A Detailed Assessment of Cross‐Neutralizing Antibodies Between SARS‐CoV‐2 and Influenza A

A marked global decline in seasonal flu due to circulation of influenza virus was observed during the COVID-19 pandemic. Although public health interventions played a role, potential immunological cross-reactivity between influenza and SARS-CoV-2 viruses remains a possibility. In this article, we describe methods to determine if SARS-CoV-2 infection has resulted in the induction of antibodies that cross-react with seasonal influenza viruses. To determine if such cross-reactivity does in fact occur, we examined plasma samples from 311 individuals with confirmed SARS-CoV-2 infection for evidence of ability to neutralize influenza virus. Antibody responses against SARS-CoV-2 were estimated by enzyme-linked immunosorbent assay, and responses to seasonal influenza viral strains-A/H3N2, A/H1N1, B/Victoria, and B/Yamagata-were assessed by performing the hemagglutination inhibition assay. Sixteen samples that were positive for SARS-CoV-2 antibodies but negative for influenza antibodies were selected for further analysis by microneutralization (MN) assay to evaluate their ability to neutralize influenza viruses. Among these, five samples exhibited high MN titers (≥20), and six exhibited moderate titers (≥10) against influenza A viruses. To elucidate the molecular basis of this cross-reactivity, genomes of SARS-CoV-2 strains were sequenced by Illumina next-generation sequencing and subjected to in silico structural modeling. Antibodies generated against the SARS-CoV-2 Delta variant demonstrated strong binding affinities with influenza A glycoproteins, including hemagglutinin from H1N1 (-12.4 kcal/mol) and H3N2 (-9.3 kcal/mol) and neuraminidase from H1N1 (-10.1 kcal/mol) and H3N2 (-11.7 kcal/mol). These results indicate that SARS-CoV-2 infection, particularly with the Delta variant, can induce antibodies with cross-neutralizing activity against influenza A viruses. This immunological cross-reactivity may have contributed to the reduced seasonal influenza activity during the COVID-19 pandemic. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Hemagglutination inhibition assay Basic Protocol 2: Evaluation of SARS-CoV-2-specific antibodies via enzyme-linked immunosorbent assay Basic Protocol 3: Pseudovirus titration and neutralization assays of SARS-CoV-2 Basic Protocol 4: Microneutralization assay.