Current Issue

  • Long-term toxicity test of oral hexavalent reassortant live attenuated rotavirus vaccine(Vero cell) in SD rats

    LUAN Chunfang;JIANG Xiaoming;WANG Xiaoxin;XU Shuzhen;ZHAO Zhenbo ;FAN Yanming ;WANG Jidong;TIAN Lei;MENG Fanhong;Sinovac (Dalian) Vaccine of Technology Co., Ltd.;

    ObjectiveTo evaluate the long-term toxicity of oral hexavalent reassortant live attenuated rotavirus(RV)vaccine(Vero cell) in SD rats, so as to provide theoretical basis for the clinical application of the vaccine.MethodsA total of 150 SPF healthy rats with a male-to-female ratio of 1∶1 were selected and divided into experimental groups and satellite groups, 120 rats in experimental groups and 30 rats in satellite groups. The experimental groups included high-dose group[oral hexavalent reassortant live attenuated rotavirus vaccine(Vero cell), 2 doses/rat], low-dose group [oral hexavalent reassortant live attenuated rotavirus vaccine(Vero cell), 1 dose/rat], negative control group(0. 9% sodium chloride, 4 mL/rat)and vector control group(vector control, 4 mL/rat), with 30 rats in each group, and the rats were administered by gavage, once every 2 weeks, for a total of 4 doses. The clinical characterization of the rats was observed, and the weight, body temperature,ophthalmologic examination, clinical pathology(blood count, coagulation function, blood biochemistry and urinalysis), virus absorption, tissue distribution, gross autopsy, weighing of major organs, and histopathological examination were performed.The satellite groups randomly divided 30 rats into high-dose, low-dose, and negative control groups, with the same dosage as the experimental groups, for immunological parameter assessment and virus shedding studies.ResultsThere were no regular changes with toxicological significance in clinical characterization, body weight, body temperature, ophthalmic examination,hematology, coagulation function, blood biochemistry, urinalysis and other indicators in each group. The RV-specific IgA antibodies could be detected in rats of both high-dose and low-dose groups, and the highest antibody titer reached 1∶80. No administration-related changes were observed in organ weights and organ coefficients of rats in each group, and no administration-related systemic toxicity pathological changes were found in histopathological examination.ConclusionNo systemic toxicity was observed in the repeated dose toxicity test, and the no observed adverse effect level(NOAEL) dose was considered to be 2 doses/rat.

    2026 03 v.39 [Abstract][OnlineView][Download 954K]

  • Construction and verification of recombinant vesicular stomatitis virus expressing tick-borne encephalitis virus prM-E protein

    NING Yuying;ZHANG Luomiaoyuan ;YANG Xiaojing ;CUI Yun;ZHANG Youdi;YE Wei ;YUAN Ruodong ;CAI Xinyu ;ZHANG Hui ;LEI Yingfeng ;DONG Yangchao ;ZHANG Qianqian;Yan'an Medical College of Yan'an University;

    ObjectiveTo construct a recombinant virus expressing tick-borne encephalitis virus(TBEV) prM-E protein using vesicular stomatitis virus(VSV) as a vector, and to identify it, so as to provide a basis for the research of TBEV vaccines based on VSV vector.MethodsThe prM-E gene of the TBEV Senzhang strain was inserted between the G and L genes of the VSV vector. The recombinant virus was rescued by co-transfecting BHK-21 cells infected by poxvirus containing T7 RNA polymerase with helper plasmids expressing VSV N, P, L, and G proteins. The supernatant was collected, and the recombinant virus rVSV-TBEVprM-E stably expressing prM-E was obtained after multiple passages. Western blot, immunofluorescence assay(IFA) and RT-PCR were used to identify the expression of prM-E protein and gene of rVSV-TBEVprM-E in cells. The viral titers of rVSV-TBEVprM-E at different time points were determined by plaque assay and the growth curve was plotted.ResultsTBEV prM-E gene was successfully inserted into the genome of recombinant virus rVSV-TBEVprM-E, and the expression of prM-E protein in BHK-21 cells was detectable. After serial passages, rVSV-TBEVprM-E achieved a viral titer of 6. 75 × 10~5 PFU/mL.ConclusionA recombinant virus rVSV-TBEVprM-E expressing prM-E protein was successfully constructed, which lays a solid experimental foundation for the related research of TBEV.

    2026 03 v.39 [Abstract][OnlineView][Download 1089K]

  • Construction of lentiviral vectors and transfection of THP-1 cell lines to achieve stable low expression of sphingosine-1-phosphate receptor 1

    TAN Jiajia;ZHAO Ling;SU Qiuyuan;ZHOU Haiqin;MO Shien;LU Fangfang;ZHOU Yang;WEI Yi;KUANG Yan;The First Affiliated Hospital of Guangxi Medical University;

    ObjectiveTo construct lentiviral vectors that interferes with the expression of sphingosine-1-phosphate receptor 1(S1 PR1) and stably transfect the lentiviral vectors into THP-1 cells, in order to study the effect of S1 PR1 on macrophage function and the mechanism of S1 PR1 in tumor development at the cellular level.MethodsAccording to the sequence of S1 PR1 gene(1901) registered in NCBI database, three pairs of primers were designed for the shRNA sequence of this gene. The target gene was amplified by PCR, inserted into vector pLKO.1-puro, and the corresponding lentiviral plasmids were constructed,which were identified by enzymatic digestion electrophoresis and sequencing. The correct lentiviral vector plasmids were named pLKO.1-S1 PR1-shRNA1, pLKO.1-S1 PR1-shRNA2, and pLKO.1-S1 PR1-shRNA3, and the vector pLKO.1-puro containing stuffer sequence was used as control(named pLKO.1-S1 PR1-shNC). The lentiviral vector plasmids and lentivirus packaging plasmids were co-transfected into 293T cells for virus packaging, and the titer of virus solution was determined after concentration. The screening concentration of puromycin(0, 0. 5, 1. 0, 1. 5, 2. 0, 2. 5 μg/mL), culture time(0, 24, 48, 72 h)and MOI(10, 20, 30, 40, 50) of THP-1 cells were determined. THP-1 cells were infected with the lentivirus under the optimum conditions and screened by puromycin. The relative mRNA and protein expression of S1 PR1 in THP-1 cells of each group were detected by RT-qPCR and Western blot respectively.ResultsEnzymatic digestion electrophoresis identification and sequencing indicated that pLKO.1-S1 PR1-shRNA1, pLKO.1-S1 PR1-shRNA2, and pLKO.1-S1 PR1-shRNA3 lentiviral vectors were correctly constructed. The lentivirus titers of shNC, shRNA1, shRNA2 and shRNA3 groups were 9. 5 × 10~9, 4. 25 × 10~9,2 × 10~9 and 4. 4 × 10~9 TU/mL, respectively. THP-1 cells were infected with the lentivirus at the optimum MOI of 50 for 72 h.After screening with 1. 5 μg/mL puromycin, the relative expression levels of S1 PR1 mRNA in shRNA1, shRNA2 and shRNA3 groups were significantly lower than that in shNC group(F = 44. 916, P < 0. 001); the relative expression level of S1 PR1 protein in shRNA1 group decreased with no significant difference(t = 1. 921, P > 0. 05), while the relative expression levels of S1 PR1 protein in shRNA2 and shRNA3 groups decreased significantly(t = 8. 730 and 6. 957, respectively, each P < 0. 05).ConclusionThe lentiviral vectors interfering with S1 PR1 expression and THP-1 cell lines stably expressing the vectors were successfully constructed, which can be used for further related research.

    2026 03 v.39 [Abstract][OnlineView][Download 1089K]

  • Glycan receptor binding characteristics of GⅡ.5 norovirus capsid proteins

    CONG Xin;LI Hanbo;HUANG Lisu;DUAN Zhaojun;Department of Infectious Disease,Children's Hospital,Zhejiang University School of Medicine,National Clinical Research Center for Child Health;

    ObjectiveTo analyze the glycan receptor binding characteristics of capsid proteins of GⅡ.5 norovirus(NoV) in order to lay a foundation for the development of antiviral drugs and vaccines for NoV.MethodsThe P proteins of GⅡ.5 N490 and GⅡ.5 12X were expressed in prokaryotic system respectively, purified using glutathione affinity chromatography, and then the binding characteristics of P proteins with saliva and glycan were detected by ELISA. The structure of two GⅡ.5 strains was modeled and the binding amino acids between the P protein and glycan were analyzed based on the epidemic GⅡ.17 KW308.ResultsThe relative molecular mass of the both P proteins of GⅡ.5 N490 and GⅡ.5 12X was 61 000,which were expressed in soluble from in the supernatant, and the purified concentration was 0. 2 mg/mL. The P proteins of two GⅡ.5 strains could bind to most of A, B, O secretory and non-secretory saliva. The P protein of GⅡ.5 N490 exhibited binding to H disaccharide, while the P protein of GⅡ.5 12X showed specific binding to H disaccharide and B trisaccharide. In addition, the model of two GⅡ.5 strains displayed a conformation similar to that of GⅡ.17 KW308. Among the six amino acid sites interacting with glycan, Arg349, Asp378, Gly443 of GⅡ.5 N490 and GⅡ.5 12X were the same as those of G Ⅱ.17 KW308, while Asn375, Glu380 and Phe444 were different from those of G Ⅱ.17 KW308.ConclusionThe two strains of G Ⅱ. 5 exhibit similar binding characteristics in saliva binding patterns with P proteins and both have a wide susceptible population, while the glycan binding patterns are slightly different.

    2026 03 v.39 [Abstract][OnlineView][Download 1022K]

  • Expression and purification of Bacillus anthracis phage endolysin PlyL and its role in Bacillus anthracis detection

    XU Minghan;LU Gejin ;JING Jie ;LIANG Bing ;ZHENG Lin ;WANG Zixian ;LI Jiannan;ZHU Lingwei ;GUO Xuejun ;HAN Peng;College of Life Sciences, Jilin Agricultural University;

    ObjectiveTo express and purify the endolysin PlyL from the Bacillus anthracis prophage LambdaBa02 in prokaryotic cells, and to explore its role in the detection of Bacillus anthracis, so as to provide a novel strategy for the specific and rapid detection of Bacillus anthracis.MethodsAccording to the sequence of Bacillus anthracis vaccine strain CVCC40205 registered in GenBank, the plyL gene was amplified by PCR with plyL gene-specific primers and inserted into the prokaryotic expression vector pET28a(+) to construct the recombinant expression plasmid pET28a(+)-His-plyL, which was then transformed into E.coli BL21(DE3) and induced by IPTG. The induced expression conditions were optimized to enhance soluble expression, and the recombinant PlyL protein was purified via nickel-affinity chromatography. The lytic activity of recombinant PlyL protein was assessed by measuring absorbance values, and an ELISA method was established to evaluate the sensitivity and specificity of Bacillus anthracis detection.ResultsThe recombinant expression plasmid pET28a(+)-His-plyL was confirmed to be correctly constructed by NdeⅠ and XhoⅠ double digestion and sequencing. The optimal induction conditions were 0. 5 mmol/L IPTG at 16 ℃ for 16 h. The relative molecular mass of the expressed recombinant PlyL protein was about 28 600. After purification and concentration, the final concentration of the protein was2. 55 mg/mL, and the purity reached 80. 6%. The purified recombinant PlyL protein could reduce the A_(600)value of Bacillus anthracis culture solution. The sensitivity of the preliminarily established ELISA method for detecting Bacillus anthracis was1 × 10~3 CFU/mL, and there was no cross-reactivity against closely related strains such as Bacillus cereus and Bacillus thuringiensis(P < 0. 000 1).ConclusionThe recombinant PlyL protein was successfully expressed and purified in prokaryotic cells, which exhibits both efficient lytic activity and species specificity, making it a promising novel tool for the rapid detection of Bacillus anthracis.

    2026 03 v.39 [Abstract][OnlineView][Download 882K]

  • Expression of antimicrobial peptide CM25-6 in two yeast species and comparison of its antimicrobial activity

    GU Hanshi;ZHAO Zihan;HU Haiyan ;ZHANG Aizhong;JIANG Ning;College of Animal Science and Technology, Heilongjiang Bayi Agricultural University,Key Laboratory of Efficient Utilization of Feed Resources and Nutritional Regulation in Cold Regions of Heilongjiang Province;

    ObjectiveTo compare the expression of antimicrobial peptide(AMP) CM25-6 in two expression systems of Pichia pastoris and Saccharomyces cerevisiae and its antimicrobial activity, so as to provide a basis for the screening of its efficient heterologous expression system.MethodsThe CM25-6 gene was subcloned into the Pichia pastoris expression vector pGAPZαA and the Saccharomyces cerevisiae expression vector pYES2/CT/α-factor, and the recombinant plasmids were constructed and transformed into Pichia pastoris SMD1168 and Saccharomyces cerevisiae INVSc1, respectively. The recombinant proteins were identified for specificity by Western blot. The culture was expanded in YPD medium, the expression of the recombinant proteins was dynamically detected by SDS-PAGE and BCA method, and the antibacterial activity of the fermentation supernatant was determined by Oxford cup method. After the recombinant proteins were purified by Ni~(2+)chelating affinity chromatography, the minimum inhibitory concentration(MIC) of the purified products was determined by micro broth dilution method.ResultsThe two recombinant yeasts were successfully constructed as identified by PCR and sequencing. The recombinant proteins secreted by recombinant Pichia pastoris and recombinant Saccharomyces cerevisiae exhibited specific binding to mouse anti-His-tag monoclonal antibody. The relative molecular mass was about 8 480 for both recombinant proteins after expanded expression in YPD medium, and the expression reached the highest levels of 2. 13 and1. 02 g/L at 60 h of fermentation, respectively. The purified products of the recombinant proteins showed significant antibacterial activity against Escherichia coli, Pseudomonas aeruginosa and Streptococcus agalactiae, and the purified product derived from Pichia pastoris had higher antibacterial activity, with the MIC value(7. 85-15. 7 μmol/L) lower than that of the purified product derived from Saccharomyces cerevisiae(15. 05-30. 2 μmol/L).ConclusionThe expression level and antibacterial activity of AMP CM25-6 in Pichia pastoris system are better than those in Saccharomyces cerevisiae system, indicating that Pichia pastoris is more suitable for heterologous expression and production development of AMP CM25-6.

    2026 03 v.39 [Abstract][OnlineView][Download 967K]

  • Preparation of Mycobacterium tuberculosis ESAT6-Fc fusion protein and evaluation of its efficacy and safety in allergic rhinitis mice

    HAI Maiyan;WANG Jing;YANG Maosheng ;HE Haiyan;ZHANG Wenxuan;YANG Yuxin;YANG Yuan;DONG Zihan;ZHANG Wei;WAN Qiaofeng;Department of Pathogen Biology and Immunology, School of Basic Medicine, Ningxia Medical University;

    ObjectiveTo prepare Mycobacterium tuberculosis ESAT6-Fc fusion protein and evaluate its efficacy and safety on ovalbumin(OVA)-induced allergic rhinitis(AR) in mice, so as to provide experimental basis for the development of novel AR immunothera-peutic agents.MethodsThe recombinant plasmid pcDNA3.1(+)-Rv3875-Fc was transfected into CHO-K1 cells to obtain ESAT6-Fc fusion protein, which was purified by Protein G affinity chromatography. The female C57 BL/6J mice of AR model sensitized by OVA were randomly divided into five groups: normal control, OVA, CpG, ESAT6-Fc + CpG and dexamethasone(DEX) group, six mice each group, which were challenged with atomized OVA after the corresponding preparation was given to the nasal cavity for intervention. HE staining method was used to evaluate the inflammatory infiltration of mouse nasal mucosa, and ELISA method was used to detect the content of histidine(HIS), leukotriene B4(LTB4), IgE, IL-4,IL-13 and IL-17A in the nasal lavage fluid. The structural changes of heart, liver, spleen and kidney tissues were observed by HE staining, and the levels of glutamic oxaloacetic transaminase(AST) and lactate dehydrogenase(LDH) in mouse heart,liver, spleen and kidney were measured by ELISA.ResultsESAT6-Fc fusion protein with high purity(> 95%) was successfully obtained. Compared with the normal control group, the inflammatory cell infiltration in nasal mucosa of the OVA group was significantly aggravated(t = 11. 180, P < 0. 001), and the levels of HIS, LTB4, IgE, IL-4, IL-13 and IL-17A in the nasal lavage fluid significantly increased(t = 3. 843, 4. 237, 5. 996, 3. 561, 4. 544, 5. 773, respectively, each P < 0. 05). Compared with the OVA group, the infiltration of inflammatory cells in nasal mucosa of mice in the ESAT6-Fc + CpG and DEX groups was significantly reduced(t = 4. 472 and 5. 582, respectively, each P < 0. 001), and the levels of HIS, IgE, LTB4, IL-4, IL-13 and IL-17A in the nasal lavage fluid significantly decreased(t = 0. 000-1. 061, each P < 0. 05). There was no significant difference in the levels of HIS, IgE, LTB4, IL-4, IL-13 and IL-17A between the ESAT6-Fc + CpG and DEX groups(t = 0. 048, 0. 000,0. 968, 1. 061, 0. 375 and 0. 638, respectively, each P > 0. 05). Intranasal immunization with ESAT6-Fc did not cause pathological damage to the heart, liver, spleen, and kidney of mice, and the levels of AST and LDH in the above organs showed no significant difference from those in the normal control group(t = 0. 001-1. 036, each P > 0. 05).ConclusionThe prepared ESAT6-Fc fusion protein has high purity, and intranasal immunization with the fusion protein can safely and effectively alleviate OVA-induced AR symptoms in mice, and inhibit the expression of related inflammatory factors, without obvious organ toxicity observed, indicating that ESAT6-Fc fusion protein is expected to become a new type of immunotherapeutic agent for AR.

    2026 03 v.39 [Abstract][OnlineView][Download 1229K]

  • Safety and immunogenicity of live attenuated varicella vaccine in healthy individuals aged 13 years and older

    LI Qiong;SHUANG Hui ;QIAN Xiaoai;XIAO Jinsong ;LI Zhaoxia ;KE Changxian ;LI Chaohong ;YANG Beifang;CAI Yan ;XU Junfeng;Hubei Provincial Center for Disease Control and Prevention;

    ObjectiveTo evaluate the safety and immunogenicity of two doses of live attenuated varicella vaccine in individuals aged 13 years and older, so as to provide a scientific basis for optimizing the varicella vaccination strategy for people aged 13 years and above.MethodsA total of 1 680 healthy adolescents and adults aged 13 to 50 years were selected from two sub-centers of the Hubei Provincial Center for Disease Control and Prevention, and were stratified by age between those aged 13 to 16 years and those aged 17 to 50 years. Subjects in each age group were randomly assigned in a 1∶1 ratio to receive two doses of the test vaccine according to the immunization schedules of 0, 4 or 0, 8 weeks, respectively. Adverse events were collected within 30 minutes and 0 to 14 days after each vaccine dose, as well as all adverse events within 28 days after each dose, and serious adverse events(SAEs) from the first dose to six months after full immunization. Serum varicella virus-specific antibodies were detected before immunization, before the second dose and 28 days after full immunization, and the antibody positive rates(titer ≥ 1∶8), seroconversion rates(antibody titer of susceptible individuals after immunization ≥ 1∶8 or antibody titer of non-susceptible individuals increased ≥ 4 times after immunization), geometric mean titers(GMTs) and geometric mean increases(GMIs) were calculated.ResultsAmong the 1 680 subjects,1 606 subjects completed the study,including 807 in the 13-16 age group and 799 in the 17-50 age group. There were 288 and 340 adverse events in the 13-16 and 17-50 age groups, respectively, mainly grade 1-2. A total of four grade 3 adverse events occurred(two cases of fever, one case of pruritus at the vaccination site, and one case of vaccination site swelling). Adverse events after vaccination mostly occurred within 0-14 days, and no vaccine-related SAE occurred within six months after full immunization. The antibody positive rates before the second dose and 28 days after full immunization were both 100. 00%(1 616/1 616). The seroconversion rates were 55. 32% and 80. 57%, respectively, and the latter was significantly higher than the former(χ~2= 235. 325, P < 0. 000 1).The GMTs were 310. 77 and 626. 63, respectively, and the latter was significantly higher than the former(t =-21. 383, P <0. 000 1). The GMIs were 3. 68 and 7. 42, respectively, and the latter was significantly higher than the former(t =-4. 387, P =0. 012). At 28 days after the full immunization, the seroconversion rates of the two immunization schedules at 0, 4 and 0, 8 weeks were 80. 52% and 80. 62%(χ~2= 0. 003 and 3. 811, P < 1. 000 and 0. 057, respectively), the GMTs were 628. 57 and624. 67(t =-2. 134 and 0. 232, P = 0. 102 and 0. 828, respectively), and the GMIs were 7. 49 and 7. 34(t =-0. 366, P =0. 733), respectively.ConclusionLive attenuated varicella vaccine demonstrates good safety and immunogenicity in individuals aged ≥13 years when administered according to a two-dose immunization schedule, with equivalent immune effects between the two immunization schedules of 0, 4 and 0, 8 weeks, and the vaccination interval can be flexibly selected according to the actual situation.

    2026 03 v.39 [Abstract][OnlineView][Download 852K]

  • Evaluation of safety, efficacy and economy of equine tetanus immunoglobulin F(ab′)2

    LV Hui;WANG Ruilan;LEI Ming ;ZHOU Huifang ;GUAN Jian ;YE Shupei ;REN Zhaojun ;HUANG Zhongyi;Department of Emergency Critical Care Medicine,Shanghai General Hospital;

    ObjectiveTo evaluate the safety, efficacy and economy of equine tetanus immunoglobulin F(ab')_2, so as to provide reference for its clinical application.MethodsPatients who were admitted to six research centers for trauma from November 23, 2021 to October 31, 2022 and required a tetanus immunoglobulin injection were selected as the research subjects to observe the positive rate of skin sensitization test of equine tetanus immunoglobulin F(ab')_2 and the incidence of adverse events(AEs) after injection. In addition, one of the study centers was selected to evaluate the incidence of tetanus in patients receiving equine tetanus immune globulin F(ab')_2 injection in this center. Meanwhile, a pharmacoeconomic model per 10 000 patients was constructed to compare the difference in drug costs of switching to human tetanus immunoglobulin(TIG) due to a positive skin sensitization test when patients preferred tetanus antitoxin(TAT) or equine tetanus immunoglobulin F(ab')_2.ResultsA total of 422 patients were enrolled in the study, of whom 419 underwent an equine tetanus immunoglobulin F(ab')_2 skin sensitization test and 416 received a test result, with a positive skin sensitization test rate of 5. 05%(21/416). Among the 395 patients who had a negative skin sensitization test and completed equine tetanus immunoglobulin F(ab')_2 injection, five(1. 27%) patients developed AEs related to equine tetanus immunoglobulin F(ab')_2, mainly including rash and pruritus, and two(0. 51%) patients had grade ≥ 3 AEs related to equine tetanus immunoglobulin F(ab')_2, including one case of immediate anaphylaxis and one case of pruritus. None of the patients developed anaphylactic shock or death related to equine tetanus immunoglobulin F(ab')_2. None of the patients in the selected research center developed tetanus after administration of equine tetanus immunoglobulin F(ab')_2. In addition, the pharmacoeconomic evaluation model showed that equine tetanus immunoglobulin F(ab')_2 had a higher drug economic value compared to TAT and TIG.Conclusion Equine tetanus immunoglobulin F(ab')_2 has shown good efficacy and safety in preventing tetanus in the real world with high pharmacoeconomic value.

    2026 03 v.39 [Abstract][OnlineView][Download 882K]

  • Optimization of Coxsackievirus A6 production process by Quality by Design concept

    WAN Xin;DAI Hanyu;XIAO Ao;LI Wei;MENG Shengli;WANG Zejun;GUO Jing;SHEN Shuo;Viral Vaccine Research Laboratory, Wuhan Institute of Biological Products Co., Ltd., National Engineering Technology Research Center of Combined Vaccines,Vaccine Technology Innovation Center of Hubei Province, National Key Laboratory for Novel Vaccines Research and Development of Emerging Infectious Diseases;

    ObjectiveTo optimize the production process of Coxsackievirus A6(CV-A6), establish the design space for critical process parameters and improve the yields of CV-A6 virus, based on the concept of Quality by Design(QbD).MethodsRisk assessment analysis of process parameters was performed using Ishikawa diagram combined with failure mode effects analysis(FMEA) to identify potential key or critical process parameters that require experimental research. The full factorial experiment design was applied to study the effects of potential critical process parameters on critical quality attributes and to screen out the critical process parameters in the validated scale-down model. The central composite facecentered design was used to optimize the critical process parameters, and the design space of critical process parameters was determined based on Monte Carlo simulation. The design space was verified in both scale-down scale and expanded scale.ResultsThe CV-A6 virus production scale, types of virus culture medium, sodium bicarbonate(NaHCO_3) concentration in virus culture medium, serum concentration in virus culture medium, multiplicity of infection(MOI), virus culture temperature and virus harvest time were considered to be the potential key or critical process parameters based on risk priority number(RPN) of above 27. The virus proliferation curves of the 6-well cell culture plate and 5-layer cell factory were consistent, and the antigen yields and virus titers were equivalent. The results of full factorial experiment design showed that virus culture temperature, DMEM ratio and NaHCO_3 concentration in virus culture medium had significant effects on antigen yields and virus titers(P < 0. 05), while MOI and serum concentration in virus culture medium had no significant effects(P > 0. 05).The design space of virus culture temperature, DMEM ratio and NaHCO_3 concentration in virus culture medium determined by Monte Carlo simulation was 32. 33-34. 33 ℃, 83%-100% and 2-4 g/L, respectively. The validated values of antigen yields and virus titers for both scale-down scale and expanded scale design space met the specification limits and fell within the 95% confidence interval and 95% prediction interval of the fitted values. After the process optimization, the harvest time of CV-A6 was 11-12 d. Compared with the yields before optimization, the antigen yields had increased by approximately five times and the virus titers had increased by 0. 5-1. 0 LgCCID_(50)/mL.ConclusionThe production process of CV-A6 was optimized, the design space for critical process parameters was established and the yield of CV-A6 virus was significantly increased, based on the concept of QbD.

    2026 03 v.39 [Abstract][OnlineView][Download 1080K]

  • Application of Poisson distribution method in determination of herpes simplex virus 2 viral titer

    WANG Guangyu;YU Lei;PEI Dening;SHI Xinchang;WEI Changlong;ZHOU Yong;LIANG Chenggang;National Key Laboratory of Drug Regulatory Science, National Institutes for Food and Drug Control;

    ObjectiveTo develop an accurate quantitative method of herpes simplex virus 2(HSV2) oncolytic viruses(OVs)viral titer based on Poisson distribution principle, aiming at the limitation of high variability and complicated operation of traditional half cell culture infectious dose method(CCID_(50)), so as to improve the accuracy and stability of quality control.MethodsA robust detection system was established by optimizing Vero cell culture conditions(serum concentration and incubation time after virus infection). HSV2 titer was determined simultaneously by traditional CCID_(50)method and new Poisson distribution method(maximum likelihood estimation using negative well data based on high-density 96-well microplate).ResultsThe mean virus titer measured by CCID_(50)method was 1. 75 × 10~6 CCID_(50)/mL, with relative standard deviation(RSD) of 36. 62%, while that measured by Poisson distribution method was 7. 94 × 10~5 ifu/mL, with significantly reduced variability(RSD of 12. 59%).ConclusionPoisson distribution method significantly improves the accuracy and efficiency of virus titer determination by fully integrating microplate data to reduce random error, and provides a standardized and reliable solution for OVs quality evaluation, which has broad clinical application prospects.

    2026 03 v.39 [Abstract][OnlineView][Download 799K]

  • Development and verification of a double antibody sandwich ELISA for quantitative detection of recombinant trivalent poliomyelitis vaccine antigen

    SHA Fangfang;SUI Xiuwen;YAN Qiaoling;XU Lishuang;DUAN Zhuojun;MA Lala;MA Wei;ZHU Tao;CanSino Biologics Inc.;

    ObjectiveTo develop a double antibody sandwich ELISA method for the detection of types Ⅰ, Ⅱ and Ⅲ antigen content of recombinant trivalent poliomyelitis vaccine, and to optimize, verify and preliminarily apply the method, in order to provide a quality control method for vaccine development.MethodsMale New Zealand white rabbits were immunized with types Ⅰ, Ⅱ and Ⅲ recombinant poliomyelitis vaccine antigens, and the corresponding polyclonal antibodies were prepared.The polyclonal antibodies were used as coating antibodies and HRP-labeled antibodies as detection antibodies to establish a double antibody sandwich ELISA method for detecting the content of three types of antigens in recombinant trivalent poliomyelitis vaccine. The checkerboard titration method was used to determine the coating antibody concentration and the detection antibody dilution. The single factor experiments were used to optimize the types of blocking solution, antibody lyophilization time, enzyme-labeled antibody diluents and chromogenic solution formulations. The established method was verified for linear range, accuracy, specificity, precision, lower limit of quantification, robustness and stability, and was used to detect the content of types Ⅰ, Ⅱ and Ⅲ antigens in recombinant trivalent poliovirus vaccine.ResultsThe optimal coating antibody concentration was 5 μg/mL, and the optimal dilutions of enzyme-labeled antibodies were 4 000, 9 000 and 5 000, respectively,for types Ⅰ, Ⅱ and Ⅲ antigens. The optimal conditions were as follows: blocking solution of 1% BSA solution, lyophilization time of 2 h, enzyme-labeled antibody dilution of 1% BSA + 1% sucrose + 1% trehalose + 0. 01% sodium thimerosal, and chromogenic solution of recipe 2 [Solution A: 13. 6 g sodium acetate, 1. 6 g citric acid, 0. 3 mL of 30% hydrogen peroxide,adding distilled water to a total volume of 500 mL. Solution B: 0. 2 g disodium ethylenediaminetetraacetate, 0. 95 g citric acid, 50 mL glycerol, 0. 15 g TMB(dissolved in DMSO before slowly adding to distilled water), adding distilled water to a total volume of 500 mL]. TypesⅠ and Ⅱ antigens showed a good linear relationship with A_(450)in the concentration range of0. 78-25 DU/mL, and type Ⅲ antigen exhibited a good linear relationship with A_(450)in the concentration range of 1. 56-50 DU/mL,each R~2> 0. 99. The recovery rates of spiked samples at high, medium and low concentration of the three types of antigen content detection methods were all between 80% and 120%. All three types of antigen detection methods detected their corresponding specific antigens, and there was no cross-reaction with the other two antigens. The lower limits of quantification of types Ⅰ, Ⅱ and Ⅲ antigen detection methods were 1. 56, 1. 56 and 3. 13 DU/mL, respectively. The CVs of precision and robustness verification were both less than 20%. The antibody-coated plate, detection antibody working solution and chromogenic solution were stored stably at 4 ℃ for six months, and the recovery rates of the three types of antigens were all within the range of 80%-120%. The CVs of harvest solution, clarified solution, concentrated solution, ion exchange chromatography solution, recovery solution and bulk solution samples in the vaccine process were all not more than 15% by the established method.ConclusionThe established double antibody sandwich ELISA quantitative detection method has good specificity,accuracy, precision, robustness and stability, and can be used to detect the content of typesⅠ, Ⅱ and Ⅲ antigens in the development of recombinant trivalent poliovirus vaccines.

    2026 03 v.39 [Abstract][OnlineView][Download 969K]

  • Advances in mucosal vaccine and immunoprotective efficacy evaluation

    WANG Hongyu;LIANG Jinghui;ZHANG Yuan;HE Qing;National Institutes for Food and Drug Control;

    Mucosae are the primary sites of causing infectious diseases in human beings. The high transmissibility and mortality rates of infectious diseases pose a serious threat to human health. Mucosal vaccines, which induce both mucosal and systemic immune responses, have emerged as an effective method for preventing or treating infectious diseases. This paper provides an overview of the structure and response characteristics of the mucosal immune system, as well as adjuvants and delivery vectors in vaccine design. It focuses on the multi-dimensional evaluation system of immunoprotective efficacy,covering key indicators such as antibody response, neutralization ability, antibody affinity and tissue-resident memory cells,providing reference for the future development of more effective mucosal vaccines.

    2026 03 v.39 [Abstract][OnlineView][Download 958K]

  • Research progress on T-cell engager drugs

    CHANG Dongfeng;SUN Zhaopeng;CSPC Megalith Biopharmaceutical Co., Ltd.;

    T-cell engager(TCE) bridges T cells to target cells via dual-targeting, activating specific cytotoxicity of T cells,revolutioni-zing oncology and autoimmune therapies. In the field of hematological tumor treatment, TCE drug CD19/CD3 bispecific antibody Blinatumomab improves outcomes in relapsed/refractory B-cell malignancies. For solid tumors, cha llenges include antigen off-target toxicity and immunosuppressive microenvironments(such as insufficient T-cell infiltration and enrichment of inhibitory factors). Strategies prioritize high-specificity targets [such as delta-like protein 3(DLL3) and claudin 18.2(CLDN18.2)], exemplified by Tarlatamab that has been approved for small cell lung cancer. In the field of autoimmune disease treatment, TCE can accurately deplete pathological cell subsets [such as CD3/CD19 for B/pre-plasma cells,CD3/B-cell maturation antigen(BCMA)for plasma cells], breaking through the limitations of traditional therapies. In this paper, the mechanism of TCE drugs, the role of CD3 as a signal hub in TCE targeted therapy and the research progress on TCE drugs are reviewed in order to provide references for optimizing the clinical transformation pathway of TCE and developing a new generation of immune reprogramming technology.

    2026 03 v.39 [Abstract][OnlineView][Download 902K]

  • Research progress on epigenetic regulation in invasion and metastasis of hepatocellular carcinoma

    DONG Wenhao;WANG Huanhuan;LIU Fang;CHEN Che;School of Public Health, Gansu University of Chinese Medicine;

    Hepatocellular carcinoma(HCC) is the most common type of primary liver cancer and one of the leading causes of cancer deaths worldwide. The incidence and lethality of HCC are on the rise, and its invasiveness and metastasis are the key factors affecting patients' prognosis. In recent years, studies in epigenetics have provided new perspectives to unravel the molecular mechanisms of HCC occurrence and development, and epigenetic alterations(such as histone modification and DNA methylation) are closely associated with various invasion and metastasis mechanisms in HCC. Therefore, this paper elucidates the epigenetic modifications and molecular mechanisms related to HCC invasion and metastasis, and explores the application of HCC epigenetic modifications in biomarkers, in order to provide valuable insights for the early diagnosis and treatment of HCC metastasis.

    2026 03 v.39 [Abstract][OnlineView][Download 967K]

  • Discussion on aseptic assurance issues of biologics manufacturing enterprises in Jilin Province in recent years

    YANG Xifan;YUAN Guohui;LI Na;SUN Wen;WANG Han;YAN Weiwen;WANG Lin;Center for Inspection of JLMPA;

    During routine Good Manufacturing Practice(GMP) compliance supervision, periodic inspections based on risk control concepts have revealed gaps between the current practices of biologics manufacturers in Jilin Province and the latest domestic and international regulatory standards. These gaps span multiple areas, including facility and equipment design for aseptic product manufacturing, airflow pattern control, cleaning and disinfection practices, environmental monitoring, aseptic process simulation, and personnel gowning procedures. This paper compiles and analyzes these identified issues. Considering the inherent tendency of biological product components to support microbial growth, and based on recent regulatory updates alongside a statistical analysis of aseptic manufacturing deficiencies observed during GMP compliance inspections in Jilin's biologics sector, this paper discusses the future direction for improving aseptic assurance levels. The aim is to provide theoretical support for both future regulatory efforts and the enhancement of aseptic assurance capabilities within enterprises.

    2026 03 v.39 [Abstract][OnlineView][Download 802K]

  • Analysis and application of Article 59 stipulated in the appendix for biological products in China's Good Manufacturing Practice

    KANG Ying;XU Qin ;MA Yansong;Center for Food and Drug Inspection of NMPA;

    The Article 59 stipulated in the appendix for Biological Products in China's Good Manufacturing Practice(GMP)mandates that vaccine manufacturers adopt information technology systems for the recording of all production and quality control data, thereby ensuring comprehensive traceability and compliance throughout the entire manufacturing lifecycle.The regulation is designed to mitigate the significant risks inherent in traditional paper-based documentation systems,including their susceptibility to loss, inadvertent damage, and deliberate tampering, with the ultimate goal of ensuring robust data integrity. This paper provides a critical analysis of this regulatory provision The implementation of Manufacturing Execution System(MES) and Laboratory Information Management System(LIMS) serves as the foundational approach to facilitate real-time monitoring, enforce automated data capture and archiving, and implement stringent access controls. Such technological infrastructure is essential for enhancing the management of Critical Process Parameters(CPPs) and Critical Quality Attributes(CQAs). This paper further examines the principal implementation challenges and proposes corresponding solutions for MES and LIMS deployment, with a focus on data acquisition interoperability, cryptographic assurance of data integrity, and necessary business process re-engineering. Additionally, the paper delineates key focus areas for regulatory inspections, including data integrity and validation of computerized systems. The findings offer a systematic framework to guide vaccine manufacturers in achieving regulatory compliance and advancing their digital maturity.

    2026 03 v.39 [Abstract][OnlineView][Download 1022K]
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@2012 Editorial Department of "Chinese Journal of Biologicals"