Currently licensed vaccines against the influenza A virus (IAV) need to be updated annually to match the constantly evolving antigenicity of the influenza virus glycoproteins， hemagglutinin (HA)， and neuramidiase (NA). Attempts to develop universal vaccines that provide broad protection have resulted in some success. Herein， we have shown that a replication-deficient adenovirus expressing H5/M2e induced significant humoral immunity against the conserved HA stalk. Compared to the humoral responses induced by an inactivated influenza vaccine， the humoral responses induced by the adenovirus-vectored vaccine against the conserved stalk domain mediated cross-protection against heterosubtypic influenza viruses. Importantly， virus inactivation by formaldehyde significantly reduced the binding of monoclonal antibodies (mAbs) to the conserved nucleoprotein (NP)， M2e， and HA stalk. These results suggest that inactivation by formaldehyde significantly alters the antigenicity of the HA stalk， and suggest that the conformation of the intact HA stalk provided by vector-based vaccines is important for induction of HA stalk-binding Abs. Our study provides insight into the mechanism by which a vector-based vaccine induces broad protection by stimulation of cross-protective Abs targeting conserved domains of viral proteins. The findings support further strategies to develop a vectored vaccine as a universal influenza vaccine for the control of influenza epidemics and unpredicted pandemics.