Journal: bioRxiv

Article Title: A 10-valent composite mRNA vaccine against both influenza and COVID-19

doi: 10.1101/2024.03.05.583547

Figure Lengend Snippet: FLUCOV-10 protects mice from homologous or heterologous challenge with influenza viruses. A. Schematic diagram of the experimental design. BALB/c Mice were immunized with 50 μg of FLUCOV-10 or each volume of a placebo and boosted with the same dose after three weeks. Serum samples were collected 14 days post the second immunization. The mice were challenged 3 weeks post second immunization with 10 × mLD50 of A/California/04/2009 (H1N1) (B-D) or 10 × mLD50 of rgA/Guangdong/17SF003/2016 (H7N9) (E-G) or 3 × mLD50 of B/Florida/4/2006 (B/Yamagata) (H-J). B, E, and H, Weight changes and survival rates were recorded for 14 days (n = 7). B, E, and H, Viral titers in the turbinate or lung tissues from influenza-infected mice (n = 5 at each indicated day). C, F, and I, H&E staining of lung tissues from influenza-infected mice.

Article Snippet: 96-well plates (JET BIOFIL) were coated with recombinant spike proteins of Wuhan-hu-1 (ACROBiosystems, SPN-C52H4), BA.2.75.2 (ACROBiosystems, SPN-C522r), BQ.1.1 (ACROBiosystems, SPN-C522), or XBB.1.5 variant (ACROBiosystems, SPN-C524i), or recombinant Influenza HA proteins of A/Wisconsin/588/2019/A/Victoria/2570/2019 (H1N1) (Sinobiological, 40787-V08H1), A/Darwin/6/2021 (H3N2) (Sinobiological, 40868-V08H), B/PHUKET/3073/2013 (Sinobiological, 40498-V08B), B/Austria/1359417/2021 (Sinobiological, 40862-V08H), A/Vietnam/1194/2004 (H5N1) (Sinobiological, 11062-V08H1), or A/Hangzhou/3/2013 (H7N9) (Sinobiological, 40123-V08B) with a concentration of 2 μg/ml at 4°C overnight.

Techniques: Infection, Staining

Journal: bioRxiv

Article Title: Amplified visual immunosensor integrated with nanozyme for ultrasensitive detection of avian influenza virus

doi: 10.1101/128538

Figure Lengend Snippet: Optical study of Au nanostructure and antibody specificity towards target virus. (A) X-ray diffraction spectra of Au nanostructure. (B) ELISA results of antibody specificity towards target virus.

Article Snippet: Influenza A (H5N2) hemagglutinin antibodies (Anti-H3N2 antibodies HA MAb, Lot: HB05AP2609), Influenza A (H7N9) hemagglutinin antibodies (Anti-H7N9 antibody HA MAb, Lot: HB05JA1903), recombinant influenza virus A (H5N2) HA1 (A/Ostrich/South Africa/A/109/2006)(lot: LC09AP1021), recombinant influenza virus A (H7N8) HA1 (A/Mallard/Netherlands/33/2006) (lot: LC09AP1323) and recombinant influenza virus A (H7N9) HA1 (A/Shanghai/1/2013) (lot: LC09JA2702) were purchased from Sino Biological, Inc. (Beijing, China).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Amplified visual immunosensor integrated with nanozyme for ultrasensitive detection of avian influenza virus

doi: 10.1101/128538

Figure Lengend Snippet: Detection of avian influenza virus A (H5N1). (A) The calibration curve of the absorbance corresponding to the concentration of avian influenza virus A (H5N1). BSA was used as a negative control. Squares (red line) and circles (black line) denote proposed and conventional ELISA sensing results, respectively. (B) ELISA results for selectivity of the present study with different influenza viruses. Error bars in (A) and (B) denote standard deviations (n=3).

Article Snippet: Influenza A (H5N2) hemagglutinin antibodies (Anti-H3N2 antibodies HA MAb, Lot: HB05AP2609), Influenza A (H7N9) hemagglutinin antibodies (Anti-H7N9 antibody HA MAb, Lot: HB05JA1903), recombinant influenza virus A (H5N2) HA1 (A/Ostrich/South Africa/A/109/2006)(lot: LC09AP1021), recombinant influenza virus A (H7N8) HA1 (A/Mallard/Netherlands/33/2006) (lot: LC09AP1323) and recombinant influenza virus A (H7N9) HA1 (A/Shanghai/1/2013) (lot: LC09JA2702) were purchased from Sino Biological, Inc. (Beijing, China).

Techniques: Concentration Assay, Negative Control, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Suv4-20h2 protects against influenza virus infection by suppression of chromatin loop formation

doi: 10.1016/j.isci.2021.102660

Figure Lengend Snippet: Deletion of Suv4-20h2 enhances influenza viral replication (A) WT mice were intratracheally infected with influenza virus (PR8 virus, 100 FFU), and lung tissues were sampled at the indicated time points. Histone was extracted from the lung tissues and was subjected to H4-tail proteomics analysis. Relative expression levels of H4K20me1, H4K20me2, and H4K20me3 to H4 were shown (n = 5 per group). (B–D) MEFs obtained from WT, Suv4-20h1 KO (h1 KO), Suv4-20h2 KO (h2 KO) and Suv4-20h1/Suv4-20h2 double KO (dKO) mice were infected with mock (Flu-) or influenza virus (MOI: 0.5) (Flu+). Cells were fixed and stained with either H4K20me1, H4K20me2 or H4K20me3 Abs, and Hoechst. Data are from three separate experiments. Representative staining is shown. Scale bars: 10 μm (B). Culture supernatant and cells were sampled at 8 hr after infection. Virus titer assessed by FFU in supernatant is shown (C, upper panel). NP mRNA expression of the cells is shown (C, lower panel). Data are from three separate experiments. ∗p < 0.05 compared to WT. Infected cells were fixed, and virus NP protein was stained (green). Nuclei were counterstained with Hoechst (blue). Representative staining is shown. Scale bars: 50 μm (D, left panel). The percentages of NP-positive in total of Hoechst positive are shown (D, right panel). ∗∗p < 0.01 compared to WT. (E) WT and dKO MEFs were infected with influenza virus (MOI: 0.5) and sampled at the indicated time point. Western blots of virus PB2, NP and M1 proteins, along with GAPDH are shown. Data were reproduced in three experiments. The data in (A, C and D) (right panel) are presented as means ± s.e.m.. Statistical analysis were carried out using analysis of variance with Bonferroni post t tests.

Article Snippet: Human SET domain of SUV4-20h2 (2-281) (Active motif) was pre-incubated with virus NP or M1 protein (SinoBiological) (0, 0.05, 0.5, or 1.0 μg) at room temperature for 1 hr and then incubated with polynucleosomes (Active motif) (1.0 μg) for additional 3 hr.

Techniques: Infection, Expressing, Staining, Western Blot

Journal: iScience

Article Title: Suv4-20h2 protects against influenza virus infection by suppression of chromatin loop formation

doi: 10.1016/j.isci.2021.102660

Figure Lengend Snippet: Suv4-20h2 interacts with virus and host nuclear proteins (A) dKO cells transduced with EGFP-tagged empty vector (EV), Suv4-20h2 full length (h2-FL), SET domain (h2-SET) or Clamp domain (h2-Clamp; left panel) were infected with influenza virus (MOI: 0.5) for 8 hr h2-EGFP and virus NP protein were co-stained. Nuclei were counterstained with Hoechst. The percentages of NP-positive in total of the h2 EGFP-positive are shown. Data are from three separate experiments. ∗∗P < 0.01 between the groups. (B) WT, dKO, and dKO MEFs overexpressed with Suv4-20h2 were infected with influenza virus (MOI: 0.5). Culture supernatant and cells were sampled at 8 hr after infection. Virus titer of FFU in supernatant is shown (B, left panel). NP mRNA expression of the cells is shown (B, right panel). Data are from three separate experiments. ∗p < 0.05, ∗∗p < 0.01 between the groups. (C) dKO MEFs transduced with AM-tagged Suv4-20h2 were infected with influenza virus (MOI: 0.5) for 8 hr. Nuclear extracts were prepared from the cells, and were immunoprecipitated (IP) with AM Ab, and IP product was subjected to a stable isotope labeling (SILAC) & LC-MS/MS analysis. Virus NP and PB2 proteins were identified in this analysis. (D) Suv4-20h2 Flag knock-in mouse embryonic stem (mES) cells were mock (Flu-) or infected with influenza virus (MOI: 0.5) (Flu+) for 8 hr. Nuclear extracts were prepared from the cells, and were immunoprecipitated with Flag Ab. Western blotting shows that virus NP and PB2 proteins were co-precipitated with Suv4-20h2. Data were reproduced in three experiments. (E) Purified recombinant viral NP and PB2 proteins and GST-fusion proteins to full length (FL), SET, and Clamp domain of Suv4-20h2, were used in a pull-down assay. Virus NP and PB2 proteins interacted with FL and SET domain of Suv4-20h2. Data were reproduced in three experiments. (F) Human SET domain of SUV4-20H2 (2-281) was pre-incubated with virus NP protein (0, 0.05, 0.5, or 1.0 μg) (left panel) or M1 protein (0, 0.05, 0.5, or 1.0 μg) (right panel) at room temperature for 1hr and then incubated with polynucleosomes (1.0 μg) for additional 3hr. Protein samples were subjected to Western blot analysis using specific antibodies. H4K20me3 was suppressed in a dose-dependent manner by NP protein. Data were reproduced in three experiments. The data in (A and B) are presented as means ± s.e.m.. Statistical analysis were carried out using analysis of variance with Bonferroni post t tests.

Article Snippet: Human SET domain of SUV4-20h2 (2-281) (Active motif) was pre-incubated with virus NP or M1 protein (SinoBiological) (0, 0.05, 0.5, or 1.0 μg) at room temperature for 1 hr and then incubated with polynucleosomes (Active motif) (1.0 μg) for additional 3 hr.

Techniques: Transduction, Plasmid Preparation, Infection, Staining, Expressing, Immunoprecipitation, Labeling, Liquid Chromatography with Mass Spectroscopy, Knock-In, Western Blot, Purification, Recombinant, Pull Down Assay, Incubation

Journal: iScience

Article Title: Suv4-20h2 protects against influenza virus infection by suppression of chromatin loop formation

doi: 10.1016/j.isci.2021.102660

Figure Lengend Snippet:

Article Snippet: Human SET domain of SUV4-20h2 (2-281) (Active motif) was pre-incubated with virus NP or M1 protein (SinoBiological) (0, 0.05, 0.5, or 1.0 μg) at room temperature for 1 hr and then incubated with polynucleosomes (Active motif) (1.0 μg) for additional 3 hr.

Techniques: Recombinant, Transfection, Protease Inhibitor, Western Blot, Purification, Clone Assay, Fractionation, Sequencing, Software

Journal: NPJ Vaccines

Article Title: Chimeric hemagglutinin and M2 mRNA vaccine for broad influenza subtype protection

doi: 10.1038/s41541-025-01178-x

Figure Lengend Snippet: A mRNA constructs of Fluaxe expressing the two chimeric HA and M2. Created in BioRender. Li (2025) https://BioRender.com/bithl81 . B Monomeric structures of HA for four subtypes of IAV (H1N1, H5N1, H3N2, and H7N9) and IBV/Victoria. The disulfide bonds presented at the junction between the head and stem domains in all four IAV HA proteins are highlighted. For IBV/Victoria HA A56 and G302 residues are situated at the two equivalent structural positions instead of cysteines. C The predicted three-dimensional structure of Fluaxe chimeric sequences. The disulfide bond connecting the original head and stem fragments persists in the chimera. All structural figures were prepared using the PyMOL Molecular Graphics System, Version 3.0 (Schrödinger, LLC).

Article Snippet: Briefly, ELISA plates were coated with 2 μg/well recombinant HA proteins (Recombinant H1N1 HA (40787-V08H), H3N2 HA (40789-V08H, H5N1 HA (40022-V08H), H7N9 HA (40325-V08H), IBV (B/Washington/02/2019) HA (40722-V08H), IBV (B/Phuket/3037/2013) HA (40722-V08H)) from homologous wild-type viruses (Sino Biological) at 4 °C overnight.

Techniques: Construct, Expressing

Journal: NPJ Vaccines

Article Title: Chimeric hemagglutinin and M2 mRNA vaccine for broad influenza subtype protection

doi: 10.1038/s41541-025-01178-x

Figure Lengend Snippet: A A schematic diagram of mice immunization and prime-boost vaccination was executed on Days 0 and 14. Female BALB/c mice were injected via i.m. with Fluaxe (5 μg for each mRNA) or empty LNP per mouse ( n = 5) and boosted with an equivalent dose. Sera were collected on Days 7, 14, 21, and 28, respectively, for antibody analysis. Lungs and spleens were harvested on Day 28 to analyze cellular immunity. Splenocytes were collected on Day 104 for memory T or B cell detection. Created in BioRender. Li (2025) https://BioRender.com/bithl81 . B Specific IgG antibodies against H1N1, H3N2, H5N1, H7N9, IBV/Victoria, and IBV/Yamagata were measured by ELISA. Data were shown as Mean ± SEM. Significance was determined by two-way ANOVA with Bonferroni’s test for multiple comparisons (** p < 0.01, *** p < 0.001). Neutralizing antibodies of sera collected on Day 28 against H1N1, H3N2, IBV/Victoria, and IBV/Yamagata influenza viruses were detected by C MN and D HAI assays, respectively. E Neutralizing responses against H5- and H7-containing viruses were measured by HAI assay. Each symbol represents one mouse, and sera from five mice were assessed. Multiple t -tests with correction for multiple comparisons by the Holm-Šídák method were used to calculate p values relative to the control LNP group (n.s. not significant; * p < 0.05, ** p < 0.01, *** p < 0.001). F Pseudotype neutralizing assays were performed using influenza pseudovirus bearing multiple group 1 and group 2 HAs (HA1-16). Detection of 50% pseudovirus neutralization titer (PNT 50 ) of sera from immunized mice. The sera from five mice per group were pooled and tested in triplicate. Significance was calculated using two-tailed unpaired t -tests.

Article Snippet: Briefly, ELISA plates were coated with 2 μg/well recombinant HA proteins (Recombinant H1N1 HA (40787-V08H), H3N2 HA (40789-V08H, H5N1 HA (40022-V08H), H7N9 HA (40325-V08H), IBV (B/Washington/02/2019) HA (40722-V08H), IBV (B/Phuket/3037/2013) HA (40722-V08H)) from homologous wild-type viruses (Sino Biological) at 4 °C overnight.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, HAI Assay, Control, Neutralization, Two Tailed Test

Journal: NPJ Vaccines

Article Title: Chimeric hemagglutinin and M2 mRNA vaccine for broad influenza subtype protection

doi: 10.1038/s41541-025-01178-x

Figure Lengend Snippet: Spleens of prime-boost vaccinated mice were collected and homogenized on Day 104, lymphocytes were stimulated with a peptide cocktail containing cH5/1-BV, cH7/3, and M2 peptide pools. Memory cells were stained for intracellular IFN-γ. A Frequency of antigen-specific CD4 + and CD8 + Tcm (CD44 + CD62L + IFN-γ + ) in splenocytes. B Frequency of antigen-specific CD4 + and CD8 + Tem (CD44 + CD62L − IFN-γ + ) in splenocytes. C Frequency of antigen-specific CD4 + and CD8 + Trm (CD44 + CD62L − CD69 + CD103 + IFN-γ + ) in splenocytes. D Frequency of antigen-specific CD4 + and CD8 + CXCR3 + Tm (CD44 + CD62L − CD69 − CXCR3 + IFN-γ + ) in splenocytes. E Frequency of H1N1 HA, H3N2 HA, H5N1 HA, H7N9 HA, IBV/Victoria HA, and IBV/Yamagata HA-specific MBC in splenocytes. Five mice per group, data are presented as Mean ± SEM. p values were determined using multiple t -tests followed by Holm-Šídák’s multiple comparisons.

Article Snippet: Briefly, ELISA plates were coated with 2 μg/well recombinant HA proteins (Recombinant H1N1 HA (40787-V08H), H3N2 HA (40789-V08H, H5N1 HA (40022-V08H), H7N9 HA (40325-V08H), IBV (B/Washington/02/2019) HA (40722-V08H), IBV (B/Phuket/3037/2013) HA (40722-V08H)) from homologous wild-type viruses (Sino Biological) at 4 °C overnight.

Techniques: Staining