Abstract
We have developed a novel cross-immune antigen vaccine platform for influenza A virus. The vaccine antigen is a fusion protein of headless hemagglutinin (HA) and matrix protein 1 (M1), which possess B-cell and T-cell epitopes, respectively, that are conserved among subgroup A viruses. The single molecule of headless HA and M1 fusion protein forms an oligomer by self-assembly of M1. T-Helper 1 (Th1) and Th2 cells were activated in an antigen-specific manner in lymphocyte cultures of antigen-immunized mice. The antigen-immunized antiserum neutralized influenza virus A subtypes H1N1 and H3N2, and intranasal administration of the antigen reduced mortality to less than 30% in a protection assay.
INTRODUCTION
Hemagglutinin (HA), the major envelope protein of influenza virus, comprises a cell receptor binding domain and a cell membrane fusion domain. HA is a major vaccine antigen in current vaccines because the cellular receptor-binding domain possesses neutralizing antibody epitopes and is immunodominant. However, influenza viruses have a high mutation frequency, and mutant strains constantly appear due to the selective pressure of antibodies against the receptor binding domain. Furthermore, cell membrane fusion peptide domains are weakly immunogenic and inefficient at eliciting antibodies, because the pre-fusion domain structure forms a conformational epitope.1–3) After HA is cleaved by proteases, the fusion domain loses its epitope as a result of conformational changes. Therefore, the B-cell immune response to conformational epitopes must directly activate B-cells without T-cell help. The mechanisms of membrane fusion and entry into the cytoplasm of class I enveloped viruses are thought to be conserved and mutation-intolerant in each virus group. If the immunogenicity of the membrane fusion peptide domain can be enhanced, cross-neutralizing antibodies could be elicited against the mutant viruses.
The M1 protein is a major structural protein of virus particles and possesses T-cell epitopes recognized by cytotoxic T-cells,4–7) and is therefore considered a cell-mediated immune antigen. M1 can oligomerize during self-assembly8,9) and it is therefore expected that the fusion protein of headless HA and M1 will oligomerize and enhance the immunogenicity of the pre-fusion peptide domain epitope.
Bachman et al. reported that organized T-cell-dependent B-cell antigens fully and strongly elicit B-cell responses.10,11) T-cell dependency means that antigen-presenting cells take up B-cell antigens by endocytosis, digest them into peptides, bind major histocompatibility complex (MHC) class II, and present them to T-cells. It is strongly suggested that the organized T-cell antigen is also taken up efficiently by antigen-presenting cells, digested into peptides, bound to MHC class I, and presented to T-cells via the mechanism of cross-presentation.12,13) The dl HA-M1 fusion protein is a fusion protein of dl HA, which lacks the global head region and the transmembrane domain of HA, and the M1 protein expected to elicit sufficient B-cell- and T-cell-mediated cross-immune responses for influenza virus A subtypes.
MATERIALS AND METHODS
Preparation of AntigenThe amino acid sequences of the HA protein and M1 from influenza A virus H1N1 strain Michigan were retrieved from the GenBank database (Accession Nos. APC60198.1 and APC60201.1, respectively). Recombinant antigen proteins were prepared as described in Supplementary materials. Recombinant protein extracts were precipitated by dialysis and solubilized by sonication with 3 mM n-dodecanoylsucrose. Three dimensional (3D) structural models were predicted using AlphaFold2.2.0 software with default parameters (on the SHIHO-computer of National Agriculture and Food Research Organization).
Immunological AssayMice were immunized via the footpad with sonicated dl HA-M1 oligomers and adjuvant. Lymphocytes collected from the lymph nodes were cultured with solubilized dl HA-M1 for an antigen-dependent proliferation assay and a cytokine release assay.14)
The serum obtained after intramuscular and subcutaneous immunization of rabbits was used to determine the serum dilution factor that inhibited the growth of 100 TCID50 influenza virus in Madin-Darby canine kidney (MDCK) cell cultures.15)
Mouse Infection Protection AssayInfluenza A virus H1N1 (1 × 104 TCID50/mouse) or H3N2 (1 × 105 TCID50/mouse) was intranasally infected into female BALB/c mice that had previously been administered antigen nasally at 14 and 7 d prior to virus infection. Mice that had not been administered antigen prior to infection were included as a control. The mortality rates of the mice were determined. The most effective, currently available, live vaccine, FluMist, was administered intranasally as a control. The details of this assay are described in Supplementary Materials.
The experiments were carried out by Hamri Co., Ltd. according to the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International and the committee of the company approved all animal protocols used in the present study under Approval No. ID 21-H034.
RESULTS AND DISCUSSION
Construction and Production of Influenza Virus dl HA-M1 Fusion ProteinHA protein is composed of HA1, the global head, and H2, the stem region. HA1 possesses the receptor binding site and HA2 possesses the membrane fusion site (Fig. 1A). After HA binds to cellular receptors, it is cleaved by endosomal proteases, exposing the fusion peptide domain of HA2. The structure of HA2 prior to cleavage reveals that the fusion peptide (FP) comprises helix A, loop B and helix C. The A and C helices form an antiparallel fold and the pre-fusion peptide domain folds into the stem region (Fig. 1C). The folded structure of the A, B, C array is necessary for the pre-fusion domain to form a structural epitope. The dl HA-M1 protein is a fusion protein of dl HA and M1 protein (Fig. 1B). The 3D structure of the antigen was predicted using AlphaFold2.2.0 software.16) The dl HA maintains an antiparallel folded structure comprising the A and C helices, which is the same as the reported structure of No 2VIU from the Protein Data Bank (PDB). M1 maintains the same structure as its reported 3D structure. Fusion of M1 to the dl HA molecule did not change the 3D structure of dl HA, suggesting that the C-terminal helix of M1 interacts with the C-helix of dl HA to form a compact 3D structure (Fig. 1D).
Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting with M1 antibody revealed a band with a calculated molecular weight corresponding to dl HA-M1. Native PAGE detected a band larger than the monomer and a smeared band (Fig. 1E).
HA forms a stable structure as a trimer, but it is not yet known whether HA trimerizes when fused to M1. The dl HA-M1 protein retains the epitope structure of the prefusion domain and is likely assembled into helical multi-layer oligomers by M1.
Immunogenicity of the dl HA-M1 OligomerAntigen-dependent proliferation was demonstrated in a lymphocyte proliferation assay (Fig. 2), and high levels of interleukin (IL)-10 and interferon-γ (IFN-γ) cytokines were detected in the culture supernatant in an antigen-dependent manner. These results indicated that dl HA-M1 potently activates Th1 and Th2 cells (Fig. 2).
Our findings indicated that the neutralizing antibody titer is sufficient to neutralize both H1N1 and H3N2 influenza viruses (Table 1). Headless HA alone was weakly immunogenic, but oligomerization of the M1 protein likely enhanced the immunogenicity of the pre-fusion peptide domain epitope. Mice intranasally administered sonicated dl HA-M1 antigen combined with cyclic GAMP, as adjuvant, showed significantly reduced mortality following infection with influenza A virus H1N1 and H3N2 (Fig. 3). Comparable efficacy was obtained with a control trial using the live vaccine FluMist containing attenuated strains of H1N1 and H3N2 (Fig. 3).
Table 1. Neutralizing Antibody Titer of Rabbit Immune Serum
| JW Rabbit (♀) | Antigen dose (µg) | Immunized (d) | H1N1 A/Michigan/45/2015 | H3H2 A/Hong Kong/4801/2014 |
|---|
| R1 | 300 | 21 | 1 : 320 | 1 : 160 |
| 70 | 1 : 320 | 1 : 160 |
| R2 | 300 | 21 | 1 : 320 | 1 : 320 |
| 70 | 1 : 320 | 1 : 320 |
Influenza virus neutralization tests were performed using MDCK cells by infection and proliferation inhibition of 100 TCID50 influenza virus. Neutralizing antibody titers are shown as serum dilution rates.
CONCLUSION
In this report, we demonstrate the cross-immunity conferred by a B-cell and T-cell antigen. Anti-pre-fusion antibodies are not induced by vaccination with inactivated and split vaccines. By contrast, in natural infections, anti-pre-fusion antibodies are detected at low levels using an antibody gene library.17) Because the human population is exposed to infection periodically, pre-fusion antibody-producing memory B-cells may be maintained. Therefore, even if the presented vaccine neutralizing antibody titer is low (Table 1), a booster effect may be observed following inoculation in humans.18) If this vaccine candidate is administered as a seasonal influenza virus vaccine, it may help to protect against future pandemics caused by highly pathogenic human influenza viruses. The cross-immunizing antigen platform presented here may also be applicable for class I enveloped viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).19)
Acknowledgments
Kenji Sekikawa would like to thank Dr. Yukio Shimizu, who developed hog cholera live vaccine, for his encouragement regarding vaccine development over many years.
Conflict of Interest
The authors declare no conflict of interest.
Supplementary Materials
This article contains supplementary materials.
REFERENCES
- 1) Sagawa H, Ohshima A, Kato I, Okuno Y, Isegawa I. The immunological activity of a deletion mutant of influenza virus hemagglutinin lacking the globular region. J. Gen. Virol., 77, 1483–1487 (1996).
- 2) Steel J, Lowen AC, Wang TT, Yondola M, Gao Q, Haye K, García-Sastre A, Palese P. Influenza virus vaccine based on conserved hemagglutinin stalk domain. MBio, 1, e00018-10 (2010).
- 3) Mallajosyula VVA, Citron M, Ferrara F, Lu X, Callahan C, Heidecker GJ, Sarma SP, Flynn JA, Temperton NJ, Liang X, Varadarajan R. Influenza hemagglutinin stem-fragment immunogen elicits broadly neutralizing antibodies and confers heterologous protection. Proc. Natl. Acad. Sci. U.S.A., 111, E2514–E2523 (2014).
- 4) Berkhoff EG, de Wit E, Geelhoed-Mieras MM, Boon AC, Symons J, Fouchier RA, Osterhaus AD, Rimmelzwaan GF. Functional constraints of influenza A virus epitopes limit escape from cytotoxic T lymphocytes. J. Virol., 79, 11239–11246 (2005).
- 5) Rimmelzwaan GF, Kreijtz JH, Bodewes R, Fouchier RA, Osterhaus AD. Influenza virus CTL epitopes, remarkably conserved and remarkably variable. Vaccine, 27, 6363–6365 (2009).
- 6) van de Sandt CE, Kreijtz JH, Geelhoed-Mieras MM, Nieuwkoop NJ, Spronken MI, van de Vijver DA, Fouchier RA, Osterhaus AD, Rimmelzwaan GF. Differential recognition of influenza A virus by M158-66 epitope-specific CD8+ T cells is determinant by extraepitopic amino acid residues. J. Virol., 90, 1009–1022 (2015).
- 7) Machkovech HM, Bedford T, Suchard MA, Bloom JD. Positive selection in CD8+ T-Cell epitopes of influenza virus nucleoprotein revealed by a comparative analysis of human and swine viral lineages. J. Virol., 89, 11275–11283 (2015).
- 8) Noton SL, Medcalf E, Fisher D, Mullin AE, Elton D, Digard P. Identification of the domains of the influenza A virus M1 matrix protein required for NP binding, oligomerization and incorporation into virions. J. Gen. Virol., 88, 2280–2290 (2007).
- 9) Zhang K, Wang Z, Liu X, Yin C, Basit Z, Xia B, Liu W. Dissection of influenza A virus M1 protein: pH-dependent oligomerization of N-terminal domain and dimerization of C-terminal domain. PLOS ONE, 7, e37786 (2012).
- 10) Bachmann MF, Rohrer UH, Kündig TM, Bürki K, Hengartner H, Zinkernagel RM. The influence of antigen organization on B cell responsiveness. Science, 262, 1448–1451 (1993).
- 11) Bachmann MF, Zinkernagel RM. Neutralizing antiviral B cell responses. Annu. Rev. Immunol., 15, 235–270 (1997).
- 12) Rock KL, Shen L. Cross-presentation: underlying mechanisms and role in immune surveillance. Immunol. Rev., 207, 166–183 (2005).
- 13) Embgenbroich M, Burgdorf S. Current concepts of antigen cross-presentation. Front. Immunol., 9, 1643 (2018).
- 14) Sato M, Tsuji NM, Gotoh H, Yamashita K, Hashimoto K, Tadotsu N, Yamanaka H, Sekikawa K, Hashimoto Y. Overexpression of the Wiskott-Aldrich syndrome protein N-terminal domain in transgenic mice inhibits T Cell proliferative responses via TCR signaling without affecting cytoskeletal rearrangements. J. Immunol., 167, 4701–4709 (2001).
- 15) Cuevas F, Kawabata H, Krammer F, Carreño JM. An in vitro microneutralization assay for influenza virus serology. Curr. Protoc., 2, e465 (2022).
- 16) Jumper J, Evans R, Pritzel A, et al. Highly accurate protein structure prediction with AlphaFold. Nature, 596, 583–589 (2021).
- 17) Wrammert J, Smith K, Miller J, Langley WA, Kokko K, Larsen C, Zheng NY, Mays I, Garman L, Helms C, James J, Air GM, Capra JD, Ahmed R, Wilson PC. Rapid cloning of high-affinity human monoclonal antibodies against influenza virus. Nature, 453, 667–671 (2008).
- 18) Henry C, Palm AKE, Krammer F, Wilson PC. From original antigenic sin to the universal influenza virus vaccine. Trends Immunol., 39, 70–79 (2018).
- 19) Yamamoto Y, Inoue T. Current status and perspective of therapeutic antibodies targeting the spike protein S2 subunit against SARS-CoV-2. Biol. Pharm. Bull., 47, 917–923 (2024).
