Identification and characterization of epitopes from influenza A virus hemagglutinin that induce broadly cross-reactive antibodies

Abstract

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.

Localization Analysis of Heterophilic Antigen Epitopes of H1N1 Influenza Virus Hemagglutinin

“…Short peptides are good immunogens to study the immunogenicity and reactivity of virus heterophilic epitopes. For example, Li et al (2018) directly immunised mice with short peptides of the influenza virus and successfully screened and prepared mAbs. Gong et al (2016) coupled the short peptides P1-P6 of influenza virus H3N2 with keyhole limpet hemocyanin carrier proteins to increase the immunogenicity of peptides and induce a strong immune response.…”

“…Polyglutamic acid was added to the carboxy terminus of positive peptides to facilitate their coating of ELISA plates (Li et al 2018). Each well was coated with 2 lg/mL of the peptide and incubated at 4°C overnight.…”

H1N1 influenza virus epitopes classified by monoclonal antibodies

“…Li et al (31) applied short-peptide immunization to the mice directly, and screened the prepared mAbs. In order to enhance immunogenicity, connection of polypeptides and macromolecular protein can also be used.…”

“…These short peptides can be also used as good immunogens to research different subtypes of influenza virus epitope vaccines. Li et al ( 31 ) applied short-peptide immunization to the mice directly, and screened the prepared mAbs. In order to enhance immunogenicity, connection of polypeptides and macromolecular protein can also be used.…”

Autophagy‐Dependent Ferroptosis as a Therapeutic Target in Cancer

“…Free fatty serves as a fuel for mitochondrial beta-type oxidation and can be esterified into triglycerides and cholesterol esters to form a unique spherical organelle named the lipid droplet. [39] Lipophagy is an unique selective autophagy that targets lipid droplets to regulate cellular lipid levels. [40] A recent study showed that lipophagy promotes ferroptosis by decreasing lipid storage and promoting lipid peroxidation.…”