Overview of experiment workflow
Conservation Analysis of Influenza Virus Sequences
1. Sequence Collection:
We obtained all HA2 protein sequences of H1N1 and B-type influenza viruses from the NCBI Influenza Virus Sequence Database (link), and saved them in FASTA format.
Fig1. Sequence Collection from NCBI
2. Sequence Alignment:
Using MEGA 10 software, we performed multiple sequence alignment of all collected sequences. The alignment included between 76 and 130 sequences for each analysis.
3. Conservation Scoring:
We used EXCEL and BioEdit software to score the conservation of each amino acid position. Conservation scores were visualized as conservation matrices.
4. Identification of Highly Conserved Sites:
Based on the conservation matrices, we identified highly conserved regions and selected the top 16 amino acid sites with the highest conservation for both H1 (as an example) and B types.
5. Print results:
Fig2. Sequence Logo Depicting Conservation of H1 Hemagglutinin (HA) Proteins from 1934 to 2020. Amino acid sequence conservation of influenza A H1 hemagglutinin (HA) proteins was analyzed using sequences collected from 1934 to 2020. Multiple sequence alignment was performed and visualized as a sequence logo using MEGA10 and the WebLogo tool. In this plot, the overall height of each stack indicates the sequence conservation at that position, while the height of individual letters represents the relative frequency of each amino acid. Amino acids are color-coded by chemical properties: polar residues (green), basic residues (blue), acidic residues (red), and hydrophobic residues (black). The red box highlights a region of interest with particularly high conservation, selected for further analysis in this study.
Fig3. Sequence Logo Depicting Conservation of Influenza B Virus Hemagglutinin (HA) Proteins from 1940 to 2020. Amino acid sequence conservation of influenza B virus HA proteins was assessed using sequences collected between 1940 and 2020. Multiple sequence alignment was performed, and the degree of conservation at each residue was visualized as a sequence logo using MEGA10 and the WebLogo tool. In the sequence logo, the total height of each stack reflects the conservation level at that position, while the height of individual amino acid letters indicates their relative frequency. Amino acids are color-coded according to their chemical properties: polar residues (green), basic residues (blue), acidic residues (red), and hydrophobic residues (black). The red box highlights a highly conserved motif identified for further analysis in this study.
B Cell Epitope Prediction
The Kolaskar & Tongaonkar antigenicity prediction method (IEDB: tools.immuneepitope.org) was used to assess the antigenic potential of conserved peptide regions. This method evaluates factors such as accessibility, flexibility, and hydrophilicity, assigning an antigenicity score to each amino acid. Regions with consecutively high scores were considered potential B cell epitopes.
1. Identification of Candidate Epitopes
Based on the B cell epitope prediction results, the following conserved region was identified as a promising B cell epitope for B-type influenza HA:
- B: TISSQIELAVLLSNEC
- H1: DIWTYNAELLVLLENE
2. Immunogen Design
The identified candidate epitopes were used for subsequent immunogen design and synthesis.
Fig4. Kolaskar & Tongaonkar Antigenicity Prediction Plot for a Conserved Influenza B HA Peptide. Antigenic propensity of a conserved peptide region from influenza B virus hemagglutinin (HA) was predicted using the Kolaskar & Tongaonkar method. The y-axis represents antigenic propensity, while the x-axis denotes sequence position. The red line indicates the antigenicity threshold (set at 1.0). Regions above the threshold (shaded yellow) are predicted to be antigenic and potential B cell epitopes, while regions below the threshold (shaded green) are considered non-antigenic. This analysis was used to identify peptide segments suitable for immunogen design.
Fig5. Structural Extraction and Modeling of Influenza HA2 Conserved Region. The schematic illustrates the stepwise extraction and modeling of the conserved region from the influenza hemagglutinin (HA) trimer. The left panel shows the full HA trimer structure, with the conserved HA2 domain highlighted by a black box. The middle panel represents the isolated HA2 subunit, and the right panel shows the further refined model of the conserved region, highlighting the secondary structure elements.
Molecular Cloning of expression vetor
All designed immunogen sequences were codon-optimized and synthesized. The recombinant genes were cloned into the prokaryotic expression vector pET28a-sumo for high-level expression in E. coli. Gene synthesis and optimization were performed by a commercial provider (Tianjin Tianshuo Gene Company).
Fig6. Schematic Design of Recombinant Ferritin Nanoparticle Immunogen Displaying Conserved Influenza HA Epitopes.
Fig7. Schematic Diagram of Recombinant Ferritin Nanoparticle Immunogen Constructs and Expression Vector.
Verify of plasmid conctruction
Fig8. Transformed colony in BL21(DE3)
Fig9. Sequence for plasmid B-(16)(4)-F、 H3-H1-B-F、H1-(16)(4)-F were vertified using restriction digest
Verify of protein expression
Fig10. Raw AKTA protein purification result
Fig11. SDS-PAGE Analysis of Soluble Expression of Recombinant Ferritin-Based Immunogens. SDS-PAGE analysis of soluble protein expression for three recombinant ferritin nanoparticle immunogens (B-(16)₄-F, H1-(16)₄-F, and H3-H1-B-F) in E. coli under different IPTG induction concentrations. Lanes 1–3: B-(16)₄-F with 0, 0.2, and 0.5 mM IPTG; lanes 4–6: H1-(16)₄-F with 0, 0.2, and 0.5 mM IPTG; lanes 7–10: H3-H1-B-F with 0, 0.2, 0.5, and 1 mM IPTG. The molecular weight markers (Marker) are shown on the left. No significant soluble expression of the target proteins was detected in the supernatant under the tested induction conditions.
The target proteins were successfully expressed and purified after inclusive body in vitro folding, with observed molecular weights matching theoretical values.
Digestion protection function of ferritin
Fig12. Assessment of Degradation Stability of Ferritin-Based Protein by SDS-PAGE. SDS-PAGE analysis of ferritin-based protein samples after overnight incubation at room temperature. M: protein marker; lanes 1–3: ferritin-based protein samples collected from elution fractions. Target protein bands are still detected in the elution fractions after incubation, although partial degradation is observed. The main protein band remains present, indicating that the ferritin-based protein retains stability under these conditions.
Nano FCM measurement of diameter
Fig13. Nano Flow cytometry measurement of ferritin particles
TEM image of Ferritin particle
Fig14. Transmission Electron Microscopy (TEM) Analysis of Recombinant Ferritin Nanoparticles. Representative TEM images showing the morphology of recombinant ferritin-based nanoparticles. The particles exhibit uniform, spherical structures with diameters ranging from 12 to 16 nm, consistent with the expected theoretical size for ferritin nanoparticles.