Objective:To investigate the effects of poly(A) tails with different lengths on mRNA expression in vitro and the passage stability of transcription template with poly (A) tail in Escherichia coli ( E. coli). Methods:Plasmids with poly(A) tails of 38, 60, 103, 125 and 126 (60 nt+ 6 nt spacer+ 60 nt) nt were designed and constructed. Then the plasmids were linearized by single enzyme digestion and used as transcription template for preparing enhanced green fluorescent protein (EGFP)-mRNA. EGFP-mRNA containing poly(A) tails of different lengths were transfected into 293T cells and the expression of EGFP was detected by flow cytometry. As to stability test, the template plasmids with poly (A) tail of 125 and 126 nt were transformed into E. coli TransStbl3 and Top10 competent cells. Seven clones were selected for culture and plasmid extraction, and then the plasmids were digested by restriction enzyme and detected by capillary electrophoresis. For passage stability, three correctly sequenced clones of each group were selected for continuous passage at 37℃, and the plasmids were extracted and digested every two generations for capillary electrophoresis. At the same time, the correctly sequenced clones of 125 nt group were also passaged at 30℃, and the plasmids were also extracted and digested every two generations for capillary electrophoresis. Results:The transcription templates with poly(A) tail of different lengths were successfully constructed. Flow cytometry showed that the fluorescence expression of the template plasmids with poly (A) tail of 103 and 125 nt were significantly higher than that of 38 and 60 nt. The fluorescence expression of the plasmid with poly (A) tail of 126 nt was significantly higher than that of all other groups. The percentages of stable sequences of the template plasmid with poly(A) tail of 125 nt in TransStbl3 and Top10 competent cells were 76% and 91%, respectively. The results of continuous passage showed that poly(A) tail of 125 nt could be stable to the 4th generation at 37℃ in both TransStbl3 and Top10 competent cells, and stable to the 16th and 10th generations at 30℃. The percentages of stable sequences of the template plasmid with poly(A) tail of 126 nt in TransStbl3 and Top10 competent cells were 95% and 48%, respectively. The results of continuous passage showed that poly(A) tail of 126 nt could be stable to the 12th generation at 37℃ in both TransStbl3 and Top10 competent cells.Conclusions:The length and composition of poly(A) tail in mRNA affected the expression of target protein. Adding a spacer with a length of 6 nt to poly(A) tail and low temperature culture were both helpful to improve the stability of the template plasmid, which provided a reference for the design and preparation of in vitro transcription template of mRNA vaccine.

Objective:To prepare a recombinant hemagglutinin trimer (HA-Tri) vaccine against influenza viruses and to study its immunogenicity in a mouse model.Methods:A stable CHO cell line that could express HA-Tri was constructed. Western blot, single radial immunodiffusion, protein particle size detection and N-glycosylation site analysis were performed for qualitative and quantitative analysis of the recombinant protein. According to the different treatment conditions such as dosage and adjuvant, BALB/c mice were divided into 11 groups and subjected to consistent immunization procedures. Serum neutralizing antibody titers were measured on 56 d after the first immunization to evaluate the immunogenicity of HA-Tri.Results:The constructed CHO cells could secret and express HA-Tri proteins. The HA-Tri proteins were biologically active and capable of forming precipitation rings in the single radial immunodiffusion. The particle size of HA-Tri was approximately 18.79 nm and 10 N-glycosylation sites were detected, including high mannose, complex glycoforms and heterozygous glycoforms. After prime-boost immunization, there was no statistically significant difference in the titers of neutralizing antibodies induced in mice by 3.75 μg of HA-Tri in combination with RFH01 adjuvant and 15 μg of monovalent vaccine stock solution ( P=0.431 2, U=36). Serum antibody titers in the HA-Tri+ RFH01 groups were higher than those in the corresponding HA-Tri groups without RFH01 adjuvant, and the highest titer was induced in the 15 μg HA-Tri+ RFH01 group, which was 1 280. Conclusions:The recombinant HA-Tri protein was successfully prepared. HA-Tri in combination with RFH01 adjuvant could induce humoral immune responses against influenza viruses in BALB/c mice, which would provide reference for the development of influenza virus recombinant subunit vaccines.

Objective:To purify H5N1 influenza virus concentrate prepared by MDCK cells with a new mixed-mode chromatography medium Capto Core700 and the traditional medium Sepharose 4FF, and to compare the separation and purification efficacy of the two media.Methods:Capto Core700 and Sepharose 4FF were used to purify inactivated H5N1 influenza virus concentrate. The morphology of virus particles in different samples was then observed under a transmission electron microscope. Single radial immunodiffusion (SRID), Folin-Phenol (Lowry) method, double-antibody sandwich ELISA and qPCR were used to detect hemagglutinin, total protein, host cell protein (HCP) and host cell DNA (HCD) before and after purification. The recovery rate of virus antigen and the removal rate of impurities were calculated. The immunogenicity of the viruses purified with different media was analyzed using animal experiments. Difference in the purification efficacy of the two chromatography media was analyzed by t-test. Results:H5N1 influenza viruses purified by Capto Core700 or Sepharose 4FF showed the typical influenza virus morphology under transmission electron microscope. There was no significant difference in the recovery rate of hemagglutinin between the two chromatography media ( P>0.05), but compared with Sepharose 4FF, Capto Core700 had a higher removal rate of impurities (total protein, HCP, HCD) and the difference was statistically significant ( P<0.05). Animal experiments showed that the viruses purified by the two chromatography media had good immunogenicity. Conclusions:Compared with Sepharose 4FF chromatography medium, Capto Core700 could more effectively remove process-related impurities such as HCP, HCD and total protein without affecting the recovery rate of viral antigen. This study provided reference for the development of purification technology in the production of H5N1 influenza virus vaccine in MDCK cells.

Objective:To evaluate the immunogenicity of a quadrivalent subunit vaccine combined with RFH01 adjuvant in a mouse model.Methods:Identification tests were performed on four monovalent influenza virus subunit vaccine stock solutions according to the methods described in Part 3 of the Chinese Pharmacopoeia 2020 Edition. In the study of the quadrivalent subunit vaccine combined with RFH01 adjuvant, 460 female BALB/c mice (6-8 weeks old) were randomly divided into 46 groups including experimental groups, vaccine control group, negative control group and blank group with 10 mice in each group. In the study of the quadrivalent subunit vaccine in old and young mice, 80 female 10-month-old and 80 female 10-week-old BALB/c mice were randomly divided into 16 groups ( n=10) including monovalent influenza virus vaccine group, quadrivalent subunit vaccine group, quadrivalent subunit vaccine+ RFH01 adjuvant group, chicken embryo quadrivalent split vaccine control group and PBS group. All mice were immunized by intramuscular injection. At 21 d after the primary immunization, a booster immunization was conducted using the same strategy. Blood samples were collected at 21 d and 42 d after the primary immunization for serum separation. Haemagglutination inhibition (HI) test was performed to detect the antibody levels in mouse serum samples. Results:After the booster immunization, the positive conversion rates in all vaccine+ RFH01 adjuvant groups reached 100%, and the geometric mean titers (GMTs) of serum antibodies were significantly higher than those of the vaccine groups without RFH01 adjuvant. There were significant differences in serum antibody titers between the monovalent/quadrivalent subunit vaccine groups with and without RFH01 adjuvant. After the booster immunization, the titers of serum antibodies against H1N1, H3N2, B/Victoria and B/Yamagata in the 10-week-old mice were significantly higher than those in the 10-month-old mice.Conclusions:The monovalent and quadrivalent influenza virus vaccines in combination with RFH01 adjuvant could elicit higher antibody titers in young (6-10 weeks old) and old (10 months old) mice, showing good immunogenicity.

Influenza is a worldwide infectious disease caused by influenza virus. It has posed great challenges on public health and social stability since 1918. At present, vaccination is the most effective way to prevent and control influenza epidemics. Broad-spectrum antiviral drugs and neutralizing antibodies against influenza virus have been widely studied in recent years. Hemagglutinin (HA), which is on the surface of influenza virus, plays an important role in the stage of viral invasion into host cells. It is the main effective antigenic component of current influenza vaccines, as well as the main target of broad-spectrum neutralizing antibodies and broad-spectrum antiviral drugs. This review summarized the progress in the development of novel influenza vaccines, neutralizing antibodies, and antiviral drugs based on influenza virus HA, as well as other prevention and control measures, hoping to present new ideas for future influenza prevention and control.

Objective:To explore the values of albumin-bilirubin (ALBI) score, carcinoembryonic antigen (CEA) and combination of the two in the prognostic assessment of colorectal cancer patients with postoperative liver metastasis.Methods:The clinicopathological data of 98 colorectal cancer patients with postoperative liver metastasis who were admitted to Lianyungang Oriental Hospital and receiving adjuvant chemotherapy from January 2016 to March 2020 were retrospective analyzed. The data of serum protein, bilirubin, and CEA before chemotherapy were obtained, the relationship between serum protein and bilirubin was analyzed, and the ALBI score was calculated. The ALBI-CEA score was judged according to the ALBI score and the CEA level. ALBI score > -2.60 points was categorized as high ALBI group, and ALBI score ≤ -2.60 points was categorized as low ALBI group; CEA >5 ng/ml was categorized as high CEA group, and CEA ≤5 ng/ml was categorized as low CEA group; patients were categorized into 0, 1, and 2 points groups based on ALBI-CEA score. Overall survival (OS) and progression-free survival (PFS) of ALBI score, CEA and ALBI-CEA score subgroups were analyzed by Kaplan-Meier method; with the actual survival and progress status of the patients as the gold standard, receiver operating characteristic (ROC) curve was used to analyze the effect of 3 indicators to assess patients' OS and PFS, and area under the curve (AUC) was compared; Cox proportional hazards model was used to analyze the influencing factors of OS and PFS.Results:The median albumin and bilirubin levels of the 98 patients were 34.4 g/L (26.8-42.8 g/L) and 16.6 μmol/L (7.6-44.6 μmol/L), and the result of Pearson correlation analysis showed a negative correlation between the levels of albumin and bilirubin ( r = -0.282, P < 0.001). The 3-year OS and PFS rates in the high ALBI group were lower than those in the low ALBI group (OS rate: 9.2% vs. 33.3%, PFS rate: 7.7% vs. 18.2%), and the differences in OS and PFS between the two groups were statistically significant ( χ2 values were 27.64, 23.30, both P < 0.001). The 3-year OS and PFS rates in the high CEA group were lower than those in the low CEA group (OS rate: 7.1% vs. 42.9%, PFS rate: 7.1% vs. 21.4%), and the differences in OS and PFS between the two groups were statistically significant ( χ2 values were 23.71, 17.14, both P < 0.001). The 3-year OS rates in the ALBI-CEA score 0, 1 and 2 points groups were 77.8%, 20.9% and 2.2%, and the 3-year PFS rates were 44.4%, 9.3% and 6.5%, and there were statistical differences in OS and PFS among the three groups ( χ2 values were 102.36, 76.55, both P < 0.001). The ROC curve analysis showed that the AUC of ALBI score, CEA and ALBI-CEA score for assessing OS were 0.688 (95% CI 0.544-0.832), 0.754 (95% CI 0.618-0.890) and 0.828 (95% CI 0.723-0.933) (all P < 0.05), and the AUC for assessing PFS were 0.618 (95% CI 0.436-0.799), 0.646 (95% CI 0.464-0.829) and 0.682 (95% CI 0.494-0.870) (all P > 0.05). Multivariate Cox regression analysis showed that ALBI-CEA score was an independent influencing factor for OS (2 points vs. 0 point: HR = 17.254, 95% CI 8.385-35.504, P < 0.001) and PFS (2 points vs. 0 point: HR = 6.144, 95% CI 3.725-10.134, P < 0.001) of patients. Conclusions:The colorectal cancer patients with liver metastasis and high ALBI-CEA score are at high risk of death and disease progression and have a poor prognosis, and they are recommended to receive intensive treatment.

Transglutaminase 2 (TGM2) is a ubiquitous multifunctional protein, which is related to the adhesion of different cells and tumor formation. Previous studies found that TGM2 is involved in the interaction between host cells and viruses, but the effect of TGM2 on the proliferation of influenza virus in cells has not been reported. To explore the effect of TGM2 during H1N1 subtype influenza virus infection, a stable MDCK cell line with TGM2 overexpression and a knockout cell line were constructed. The mRNA and protein expression levels of NP and NS1 as well as the virus titer were measured at 48 hours after pot-infection with H1N1 subtype influenza virus. The results showed that overexpression of TGM2 effectively inhibited the expression of NP and NS1 genes of H1N1 subtype influenza virus, while knockout of TGM2 up-regulated the expression of the NP and NS1 genes, and the expression of the NP at protein level was consistent with that at mRNA level. Virus proliferation curve showed that the titer of H1N1 subtype influenza virus decreased significantly upon TGM2 overexpression. On the contrary, the virus titer in TGM2 knockout cells reached the peak at 48 h, which further proved that TGM2 was involved in the inhibition of H1N1 subtype influenza virus proliferation in MDCK cells. By analyzing the expression of genes downstream of influenza virus response signaling pathway, we found that TGM2 may inhibit the proliferation of H1N1 subtype influenza virus by promoting the activation of JAK-STAT molecular pathway and inhibiting RIG-1 signaling pathway. The above findings are of great significance for revealing the mechanism underlying the interactions between host cells and virus and establishing a genetically engineering cell line for high-yield influenza vaccine production of influenza virus.
Animals ; Cell Proliferation ; Dogs ; Humans ; Influenza A Virus, H1N1 Subtype/genetics* ; Influenza, Human ; Madin Darby Canine Kidney Cells ; Protein Glutamine gamma Glutamyltransferase 2

Objective:To express the head domain of influenza A virus hemagglutinin (HA) in a prokaryotic expression system and to evaluate its immunogenicity.Methods:The genes encoding the HA head domains of H1N1 and H3N2 influenza viruses were cloned into pET-22b(+ ) prokaryotic expression plasmid. After the induction with IPTG, the fusion proteins rH1N1-HA and rH3N2-HA containing HA head domain and His-tag were expressed and obtained from E. coli BL21. SDS-PAGE and Western blot was used to verify the expression of the recombinant proteins. Rabbits were immunized with multiple doses of the purified recombinant proteins to obtain polyclonal antibodies against the HA head domains of H1N1 and H3N2. The immunogenicity of the recombinant proteins was evaluated in BALB/c mice. Results:rH1N1-HA and rH3N2-HA induced protective antibodies (geometric mean titer ≥40) in mice and could be used as protective antigens. Polyclonal antibodies against rH1N1-HA and rH3N2-HA could be used as important materials for Western blot, ELISA and other immunological assays.Conclusions:The HA head domains prepared in this study could be used as protective antigens to induce protective antibodies in mice. Polyclonal antibodies against the HA head domains could be used for immunological and serological studies of influenza A viruses.

Objective:To evaluate the immunogenicity of Madin-Darby canine kidney (MDCK) cell-based quadrivalent influenza split vaccine (MDCK-Va) combined with different adjuvants.Methods:Different doses of MDCK-Va and chicken embryo-based quadrivalent influenza split vaccine (egg-Va) were intramuscularly immunized BALB/c mice twice with an interval of three weeks. Serum samples were collected to detect antibody titers using hemagglutination inhibition (HI) assay. BALB/c mice were immunized with different doses of MDCK-Va combined with QS21, AddVax, PolyI∶C, CpG ODN 1826 and AddVax/PolyI∶C (Add/Poly), respectively. HI and microneutralization assays were used to detect antibody titers 21 d after the first and booster immunization. Spleen tissues were collected from the mice immunized with 10 μg MDCK-Va combined with the above adjuvants 5 d after the booster immunization to analyze spleen index and the types of spleen cells.Results:The immunoprotective effect of MDCK-Va was not inferior to that of egg-Va. MDCK-Va combined with each of the above adjuvants could induce higher HI antibody titer than MDCK-Va alone, especially the QS21/Va and Add/Poly/Va groups, and the differences were statistically significant. For H1N1 vaccine, the Pearson′s correlation coefficient ( r) between HI antibody and neutralizing antibody was 0.737-0.910, and for H3N2 subtype vaccine, the value of r was 0.839-0.947. Compared with the MDCK-Va group, the QS21/Va group showed significantly increased spleen index and decreased proportion of single lymphocytes. QS21 and Add/Poly were much better than other adjuvants in stimulating mouse splenic neutrophils and CD4/CD8 cells. Conclusions:Add/Poly had a stronger immune enhancement effect on MDCK-Va, suggesting that it was a potential adjuvant for MDCK-Va. The antibody titer detected by HI and MN assays had a strong positive correlation.

Objective:To establish a method for isolation and purification of neuraminidase from influenza vaccine and to prepare reference substance for quantitative detection of neuraminidase.Methods:Functional magnetic particles with specific affinity for neuraminidase were prepared. The method for separation and purification of neuraminidase was established based on the magnetic particles. The separation and purification conditions were optimized. The purity of neuraminidase was analyzed and the specificity was verified. The enzyme activity was determined and the protein was quantified.Results:The functional magnetic particles modified with 4-aminophenanthroline were successfully prepared and the method for isolation and purification of neuraminidase based on the magnetic particles was established. The purity of neuraminidase was 98.7%. The concentrations of neuraminidase isolated and purified from the monovalent stock solution of H1N1, H3N2, B/Victoria and B/Yamagate vaccines were 71.50, 100.58, 64.11 and 37.68 μg/ml, respectively, and the enzyme activity remained.Conclusions:The method for isolation and purification of influenza virus neuraminidase was established and the corresponding reference substance was prepared.