Hemagglutinin (HA) is the major influenza virus target antigen recognized by neutralizing antibodies. It is a surface glycoprotein, synthesized as a single polypeptide, which is trimerized. Each monomer of HA is synthesized as a precursor molecule HA0 post-translationally cleaved by host proteases into two subunits, HA1 and HA2 linked by a single disulfide bond [
16]. Cleavage into HA1 and HA2 gp is essential for the infectivity of the virus particle and spread of the infection in the host organism [
44].
The HA1 of influenza A virus forms a membrane-distal globular domain that contains the receptor-binding site and most antigenic sites recognized by virus-neutralizing antibodies preventing attachment of virus to the host cell. Escape variants with mutation in the antigenic site easily avoid neutralization by existing host antibodies, leading to seasonal influenza outbreaks [
45]. In spite of continual antigenic changes of hemagglutinin, common epitopes shared by various strains were identified. Although the degree of sequence diversity between HA subtypes is great, particularly in the HA1 glycopolypeptides, HA2 is its rather conserved part. According to documented results, HA2 has the prerequisite to be one of the potential inductors of protective heterosubtypic immunity [
1,
28,
29,
46‐
48]. HA2 represents the smaller C-terminal portion of hemagglutinin, which forms a stem-like structure that mediates the anchoring of the globular domain to the cellular or viral membrane. N-terminal part of HA2 gp, termed the fusion peptide, plays a substantial role in the fusion activity of influenza virus. It was demonstrated that the rearrangements of HA as well as the fusion process is temperature- and pH-dependent [
49,
50]. At neutral pH, the N-terminus of the fusion peptide is inserted into the inter-space of HA trimer. At low pH, which triggers the fusion process, N-terminus of the fusion peptide is exposed and inserted into the target membrane, allowing the release of the ribonucleoprotein complex into the cytoplasm [
51,
16]. Although the epitopes of the HA2 gp are less accessible for interaction with antibodies in native virus than those of HA1 gp, HA2-specific antibodies are induced during natural infection in humans [
52] as well as in mice [
53]. Significance of HA2-specific antibodies for the heterosubtypic immunity lies in their broad cross-reactivity [
1,
31,
48,
54,
55]. While HA2-specific antibodies do not act by obstructing the binding of the virus to the host cells [
56‐
58] it should be emphasized that HA2-specific antibodies are able to reduce the replication of influenza viruses of various HA subtypes by several ways: binding of antibody can inhibit the fusion of viral and endosomal membranes [
59,
60] by preventing the conformation change of HA induced by low pH [
20,
21,
61] or by blocking the insertion of the fusion peptide into the endosomal membrane [
62,
63]. Moreover, it was shown that passive immunization with monoclonal antibodies against HA2 gp, as well as active immunization with recombinant vaccinia virus expressing chimeric molecules of HA, improve the recovery from influenza infection and contribute to a milder course of infection [
28,
29]. A recent study showed that increased immunogenicity of HA2 gp could be achieved by unmasking of HA2 gp after removing the highly immunogenic globular head domain of HA1 gp. Headless HA trimers form the conserved HA stalk domain, on which HA2 epitopes are more accessible for B cells than in the native HA. Vaccination of mice with this headless HA immunogen elicited antibodies cross-reactive with multiple subtypes of hemagglutinin and provide protection against lethal influenza virus infection [
31].
Hemagglutinin HA1-HA2 connecting region, as well as N-terminal fusion peptide of HA2, are the broadly conserved parts of HA, the latter conserved even among all 16 subtypes of influenza A viruses [
1,
47,
61,
64]. Protective potential of the fusion peptide or HA1-HA2 cleavage site of influenza A viruses were investigated by several groups. They found that mice vaccinated with a peptide spanning the HA1-HA2 connecting region exhibited milder illness and fewer deaths upon virus challenge [
64,
65].
Generation of monoclonal antibodies against universally conserved fusion peptide has attracted interest in the recent past, as such antibodies are known to inhibit the HA fusion activity and to reduce virus replication
in vitro and also
in vivo [
28,
30,
54,
62,
63]. Additionally, passive immunotherapy with Abs reactive with all strains of influenza A could be an alternative for some populations at high risk of infection, like infants, the elderly and the immunocompromised patients, who may not benefit from active vaccination. Several groups described the potential of human monoclonal antibodies against HA2 subunit and its fusion peptide with broad-spectrum protection as a universal passive immunotherapeutic agent against seasonal and pandemic influenza viruses [
66‐
69]. Sui et al. [
70] obtained a panel of high-affinity human antibodies that bind to the highly conserved pocket in the stem region of hemagglutinin, comprising part of the fusion peptide and several residues of the HA1 subunit. These antibodies showed a broad degree of cross-reactivity. Moreover, it was suggested that the conformational epitope on HA recognized by one of these neutralizing antibodies (F10) is recalcitrant to the generation of escape mutants [
70].
Thus, identification of antibodies against conserved epitopes of hemagglutinin shows the way for their use in passive immunotherapy, designing of antivirals and represents an important step towards development of cross-protective universal vaccine against influenza virus that potentially does not require annual adjustment.