As shown in Fig.?1H, compared with total IgG, HA7-CT-specific IgG showed significantly low binding affinity with the HA7 extracellular domain name peptide ( em P /em ? ?0.01), but markedly high binding affinity with the HA7-CT peptide ( em P /em ? ?0.05), indicating that HA7-CT-specific IgG can be specifically recognized by AL 8697 the HA7-CT peptide. virus entry into host cells (Scolari em et al. /em 2016). Although its functions have been well studied, little is known about the immunogenicity and sub-localization of HA-CT. Previous studies have identified a specific neutralizing antibody against the CT of human immunodeficiency virus glycoprotein 41 (Chen em et al. /em 2015), which led us to hypothesize that HA-CT may function in a similar manner. To investigate the immunogenicity of HA7-CT epitope further, we pulled down CT-specific antibody from total IgG in the plasma samples from five H7N9 patients using biotinylated HA7-CT peptide. We analyzed the relationship between the titer of HA7-CT-specific IgG (measured photometrically) and the total IgG titer in H7N9 patient plasma (measured by virus neutralization assay in H7N9-infected MadinCDarby canine kidney (MDCK) cells or by hemagglutination inhibition (HI) assay). As shown in Fig.?1D, the HA7-CT-specific IgG titer showed a pattern similar to that of the plasma neutralization titer. Furthermore, the HA7-CT-specific IgG titer was significantly positively correlated with the plasma neutralization titer ( em P /em ? ?0.0001; Fig.?1E) and the plasma HI titer ( em P /em ?=?0.0303; Fig.?1F), but not with the antibody-dependent cell-mediated cytotoxicity percentage ( em P /em ?=?0.5318; Fig.?1G). The IgG titers of the other candidate epitopes, i.e., P15, P21, P30, P63, P76, and P98 (data not shown), were not correlated with the plasma neutralization titer. These results suggested that HA7-CT possesses an antigenic epitope that is capable of blocking virus contamination by neutralization, but not of the antibody-dependent cell-mediated cytotoxic AL 8697 effects. Because surface protein of the influenza virus is a major antibody target, we next sought to investigate whether HA7-CT is usually displayed around the H7N9 surface and AL 8697 thus can be recognized by antibodies. We pulled down HA7-CT-specific IgG from total IgG in plasma samples of H7N9 patients and tested its affinity with HA7-CT peptide, HA7 extracellular domain name peptide, and inactivated H7N9 virion by ELISA. MDCK cell culture supernatant was used as a blank control. As shown in Fig.?1H, compared with total IgG, HA7-CT-specific IgG showed significantly low binding affinity with the HA7 extracellular domain name peptide ( em P /em ? ?0.01), but markedly high binding affinity with the HA7-CT peptide ( em P /em ? ?0.05), indicating that HA7-CT-specific IgG can be specifically recognized by the HA7-CT peptide. On the other hand, there was no difference in binding affinity for inactivated virions between HA7-CT-specific and total IgG, suggesting that HA7-CT-specific and total IgG can bind to whole-virus particles with comparable affinity. These results suggested that HA7-CT is usually displayed around the virus surface, at least occasionally. The CT of HA consists of 11 AAs. Based on HA and CT sequences of various subtypes of influenza A virus retrieved from the influenza database (https://www.fludb.org) and weblogo3.5 (http://weblogo.threeplusone.com/), respectively, we found that the CT sequence was highly conserved, especially within virus clades. As shown in Fig.?1I, all sequences analyzed shared a CXICI motif at the C terminus. The CT sequences of HAs in group 1 were more comparable than those in group 2. Of note, H1, H2, and H5 in clade H1a had identical CT CD244 sequences. To explore whether the CTs of different influenza A subtypes commonly possess antigenicity, we analyzed the antigenic indexes of the CTs predicted by the Jameson-Wolf model. The CTs of clades H9, H3, and H7 had significantly higher antigenic index values than those of clade H1a (H1, H2, H5), consistent with our experimental results. Currently available influenza vaccines are mainly HA- or NA-specific neutralizing antibodies (Gerhard em et al. /em 2006). However, because of the high antigenic variability of HA and NA, it is difficult to produce a universal and effective vaccine to prevent epidemics (Carrat and Flahault 2007; Deng em et al. /em 2015). We found more than 50% sequence similarity in the CT sequences of different HA subtypes (Fig.?1I). The highly conserved CT sequence and high antigenic index scores indicated that HA-CT may serve as a potential antigenic epitope for the development of a broad-spectrum influenza antibody. Our study had some limitations. First, we used short linear peptides to coat the ELISA plates, and we cannot guarantee consistent coating efficiency. Second, total IgG in the plasma may recognize conformational epitopes on HA, but the linear peptide-based ELISA is usually.