Influenza outbreaks arise from viruses that evade human immunity. Advances in influenza virus structural biology, nanotechnology, and gene delivery offer new opportunities to develop improved vaccines that can confer more broadly protective immunity against diverse influenza viruses^4–6,8,9. Among recent innovations, several natural proteins have shown the ability to form nanoparticles well-suited for antigen presentation and immune stimulation¹⁰. One such protein is ferritin, a ubiquitous iron storage protein that self-assembles into nanoparticles³. Though ferritin has been used to display exogenous peptides¹¹, it has not been possible to display viral glycoproteins because of their complexity and requirements for trimerization. Additionally, recombinant ferritins made in prokaryotic cells were not subjected to mammalian glycosylation and other posttranslational modifications typical of viral proteins^11–13. Structural analysis of ferritin suggested that it would be possible to insert a heterologous protein, specifically influenza virus HA, so that it could assume the physiologically relevant trimeric viral spike (Fig. 1a). Ferritin forms a nearly spherical particle composed of 24 subunits arranged with octahedral symmetry around a hollow interior. The symmetry includes eight three-fold axes on the surface. The aspartic acid (Asp) at residue 5 near the NH₂ terminus is readily solvent accessible, and the distance (28 Å) between each Asp5 on the three-fold axis is almost identical to the distance between the central axes of each HA2 subunit of trimeric HA (Fig. 1a, right). We therefore hypothesized that HA would trimerize properly if inserted into this structure.

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Figure 1.

Molecular design and characterization of ferritin nanoparticles displaying influenza virus HA.

a, A subunit of H. pylori nonheme ferritin (PDB: 3bve) (left). The NH₂- and COOH-termini are labeled as N and C, respectively. Three subunits surrounding a three-fold axis are shown (middle) and the Asp5 is colored in red. An assembled ferritin nanoparticle and an HA trimer (PDB: 3sm5) (viewed from membrane proximal end) (right). A triangle connecting the Asp 5 residues at the three-fold axis is shown in red. The same triangle is drawn on the HA trimer (right). A schematic representation of the HA-ferritin fusion protein is shown (bottom). b, Negatively stained TEM images of nanoparticles (np) (left and middle). Computational models and observed TEM image (right, top and bottom panels) representing octahedral 2-, 3- and 4-fold symmetries of HA-nanoparticles are shown as indicated. Visible HA spikes are numbered in the images.

To test this hypothesis, we genetically fused the ectodomain of A/New Caledonia/20/1999 (1999 NC) HA to Helicobacter (H.) pylori nonheme ferritin¹⁴ (Fig. 1a, bottom), a ferritin that diverges highly from its mammalian counterparts (Supplementary Fig. 1). This fusion protein was expressed in mammalian cells, and self-assembly of ferritin and HA-ferritin nanoparticles was confirmed by size exclusion chromatography and dynamic light scattering (Supplementary Fig. 2a, b). HA-ferritin also had the expected apparent molecular weight of 85 kDa (Supplementary Fig. 2c). While ferritin alone formed smooth spherical particles as visualized by transmission electron microscopy (TEM), HA-ferritin exhibited clearly visible spikes protruding from the spherical core (Fig. 1b, Ferritin np vs. HA-np). Remarkably, the placement of these spikes illustrated the octahedral symmetry of the HA-nanoparticle design. Octahedral two-, three- and four-fold axes were distinctly observed in the TEM image (Fig. 1b, right). These data demonstrated the formation of trimeric HA spikes on self-assembling nanoparticles.

To verify the antigenicity of the HA spikes on the nanoparticles, we analyzed their reactivity with an anti-HA head monoclonal ab (mAb) and a conformation-dependent mAb, CR6261, which recognizes a conserved structure on the HA stem⁴. The HA-nanoparticle binds to anti-head and anti-stem mAbs with affinities similar to trimeric HA or trivalent inactivated influenza vaccine (TIV) containing the same 1999 NC HA at equimolar concentrations of HA (Supplementary Fig. 3a). Analogous to trimeric HA, the HA-nanoparticle also blocked neutralization by CR6261 and another stem-directed mAb, F10 (ref. 5) (Supplementary Fig. 3b). These results confirmed that HA molecules on the HA-nanoparticle antigenically resembled the physiological HA viral spike.

To assess the immunogenicity of the HA-nanoparticle, mice were immunized twice with TIV or HA-nanoparticles with or without Ribi adjuvant. The HA-nanoparticles induced 1.6-fold higher HAI titers than TIV in the absence of adjuvant. While this increase did not reach statistical significance, HAI titers were 7.2-fold higher in animals receiving HA-nanoparticles when Ribi was used (Fig. 2a, left; p<0.0001), and a similar effect was observed in the neutralization and ELISA titers (Fig. 2a, middle and right; p<0.0001). For example, neutralization titers elicited by HA-nanoparticles as assessed by the concentration of ab needed to inhibit viral entry by 90% (IC₉₀) were ~34 times higher than TIV (Fig. 2a, middle). We also evaluated whether a similar immune response can be induced by HA-nanoparticles with MF59, an adjuvant that has been used in humans¹⁵. Similarly high HAI and IC₉₀ titers were observed in mice receiving MF59-adjuvanted HA-nanoparticles (4,608±512 and 47,140±22,561, respectively), and the titers were significantly higher than those induced by either non-adjuvanted HA-nanoparticles (p=0.0005 and 0.0311 for HAI and IC₉₀, respectively) or MF59-adjuvanted TIV (p=0.0016 and 0.0388 for HAI and IC₉₀, respectively) (Fig. 2b). These results demonstrated the feasibility of using the HA-nanoparticles with an adjuvant suitable for humans¹⁶. Because higher titers were observed using either adjuvant, further comparisons were performed with adjuvant (Ribi). Neutralization against a panel of H1N1 strains revealed not only increased potency, but also enhanced breadth, stimulated by HA-nanoparticles compared with TIV or trimeric HA (Fig. 2c). Neutralization against two unmatched, highly divergent H1N1 strains, A/Puerto Rico/8/1934 (1934 PR8) and A/Singapore/6/1986 (1986 Sing), was only observed in mice immunized with the HA-nanoparticles, and the titer against the contemporary strain A/Brisbane/59/2007 (2007 Bris) was more than 10-fold higher in mice immunized with HA-nanoparticles than with TIV (Fig. 2c).

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Figure 2.

Immune responses in HA-nanoparticle-immunized mice.

a, HAI (left), IC₉₀ neutralization (middle), and anti-HA ab endpoint titers (right) after two immunizations of TIV or HA-nanoparticles with or without (−) Ribi. b, HAI (left) and IC₉₀ (right) titers after two immunizations of TIV with MF59 or HA-nanoparticles with or without (−) MF59. Two of five mice immunized with MF59-adjuvanted HA-nanoparticles exhibited IC₉₀ titer >51,200 and were plotted as 51,200. The data are presented as box-and-whiskers plots (boxed from lower to upper quartile with whiskers from minimum to maximum) with lines at the mean (n=5). c, Neutralization breadth of the immune sera (with Ribi). IC₅₀ titers against a panel of H1N1 pseudotyped viruses were determined. Heat map is colored in gradient from green to yellow to red reflecting the neutralization strength. d, Cellular (left and middle) and humoral (right) immune responses against H. pylori and mouse ferritins. Cells expressing IFN-γ, TNFα, or IL-2 upon stimulation with peptides covering H. pylori or mouse ferritins were combined and plotted as cytokine⁺. The data are presented as box-and-whiskers plots with lines at the mean (n=5).

We next examined whether preexisting immunity to ferritin or to other HA subtypes would interfere with subsequent immunization using HA-nanoparticles. Mice pre-immunized with either H3 (A/Perth/16/09, 2009 Perth) HA-nanoparticles or empty ferritin nanoparticles generated substantial anti-H3 HA and/or anti-H. pylori ferritin ab responses (Supplementary Fig. 4a). They were then immunized with H1 (1999 NC) HA-nanoparticles. Comparable HAI, IC₉₀ and ELISA titers against 1999 NC HA were observed in naïve animals as well as in groups pre-immunized with H3 HA-nanoparticles or empty ferritin nanoparticles (Supplementary Fig. 4b). These results indicated that preexisting anti-H. pylori ferritin immunity did not diminish the HA-specific ab response.

To address the concern that immunization with ferritin might abrogate immune tolerance and induce autoimmunity, we analyzed T-cell and ab responses against murine and H. pylori ferritins in HA-nanoparticle-immunized mice. While we found an increase in intracellular cytokine staining (ICS) of CD4⁺ T cells stimulated with H. pylori ferritin peptides (Fig. 2d, left), no increase in CD4⁺ or CD8⁺ ICS responses to murine ferritin peptides were observed (Fig. 2d, left and middle). In addition, abs to H. pylori ferritin, but not to mouse ferritin, were detected in the immune sera (Fig. 2d, right). We therefore found no evidence of immunity to autologous ferritin in mice. Moreover, the HA-nanoparticles are unlikely to affect iron homeostasis in vivo because of their minimal iron incorporation activity (Supplementary Fig. 5).

We next generated a trivalent vaccine comprising three, separately purified HA-nanoparticles formulated into a single vaccine dose, analogous to a standard TIV. The strains chosen were H1 (A/California/04/09, 2009 CA), H3 (2009 Perth) and influenza B (B/Florida/04/06, 2006 FL). All three HA-nanoparticles self-assembled and displayed morphology similar to 1999 NC HA-nanoparticles (Supplementary Fig. 6a). Immunogenicity of multispecific HA-nanoparticles (combination of three monospecific HA-nanoparticles) was compared to a seasonal TIV containing the same H1 and H3 strains and a mismatched influenza B (B/Brisbane/60/08). HAI titers against homologous H1N1 and H3N2 viruses were significantly increased in animals immunized with multispecific HA-nanoparticles relative to TIV (Supplementary Fig. 6b; p=0.0125 and 0.0036, respectively). When compared to animals immunized with the corresponding monospecific HA-nanoparticles, HAI titers against H1N1 and H3N2 viruses induced by multispecific HA-nanoparticles were comparable (Supplementary Fig. 6b). Therefore no substantial antigenic competition was observed with the multispecific HA-nanoparticle vaccine.

We next examined the immunogenicity of HA-nanoparticles (1999 NC) in ferrets. Three weeks after the first immunization, all ferrets receiving Ribi-adjuvanted HA-nanoparticles generated protective HAI titers against homologous virus (>40), while only 50% (3/6) of Ribi-adjuvanted TIV-immunized ferrets induced titers greater than 40 (Fig. 3a, left; p=0.0056). The same difference was also observed for both neutralization and ELISA titers (Fig. 3a, middle and right; p=0.0047 and p=0.0045, respectively), documenting the potency of HA-nanoparticles in a second species. After boosting, the HAI, IC₉₀ and ELISA titers of the HA-nanoparticle-immune sera were ~10-fold higher than those of TIV-immune sera (Fig. 3a, left, middle and right; 457±185 vs. 5,760±1,541, p=0.0066; 598±229 vs. 5,515±1,074, p=0.0012; and 5,902±1,851 vs. 55,105±13,018, p=0.0038, respectively). Remarkably, a single immunization of HA-nanoparticles induced immune responses comparable to two immunizations of TIV (Fig. 3a).

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Figure 3.

Protective immunity induced in ferrets immunized with the HA-nanoparticles.

a, HAI (left), IC₉₀ (middle), and anti-HA ab endpoint titers (right) against 1999 NC HA. Immune sera were collected after the first (1) and second (2) immunizations. The data are presented as box-and-whiskers plots with lines at the mean (n=6). b, Protection of immunized ferrets from 2007 Bris virus challenge. Challenge was performed with 10^6.5 EID₅₀ via intranasal inoculation. Virus titers in the nasal washes were determined by TCID₅₀ assay (left). The mean viral loads with s.d. at each time point were plotted (n=6). Change in body weight after virus challenge was monitored (right). Each data point represents the mean percent change in body weight from day 0 (pre-challenge) with s.e.m. (n=6).

To determine whether HA-nanoparticles could confer protection against an unmatched H1N1 virus, immunized ferrets were challenged with 2007 Bris virus, which had not yet evolved when 1999 NC circulated and required a different seasonal vaccine to confer protection in humans. Ferrets immunized with HA-nanoparticles showed a significant reduction in viral shedding beginning 1 day after challenge compared to the sham control group (Fig. 3b, left; p=0.0259). At the same time point, no significant reduction in viral shedding was seen in the TIV-immunized group (Fig. 3b, left; p=0.4665). In addition, HA-nanoparticle-immunized ferrets suffered less weight loss compared to the TIV-immunized and sham control animals (Fig. 3b, right), further demonstrating the protective efficacy of HA-nanoparticles.

Interestingly, unlike the TIV-immune sera which preferentially neutralized homologous 1999 NC and another closely related strain (A/Beijing/262/1995, 1995 Beijing), sera from HA-nanoparticle-immunized ferrets broadly neutralized heterologous 1986 Sing, 1995 Beijing, A/Solomon Islands/3/2006 (2006 SI) and 2007 Bris viruses (Fig. 4a, left). The recent identification of two classes of broadly nAbs (bnAbs) that target the highly conserved but vulnerable regions of HA suggests a potential pathway to develop influenza vaccines with broad coverage¹⁷. One class of bnAbs recognizes a hydrophobic groove on the HA stem and neutralizes virus by inhibiting membrane fusion^4,5,18–21. The second class recognizes the RBS on the HA head and inhibits viral entry^6,7,22. To determine whether the cross-reactivity across diverse H1N1 strains induced by HA-nanoparticles included nAbs to the HA stem epitope, ferret immune sera were pre-absorbed with cells expressing a stem mutant (ΔStem)⁸ HA to remove non-stem-directed abs and analyzed for binding to wild-type (WT) or ΔStem HA as previously described⁸. Stem-specific abs were detected in HA-nanoparticle-immunized ferrets (6/6) in greater frequency and magnitude than TIV-immunized ferrets (2/6) (Fig. 4b, left; p=0.0056). Moreover, binding of these pre-absorbed sera to HA was reduced by CR6261 (Fig. 4b, right; p=0.0019), further documenting the presence of abs targeting the same epitope as CR6261. The HAI titers against heterologous 2007 Bris virus were also significantly higher in ferrets immunized with HA-nanoparticles (6/6) than with TIV (3/6) (Fig. 4a, right; p=0.0054). Interestingly, HA-nanoparticle-immune sera have HAI responses against a highly divergent 1934 PR8 strain, with titers ≥40 in all ferrets. However, no HAI titers against 1934 PR8 were detected in TIV-immunized ferrets (Fig. 4a, right). These data suggested that the HA-nanoparticles might elicit another class of nAb directed towards the conserved RBS in the HA head. To dissect the specificity of the RBS-directed ab response, we generated an RBS mutant HA (ΔRBS) by introducing a glycosylation site in the sialic acid binding pocket at residue 190 (Supplementary Fig. 7)²³. Ferret immune sera were absorbed with ΔRBS HA-expressing cells to remove abs to HA outside of this region and tested for binding against WT or ΔRBS HA. RBS-directed abs were detected with titers of >2,000 in all HA-nanoparticle-immunized ferrets, but only in 1 of 6 ferrets that received TIV (Fig. 4b, middle).

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Figure 4.

Improved neutralization breadth and detection of stem- and RBS-directed abs.

a, Breadth of serum neutralization in immune ferrets. IC₅₀ titers against a panel of H1N1 pseudotyped viruses (left) and HAI titers against 1934 PR8 and 2007 Bris H1N1 viruses (right) were determined. Heat map is colored as in Figure 2. HAI titers are presented as box-and-whiskers plots with lines at the mean (n=6). b, Stem- and RBS-directed abs elicited by HA-nanoparticle immunization. Immune sera were pre-absorbed with ΔStem (left) and ΔRBS (middle) HA-expressing cells and analyzed for their binding to WT and a respective mutant (Δ) HA. The mean endpoint titers were plotted with s.d. (n=6). Binding of ΔStem HA pre-absorbed immune sera to HA pre-incubated with a control or CR6261 mAbs (right). Each symbol represents the titer of an individual ferret (n=6). c, Neutralization competition with WT, ΔStem or ΔRBS HA (left). The neutralization of HA-nanoparticle-immune sera was measured in the presence of indicated competitor proteins. Percent neutralizations at serum dilution 1/200 (1986 Sing and 2007 Bris), 1/800 (1995 Beijing) or 1/3,200 (1999 NC) were plotted. Each symbol represents an individual ferret and mean is indicated as a red line with s.d. (n=6 except for 2007 Bris (n=3)). The relative contributions of stem- and RBS-directed neutralization were calculated and plotted as mean percent (n=6).

To define the relative contributions of stem- and RBS-directed abs to the breadth of neutralization, we performed neutralization assays in the presence of competitor proteins: WT, ΔStem or ΔRBS HA. In the presence of excess ΔStem HA, only stem-directed abs can neutralize viruses; similarly, ΔRBS HA interferes with all abs except those targeting the RBS. Four H1N1 strains were tested in this assay and the pattern of neutralization inhibition varied by strain. Neutralization of 1999 NC or 2007 Bris was mediated predominantly by RBS-directed abs. However, neutralization of 1986 Sing was due mainly to stem-directed abs. Interestingly, both stem- and RBS-directed abs contributed to neutralize 1995 Beijing virus (Fig. 4c). While the neutralization specificities of mouse and ferret abs differ somewhat from previously described human abs, we have observed these differences previously⁸. This variation is most likely due to differences in the origins of the VH genes that give rise to them and affect their fine specificity²³. In particular, anti-stem abs isolated from humans derive predominantly from the VH1-69 gene¹⁹, which is not present in other species. Even different human VH1-69 abs show differences in breadth and fine specificity among influenza subtypes¹⁹.

Based on the premise that highly ordered repetitive arrays induce yet stronger immune responses²⁴, we have successfully designed an HA-nanoparticle to present trimeric HA spike in its native fold, rigidly and symmetrically, with sufficient spacing to ensure optimal access to potential bnAbs directed to the stem. These nanoparticles not only had the desired physical properties but also enhanced the potency and breadth of nAb responses compared to TIV, the current commercial vaccine, directed to two independent highly conserved epitopes. Although not yet a universal influenza vaccine, these nanoparticles provide a major increment in influenza protection by eliciting potent nAbs against a broad spectrum of H1N1 viruses. Moreover, the synthetic nanoparticles are fully recombinant, eliminating the need to produce potentially dangerous virus in eggs or in cell culture, and allowing for modifications that improve immunogenicity which would otherwise not be tolerated in replication-competent viruses currently used for vaccines. HA-nanoparticle technology therefore represents a foundation for a new generation of influenza vaccines and could be adapted to create analogous vaccines for a wide variety of pathogens.