Incorporation of Membrane-Anchored Flagellin into Influenza Virus-Like Particles Enhances the Breadth of Immune Responses

Since the outbreak of a highly pathogenic avian influenza virus (HPAI) H5N1 variant in 1997 in Hong Kong, there have been increased concerns about the threat of a new pandemic that may cause widespread fatal infection in humans. Although the transmission of avian influenza viruses from birds to humans is a rare event, both the continuing increase of infected human cases and the high mortality rates suggest the persisting threat of an H5N1 pandemic (6). There is evidence that the 1918 pandemic virus, which caused an estimated 40 million deaths, was an avian virus directly adapted to humans (53). Although two classes of antiviral drugs targeting the viral matrix protein M2 and neuraminidase, respectively, are available against influenza A viruses, financial and supply limitations as well as frequent drug resistance may limit the ability to utilize these drugs for preventing a new pandemic (6, 36, 38, 66). It is well recognized that an effective vaccine is the primary strategy for protection against an emerging pandemic (37, 51, 61). Currently, an inactivated influenza vaccine is the dominant form used, although a live attenuated (cold-adapted) influenza virus vaccine was also recently licensed (22). However, the emergence of a new pandemic strain could easily overwhelm the present capacity of vaccine production, which is based on embryonic hens’ eggs. There are additional concerns that biosafety containment facilities may be needed for virus-based vaccine production, and a period of 6 to 9 months would be required. A safe, convenient, and more reliable alternative is needed as a countermeasure to the emerging challenge. As a new form of vaccine candidate, virus-like particles (VLPs) have been reported to be potent vaccines for a variety of pathogenic viruses (24, 25, 44, 47, 65). VLPs elicit immune responses including both B-cell-mediated antibody and specific T-cell-mediated cellular responses to protect experimental animals against lethal influenza virus challenge (3, 13, 30, 40, 42). However, though VLPs provide an attractive platform for designing vaccines against a possible new influenza virus pandemic strain, they resemble the current vaccines in inducing immune responses that are predominantly subtype specific (23). An important advance would be the development of new vaccines with enhanced breadth of immunity, which could potentially be used to prevent infection by newly emerging variants, including influenza viruses of other subtypes. To develop a more effective influenza virus vaccine, we have designed a chimeric influenza VLP (cVLP) vaccine candidate by incorporating flagellin, the Toll-like receptor 5 (TLR-5) ligand, into VLPs as a molecular adjuvant. Flagellin is the primary protein component of the highly complex flagellar structures that extend from the outer membrane of gram-negative organisms. It has been well documented that TLR-5 recognizes a conserved site on flagellin (15, 33, 48, 49). As a natural agonist of TLR-5, flagellin is an effective inducer of innate immune effectors such as cytokines and nitric oxide, thereby stimulating the activation of adaptive immune responses (34, 35). Furthermore, flagellin-induced enhancement of adaptive immune responses is known to influence the presentation of antigens and the activation of cellular immune responses (10, 32, 56). In the present study, we designed a modified membrane-anchored form of flagellin for incorporation into influenza VLPs. We determined the immune responses to these VLPs in a mouse model, including their ability to protect against challenge infection with an influenza A virus of a different subtype.