Here, we engineered BOAS that included tandemly linked, antigenically distinct HA heads as a single construct. This platform allows a mixing-and-matching of up to eight distinct HA heads from both influenza A and B viruses. Furthermore, we showed that the order and number of HA heads can vary without losing reactivity to conformation-specific mAbs in vitro, highlighting the flexibility of this platform. Mice immunized with BOAS had comparable serum reactivity to each individual component though relative binding and neutralization titers varied between immunogens; this is likely a consequence of length and/or composition. Further oligomerization for increased multivalent display was accomplished by conjugating two 4mer BOAS inclusive of eight distinct HA heads to a ferritin nanoparticle via SpyTag/SpyCatcher ligation. Similar to the BOAS, these conjugated nanoparticles elicited similar titers to all eight HA components and could neutralize matched viruses.
Thus, tandemly linking HA heads is a robust method for displaying multiple influenza HA subtypes in a single protein-based immunogen. Binding titers were elicited to all components present in the immunogen, and there was no significant correlation between HA position within the BOAS (i.e. internal or terminal) and immunogenicity. However, the relative immunogenicity of each HA varied despite equimolar display of each HA subtype. There were qualitatively immunodominant HAs, notably H4 and H9, and these were relatively consistent across BOAS in which they were a component; this effect was reduced in the mix cohort. Further studies using the modularity of the BOAS could further deconvolute relative immunodominances of HA subtypes.
Despite similar binding titers across multiple BOAS lengths, expression levels and neutralization titers were quite variable. While all 3mer to 8mer BOAS could be overexpressed, expression inversely correlated with overall length. To mitigate this, multiple BOAS (e.g. two 4mers) or conjugation to protein-based nanoparticles, as was done here, could be used to ensure coverage of each desired HA subtype. Furthermore, neutralization titers were quite variable across different BOAS lengths despite similar binding titers. This may be related to multiple factors, including homology, stability, and accessibility of neutralizing epitopes for different BOAS lengths. Notably, for longer BOAS, we observed degradation following longer term storage at 4° C, which may reflect their overall stability. Studies manipulating BOAS composition at intermediate lengths could optimize neutralizing responses to particular influenzas of interest.
Based on the immunogenicity of the various BOAS and their ability to elicit neutralizing responses, it may not be necessary to maximize the number of HA heads into a single immunogen. Indeed, it qualitatively appears that the intermediate 4-, 5-, and 6mer BOAS were the most immunogenic and this length may be sufficient to effectively engage and crosslink B cell receptors (BCRs) for potent stimulation. These BOAS also had similar or improved binding cross-reactivity to mismatched HAs as compared to longer 7- or 8mer BOAS. Notably, the 3mer BOAS elicited detectable cross-reactive binding titers to H4 and H5 mismatched HAs. This observed cross-reactivity could be due to sequence conservation between the HAs, as H3 and H4 share ~51% sequence identity, and H1 and H2 share ~46% and~62% overall sequence identity with H5, respectively (Figure 4—figure supplement 1). Additionally, the degree of surface conservation decreased considerably beyond the 5mer as more antigenically distinct HAs were added to the BOAS. These data suggest that both antigenic distance between HA components and BOAS length play a key role in eliciting cross-reactive antibody responses, and further studies are necessary to optimize BOAS valency and antigenic distance for a desired humoral response.
Potential enrichment of serum antibodies targeting the conserved RBS and TI epitopes may also be contributing to observed cross-reactivity. Both epitopes are relatively conserved across all BOAS (Figure 4C), and the two BOAS showing the most cross-reactivity, the 3mer and 5mer, elicit a significant portion of the serum response toward both RBS and TI epitopes as determined via a serum competition assay with available epitope-directed mAbs (Figure 4B). Notably, this proportion is approximate, as at the time of reporting, mAbs that bind the receptor binding site of all components were not available. RBS-directed mAbs to the H4 and H9 components were not available, and the RBS-directed antibodies used targeting the other HA components have different footprints around the periphery of the RBS. Additionally, there are currently no reported influenza B TI-directed mAbs in the literature. Therefore, this may be an underestimate of the serum proportion focused on the conserved RBS and TI epitopes. Isolated TI-directed mAbs, in particular, can engage more than nine unique subtypes across both group 1 and 2 influenzas (McCarthy et al., 2021; Watanabe et al., 2019), and our monomeric head-based BOAS immunogens have the otherwise occluded TI epitope exposed (McCarthy et al., 2021; Bangaru et al., 2019; Watanabe et al., 2019). Furthermore, we have previously shown that this TI epitope, when exposed, is immunodominant in the murine model (Bajic et al., 2019). Further studies with different combinations of HAs could aid in understanding how length and composition influences epitope focusing. For example, a BOAS design with a cluster of group 1 HAs followed by a cluster of group 2 HAs, rather than our roughly alternating pattern could influence which HAs are in close proximity to one other or could be potentially shielded in certain conformations and thus could affect antigenicity. Combining the BOAS platform with other immune-focusing approaches (Dosey et al., 2023), such as hyperglycosylation (Bajic et al., 2019; Thornlow et al., 2021; Ingale et al., 2014; Eggink et al., 2014) or resurfacing (Bajic et al., 2020; Hai et al., 2012) could enhance cross-reactive responses. Additionally, modifying linker spacing and rigidity can also be used as a mechanism to enhance BCR cross-linking and thus enhance cross-reactive B cell activation and elicitation (Veneziano et al., 2020).
BOAS can be further multimerized via conjugation to a surface of a NP. Interestingly, this only had a marginal effect on immunogenicity. The BOAS NP elicited titers of ~105 (Figure 6B), whereas the best BOAS alone reached an order of magnitude greater (Figure 3C). This appears in contrast with other studies where attaching an antigen to a NP scaffold enhanced immunogenicity and neutralization potency (Kanekiyo et al., 2013; Jardine et al., 2013; Tokatlian et al., 2019; Kato et al., 2020; Marcandalli et al., 2019). One recent example designed quartets of antigenically distinct SARS-like betacoronavirus receptor binding domains (RBDs) coupled to an mi3-VLP scaffold via a similar SpyTag-SpyCatcher system and showed increased binding and neutralization titers following conjugation to the NP compared to quartet alone (Hills et al., 2024). This discrepancy may be in part due to the larger mi3 NP (Bruun et al., 2018) which displays 60 copies of the antigen rather than the 24 copies displayed on the ferritin NP used in this study. It is also possible that the difference in immunogenicity could arise from the increased molecular weight of the BOAS NP immunogen compared to the BOAS alone, leading to a difference in moles of BOAS antigen in each cohort. However, due to the large size of the BOAS, the addition of the ferritin NP does not add a large amount of mass. 20 µg of BOAS NP or an 8mer BOAS equates to ~64 and ~83 µmoles of each HA component, respectively. This ~30% greater amount of HA in the 8mer BOAS, however, does not account for the observed difference in serum binding titers. Nevertheless, HA-specific responses were similar whether the BOAS were conjugated to the nanoparticle or not, indicating that HA proximity to the NP surface did not impact responses to each component. This observation is consistent with betacoronavirus quartet NPs as well. Additionally, BOAS conjugation to the NP significantly reduced the scaffold-directed response. The addition of the large BOAS projections to the NP surface likely masked the immunogenic scaffold epitopes (Kraft et al., 2022).
Collectively, this study demonstrates the versatility of the BOAS platform to present multiple HA subtypes as a single immunogen. This ‘plug-and-play’ approach can readily exchange HAs to elicit desired immune responses. BOAS are potentially advantageous over other multivalent display platforms, such as protein-based NPs, which can produce off-target responses due to their inherent immunogenicity (Kanekiyo et al., 2013). Furthermore, when genetic fusions of the antigen to nanoparticles is not possible, SpyTag-SpyCatcher (or another suitable conjugation approach) must be used, further contributing to scaffold-specific responses as well as additional multi-step manufacturing and purification challenges. Not only does our BOAS platform circumvent these potential caveats, but because this is a single polypeptide chain, this immunogen could readily be formulated as an mRNA lipid nanoparticle (LNP) (Chaudhary et al., 2021). The BOAS platform forms the basis for next-generation influenza vaccines and can more broadly be readily adapted to other viral antigens.