Because it is highly conserved in all A-type influenza viruses, M2e has been studied as a universal influenza vaccine (8
). Here we used a combined approach to target M2e for the development of such a vaccine. First, multiple copies of M2e (4.M2e) in our vaccine candidates increased the density of epitopes. Second, replacement of the highly immunogenic variable region of FliC with 4.M2e may endow M2e with high immunogenicity because the resulting 4.M2e-tFliC fusion protein retains the TLR5 ligand property of FliC. VLPs are advantageous as immunogens in inducing strong immune responses since they mimic the structures of viruses which the immune system has evolved to fight. The HA cytoplasmic domain contains signals guiding the envelope glycoprotein to assemble into VLPs (7
). Previously, we have found that a fusion protein with an HA membrane anchor can be incorporated into M1 VLPs (40
). The VLP approach enables M2e to be delivered in a particulate form and to be presented in its native external membrane microenvironment. Particulate repetitive epitopes are more effective antigens because of their increased epitope density (13
). Several groups have also reported enhanced immune responses induced by physical repeats of M2e (11
). The potential of this approach has been demonstrated by inducing a high level of M2-specific immunity and complete protection against lethal live virus challenge, as shown in the present study.
As the natural ligand of TLR5, flagellin has been found to be an effective adjuvant. It has been used to enhance the immunogenicity of antigens in mixtures with antigens and the physical association as fusion proteins, and it has been coincorporated into the same particles with antigens (16
). The replacement of the variable region of FliC with 4.M2e should not impair its innate TLR signaling because flagellins from different bacterial strains show significant differences in their variable regions (2
). A fusion protein composed of 4 repeating M2e peptides attached to the C terminus of the phase II flagellin (FljB) was reported to induce partial protection in mice against a lethal influenza virus infection with reduced clinical symptoms (16
). Our results demonstrate that the membrane-anchored 4.M2e-tFliC in VLPs retains innate TLR5 signaling activity. As expected, these VLPs induced high levels of M2e-specific immune responses, in particular when they were given by i.n. immunization, in which complete protection against either A/PR8 or A/Philippines virus with little body weight loss was induced. These promising results are probably derived from the advantages of our approach: the physical association of the FliC TLR5 binding domains with 4.M2e and the replacement of the FliC hyperimmunogenic central region with 4.M2e. Also, these results are consistent with results reported in other studies which indicate that the TLR5 recognition domains of flagellin are not associated with the central variable region (2
) and that a variable region-truncated FliC is more effective as a mucosal adjuvant (27
). Because the variable region of FliC is hyperimmunogenic, another advantage of the approach is that replacement of the variable region of FliC with M2e sequences minimizes antibody responses to FliC (27
), although other work showed that induction of anti-FliC antibody is not a limitation for its adjuvant properties (3
Several different immunization routes have been used to deliver M2e-derived antigens for inducing protective immunity. i.n. immunization was reported to be an advantageous approach in inducing protective immunity compared to other routes (9
). Our results also demonstrated that i.n. immunization with 4.M2e-tFliC/M1 VLPs induces high systemic immune responses as well as strong mucosal immunity and confers more effective protection against live virus challenge, as demonstrated by the complete protection against challenge with H1N1- or H3N2-subtype viruses with minimal body weight loss. Also, virus replication in the lungs of immunized mice is restricted, as revealed by lower lung virus titers, although they are still higher than those in the lungs of mice immunized with HA-containing VLPs (40
). i.n. immunization stimulates the nasopharynx-associated lymphoreticular tissue and induces local mucosal immunity (19
). Furthermore, the variable region-truncated flagellin has been reported to be more efficient as a mucosal adjuvant, as discussed above. These observations provide a basis for the finding that the M2e-tFliC/M1 VLPs induce enhanced mucosal immunity.
Because the M2-specific antibodies are not neutralizing and recent work demonstrated that M2 VLP antigens used in combination with the trivalent influenza vaccines or inactivated viral vaccines showed enhanced subtypic protection (36
), the approaches described here provide new alternatives for such combinations. Thus, a fusion protein containing a replacement of the variable region of FliC with repetitive M2e in VLPs is promising for further development of universal influenza A virus vaccines.