To characterize the antigenic epitopes of the hemagglutinin (HA) protein of H1N1 influenza virus, a panel consisting of 84 clones of murine monoclonal antibodies (mAbs) were generated using the HA proteins from the 2009 pandemic H1N1 vaccine lysate and the seasonal influenza H1N1(A1) vaccines. Thirty-three (39%) of the 84 mAbs were found to be strain-specific, and 6 (7%) of the 84 mAbs were subtype-specific. Twenty (24%) of the 84 mAbs recognized the common HA epitopes shared by 2009 pandemic H1N1, seasonal A1 (H1N1), and A3 (H3N2) influenza viruses. Twenty-five of the 84 clones recognized the common HA epitopes shared by the 2009 pandemic H1N1, seasonal A1 (H1N1) and A3 (H3N2) human influenza viruses, and H5N1 and H9N2 avian influenza viruses. We found that of the 16 (19%) clones of the 84 mAbs panel that were cross-reactive with human respiratory pathogens, 15 were made using the HA of the seasonal A1 (H1N1) virus and 1 was made using the HA of the 2009 pandemic H1N1 influenza virus. Immunohistochemical analysis of the tissue microarray (TMA) showed that 4 of the 84 mAb clones cross-reacted with human tissue (brain and pancreas). Our results indicated that the influenza virus HA antigenic epitopes not only induce type-, subtype-, and strain-specific monoclonal antibodies against influenza A virus but also cross-reactive monoclonal antibodies against human tissues. Further investigations of these cross-reactive (heterophilic) epitopes may significantly improve our understanding of viral antigenic variation, epidemics, pathophysiologic mechanisms, and adverse effects of influenza vaccines.
Influenza is the most common infectious disease and is caused by influenza A virus (IAV) infection. Hemagglutinin (HA) is an important viral protein of influenza A and is a major component of current IAV vaccines. The side effects associated with IAV vaccination are well studied; however, the HA‑induced immunopathological changes have remained largely elusive. The primary objective of the present study was to determine the tissue cross‑reactive epitopes of HA proteins. Monoclonal antibodies (McAbs) were generated according to traditional methods using purified HA proteins from influenza vaccine lysates. The specificity of these McAbs was analyzed using western blot analysis and ELISA. Human tissue microarrays were employed for immunohistochemical staining to screen these McAbs. Rat brain tissues were subjected to immunohistochemical staining and electron microscopy to demonstrate the subcellular localization of antibodies targeting specific antigens. A total of 67 hybridoma cell lines positive for McAb against HA antigen were obtained. Three cross‑reactive McAbs (H1‑13, H1‑15 and A1‑10) were discovered through tissue screening. Based on the 3 cross‑reactive McAbs and the amino acid sequence of HA, the presence of two broadly cross‑reactive HA epitopes, 194‑WGIHH‑198 and 365‑WYGYHH‑370, was assumed. McAbs against these synthetic epitope peptides were obtained. They reacted with porphyrin ring‑containing molecules, including hemoglobin (Hb) and protoporphyrin, and with numerous types of normal tissue. In conclusion, the present study identified two broadly cross‑reactive epitopes on HA (194‑WGIHH‑198 and 365‑WYGYHH‑370). Antibodies against these epitopes react with Hb and numerous types of important normal tissues/organs. These newly identified cross‑reactive epitopes from IAV HA may provide crucial information for influenza research.
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