Influenza is a substantial global clinical burden, with over 1 billion cases of seasonal influenza occurring annually. Vaccination serves as a cornerstone in controlling influenza-related diseases.
However, the seasonal nature of influenza means that each year we need to develop and make new vaccines. When developing these vaccines, it is essential to evaluate active ingredients such as antigens and antibodies.
Commercially available vaccines include egg- or cell-based inactivated, live attenuated and recombinant influenza vaccines. To develop more effective vaccines, a deeper understanding of influenza virus infection, as well as pathogenic and transmission mechanisms is required, with the ultimate goal of discovering of a universal influenza vaccine.
Influenza viruses are part of the RNA virus Orthomyxoviridae family, comprising four genera: influenza A, B, C and D. Influenza A and B are responsible for the majority of annual human influenza outbreaks.
The influenza A virus genome consists of eight segments of single-stranded, negative-sense viral RNA, which encode multiple proteins, including hemagglutinin (HA), neuraminidase (NA), matrix protein 1, M2 proton channel, nucleoprotein, non-structural protein 1, nuclear export protein, polymerase acid protein, polymerase basic proteins and PB1-F2.
Of these proteins, the surface glycoproteins HA and NA display distinct antigenic properties linked to specific influenza strains. The trimeric HA glycoprotein plays a crucial role in virulence by facilitating viral attachment to a host cell's surface via a sialic acid-containing protein. There are 18 recognized HA subtypes (H1–H18).1,2,5
Antigenic drift and antigenic shift, which lead to variations in HA subtypes, drive the seasonal patterns of human influenza outbreaks.3
Recent research on HA as an antigen emphasizes the importance of its natural trimeric structure and hemagglutination activity. Anti-HA antibodies are primarily focused on subtype and strain specificity.
The study discussed in this article examines the characteristics of trimeric influenza HA protein and trimeric-derived anti-HA antibodies while exploring methods for evaluating vaccine efficacy.
Figure 1. The influenza A and B virion.4 Image Credit: ACROBiosystems
Results and discussion
Characterization of HA as a bioactive ingredient
Influenza HA naturally exists in a trimeric conformation consisting of a head and stem region. Each chain is initially synthesized as a precursor polypeptide, which is then cleaved into two parts: HA1 (328 amino acids) and HA2 (221 amino acids), connected by disulfide bonds.5
In this study, HA strain A was used to compare the natural trimeric structure with the monomeric form. Conformational changes and activity differences were analyzed using SEC-MALS, ELISA, and hemagglutination assays.
Structural and binding characterization of monomeric and trimeric HA
SEC-MALS confirmed the polymeric state of HA between trimeric and monomeric HA, with a significant shift in peak retention time reflected in the chromatogram. This indicated a higher molecular weight for the trimeric HA protein. Similarly, reducing SDS-PAGE revealed several bands corresponding to monomeric components HA1 and HA2 after disulfide bond cleavage.
Figure 2. (a) Chromatogram collected through SEC-MALS using standard solutions of HA proteins. Retention time (RT) information is collected from UV detector, while molecular weight (MW) is collected from MALS. (b) Reducing SDS-PAGE of trimeric HA. Image Credit: ACROBiosystems
Table 1. Molecular Analysis Results of Monomer and Trimer HA Protein. Source: ACROBiosystems
| |
Monomer |
Trimer |
| Theoretical MW |
58.67 kDa |
62.1 kDa |
| SEC-MALS |
78-84 kDa |
214-234 kDa |
| Peak Retention Time (TUV) |
14.995 min |
13.508 min |
Indirect ELISA was performed to compare the binding activity of trimer and monomer HA. The trimeric HA exhibited enhanced binding kinetics, with a half-maximal effective concentration (EC50) of 1.62 ng/mL compared with 2.01 ng/mL for monomeric HA.
This finding highlights the role of conformation in improving antigen binding and further underscores the importance of native-conformation antigens for vaccine development.
Evaluating bioactivity of trimeric HA by hemagglutination assay
Binding activity provides only one aspect of an antigenic protein’s bioactivity. On influenza particles, surface HA functions by binding to N-acetylneuraminic acid proteins on erythrocytes. Functionally, HA should induce an agglutination reaction, causing erythrocytes to form a diffuse lattice structure.
To confirm HA’s agglutination activity on erythrocytes, a hemagglutination assay using rooster red blood cells (RBCs) was performed. The results demonstrated strong hemagglutination activity even at low trimeric HA concentrations.
![Influenza B virus [Austria/1359417/2021 (B/Victoria lineage)] Hemagglutinin (HA) Protein, His Tag (Cat. No. HAE-V52H8) binding to RBCs (Rooster red blood cells) at a final concentration of 0.4688 μg/mL can lead to complete hemagglutination. The final concentration of the sample in the first well was 15 μg/mL](https://d2jx2rerrg6sh3.cloudfront.net/images/appnotes/ImageForAppNote_5541_17389285425194856.png)
Figure 4. Influenza B virus [Austria/1359417/2021 (B/Victoria lineage)] Hemagglutinin (HA) Protein, His Tag (Cat. No. HAE-V52H8) binding to RBCs (Rooster red blood cells) at a final concentration of 0.4688 μg/mL can lead to complete hemagglutination. The final concentration of the sample in the first well was 15 μg/mL. Image Credit: ACROBiosystems
Immunogenicity of trimeric HA
By replicating the structure of HA found on the surface of influenza particles, trimeric HA can be used for immunization, eliciting an immune response similar to natural exposure. In this study, three Balb/c mice were immunized, with all antibody titers recorded at over 1:2,048,000.
Table 2. Antibody titer in Balb/c mice immunized with HA protein. Source: ACROBiosystems
| Antibody Titer |
OD Value |
| 6# |
7# |
8# |
| 1:500 |
3.084 |
3.09 |
3.075 |
| 1:1000 |
3.146 |
3.121 |
3.072 |
| 1:2000 |
3.077 |
3.093 |
3.013 |
| 1:4000 |
3.154 |
3.133 |
3.09 |
| 1:8000 |
3.032 |
3.002 |
3.039 |
| 1:16000 |
3.083 |
2.992 |
2.976 |
| 1:32000 |
3.039 |
3.093 |
2.987 |
| 1:64000 |
3.143 |
3.144 |
3.124 |
| 1:128000 |
3.018 |
2.913 |
2.977 |
| 1:256000 |
2.621 |
2.495 |
2.623 |
| 1:512000 |
1.723 |
1.442 |
1.727 |
| 1:1024000 |
1.038 |
0.856 |
1.041 |
| 1:2048000 |
0.587 |
0.461 |
0.578 |
| NC |
0.019 |
0.019 |
0.023 |
| Blank |
0.021 |
0.019 |
0.022 |
| Blank0 |
0.019 |
0.017 |
0.019 |
Method development for vaccine evaluation
In addition to the antibodies generated after immunization, specific antibodies recognizing trimeric HA were screened for quantitative detection of the trimeric HA antigen. Using the hybridoma antibody discovery technique, these antibodies were selected and later verified through ELISA, followed by classification based on subtype and strain-specific binding.
Several ELISA assays were developed to assess vaccine effectiveness and quantify antigen levels.
Table 3. Cross-reactivity of HA antibodies against HA proteins of varying influenza strains by ELISA (partial display). Source: ACROBiosystems
Development of indirect ELISA for vaccine effectiveness evaluation
Indirect ELISA can be used to evaluate the effectiveness of influenza vaccines and measure antibody titers following immunization. Figure 5 illustrates the detection of known antibodies based on the natural trimeric HA protein structure.
In this assay, immobilized Influenza A (H3N2) Virus HA Protein at 1 µg/mL (100 µL/well) binds to the broadly neutralizing anti-HA antibody CR8020, with a linear detection range of 0.2–10 ng/mL.
Figure 5. Influenza vaccine efficacy evaluation method based on HA protein with natural trimer structure (indirect ELISA). Image Credit: ACROBiosystems
Development of sandwich ELISA for antigen quantification
A sandwich ELISA method was developed to identify the active components of the HA protein in the H1N1 subtype. This assay specifically detects the H1N1 subtype but does not recognize H3N2 or influenza B.
This ELISA can be used to assess the active ingredients in influenza vaccines and verify strain- and subtype-specific formulations.
Figure 6. A subtype-specific anti-HA antibodies based method for the detection of influenza vaccine active ingredients (sandwich ELISA). Image Credit: ACROBiosystems
HA1-V52H3 Influenza A/Wisconsin/588/2019(H1N1)
HA1-V52H8 Influenza A/Victoria/4897/2022(H1N1)
HA1-V52H7 Influenza A/Wisconsin/67/2022(H1N1)
H32-V52H3 Influenza A/Thailand/8/2022 & Massachusetts/18/2022 (H3N2)
HA2-V52H5 Influenza A/Darwin/6/2021(H3N2)
HA2-V52H6 Influenza A/Darwin/9/2021(H3N2)
HAE-V52H3 Influenza B/Austria/1359417/2021(B/Victoria lineage)
HAE-V52H4 Influenza B/Phuket/3073/2013(B/Yamagata lineage)
Conclusions
HA on the surface of influenza virus particles naturally exists in a trimeric structure. Preserving this structure through recombinant HA expression helps ensure that its biological activity closely mimics natural conditions.
The use of naturally active trimeric HA protein, along with specific antibodies screened against it, plays a crucial role in influenza vaccine development. These components support the precise and effective formulation of influenza vaccines.
References and further reading
- Wu, N.C. and Wilson, I.A. (2020). Influenza Hemagglutinin Structures and Antibody Recognition. Cold Spring Harbor Perspectives in Medicine, (online) 10(8), p.a038778. https://doi.org/10.1101/cshperspect.a038778.
- Fodor, E. and te Velthuis, A.J.W. (2019). Structure and Function of the Influenza Virus Transcription and Replication Machinery. Cold Spring Harbor Perspectives in Medicine, 10(9), p.a038398. https://doi.org/10.1101/cshperspect.a038398.
- Gomez Lorenzo, M.M. and Fenton, M.J. (2013). Immunobiology of Influenza Vaccines. Chest, 143(2), pp.502–510. https://doi.org/10.1378/chest.12-1711.
- Petrova, V.N. and Russell, C.A. (2017). The evolution of seasonal influenza viruses. Nature Reviews Microbiology, [online] 16(1), pp.47–60. doi:https://doi.org/10.1038/nrmicro.2017.118.
- Du, J., Cross, T.A. and Zhou, H.-X. (2012). Recent progress in structure-based anti-influenza drug design. Drug Discovery Today, (online) 17(19-20), pp.1111–1120. https://doi.org/10.1016/j.drudis.2012.06.002.
About ACROBiosystems
ACROBiosystems is a cornerstone enterprise of the pharmaceutical and biotechnology industries. Their mission is to help overcome challenges with innovative tools and solutions from discovery to the clinic. They supply life science tools designed to be used in discovery research and scalable to the clinical phase and beyond. By consistently adapting to new regulatory challenges and guidelines, ACROBiosystems delivers solutions, whether it comes through recombinant proteins, antibodies, assay kits, GMP-grade reagents, or custom services. ACROBiosystems empower scientists and engineers dedicated towards innovation to simplify and accelerate the development of new, better, and more affordable medicine.
Sponsored Content Policy: News-Medical.net publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.Net which is to educate and inform site visitors interested in medical research, science, medical devices and treatments.