Identification and In Silico Characterization of a Conserved Peptide on Influenza Hemagglutinin Protein: A New Potential Antigen for Universal Influenza Vaccine Development

Abstract

Antigenic changes in surface proteins of the influenza virus may cause the emergence of new variants that necessitate the reformulation of influenza vaccines every year. Universal influenza vaccine that relies on conserved regions can potentially be effective against all strains regardless of any antigenic changes and as a result, it can bring enormous public health impact and economic benefit worldwide. Here, a conserved peptide (HA2_88-107) on the stalk domain of hemagglutinin glycoprotein is identified among highly pathogenic influenza viruses. Five top-ranked B-cell and twelve T-cell epitopes were recognized by epitope mapping approaches and the corresponding Human Leukocyte Antigen alleles to T-cell epitopes showed high population coverage (>99%) worldwide. Moreover, molecular docking analysis indicated that VLMENERTL and WTYNAELLV epitopes have high binding affinity to the antigen-binding groove of the HLA-A*02:01 and HLA-A*68:02 molecules, respectively. Theoretical physicochemical properties of the peptide were assessed to ensure its thermostability and hydrophilicity. The results suggest that the HA2_88-107 peptide can be a promising antigen for universal influenza vaccine design. However, in vitro and in vivo analyses are needed to support and evaluate the effectiveness of the peptide as an immunogen for vaccine development.

Keywords: epitope mapping; hemagglutinin; immunoinformatic; nanoparticle; peptide-based vaccine; universal influenza vaccine.

Figure 1

Workflow for in silico prediction and characterization analysis of a conserved peptide on HA glycoprotein as a potential antigen for universal influenza vaccine design.

Figure 2

Multiple sequence alignment of HA2 amino acid sequences from six different strains of influenza virus using Multalin online software. The red color demonstrates the full conservancy, the blue color indicates one amino acid alternation and the black color represents the alternation in more than one amino acid at the specific region for different strains. The green (fusion peptide) and yellow (HA2_88–107) boxes show the regions with the highest conservation distribution. The letter in the consensus line means conservative and the dot indicates non-conservative amino acid substitution.

Figure 3

Location of the conserved HA2_88–107 peptide on influenza HA protein. (A) Sequence diagram showing the polypeptide segment of the HA2 domain (222 amino acids) in its primary structure, indicating the HA2_88–107 peptide is not associated with the membrane. (B) The position of HA2_88–107 peptide (pink color) on the 3D full structure of HA protein (yellow color) using iCn3D online software, access date: 1 July 2023). The green color represents the carbohydrate components of the protein.

Figure 4

Linear B-cell epitope identification on HA2_88–107 peptide. The most antigenic epitopes are shown in yellow color above the threshold value (red line) and green areas are not considered as an epitope. (A): Emini surface accessibility prediction with a threshold value of 1.000; (B): Karplus and Schulz flexibility prediction with a threshold value of 0.968; (C): Chou and Fasman secondary structure prediction with a threshold value of 0.834; (D): Parker hydrophilicity prediction with the threshold value of 0.064; (E): Kolaskar and Tongaonkar antigenicity prediction with the threshold value of 1.020.

Figure 5

Population coverage of the HA2_88–107 peptide. The world populations were evaluated for the peptide using IEDB online population coverage analysis. (A) The graph shows a population coverage of 79.52% for MHC I epitopes. (B) The graph indicates population coverage of 99.88% for MHC II epitopes. The line (-o-) represents the cumulative percentage of population coverage of the epitopes; the bars represent the population coverage for each epitope; the red line represents PC90.

Figure 5

Population coverage of the HA2_88–107 peptide. The world populations were evaluated for the peptide using IEDB online population coverage analysis. (A) The graph shows a population coverage of 79.52% for MHC I epitopes. (B) The graph indicates population coverage of 99.88% for MHC II epitopes. The line (-o-) represents the cumulative percentage of population coverage of the epitopes; the bars represent the population coverage for each epitope; the red line represents PC90.

Figure 6

Cluster analysis of the HLA alleles. (A) Representing the cluster of MHC-I alleles and (B) Representing the cluster of MHC-II alleles. The red color indicates strong interaction and the yellow zone indicates weaker interaction with appropriate annotation.

Figure 7

Visualization of ClusPro molecular docking of (A) VLMENERTL epitope interaction with HLA-A*02:01 molecule and (B) WTYNAELLV epitope interaction with HLA-A*68:02 molecule. Blue color indicates epitopes and magenta color indicates corresponding molecules (receptors).