Our results demonstrate the efficacy of cHA constructs in eliciting broad-spectrum immunity against group 1 influenza viruses. cHA constructs not only allow for the sequential exposure of the same stalk in the context of an irrelevant globular head domain but also provide for the use of challenge viruses with globular heads to which animals have never been exposed. In this study, all animals were naive to the HA globular head domains of the challenge viruses used. This design demonstrates that protection from challenge following vaccination is based primarily on an immune response toward the HA stalk. Because cross-reactive antibodies toward the receptor binding site could possibly play a role in the protection seen here (2
), we confirmed that all mice were HI negative for their respective challenge viruses. It is also possible that low levels of cross-reactive anti-globular head antibodies (not detected by HI assay) contributed to the neutralizing effect seen herein. This would, however, be considered an unexpected boon of our vaccination strategy, as this would supplement the protection already provided by antibodies with stalk specificities. Nevertheless, protection was impressively conferred following each virus challenge, even though animals were never exposed to the globular head of that particular virus subtype.
We hypothesize that the sequential exposure of the same H1 stalk stimulates adaptive immunity to this region. Note that cHA constructs contain a trimerization domain that maintains a correctly folded stalk structure in the absence of a viral membrane that normally stabilizes this interaction (27
). As such, the unfettered exposure to a correctly formed HA stalk could also drive the boost in stalk titer that was seen following vaccination.
We conclusively showed that the protection induced by our vaccine was mediated by broadly neutralizing antibodies. However, the contributions of alternative mechanisms of action remain to be elucidated. We believe that processes such as antibody-dependent cell-mediated cytotoxicity and complement activation may play an important role. Other factors, such as temperature, might influence the efficacy of stalk-antibody binding. All in vitro
binding assays we describe were carried out at room temperature. Even though influenza virus infection in mice induces hypothermia, prophylactic and therapeutic efficacy of stalk-reactive antibodies has been shown in ferrets as well (28
). These animals develop fever upon influenza virus infection, similar to humans, suggesting that the neutralizing ability of these antibodies is not highly temperature dependent.
It is possible that a similar outcome could be achieved by using different vaccination schemes or employing other constructs that express conserved epitopes of influenza virus proteins (11
), including those within the receptor binding site (32
) or the extracellular domain of the M2 protein (34
). While we focused on a proof-of-principle experiment with group 1 influenza viruses, we believe that a similar approach could be employed for group 2 and influenza B viruses, leading to the development of a trivalent universal influenza virus vaccine.
In conclusion, we definitively demonstrated that sequential vaccination with cHA constructs can elicit polyclonal humoral responses to the HA stalk domain which are protective against heterologous and heterosubtypic challenges. The stimulation of these responses following previous influenza virus exposure supports the potential of a similar vaccine protocol in humans to provide protection against a broad range of influenza virus strains.