Heterosubtypic protection against influenza A induced by adenylate cyclase toxoids delivering conserved HA2 subunit of hemagglutinin.

The protective efficacy of currently available influenza vaccines is restricted to vaccine strains and their close antigenic variants. A new strategy to obtain 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 2days 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 an HA2-based vaccine that can induce both humoral and cellular immune responses without the need of adjuvant.

Epitope specificity of anti-HA2 antibodies induced in humans during influenza infection.

BACKGROUND: The conserved, fusion-active HA2 glycopolypeptide (HA2) subunit of influenza A hemagglutinin comprises four distinct antigenic sites. Monoclonal antibodies (MAbs) recognizing three of these sites are broadly cross-reactive and protective. OBJECTIVES: This study aimed to establish whether antibodies specific to these three antigenic sites were elicited during a natural influenza infection or by vaccination of humans. METHODS: Forty-five paired acute and convalescent sera from individuals with a confirmed influenza A (subtype H3) infection were examined for the presence of HA2-specific antibodies. The fraction of antibodies specific to three particular antigenic sites (designated IIF4, FC12, and CF2 here) was investigated using competitive enzyme immunoassay. RESULTS: Increased levels of antibodies specific to an ectodomain of HA2 (EHA2: N-terminal residues 23-185 of HA2) were detected in 73% of tested convalescent sera (33/45), while an increased level of antibodies specific to the HA2 fusion peptide (N-terminal residues 1-38) was induced in just 15/45 individuals (33%). Competitive assays confirmed that antibodies specific to the IIF4 epitope (within HA2 residues 125-175) prevailed in 86% (13/15) over those specific to the other two epitopes during infection. However, only a negligible increase in HA2-specific antibodies was detectable following vaccination with a current subunit vaccine. CONCLUSIONS: We observed that the antigenic site localized within N-terminal HA2 residues 125-175 was more immunogenic than that within residues 1-38 (HA2 fusion protein), although both are weak natural immunogens. We suggest that new anti-influenza vaccines should include HA2 (or specific epitopes localized within this glycopolypeptide) to enhance their cross-protective efficacy.

Conserved epitopes of influenza A virus inducing protective immunity and their prospects for universal vaccine development.

Influenza A viruses belong to the best studied viruses, however no effective prevention against influenza infection has been developed. The emerging of still new escape variants of influenza A viruses causing epidemics and periodic worldwide pandemics represents a threat for human population. Therefore, current, hot task of influenza virus research is to look for a way how to get us closer to a universal vaccine. Combination of chosen conserved antigens inducing cross-protective antibody response with epitopes activating also cross-protective cytotoxic T-cells would offer an attractive strategy for improving protection against drift variants of seasonal influenza viruses and reduces the impact of future pandemic strains. Antigenically conserved fusion-active subunit of hemagglutinin (HA2 gp) and ectodomain of matrix protein 2 (eM2) are promising candidates for preparation of broadly protective HA2- or eM2-based vaccine that may aid in pandemic preparedness. Overall protective effect could be achieved by contribution of epitopes recognized by cytotoxic T-lymphocytes (CTL) that have been studied extensively to reach much broader control of influenza infection. In this review we present the state-of-art in this field. We describe known adaptive immune mechanisms mediated by influenza specific B- and T-cells involved in the anti-influenza immune defense together with the contribution of innate immunity. We discuss the mechanisms of neutralization of influenza infection mediated by antibodies, the role of CTL in viral elimination and new approaches to develop epitope based vaccine inducing cross-protective influenza virus-specific immune response.