Objective To screen candidate preparations of live rotavirus(RV) vaccine using stability data modeling.Methods The citric acid, sodium citrate and disodium hydrogen phosphate buffer bulk solutions were prepared, and sucrose and RV bulk solution were added after sterile filtration to prepare three RV liquid formulations: FA(5 × sucrose-1 × citric acid-1 × sodium citrate), FB(5 × sucrose-1 × citric acid-1 × disodium hydrogen phosphate) or FC(1 × sucrose), with MEM as the diluent and virus titer of 7. 5 logCCID_(50)/mL. The stability data of the three formulations were linearly extrapolated using 2-8 ℃ data extrapolation model and Arrhenius model, and compared with the actual stability curves at 2-8 ℃ for36 months to evaluate the predictive models and identify the optimal formulation.Results The two model analysis indicated that at temperatures ranging from 2 to 8 ℃, formulations FA and FB exhibited slower declines in virus titer, demonstrating significantly superior stability compared to FC. Both models confirmed the stability hierarchy as FA = FB > FC, with predictions based on medium-to-long-term data showing good agreement with 36-month real-time stability results.Conclusion This study successfully identified the more stable RV vaccine candidates FA and FB by establishing and validating two stability predictive models. These two models provide reliable tools for assessing the stability and efficiently screening RV vaccines.

Objective To establish two models of pertussis infection in mice through aerosol and intratracheal nebulization,and compare and evaluate the infection effects.Methods Using fermale NIH mice with five for each group, the aerosol infection model was set up with 10~8, 10~9, 10~(10)CFU/mL and initial concentration(1. 42 × 10~(11)CFU/mL) groups, and the infection was carried out by nebulization inhalation in a closed space for 30 minutes. Intratracheal nebulization infection model adopted a fixed nebulization dose of 25 ??L, including 10~3, 10~4 and 10~5 CFU/25 ??L infected bacteria groups, by inserting into the trachea for nebulization infection. On the 3 rd, 7 th, 14 th, 21 st and 28 th days after infection, three mice in each group were selected for lung specific gravity detection, and one mouse was randomly selected for pathological section analysis of lung tissue. Finally, the mice were infected by aerosol or intratracheal nebulization, and observed for 14 days to calculate the half lethal dose(LD_(50)).Results The lung specific gravity of mice significantly increased when aerosol infection occurred at a bacterial concentration of 10~(10)CFU/mL, or when nebulization infection occurred in the trachea with a bacterial count of 10~4 CFU/25 ??L or above. Lung slices showed that intratracheal nebulization infection caused more significant pulmonary inflammatory infiltration than aerosol infection, and was more correlated with infection time and infection dose.The LD_(50)of intratracheal nebulization infection was 1. 88 × 10~8 CFU/25 ??L, while the aerosol infection did not reach the lethal dose.Conclusion Both respiratory tract infections can successfully establish a pertussis mouse infection model. The intratracheal nebulization infection model was more relevant to the dose and time of infection, and can be used as a lethal model, which has the potential to detect the potency of pertussis vaccine.

Objective To identify the key sites of Rift Valley fever virus(RVFV) envelope protein Gn binding to the receptor,in order to provide a basis for the development of RVFV infection inhibitors.Methods The eukaryotic cell expression system was used to express RVFV envelope protein Gn, RVFV positive antibodies(antibodies 140, 144, 268, R12 and R17),low density lipoprotein receptor-related protein 1(LRP1) receptor domains(ClusterⅡ-Fc and ClusterⅣ-Fc) and six Gn mutants(T173A, Q174A, D176A, K180A, S291A and K294A). ELISA method or competitive ELISA were used to detect the binding activity of LRP1 receptor domain and RVFV Gn, the effect of RVFV positive antibody on the binding ability of LRP1 receptor domain and RVFV Gn, and the effect of Gn point mutation on its binding ability to RVFV positive antibody and LRP1 receptor domain. The inhibitory effect of LRP1 domain on RVFV infection was detected by pseudovirus neutralization assay.Results ClusterⅡ-Fc and ClusteⅣ-Fc domains of LRP1 exhibited specific binding to RVFV Gn protein. The binding ability of antibodies 268, R12 and R17 to Gn protein was dose-dependent, and no significant binding activity was detected for antibodies 140 and 144. Antibodies 268, R12 and R17 showed a certain competitive inhibitory effect on the binding ability of ClusterⅡ-Fc to Gn protein, and the competitive effect of antibody 268 was the most significant. Only antibody 268 showed competitive effect on ClusterⅣ-Fc. The binding activity of Gn mutants to RVFV positive antibodies was lower than that of Gn. Except for Q174A, the binding ability of other Gn mutants to ClusterⅡ-Fc and ClusterⅣ-Fc was all lower than that of Gn. Among them, the binding ability of T173A, K180A, S291A and K294A to ClusterⅡ-Fc was significantly lower than that of Gn(F = 15. 83, P < 0. 05), and the binding ability of K180A, S291A and K294A to ClusterⅣ-Fc was significantly lower than that of Gn(F = 8. 75, P < 0. 05).Conclusion K180, S291 and K294 are the key amino acid sites for RVFV Gn protein to mediate the interaction with the host receptor LRP1, revealing part of the molecular basis of the binding of Gn to LRP1 and laying the foundation for the development of inhibitors targeting virus adsorption.

Objective To establish a drug quality control method for anti-interleukin-5 receptor(IL-5R) monoclonal antibody, in order to provide reference for the quality control of anti-IL-5R monoclonal antibody and other monoclonal antibodies targeting cytokine receptors.Methods The monoclonal antibody was determined for the peptide mapping by reversed-phase ultra-performance liquid chromatography(RP-UPLC), for the charge variants by imaged capillary isoelectric focusing(iCIEF), for the molecular size variants by reduced/non-reduced capillary electrophoresis sodium dodecyl sulfate(CESDS) and size exclusion chromatography(SEC), for the antibody-dependent cell-mediated cytotoxicity(ADCC) activity by reporter gene assay, and for the N-glycan profile by 2-aminobenzamide(2-AB) labeling method.Results The peptide mapping of the anti-IL-5R monoclonal antibody exhibited visual similarity to the reference standard. The iCIEF main peak content was(66. 94 ± 1. 52)%. The reduced CE-SDS heavy chain plus light chain content reached(96. 39 ± 0. 11)%, while the non-reduced main peak content was(96. 98 ± 0. 13)%. The SEC monomer content was(99. 62 ± 0. 06)%. The ADCC biological activity showed a relative potency of(90. 33 ± 15. 42)%. For the N-linked glycan profile, the G1/G0 ratio was(0. 14 ± 0. 00)%, and the high-mannose content was(1. 37 ± 0. 05)%.Conclusion A comprehensive quality control methodology was established for the anti-IL-5R monoclonal antibody, encompassing identity verification, purity analysis, biological activity, and N-linked glycosylation profiling, which provides reference for the development and quality control of therapeutic antibodies against this target and other cytokine receptors.

Objective To analyze the use of non-national immunization program(nNIP) vaccines in Liaoning Province from2018 to 2024, evaluate the current situation of nNIP vaccination in Liaoning Province, and to provide scientific basis for formulating public health policies and optimizing vaccination strategies.Methods The nNIP vaccination data from 2018 to2022 and 2023 to 2024 were extracted through “China Immunization Program Information Management System” and“Liaoning Province Immunization Program Comprehensive Information Platform-Vaccination Information System”, including nNIP vaccine replacement records. Pearson correlation coefficient matrix analysis was performed using SPSS 13.0 software,and descriptive analysis method was used to comprehensively analyze the data.Results From 2018 to 2024, a total of25 726 024 doses of nNIP vaccines were administered in Liaoning Province, with per capita dose of 866 doses per 10 000 people. The per capita doses of nNIP vaccine and the proportion in total doses of all vaccines were increasing year by year.The top three nNIP vaccine varieties used were rabies vaccine for human use(Rab), influenza vaccine(InfV) and varicella attenuated live vaccine(VarV). Among the NIP, poliomyelitis vaccine(PV) showed the highest substitution rate(30. 40%),followed by hepayitis A attenuated live vaccine(HepA-L)(29. 08%) and diphtheria,tetanus and acellular pertussis combined vaccine(DTaP)(16. 37%).Conclusion The importance of nNIP vaccines in public health has become increasingly prominent. As an important part of vaccination, the quality of surveillance needs to be further strengthened. The nNIP vaccines can make up for the lack of NIP vaccine coverage, and can also play an important role when some NIP vaccines are temporarily in short supply. On the basis of optimizing the supply and distribution of NIP vaccines, increasing the level of nNIP vaccination can effectively prevent and control the epidemic of vaccine-preventable infectious diseases.

Objective To explore the epidemiological and etiological characteristics of hand, foot and mouth disease(HFMD)in the surrounding areas of Shijiazhuang from 2014 to 2023, so as to provide a basis for timely detection of changes in pathogen spectrum, further optimization of treatment plans, and taking targeted prevention and control measures in advance.Methods A retrospective analysis was conducted on the clinical data of children with HFMD who visited Hebei Children's Hospital from January 1, 2014 to December 31, 2023. PCR was used to classify and identify enterovirus 71(EV71), Coxsackievirus A group 16 strain(CA16), and enterovirus undefined(EVU). Descriptive epidemiological statistics and pathogen characteristics analysis were performed on age, gender, season, and other data.Results A total of 26 563 specimens were tested, with a positive rate of 48. 95%(13 003/26 563) and a male to female ratio of 1. 50∶1(15 950 vs.10 613), there was a statistically significant difference in gender ratios between different years(χ~2= 19. 61, P < 0. 05), and the male positive detection rate was higher than that of females(50. 69% vs. 46. 34%), with a statistically significant difference(χ~2= 48. 26,P < 0. 05). The positive rate was highest in 2016(69. 84%) and lowest in 2020(10. 64%). There was a statistically significantly difference in the positive rate of enterovirus detection between different years(χ~2= 2 648. 40, P < 0. 05).Overall, EVU type was the dominant strain of infection in this region, accounting for 75. 37% of cases, followed by EV71 type(18. 42%). After 2018, EVU type has become the absolute dominant strain with a composition rate exceeding 89%. There was a statistically significant difference in the composition of enteroviruses among different years(χ~2= 2 217. 44, P < 0. 05).HFMD occurred throughout the year, with a single peak concentrated in June to August, with a statistically significant difference in the positive detection rate among different months(χ~2= 4 050. 78, P < 0. 05). The composition of enteroviruses in different months was as follows: with CA16 having a higher composition from May to July(7. 11% to 7. 96%), EV71 having a higher composition from March to May(32. 02% to 52. 16%), and EVU having a higher composition from June to December(71. 75% to 82. 96%), with a statistically significant difference(χ~2= 541. 67, P < 0. 05). Among different age groups, the highest positive detection rate(55. 53%) was in the age group of 3-6 years and the lowest rate(33. 42%) was in the age group of older than 6 years, with a statistically significant difference(χ~2= 842. 33, P < 0. 05). The composition of different enteroviruses in different age groups was as follows: with CA16 and EV71 having the highest composition in the age group of1-3 years(8. 33%, 23. 77%), and EVU having the highest composition in the age group of older than 6 years(91. 99%), also with a statistically significant difference(χ~2= 449. 69, P < 0. 05).Conclusion EVU is a common infectious pathogen in the surrounding areas of Shijiazhuang, with a high incidence in children under 3 years old from June to August. This suggests that the epidemic trend of HFMD should be closely monitored, vaccination should be strengthened, and prevention and control measures should be strengthened for key populations.

Objective To develop and verify an analytical method for the size of host cell residual DNA(HCD) fragments in MDCK cells based on magnetic bead method and quantitative real-time PCR(qPCR), so as to use it to evaluate the removal effect of HCD during the production process of pandemic influenza vaccine(H5N1).Methods The HCD of samples was extracted by magnetic bead method, and specific primers and probes were designed to establish qPCR analytical method that can quantify 84, 142, 204 and 504 bp DNA fragments, respectively. The established method was verified for the linear range,limit of quantitation(LOQ),precision, accuracy, robustness and specificity, and used to analyze the proportion of HCD fragments in the process samples of pandemic influenza vaccine(H5N1), and the HCD content was simultaneously detected.Results The linear ranges of 84, 142, 204 and 504 bp standards were 0. 003-300 pg/??L, showing a good linear relationship with Ct, and the coefficients of determination(R2) of the four standard curves were all greater than 0. 99. The LOQs of 84, 142 and204 bp were 3 fg/??L, and the LOQ of 504 bp was 1 fg/??L. The recovery rates of spiked samples at high, medium and low concentrations corresponding to 84, 142, 204 and 504 bp were all within the range of 50% to 150%. The CVs of precision,robustness and specificity verification were not greater than 40%. The proportion of large HCD fragments in the harvest solution of pandemic influenza vaccine(H5N1) was relatively high(≥ 204 bp fragments accounted for 81. 0%). After enzymatic hydrolysis and purification, the proportion of ≥ 204 bp fragments in the bulk solution was reduced to 8. 9%, and no ≥ 504 bp fragments were detected. At the same time, the total HCD content was reduced from 689. 9 ng/m L to 10. 2 ng/m L.ConclusionThe q PCR analytical method for HCD fragment size was developed with good precision, accuracy, robustness and specificity,which can be used for the analysis and detection of HCD fragments of pandemic influenza vaccine(H5N1) and the quality control in the production process.

Objective To develop a size exclusion-high-performance liquid chromatography(SEC-HPLC) method for the simultaneous determination of free proteins and residual 4-dimethylaminopyridine(DMAP) in the bulk solution of pneumococcal polysaccharide-protein conjugate, and to verify and preliminarily apply the method.Methods By studying the characteristic ultraviolet absorption peak of DMAP and optimizing the separation method of chromatographic column, the SEC-HPLC method was established to realize the retention and separation of free proteins and DMAP on the same chromatographic column and quantify them. The method was verified for specificity(system suitability), limit of quantification(LOQ), accuracy, precision, linearity and range, and robustness, and was used to detect the free proteins and DMAP residues in the test sample.Results The chromatographic conditions were determined as follows: TSKgel G5000 PWXL analytical column was used as the column(7. 8 mm × 300 mm, 10 ??m), the column temperature was set at 35 ℃, the mobile phase was0. 85% sodium chloride solution, the pH was(6. 2 ± 0. 2), the flow rate was 0. 5 mL/min, and the detection wavelength was280 nm. The resolution of free protein peaks of diphtheria toxoid(DT) or tetanus toxoid(TT) and DMAP peak from adjacent peaks was greater than 1. 5. The LOQs of TT, DT and DMAP were 0. 50, 1. 20, and 0. 02 ??g/mL, respectively. The spiked recovery rates of TT, DT and DMAP were 98. 11%-104. 60%, 95. 43%-103. 26% and 97. 11%-98. 42%, respectively.The relative standard deviations(RSDs) of intermediate precision of TT, DT and DMAP were all less than 5%. The R~2 values of TT and DT were greater than 0. 999 0 in the range of 6-24 ??g/mL, and the R~2 value of DMAP was 1. 000 in the range of2-10 ??g/mL. The RSDs of TT, DT and DMAP content were all less than 3% at the injection temperature of 30, 35 and 40 ℃.The established method was used to determine the content of free proteins and DMAP in the test sample, and rapid and accurate results were obtained.Conclusion The established SEC-HPLC method has good system suitability, accuracy,precision, linear relationship and robustness, which can realize the dual quantification of free proteins and DMAP in the same experiment, and can be used to monitor the impurities in the binding process.

Objective To prepare mouse monoclonal antibodies(mAbs) against ovalbumin(OVA), and to establish and verify a double antibody sandwich ELISA quantitative detection method for the detection of OVA residues in influenza virus split vaccine.Methods Using OVA standard as immunogen, three positive hybridoma cell lines were screened by mouse hybridoma fusion technique. The OVA mAb ascites was prepared by in vivo induction method, and then purified by ammonium sulfate precipitation and Protein A affinity chromatography. The specificity of mAb was detected by Western blot, and the relative affinity was determined by indirect ELISA. Using the overlapping coefficient as the index, the optimal paired mAbs of three hybridoma cell lines were screened, and the OVA double antibody sandwich ELISA detection method was established. The concentration of coating antibody(8. 0, 4. 0, 2. 0, 1. 0, 0. 5 ??g/mL) and the dilution of detection antibody(1∶100, 1∶500, 1∶1 000, 1∶2 000) were optimized by checkerboard titration, and then the limit of quantitation(LOQ),linear range, specificity, accuracy, precision, applicability and stability of the method were verified. The OVA content of seven batches of influenza virus split vaccine bulk solutions was determined by the established method and imported OVA detection kit, and the coincidence rate was calculated.Results A total of three hybridoma cell lines that could be stably passaged and produce OVA-specific mAb were selected, named 3D6, 2B11 and 3H8, respectively, and the ascites titers were all higher than 10~7. After purification, the purity was over 95%, with good specificity, and the relative affinity ranking was2B11 > 3D6 > 3H8. After pairing screening and optimization, it was determined that 2B11 was used as the coating antibody with the optimal concentration of 4 ??g/mL, and biotin-labeled 3D6 was used as the detection antibody with the optimal dilution of 1∶100. The method had a limit of quantitation(LOQ) of 3 ng/mL. In the concentration range of 3. 13-50. 00 ng/mL,OVA standard exhibited a good linear relationship with A_(450/630), and the linear regression equation was y = 0. 004 9 x + 0. 088 7,R~2= 0. 987 9. This method had no cross-reaction with fetal bovine serum and human serum albumin. The spiked recovery rates of three influenza virus split vaccine bulk solutions were all within the range of 90% to 110%. The CVs of precision verification were less than 10%. The dilution of three batches of influenza virus split vaccine bulk solutions showed a good dose-dependent relationship with A_(450/630). Compared with the detection reagents stored at 4 ℃ for 6 days, the detection results of the detection reagents stored at 37 ℃ for 3 and 6 days decreased by less than 20%, and each R~2 was more than 0. 97.The coincidence rates between the detection results of the established method and imported OVA detection kit were between93. 96% and 100. 89%.Conclusion The OVA double antibody sandwich ELISA quantitative detection method was established by using the prepared OVA mAb, with good specificity, accuracy, precision, applicability and stability, which provides a reliable detection method for the determination of OVA residues in the verification requirements of chicken embryo-derived vaccine finished products.

Objective To selectively purify antibodies with high specificity for the C subunit of tetanus toxin(TT) from traditional tetanus antitoxins(TATs).Methods In this study, NHS-activated Sepharose 4 FF resin was used in combination with the TT TeNT-Hc-C869A subunit as a ligand to develop an immunoaffinity chromatography method. The stability and durability of the resin were tested by storing it under different conditions(at 4, 25 and 37 ℃ for 3, 10 and 30 d) and monitoring its reusability. The changes in the potency of the purified fractions by immunoaffinity chromatography were evaluated both in vitro and in vivo.Results Storage at 4 and 25 ℃ for up to 30 d did not significantly affect column performance. F(ab') 2 was purified by immunoaffinity column for five times continuously, and the loading capacity, elution efficiency and purity of eluted antibody were consistent each time, with the protein purity greater than 95%. After five times of continuous alkaline washing and the following purification, the purity of the protein was above 95%. The specific activities of purified equine plasma, enzymatically digested equine tetanus immunoglobulin(TIG) F(ab')2 and human TIG increased by5, 5 and 30 times respectively. And in the mouse neutralization assay, the specific activity of F(ab')2 increased significantly after purification, reaching 243 902 IU/g.Conclusion The developed immunoaffinity chromatography column shows broad applicability, and the chromatography matrix used is simple, cost-effective, and easy to replicate, making it suitable for largescale industrial applications, greatly enhancing safety, and reducing the risk of common clinical allergic reactions.