The protective efficacy of currently available influenza vaccines is restricted to vaccine strains and their close antigenic variants. A new strategy to obtain a cross-protection against influenza is based on conserved antigens of influenza A viruses (IAV), which are able to elicit a protective immune response. Here we describe a vaccination approach involving the conserved stem part of hemagglutinin, the HA2 subunit, shared by different HA subtypes of IAV. To increase its immunogenicity, a novel strategy of antigen delivery to antigen presenting cells (APCs) has been used. The HA2 segment (residues 23-185) was inserted into a genetically detoxified adenylate cyclase toxoid (CyaA-E5) which specifically targets and penetrates CD11b-expressing dendritic cells. The CyaA-E5-HA2 toxoid induced HA2(93-102), HA2(96-104) and HA2(170-178)-specific and Th1 polarized T- cell responses, and also elicited strong broadly cross-reactive HA2-specific antibody response. BALB/c mice immunized with three doses of purified CyaA-E5-HA2 without any adjuvant recovered from influenza infection 2 days earlier than the control mock-immunized mice. More importantly, immunized mice were protected against a lethal challenge with 2LD(50) dose of a homologous virus (H3 subtype), as well as against the infection with a heterologous (H7 subtype) influenza A virus. This is the first report on heterosubtypic protection against influenza A infection mediated by HA2-based vaccine that can induce both humoral and cellular immune responses without the need of adjuvant.