and C8166 cells. The linear range of the assay was 1×10 2 copies/test-1×10 9 copies/test with a R2 value more than 0.998 and amplification efficiency between 93% and 98%. The precision (relative standard deviation) of the assay was less than 12% and the trueness (the rate of recovery) of the assay was between 85% and 106%. The limit of detection (LOD) and limit of quantification (LOQ) of the assay was 5 copies/test and 40 copies/test. In addition, the robustness of the assay was also well. All the results of validation illustrated that the assay could meet the detection requirements. All of the 54 samples including CAR-T cells, lentivirus vector and end of product cells after amplification and passage on C8166 cells were negative of RCL by using the established assay. Conclusions:The real-time fluorescent quantitative PCR for VSV-G were established successfully. All of the validation results illustrated that the assay could meet the detection requirements. The application of the assay was conducive to further enhance the safety of the lentivirus vector related products.
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Objective To optimize and compare four in vitro cytotoxicity assays in order to find a relatively rapid assay that can replace the traditional 51Cr release assay to evaluate the cytotoxicity of immune cells prepared for immunotherapy.Methods Four assays including BATDA, CAM (calcein acetoxymethyl ester), CytoTox-Glo and PKH were optimized and used to measure the in vitro cytotoxicity of NK-92 cells to K562 cell line.Intra-and inter-assay reproducibility of these assays and their correlation with 51Cr release assay were analyzed.Results After optimization, all of the four cytotoxicity assays showed good correlation with effector to target (E/T) ratio in a certain range.Compared with the other three assays, CytoTox-Glo assay showed obvious hook effect at a high E/T ratio of 40∶1.BATDA assay could detect the significant cytotoxicity of NK-92 cells to K562 cells after incubating them for one hour and that was the shortest time taken by the four assays to detect in vitro cytotoxicity.Both CAM and PKH assays took about four hours and CytoTox-Glo assay took six to eight hours to detect the significant cytotoxic potency.All of the four assays, especially BATDA and CAM assays, showed good intra-and inter-assay reproducibility.Among these assays, BATDA assay showed the best correlation with the traditional 51Cr release assay.BATDA assay, as compared with the other three assays, could be used to detect the cytotoxicity of Caov3 cells, an adherent cell line, and showed good results in evaluating the cytotoxic potency of autologous primary NK cells and CD19-CAR T cells.Conclusion Compared with the other three assays, BATDA assay shows the best linear correlation with 51Cr release assay and has the advantages of time-saving and good reproducibility.Besides, it is a better assay for detecting the cytotoxicity of adherent cells.BATDA assay is a promising substitute for 51Cr release assay in evaluating the in vitro cytotoxic potency of NK cells and other immune cells.
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Abstract] Objective To establish an assay for the detection of porcine parvovirus ( PPV) and to verify its application for monitoring cells used for production.Methods A pair of primers and one probe were designed according to the conserved sequence encoding non-structural protein 1 (NS1).Based on the designed primers, a real-time fluorescent quantitative PCR assay for the detection of PPV was developed. Several parameters including the linearity, precision, minimum detection limit and anti-interference of the established assay were evaluated.A stock of PPV strains was prepared by infecting swine testicle ( ST) cells with PPV strains.An assay for the detection of PPV infection was developed by using ST cells as sensitive cells.A combined ST cell infection-PCR test was developed by combining the ST cell infection assay with the real-time fluorescent quantitative PCR assay.The sensitivity of ST cell infection-PCR test was analyzed.The cell samples used for production of biological products were detected by using the established assay.Results The real-time fluorescent quantitative PCR assay was specific for the detection of PPV without cross-reaction to other species of parvovirus virus, SV40 virus and other porcine viruses.The linear range of the assay was 1×109-1×104 copies/μl with a R2 value more than 0.98.The sensitivity of the real-time quantitative PCR assay was 1×104 copies/μl.Both of the intra-and inter-coefficient of variation (CV) were less than 5%in Ct values.The intra-and inter-CV in copies of detection were 5%-15% and 30%-40% respectively.The minimum detection limit of the real-time fluorescent quantitative PCR assay was 1CCID50/ml.The PPV strains were detected in cell samples with no interference.The sensitivity of ST cells infection-PCR test was 0.01CCID50/ml.All of the 22 cell samples were negative for PPV by using the real-time fluorescent quanti-tative PCR assay.Conclusion The real-time fluorescent quantitative PCR and the ST cell infection-PCR test for the detection of PPV in cells were established successfully.The application of the two assays was conducive to further enhance the safety of using cells for production and therapy.
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Objective To establish an assay for the detection of Torque teno sus virus ( TTSuV) strains and to analyze its preliminary application to biologics.Methods Primers and probe were designed according to the conserved sequences.A fluorescent PCR assay for the detection of TTSuV strains was estab-lished.Several parameters including the specificity, linearity, accuracy, sensitivity and anti-interference of the established assay were verified.The fluorescent PCR assay was performed to detect the samples of por-cine blood, cell substrate and rotavirus vaccine.The porcine blood samples positive for TTSuV strains were further genotyped.Results The established fluorescent PCR assay was confirmed to have high specificity as no cross-reactions with parvovirus virus of various species, SV40 and porcine circovirus strains were detec-ted.The linear range of the assay was 1×109-1×103 copies/μl for TTSuV1 genotype and 1×109-1×102 cop-ies/μl for TTSuV2 genotype with a R2 value more than 0.993.The sensitivity of the fluorescent PCR assay was 1×103 copies/μl for TTSuV1 genotype and 1×102 copies/μl for TTSuV2 genotype.The intra-and inter-CVs were both less than 7%in Ct values and less than 25% and 45% respectively in copies.No interfer-ence was found in the detection of TTSuV nucleic acids from cell samples.8 out of 20 porcine blood samples were positive for TTSuV strains, among which one sample was positive for TTSuV1 genotype, four samples were positive for TTSuV2 genotype and the rest were positive for both TTSuV1 and TTSuV2 genotypes.Com-pared with the reference strain, strains genotyped as TTSuV1 and TTSuV2 were respectively shared 98%-99%and 98%homologies in sequences.All of the cell substrate and rotavirus vaccine samples detected by the fluorescent PCR assay were negative for TTSuV strains.Conclusion The fluorescent PCR assay for the detection of TTSuV was established successfully, the application of which would further improve the safety of biologics.
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Objective To construct the bait plasmid of pSos-single immunoglobin IL-1 receptor related protein (SIGIRR) in Cy-toTrap yeast two hybrid system ,and to test its self-activation .Methods The cDNA fragments of SIGIRR(480 -1 230 bp) were amplified from pReceiver-LV19-SIGIRR and ligated into the bait plasmid pSos to generate the plasmid pSos-SIGIRR .The pSos-SI-GIRR was identified by DNA sequencing and dual-site endonuclease digestion .Then the recombinant plasmid and control plasmid were introduced into the yeast cell cdc25H .The transformants were inoculated on plates of 25 ℃ /SD/Glucose(-UL) ,25 ℃/SD/Ga-lactose(-UL) ,37 ℃ /SD/Glucose(-UL) and 37 ℃ /SD/Galactose ,respectively and the proliferation ability of transformant was ob-served for 6 d .The Western blot was adopted to detect the expression of target protein .Results The pSos-SIGIRR vector was cor-rectly constructed and proved of no self-activation and toxic action .The Western blot showed that the target protein was expressed in a form of fusion protein of 170KD .Conclusion The bait plasmid containing SIGIRR cytoplasmic tail can be applied to the yeast two-hybrid system and lays the important foundation for seeking the interacting protein with SIGRR from the human lung cDNA li-brary in .
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Objective To conduct extensive quality control tests on Madin-Darby Canine Kidney ( MDCK) cells used for the production of influenza vaccine .Methods Tests for characteristics , extraneous agents, endogenous agents and tumorigenicity were performed on MDCK cells according to Chinese Pharma -copeia Book III .Cell lysate and DNA of MDCK cells were tested for oncogenicity in the light of new interna -tional requirements .Results The MDCK cells extracted from canis were adherent cells with an epithelial morphology, whose average number of chromosome was 80±1.No bacteria, fungi and mycoplasma contami-nation were detected . The detection for extraneous and endogenous virus showed that there was no nonspecific virus causing cytopathic effect , hemadsorption , hemagglutination or animal death .Tests for re-verse transcriptase , bovine viruses and canine viruses were all negative .Each nude mouse was injected with 107 viable cells to observe their tumorigenicity .Twelve weeks after cell injection , no node was found at the injection site and in large organs by gross anatomy .There was no significant difference between test group and negative control group .The test for tumorigenicity of viable cells was negative .Cell lysate and cellular DNA collected from equivalent amount of cells were respectively injected into nude mice , and no node forma-tion was found.There was no significant difference between the test cells and negative controls in pathology indicating that the tested MDCK cells were non-oncogenic .Conclusion It showed the possibility of using MDCK cells for the production of influenza vaccine .
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The serum samples and corresponding cervical swabs were collected fi'om 50 women with genital warts from Tianjin city,China.The neutralizing antibodies against HPV-16,-18,-58,-45,-6 and-11 in serum samples were tested by using pseudovirus-based neutralization assays and HPV DNAs in cervical swabs were also tested by using a typing kit that can detect 21 types of HPV.The results revealed that 36%(18/50)of sera were positive for type-specific neutralizing antibodies with a titer range of 160-2560,of which 22%(11/50),12%(6/50),10%(5/50),4%(2/50),4%(2/50)and 2%(1/50)were against HPVs-6,-16,-18,-58,.45 and-11,respectively.Additionally,60%(30/50)of samples were HPV DNA-positive,in which the most common types detected were HPV-68(18%),HPV-16(14%),HPV-58(12%),HPV-33(8%)and HPV-6,HPV-11,HPV-18 and HPV-52(6% each).The concordance between HPV DNA and corresponding neutralizing antibodies was 56%(28/50)with a significant difference(P＜0.05).The full-length sequences of five HPV types(HPV-42,-52,-53,-58 and-68)were determined and exhibited 98%-100% identities with their reported genomes.The present data may have utility for investigating the natural history of HPV infection and promote the development of HPV vaccines.
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Objective To study the profiling and authentication of human cell lines in cell bank of our department using short tandem repeat (STR) loci and to analyze the situation of cell contamination and misjudgment. Methods Sixty-one human cells including cells collected and preserved by our cell bank and cells from the other departments were detected by the 16 STR loci-method. To analyze the cross contamina-tion between human cell lines, the results were aligned with profiles published by international cell banks. Isoenzyme detection was employed to authenticate the cell species when the STR signal can not be detected. Results Among the 61 cells, specific profiles were produced by 41 ceils and there was no cross contamina-tion. Thirty-six cells had the completely same STR profiles in 9 STR loci with the same cells preserved by ATCC or JCRB, while 5 cells have different profiling just in the vWA loci. The cells referred above can be recognized as correct cells; Eleven cells (18.0%) were the false cells. Among them, cancer cells of tongue named Tca8113 and cancer cell of liver named HHCC(changed to FHCC98 now) had the same profile with HeLa and HeLa S3 respectively; Two ceils both named HUT-102 have the completely different profiles with ATCC; The signal of 4 cells was not be detected, and all of them were determined as hamster cell lines by u-sing isoenzyme detection. Also 2 cells were identified to be mixed cells. Conclusion The phenomenon of cell misjudgment and cross contamination between cells is serious. Authentication of cell lines correctly, es-pecially for the re-authenticatian of domestic self-established cells, is very important for the guarantee of the reliability and reproducibility for scientific researches.
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Objective To detect the protection induced by HPV-58 L2 11-200 AA in animal, and analyze the relationship between antibody or neutralizing antibody titers and the protection generated by the immunizmg agent. Methods The peptide of HPV-58 L2 11-200 AA was expressed in E. coli and the mice were immunized with the peptide after purification and adsorption with aluminum adjuvant. The protection provided by different immunizing doses was detected in the mouse model against the challenge of the pseud-ovirions of human papiilomavirus types 58. The total antibodies and neutralizing antibody titers of serum were tested with ELISA and neutralization assay against HPV-58 pseudovirus, respectively. The total antibodies or neutralizing antibody titers that can protect the mouse from infection were analyzed. Results The mice can be protected from the challenge with HPV pseudovirus when the immunizing dose was 8 μg. The neutralizing antibody can not be detected in the immune serum by neutralization assay against pseudovirus. The total anti-body level has a corresponding relationship with the protection showed in mouse model. The results of total antibodies detected by ELISA showed that when the titer of total antibodies was ≥25 000, luminescent signal can not be detected and the mice can be protected from pseudovirus infection. Conclusion HPV-58 L2 11-200 AA peptide can protect mice from pseudovirus infection. L2 peptide has a promising perspective to be a candidate vaccine and the level of total antibodies in the immune serum can be used as a surrogate for the evaluation of protection against HPV infection.
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Objective To study the influence of the modification of the special neutralizing epitopes of the HIV-1 envelope (Env) on its assembly of functional pseudovirus and neutralizing activity. Methods Site-directed mutations were performed using cycling mutagenesis and selection of mutants with Dpn I . With this method, the 2G12 and 2F5 neutralizing epitopes were integrated into Env of subtype BC which was without the two epitopes, then the capability of forming pseudovirus and the neutralizing activity against 2G12 and 2F5 were compared with pre-modified Env. Results The special Env neutralizing epitopes of five HIV pseudovirus (BC02, BC03, BC04, BC05 and BC12) were modified. Among the five pseudovirus, BC04 and BC12 pseudovirus can't be formed after the 2G12 epitope was modified, whilst the BC02, BC03 and BC05 pseudovirus can be formed after the 2G12 and 2F5 epitopes were added, and there was no variation of the pseudovirus titer; On the aspect of neutralizing activity, BG03 pseudovirus against 2G12 and 2F5 was enhanced, BC02 and BC05 pseudovirus against 2F5 was enhanced while which against 2G12 was not changed. Conclusion The modification of 2G12 epitope influences the forming of pseudovirus and the addition of neutralizing epitopes can enhance the neutralizing activity of pseudovirus, which offers new approach for the optimization of HIV immunogen.
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Objective To study the application of short tandem repeat (STR) profiling in quality control of human cell lines used for biological production. Methods The methods detecting 9 and 16 STR loci to identify human cell lines by PCR-capiilary electrophoresis were established respectively. Human cell lines, which were derived from many corporations and including diploid cell strains used for virus-vaccine production and 293 cell lines used for gene therapy products, were analyzed and compared by these two methods. Results The STR profiling methods used for authentication of human cell lines were established. Most of human diploid cell strains(20/21 ) used for virus-vaccine production from 13 corporations were iden-tiffed as the intended cells and no cross-contamination was found. However, one MRC-5 cells was identified as a false cell line and one MRC-5 had 3 alleles in D13S317 locus. For 12 strains of 293 cell lines, there were significant differences in STR profiling from different manufactures, which was likely be explained that the sources and gene modifications of these 293 cell lines are not well known and their genes are unstable during passage. Conclusion The STR profiling method has the advantages of high sensitivity and specifici-ty, and can be used for authentication of each of human cell lines for biological production.
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Eleven env mutants were designed and generated by site-directed mutagenesis of the regions around Nab epitopes and deletions of variable regions in env.The immunogenicities of the generated mutants were evaluated using single-cycle infection neutralization assays with two pseudoviruses and IFN-γELISPOT.Overall,five mutants(dWt,M2,M5-2,M5-1 and dM7)induced highed neutralization activities for both pseudoviruses than plasmid Wt,while only two of the mutants(dWt and M5-2)showed significant differences(P<0.05).Two mutants(M2 and dM2)induced more Env-specific T cells than plasmid Wt.Statistically however,significance was only reached for mutant M2.Thus,properly modified HIV-1 Env may have the potential to induce potent cellular and humoral immune responses.
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Objective To study the influence of site-directed deglycosylation of the HIV-1 envelope (Env) on its immunogenicity and assembly of functional pseudovirus. Methods Site-directed deglycosylation were performed using cycling mutagenesis and selection of mutants with DpnⅠ. Single-cycle infection assay was employed to analyze the effect of the mutations on the ability of functional pseudovirus assembly. The influence of deglycosylations on the immunodeficiency of Env was evaluated using pseudovirusbased neutralization assay and ELISPOT assay. Results Mutant N197Q induced higher neutralization activities for both pseudoviruses, but lower Env-specific T-cell response. And N197Q rendered the Env to lose the ability of functional pseudovirus assembly. Mutant G2 induced higher neutralization activities for pseudovirus 74-2 but lower for pseudovirus Wt, and had almost no influence on Env-specific T-cell response and functional pseudovirus forming. Conclusion The site-directed deglycosylation of the HIV-1 Env affects the pseudovirus forming and its immunogenicity.
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Objective To establish the mouse model for human papillomavirus types 16, 45 and 58 by the corresponding pseudovirions. Methods The 293FT cells were co-transfected with eodon-modified HPV eapsids genes together with a reporter plasmid containing the luciferase gene. The cells were collected and lysed, then the pseudovirus was collected and the titration was performed. The mouse was subcutaneous-ly injected with Depo-Provera. After 4 d and intravaginally injected with nonoxynol-9, and 6 h later pseud-oviruses were inoculated in intravaginal. After 7 d, the mouse was instilled luciferin substrate intravaginally, and the expression level of the lueiferase gene was detected by the in vivo Imaging System (IVIS). Results Three types (HPV16, HPV45 and HPV58) of pseudoviruses had been produced and the titer was 3.7×108 TU/ml, 1.5×108 TU/ml and 1.2×108 TU/ml, respectively. The luminescent regions could be detected in the mice which were infected with the pseudovirions, the luminescent signal intensity for types 16,45 and 58 was 1.779×106p/s, 5.738×105×p/s and 1.829×106p/s, respectively. Conclusion The mouse models for HPV16, 45 and 58 have been successfully established based on pseudovirions, which will be very useful for the research of HPV infection intervention, the evaluation of HPV vaccines and the screening of the prophylactic agents.
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Clinical research is to provide guidelines for the clinical practice.First clinical re search should follow the ethics,and the researcher should have correct scientific attitude.Furthermore clinical research should be closely integrated with clinical practice and innovated with its feasibility taken into account.
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Objective:To investigate the effects of nitric oxide donor,sodium nitroprusside(SNP)on the expression and production of TLR4 in U937 induced by LPS.Methods:U937 was induced to maturation by PMA and then stimulated by LPS.Then the cells were treated with SNP and divided into four groups:control group without stimulation of LPS,LPS group(10 ng/ml LPS+100 ng/ml rhLBP),low dose SNP group and high dose SNP group(50,500 ?mol/L SNP respectly).The mRNA and protein of TLR4 was determined by RT-PCR and Western blot.Results:The mRNA of TLR4 in SNP groups was lower than those in LPS group(0.308?0.050 and 0.138?0.0044 vs 0.342?0.098,P0.05).Conclusion:SNP might have a potential protective role in LPS induced inflammation such as sepsis and acute lung injury through inhibiting the mRNA and protein expression of TLR4.
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AIM:To investigate the inhibitory effect of P12,a kind of lipopolysaccharide(LPS)-binding protein(LBP) inhibitory peptide,on the binding of LPS to macrophage in vitro.METHODS:Human monocyte-like cell line(U937 cells) was grown in RPMI-1640 and stimulated with PMA in order to induce their differentiation to macrophage stage.The relative affinity of P12 to LPS was determined by enzyme-linked immunosorbent assay(ELISA).The effects of P12 on the binding of LPS to U937 cells were determined by flow cytometry analysis.The production of tumor necrosis factor-alpha(TNF-?) was measured by ELISA.RESULTS:The relative binding activity of P12 to LPS was higher than that of LBP in the same mass concentration.P12 inhibited the binding of FITC-conjugated LPS(FITC-LPS) to U937 cells.The productions of TNF-? was also significantly suppressed by P12.CONCLUSION:The results suggest that blockage of LBP at the inflammatory sites might attenuate LPS-induced circulatory shock.