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.

Currently, influenza vaccination is the most effective measure to control the epidemic of influenza. The addition of adjuvant in a vaccine can reduce the dose of antigen required, enhance the immunogenicity, and produce cross-protection. This review summarized the literature on influenza-related adjuvants and outlined the mechanism and safety of vaccine adjuvants approved and under development in order to provide reference for the development of new influenza vaccines.

Objective:To compare the optimal conditions, virus yield, viral titer and cell metabolism between culturing influenza virus H1N1 vaccine strain in MDCK and MDCK-G1 cells.Methods:The optimal culture conditions were investigated using chessboard method. The hemagglutination titer, half of the tissue infection dose (TCID 50) and the metabolism of glucose and lactic acid were monitored and compared between the two cell lines. Results:After MDCK-G1 cells were inoculated with H1N1 at the multiplicity of infection (MOI) of 0.001 with the presence of 1 μg/ml of trypsin, the hemagglutination titer reached the peak of 1∶512 at 72 h and the viral titer was 10 7.4TCID 50/ml. In the MDCK cell line group, the hemagglutination titer reached the peak of 1∶256 at 72 h and the viral titer was 10 6.6TCID 50/ml when using H1N1 at MOI=0.0001 and 1 μg/ml of trypsin. Conclusions:MDCK-G1 cells were more suitable than MDCK cells for the proliferation of influenza virus. This study provided reference data for further research on cell-derived influenza vaccine.

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 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 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.