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