Objective To prepare an emulsion with enhanced stability and application potential by optimizing the formulation ratios of key components in squalene-based emulsion adjuvants, and to evaluate the immune effects of its compatibility with respiratory syncytial virus(RSV) recombinant subunit antigen pre-fusion(PreF).MethodsTwo squalene-based emulsions,designated WS02 and WS03, were prepared by optimizing hydrophilic-lipophilic balance(HLB) values and using an orthogonal design approach. The physicochemical properties of these two emulsion adjuvants were subsequently evaluated,along with their long-term stability under storage condition at 4 ℃. These two adjuvants were formulated with RSV PreF, and were used to immunize 11 female BALB/c mice intramuscularly twice at an interval of 21 days. The humoral immunity, cellular immunity and immune memory were evaluated.ResultsThe two adjuvants, WS02 and WS03, maintained good stability under 4 ℃ storage condition, with no significant stratification, precipitation, or other adverse changes. However, WS03 exhibited a gradual decrease in pH, whereas WS02 maintained stable pH levels throughout. Both WS02 and WS03 adjuvants demonstrated substantial application potential in PreF immunoenhancement, with WS02 proving more effective in sustaining long-term immune memory and offering broader, more durable protection.ConclusionOptimization of formulation and processing parameters for emulsion adjuvants can significantly enhance their stability and immunological efficacy. WS02,with its superior stability and heightened immunogenic potential, represents a promising candidate for application in recombinant protein-based RSV vaccines.

Objective To optimize the filtration process before inactivation in the preparation of enterovirus 71(EV71)inactivated vaccine, screen high recovery filters, and evaluate their effects on viral structural proteins and immunogenicity.MethodsBy comparing the recovery rates of feed solution, antigen, and protein processed by four different filters, the optimal filter was selected. SDS-PAGE and Western blot were used to analyze the changes in viral structural proteins before and after filtration. The prepared bulk solution and aluminum hydroxide adjuvant were adsorbed and divided into five groups to intraperitoneally immunize female BALB/c mice, including unfiltered high-dose group(4. 5 μg/dose), unfiltered low-dose group(1. 5 μg/dose), filtered high-dose group, filtered low-dose group and blank control group(1. 05 mg/dose aluminum hydroxide adjuvant), with 10 mice in each group. The mice were immunized twice on day 0 and day 14. Blood samples were collected on day 0(before immunization), day 14 and day 28. The neutralizing antibody titer of immunized animals was detected by micro cytopathic inhibition method, and then the effect of filtration process on virus immunogenicity was evaluated.ResultsFilter 2 exhibited the highest antigen and protein recovery rates among the four filters, at 96. 10% and94. 64%, respectively. There was no significant difference in the proportion of viral structural proteins(VP0, VP1, VP2, VP3)before and after filtration(t = 1. 527, 0. 693, 1. 113 and 2. 416, P = 0. 201, 0. 526, 0. 328 and 0. 073, respectively). There was no significant difference in immunogenicity of EV71 vaccine before and after filtration on day 14 and day 28 between the low-dose groups as well as the high-dose groups(low-dose groups: t = 1. 067 and 0. 574, P = 0. 300 and 0. 573, respectively;high-dose groups: t = 0. 249 and 0. 110, P = 0. 806 and 0. 914, respectively), indicating that the addition of filtration process had no effect on the immunogenicity of EV71 vaccine.ConclusionAdding a filtration process before inactivation has no effect on virus structure and immunogenicity, which meets the requirements of WHO for vaccine safety.

Objective To identify and verify five strains of human embryonic lung diploid cells independently isolated and cultured, and to explore their sensitivity to encephalomyocarditis virus(EMCV), so as to screen for promising human embryonic lung diploid cells with application prospects.MethodsFive human embryonic lung cell strains were successfully isolated from artificially aborted embryos using the trypsin digestion method and named MU-L6, MU-L9, MU-L10, MU-L11 and MUL12. The five cell strains were identified by growth curve analysis, chromosomal karyotype analysis, immunofluorescence,nucleic acid electrophoresis, and short tandem repeat(STR) profiling. Their safety was assessed through sterility testing,mycoplasma detection, examination for endogenous and exogenous viral agents, and tumorigenicity testing. The sensitivity of the five cell strains to EMCV was determined by the 50% tissue culture infective dose(TCID_(50)) assay.ResultsAfter continuous subculturing, the MU-L10 cell strain could be passaged up to 81 generations, while the other four strains reached between 30 and 45 generations. The maximum proliferation density of MU-L10 cells was(44. 07 ± 2. 57) × 10~4 cells/mL, with a population doubling time of(25. 77 ± 1. 00) hours. All five human embryonic lung diploid cell strains exhibited fibroblast-like morphology and diploid karyotypes. No interspecies cross-contamination was detected. MU-L9, MU-L10, MU-L11 and MUL12 possessed unique cellular identities, indicating they are novel cell lines. Safety evaluations revealed no contamination with exogenous bacteria or viruses in any of the five cell strains. MU-L6, MU-L9, MU-L10 and MU-L11 were free of mycoplasma contamination. No colony formation was observed in the in vitro tumorigenicity assay. All five cell strains demonstrated sensitivity to EMCV infection. At 48 hours post-infection, the TCID_(50)of MU-L10 cells was 10~((-8.22 ± 0.14))/0.1 mL,showing no significant difference compared to the control MRC-5 cells(P = 0. 317 7).ConclusionThe independently isolated and cultured MU-L10 cell strain exhibits high passage longevity, excellent biosafety and genetic stability, making it a promising candidate for research on viral vaccines.

Objective To investigate the effects of thioridazine(TZ) on the proliferation, adhesion and migration of human esophageal cancer cells and the mechanism, with the aim of providing new evidence and potential drug candidates for targeted therapy of esophageal cancer.MethodsHuman esophageal carcinoma OE19 cell line was cultured in vitro and divided into control group(without intervention), positive drug group(6 μmol/L doxorubicin), TZ + inhibitor group(80 μg/mL TZ + 2 μmol/L XAV939), TZ + activator group(80 μg/mL TZ + 20 μmol/L SKL2001) and TZ groups with different concentrations(40, 80 and 160 μg/mL), which was treated for at 37 ℃ 24 h. The cell viability of human esophageal carcinoma OE19 cells was determined by CCK-8 assay. The cell proliferation was measured by 5-acetylidene-2 'deoxyuracil nucleoside(EdU)method. The adhesion ability was determined by cell adhesion test. The migration ability was measured in Transwell chamber. The telative expression leaves of Wnt/β-catenin pathway-associated proteins were measured by Western blot.ResultsCompared with the control group, the cell viability of the 80, 160 μg/mL TZ and the positive drug groups significantly decreased(t = 8. 401, 9. 637 and 9. 466, respectively, each P < 0. 05). In the subsequent experiments, compared with the control group, the cell proliferation rate, the number of adhering cells, the number of migrating cells, and the protein expression levels of Wnt and β-catenin in the 80 μg/mL TZ and the positive drug groups all decreased significantly(t = 2. 819-17. 612,each P < 0. 05). Additionally, the cell proliferation rate, the number of adhering cells, the number of migrating cells, and the relative protein expression levels of Wnt and β-catenin in the TZ + inhibitor group were fsignificantly reduced compared with the 80 μg/mL TZ group(t = 3. 098-15. 105, each P < 0. 05), whereas the above indicators significantly increased in the TZ +activator group(t = 2. 449-5. 502, each P < 0. 05).ConclusionTZ can inhibit the proliferation, adhesion and migration of OE19 cells, and the mechanism may be related to the inhibition of Wnt/β-catenin pathway transduction.

Objective To evaluate the post-vaccination safety of Sabin and Salk strain inactivated poliovirus vaccines(IPVs)in the real-world settings, and to provide evidence-based basis for scientific formulation of vaccination strategies and subsequent development of safer and more effective polio vaccines.MethodsThe study followed the PRISMA statement,searched multiple databases including China National Knowledge Infrastructure(CNKI), Wanfang Database, Embase, Web of Science, PubMed, etc., and collected surveillance data on adverse events following immunization(AEFI) from the Chinese Centers for Disease Control and Prevention(CDCs) at or above the provincial level. The included studies were able to extract data to display safety information of different AEFI classification data. After screening, a total of 19 documents were included in the final analysis, all of which were cross-sectional studies.ResultsThe included studies showed that the overall AEFI incidence of the two strains of IPV vaccines used in China was 29. 9/10~5 doses[95% CI:(22. 4-40. 0)/10~5 doses]. Comparing the national and provincial monitoring studies, there were significant differences in incidence rates of fever, thrombocytopenic purpura(TP), and febrile convulsions(P = 0. 002, 0. 016 and 0. 001, respectively), which were 6. 2/10~5 doses[95% CI:(4. 6-8. 4)/10~5 dose]vs 22. 1/10~5 doses[95% CI:(14. 4-34. 1)/10~5 doses], 0. 08/10~5 doses[95% CI:(0. 07-0. 11)/10~5 doses]vs 0. 29/10~5 doses[95% CI:(0. 20-0. 42)/10~5 doses], and 0. 03/10~5 doses[95% CI:(0. 02-0. 05)/10~5 doses]vs0. 25/10~5 doses[95% CI:(0. 16-0. 40)/10~5 doses], respectively. There was no significant difference in the incidence of AEFI, general reac-tions, abnormal reactions, febrile reactions, allergic rashes, TP, and febrile convulsions between Sabin and Salk strains in the provincial study. Sensitivity analysis showed that the results were robust and publication bias was not obvious.ConclusionBoth strains of IPV vaccines used in the Chinese market have good safety, and China's domestic Sabin strain IPV and imported Salk strain IPV vaccines have similar safety profiles.

Objective To evaluate the purification effects of five pre-purification processes on human prothrombin complex(PCC), in order to screen a better PCC pre-purification process.MethodsThe dilution plasma adsorption process, semicontinuous adsorption process, cyclic adsorption process, low-temperature batch adsorption process and temperature-rising batch adsorption process were used to separate and purify PCC in cryoprecipitate-removed supernatant plasma, and the titers of human coagulation factor Ⅶ(FⅦ) and FⅨ were detected according to the method specified in the Chinese Pharmacopoeia(Volume Ⅲ, 2025 edition).ResultsUnder three different plasma conductivity conditions, the yield of F Ⅶ and F Ⅸ in dilution plasma process was lower than that of the temperature-rising batch adsorption process without changing plasma conductivity. The adsorption effect of semi-continuous flow adsorption process on FⅦ and FⅨ at different temperatures was inferior to that of temperature-rising batch adsorption process. The yield of FⅦ and FⅨ in the eluent of cyclic adsorption process was also much lower than that of temperature-rising batch adsorption process. There was no significant difference in FⅨ yield between the two adsorption processes, but the FⅦ yield in the temperature-rising batch adsorption process was much higher than that in the low-temperature batch adsorption process. The adsorption capacity of the two adsorption processes for protein C(PC) and protein S(PS) was superior to that of foreign products.ConclusionThe temperature-rising batch adsorption process is superior to the other four PCC pre-purification processes in terms of adsorption time, FⅦ and FⅨ yields, and PC and PS purification effects, and has high potential for processability.

Objective To optimize the animal-free fermentation medium and fermentation conditions of pneumococcus, and make it suitable for fermentation production.MethodsThe orthogonal method was used to optimize the carbon source(glucose)(12. 5, 18. 75, 25 g/L), nitrogen source(soybean peptone)(30, 40, 50 g/L) and uracil(10, 20, 30 mg/L) concentrations of fermentation medium for type 1, 3, 5, 9N and 33F pneumococci, as well as fermentation culture conditions such as strain concentration(A_(694))(0. 2, 0. 4, 0. 6), fermentation temperature(35, 36, 37 ℃) and pH(6. 8, 7. 2, 7. 6).ResultsThe optimal fermentation medium compositions for pneumococcus were as follows: for type 1, soybean peptone at 50 g/L, initial glucose concentration at 18. 75 g/L, and uracil at 30 mg/L; the optimal fermentation conditions were A_(694)at 0. 6, temperature at 35 ℃,and pH at 7. 6. For type 3, the optimal medium consisted of soybean peptone at 30 g/L, initial glucose concentration at 12. 5 g/L,and uracil at 10 mg/L; the optimal fermentation conditions were A_(694)at 0. 6, temperature at 35 ℃, and pH at 7. 2. For type 5,the optimal medium comprised soybean peptone at 50 g/L, initial glucose concentration at 12. 5 g/L, and uracil at 20 mg/L;the optimal conditions were A_(694)at 0. 6, temperature at 36 ℃, and pH at 6. 8. For type 9N, the optimal medium consisted of soybean peptone at 50 g/L, initial glucose concentration at 18. 75 g/L, and uracil at 10 mg/L; the optimal conditions were A_(694)at 0. 6, temperature at 36 ℃, and pH at 7. 2. For type 33F, the optimal medium comprised soybean peptone at 50 g/L, initial glucose concentration at 12. 5 g/L, and uracil at 30 mg/L; the optimal conditions were A_(694)at 0. 4, temperature at 37 ℃, and pH at 6. 8.ConclusionThe optimized fermentation medium and fermentation conditions can be applied to the research and production of 24-valent pneumococcal polysaccharide vaccine.

Objective To develop a desorption method for aluminum-adjuvanted recombinant protein vaccines and apply the method to the pre-treatment process for adsorption rate determination of respiratory syncytial virus(RSV) recombinant vaccine intermediates, involving antigen content quantification.MethodsTwo aluminum adjuvants, aluminum hydroxide(AH) and amorphous aluminum hydroxyphosphate sulfate(AAHS), were combined respectively with RSV preF protein to develop a desorption agent formulation(a mixture of 0. 2 mol/L potassium dihydrogen phosphate and 0. 8 mol/L dipotassium hydrogen phosphate). The effects of different treatment methods on desorption efficiency were investigated. The optimal desorption method was validated for its effectiveness in releasing RSV preF recombinant protein from antigen-adjuvant complexes with different formulations and different protein concentrations. Its applicability was also extended to antigenadjuvant complexes of different proteins.ResultsA desorption method was determined: antigen-adjuvant complex + 1%BSA/PBST + high-concentration phosphate(mixed solution of 0. 2 mol/L potassium dihydrogen phosphate and 0. 8 mol/L dipotassium hydrogen phosphate) mixed at a volume ratio of 1∶1∶2, followed by incubation at room temperature for 0. 5 h.This method combined the interfering effects of high-concentration phosphate ions, bovine serum albumin(BSA), and the nonionic surfactant polysorbate 20 on antigen-adjuvant binding, and achieved complete desorption for four RSV preF antigenadjuvant complexes and antigen-AH adjuvant complexes with three different protein concentrations(60-240 μg/mL). The antigen recovery rates detected by ELISA were all within the range of(100 ± 10)%. Compared to the non-optimized common phosphate desorbent, this method significantly improved the post-desorption antigen recovery rate, verifying its high efficiency and stability. It also demonstrated the important synergistic role of BSA and polysorbate 20 in assisting phosphate during the desorption process. This method was also successfully applied to complexes of another RSV preF recombinant protein(DS-CAV1) and human metapneumovirus(HMPV) preF recombinant protein with AH adjuvant, achieving antigen recovery rates above 90%, proving its certain universality and suggesting its potential application in combined respiratory virus vaccines.ConclusionThe established desorption method achieves highly efficient and stable desorption for recombinant RSV preF antigen-adjuvant complexes with different aluminum adjuvants, different buffer systems, and different protein concentrations. It is also applicable to other RSV preF variants and HMPV preF protein, effectively solving the interference problem of aluminum adjuvants in antigen content detection. This provides technical support for the quality control and production process optimization of recombinant RSV vaccines, and also serves as reference for the development of combined respiratory vaccines and other aluminum-adjuvanted vaccines.

Objective To develop a modified Lowry method for the detection of protein content of Sabin strain inactivated poliovirus vaccine(sIPV)(Vero cells), and compare it with the Lowry2 method after verification.MethodsThe specificity,linear range, repeatability, intermediate precision, accuracy and robustness of the developed modified Lowry method were verified, and the detection results of protein content of sIPV typesⅠ,Ⅱand Ⅲ monovalent bulk solution and finished products were compared with those of Lowry2 method, and the applicability research of the modified Lowry method was conducted.ResultsThe total RSD of the detected protein content of the standard at the same concentration diluted with two diluents was 2. 1%. The protein content showed good linearity in the range of 0-50 μg/mL, R2 > 0. 99, and the recovery rates of30 μg protein were 101%-103%. The total RSDs of six repeated tests of sIPV typesⅠ,Ⅱand Ⅲ monovalent bulk solutions and finished products by different experimenters on the same equipment were 1. 2%, 0. 0%, 1. 9% and 2. 4%, and the total RSDs of six repeated tests by the same experimenter on different equipment were 1. 4%, 0. 0%, 1. 9% and 2. 4%, respectively.The recovery rates of spiked sIPV typesⅠ,Ⅱand Ⅲ monovalent bulk solutions and finished products were 96%-102%,99%-101%, 96%-101% and 100%-103%, respectively. After being placed at room temperature for 30 min and 50 min,the total RSDs of sIPV typesⅠ,Ⅱand Ⅲ monovalent bulk solutions and finished products were 2. 1%, 1. 1%, 2. 2% and2. 4%, respectively. The modified Lowry method was simpler in operation steps and had similar recovery rate with the Lowry2 method(F = 1. 655, P > 0. 05), and both could remove impurities in the sIPV vaccine. Samples from three batches of sIPV types Ⅰ, Ⅱ and Ⅲ monovalent bulk solutions and finished products for 12 times of applicability tests were all qualified.ConclusionThe modified Lowry method can be used to detect the protein content of sIPV(Vero cells) as an indicator of product effectiveness evaluation.

Objective To establish and verify a pseudovirus detection method for respiratory syncytial virus(RSV) neutralizing antibodies, in order to provide a rapid, high-throughput and safe method for RSV vaccine development and antiviral drug screening.MethodsThe RSV A2 pseudovirus was prepared, and the pseudovirus detection method for RSV neutralizing antibody was established by optimizing the pseudovirus concentration factor(0, 2, 4, 8 and 16 times), infected cell type(HEK293T, Hep-2, Huh7.5.1, HeLa and Vero cells), cell inoculation density(0. 5 × 10~4, 1. 0 × 10~4, 1. 5 × 10~4, 2. 0 × 10~4 and2. 5 × 10~4 cells/well), cell generation(1, 5, 10, 15, 20 and 25 generations), detection time(6, 12, 24, 36, 48, 60 and 72 h) and virus inoculation amount(5. 0 × 10~3, 2. 5 × 10~3, 1. 25 × 10~3, 6. 25 × 10~2, 3. 125 × 10~2, 1. 562 5 × 10~2 TCID50/mL). In addition, the precision, specificity, accuracy and edge effects of the method were verified. Finally, the established method and plaque-reduction neutralization test(PRNT) were used to detect 30 mouse positive sera simultaneously, and the correlation between the detection results was analyzed.ResultsThe optimal pseudovirus concentration factor was 8, the infected cells were HEK293T cells of 1-10 passages after resuscitation with the inoculation density of 2. 0 × 10~4 cells/well, the detection duration was 48 h, and the virus inoculation amount was 3. 125 × 10~2-2. 5 × 10~3 TCID50/mL. The coefficients of variation(CVs) of repeatability and intermediate precision verification were both less than 15%. No RSV neutralizing activity was detected in normal mouse serum, and high RSV neutralizing activity was detected in positive mouse serum and Nirsevimab monoclonal antibody. The recovery rates of accuracy verification were within the range of 80% to 120%. The ratios of the detection values of edge holes to non-edge holes were 0. 5-0. 7, indicating that there was edge hole effects, and non-edge holes should be used for detection. The results of 30 mouse positive sera detected by the two methods showed a good positive correlation(r = 0. 914 5, P < 0. 000 1).ConclusionThe established RSV neutralizing antibody titer pseudovirus detection method has good precision, specificity, accuracy, with simple and fast operation, which can be used for the evaluation of RSV vaccine immunogenicity and the high-throughput detection of serum neutralizing antibody potency.