Neuraminidase-on-a-string nanoparticles probe how antigenic distance shapes elicited humoral immunity

Neuraminidase-on-a-string nanoparticles probe how antigenic distance shapes elicited humoral immunity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Neuraminidase-on-a-string nanoparticles probe how antigenic distance shapes elicited humoral immunity Rochel Hecht, Faez Amokrane Nait Mohamed, Daniel Lingwood, Aaron Schmidt This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7530142/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Understanding how antigenic distance influences cross-reactive responses can inform vaccine design. Multivalent displays of viral proteins can improve B cell activation due to receptor cross-linking, and mosaic nanoparticles that incorporate variants can lead to cross-reactive B cell responses recognizing conserved epitopes. Here, we used the influenza virus neuraminidase to develop a neuraminidase-on-a-string platform displaying neuraminidase dimer pairs conjugated to a nanocarrier To systematically assess the influence of antigenic distance on humoral immunity, we paired H2N2 neuraminidase with either divergent H3N2 or H11N9 neuraminidases. We found that nanoparticle immunizations with heterologous antigens elicited sera with greater breadth and enhanced enzymatic inhibition relative to immunizations that incorporated a single neuraminidase strain. While sera reactivity for H2N2 neuraminidase was not impacted by inclusion of a second strain, strain-specific responses correlatively increased with the antigenic distance between neuraminidase components. These data show how neuraminidase strain selection for multivalent display immunizations influences elicited breadth and cross-reactivity, highlighting findings that may extend to other viral antigens. Biological sciences/Biochemistry Biological sciences/Biotechnology Biological sciences/Immunology protein engineering neuraminidase nanoparticle immunogens antigenic distance breadth next-generation vaccines Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Humoral responses directed at surface-exposed viral proteins ( e.g. , coronavirus spike and influenza hemagglutinin) drive viral evolution and escape 1 – 3 . However, viruses cannot readily mutate conserved regions without impairing viral fitness 4 . A goal of rational immunogen design strategies, such as hyperglycosylation, epitope grafting and multivalent display aim to focus humoral immunity toward conserved epitopes to limit viral escape 5 – 10 . Multivalent immunogen displays increase B cell receptor crosslinking and subsequent activation 15 . Indeed, in vivo studies comparing monomeric versus multimeric antigens show increased antibody titers, B cell activation, and T cell engagement with multimerization 16 – 24 . Mechanistically, in vivo studies also showed that restriction to germinal center (GC) entry imposed by B cell affinity and precursor frequency thresholds are mitigated through increased avidity on an immunogen 25 . The lowered thresholds for GC entry may, in turn, increase B cell clonal diversity and breadth 22 . The size of multivalent immunogens is important, as larger particles induce slower trafficking within the lymph node 20 , 22 , 23 , leading to a prolonged antigen exposure and duration of GCs 18 . Thus, multivalent display, including viral-like particles 26 , self-assembling protein nanoparticles 27 , and DNA-origami 15 , are promising platforms for next-generation viral vaccines 20 . Mosaic displays of distinct viral protein variants on the same nanoparticle (NP) enhanced breadth and neutralization relative to NP displays of a single protein 28 – 30 . This is likely due to a selective advantage for B cell receptors that engage conserved epitopes shared between the displayed antigens relative to the receptors that engage less conserved regions. However, further studies are needed to clarify the mechanisms involved, such as whether a minimum level of diversity is required to promote this advantage and whether significant variation between antigens might elicit strain-specific responses at the expense of cross-reactive responses. For example, mosaic hemagglutinin NPs showed increased proportions of cross-reactive B cells using mosaic displays only when including 6 or more strains on the same NP 29 . Additionally, whether mosaic displays stimulate cross-reactivity better than a homologous “cocktail” of the same strains has yet to be clarified. Indeed, studies incorporating antigenically equivalent cocktail versus mosaic immunogens have indicated mixed outcomes in terms of benefits 29 – 33 . Moreover, for both mosaic and homologous cocktail experiments, data indicate that different combinations of 2 or 4 heterologous strains resulted in different sera cross-reactivity profiles 28 , 34 . Here, we addressed the potential mechanism(s) underlying these observations, and to understand how antigenic distance between displayed proteins on a NPs influenced elicited sera breadth and cross-reactivity. We chose the influenza neuraminidase (NA) as our prototypic antigen. NA catalyzes the cleavage of terminal sialic acids, and its function is essential for influenza viral egress and spread 35 – 37 . There is growing interest in NA as a vaccine target 38 – 41 , as antibodies targeting NA can be broadly reactive and protective against influenza infection 42 – 44 . Thus, inclusion of an NA component in next-generation influenza vaccines may provide an independent arm of immune protection against influenza, contributing to seasonal vaccine effectiveness 45 , 46 . Briefly, we engineered a series of dimeric NA head constructs tandemly linked by a rigid peptide to create neuraminidase-on-a-string (NoaS) immunogens, which were then conjugated to ferritin NPs via SpyTag/SpyCatcher 47 . Each dimer contained the NA from H2N2 Japan 1957, but the second NA component was a distinct strain, which varied the antigenic distance up to ~ 50% between the NA pairs. Homologous dimers of each NA were used as controls. The dimeric design enabled consistent molar ratios and spatial orientations of the NA components. Mice were immunized with either heterologous ( i.e. , mosaic) NoaS NPs or an equivalent equimolar homologous cocktail of NoaS NPs. Serum samples were collected before and after boosting and evaluated for anti-NA breadth, strain-specificity, and NA enzymatic inhibition. We found that including a second NA strain in immunizations enhanced both anti-NA breadth and NA enzymatic inhibition. Notably, the proportion of strain-specific antibodies for each NA component increased with the antigenic distance of the NoaS pairing, even as overall anti-NA reactivity remained comparable. These findings guide next-generation viral vaccine development and underscore the importance of strain selection in multivalent vaccine platforms designed to maximize elicited cross-reactive immune responses. RESULTS Design of neuraminidase-on-a-string nanoparticles We selected four influenza neuraminidase (NA) strains to be incorporated in our immunogens: H2N2 A/Japan/305/1957 (J’57), H3N2 A/Bilthoven/21438/1971 (B’71), H3N2 A/Darwin/6/2021 (D’21), and H11N9 A/tern/Australia/G70C/1975 (Au’75). We chose these strains primarily based on their amino acid diversity relative to the J’57 NA head: B’71, D’21, and Au’75 differed from J’57 by ~ 8%, ~ 17%, and ~ 50%, respectively (Fig. 1 A,B and fig. S1 C ). Additionally, the H2N2 as well as both H3N2s previously circulated within the human population, while H11N9 represents a potentially zoonotic influenza that is within the avian population. To understand how antigenic distance between NAs on a multivalent nanoparticle (NP) immunogen influenced sera cross-reactivity and breadth, we designed neuraminidase-on-a-string (NoaS) NPs. We tandemly linked pairs of monomeric NA heads together using a rigid, proline-rich L3 amino acid peptide linker 48 to make NoaS and subsequently conjugated them to the Helicobacter Pylori ferritin NP 49 using SpyTag/SpyCatcher 47 (Fig. 1 B, C and fig. S1 A-B ). We designed homodimeric NoaS NPs of each of these four strains (groups 1, 2, 4, and 6; Fig. 1 C) and made heterodimeric NoaS NPs by pairing J’57 with either B’71, D’21, or Au’75 (groups 3, 5, and 7; Fig. 1 C). Thus, using ‘percent amino acid difference’ as a proxy for antigenic distance, we varied the presented antigenic distance on the NoaS NPs from 0% up to ~ 50%. Each NoaS NP displayed a total of 48 copies of the NA head, with one NoaS dimer per ferritin protomer. In the heterologous NoaS, the J’57 NA strain was conjugated adjacent to the ferritin protomer with the second NA component projecting outwardly (Fig. 1 C). Biochemical characterization of NoaS NPs NoaS and NoaS NPs were recombinantly expressed from mammalian cells and purified with affinity and size exclusion chromatography (SEC). The C-terminal affinity tags were removed by enzymatic cleavage and re-purified by SEC. The NoaS, in molar excess, were then conjugated to the NPs and purified again over SEC. The resulting NoaS NPs were monodisperse (Fig. 2 A and fig. S2B ) and homogeneous as assayed by SDS-PAGE analysis (Fig. 2 B). All NoaS NPs were tested for reactivity to conformation-specific monoclonal antibodies (mAbs) NA73 50 , 3A10 51 , 1G01 42 , and NDS.1 52 to assess structural integrity of the NA components (Fig. 2 C and fig. S2A ). NoaS dimers and NoaS NPs had comparable reactivity to the diagnostic mAbs, indicating that each NA component was in its native conformation and accessible after conjugation to the ferritin NP. NoaS NPs were visualized using negative stain electron microscopy and showed uniform NoaS NP sizes of ~ 60nm, corresponding well to the approximate additive size of all components (Fig. 2 D). The L3 linker 48 spaced each of the ~ 5nm-wide NA heads about 10nm apart ( fig. S2C ). This approximate distance between the two NA heads is optimal to engage both antibody arms of a B cell receptor (~ 13nm distance) 53 . Group design and vaccine regimen Seven groups of C57BL/6 mice (n = 5 per group) were immunized using a homologous prime-boost regimen (Fig. 3 ). Mice received 100µL of inoculum intraperitoneally, containing 20µg of total protein adjuvanted with 50% w/v Sigma adjuvant. Group 1, which received J’57/J’57 NoaS NPs, served as the control group. Groups 3, 5, and 7 received heterologous NoaS NPs displaying J’57 with either B’71, D’21, or Au’75, respectively. Groups 2, 4, and 6 received a cocktail of homologous J’57/J’57 NPs with either B’71/B’71, D'21/D’21, or Au’75/Au’75 NPs, respectively. The cocktails were prepared in equimolar ratios to their heterologous counterparts. Mice were bled weekly, and the sera were collected 14–27 days post-prime and 41–62 days post-prime ( i.e. , 14–35 days post-boost) to be evaluated for NA breadth, NA strain-specificity, and NA enzymatic inhibition. Assessment of elicited sera breadth in NoaS NP groups A panel of six historical N2 heads (A/Japan/305/1957 (J’57), A/Bilthoven/21438/1971 (B’71), A/Aichi/2/1968 (Ai’68), A/Moscow/10/1999 (M’99), A/Texas/50/2012 (Tx’12), and A/Darwin/6/2021 (D’21)) and one H11N9 (A/tern/Australia/G70C/1975 (Au’75)), which included the four NA strains incorporated into the immunogens, were recombinantly expressed as monomers. The amino acid differences between the NA strains in the panel varied from ~ 4.4–53.5% ( fig. S3A ) and the number of predicted-N-linked glycosylation sites in the panel ranged from 3–7 ( fig. S3B ). Sera from d20 and d48 were tested for reactivity to the entire NA panel in ELISA. Absorbance values were normalized per antigen, and the degree of normalized area under the curve (nAUC) was plotted for each mouse serum sample ( figs. S4A-B and S5A-B ). Except for the reactivity of group 4 versus group 5 to the Tx’12 antigen on d20 ( fig. S6B ), there were no statistically significant differences in the observed reactivity to the NA panel when comparing the homologous cocktail groups ( i.e. , groups 2, 4, and 6) to their heterologous NoaS counterparts ( i.e. , groups 3, 5, and 7) ( fig. S6A-C ). This suggested that any effect of heterologous strain presentation versus homologous cocktail presentation was likely too small to be observed with our n = 5 group size. We therefore combined the data from groups 2 and 3, groups 4 and 5, and groups 6 and 7, respectively, to probe how the antigenic distance within a two-strain immunization influenced elicited breadth. (Note: In this study, serum breadth refers to the overall extent of unique NA strains recognized by the serum in our NA panel. Serum cross-reactivity describes when individual antibodies within the serum recognize more than one antigenically distinct NA strain. Serum breadth can result either from cross-reactive serum antibodies ( i.e. , the same serum antibodies binding multiple NA strains) or from a more diverse serum antibody pool ( i.e. , different serum antibodies recognizing different NA strains). Thus, while serum that recognizes distinct NA strains has greater breadth than serum recognizing only one strain, this does not necessarily imply that it contains more cross-reactive serum antibodies.) We found that all groups had comparable reactivity to the J’57 antigen (Fig. 4 A), which indicated that J’57 immunogenicity was not affected by the inclusion of a second NA strain. Additionally, all experimental groups had greater anti-NA breadth than the control group, implying that inclusion of a second strain increased the elicitation of NA breadth (Fig. 4 B-G). On d20, J’57/D’21 groups had the broadest reactivity across the tested N2 NA panel (Fig. 4 C-F). On d48, although the groups receiving J’57/D’21 still showed broad N2 NA reactivity, the groups receiving J’57/B’71 developed statistically significant reactivity to earlier N2 NA strains ( i.e. , Ai’68 and B’71; Fig. 4 B-C). These data suggested that the inclusion of two antigenically similar NA strains ( i.e. , J’57 and B’71) could enhance the reactivity to each other as well as to strains antigenically like each other ( i.e. , Ai’68). Finally, the groups receiving J’57/Au’75 had N2 NA breadth that was comparable to the control group across the N2 panel but also had reactivity to the N9 strain included in the NoaS (Fig. 4 A-G ) . These data suggested that the antigenic distance between J’57 and Au’75 was too great to elicit broader N2 reactivity. Nonetheless, including the N9 NA allowed strain-specific recognition of each component. To assess whether serum antibodies from the groups acquired breadth by focusing on the conserved NA catalytic site (CS), we tested sera reactivity to a modified J’57 NA with a glycan projecting into the CS. This ‘ΔCS’ J’57 version abrogated binding to all CS-directed mAbs assayed ( fig. S7A ). By comparing the sera reactivity to the wildtype J’57 versus ΔCS J’57 antigens, we could compute the percent loss of signal due to the glycan and assess how much of the sera was directed at or near the CS. We did not observe any differences in ΔCS J’57 reactivity in the cocktail versus heterologous groups, so we analyzed groups based on the pair of NA strains received in the immunizations ( fig. S7B-C ). On both d20 and d48, there were statistically comparable reactivities to the ΔCS J’57 antigen across all paired groups ( fig. S7D ). On d20, reduced reactivity to the ΔCS J’57 antigen compared to the wildtype J’57 antigen was significant for the J’57/J’57, J’57/B’71, and J’57/Au’75 groups. By d48, the reduced reactivity was significant for all groups except J’57/J’57, however this group trended similarly ( fig. S7E ). These results suggest that, across groups, the inclusion of a second, antigenically distinct NA strain contributed to focusing the sera responses on the CS, which is conserved across NA strains. Lastly, scaffold-specific sera responses to the ferritin NP were assayed on d27 and d62 ( fig. S8A-C ). On d27, the NP reactivity was similar across groups. However, on d62, the J’57/B’71 and J’57/Au’75 had enhanced reactivity toward the naked NP relative to the J’57/J’57 control group ( fig. S8C ). Strain-specificity of elicited sera responses We developed a serum depletion assay to evaluate whether NA pairs elicited strain-specific or cross-reactive antibodies ( fig. S9-19 ). Each serum sample was first tested by ELISA for reactivity against both NA components used in each immunization. For example, serum from mouse 30 (m30) in group 6 (which received both J’57 and Au’75 NAs) was first assayed for binding to J’57 and Au’75 NAs ( fig. S12A ). The m30 serum was then incubated over a column containing J’57 NA, thereby depleting the sample of J’57-reactive antibodies. The J’57-depleted serum was subsequently tested for reactivity to both J’57 NA ( fig. S12B ) and Au’75 NA ( fig. S12C ) by ELISA. The absence of any remaining J’57 NA reactivity confirmed successful depletion, and the remaining Au’75 NA reactivity was compared to the pre-depletion level. The ratio of Au’75 NA reactivity post- vs. pre-J’57 depletion reflected the degree of strain-specificity for Au’75 ( fig. S13E ). Importantly, this assay quantified how much of the serum antibody response to a given NA was specific to that NA. It did not directly measure the percentage of antibodies that were cross-reactive. For instance, a strain-specificity value of ~ 79% for the Au’75 NA in m30 did not imply that ~ 21% of total serum antibodies were cross-reactive. Rather, it meant that of the antibodies binding Au’75 NA, ~ 21% also recognized J’57 NA. In this way, the assay measured how much of the response to strain A was unique to strain A, and it only indirectly reflected cross-reactivity with strain B. Using this approach and analysis, sera-depletion assays were performed on each serum sample collected on d13 and d55. The homologous cocktail groups 2, 4, and 6 had similar strain-specificity profiles for their counterpart heterologous groups 3, 5, and 7 on both d13 and d55 ( fig. S20A-B ). We therefore analyzed data from groups 2 and 3, 4 and 5, and 6 and 7 as combined cohorts, respectively. The J’57/J’57 group acquired some breadth for B’71, D’21, and Au’75 NAs ( fig. S22A-B ). However, after sera depletion for J’57-reactive antibodies, the remaining sera no longer recognized B’71, D’21, and Au’75 NAs ( figs. S13 and S18 ). Thus, we conclude that none of the serum antibodies elicited in the J’57/J’57 group were strain-specific for B’71, D’21, or Au’75 NAs. The remaining groups were depleted for both J’57 NA reactivity and the second NA component in their immunogens ( figs. S9-S18 ). On d13, all groups had comparable reactivity to J’57 NA (Fig. 5 A), and most groups had comparable reactivity to J’57 NA on d55. Nonetheless, the degree of strain-specificity for J’57 NA increased in the groups in correlation with the antigenic distance of their second NA component (Fig. 5 B). This same phenomenon was observed for the NA strain that J’57 was paired with (Fig. 5 C). Thus, groups that received J’57/B’71 had substantially less strain-specificity toward the J’57 NA than did the groups that received J’57/Au’75. Similarly, the calculated strain-specificities toward B’71 NA in groups 2 and 3 were substantially lower than the calculated strain-specificities toward Au’75 NA in groups 6 and 7. Thus, pairing the J’57 NA with an NA strain that had increased antigenic distance elicited a greater proportion of serum antibodies specific to each NA component. When comparing the strain-specificity on d55 versus d13, all groups showed a decrease in the magnitude of specificity for both the J’57 NA ( fig. S21A ) and their paired NA component ( fig. S21B ), with varying statistical significance across groups. Sera inhibition of NA enzymatic activity We next assessed whether the elicited sera from the NoaS NPs inhibited NA enzymatic activity using the enzyme-linked lectin assay (ELLA), which uses a large fetuin substrate 55 . Antibodies that bind directly to the CS block enzymatic activity even with small substrates, but antibodies that bind proximal to the CS or on the NA periphery can sterically obstruct the CS. The ELLA inhibition assay is therefore considered more reflective of the broader potential to inhibit NA activity over other assays that use smaller substrates , 42 , 52 , such as NA-Star™ or MUNANA 56 . We assayed d20 and d62 sera for enzymatic inhibition of the NA strains used in each immunization ( figs. S23-S24 ). We first determined the EC 50 concentration for a given NA strain and then incubated two times the EC 50 with two times the serial serum dilution and measured the resulting NA activity. Using d0 serum as a negative control, we identified the dilution factor at which a given serum sample inhibited NA enzymatic activity more than the highest concentration of d0 serum ( i.e. , the maximum serum dilution factor with inhibitory activity, or MDFI). Homologous cocktail groups 2, 4, and 6 showed similar inhibition profiles to their counterpart heterologous groups 3, 5, and 7, respectively ( fig. S25A-B ). Groups were therefore evaluated based on immunizing strains. All groups had comparable enzymatic inhibition of J’57 NA activity on both d20 and d62, indicating that the inclusion of a second NA strain did not reduce serum antibodies with J’57 CS-inhibiting activity (Fig. 6 A and figs. S23A and S24A ). We then tested B’71 NA enzymatic inhibition in groups 2 and 3, D’21 NA enzymatic inhibition in groups 4 and 5, and Au’75 NA enzymatic inhibition in groups 6 and 7 ( figs. S23B-D and S24B-D ). Each NA strain was also tested against sera from the control, group 1. Sera from d62 showed substantially stronger inhibition of B’71 in groups 2 and 3 compared to the control group (Fig. 6 B). Surprisingly, at both time points, despite the greater antigenic distance between D’21 and J’57 NAs than the B’71 and J’57 NAs, inhibition of D’21 NA activity was not significantly improved in groups 4 and 5 sera compared to the control group sera (Fig. 6 B). Lastly, sera from groups 6 and 7 inhibited the Au’75 NA activity substantially better than sera from the control group (Fig. 6 B). These data show that including a second NA component in NoaS NPs with J’57 NA does not detract from J’57 NA activity inhibition and, moreover, better ensures the production of antibodies with inhibitory potential to a second NA. DISCUSSION Here, we engineered a multivalent, modular neuraminidase-on-a-string (NoaS) nanoparticle (NP) platform to assess the effects of varying neuraminidase (NA) antigenic distance on the elicited humoral response. Sera reactivity to the NA panel yielded two key observations. First, J’57 NA immunogenicity was not reduced when a second NA strain was included in the NoaS NPs, regardless of the antigenic distance relative to J’57 NA. Second, anti-NA breadth is augmented when a second, antigenically distinct NA strain is included. Additionally, the proportion of strain-specific serum antibodies increases with the antigenic distance between the NA components, and that this relationship remains consistent after boosting. We also found that inhibition of J’57 NA enzymatic activity by sera was not impacted by including a second NA strain in NoaS NPs; this observation may correlate with protection. Interrogating scaffold-specific responses is necessary to ensure that next-generation vaccine platforms do not abrogate desired immune responses to the viral immunogens 57 . Since each NoaS dimers were approximately similar in size across experimental groups, we anticipated that the NoaS dimers would sterically occlude access to the ferritin surface equivalently and thus have comparable scaffold-specific responses. While this was generally observed, at d62 ( i.e. , 35 days post-boost) the J’57/B’71 and J’57/Au’75 experimental groups elicited stronger ferritin-specific responses relative to the J’57/J’57 control group ( fig. S8C ). It is unclear why these NA pairings resulted in a more robust scaffold response however, these experimental groups also had stronger reactivity to both NA components on d20 and d48 (Fig. 4 A, C, and G ). Importantly, the observed scaffold-specific responses did not appear to reduce sera responses to the NA components. Thus, using the ferritin NP to increase multivalency is a possible platform for NA-based vaccines. Incorporating diverse strains of the same viral protein into a vaccine can induce humoral responses to each individual strain 12 , 13 . This strategy of enhancing breadth through multi-strain immunization is supported by numerous studies 28 – 31 . Therefore, our finding that NoaS NPs elicited serum responses to each included NA component is consistent with prior observations. Notably, the combination of J’57 and D’21 NAs elicited broad reactivity to a panel of historical N2 NAs spanning > 60 years of antigenic distance. The observed reactivity may be due to cross-reactive serum antibodies recognizing both J’57 and D’21 NAs, or from distinct serum antibody populations targeting each NA strain. Data from our serum-depletion assay supported the latter: we observed that serum strain-specificity increased with antigenic distance between paired NAs on the NoaS NPs, suggesting that the J’57/D’21 group achieved breadth primarily through separately affinity-matured serum antibody pools. While some cross-reactive serum antibodies may have been elicited, they appeared to represent a minority of the response, particularly in the J’57/D’21 and J’57/Au’75 groups. In general, we did not find that the mosaic displays of the heterologous strains resulted in statistically significant greater NA breadth, cross-reactivity, or activity inhibition than their homologous cocktail counterparts. Previous studies also showed “cocktails” of individual components were generated comparable 32 or even greater 30 cross-reactivity within the sera than the mosaic displays. Considering our data and these discrepant studies, we suspect that it is possible (if not likely) that the benefits of enriching for conserved epitopes through the display of heterologous strains on the same NP is best stimulated using a greater number of strains while still covering a well-constrained antigenic distance 29 . While the heterologous NP groups in our study had comparable or qualitatively greater sera cross-reactivity than did the homologous cocktail groups, statistical significance might require a greater number of incorporated NA strains ( e.g. , > 2). We also observed that all groups had decreased strain-specificity at d55 ( i.e. , 28 days post-boost) compared to d13 ( fig. S21A-B ). This observation may be due to prolonged duration of somatic hypermutation 58 , 59 , where antigen-reactive B cells in germinal centers generated increased cross-reactivity over time through modulating affinity for the strains incorporated in the immunization 60 – 62 . Previous studies showed that NA immunizations protected against viral challenge better when the NA strain was matched rather than heterologous 63 – 65 . Our NA enzymatic inhibition data used as a proxy for protection are consistent with these observations: experimental groups that received B’71 or Au’75 NA strains outperformed the control J’57/J’57 group when testing for NA enzymatic inhibition against B’71 or Au’75 NAs, respectively. However, the results from groups receiving the D’21 NA, which did not inhibit D’21 NA enzymatic activity better than the control J’57/J’57 group, highlights a greater nuance. Structural analysis of D’21 and J’57 NAs suggest that it may be due to the unique glycosylation patterns on D’21 NA. D’21 NA has three different and two additional predicted N-linked glycosylation sites (PNGs) compared to the J’57 NA. In particular, the N245 and N367 PNGs are adjacent to the CS and may have therefore affected the CS-directed antibody responses to D’21 NA. Indeed, some previously characterized broadly reactive CS-directed antibodies have reduced binding to NA strains with the N245 glycan 66 . Our observations suggest that eliciting sera antibodies against specific epitopes across diverse NA strains might require additional design strategies for epitope-focusing. Rational design strategies like hyperglycosylation and epitope grafting are compatible with these multivalent NoaS NP designs and may be used r to improve humoral focusing to desired a desired NA epitope(s) 8 , 10 . The immune response to homologous boosts is often dominated by antibodies generated during the prime immunization 67 , and early influenza exposures can imprint long-lasting immune biases 68 . Therefore, we specifically evaluated the NoaS NPs in an immunologically naïve cohort to understand how to elicit breadth in the context of primary imprinting. Understanding how complex immune histories, whether initiated through vaccination or infection, influence subsequent responses to the NoaS NPs is necessary. Additionally, understanding how NA antigenic distance influences single B cell responses or memory B cell formation may guide vaccine design. Nevertheless, these data show how rationally designed NoaS NP can advance our understanding of how antigenic distance influences elicited humoral immunity and lays a foundation for next-generation influenza vaccines that incorporate NA strains for improved protection against seasonal and potentially pre-pandemic influenzas. MATERIALS AND METHODS Strain Selections The four NA strains used in the Neuraminidase-on-a-String (NoaS) nanoparticles (NPs) were A/Japan/305/1957, A/Bilthoven/21438/1971 (H3N2), A/Darwin/6/2021 (H3N2), and A/tern/Australia/G70C/1975 (H11N9). Strains were selected by performing amino acid alignments of many NA heads and finding strains that together represented a rational distribution of antigenic distances. Strains were then tested for expression as both monomers and paired NoaS dimers. In total, 20 different strains of NA and many neuraminidase-on-a-string combinations were tested. Final selections for the NoaS were based on ease of expression, distribution of antigenic distance, and structural integrity. Cloning of NoaS NPs The NoaS plasmids were engineered using type IIS cloning 69 (with a SAPI enzyme) and cloned into pVRc8400 protein expression vectors. The plasmids coded for the following: t-PA signal sequence, Histidine tag (for affinity purification), HRV 3C protease site (to cleave off the HIS tag), SpyTag sequence 47 , first NA head, L3 rigid amino acid linker 48 , and, finally, the second NA head (Fig. B.1B). The Helicobacter Pylori ferritin nanoparticle 49 plasmid coded for an N-terminus Histidine affinity tag, SpyCatcher domain ( fig. S1 A ), and ferritin protomer ( fig. S1 A ). All nucleotide sequences were codon optimized and synthesized by IDT. The monomeric versions of these four strains of NA as well as the NA heads of A/Aichi/2/1968 (H3N2), A/Moscow/10/1999 (H3N2), and A/Texas/50/2012 (H3N2) used in the ELISA breadth assay panel, were also codon-optimized and cloned into pVRc vectors. Expression and purification of proteins SpyTag NoaS dimers and SpyCatcher NP plasmids were transiently expressed with Expi293F cells (ThermoFisher). After 5–7 days, the cell culture supernatants were harvested by centrifugation. Proteins were purified by affinity chromatography using Cobalt TALON resin (TAKARA) followed by size exclusion chromatography on a Superdex 200 Increase 10/300 GL column (GE Healthcare). The NoaS Dimers were then cleaved using HRV 3C protease (ThermoFisher Pierce) with an overnight incubation at 4ºC. They were eluted over Cobalt TALON resin to remove Histidine tags and HRV 3C protease, repurified over a Superdex 200, and then conjugated in 40x molar excess to previously purified SpyCatcher NPs overnight at 4ºC. The conjugated NoaS NPs were purified a final time using size exclusion chromatography. (IgGs referenced in Fig. 2 C (including the heavy- and light-chain variable domains were synthesized and codon optimized by IDT, then subcloned into pVRc8400 protein expression vectors with human constant regions. They were expressed and purified using the same workflow as the NA antigens.) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) 3µg of a protein sample, diluted to a final volume of 10µL, were mixed with 10µL of non-reducing 2x Laemmli Sample Buffer (Bio-Rad, Cat#: 1610737), then boiled for 10min at 100ºC. 12.5µL of sample were loaded into wells of a Mini-PROTEAN TGX Stain-Free precast gels (Bio-Rad, Cat#: 456–8026), then electrophoresed for 18-20min at 280V. Gels were imaged using a ChemiDoc (Bio-Rad) with appropriate standard imaging protocols. For the sera-depletion serum samples in fig. S19 , 10µL of concentrated sample were used without prior standardization for protein amount. Negative Stain Electron Microscopy 5µl of the sample (at 5–10µg/ml) were adsorbed for 60 seconds to a carbon-coated grid (EMS www.emsdiasum.com order# CF400-CU). The grid was rendered hydrophilic via a 20 second exposure to a glow discharge (25mA). Excess liquid was removed with a filter paper (Whatman #1). The grid was then exposed briefly to a water droplet to wash away ions and salts and blotted once again on a filter paper. The grid was then stained with 0.75% Uranyl Formate (EMS catalog # 22451) for 20 seconds. After removing the excess stain with filter paper, the grids were examined in a TecnaiG² Spirit BioTWIN and images were recorded with an AMT NanoSprint43 CCD camera. In vivo immunizations All animal experiments were approved by the Institutional Animal Care and Use Committees (IACUC) of Harvard University and Massachusetts General Hospital (Protocol #2014N000252) and were conducted in compliance with the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) guidelines. Mice were used at 8–10 weeks of age. The immunizations were performed using female C57BL/6 mice purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were divided randomly in 7 groups of n = 5 and received a prime immunization intraperitoneally on day 0, which consisted of 100uL of inoculum containing 20ug of protein (J’57/J’57 NoaS NPs as control, NoaS NPs displaying J’57 with either B’71, D’21, or Au’75 and a cocktail of homologous J’57/J’57 NPs with either B’71/B’71, D'21/D’21, or Au’75/Au’75 NPs, respectively) adjuvanted with 50% v/v Sigma adjuvant (Sigma, Cat#S6322) in sterile PBS. The boost occurred on day 28. Mice were bled from the submandibular vein for sera analyses on day − 1 and then every 7 days thereafter (until day 70, when they were euthanized). ELISA Assays Sera and monoclonal antibody reactivity to NA antigens were assayed by ELISA. 96-well high binding plates (Corning) were coated with 3µg/ml of NA antigens in PBS buffer at 100µl/well and incubated overnight at 4ºC. Plates were blocked with 1% BSA in PBS containing 0.1% Tween-20 (PBS-T) for 1–2 hours at room temperature (RT). The blocking solution was then discarded. Plates were coated with sera or monoclonal antibody solutions and incubated at RT for 1.5 hours. (Sera were serially diluted into 1xPBS with a 10 0.5 dilution factor (DF), starting from a dilution of 1:40. Monoclonal antibodies were serially diluted by a 10 0.5 DF into 1xPBS. Starting concentrations for mAbs and sera are noted on ELISA graphs. The final well for each sample contained 1xPBS alone and guided the subtraction of the background signal calculation for each experiment.) Plates were washed 3x with PBS-T. For ELISAs that used murine sera as the source for the primary antibodies, the secondary antibody was rabbit pAb anti-mouse IgG-HRP (Abcam, AB97046). The secondary was added at 1:20,000 dilution (in PBS) on d34, d48, and d55, and it was added at 1:16000 on d13, d20, and d62. The secondary antibody solution was coated onto the plates for 1 hour at RT. For monoclonal antibodies, secondary goat pAb anti-human IgG-HRP (Abcam, AB97225) was added at 1:20,000 dilution (in PBS) for 1 hour at RT. Plates were washed three times with PBS-T and developed with 1-step ABTS Substrate Solution (ThermoFisher) for 45 minutes. 405nm absorbance values were measured using a plate reader. EC 50 values were determined by non-linear regression (sigmoidal) using GraphPad Prism 10.2.3 software. Sera Depletion Assays A/Japan/305/1957 (J’57), A/Bilthoven/21438/1971 (B’71), A/Darwin/6/2021 (D’21), and A/tern/Australia/G70C/1975 (Au’75) strains were cloned with N-terminus StrepII, i.e., TwinStrep, sequences, transiently expressed, and purified as described above. For the sera depletion assays, 65 columns of 500uL of Strep-Tactin®XT resin were prepared in 15mL gravity-flow columns and washed with PBS buffer. Based on prior studies related to antibody production kinetics 70 , excess StrepII tagged antigen was incubated with the Strep-Tactin resin for 1 hour, then eluted. Columns 1–35 were incubated with J’57 NA antigen. Columns 36–45 were incubated with B’71 NA antigen. Columns 46–55 were incubated with D’21 NA antigen. Columns 56–65 were incubated with Au’75 NA antigen. Separately, 555uL of PBS were mixed with 45µL of sera collected on d13 from each mouse for a 1/13.3 sera dilution. This was used as the stock of sera for each mouse for a given depletion experiment to minimize any experimental fluctuation due to pipetting. For mice m1-m35, 200uL of the diluted sera were incubated with the washed J’57-tagged Strep-Tactin resin columns 1–35, respectively. 200µL of m6-m15 sera mixtures were incubated with columns 36–45, respectively. 200µL of m16-m25 sera mixtures were incubated with columns 46–55, respectively. Lastly, 200µL of m26-m55 sera mixtures were incubated with columns 56–65, respectively. After 2–4 hours at 4ºC incubation, serum was eluted from each column and collected along with a 100µL of PBS column wash into the first rows of a 96-well PCR plate. The final concentration of the strain-depleted sera was therefore 1/20. These depleted sera were then serially diluted using a factor of 10 0.5 for ELISA testing. Pre-depletion serum was taken from the same 1/13.3 diluted serum stock by taking 200µL of diluted serum and further diluting it with 100uL PBS for a final dilution of 1/20. The same was done for the sera that were collected on d55. Pre-depletion serum and post J’57-depletion serum from m1-m5 were tested for reactivity against the J’57 strain ( figs. S9, S14 ). The pre-depletion reactivity established the baseline J’57 reactivity value, and the post J’57 depletion reactivity was used as an internal control to verify successful J’57 depletion ( e.g. , fig. S13E ). Mice m1-m5 were also tested pre-depletion for reactivity to B’71, D’21, and Au’75 ( fig. S22 ), respectively, to establish baseline levels of cross-reactivity in the control group. Mice m1-m5 were tested post J’57-depletion for any residual reactivity to B’71, D’21, and Au’75 ( figs. S9, S14 ). Mice m6-m5 were tested pre-depletion for their reactivity to J’57 and B’71, post J’57-depletion for their reactivity to J’57 (internal control) and B’71 ( i.e. , B’71 strain-specificity), and post B’71-depletion for their reactivity to J’57 ( i.e. , J’57 strain-specificity) and B’71 (internal control). Analogous experiments were done for mice m16-m25 using J’57 and D’21 NAs, and for mice m26-m35 using J’57 and Au’75 NAs. To minimize any variation in OD 405nm absorbance due to antigen coating, the same stock of 3µg/mL antigen concentration was used for all tested groups. Additionally, the 96-well (Corning) plates were organized to have all samples tested for reactivity against a given antigen on the same plates. For example, for the m6 serum that was tested against B’71, an entire plate was coated using the same B’71 3µg/mL diluted stock. Column 1 tested m6 pre-depletion serum vs. B’71, Column 2 tested m6 post-J’57 depletion vs. B’71, and Column 3 tested m6 post-B’71 depletion vs. B’71, etc. Since the number of tested samples for a given antigen exceeded one 96-well plate, the OD 405nm absorbance values were furthermore normalized for each antigen by dividing each OD 405nm data point by the average of the top 10 absorbance values for a given antigen. ELLA inhibition Assays To test the ability of the sera to inhibit the NA activity of a given strain, tetramers of each strain were made, as NA tetramers have much greater catalytic activity than NA monomers 71 , 72 . J’57 NA was tetramerized using the tetrabrachion 73 domain. B’71, D’21, and Au’75 NAs were tetramerized using VASP 74 domains. (Choice of tetramerization domain was based solely on which domain helped the protein express better and more homogeneously.) Proteins were transiently expressed and purified as described above for the monomeric antigens. The EC 50 of each NA activity was then determined. This was done through the following experiment setup: 100µL of 25µg/mL of fetuin (Sigma Aldrich, F3004) were coated onto 96-well plates and incubated overnight at 4°C. Plates were blocked with PBS, 1% BSA, 0.1% Tween-20 solutions and put on a shaker for 2 hours at RT. Plates were then washed 6x. Next, 100µL of recombinant NA TB , serially diluted 1:2 in DPBS with Ca/Mg (Gibco, Cat#: 14040117) with a starting concentration of 0.005µg/mL were added to the plate in columns 1–11. Column 12 was plated with DPBS alone. Plates were then placed in a 37°C incubator for 2 hours and subsequently washed with ELISA buffer 6x. Plates were incubated with 100µL of either 1 or 5µg/mL of HRP-conjugated lectin from Arachis hypogaea (peanut) (Sigma Aldrich, L7759) and incubated for 75-100min at RT on a plate shaker. Plates were then washed 6x and coated with 100uL of ABTS. After 45 min, plates were imaged. EC 50 values of NA tetramers were determined by non-linear regression (sigmoidal) using GraphPad Prism 10.2.3 software. On d20, monomeric B’71 was used instead tetrameric B’71 due to poor tetramer expression, but the EC 50 of the monomer was determined in the exact same way. The ELLA inhibition assay was based on a standard protocol 55 . 100µL of 25µg/mL of fetuin (Sigma Aldrich, F3004) were coated onto 96-well plates and incubated overnight at 4°C. Plates were blocked with PBS, 1% BSA, 0.5% Tween-20 solutions and put on a shaker for 2 hours at RT. Plates were then washed 6x. A stock of 2x the EC 50 of each strain was prepared and 65µL of solution was added to each well of a 96well PCR plate. Separately, 28µL of each tested mouse serum sample were added to 252µL of PBS with calcium, for a serum concentration of 1/10th. Serum was then serially diluted 1:2 down columns 1–11. Column 12 contained PBS with calcium. 65µL were aliquoted into the same 96 well plate as the 65µL of NA solution and pipetted 3x. After 5–10 minutes, 120µL of the sera/NA mixture were added to the fetuin-coated plate. [Sera from m1-m5 and a d0 sera negative control were tested against J’57, B’71, D’21, and Au’75. Sera from m6-m15 and a d0 sera negative control were tested against J’57 and B’71. Sera from m16-m25 and a d0 sera negative control were tested against J’57 and D’21. Finally, sera from m26-m35 and a d0 sera negative control were tested against J’57 and A’75.] Plates were placed into a 37°C for 90min, except for plates testing strain B’71, which incubated for 2hrs to give more time for NA activity to cleave the substrate. Plates were then washed 6x and coated with 100µL of HRP-lectin at 2µg/ml. They were then put on a RT shaker for 90 min. After washing the plates 6x, plates were coated with 150µL of ABTS. Plates were then imaged after exactly 45 min. Non-linear (sigmoidal) curves were generated for each serum sample using GraphPad Prism 10.2.3 software The loss of OD 405nm signal observed at the highest sera concentration ( i.e. , dilution factor of 20) for the d0 negative control was used as the threshold for what would be considered ‘real’ signal due to specific sera antibody inhibition of NA rather than just the sera providing diffusion/viscosity inhibition of the NA activity. Sera from mice were then evaluated for the highest dilution factor at which the sera still outperformed the maximum inhibition of the d0 negative control. This was termed the sample’s MDFI, or the maximal dilution factor at which there was observable inhibition. Experimental group MDFIs were compared to the control. To avoid an observable hook-effect that impacted the regression analyses, all experiments for all samples were evaluated up until a dilution factor of 1280. Statistics All analyses that compared data between only 2 groups of mice were performed using the unpaired, non-parametric Mann-Whitney test with an alpha value of 0.05. Analyses that compared the mean rank of one group to the mean rank of more than one group used the unpaired, non-parametric Kruskal-Wallis test with Dunn correction for multiple comparisons. The p-values cutoffs for significance were the following: 0.0332 (*), 0.0021 (**), 0.0002 (***), < 0.0001(****). Declarations ACKNOWLEDGMENTS We thank Maria Ericsson and the Harvard Electron Microscopy Core for collection of negative stain images. We thank Daniel Maurer and Emerson Glassey for helpful discussions. We acknowledge support from NIGMS T32 GM0008313, NIH 1F30 AI181355 (to R.H.), NIH P01 AI089618 (A.G.S.), NIH AI155447, AI137057, AI153098 AI193280 (DL). This research has been funded in whole or part with federal funds under a contract from the National Institute of Allergy and Infectious Diseases, NIH contract 75N93019C00050 (A.G.S.). AUTHOR INFORMATION Author Contributions: R.H., A.G.S., and D.L. designed research; R.H. and F.A.N.M. performed research; R.H. and A.G.S. analyzed data; R.H. and A.G.S. wrote the paper. All authors commented on the manuscript. Correspondence and requests for materials should be addressed to: Aaron G. Schmidt ( [email protected] ) Competing financial interest: No competing financial interests. Author Contribution R.H., A.G.S., and D.L. designed research; R.H. and F.A.N.M. performed research; R.H. and A.G.S. analyzed data; R.H. and A.G.S. wrote the paper. All authors commented on the manuscript. References Abduljaleel, Z. Decoding SARS-CoV-2 variants: Mutations, viral stability, and breakthroughs in vaccines and therapies. Biophys. Chem. 320–321, 107413 (2025). Maurer, D. P., Vu, M. & Schmidt, A. G. 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Biol. Commun. 75, 89–97 (2019). Kü, K. et al. The VASP tetramerization domain is a right-handed coiled coil based on a 15-residue repeat. PNAS 101, 17027–17032 (2004). Additional Declarations No competing interests reported. Supplementary Files SuppNoaS82125.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7530142","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":538530929,"identity":"d2f4b232-a76b-4cdf-923d-bbbf58331211","order_by":0,"name":"Rochel Hecht","email":"","orcid":"","institution":"Ragon Institute of MGH, MIT and Harvard","correspondingAuthor":false,"prefix":"","firstName":"Rochel","middleName":"","lastName":"Hecht","suffix":""},{"id":538530930,"identity":"f599bb5c-a7d3-40e3-9054-f73a1c58eb93","order_by":1,"name":"Faez Amokrane Nait Mohamed","email":"","orcid":"","institution":"Ragon Institute of MGH, MIT and Harvard","correspondingAuthor":false,"prefix":"","firstName":"Faez","middleName":"Amokrane Nait","lastName":"Mohamed","suffix":""},{"id":538530931,"identity":"334b9f3a-d559-4aef-a3cb-093cbe4fafcb","order_by":2,"name":"Daniel Lingwood","email":"","orcid":"","institution":"Ragon Institute of MGH, MIT and Harvard","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Lingwood","suffix":""},{"id":538530932,"identity":"acb6bc9d-ab12-4bc7-be8e-e6abbdbfc2e2","order_by":3,"name":"Aaron Schmidt","email":"data:image/png;base64,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","orcid":"","institution":"Harvard Medical School","correspondingAuthor":true,"prefix":"","firstName":"Aaron","middleName":"","lastName":"Schmidt","suffix":""}],"badges":[],"createdAt":"2025-09-03 20:38:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7530142/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7530142/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":95050898,"identity":"a16e4976-5c8d-4248-8c19-0367ae82844c","added_by":"auto","created_at":"2025-11-03 18:22:41","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6953513,"visible":true,"origin":"","legend":"","description":"","filename":"MSFigsNoaSv69325.docx","url":"https://assets-eu.researchsquare.com/files/rs-7530142/v1/37d05ae412839ba4b8c29581.docx"},{"id":95050879,"identity":"4dd9e6e1-cbf0-48d5-9674-f4d03688cb8d","added_by":"auto","created_at":"2025-11-03 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18:22:41","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":57463,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7530142/v1/19ecc75eae088486c27978a2.png"},{"id":95050900,"identity":"23b2709c-5e42-4a1c-a51d-824f700d9e53","added_by":"auto","created_at":"2025-11-03 18:22:41","extension":"xml","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":155429,"visible":true,"origin":"","legend":"","description":"","filename":"b8859259d69a471192c780ce115a98d41structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7530142/v1/8e238a78ba44129eeb5b9a26.xml"},{"id":95050899,"identity":"eb89b853-412e-4442-aa8c-529afc01c7e5","added_by":"auto","created_at":"2025-11-03 18:22:41","extension":"html","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":169182,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7530142/v1/15dd034c60b8e107097f586b.html"},{"id":95050877,"identity":"69bf7a9c-63ff-4c47-8ed1-40d6708279c4","added_by":"auto","created_at":"2025-11-03 18:22:41","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":673427,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDesign of neuraminidase-on-a-string nanoparticles. (A)\u003c/strong\u003e The four neuraminidase (NA) strains included in the experiment were A/Japan/305/1957 (J’57), A/Bilthoven/21438/1971 (B’71), A/Darwin/6/2021 (D’21), and A/tern/Australia/G70C/1975 (Au’75). A monomer cartoon of the J’57 strain (PDB 6Q20\u003csup\u003e42\u003c/sup\u003e) is shown in gray, with a direct view of the catalytic site. Amino acid residues that differ between J’57 and B’71, D’21, and Au’75 NA strains are highlighted in pink, green, and blue, respectively. \u003cstrong\u003e(B)\u003c/strong\u003e Schematic of the homo- and heterodimer NA antigens. A rigid “L3” protein linker\u003csup\u003e48\u003c/sup\u003e (gray curved line) separates the NAs in each dimer. Approximate percent identity amino acid difference is noted for the between the NA monomers in the heterodimers; this is relative to the J’57 NA sequence. \u003cstrong\u003e(C)\u003c/strong\u003e The NoaS NP groups used \u003cem\u003ein vivo\u003c/em\u003e are shown schematically. 24 NA dimers (48 total NAs) are conjugated via a SpyTag to SpyCatcher on the N-termini of the ferritin protomers. J’57/J’57 NA homodimer (Group 1) is the control immunogen. Groups 2, 4, and 6 consisted of an equimolar cocktail of homologous J’57/J’57 NPs with B’71/B’71, D’21/D’21, or Au’75/Au’75 NPs, respectively. Groups 3, 5, and 7 display 24 copies of NoaS heterodimers on each NP. Each heterodimer contained two strains: J’57 and B’71 (Group 3), D’21 (Group 5), or Au’75 (Group 7).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7530142/v1/728e29e7e38d07a2432fb682.jpeg"},{"id":95223604,"identity":"657819ef-f4bd-4dcd-9dd8-6f826d4c4aa0","added_by":"auto","created_at":"2025-11-05 16:22:32","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":858603,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiochemical characterization of NoaS and NoaS NPs. (A)\u003c/strong\u003e Size exclusion chromatography traces of NoaS NPs with excess NoaS dimers after conjugation. \u003cstrong\u003e(B)\u003c/strong\u003e SDS PAGE with NoaS dimers conjugated to NP protomers and NoaS dimers. (Note: ferritin NP is non-covalently assembled and dissociates into protomers in SDS-PAGE).\u0026nbsp; \u003cstrong\u003e(C)\u003c/strong\u003e Reactivity of strain- and conformation-specific mAbs to NoaS NP. NA73 mAb is N9-specific, used for Au’75 NA detection. NDS.1 mAb binds J’57, B’71, and D’21 NAs but does not bind Au’75 NA. It was used in combination with NA73 mAb to verify the J’57/Au’75 NA components. 3A10 mAb binds B’71 and D’21 NAs but does not bind J’57 NA. 1G01 mAb does not bind D’21 NA. 3A10 mAb was used with 1G01 mAb to detect J’57/D’21 NAs. 3A10, NDS.1, and 1G01 mAbs detected J’57/B’71 NAs. 641-I9 mAb\u003csup\u003e54\u003c/sup\u003e is influenza HA-specific and was used as a negative control. \u003cstrong\u003e(D)\u003c/strong\u003e Representative negative stain micrographs of NoaS NP from each group.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7530142/v1/4628bf894da77fbedec04be0.jpeg"},{"id":95050878,"identity":"40ede027-9dfb-4b50-af26-29d334db4682","added_by":"auto","created_at":"2025-11-03 18:22:41","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":396263,"visible":true,"origin":"","legend":"\u003cp\u003eNoaS NP groups and immunization and assessment schema. Seven groups of C57BL/6 mice (n=5 per group) were immunized intraperitoneally on day (d) 0 and d28 and euthanized on d70. Serum from each mouse was collected weekly. Sera were evaluated on either the second or third week after the prime and up to 5 weeks after the boost for: breadth elicited against NA strains, degree of developed strain-specificity, and ability to inhibit NA enzymatic activity.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7530142/v1/5536e55829d0bbcd8847ad13.jpeg"},{"id":95222427,"identity":"c9d6beb5-4cca-4f85-83d2-f5ecb70752f6","added_by":"auto","created_at":"2025-11-05 16:20:39","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":601739,"visible":true,"origin":"","legend":"\u003cp\u003eNA breadth of d20 and d48 sera. (A) Sera reactivity to the J’57 NA monomer. No statistically significant differences in reactivity were observed. (B) Sera reactivity to the Ai’68 NA monomer. Statistically greater reactivity to the Ai’68 NA strain on d48 is noted for groups receiving J’57/B’71 NoaS NPs over groups receiving J’57/D’21 NoaS NPs (*0.0122). (C) Sera reactivity to the B’71 NA monomer. On d20, groups that received either B’71 or D’21 NA as their second NA component in immunizations had statistically greater reactivity to B’71 NA over the control group (*0.0224, **0.0064). On d48, groups that received B’71 NA had greater reactivity to the B’71 NA than the remaining experimental groups (*0.0150, **0.0052). (D) Sera reactivity to the M’99 NA monomer. Groups receiving D’21 NA in their immunizations had statistically greater reactivity to the M’99 NA on d20 (*0.0371, ***0.0005 for control, ***0.0003 for Au’75) and on d48 (*0.0203 for control, *0.0110 for Au’75). (E) Sera reactivity to the Tx’12 monomer. On both d20 and d48, groups that received D’21 NA in immunizations had statistically greater reactivity to the Tx’12 NA strain than the control group or groups receiving Au’75 NA (d20: ***0.0001 for control, ***0.0004 for Au’75. d48: **0.0023, ***0.0004). (F) Sera reactivity to the D’21 NA monomer. Groups receiving D’21 NA in their immunizations showed statistically greater reactivity to D’21 NA than all other groups on both d20 and d48. (d20: ***0.0007 for control, *0.0279, ***0.0004 for Au’75. d48: **0.0037 for control, *0.0368, **0.0018 for Au’75.) (G) Sera reactivity to the Au’75 NA monomer. Groups that received Au’75 NA in their immunizations had statistically greater reactivity to the Au’75 NA strain than other groups. (d20: ***0.0003, ****\u0026lt;0.0001. d48: **0.0043, ***0.0009.) Statistical differences were assessed using unpaired, non-parametric Kruskal-Wallis tests with Dunn correction for multiple comparisons. Horizontal lines represent the median. Data from mice (m) m21, m28, and m31 were removed from all statistical analyses due to lack of response to the prime immunization.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7530142/v1/3a77876c494cbda9cba30c42.jpeg"},{"id":95222330,"identity":"197e1998-5b37-479e-b260-71c6af9e6041","added_by":"auto","created_at":"2025-11-05 16:20:27","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":439777,"visible":true,"origin":"","legend":"\u003cp\u003eStrain-specificity toward J’57 NA and the second NA component. (A) d13 and d55 sera reactivities toward the J’57 NA. On d13, reactivities toward the J’57 strain were not statistically different across groups. On d55, reactivity toward the J’57 strain was significant between groups receiving B’71 NA versus D’21 NA as their second NA component (*0.0408). (B) Strain-specificity for the J’57 NA on d13 (p-values: **0.0081 and ****\u0026lt;0.0001) and d55 (p-values: *0.0357 and ****0.0001). (C) Strain-specificity for the second NA component on d13 (p-values: *0.0276 and ****\u0026lt;0.0001) and d55 (p-values: *0.0213 and ****\u0026lt;0.0001). Differences in groups were assessed using the Kruskal-Wallis test with Dunn correction for multiple comparisons. Lines and bars represent the median with a 95% confidence interval.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7530142/v1/a5dda6fbed93d2963934382b.jpeg"},{"id":95050887,"identity":"39d3fa14-8d32-4ce4-8ef5-ef6cd2382d09","added_by":"auto","created_at":"2025-11-03 18:22:41","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":270023,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMaximum dilution factor of inhibition (MDFI) of sera as assessed in ELLA inhibition assays.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e ELLA inhibition of J’57 NA. Sera from all groups showed statistically comparable inhibition of J’57 NA activity on d20 and d62.\u003cstrong\u003e \u003c/strong\u003eData were analyzed using the Kruskal-Wallis test with Dunn corrections for multiple comparisons. \u003cstrong\u003e(B)\u003c/strong\u003e ELLA inhibition on d20 and d62 of B’71, D’21, and Au’75 NAs. Statistical significances are noted (B’71 groups: **0.007, Au’75 groups: **0.002).\u003cstrong\u003e \u003c/strong\u003eData were assessed using Mann-Whitney tests. Lines reflect the median MDFI values.Serum from d0 was used as the negative control.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7530142/v1/caff24e5dba60f620822770e.jpeg"},{"id":104779539,"identity":"ec05017a-c23a-482a-9b20-b97e79e410f0","added_by":"auto","created_at":"2026-03-17 07:41:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4349694,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7530142/v1/d4421afb-d1cd-42c9-9e0e-93bffe2cf9ae.pdf"},{"id":95050902,"identity":"f16789df-2e6c-4025-890f-94ae6ffb7dba","added_by":"auto","created_at":"2025-11-03 18:22:43","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":57506065,"visible":true,"origin":"","legend":"","description":"","filename":"SuppNoaS82125.docx","url":"https://assets-eu.researchsquare.com/files/rs-7530142/v1/7a1a504ccc07ae15815725d5.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Neuraminidase-on-a-string nanoparticles probe how antigenic distance shapes elicited humoral immunity","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eHumoral responses directed at surface-exposed viral proteins (\u003cem\u003ee.g.\u003c/em\u003e, coronavirus spike and influenza hemagglutinin) drive viral evolution and escape\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. However, viruses cannot readily mutate conserved regions without impairing viral fitness\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. A goal of rational immunogen design strategies, such as hyperglycosylation, epitope grafting and multivalent display aim to focus humoral immunity toward conserved epitopes to limit viral escape\u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Multivalent immunogen displays increase B cell receptor crosslinking and subsequent activation\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Indeed, \u003cem\u003ein vivo\u003c/em\u003e studies comparing monomeric versus multimeric antigens show increased antibody titers, B cell activation, and T cell engagement with multimerization\u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18 CR19 CR20 CR21 CR22 CR23\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Mechanistically, \u003cem\u003ein vivo\u003c/em\u003e studies also showed that restriction to germinal center (GC) entry imposed by B cell affinity and precursor frequency thresholds are mitigated through increased avidity on an immunogen\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The lowered thresholds for GC entry may, in turn, increase B cell clonal diversity and breadth\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The size of multivalent immunogens is important, as larger particles induce slower trafficking within the lymph node\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, leading to a prolonged antigen exposure and duration of GCs\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Thus, multivalent display, including viral-like particles\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, self-assembling protein nanoparticles\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, and DNA-origami\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, are promising platforms for next-generation viral vaccines\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMosaic displays of distinct viral protein variants on the same nanoparticle (NP) enhanced breadth and neutralization relative to NP displays of a single protein\u003csup\u003e\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. This is likely due to a selective advantage for B cell receptors that engage conserved epitopes shared between the displayed antigens relative to the receptors that engage less conserved regions. However, further studies are needed to clarify the mechanisms involved, such as whether a minimum level of diversity is required to promote this advantage and whether significant variation between antigens might elicit strain-specific responses at the expense of cross-reactive responses. For example, mosaic hemagglutinin NPs showed increased proportions of cross-reactive B cells using mosaic displays only when including 6 or more strains on the same NP\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Additionally, whether mosaic displays stimulate cross-reactivity better than a homologous \u0026ldquo;cocktail\u0026rdquo; of the same strains has yet to be clarified. Indeed, studies incorporating antigenically equivalent cocktail versus mosaic immunogens have indicated mixed outcomes in terms of benefits\u003csup\u003e\u003cspan additionalcitationids=\"CR30 CR31 CR32\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Moreover, for both mosaic and homologous cocktail experiments, data indicate that different combinations of 2 or 4 heterologous strains resulted in different sera cross-reactivity profiles\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eHere, we addressed the potential mechanism(s) underlying these observations, and to understand how antigenic distance between displayed proteins on a NPs influenced elicited sera breadth and cross-reactivity. We chose the influenza neuraminidase (NA) as our prototypic antigen. NA catalyzes the cleavage of terminal sialic acids, and its function is essential for influenza viral egress and spread\u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. There is growing interest in NA as a vaccine target\u003csup\u003e\u003cspan additionalcitationids=\"CR39 CR40\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, as antibodies targeting NA can be broadly reactive and protective against influenza infection\u003csup\u003e\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Thus, inclusion of an NA component in next-generation influenza vaccines may provide an independent arm of immune protection against influenza, contributing to seasonal vaccine effectiveness\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eBriefly, we engineered a series of dimeric NA head constructs tandemly linked by a rigid peptide to create neuraminidase-on-a-string (NoaS) immunogens, which were then conjugated to ferritin NPs via SpyTag/SpyCatcher\u003csup\u003e47\u003c/sup\u003e. Each dimer contained the NA from H2N2 Japan 1957, but the second NA component was a distinct strain, which varied the antigenic distance up to ~\u0026thinsp;50% between the NA pairs. Homologous dimers of each NA were used as controls. The dimeric design enabled consistent molar ratios and spatial orientations of the NA components. Mice were immunized with either heterologous (\u003cem\u003ei.e.\u003c/em\u003e, mosaic) NoaS NPs or an equivalent equimolar homologous cocktail of NoaS NPs. Serum samples were collected before and after boosting and evaluated for anti-NA breadth, strain-specificity, and NA enzymatic inhibition. We found that including a second NA strain in immunizations enhanced both anti-NA breadth and NA enzymatic inhibition. Notably, the proportion of strain-specific antibodies for each NA component increased with the antigenic distance of the NoaS pairing, even as overall anti-NA reactivity remained comparable. These findings guide next-generation viral vaccine development and underscore the importance of strain selection in multivalent vaccine platforms designed to maximize elicited cross-reactive immune responses.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eDesign of neuraminidase-on-a-string nanoparticles\u003c/h2\u003e\u003cp\u003eWe selected four influenza neuraminidase (NA) strains to be incorporated in our immunogens: H2N2 A/Japan/305/1957 (J\u0026rsquo;57), H3N2 A/Bilthoven/21438/1971 (B\u0026rsquo;71), H3N2 A/Darwin/6/2021 (D\u0026rsquo;21), and H11N9 A/tern/Australia/G70C/1975 (Au\u0026rsquo;75). We chose these strains primarily based on their amino acid diversity relative to the J\u0026rsquo;57 NA head: B\u0026rsquo;71, D\u0026rsquo;21, and Au\u0026rsquo;75 differed from J\u0026rsquo;57 by ~\u0026thinsp;8%, ~\u0026thinsp;17%, and ~\u0026thinsp;50%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA,B and \u003cb\u003efig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC\u003c/b\u003e). Additionally, the H2N2 as well as both H3N2s previously circulated within the human population, while H11N9 represents a potentially zoonotic influenza that is within the avian population. To understand how antigenic distance between NAs on a multivalent nanoparticle (NP) immunogen influenced sera cross-reactivity and breadth, we designed neuraminidase-on-a-string (NoaS) NPs. We tandemly linked pairs of monomeric NA heads together using a rigid, proline-rich L3 amino acid peptide linker\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e to make NoaS and subsequently conjugated them to the \u003cem\u003eHelicobacter Pylori\u003c/em\u003e ferritin NP\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e using SpyTag/SpyCatcher\u003csup\u003e47\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C and \u003cb\u003efig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-B\u003c/b\u003e). We designed homodimeric NoaS NPs of each of these four strains (groups 1, 2, 4, and 6; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) and made heterodimeric NoaS NPs by pairing J\u0026rsquo;57 with either B\u0026rsquo;71, D\u0026rsquo;21, or Au\u0026rsquo;75 (groups 3, 5, and 7; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Thus, using \u0026lsquo;percent amino acid difference\u0026rsquo; as a proxy for antigenic distance, we varied the presented antigenic distance on the NoaS NPs from 0% up to ~\u0026thinsp;50%. Each NoaS NP displayed a total of 48 copies of the NA head, with one NoaS dimer per ferritin protomer. In the heterologous NoaS, the J\u0026rsquo;57 NA strain was conjugated adjacent to the ferritin protomer with the second NA component projecting outwardly (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eBiochemical characterization of NoaS NPs\u003c/h3\u003e\n\u003cp\u003eNoaS and NoaS NPs were recombinantly expressed from mammalian cells and purified with affinity and size exclusion chromatography (SEC). The C-terminal affinity tags were removed by enzymatic cleavage and re-purified by SEC. The NoaS, in molar excess, were then conjugated to the NPs and purified again over SEC. The resulting NoaS NPs were monodisperse (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cb\u003efig. S2B\u003c/b\u003e) and homogeneous as assayed by SDS-PAGE analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). All NoaS NPs were tested for reactivity to conformation-specific monoclonal antibodies (mAbs) NA73\u003csup\u003e50\u003c/sup\u003e, 3A10\u003csup\u003e51\u003c/sup\u003e, 1G01\u003csup\u003e42\u003c/sup\u003e, and NDS.1\u003csup\u003e52\u003c/sup\u003e to assess structural integrity of the NA components (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cb\u003efig. S2A\u003c/b\u003e). NoaS dimers and NoaS NPs had comparable reactivity to the diagnostic mAbs, indicating that each NA component was in its native conformation and accessible after conjugation to the ferritin NP. NoaS NPs were visualized using negative stain electron microscopy and showed uniform NoaS NP sizes of ~\u0026thinsp;60nm, corresponding well to the approximate additive size of all components (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The L3 linker\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e spaced each of the ~\u0026thinsp;5nm-wide NA heads about 10nm apart (\u003cb\u003efig. S2C\u003c/b\u003e). This approximate distance between the two NA heads is optimal to engage both antibody arms of a B cell receptor (~\u0026thinsp;13nm distance)\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eGroup design and vaccine regimen\u003c/h3\u003e\n\u003cp\u003eSeven groups of C57BL/6 mice (n\u0026thinsp;=\u0026thinsp;5 per group) were immunized using a homologous prime-boost regimen (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Mice received 100\u0026micro;L of inoculum intraperitoneally, containing 20\u0026micro;g of total protein adjuvanted with 50% w/v Sigma adjuvant. Group 1, which received J\u0026rsquo;57/J\u0026rsquo;57 NoaS NPs, served as the control group. Groups 3, 5, and 7 received heterologous NoaS NPs displaying J\u0026rsquo;57 with either B\u0026rsquo;71, D\u0026rsquo;21, or Au\u0026rsquo;75, respectively. Groups 2, 4, and 6 received a cocktail of homologous J\u0026rsquo;57/J\u0026rsquo;57 NPs with either B\u0026rsquo;71/B\u0026rsquo;71, D'21/D\u0026rsquo;21, or Au\u0026rsquo;75/Au\u0026rsquo;75 NPs, respectively. The cocktails were prepared in equimolar ratios to their heterologous counterparts. Mice were bled weekly, and the sera were collected 14\u0026ndash;27 days post-prime and 41\u0026ndash;62 days post-prime (\u003cem\u003ei.e.\u003c/em\u003e, 14\u0026ndash;35 days post-boost) to be evaluated for NA breadth, NA strain-specificity, and NA enzymatic inhibition.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eAssessment of elicited sera breadth in NoaS NP groups\u003c/h3\u003e\n\u003cp\u003eA panel of six historical N2 heads (A/Japan/305/1957 (J\u0026rsquo;57), A/Bilthoven/21438/1971 (B\u0026rsquo;71), A/Aichi/2/1968 (Ai\u0026rsquo;68), A/Moscow/10/1999 (M\u0026rsquo;99), A/Texas/50/2012 (Tx\u0026rsquo;12), and A/Darwin/6/2021 (D\u0026rsquo;21)) and one H11N9 (A/tern/Australia/G70C/1975 (Au\u0026rsquo;75)), which included the four NA strains incorporated into the immunogens, were recombinantly expressed as monomers. The amino acid differences between the NA strains in the panel varied from ~\u0026thinsp;4.4\u0026ndash;53.5% (\u003cb\u003efig. S3A\u003c/b\u003e) and the number of predicted-N-linked glycosylation sites in the panel ranged from 3\u0026ndash;7 (\u003cb\u003efig. S3B\u003c/b\u003e). Sera from d20 and d48 were tested for reactivity to the entire NA panel in ELISA. Absorbance values were normalized per antigen, and the degree of normalized area under the curve (nAUC) was plotted for each mouse serum sample (\u003cb\u003efigs. S4A-B\u003c/b\u003e and \u003cb\u003eS5A-B\u003c/b\u003e). Except for the reactivity of group 4 versus group 5 to the Tx\u0026rsquo;12 antigen on d20 (\u003cb\u003efig. S6B\u003c/b\u003e), there were no statistically significant differences in the observed reactivity to the NA panel when comparing the homologous cocktail groups (\u003cem\u003ei.e.\u003c/em\u003e, groups 2, 4, and 6) to their heterologous NoaS counterparts (\u003cem\u003ei.e.\u003c/em\u003e, groups 3, 5, and 7) (\u003cb\u003efig. S6A-C\u003c/b\u003e). This suggested that any effect of heterologous strain presentation versus homologous cocktail presentation was likely too small to be observed with our n\u0026thinsp;=\u0026thinsp;5 group size. We therefore combined the data from groups 2 and 3, groups 4 and 5, and groups 6 and 7, respectively, to probe how the antigenic distance within a two-strain immunization influenced elicited breadth. (Note: In this study, \u003cem\u003eserum breadth\u003c/em\u003e refers to the overall extent of unique NA strains recognized by the serum in our NA panel. \u003cem\u003eSerum cross-reactivity\u003c/em\u003e describes when individual antibodies within the serum recognize more than one antigenically distinct NA strain. Serum breadth can result either from cross-reactive serum antibodies (\u003cem\u003ei.e.\u003c/em\u003e, the same serum antibodies binding multiple NA strains) or from a more diverse serum antibody pool (\u003cem\u003ei.e.\u003c/em\u003e, different serum antibodies recognizing different NA strains). Thus, while serum that recognizes distinct NA strains has greater breadth than serum recognizing only one strain, this does not necessarily imply that it contains more cross-reactive serum antibodies.)\u003c/p\u003e\u003cp\u003eWe found that all groups had comparable reactivity to the J\u0026rsquo;57 antigen (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), which indicated that J\u0026rsquo;57 immunogenicity was not affected by the inclusion of a second NA strain. Additionally, all experimental groups had greater anti-NA breadth than the control group, implying that inclusion of a second strain increased the elicitation of NA breadth (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-G). On d20, J\u0026rsquo;57/D\u0026rsquo;21 groups had the broadest reactivity across the tested N2 NA panel (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-F). On d48, although the groups receiving J\u0026rsquo;57/D\u0026rsquo;21 still showed broad N2 NA reactivity, the groups receiving J\u0026rsquo;57/B\u0026rsquo;71 developed statistically significant reactivity to earlier N2 NA strains (\u003cem\u003ei.e.\u003c/em\u003e, Ai\u0026rsquo;68 and B\u0026rsquo;71; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C). These data suggested that the inclusion of two antigenically similar NA strains (\u003cem\u003ei.e.\u003c/em\u003e, J\u0026rsquo;57 and B\u0026rsquo;71) could enhance the reactivity to each other as well as to strains antigenically like each other (\u003cem\u003ei.e.\u003c/em\u003e, Ai\u0026rsquo;68). Finally, the groups receiving J\u0026rsquo;57/Au\u0026rsquo;75 had N2 NA breadth that was comparable to the control group across the N2 panel but also had reactivity to the N9 strain included in the NoaS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-G\u003cb\u003e)\u003c/b\u003e. These data suggested that the antigenic distance between J\u0026rsquo;57 and Au\u0026rsquo;75 was too great to elicit broader N2 reactivity. Nonetheless, including the N9 NA allowed strain-specific recognition of each component.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo assess whether serum antibodies from the groups acquired breadth by focusing on the conserved NA catalytic site (CS), we tested sera reactivity to a modified J\u0026rsquo;57 NA with a glycan projecting into the CS. This \u0026lsquo;ΔCS\u0026rsquo; J\u0026rsquo;57 version abrogated binding to all CS-directed mAbs assayed (\u003cb\u003efig. S7A\u003c/b\u003e). By comparing the sera reactivity to the wildtype J\u0026rsquo;57 versus ΔCS J\u0026rsquo;57 antigens, we could compute the percent loss of signal due to the glycan and assess how much of the sera was directed at or near the CS. We did not observe any differences in ΔCS J\u0026rsquo;57 reactivity in the cocktail versus heterologous groups, so we analyzed groups based on the pair of NA strains received in the immunizations (\u003cb\u003efig. S7B-C\u003c/b\u003e). On both d20 and d48, there were statistically comparable reactivities to the ΔCS J\u0026rsquo;57 antigen across all paired groups (\u003cb\u003efig. S7D\u003c/b\u003e). On d20, reduced reactivity to the ΔCS J\u0026rsquo;57 antigen compared to the wildtype J\u0026rsquo;57 antigen was significant for the J\u0026rsquo;57/J\u0026rsquo;57, J\u0026rsquo;57/B\u0026rsquo;71, and J\u0026rsquo;57/Au\u0026rsquo;75 groups. By d48, the reduced reactivity was significant for all groups except J\u0026rsquo;57/J\u0026rsquo;57, however this group trended similarly (\u003cb\u003efig. S7E\u003c/b\u003e). These results suggest that, across groups, the inclusion of a second, antigenically distinct NA strain contributed to focusing the sera responses on the CS, which is conserved across NA strains.\u003c/p\u003e\u003cp\u003eLastly, scaffold-specific sera responses to the ferritin NP were assayed on d27 and d62 (\u003cb\u003efig. S8A-C\u003c/b\u003e). On d27, the NP reactivity was similar across groups. However, on d62, the J\u0026rsquo;57/B\u0026rsquo;71 and J\u0026rsquo;57/Au\u0026rsquo;75 had enhanced reactivity toward the naked NP relative to the J\u0026rsquo;57/J\u0026rsquo;57 control group (\u003cb\u003efig. S8C\u003c/b\u003e).\u003c/p\u003e\n\u003ch3\u003eStrain-specificity of elicited sera responses\u003c/h3\u003e\n\u003cp\u003eWe developed a serum depletion assay to evaluate whether NA pairs elicited strain-specific or cross-reactive antibodies (\u003cb\u003efig. S9-19\u003c/b\u003e). Each serum sample was first tested by ELISA for reactivity against both NA components used in each immunization. For example, serum from mouse 30 (m30) in group 6 (which received both J\u0026rsquo;57 and Au\u0026rsquo;75 NAs) was first assayed for binding to J\u0026rsquo;57 and Au\u0026rsquo;75 NAs (\u003cb\u003efig. S12A\u003c/b\u003e). The m30 serum was then incubated over a column containing J\u0026rsquo;57 NA, thereby depleting the sample of J\u0026rsquo;57-reactive antibodies. The J\u0026rsquo;57-depleted serum was subsequently tested for reactivity to both J\u0026rsquo;57 NA (\u003cb\u003efig. S12B\u003c/b\u003e) and Au\u0026rsquo;75 NA (\u003cb\u003efig. S12C\u003c/b\u003e) by ELISA. The absence of any remaining J\u0026rsquo;57 NA reactivity confirmed successful depletion, and the remaining Au\u0026rsquo;75 NA reactivity was compared to the pre-depletion level. The ratio of Au\u0026rsquo;75 NA reactivity post- vs. pre-J\u0026rsquo;57 depletion reflected the degree of strain-specificity for Au\u0026rsquo;75 (\u003cb\u003efig. S13E\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eImportantly, this assay quantified how much of the serum antibody response to a given NA was specific to that NA. It did not directly measure the percentage of antibodies that were cross-reactive. For instance, a strain-specificity value of ~\u0026thinsp;79% for the Au\u0026rsquo;75 NA in m30 did not imply that ~\u0026thinsp;21% of total serum antibodies were cross-reactive. Rather, it meant that \u003cem\u003eof\u003c/em\u003e the antibodies binding Au\u0026rsquo;75 NA, ~\u0026thinsp;21% also recognized J\u0026rsquo;57 NA. In this way, the assay measured how much of the response to strain A was unique to strain A, and it only indirectly reflected cross-reactivity with strain B. Using this approach and analysis, sera-depletion assays were performed on each serum sample collected on d13 and d55.\u003c/p\u003e\u003cp\u003eThe homologous cocktail groups 2, 4, and 6 had similar strain-specificity profiles for their counterpart heterologous groups 3, 5, and 7 on both d13 and d55 (\u003cb\u003efig. S20A-B\u003c/b\u003e). We therefore analyzed data from groups 2 and 3, 4 and 5, and 6 and 7 as combined cohorts, respectively. The J\u0026rsquo;57/J\u0026rsquo;57 group acquired some breadth for B\u0026rsquo;71, D\u0026rsquo;21, and Au\u0026rsquo;75 NAs (\u003cb\u003efig. S22A-B\u003c/b\u003e). However, after sera depletion for J\u0026rsquo;57-reactive antibodies, the remaining sera no longer recognized B\u0026rsquo;71, D\u0026rsquo;21, and Au\u0026rsquo;75 NAs (\u003cb\u003efigs. S13\u003c/b\u003e and \u003cb\u003eS18\u003c/b\u003e). Thus, we conclude that none of the serum antibodies elicited in the J\u0026rsquo;57/J\u0026rsquo;57 group were strain-specific for B\u0026rsquo;71, D\u0026rsquo;21, or Au\u0026rsquo;75 NAs.\u003c/p\u003e\u003cp\u003eThe remaining groups were depleted for both J\u0026rsquo;57 NA reactivity and the second NA component in their immunogens (\u003cb\u003efigs. S9-S18\u003c/b\u003e). On d13, all groups had comparable reactivity to J\u0026rsquo;57 NA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), and most groups had comparable reactivity to J\u0026rsquo;57 NA on d55. Nonetheless, the degree of strain-specificity for J\u0026rsquo;57 NA increased in the groups in correlation with the antigenic distance of their second NA component (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). This same phenomenon was observed for the NA strain that J\u0026rsquo;57 was paired with (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Thus, groups that received J\u0026rsquo;57/B\u0026rsquo;71 had substantially less strain-specificity toward the J\u0026rsquo;57 NA than did the groups that received J\u0026rsquo;57/Au\u0026rsquo;75. Similarly, the calculated strain-specificities toward B\u0026rsquo;71 NA in groups 2 and 3 were substantially lower than the calculated strain-specificities toward Au\u0026rsquo;75 NA in groups 6 and 7. Thus, pairing the J\u0026rsquo;57 NA with an NA strain that had increased antigenic distance elicited a greater proportion of serum antibodies specific to each NA component. When comparing the strain-specificity on d55 versus d13, all groups showed a decrease in the magnitude of specificity for both the J\u0026rsquo;57 NA (\u003cb\u003efig. S21A\u003c/b\u003e) and their paired NA component (\u003cb\u003efig. S21B\u003c/b\u003e), with varying statistical significance across groups.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eSera inhibition of NA enzymatic activity\u003c/h2\u003e\u003cp\u003eWe next assessed whether the elicited sera from the NoaS NPs inhibited NA enzymatic activity using the enzyme-linked lectin assay (ELLA), which uses a large fetuin substrate\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Antibodies that bind directly to the CS block enzymatic activity even with small substrates, but antibodies that bind proximal to the CS or on the NA periphery can sterically obstruct the CS. The ELLA inhibition assay is therefore considered more reflective of the broader potential to inhibit NA activity over other assays that use smaller substrates\u003csup\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, such as NA-Star\u0026trade; or MUNANA\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. We assayed d20 and d62 sera for enzymatic inhibition of the NA strains used in each immunization (\u003cb\u003efigs. S23-S24\u003c/b\u003e). We first determined the EC\u003csub\u003e50\u003c/sub\u003e concentration for a given NA strain and then incubated two times the EC\u003csub\u003e50\u003c/sub\u003e with two times the serial serum dilution and measured the resulting NA activity. Using d0 serum as a negative control, we identified the dilution factor at which a given serum sample inhibited NA enzymatic activity more than the highest concentration of d0 serum (\u003cem\u003ei.e.\u003c/em\u003e, the maximum serum dilution factor with inhibitory activity, or MDFI). Homologous cocktail groups 2, 4, and 6 showed similar inhibition profiles to their counterpart heterologous groups 3, 5, and 7, respectively (\u003cb\u003efig. S25A-B\u003c/b\u003e). Groups were therefore evaluated based on immunizing strains.\u003c/p\u003e\u003cp\u003eAll groups had comparable enzymatic inhibition of J\u0026rsquo;57 NA activity on both d20 and d62, indicating that the inclusion of a second NA strain did not reduce serum antibodies with J\u0026rsquo;57 CS-inhibiting activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cb\u003efigs. S23A\u003c/b\u003e and \u003cb\u003eS24A\u003c/b\u003e). We then tested B\u0026rsquo;71 NA enzymatic inhibition in groups 2 and 3, D\u0026rsquo;21 NA enzymatic inhibition in groups 4 and 5, and Au\u0026rsquo;75 NA enzymatic inhibition in groups 6 and 7 (\u003cb\u003efigs. S23B-D\u003c/b\u003e and \u003cb\u003eS24B-D\u003c/b\u003e). Each NA strain was also tested against sera from the control, group 1. Sera from d62 showed substantially stronger inhibition of B\u0026rsquo;71 in groups 2 and 3 compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Surprisingly, at both time points, despite the greater antigenic distance between D\u0026rsquo;21 and J\u0026rsquo;57 NAs than the B\u0026rsquo;71 and J\u0026rsquo;57 NAs, inhibition of D\u0026rsquo;21 NA activity was not significantly improved in groups 4 and 5 sera compared to the control group sera (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Lastly, sera from groups 6 and 7 inhibited the Au\u0026rsquo;75 NA activity substantially better than sera from the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). These data show that including a second NA component in NoaS NPs with J\u0026rsquo;57 NA does not detract from J\u0026rsquo;57 NA activity inhibition and, moreover, better ensures the production of antibodies with inhibitory potential to a second NA.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eHere, we engineered a multivalent, modular neuraminidase-on-a-string (NoaS) nanoparticle (NP) platform to assess the effects of varying neuraminidase (NA) antigenic distance on the elicited humoral response. Sera reactivity to the NA panel yielded two key observations. First, J\u0026rsquo;57 NA immunogenicity was not reduced when a second NA strain was included in the NoaS NPs, regardless of the antigenic distance relative to J\u0026rsquo;57 NA. Second, anti-NA breadth is augmented when a second, antigenically distinct NA strain is included. Additionally, the proportion of strain-specific serum antibodies increases with the antigenic distance between the NA components, and that this relationship remains consistent after boosting. We also found that inhibition of J\u0026rsquo;57 NA enzymatic activity by sera was not impacted by including a second NA strain in NoaS NPs; this observation may correlate with protection.\u003c/p\u003e\u003cp\u003eInterrogating scaffold-specific responses is necessary to ensure that next-generation vaccine platforms do not abrogate desired immune responses to the viral immunogens\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Since each NoaS dimers were approximately similar in size across experimental groups, we anticipated that the NoaS dimers would sterically occlude access to the ferritin surface equivalently and thus have comparable scaffold-specific responses. While this was generally observed, at d62 (\u003cem\u003ei.e.\u003c/em\u003e, 35 days post-boost) the J\u0026rsquo;57/B\u0026rsquo;71 and J\u0026rsquo;57/Au\u0026rsquo;75 experimental groups elicited stronger ferritin-specific responses relative to the J\u0026rsquo;57/J\u0026rsquo;57 control group (\u003cb\u003efig. S8C\u003c/b\u003e). It is unclear why these NA pairings resulted in a more robust scaffold response however, these experimental groups also had stronger reactivity to both NA components on d20 and d48 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, C, and \u003cb\u003eG\u003c/b\u003e). Importantly, the observed scaffold-specific responses did not appear to reduce sera responses to the NA components. Thus, using the ferritin NP to increase multivalency is a possible platform for NA-based vaccines.\u003c/p\u003e\u003cp\u003eIncorporating diverse strains of the same viral protein into a vaccine can induce humoral responses to each individual strain\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. This strategy of enhancing breadth through multi-strain immunization is supported by numerous studies\u003csup\u003e\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Therefore, our finding that NoaS NPs elicited serum responses to each included NA component is consistent with prior observations. Notably, the combination of J\u0026rsquo;57 and D\u0026rsquo;21 NAs elicited broad reactivity to a panel of historical N2 NAs spanning\u0026thinsp;\u0026gt;\u0026thinsp;60 years of antigenic distance. The observed reactivity may be due to cross-reactive serum antibodies recognizing both J\u0026rsquo;57 and D\u0026rsquo;21 NAs, or from distinct serum antibody populations targeting each NA strain. Data from our serum-depletion assay supported the latter: we observed that serum strain-specificity increased with antigenic distance between paired NAs on the NoaS NPs, suggesting that the J\u0026rsquo;57/D\u0026rsquo;21 group achieved breadth primarily through separately affinity-matured serum antibody pools. While some cross-reactive serum antibodies may have been elicited, they appeared to represent a minority of the response, particularly in the J\u0026rsquo;57/D\u0026rsquo;21 and J\u0026rsquo;57/Au\u0026rsquo;75 groups.\u003c/p\u003e\u003cp\u003eIn general, we did not find that the mosaic displays of the heterologous strains resulted in statistically significant greater NA breadth, cross-reactivity, or activity inhibition than their homologous cocktail counterparts. Previous studies also showed \u0026ldquo;cocktails\u0026rdquo; of individual components were generated comparable\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e or even greater\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e cross-reactivity within the sera than the mosaic displays. Considering our data and these discrepant studies, we suspect that it is possible (if not likely) that the benefits of enriching for conserved epitopes through the display of heterologous strains on the same NP is best stimulated using a greater number of strains while still covering a well-constrained antigenic distance\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. While the heterologous NP groups in our study had comparable or qualitatively greater sera cross-reactivity than did the homologous cocktail groups, statistical significance might require a greater number of incorporated NA strains (\u003cem\u003ee.g.\u003c/em\u003e, \u0026gt;\u0026thinsp;2). We also observed that all groups had decreased strain-specificity at d55 (\u003cem\u003ei.e.\u003c/em\u003e, 28 days post-boost) compared to d13 (\u003cb\u003efig. S21A-B\u003c/b\u003e). This observation may be due to prolonged duration of somatic hypermutation\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, where antigen-reactive B cells in germinal centers generated increased cross-reactivity over time through modulating affinity for the strains incorporated in the immunization\u003csup\u003e\u003cspan additionalcitationids=\"CR61\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePrevious studies showed that NA immunizations protected against viral challenge better when the NA strain was matched rather than heterologous\u003csup\u003e\u003cspan additionalcitationids=\"CR64\" citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Our NA enzymatic inhibition data used as a proxy for protection are consistent with these observations: experimental groups that received B\u0026rsquo;71 or Au\u0026rsquo;75 NA strains outperformed the control J\u0026rsquo;57/J\u0026rsquo;57 group when testing for NA enzymatic inhibition against B\u0026rsquo;71 or Au\u0026rsquo;75 NAs, respectively. However, the results from groups receiving the D\u0026rsquo;21 NA, which did not inhibit D\u0026rsquo;21 NA enzymatic activity better than the control J\u0026rsquo;57/J\u0026rsquo;57 group, highlights a greater nuance. Structural analysis of D\u0026rsquo;21 and J\u0026rsquo;57 NAs suggest that it may be due to the unique glycosylation patterns on D\u0026rsquo;21 NA. D\u0026rsquo;21 NA has three different and two additional predicted N-linked glycosylation sites (PNGs) compared to the J\u0026rsquo;57 NA. In particular, the N245 and N367 PNGs are adjacent to the CS and may have therefore affected the CS-directed antibody responses to D\u0026rsquo;21 NA. Indeed, some previously characterized broadly reactive CS-directed antibodies have reduced binding to NA strains with the N245 glycan\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. Our observations suggest that eliciting sera antibodies against specific epitopes across diverse NA strains might require additional design strategies for epitope-focusing. Rational design strategies like hyperglycosylation and epitope grafting are compatible with these multivalent NoaS NP designs and may be used r to improve humoral focusing to desired a desired NA epitope(s)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe immune response to homologous boosts is often dominated by antibodies generated during the prime immunization\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e, and early influenza exposures can imprint long-lasting immune biases\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. Therefore, we specifically evaluated the NoaS NPs in an immunologically na\u0026iuml;ve cohort to understand how to elicit breadth in the context of primary imprinting. Understanding how complex immune histories, whether initiated through vaccination or infection, influence subsequent responses to the NoaS NPs is necessary. Additionally, understanding how NA antigenic distance influences single B cell responses or memory B cell formation may guide vaccine design. Nevertheless, these data show how rationally designed NoaS NP can advance our understanding of how antigenic distance influences elicited humoral immunity and lays a foundation for next-generation influenza vaccines that incorporate NA strains for improved protection against seasonal and potentially pre-pandemic influenzas.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eStrain Selections\u003c/h2\u003e\u003cp\u003eThe four NA strains used in the Neuraminidase-on-a-String (NoaS) nanoparticles (NPs) were A/Japan/305/1957, A/Bilthoven/21438/1971 (H3N2), A/Darwin/6/2021 (H3N2), and A/tern/Australia/G70C/1975 (H11N9). Strains were selected by performing amino acid alignments of many NA heads and finding strains that together represented a rational distribution of antigenic distances. Strains were then tested for expression as both monomers and paired NoaS dimers. In total, 20 different strains of NA and many neuraminidase-on-a-string combinations were tested. Final selections for the NoaS were based on ease of expression, distribution of antigenic distance, and structural integrity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eCloning of NoaS NPs\u003c/h2\u003e\u003cp\u003eThe NoaS plasmids were engineered using type IIS cloning\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e (with a SAPI enzyme) and cloned into pVRc8400 protein expression vectors. The plasmids coded for the following: t-PA signal sequence, Histidine tag (for affinity purification), HRV 3C protease site (to cleave off the HIS tag), SpyTag sequence\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, first NA head, L3 rigid amino acid linker\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, and, finally, the second NA head (Fig. B.1B). The \u003cem\u003eHelicobacter Pylori\u003c/em\u003e ferritin nanoparticle\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e plasmid coded for an N-terminus Histidine affinity tag, SpyCatcher domain (\u003cb\u003efig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA\u003c/b\u003e), and ferritin protomer (\u003cb\u003efig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA\u003c/b\u003e). All nucleotide sequences were codon optimized and synthesized by IDT. The monomeric versions of these four strains of NA as well as the NA heads of A/Aichi/2/1968 (H3N2), A/Moscow/10/1999 (H3N2), and A/Texas/50/2012 (H3N2) used in the ELISA breadth assay panel, were also codon-optimized and cloned into pVRc vectors.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eExpression and purification of proteins\u003c/h2\u003e\u003cp\u003eSpyTag NoaS dimers and SpyCatcher NP plasmids were transiently expressed with Expi293F cells (ThermoFisher). After 5\u0026ndash;7 days, the cell culture supernatants were harvested by centrifugation. Proteins were purified by affinity chromatography using Cobalt TALON resin (TAKARA) followed by size exclusion chromatography on a Superdex 200 Increase 10/300 GL column (GE Healthcare). The NoaS Dimers were then cleaved using HRV 3C protease (ThermoFisher Pierce) with an overnight incubation at 4\u0026ordm;C. They were eluted over Cobalt TALON resin to remove Histidine tags and HRV 3C protease, repurified over a Superdex 200, and then conjugated in 40x molar excess to previously purified SpyCatcher NPs overnight at 4\u0026ordm;C. The conjugated NoaS NPs were purified a final time using size exclusion chromatography. (IgGs referenced in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC (including the heavy- and light-chain variable domains were synthesized and codon optimized by IDT, then subcloned into pVRc8400 protein expression vectors with human constant regions. They were expressed and purified using the same workflow as the NA antigens.)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eSodium dodecyl sulfate\u0026ndash;polyacrylamide gel electrophoresis (SDS-PAGE)\u003c/h2\u003e\u003cp\u003e3\u0026micro;g of a protein sample, diluted to a final volume of 10\u0026micro;L, were mixed with 10\u0026micro;L of non-reducing 2x Laemmli Sample Buffer (Bio-Rad, Cat#: 1610737), then boiled for 10min at 100\u0026ordm;C. 12.5\u0026micro;L of sample were loaded into wells of a Mini-PROTEAN TGX Stain-Free precast gels (Bio-Rad, Cat#: 456\u0026ndash;8026), then electrophoresed for 18-20min at 280V. Gels were imaged using a ChemiDoc (Bio-Rad) with appropriate standard imaging protocols. For the sera-depletion serum samples in \u003cb\u003efig. S19\u003c/b\u003e, 10\u0026micro;L of concentrated sample were used without prior standardization for protein amount.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eNegative Stain Electron Microscopy\u003c/h2\u003e\u003cp\u003e5\u0026micro;l of the sample (at 5\u0026ndash;10\u0026micro;g/ml) were adsorbed for 60 seconds to a carbon-coated grid (EMS \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.emsdiasum.com\" target=\"_blank\"\u003ewww.emsdiasum.com\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.emsdiasum.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e order# CF400-CU). The grid was rendered hydrophilic via a 20 second exposure to a glow discharge (25mA). Excess liquid was removed with a filter paper (Whatman #1). The grid was then exposed briefly to a water droplet to wash away ions and salts and blotted once again on a filter paper. The grid was then stained with 0.75% Uranyl Formate (EMS catalog # 22451) for 20 seconds. After removing the excess stain with filter paper, the grids were examined in a TecnaiG\u0026sup2; Spirit BioTWIN and images were recorded with an AMT NanoSprint43 CCD camera.\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e immunizations\u003c/p\u003e\u003cp\u003e All animal experiments were approved by the Institutional Animal Care and Use Committees (IACUC) of Harvard University and Massachusetts General Hospital (Protocol #2014N000252) and were conducted in compliance with the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) guidelines. Mice were used at 8\u0026ndash;10 weeks of age. The immunizations were performed using female C57BL/6 mice purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were divided randomly in 7 groups of n\u0026thinsp;=\u0026thinsp;5 and received a prime immunization intraperitoneally on day 0, which consisted of 100uL of inoculum containing 20ug of protein (J\u0026rsquo;57/J\u0026rsquo;57 NoaS NPs as control, NoaS NPs displaying J\u0026rsquo;57 with either B\u0026rsquo;71, D\u0026rsquo;21, or Au\u0026rsquo;75 and a cocktail of homologous J\u0026rsquo;57/J\u0026rsquo;57 NPs with either B\u0026rsquo;71/B\u0026rsquo;71, D'21/D\u0026rsquo;21, or Au\u0026rsquo;75/Au\u0026rsquo;75 NPs, respectively) adjuvanted with 50% v/v Sigma adjuvant (Sigma, Cat#S6322) in sterile PBS. The boost occurred on day 28. Mice were bled from the submandibular vein for sera analyses on day \u0026minus;\u0026thinsp;1 and then every 7 days thereafter (until day 70, when they were euthanized).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eELISA Assays\u003c/h2\u003e\u003cp\u003eSera and monoclonal antibody reactivity to NA antigens were assayed by ELISA. 96-well high binding plates (Corning) were coated with 3\u0026micro;g/ml of NA antigens in PBS buffer at 100\u0026micro;l/well and incubated overnight at 4\u0026ordm;C. Plates were blocked with 1% BSA in PBS containing 0.1% Tween-20 (PBS-T) for 1\u0026ndash;2 hours at room temperature (RT). The blocking solution was then discarded. Plates were coated with sera or monoclonal antibody solutions and incubated at RT for 1.5 hours. (Sera were serially diluted into 1xPBS with a 10\u003csup\u003e0.5\u003c/sup\u003e dilution factor (DF), starting from a dilution of 1:40. Monoclonal antibodies were serially diluted by a 10\u003csup\u003e0.5\u003c/sup\u003e DF into 1xPBS. Starting concentrations for mAbs and sera are noted on ELISA graphs. The final well for each sample contained 1xPBS alone and guided the subtraction of the background signal calculation for each experiment.) Plates were washed 3x with PBS-T. For ELISAs that used murine sera as the source for the primary antibodies, the secondary antibody was rabbit pAb anti-mouse IgG-HRP (Abcam, AB97046). The secondary was added at 1:20,000 dilution (in PBS) on d34, d48, and d55, and it was added at 1:16000 on d13, d20, and d62. The secondary antibody solution was coated onto the plates for 1 hour at RT. For monoclonal antibodies, secondary goat pAb anti-human IgG-HRP (Abcam, AB97225) was added at 1:20,000 dilution (in PBS) for 1 hour at RT. Plates were washed three times with PBS-T and developed with 1-step ABTS Substrate Solution (ThermoFisher) for 45 minutes. 405nm absorbance values were measured using a plate reader. EC\u003csub\u003e50\u003c/sub\u003e values were determined by non-linear regression (sigmoidal) using GraphPad Prism 10.2.3 software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eSera Depletion Assays\u003c/h2\u003e\u003cp\u003eA/Japan/305/1957 (J\u0026rsquo;57), A/Bilthoven/21438/1971 (B\u0026rsquo;71), A/Darwin/6/2021 (D\u0026rsquo;21), and A/tern/Australia/G70C/1975 (Au\u0026rsquo;75) strains were cloned with N-terminus StrepII, i.e., TwinStrep, sequences, transiently expressed, and purified as described above. For the sera depletion assays, 65 columns of 500uL of Strep-Tactin\u0026reg;XT resin were prepared in 15mL gravity-flow columns and washed with PBS buffer. Based on prior studies related to antibody production kinetics\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e, excess StrepII tagged antigen was incubated with the Strep-Tactin resin for 1 hour, then eluted. Columns 1\u0026ndash;35 were incubated with J\u0026rsquo;57 NA antigen. Columns 36\u0026ndash;45 were incubated with B\u0026rsquo;71 NA antigen. Columns 46\u0026ndash;55 were incubated with D\u0026rsquo;21 NA antigen. Columns 56\u0026ndash;65 were incubated with Au\u0026rsquo;75 NA antigen. Separately, 555uL of PBS were mixed with 45\u0026micro;L of sera collected on d13 from each mouse for a 1/13.3 sera dilution. This was used as the stock of sera for each mouse for a given depletion experiment to minimize any experimental fluctuation due to pipetting. For mice m1-m35, 200uL of the diluted sera were incubated with the washed J\u0026rsquo;57-tagged Strep-Tactin resin columns 1\u0026ndash;35, respectively. 200\u0026micro;L of m6-m15 sera mixtures were incubated with columns 36\u0026ndash;45, respectively. 200\u0026micro;L of m16-m25 sera mixtures were incubated with columns 46\u0026ndash;55, respectively. Lastly, 200\u0026micro;L of m26-m55 sera mixtures were incubated with columns 56\u0026ndash;65, respectively. After 2\u0026ndash;4 hours at 4\u0026ordm;C incubation, serum was eluted from each column and collected along with a 100\u0026micro;L of PBS column wash into the first rows of a 96-well PCR plate. The final concentration of the strain-depleted sera was therefore 1/20. These depleted sera were then serially diluted using a factor of 10\u003csup\u003e0.5\u003c/sup\u003e for ELISA testing. Pre-depletion serum was taken from the same 1/13.3 diluted serum stock by taking 200\u0026micro;L of diluted serum and further diluting it with 100uL PBS for a final dilution of 1/20. The same was done for the sera that were collected on d55.\u003c/p\u003e\u003cp\u003ePre-depletion serum and post J\u0026rsquo;57-depletion serum from m1-m5 were tested for reactivity against the J\u0026rsquo;57 strain (\u003cb\u003efigs. S9, S14\u003c/b\u003e). The pre-depletion reactivity established the baseline J\u0026rsquo;57 reactivity value, and the post J\u0026rsquo;57 depletion reactivity was used as an internal control to verify successful J\u0026rsquo;57 depletion (\u003cem\u003ee.g.\u003c/em\u003e, \u003cb\u003efig. S13E\u003c/b\u003e). Mice m1-m5 were also tested pre-depletion for reactivity to B\u0026rsquo;71, D\u0026rsquo;21, and Au\u0026rsquo;75 (\u003cb\u003efig. S22\u003c/b\u003e), respectively, to establish baseline levels of cross-reactivity in the control group. Mice m1-m5 were tested post J\u0026rsquo;57-depletion for any residual reactivity to B\u0026rsquo;71, D\u0026rsquo;21, and Au\u0026rsquo;75 (\u003cb\u003efigs. S9, S14\u003c/b\u003e). Mice m6-m5 were tested pre-depletion for their reactivity to J\u0026rsquo;57 and B\u0026rsquo;71, post J\u0026rsquo;57-depletion for their reactivity to J\u0026rsquo;57 (internal control) and B\u0026rsquo;71 (\u003cem\u003ei.e.\u003c/em\u003e, B\u0026rsquo;71 strain-specificity), and post B\u0026rsquo;71-depletion for their reactivity to J\u0026rsquo;57 (\u003cem\u003ei.e.\u003c/em\u003e, J\u0026rsquo;57 strain-specificity) and B\u0026rsquo;71 (internal control). Analogous experiments were done for mice m16-m25 using J\u0026rsquo;57 and D\u0026rsquo;21 NAs, and for mice m26-m35 using J\u0026rsquo;57 and Au\u0026rsquo;75 NAs.\u003c/p\u003e\u003cp\u003eTo minimize any variation in OD\u003csub\u003e405nm\u003c/sub\u003e absorbance due to antigen coating, the same stock of 3\u0026micro;g/mL antigen concentration was used for all tested groups. Additionally, the 96-well (Corning) plates were organized to have all samples tested for reactivity against a given antigen on the same plates. For example, for the m6 serum that was tested against B\u0026rsquo;71, an entire plate was coated using the same B\u0026rsquo;71 3\u0026micro;g/mL diluted stock. Column 1 tested m6 pre-depletion serum vs. B\u0026rsquo;71, Column 2 tested m6 post-J\u0026rsquo;57 depletion vs. B\u0026rsquo;71, and Column 3 tested m6 post-B\u0026rsquo;71 depletion vs. B\u0026rsquo;71, etc. Since the number of tested samples for a given antigen exceeded one 96-well plate, the OD\u003csub\u003e405nm\u003c/sub\u003e absorbance values were furthermore normalized for each antigen by dividing each OD\u003csub\u003e405nm\u003c/sub\u003e data point by the average of the top 10 absorbance values for a given antigen.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eELLA inhibition Assays\u003c/h2\u003e\u003cp\u003eTo test the ability of the sera to inhibit the NA activity of a given strain, tetramers of each strain were made, as NA tetramers have much greater catalytic activity than NA monomers\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e,\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. J\u0026rsquo;57 NA was tetramerized using the tetrabrachion\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e domain. B\u0026rsquo;71, D\u0026rsquo;21, and Au\u0026rsquo;75 NAs were tetramerized using VASP\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e domains. (Choice of tetramerization domain was based solely on which domain helped the protein express better and more homogeneously.) Proteins were transiently expressed and purified as described above for the monomeric antigens. The EC\u003csub\u003e50\u003c/sub\u003e of each NA activity was then determined. This was done through the following experiment setup: 100\u0026micro;L of 25\u0026micro;g/mL of fetuin (Sigma Aldrich, F3004) were coated onto 96-well plates and incubated overnight at 4\u0026deg;C. Plates were blocked with PBS, 1% BSA, 0.1% Tween-20 solutions and put on a shaker for 2 hours at RT. Plates were then washed 6x. Next, 100\u0026micro;L of recombinant NA\u003csub\u003eTB\u003c/sub\u003e, serially diluted 1:2 in DPBS with Ca/Mg (Gibco, Cat#: 14040117) with a starting concentration of 0.005\u0026micro;g/mL were added to the plate in columns 1\u0026ndash;11. Column 12 was plated with DPBS alone. Plates were then placed in a 37\u0026deg;C incubator for 2 hours and subsequently washed with ELISA buffer 6x. Plates were incubated with 100\u0026micro;L of either 1 or 5\u0026micro;g/mL of HRP-conjugated lectin from Arachis hypogaea (peanut) (Sigma Aldrich, L7759) and incubated for 75-100min at RT on a plate shaker. Plates were then washed 6x and coated with 100uL of ABTS. After 45 min, plates were imaged. EC\u003csub\u003e50\u003c/sub\u003e values of NA tetramers were determined by non-linear regression (sigmoidal) using GraphPad Prism 10.2.3 software. On d20, monomeric B\u0026rsquo;71 was used instead tetrameric B\u0026rsquo;71 due to poor tetramer expression, but the EC\u003csub\u003e50\u003c/sub\u003e of the monomer was determined in the exact same way.\u003c/p\u003e\u003cp\u003eThe ELLA inhibition assay was based on a standard protocol\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. 100\u0026micro;L of 25\u0026micro;g/mL of fetuin (Sigma Aldrich, F3004) were coated onto 96-well plates and incubated overnight at 4\u0026deg;C. Plates were blocked with PBS, 1% BSA, 0.5% Tween-20 solutions and put on a shaker for 2 hours at RT. Plates were then washed 6x. A stock of 2x the EC\u003csub\u003e50\u003c/sub\u003e of each strain was prepared and 65\u0026micro;L of solution was added to each well of a 96well PCR plate. Separately, 28\u0026micro;L of each tested mouse serum sample were added to 252\u0026micro;L of PBS with calcium, for a serum concentration of 1/10th. Serum was then serially diluted 1:2 down columns 1\u0026ndash;11. Column 12 contained PBS with calcium. 65\u0026micro;L were aliquoted into the same 96 well plate as the 65\u0026micro;L of NA solution and pipetted 3x. After 5\u0026ndash;10 minutes, 120\u0026micro;L of the sera/NA mixture were added to the fetuin-coated plate. [Sera from m1-m5 and a d0 sera negative control were tested against J\u0026rsquo;57, B\u0026rsquo;71, D\u0026rsquo;21, and Au\u0026rsquo;75. Sera from m6-m15 and a d0 sera negative control were tested against J\u0026rsquo;57 and B\u0026rsquo;71. Sera from m16-m25 and a d0 sera negative control were tested against J\u0026rsquo;57 and D\u0026rsquo;21. Finally, sera from m26-m35 and a d0 sera negative control were tested against J\u0026rsquo;57 and A\u0026rsquo;75.] Plates were placed into a 37\u0026deg;C for 90min, except for plates testing strain B\u0026rsquo;71, which incubated for 2hrs to give more time for NA activity to cleave the substrate. Plates were then washed 6x and coated with 100\u0026micro;L of HRP-lectin at 2\u0026micro;g/ml. They were then put on a RT shaker for 90 min. After washing the plates 6x, plates were coated with 150\u0026micro;L of ABTS. Plates were then imaged after exactly 45 min. Non-linear (sigmoidal) curves were generated for each serum sample using GraphPad Prism 10.2.3 software The loss of OD\u003csub\u003e405nm\u003c/sub\u003e signal observed at the highest sera concentration (\u003cem\u003ei.e.\u003c/em\u003e, dilution factor of 20) for the d0 negative control was used as the threshold for what would be considered \u0026lsquo;real\u0026rsquo; signal due to specific sera antibody inhibition of NA rather than just the sera providing diffusion/viscosity inhibition of the NA activity. Sera from mice were then evaluated for the highest dilution factor at which the sera still outperformed the maximum inhibition of the d0 negative control. This was termed the sample\u0026rsquo;s MDFI, or the maximal dilution factor at which there was observable inhibition. Experimental group MDFIs were compared to the control. To avoid an observable hook-effect that impacted the regression analyses, all experiments for all samples were evaluated up until a dilution factor of 1280.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eStatistics\u003c/h2\u003e\u003cp\u003eAll analyses that compared data between only 2 groups of mice were performed using the unpaired, non-parametric Mann-Whitney test with an alpha value of 0.05. Analyses that compared the mean rank of one group to the mean rank of more than one group used the unpaired, non-parametric Kruskal-Wallis test with Dunn correction for multiple comparisons. The p-values cutoffs for significance were the following: 0.0332 (*), 0.0021 (**), 0.0002 (***), \u0026lt; 0.0001(****).\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Maria Ericsson and the Harvard Electron Microscopy Core for collection of negative stain images. We thank Daniel Maurer and Emerson Glassey for helpful discussions. We acknowledge support from NIGMS T32 GM0008313, NIH 1F30 AI181355 (to R.H.), NIH P01 AI089618 (A.G.S.), NIH\u0026nbsp;AI155447, AI137057, AI153098\u0026nbsp;AI193280\u0026nbsp;(DL). This research has been funded in whole or part with federal funds under a contract from the National Institute of Allergy and Infectious Diseases, NIH contract 75N93019C00050 (A.G.S.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eR.H., A.G.S., and D.L. designed research; R.H. and F.A.N.M. performed research; R.H. and A.G.S. analyzed data; R.H. and A.G.S. wrote the paper. All authors commented on the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence and requests for materials should be addressed to:\u003c/strong\u003e Aaron G. Schmidt ([email protected])\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting financial interest:\u0026nbsp;\u003c/strong\u003eNo competing financial interests.\u0026nbsp;\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eR.H., A.G.S., and D.L. designed research; R.H. and F.A.N.M. performed research; R.H. and A.G.S. analyzed data; R.H. and A.G.S. wrote the paper. All authors commented on the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbduljaleel, Z. Decoding SARS-CoV-2 variants: Mutations, viral stability, and breakthroughs in vaccines and therapies. \u003cem\u003eBiophys. Chem.\u003c/em\u003e 320\u0026ndash;321, 107413 (2025).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMaurer, D. P., Vu, M. \u0026amp; Schmidt, A. G. 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Multivalent displays of viral proteins can improve B cell activation due to receptor cross-linking, and mosaic nanoparticles that incorporate variants can lead to cross-reactive B cell responses recognizing conserved epitopes. Here, we used the influenza virus neuraminidase to develop a neuraminidase-on-a-string platform displaying neuraminidase dimer pairs conjugated to a nanocarrier To systematically assess the influence of antigenic distance on humoral immunity, we paired H2N2 neuraminidase with either divergent H3N2 or H11N9 neuraminidases. We found that nanoparticle immunizations with heterologous antigens elicited sera with greater breadth and enhanced enzymatic inhibition relative to immunizations that incorporated a single neuraminidase strain. While sera reactivity for H2N2 neuraminidase was not impacted by inclusion of a second strain, strain-specific responses correlatively increased with the antigenic distance between neuraminidase components. These data show how neuraminidase strain selection for multivalent display immunizations influences elicited breadth and cross-reactivity, highlighting findings that may extend to other viral antigens.\u003c/p\u003e","manuscriptTitle":"Neuraminidase-on-a-string nanoparticles probe how antigenic distance shapes elicited humoral immunity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-03 18:22:36","doi":"10.21203/rs.3.rs-7530142/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"feed097c-ff29-48cf-9722-4fc1f8c96c6a","owner":[],"postedDate":"November 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":57278666,"name":"Biological sciences/Biochemistry"},{"id":57278667,"name":"Biological sciences/Biotechnology"},{"id":57278668,"name":"Biological sciences/Immunology"}],"tags":[],"updatedAt":"2026-03-08T16:54:57+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-03 18:22:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7530142","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7530142","identity":"rs-7530142","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}