Objective To prepare A29L protein, A29L neutralization epitope tandem protein(Neu6) and A29L-mRNA-lipid nanoparticles(LNPs) vaccine based on A29L antigen, a key neutralizing target of monkeypox virus(MPXV), and evaluate their immunogenicity, so as to provide experimental basis for the development of MPXV vaccine and antibody drugs.Methods The A29L and Neu6 proteins were expressed by E.coli prokaryotic system and purified. Female BALB/c mice were immunized at five sites by subcutaneous injection in the extremities and intraperitoneal injection with five mice in each group. After three times of immunization, blood samples were collected from posterior orbital venous plexus and serum was separated. The template sequence of A29L-mRNA was cloned into plasmid pBluescript Ⅱ SK(+), and the template plasmid pBlueScriptⅡSK(+)-A29L-mRNA was constructed. A29L-mRNA was synthesized by transcription in vitro and purified by capping. A29L-mRNA were encapsulated by INano~(TM)L nano-drug preparation system to prepare A29L-mRNA-LNPs, which wereinjectedintramuscularlyintothebackoffivefemale BALB/c mice. After three times of immunization, blood samples were collected from orbital venous plexus and serum was separated. The serum IgG titers were detected by indirect ELISA and the serum neutralizing antibody titers were detected by plaque reduction neutralization test(PRNT).Results A29L and Neu6proteins were obtained successfully, with the purity of more than 90%, which were expressed mainly in inclusion bodies and showed specific binding to mouse anti-A29L monoclonal antibodies. The particle size of A29L-mRNA-LNPs was uniform(71. 35 nm), the polymer dispersity index(PDI) was 0. 097, and the encapsulation efficiency was 93%. HEK-293T cells transfected with A29L-mRNA-LNPs in vitro effectively expressed A29L protein. The serum IgG antibody titers of mice in A29L and Neu6 groups were significantly higher than that of mRNA group(t = 5. 692 and 5. 774, respectively, each P < 0. 001),while the serum neutralizing antibody titer of Neu6 group was significantly higher than those of A29L and mRNA groups(t =5. 202 and 4. 475, respectively, each P < 0. 01).Conclusion The immunogenicity of A29L-mRNA-LNPs is weak, suggesting that the effect of single antigen mRNA vaccine is limited. A29L and Neu6 proteins expressed in prokaryotic cells have good immunogenicity, and Neu6 can significantly improve the neutralizing antibody level, indicating that multi-antigen combination strategy should be explored in the future, and Neu6 protein is expected to become a highly effective candidate immunogen for MPXV vaccine and neutralizing antibody development.

Objective To determine the immune protection rates of the identified antigens and potential antigens against Trueperella pyogenes(T. pyogenes) in order to screen for protective antigens.Methods The ATP binding cassette(ABC)substrate-binding proteins(SBPs) were searched from the proteins annotated in the genome of T.pyogenes strain 2012CQZSH(GenBank: CP012649), and the conservation and strain distribution rate of the genes were analyzed using BLSAT. The screened proteins were expressed in the GST fusion protein expression system, purified by Glutathione-Sepharose 4B and used to immunize 20 female Kunming mice. PBS without recombinant protein was used as control group. On the 14th day after the second immunization, the mice were attacked with lethal doses of T.pyogenes strain ZSH-2020[(8. 9 ± 0. 4) × 10~8CFU]via intraperitoneal injection. The number of mice that died within 21 days after the attack was recorded, and the protein protection rates were calculated. The proteins a with protection rate greater than 80% were judged as protective antigens.Results The genes encoding ALD74485(pyolysin, PLO), ALD74170(iron-binding protein, IBP), ALD74643,ALD73390, ALD73068, ALD73591, and ALD73669 were conserved and widely distributed in the strains, and their recombinant proteins were prepared. The protective rates of recombinant proteins PLO, IBP, and ALD73390 were 87. 5%, 81. 25%,and 81. 25%, respectively, which were higher than the protective rates of other proteins.Conclusion PLO, IBP and ALD-73390 are protective antigens against T.pyogenes infection, and the multi-component vaccine composed of these proteins may enhance the effectiveness of the subunit vaccine against T.pyogenes

Objective To explore and analyze the effects of different batches of fetal bovine serum(FBS) on the quality evaluation of human mesenchymal stem cells(hMSCs) products and provide reference for the development and quality control of hMSCs products.Methods Retrospective analysis was conducted on the immune-regulatory function data of standard cell line CCRC-hMSC-S1 cultured in medium supplemented with FBS from different batches, based on the quality review data of hMSCs products. The standard cell line CCRC-hMSC-S1 was co-cultured with allogeneic peripheral blood mononuclear cells(PBMCs) from 5-6 donors respectively, and five different batches of FBS(FBS-H, FBS-A, FBS-E1, FBS-E2 and FBS-E3)were added to the co-culture system to clarify the direct effects of different batches of FBS on the detection value of immuneregulatory function by CCRC-hMSC-S1. The hMSCs from eight different individuals were cultured respectively in five medium containing different batches of FBS for two weeks, and the immune-regulatory function and osteogenic differentiation capacity were evaluated to clarify the effects of long-term culture of different batches of FBS on the key quality attributes of hMSCs.Results In the co-culture system of hMSCs and PBMC, the detection value of regulatory T-cell(Treg) promotion rate in FBS-H culture system was significantly lower than that in FBS-A, FBS-E1, FBS-E2 and FBS-E3 culture systems(t = 3. 2,3. 0, 3. 2 and 2. 9, respectively, each P < 0. 05), and the detection value of T lymphocyte proliferation inhibition rate in FBSH culture system was significantly higher than that in FBS-A, FBS-E1, FBS-E2 and FBS-E3 culture systems(t = 3. 7, 3. 4,2. 8 and 3. 0, respectively, each P < 0. 05); the inhibitory ability of hMSCs long-term cultured in FBS-A on Th1 proliferation was significantly lower than that of hMSCs long-term cultured in FBS-H, FBS-E1, FBS-E2 and FBS-E3(t = 3. 0, 2. 4, 3. 7and 3. 3, respectively, each P < 0. 05); the suppressive effect of hMSCs long-term cultured in FBS-H on T lymphocyte proliferation was significantly higher than that of hMSCs long-term cultured in FBS-A, FBS-E1, FBS-E2 and FBS-E3(t = 4. 7,2. 6, 3. 4 and 3. 9, respectively, each P < 0. 05); the alizarin red S staining area of hMSCs long-term cultured in FBS-E3 after osteogenic differentiation was significantly lower than that of hMSCs long-term cultured in FBS-A, FBS-H and FBS-E1(t =4. 1, 5. 5 and 3. 9, respectively, each P < 0. 01).Conclusion In the research and development of hMSCs cell therapy products, it is necessary to pay close attention to the effects of FBS components on the quality evaluation of hMSCs, especially the immune-regulatory function and differentiation potential, and select suitable batches of FBS for the development and quality control of hMSCs products.

Objective To investigate the effect of fucosyltransferase 7(Fut7) dysregulation on lipopolysaccharide(LPS)-stimulated inflammatory response in human monocytes THP-1, so as to provide experimental evidence for the study of inflammatory induction mechanism of clinical related diseases.Methods THP-1 cells were treated with different concentrations of LPS(0, 0. 5, 1, 2, 4, 10 μg/mL). The transcription levels of interleukin-1β(IL-1β), IL-6, IL-8 and tumor necrosis factor-α(TNF-α) in the cells were detected by RT-qPCR, and the LPS stimulation concentration for establishing inflammatory cell model was determined. THP-1 cells were stimulated with the optimum concentration of LPS to establish the inflammatory cell model. The effects of Fut7, sialyl Lewis X(sLeX) and Fut7 activity inhibitor SGN-2FF on the transcription levels of IL-1β, IL-6,IL-8 and TNF-α genes in inflammatory cell model were detected by RT-qPCR.Results The LPS stimulation concentration for establishing inflammatory cell model was 4 μg/mL. Overexpression of Fut7 and sLeX significantly increased the transcription levels of IL-1β, IL-6, IL-8 and TNF-α in LPS-induced THP-1 cell inflammatory model(t = 3. 93-30. 05, each P < 0. 05).Inhibiting the expression of Fut7 significantly decreased the transcription levels of IL-1β, IL-6, IL-8 and TNF-α in the inflammatory cell model(t = 6. 98-39. 50, each P < 0. 01). SGN-2FF significantly decreased the transcription levels of IL-1β, IL-6and IL-8(t = 5. 17-6. 44, each P < 0. 001), while significantly increased the transcription level of TNF-α(t = 12. 31, P < 0. 001).Conclusion Overexpression of FuT7 and sLeX can enhance the LPS-induced inflammatory response in THP-1 cells by promoting the expression of inflammatory cytokines. Inhibiting the expression of Fut7 and SGN-2FF, an inhibitor of Fut7enzyme activity, can effectively relieve inflammation, which provides a new strategy for the treatment of inflammatory diseases.

Objective To express M cell-targeted fusion peptide SAM-GFP in prokaryotic cells, purify it and identify its targeting properties, so as to lay a foundation for the research of M cell-targeted oral vaccine against Helicobacter pylori.Methods The GFP gene was synthesized by PCR-based accurate synthesis(PAS) and cloned into the vector pCzn1-SAM to construct the recombinant plasmid pCzn1-SAM-GFP. The recombinant plasmid was transformed into competent E.coli Arctic Express, induced by IPTG, and then purified by Ni-NTA affinity chromatography. The obtained fusion protein SAM-GFP was identified by 12% SDS-PAGE, Western blot and fluorescence microscopy. The M-cell targeting properties of SAM-GFP fusion protein was verified by immunofluorescence staining using mouse ileal loop model.Results The recombinant plasmid pCzn1-SAM-GFP was constructed correctly as identified by double digestion and sequencing. The fusion protein SAM-GFP had a relative molecular mass of about 54 900, and was mainly expressed in soluble form with a purity of 94. 5% after purification. It showed specific binding to anti-6 × His/GFP monoclonal antibody, and green fluorescence was observed under fluorescence microscope. SAM-GFP could be specifically uptaken by small intestinal M cells. Conclusion The fusion protein SAMGFP with M-cell targeted function was successfully expressed and purified, which lays a foundation for the subsequent research and development of oral vaccine against Helicobacter pylori.

Objective To evaluate the immune responses of mice immunized with different doses of therapeutic tuberculosis DNA vaccine B21, in order to provide a reference for dose selection of quality control methods for the vaccine efficacy.Methods SPF female C57BL/6 mice were immunized by intramuscular injection of 25(LD), 50(MD), 100(HD) μg/100 μL B21 DNA vaccine through the medial thigh, combined with electroporation stimulation for a total of three times at an interval of two weeks. Three weeks after the last immunization, the spleen lymphocytes and peripheral blood serum were isolated. The cellular immunity and immune memory effects were detected by enzyme-linked immunosorbent spot(ELISpot) and flow cytometry(FCM), and the humoral immunity was detected by ELISA.Results Three weeks after the last immunization, the numbers of Ag85b-specific and Rv1738-specific IFNγ spot-forming cells(SFCs) in HD group were significantly higher than those in MD and LD groups(F = 11. 22 and 15. 89, respectively,each P < 0. 001); Rv2029c-specific IFNγ SFCs were significantly higher than that of MD group(F = 5. 761, P < 0. 05), but showed no significant difference with LD group(F = 5. 761,P > 0. 05). IL-2 SFCs of the three antigens in HD group were significantly higher than those in MD and LD groups(F =8. 044-33. 07, each P < 0. 05). The proportion of CD4~+ T cells secreting IFNγ in HD group was significantly higher than those in MD and LD groups(F = 5. 976, P < 0. 01), while there was no significant difference in the proportion of CD4~+ T cells secreting IL-2 among the three groups(F = 2. 836, P > 0. 05). The proportion of IFNγ~+ memory T cells(CD44~+ CD62L~+/~-) in HD group was significantly higher than those in MD and LD groups(F = 8. 793, P < 0. 05), and the proportion of activated memory B cells(CD19~+ CD27~+) in HD group was significantly higher than those in MD and LD groups(F = 7. 958, P < 0. 01).There was no significant difference in B21-specific IgG antibody level among the three groups(F = 5. 765, P > 0. 05).Conclusion B21DNA vaccine effectively induced cellular immunity, humoral immunity and immune memory responses in a dose-dependent manner. The dose of 100 μg/100 μL induced the best immunization effect, and ELISpot could detect IFNγ/IL-2 SFCs with good stability and reproducibility, which can be used as the recommended immune dose,evaluation indicator and detection method for quality control of B21 vaccine efficacy.

Objective To evaluate the safety of age-eligible children vaccinated with vaccines containing diphtheria, tetanus and acellular pertussis combined vaccine(DTaP) in Jilin Province from 2020 to 2022, so as to provide references for the safe use of vaccines in follow-up vaccination work.Methods Data on individual cases of adverse event following immunization(AEFI) in age-eligible children vaccinated with diphtheria, tetanus and acellular pertussis combined vaccine(DTaP), diphtheria, tetanus,acellular pertussis and Haemophilus influenzae type b combined vaccine(DTaP-Hib) and diphtheria, tetanus,pertussis(acellular, component), poliomyelitis(inactivated) vaccine and Haemophilus type b conjugate vaccine, adsorbed(DTaP-IPV/Hib) in Jilin Province from January 1, 2020 to December 31, 2022 were collected through the AEFI system of the Jilin Provincial Immunization Program Information Management System and the National Immunization Program Information System. A descriptive epidemiological approach was employed for analysis, and statistical methods were applied to compare differences between groups.Results Age-eligible children in Jilin Province from 2020 to 2022 were inoculated with DTaP,DTaP-Hib and DTaP-IPV/Hib vaccines 1 301 812 doses. A total of 3 132 AEFI cases(240. 59 per 100 000 doses) were reported. 3 122 common adverse reaction cases(99. 68%, 239. 82 per 100 000 doses), 7 rare adverse reaction cases(0. 22%,0. 54 per 100 000 doses) and 3 coupling reaction cases(0. 1%, 0. 23 per 100 000 doses) were reported in the study. The incidence rates of AEFI cases of 3 vaccines containing DTaP were 257. 00 per 100 000 doses(3 025 cases), 175. 61 per 100 000doses(34 cases) and 69. 26 per 100 000 doses(73 cases), with statistically significant difference(χ~2= 145. 53, P < 0. 001).The incidence rates of AEFI cases for the booster immunization of 3 vaccines containing DTaP were higher than the basic immunization and the difference was statistically significant(χ~2= 254. 98, 18. 03 and 20. 39, respectively, each P < 0. 05).The reported AEFI cases were mainly common adverse reactions,inclucding fever, local redness and swelling, local induration and so on, and occurred mainly within 0 to 1 day after vaccination.Conclusion AEFI of age-eligible children vaccinated with DTaP, DTaP-Hib and DTaP-IPV/Hib in Jilin Province from 2020 to 2022 was mainly common adverse reaction, and the overall safety was good. It is suggested that AEFI monitoring after vaccination should be continuously strengthened and passive monitoring system should be optimized to reduce underreporting bias.

Objective To establish and verify a method for rapid testing of type D antigen in the monovalent bulk of Sabin strain inactivated polio vaccine(sIPV) using surface plasmon resonance(SPR) technology.Methods Bovine polyclonal antibodies against typesⅠ, Ⅱ and Ⅲ D antigens were labeled on the surface of an SPR chip to capture poliovirus typeⅠ, Ⅱ andⅢ D antigens. The change of signal response values on the chip surface was monitored and the relationship between D antigen content and response signals was determined to establish SPR method for the determination of D antigen content. The linearity, specificity, precision and accuracy of the method were validated. The SPR method and ELISA method were used to detect D antigen in six batches of monovalent bulks of sIPV of typeⅠ, Ⅱ and Ⅲ, separately. The results of the two methods were compared and analyzed to supplement the validation of the feasibility of this method.Results The D antigen content of the three types showed a good linear relationship with the response values, with R~2values all above 0. 99. There was some cross-reactivity between the three types, but the cross-reactivity response values were obviously smaller than the positive reaction response values. The relative standard deviations(RSDs) for the repeated measurements of the three types were3. 1%, 1. 2% and 1. 4%, respectively, and the RSDs of intermediate precision results were 5. 6%, 2. 0% and 2. 7%, all within 8%, lower than the 15% limit set by the ELISA method. The recovery rates of accuracy verification were 85. 8%,105. 1% and 88. 4%, all within the range of 80% to 120%. Compared with the ELISA results of six batches of monovalent bulks, there was no statistically significant difference in typeⅠand type Ⅱ(t = 1. 924 and-0. 901, P = 0. 083 and 0. 389,respectively), but a statistically significant difference in type Ⅲ(t =-2. 617, P = 0. 026).Conclusion A SPR method for rapid and high-throughput detection of D antigens in sIPV of typesⅠ, Ⅱ and Ⅲ was established, which has good precision and accuracy, and provides more schemes for the whole process monitoring and quality inspection of vaccine production.

Objective To optimize the fermentation process of mannatide strain and screen a more accurate method for determination of mannatide content in order to lay a foundation for its large-scale production.Methods Molecular identification of α-hemolytic streptococcus strain 33# was performed by 16S rDNA sequencing and phylogenetic tree. Using the content of mannatide as the indicator, the activation times(1-4 times), inoculation time(2, 5, 7, 10 h), and fermentation mode(standing culture, shaking once every 6 h, shaking table 70 r/min, shaking table 200 r/min) were optimized by single factor test. The culture medium composition and inoculation amount were optimized by orthogonal test. In addition, the effect of pH on mannatide content was determined by dynamically controlling pH in 10 h before fermentation. The content of mannatide in the supernatant of fermentation broth prepared by five groups of formulations in orthogonal test was determined by phenolsulfuric acid method and gel permeation chromatography(GPC) method respectively, and the results were compared.Results The mannatide strain was identified as Enterococcus faecalis by molecular identification. The optimum conditions were 3 timesofactivation,5 hofinoculation and 70r/min shakingtableculture.Theoptimum culturemedium composition was0.2%glucose,1%yeastextract,0.3%beefextract,0.6%soybeanpeptoneand0.5%sodiumchloride,andtheinoculationamount was 10%. After dynamically adjusting the pH to 6. 8-7. 4, the mannatide content increased from(0. 451 ± 0. 018) mg/mL(without pH adjustment) to(0. 642 ± 0. 007) mg/mL. Compared with phenol-sulfuric acid method, GPC method could accurately separate the target product(after ethanol precipitation) and avoid the interference of small molecular sugars.Conclusion The fermentation process of mannatide strain was optimized successfully, and GPC was determined as a more reliable method for detecting mannatide content, laying a foundation for the large-scale production of mannatide.

Objective To establish an ELISA method for the detection of mumps virus titer and verify it, so as to shorten the detection time and provide technical support for emergency inspection.Methods Vero cells were infected with reference standard of mumps vaccine virus. With mouse ascites as the primary antibody and HRP-labeled goat anti-mouse IgG as the secondary antibody, the inoculation concentration of Vero cells, centrifugation time after virus inoculation, virus infection time, antibody concentration, sealing fluid type, and fixation method and time were optimized to establish an ELISA method for detecting mumps virus titer. The specificity, accuracy, precision and linearity were verified, and the results were compared with those of micro-cytopathic method.Results The established ELISA method was as follows: adding Vero cell suspension with concentration of 3 × 10~5 cells/mL to 96-well cell culture plate for 24 h; adding samples and reference standard, centrifuging at 236 × g for 2 h, and then culturing for 48 h; fixing the cells with 4% paraformaldehyde for 10 min, then treating with PBS-0. 5% TritonX-100 for 30 min; after sealing with 5% skim milk powder, adding primary antibody(1∶1 000 diluted) and secondary antibody(1∶2 000 diluted); using TMB for color development and sulfuric acid to terminate the reaction, and detecting the A_(450) value. The established method had good specificity for mumps virus. The relative bias(RB) in detecting virus titer of different concentrations of reference standard was less than 10%. The coefficient of variation(CV) of virus titer of reference standard detected six times in different working days was 3. 9%. The linear slope of the method was 0. 920 6 and the correlation coefficient(r) was 0. 993 9. There was no significant difference in virus titer results detected by the two methods(t = 2. 049, P > 0. 05).Conclusion The established ELISA method for detecting mumps virus titer has strong specificity, good accuracy and precision, and has good consistency with the detection results of micro-cytopathic method, which can obviously shorten the detection time