Influenza A virus remains a significant global health concern, with annual infections causing substantial morbidity and mortality. The challenge in developing effective vaccines lies in the virus's ability to undergo antigenic drift, accumulating mutations that render existing vaccines less effective. Annual vaccine efficacy varies considerably, ranging from 10% to 60%, highlighting the urgent need for improved vaccine strategies. The threat is further amplified by the virus's pandemic potential, as evidenced by four major pandemics in the 20th and 21st centuries. These factors underscore the high priority assigned to universal influenza vaccine development by organizations like the National Institute of Allergy and Infectious Diseases (NIAID). One promising avenue for developing broadly protective influenza vaccines is the use of computationally optimized broadly reactive antigens (COBRAs). This approach leverages known sequence information to design composite proteins representing a wide range of viral strains. Sequences from the target antigen are subjected to iterative sequence alignments, generating consensus sequences that incorporate both conserved and divergent features. This method has yielded COBRA hemagglutinin (HA) proteins demonstrating broader protection than wild-type proteins in preclinical models. Notably, these COBRA HAs have shown protective efficacy against viruses that emerged after the design sequences were collected, suggesting resilience to antigenic drift. This study focuses on a particularly effective COBRA HA designated P1, developed using H1N1 sequences from both human and swine origins spanning several decades. P1's effectiveness has been demonstrated in preclinical studies, where it elicits a broad neutralizing antibody response including the broadly reactive antibody 1F8. Despite this promising preclinical data, the precise mechanisms driving P1's broad reactivity remain unclear. This lack of mechanistic understanding motivates the current research, aiming to elucidate the structural features responsible for P1's efficacy through high-resolution structural analysis.

Previous research established the efficacy of computationally optimized broadly reactive antigens (COBRAs) in preclinical influenza vaccine models. Studies demonstrated that COBRA hemagglutinin (HA) proteins, designed using a consensus sequence approach incorporating both conserved and variable regions from multiple influenza strains, elicited broader protective immunity than wild-type HA proteins. In particular, the COBRA HA protein P1, the subject of this study, has shown promise in pre-clinical mouse models, protecting against a broad range of both historical and contemporary H1N1 influenza strains, including pandemic strains. However, despite this success, the underlying structural and antigenic features that drive the broad reactivity of P1 remained unknown. Prior work identified some antigenic features correlated with broad protection, but a comprehensive understanding requires structural insights. The lack of a precise structural understanding of the epitope(s) recognized by broadly neutralizing antibodies elicited by P1, such as 1F8, also necessitates further investigation.

To elucidate the structural basis for the broad reactivity of COBRA HA P1, the researchers employed a combination of X-ray crystallography and cryo-electron microscopy (cryo-EM). Initial attempts to crystallize mammalian-expressed P1, which included a Foldon trimerization domain and affinity tags, proved unsuccessful. To improve crystallization, the P1 gene was cloned into a baculovirus expression vector for expression and secretion in insect cells, resulting in a simpler glycosylation pattern. A thrombin cleavage site was included to facilitate removal of the Foldon domain and tags. The majority of the purified P1 protein remained trimeric, indicating structural integrity even without the Foldon domain. High-quality crystals were obtained from this insect cell-derived P1, leading to a 3.0 Å resolution crystal structure. The crystal structure revealed the overall structure of P1 adopted a classical HA prefusion conformation, with the characteristic α-helices forming the stem and β-sheets in the head domain. A detailed analysis of the antigenic sites and the glycosylation pattern was performed by comparing the P1 structure with previously solved HA structures from strains against which P1 showed variable levels of protective efficacy (CA/04/09 and SI/3/06). The correlation between the similarity of the major head antigenic sites in P1 to those of the strains and the observed immune protection was analyzed. In parallel, cryo-EM studies were conducted to determine the structure of the P1-1F8 antibody complex, using P1 expressed and purified according to a previously established mammalian cell line protocol. The complex formed particles with various orientations, leading to a 3.1 Å resolution 3D reconstruction. This reconstruction revealed two Fab fragments of 1F8 bound to a single P1 trimer, clearly depicting the interaction interface. Biolayer interferometry (BLI) was used to quantify the binding affinity of Fab 1F8 to both wild-type P1 and a mutant with an asparagine substitution (N127D) at a potential glycosylation site. This experiment aimed to determine if glycosylation at this site directly impacted antibody binding. For comparison, structures of other RBS antibodies bound to HA were analyzed to contrast the binding mode of 1F8 to these previously known antibodies.

The X-ray crystal structure of COBRA P1 HA revealed a classical HA fold in the prefusion conformation, confirming the structural integrity of the computationally designed protein. Analysis of the antigenic sites (Sa, Sb, Ca1, Ca2, Cb) revealed a strong correlation between structural similarity to P1 and the observed protective efficacy against different influenza strains. Strains against which P1 was protective (e.g., CA/04/09) showed high similarity in these sites, while strains showing poor protection (e.g., SI/3/06) exhibited significant differences, particularly in charged and hydrophobic residues. The conserved central stem and anchor epitopes also played a role in the broad reactivity, although some minor variations in CA/04/09 may affect antibody binding to the stem. An unusual glycosylation site at Asn127 was identified near the Sa antigenic site. BLI analysis indicated that this glycosylation site did not directly contribute to 1F8 binding but influenced accessibility of the epitope by other antibodies such as CA09-15, suggesting a role in modulating the antibody response. The cryo-EM structure of the P1-1F8 complex revealed that 1F8 targets the receptor-binding site (RBS) and part of the Ca2 antigenic site. The antibody utilizes a unique binding mode, engaging the RBS primarily via its light chain (CDRL1), unlike most previously characterized RBS antibodies which primarily interact with the RBS using heavy chains. This atypical orientation is a key feature that distinguishes 1F8. Comparisons of 1F8 to other RBS-targeting antibodies highlighted its distinct angle of approach. While sharing common molecular features with other RBS antibodies, 1F8 uniquely uses its light chain CDRL1 for primary RBS engagement, while the heavy chain interacts with the Ca2 site.

The findings of this study provide crucial insights into the mechanisms underlying the broad protection afforded by the COBRA P1 vaccine. The strong correlation between structural features of the classical head antigenic sites in P1 and its protective efficacy against specific strains underscores the importance of structural similarity in determining antibody recognition. The identification of the Asn127 glycosylation site as a factor influencing epitope accessibility adds to our understanding of how glycosylation can modulate the immune response. This site's rare occurrence in contemporary strains suggests a possible evolutionary trade-off between immune evasion and receptor binding efficiency. The unique binding mode of antibody 1F8, utilizing its light chain for primary RBS engagement, represents a significant advance in our understanding of broadly neutralizing antibodies. This unexpected mode of binding suggests a novel strategy for achieving broad neutralization, possibly via a combination of conserved RBS interactions and variable Ca2 site interactions. The recent discovery of a similar antibody (12H5), with a different heavy chain but similar light chain usage, strengthens the potential for this new class of antibodies. These structural data support a paradigm shift towards structure-based vaccine design. The detailed insights into the relationships between antigenic features, antibody binding, and broad protection provide a foundation for developing next-generation influenza vaccines.

This research successfully elucidated the structural basis of the broad protective immunity elicited by the computationally designed influenza HA vaccine antigen P1. The crystal structure of P1 and the cryo-EM structure of the P1-1F8 complex reveal critical antigenic and glycosylation features that contribute to its immunogenicity. Notably, the antibody 1F8 uses a unique light chain-mediated RBS interaction not previously observed, providing new avenues for broadly neutralizing antibody design. Future work should focus on evaluating the efficacy of P1 in human clinical trials and leveraging these structural insights to design even more effective universal influenza vaccines.

The study's findings are primarily based on preclinical data from mouse models. While these models provide valuable insights, the translational relevance to human populations requires further investigation. The study's focus on a single broadly neutralizing antibody (1F8) may not fully capture the complexity of the polyclonal antibody response elicited by P1 in vivo. A more comprehensive analysis of the entire antibody repertoire would provide a more complete picture.