Influenza A viruses represent a persistent global health threat, causing significant morbidity and mortality annually. The substantial economic burden associated with seasonal influenza, caused by H1N1 and H3N2 influenza A viruses and influenza B viruses, highlights the urgent need for effective broad-protection strategies. While vaccines are the primary countermeasure, their effectiveness is limited by the rapid antigenic drift of influenza viruses, necessitating the development of next-generation vaccines offering broader protection. Antiviral drugs, while an alternative, suffer from limited therapeutic windows and the emergence of drug resistance. The unpredictable nature of influenza viruses and their potential for pandemic spread, as exemplified by the 1918 Spanish flu and the 2009 H1N1 pandemic, further emphasizes the critical need for innovative approaches to influenza prevention and treatment. The high mortality rates associated with highly pathogenic avian influenza viruses like H5N1, H5N6, and H7N9 underscore the importance of developing strategies that offer cross-protection against diverse subtypes. This study focuses on the identification and characterization of a broadly neutralizing antibody that provides a potential avenue for combating this persistent public health challenge.

Most broadly neutralizing antibodies (bnAbs) against influenza target hemagglutinin (HA), the surface glycoprotein responsible for virus attachment to host cells via its receptor-binding site (RBS). The RBS is located on the globular head (HA1) of HA, mediating binding to sialic acid receptors on the cell surface. Subsequent entry involves pH-dependent fusion mediated by the fusion peptide on the HA stem (HA2). The high antigenic variability of HA, due to antigenic drift, makes developing broadly protective vaccines challenging. Research has focused on identifying bnAbs that target conserved epitopes on either the HA stem or head region. Stem-targeting bnAbs, such as CR6261, exhibit broad protection against influenza A group 1 viruses. Other bnAbs, like CR9114, show cross-reactivity against both influenza A and B viruses. However, most cross-reactive antibodies are mapped to the HA stem region. While head-region bnAbs offer potentially higher potency and immunodominance, their cross-subtype neutralization is often limited. Several studies have described head-targeting bnAbs, including C05, S139/1, and F045-092, which show cross-reactivity against H1, H2, and H3 subtypes. However, bnAbs like CH65, 5J8, and Ab6649, while showing broad neutralizing activity against H1N1, lack cross-subtype reactivity. This research aims to explore a new head-region bnAb with broader cross-reactivity.

The study employed a multifaceted approach involving the generation and characterization of a broadly neutralizing monoclonal antibody (mAb) against H1N1 influenza viruses. Mice were immunized with live influenza A H1N1 viruses (A/Hong Kong/134801/1994, A/New Caledonia/20/1999, and A/Brisbane/59/2007) using standard hybridoma technology. The resulting mAbs were screened for neutralizing activity against a panel of H1N1, H3, and H5 influenza A viruses using hemagglutination inhibition (HAI) and microneutralization (MN) assays. The protective efficacy of the lead mAb, 12H5, was evaluated in a mouse model against lethal challenge with mouse-adapted (MA) H1N1 and H5N1 viruses. A chimeric version of 12H5, C12H5, was constructed by replacing the mouse constant regions with human IgG1 Fc, facilitating further characterization and assessment of its therapeutic potential. The binding affinity and specificity of C12H5 were assessed using ELISA and surface plasmon resonance (SPR). The neutralization mechanism was explored through cell-based entry inhibition and virus egress inhibition assays. Structural analyses of 12H5 Fab in complex with HA proteins from different strains were performed using X-ray crystallography and cryo-electron microscopy (cryo-EM). To identify key residues involved in antibody binding and cross-neutralization, escape mutant selection and site-directed mutagenesis were performed, with the effects of mutations on antibody binding analyzed through ELISA and SPR. Finally, the relevance of the C12H5 epitope to human immune responses was investigated using a blocking ELISA with human sera from H1N1-infected individuals.

A novel broadly neutralizing monoclonal antibody, C12H5, was identified and characterized. C12H5 demonstrated potent neutralizing activity against seasonal and pandemic H1N1 influenza viruses in vitro and in vivo. Notably, C12H5 also exhibited cross-neutralizing activity against multiple H5N1 strains, providing significant protection in a mouse model. Structural studies, including X-ray crystallography and cryo-EM, revealed that C12H5 binds to the receptor-binding site (RBS) of HA, overlapping with the 130-loop and uniquely encompassing the 140-loop. Eight highly conserved residues were identified as critical for C12H5 binding and neutralization: Y98, A137, H141, A142, G143, A144, W153, and D190. Interestingly, the antibody demonstrated tolerance for the Asp/Glu polymorphism at position 190, a key determinant of human or avian host specificity. The neutralization mechanism involves both inhibition of viral entry into host cells and blockage of viral egress. Analysis of human sera from H1N1-infected individuals indicated the presence of antibodies targeting the C12H5 epitope, although the prevalence was relatively moderate compared to control antibodies. Mutagenesis studies further supported the importance of the identified key residues for both H1N1 and H5N1 binding, showing that the Q142, R144, and S145 are crucial for H5N1 binding. Initial immunization experiments with synthetic peptides derived from the C12H5 epitope demonstrated immunogenicity, potentially serving as a starting point for vaccine design.

This study successfully identifies and characterizes a novel cross-neutralizing antibody, C12H5, offering significant implications for influenza prevention and treatment. C12H5’s broad neutralization of H1N1 and cross-neutralization of H5N1 viruses, both in vitro and in vivo, is remarkable. The unique binding to the RBS encompassing both the 130- and 140-loops, alongside tolerance to the D/E polymorphism at position 190, explains its broad activity. The identification of eight key residues, six of which are highly conserved across H1N1 strains, provides a structural basis for its binding and neutralization capacity. This information is highly valuable for rational drug design, targeting small molecules to interact with these key residues. The antibody’s dual mechanism of action – inhibiting both virus entry and egress – suggests its potential efficacy even against viruses with escape mutations. The findings on human serum reactivity to the C12H5 epitope highlight the presence of naturally occurring antibodies with similar specificity, suggesting the potential for eliciting a similar response through vaccination. The demonstration of immunogenicity of the C12H5 epitope using synthetic peptides provides initial evidence to support vaccine development based on this epitope.

The discovery of C12H5, a broadly neutralizing antibody with cross-subtype activity against both H1N1 and H5N1 influenza viruses, provides valuable insights for developing novel antiviral strategies. Its unique binding mode and tolerance for key mutations provide a foundation for rational drug design. Furthermore, the identification of highly conserved epitopes opens promising avenues for the development of broadly protective influenza vaccines, potentially overcoming the limitations of current seasonal vaccines.

The study primarily focuses on H1N1 and H5N1 viruses, limiting the generalization of its findings to other influenza subtypes. While the in vivo studies demonstrated protective efficacy, the long-term protective effects of C12H5 require further investigation. The relatively moderate prevalence of C12H5 epitope-specific antibodies in human sera warrants further investigation to optimize vaccine design for a stronger and broader immune response. Finally, the initial findings with synthetic peptides require further development and validation to confirm their potential as vaccine candidates.