Cutting edge:

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

Influenza and Other Respiratory Viruses, 2012;6 (6):389 – 395

Staneková Zuzana1, Mucha Vojtech1, Sládková Tatiana1, Blaškovičová Hana2, Kostolanský František1 and Varečková Eva1*

1Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 845 05 Bratislava, Slovak Republic

2 Public Health Authority of the Slovak Republic, National Influenza Centre, Bratislava, Slovak Republic



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.

PMID: 22236105



This study focuses on the analysis of antibodies specific to the HA2 gp of influenza A virus (IAV) hemagglutinin (HA) during the natural infection of humans.

The HA2 gp of IAV attracts attention of numerous research laboratories focused on the preparation of universal vaccine against influenza. The reason is that HA2 gp, a relatively conserved part of the variable surface antigen HA, is able to induce cross-protective immune response (4). Recently, several studies on mouse model have been published.  We showed that monoclonal antibodies (MAbs CF2, IIF4, FC12) specific to three out of 4 epitopes of HA2 gp belonging to different antigenic sites (Fig. 1) improved the survival of mice infected with lethal dose of IAV (3). Due to the proved protective potential of HA2-specific antibodies in mice we supposed that if HA2-specific antibodies are induced as a consequence of the natural infection or vaccination of humans, they would contribute to a better recovery from the disease. The protection mediated by HA2-specific antibodies is dependent on their epitope specificity and their binding potential (i.e. concentration and affinity of binding) in convalescent sera. As no available data analyzing the HA2-specific antibody response induced during the IAV infection in humans in details have been reported before, we scrutinized 45 pairs of acute and convalescent sera from patients with confirmed influenza infection collected during the season 2003/2004 (antigen A/Fujian/411/2002 (H3N2)-like virus). Examination of these sera showed that HA2-specific antibodies are not produced in all patients. They are induced prevalently in the older groups, which can be explained by the repeated experience with influenza infection during the lifespan of adults. Similarly, the level of HA2-specific antibodies of mice significantly increases after repeated infection with antigenically different IAV viruses (1).

Moreover, not all HA2 epitopes do induce protective antibody response. On mouse model we found one MAb (CB8)-recognizing antigenic site III (inside the aa region 38-112 of HA2 gp, Fig. 1) which enhanced the replication of virus in mouse lungs and worsened the course of the disease (3). Therefore there is a need to select suitable epitopes of HA2 gp as inducers of antibodies able to lower the replication of virus in lungs and to mitigate the course of the infection.

The increase of HA2-specific antibodies induced in humans during the acute infection is low and not universally observed. To determine the immunogenicity of particular epitopes we used competitive EIA and found that the most immunogenic HA2 epitope is localized inside the region aa125-175 of HA2 gp, recognized by MAb IIF4, which was shown to be protective in our previous studies on mice. Insufficient immunogenicity appears to be a common problem when designing cross-protective (‘universal’) influenza vaccines. In our extended new studies we tried to avoid this problem by insertion of selected parts of HA2 into adenylate-cyclase toxoid from Bordetella pertussis (CyaA) as a vector for antigen delivery to immunocompetent cells (7). This vector enables the presentation of HA2 antigen to B and also to T cells.  In comparison with stimulation of HA2-specific immune response by purified HA2 gp (applied to mice together with adjuvant), CyaA-HA2 induced the broadened protection not only against the homologous (H3), but also against the heterologous avian IAV of H7 subtype (6; 7). The advantage of using such vector is the possibility to combine various conserved IAV epitopes (i.e. from HA, nucleoprotein, M1 protein…).

Our knowledge about the epitope specificity of anti HA2-antibodies induced in humans during the acute infection is important from the point of view of the selection of HA2-epitopes as potential alternative inducers of protection especially against newly emerged IAVs, when do not work current vaccines. As after the vaccination of human volunteers with current subunit vaccine only negligible increase of HA2-specific antibodies is induced (5), we suppose that targeted immunization with selected HA2 epitopes can increase the immunogenicity of naturally weak HA2 immunogen, and induced antibodies could contribute to a better cross-protective efficacy of such newly designed vaccines in comparison to currently used ones.

Our results indicate that thoroughly selected conserved epitopes of HA2 gp or other conserved IAV antigens with protective impact on the infection could be applied in the future also for the construction of multi-epitope based vaccine with broader  efficacy as a possible, promising way of prevention against  broad spectrum of IAV viruses.

 Eva Vareckova-png

Fig. 1. Antigenic structure of the light chain of influenza virus hemagglutinin mapped into native conformation of Influenza A virus (A/X-31(H3N2)) hemagglutinin (Protein Database accession number 1HGF) or post-fusion conformation of Influenza A virus (EHA2 Influenza Recombinant HA2 Chain,  Protein Database accession number 1QU1). Antigenic sites defined on HA2 gp: I (aa1-38)-recognized by MAb CF2, II  (125-175)-MAb IIF4, III (aa38-112)- MAb CB8, IV (aa 125-175)-MAb FC12 (2).



RNDr. Varečková Eva, DrSc.

Institute of Virology,

Slovak Academy of Sciences

Dubravska cesta 9

845 05 Bratislava,

Slovak Republic




1/ Kostolanský F., Mucha V, Slováková R, Varečková E.:Natural influenza A virus infection of mice elicits strong antibody response to HA2 glycopolypeptide. Acta Virol. 2002;46(4):229-36.

2/ Varečková E., Mucha V., Wharton S., Kostolanský F.: Inhibition of fusion activity of influenza A haemagglutinin mediated by HA2-specific monoclonal antibodies. Archives of Virology , 2003; (148) 469-486.

3/ Gocník M., Fislová T., Sládková T., Mucha V., Kostolanský F., Varečková E.: Antibodies specific to the HA2 glycopolypeptide of influenza A virus haemagglutinin with fusion-inhibition activity contribute to the protection of mice against lethal infection. J. Gen. Virol. 2007; 88: 951-955.

4/ Gocník M., Fislová T., Mucha V., Sládková T., Russ G., Kostolanský F., Varečková E: Antibodies induced by the HA2 glycopolypeptide of influenza virus haemagglutinin improve recovery from influenza A virus infection. J. Gen. Virol., 2008, 89: 958-967.

5/ Staneková Z., Mucha V., Sládková T., Blaškovičová H., Kostolanský F., Varečková E: Epitope specificity of anti-HA2 antibodies induced in humans during influenza infection. Influenza and Other Respiratory Viruses, 2012;6 (6):389 – 395.

6/ Janulíková J., Staneková Z., Mucha V., Kostolanský F., Varečková E: Two distinct regions of HA2 glycopolypeptide of influenza virus hemaglutinin elicit cross-protective immunity against influenza. Acta Virologica, 2012;56(3):169 – 176.

7/ Staneková Z, Adkins I, Kosová M, Janulíková J, Sebo P, Varečková E.: Heterosubtypic protection against influenza induced by adenylate cyclase toxoids delivering conserved HA2 subunit of hemagglutinin. Antiviral Research, 2013; 97 (1) 24-35.

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