Elsevier

Vaccine

Volume 26, Issue 2, 10 January 2008, Pages 201-214
Vaccine

Potent immunogenicity and efficacy of a universal influenza vaccine candidate comprising a recombinant fusion protein linking influenza M2e to the TLR5 ligand flagellin

https://doi.org/10.1016/j.vaccine.2007.10.062Get rights and content

Summary

The recognition of specific pathogen associated molecular patterns (PAMPs) by members of the Toll-like receptor (TLR) family is critical for the activation of the adaptive immune response. Thus, incorporation of PAMPs into vaccines should result in more potent, protective antigen-specific responses in the absence of adjuvants or complex formulations. Here we describe an influenza A vaccine that is refractory to the genetic instability of hemagglutinin and neuraminidase and includes a trigger of the innate immune response to enhance immunogenicity and efficacy. A recombinant protein comprising the TLR5 ligand flagellin fused to four tandem copies of the ectodomain of the conserved influenza matrix protein M2 (M2e) was expressed in Escherichia coli and purified to homogeneity. This protein, STF2.4xM2e, retained TLR5 activity and displayed the protective epitope of M2e defined by a monoclonal antibody, 14C2. Mice immunized with STF2.4xM2e in aqueous buffer, without adjuvants or other formulation additives, developed potent M2e-specific antibody responses that were quantitatively and qualitatively superior to those observed with M2e peptide delivered in alum. The antibody response was dependent on the physical linkage of the antigen to flagellin and recognized the epitope defined by monoclonal antibody 14C2, which has been shown to protect mice from challenge with influenza A virus. Moreover, immunization with STF2.4xM2e at a dose of 0.3 μg per mouse protected mice from a lethal challenge with influenza A virus, and significantly reduced weight loss and clinical symptoms. These data demonstrate that the linkage of specific TLR ligand with influenza M2e yields a vaccine candidate that offers significant promise for widespread protection against multiple influenza A virus strains.

Introduction

Efforts to develop effective influenza vaccines are repeatedly challenged by the genetic instability of hemagglutinin and neuraminidase [1], thus requiring annual reformulation of the standard vaccine. A vaccine consisting of a genetically conserved influenza antigen would provide a second layer of protection against multiple strains, and could offer the promise of influenza vaccination in the developing world where the current seasonal strategy is not practical. One of the most promising conserved antigens is the ectodomain of the matrix 2 protein (M2e), a 24 amino acid polypeptide sequence that is primarily expressed on the surface of infected cells, where it forms an ion channel that is critical in the process of viral replication [2], [3], [4]. The sequence of M2e has remained remarkably stable across all influenza A isolates dating back to the pandemic strain of 1918 [5], thus making this epitope an attractive pan-influenza vaccine candidate. Studies evaluating the vaccine potential of M2e have reported varied results that may reflect the method of M2e delivery and presentation to the host immune system. These studies suggest that native M2e is poorly immunogenic, but the immunogenicity can be increased by delivery in multimeric form or in combination with complex adjuvants or delivery systems [6], [7], [8], [9], [10], [11], [12]. Based on these findings we investigated the potential application of recombinant protein technology in the design of a novel M2e-based vaccine candidate.

The M2 protein forms an ion channel on the surface of the virion which promotes uncoating of the virus in the endosome of the infected cell; the protein is also expressed on the infected cell surface during the budding process [13], [14], [15], [16]. The importance of the M2 protein in viral replication has been demonstrated by the ability of the anti-viral amantadine to inhibit viral propagation [17]. Similarly, antibody responses to M2 have been shown to reduce the rate of plaque formation and viral replication in vitro and in vivo, respectively [18], [19]. Based on these findings several studies have examined the use of M2 as a vaccine candidate, using various approaches including proteins, peptides, DNA vectors and attenuated viral vectors [7], [8], [9], [10], [11], [12], [20]. In these studies varying levels of immunogenicity and efficacy were reported. In studies involving delivery of synthetic M2-based peptides, immunogenicity required the use of complex adjuvants or carriers such as hepatitis B core protein (HBc) [10], [11] or the Neisseria meningitides outer membrane complex protein (OMPC) [8]. In the study of Jegerlehner et al., immunization with M2e coupled to HBc provided modest protection against a low dose viral challenge; protection was attributed to anti-M2 antibodies and NK cells [21].

To increase the immunogenicity of the M2e epitope, we utilized a strategy that actively engages both the innate and adaptive immune system in a coordinated fashion. Toll-like receptors (TLRs) have been shown to play a critical role in controlling the adaptive immune response, by properly arming dendritic cells and other antigen-presenting cells, triggering important costimulatory and regulatory mechanisms, and promoting antigen presentation [22], [23]. We and others have previously shown that genetically fusing an antigen of interest to flagellin, the ligand for TLR5, significantly increases the immunogenicity and protective capacity of the antigen. In one study, a fusion protein linking flagellin to ovalbumin yielded a vaccine that induced antibody and CD8+ T-cell responses to ovalbumin in mice immunized with the protein in PBS, while immunization with ovalbumin alone failed to induce an immune response [24]. In a different model system, a West Nile virus envelope protein subunit was fused to flagellin, producing a vaccine that induced neutralizing and protective antibodies against West Nile virus; again, immunization with the antigen subunit alone failed to induce a potent and protective antibody response [25]. These results led us to apply the same strategy to the genetically conserved M2e epitope of influenza A viruses. Four tandem copies of a consensus M2e sequence based on human H1, H2, and H3 virus isolates were genetically fused to the TLR5-specific ligand Salmonella typhimurium flagellin fljB (STF2), and expressed as a fusion protein denoted as STF2.4xM2e. The resulting recombinant fusion protein retained the ability to activate cells in a TLR5-specific manner, and was recognized by 14C2, a monoclonal antibody-specific for M2e [18]. In mice and rabbits, STF2.4xM2e administered in adjuvant-free aqueous solutions elicited potent antibody responses that were quantitatively and qualitatively superior to those observed when M2e was delivered in aqueous vehicle or on alum. The recognition of native M2e was confirmed by the ability of immune sera to react specifically with cells infected with influenza A virus. Mice immunized with STF2.4xM2e by the subcutaneous or intranasal routes survived an intranasal challenge with a lethal dose of influenza A/Puerto Rico/8/34 (PR8). In addition to exhibiting 90–100% survival compared to 0–20% survival of naïve mice, the immunized mice experienced significantly lower weight loss and clinical symptoms compared to naïve control mice. The sera from immunized animals contained antibodies that recognized a known protective epitope of M2e and its flanking sequences, and retained recognition of the epitope when single residues were substituted with alanine, suggesting that such a vaccine might provide protection against H1, H2 and H3 influenza strains that may differ in the M2e sequence by one or two residues. Collectively the results of this study demonstrate that a recombinant protein containing a consensus M2e sequence linked to the TLR5 ligand flagellin provides an effective and easily adaptable approach to developing non-egg-based vaccines against widespread epidemic and pandemic influenza.

Section snippets

Cloning and expression of recombinant STF2.4xM2e proteins

A construct encoding four tandem copies of M2e derived from the consensus sequence (see Table 1) of the human influenza A virus (H1N1, H2N1, H3N2) was chemically synthesized (DNA2GO, Menlo Park, CA) as a DNA concatemer. In this synthetic gene the eight cysteine residues (two per M2e copy) were modified to serine (SLLTEVETPIRNEWGSRSNDSSDPSR), to prevent disulfide bond formation that would be incompatible with Eschrichia coli expression. Substitution of serine for cysteine does not affect

Purification and TLR5 bioactivity of STF2.4xM2e

The ectodomain of the influenza A matrix protein (M2e) represents a highly conserved viral determinant that is expressed on the surface of influenza infected cells [28], [29]. The ectodomain of M2 comprises a relatively small polypeptide of 24 amino acids that is poorly immunogenic when delivered alone. However, studies have demonstrated that multimeric delivery of the peptide sequence resulted in enhanced immunogenicity [7], [8], [9], [11]. Previously we have demonstrated that fusing a protein

Discussion

Recognition of pathogens by specific receptors of the immune system was originally postulated by Janeway [37]. This hypothesis is supported by the identification of 11 members belonging to the Toll-like receptor family (TLR) (reviewed in [22], [38]). On the surface of APCs, the specific recognition of pathogen-derived ligands by their cognate TLR initiates a signaling cascade resulting in the recruitment of adaptor proteins including the myeloid differentiation factor 88 (MyD88) and the

Acknowledgements

The authors wish to thank Ms. Dallas Jock for expert technical assistance in the mouse studies; Ms. Darlene E. Guillen, Mr. Douglas M. Krouse, Dr. Albert Price and Mr. Bruce Weaver for supplying recombinant protein reagents; Dr. Ruslan Medzhitov, Dr. Alan Shaw and Dr. David Taylor for review of the manuscript.

References (52)

  • J.W. Huleatt et al.

    Vaccination with recombinant fusion proteins incorporating Toll-like receptor ligands induces rapid cellular and humoral immunity

    Vaccine

    (2007)
  • K. Mozdzanowska et al.

    Induction of influenza type A virus-specific resistance by immunization of mice with a synthetic multiple antigenic peptide vaccine that contains ectodomains of matrix protein 2

    Vaccine

    (2003)
  • D.F. Smee et al.

    Comparison of colorimetric, fluorometric and visual methods for determining anti-influenza (H1N1 and H3N2) virus activities and toxicities of compounds

    J Virol Methods

    (2002)
  • W. Liu et al.

    Monoclonal antibodies recognizing EVETPIRN epitope of influenza A virus M2 protein could protect mice from lethal influenza A virus challenge

    Immunol Lett

    (2004)
  • P. Zou et al.

    The epitope recognized by a monoclonal antibody in influenza A virus M2 protein is immunogenic and confers immune protection

    Int Immunopharmacol

    (2005)
  • A.A. Horner et al.

    Immunostimulatory DNA is a potent mucosal adjuvant

    Cell Immunol

    (1998)
  • A.M. Frace et al.

    Modified M2 proteins produce heterotypic immunity against influenza A virus

    Vaccine

    (1999)
  • M. De Filette et al.

    The universal influenza vaccine M2e-HBc administered intranasally in combination with the adjuvant CTA1-DD provides complete protection

    Vaccine

    (2006)
  • M. De Filette et al.

    Improved design and intranasal delivery of an M2e-based human influenza A vaccine

    Vaccine

    (2006)
  • R.G. Webster et al.

    Evolution and ecology of influenza A viruses

    Microbiol Rev

    (1992)
  • R.J. Sugrue et al.

    Specific structural alteration of the influenza hemagglutinin by amantadine

    Embo J

    (1990)
  • L.J. Holsinger et al.

    Analysis of the post-translational modifications of the influenza virus M2 protein

    J Virol

    (1995)
  • A.H. Reid et al.

    Characterization of the 1918 “Spanish” influenza virus matrix gene segment

    J Virol

    (2002)
  • S. Neirynck et al.

    A universal influenza A vaccine based on the extracellular domain of the M2 protein

    Nat Med

    (1999)
  • A.G. Bukrinskaya et al.

    Influenza virus uncoating in infected cells and effect of rimantadine

    J Gen Virol

    (1982)
  • M. Bui et al.

    Effect of M1 protein and low pH on nuclear transport of influenza virus ribonucleoproteins

    J Virol

    (1996)
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    1

    These authors contributed equally to this work.

    2

    Current address: Sanofi Pasteur, Discovery Drive, Swiftwater, PA 18370, USA.

    3

    Current address: Viron Therapeutics Inc., 700 Collip Circle, Suite 203, London, Ontario N6G 4X8, Canada.

    4

    Current address: Biologics & Vaccines, Merck & Co., WP78-302, 770 Sumneytown Pike, West Point, PA 19486, USA.

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