Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Apr;592(7855):623-628.
doi: 10.1038/s41586-021-03365-x. Epub 2021 Mar 24.

Quadrivalent influenza nanoparticle vaccines induce broad protection

Seyhan Boyoglu-Barnum #  1 , Daniel Ellis #  2   3   4 , Rebecca A Gillespie  1 , Geoffrey B Hutchinson  1 , Young-Jun Park  3 , Syed M Moin  1 , Oliver J Acton  3   5 , Rashmi Ravichandran  2   3 , Mike Murphy  2   3 , Deleah Pettie  2   3 , Nick Matheson  2   3 , Lauren Carter  2   3 , Adrian Creanga  1 , Michael J Watson  6 , Sally Kephart  6 , Sila Ataca  1 , John R Vaile  1 , George Ueda  2   3 , Michelle C Crank  1 , Lance Stewart  2   3 , Kelly K Lee  6 , Miklos Guttman  6 , David Baker  2   3   7 , John R Mascola  1 , David Veesler  3 , Barney S Graham  8 , Neil P King  9   10 , Masaru Kanekiyo  11
Affiliations

Quadrivalent influenza nanoparticle vaccines induce broad protection

Seyhan Boyoglu-Barnum et al. Nature. 2021 Apr.

Abstract

Influenza vaccines that confer broad and durable protection against diverse viral strains would have a major effect on global health, as they would lessen the need for annual vaccine reformulation and immunization1. Here we show that computationally designed, two-component nanoparticle immunogens2 induce potently neutralizing and broadly protective antibody responses against a wide variety of influenza viruses. The nanoparticle immunogens contain 20 haemagglutinin glycoprotein trimers in an ordered array, and their assembly in vitro enables the precisely controlled co-display of multiple distinct haemagglutinin proteins in defined ratios. Nanoparticle immunogens that co-display the four haemagglutinins of licensed quadrivalent influenza vaccines elicited antibody responses in several animal models against vaccine-matched strains that were equivalent to or better than commercial quadrivalent influenza vaccines, and simultaneously induced broadly protective antibody responses to heterologous viruses by targeting the subdominant yet conserved haemagglutinin stem. The combination of potent receptor-blocking and cross-reactive stem-directed antibodies induced by the nanoparticle immunogens makes them attractive candidates for a supraseasonal influenza vaccine candidate with the potential to replace conventional seasonal vaccines3.

PubMed Disclaimer

Conflict of interest statement

Competing interests S.B.B., D.E., R.A.G., G.U., B.S.G., N.P.K., and M.K. are listed as inventors on a patent application based on the studies presented in this paper. D.V. is a consultant for Vir Biotechnology Inc. The Veesler laboratory has received an unrelated sponsored research agreement from Vir Biotechnology Inc. N.P.K. is a co-founder, shareholder, and chair of the scientific advisory board of Icosavax, Inc. The King laboratory has received an unrelated sponsored research agreement from Pfizer. D.B. is a co-founder and shareholder of Icosavax, Inc. All other authors declare no competing interests.

Figures

Extended Data Fig. 1 |
Extended Data Fig. 1 |. Production and characterization of HA-I53_dn5 components and nanoparticle immunogens.
a, SEC purification of seasonal HAs fused to I53_dn5B trimeric components, using a Superdex 200 Increase 10/300 GL column. b, Reducing and non-reducing SDS-PAGE of SEC-purified trimeric HA-I53_dn5B fusions, pentameric I53_dn5A component, and I53_dn5B trimer lacking fused HA. c, SEC purification of nanoparticle immunogens after in vitro assembly, including I53_dn5 lacking displayed antigen, using a Superose 6 Increase 10/300 GL column. The nanoparticle immunogens elute at the void volume of the column, while I53_dn5 is resolved. Residual, unassembled trimeric and pentameric components elute around 15 mL and 18 mL, respectively. d, Reducing and non-reducing SDS-PAGE of SEC-purified nanoparticle immunogens and I53_dn5. e, Antigenic characterization of purified nanoparticle immunogens by ELISA. Symbols indicate the specificity of each mAb. AUC, area under the curve. f, Analytical SEC of purified nanoparticle immunogens, compared to I53_dn5 nanoparticles lacking displayed antigen and trimeric H1-I53_dn5B, using a Sephacryl S-500 HR 16/60 column. g, Dynamic light scattering (DLS) of SEC-purified nanoparticle immunogens, including I53_dn5. Dh, hydrodynamic diameter; Pd, polydispersity. h, Representative electron micrograph of H1-I53_dn5 embedded in vitreous ice. Scale bar, 100 nm. i, 2D class averages obtained using single-particle cryo-EM. Scale bar, 20 nm. j, Gold-standard Fourier shell correlation curve for the H1-I53_dn5 density map presented in Fig. 1c. k, Gold-standard Fourier shell correlation curve for the localized reconstruction of H1 MI15 presented in Fig. 1c. All experiments except for electron microscopy data collection and processing were performed at least twice.
Extended Data Fig. 2 |
Extended Data Fig. 2 |. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) of H1-foldon trimer and H1-I53_dn5 nanoparticle.
a, Amino acid sequence of H1 ectodomain expressed as a genetic fusion to both foldon and I53_dn5B. Underlined sequences correspond to peptides analyzed by HDX-MS. b, Hydrogen-deuterium exchange percentages after 20 h for both samples mapped onto the structure of H1 HA (PDB 3LZG). c, Kinetics of hydrogen-deuterium exchange for both samples at multiple timepoints up to 20 h. *, peptides where a negative percent exchange was corrected to zero (< 2% magnitude correction); **, peptides that were missing a replicate at the 30 min timepoint.
Extended Data 3 |
Extended Data 3 |. Controllable co-display of multiple antigenic variants on two-component nanoparticle immunogens.
a, Sandwich BLI comparing qsCocktail-I53_dn5 and qsMosaic-I53_dn5. Biotinylated 5J8 immobilized on streptavidin probes was used to capture H1-containing nanoparticles from each sample. The captured particles were then exposed to antibodies specific to H3 (CR8020; left) or influenza B HA (CR8071; right). b, Numerical approximation of the H1 HA content of individual qsMosaic-I53_dn5 nanoparticles assuming an equimolar quadrivalent in vitro assembly reaction (i.e., 25% of the input HA-I53_dn5B trimers bear H1 HA) and random incorporation of each HA-I53_dn5B trimer at each of the 20 trimeric positions into the nanoparticle. A distribution centered on 25% valency (5 H1 HA trimers per nanoparticle) is observed. c, Calculation of the fraction of individual mosaic nanoparticles displaying at least one H1 HA trimer as a function of the fractional concentration of H1-I53-dn5B in the in vitro assembly reaction ([H1]), expressed as: 1 - (1 - [H1]). At the 25% fractional concentration used to assemble qsMosaic-I53_dn5, 99.7% of the individual nanoparticles are expected to display at least one H1 HA trimer. d, Quantitation of HA antigen content by peptide mass spectrometry in three distinct qsMosaic-I53_dn5 nanoparticles with various antigen ratios before and after preparative SEC. Dashed lines represent the fractional concentration of each HA in the in vitro assembly reactions used to prepare the mosaic nanoparticle immunogens, main bars represent the mean values of four unique peptides from each HA, and error bars represent the standard deviation of measurements across the four unique peptides from each HA. The peptides used to quantify each HA are provided in Supplementary Table 3.
Extended Data Fig. 4 |
Extended Data Fig. 4 |. Vaccine-elicited antibody responses against vaccine-matched antigens.
HA-specific antibody titers in immunized mice (a), ferrets (b), and NHPs (c). Immunization schemes are shown at the top of each panel. All immunizations were given intramuscularly with AddaVax. Groups of BALB/cJ mice (N = 10), Finch ferrets (N = 9), and rhesus macaques (N = 4) were used in each experiment. ELISA antibody titers are expressed as endpoint dilutions. Each symbol represents an individual animal and the horizontal bar indicates the geometric mean of the group. Individual NHPs are identified by unique symbols. d, Antibody responses against unmodified I53_dn5 nanoparticles lacking displayed HA. Immunization scheme is shown at the top of the panel. Groups of NHPs (N = 4) were immunized three times with either QIV, qsCocktail-I53-dn5, or qsMosaic-I53_dn5 with AddaVax at weeks 0, 8 and 16. Serum samples were collected 2 weeks after each immunization and tested for ELISA binding antibody against unmodified I53_dn5 particles. Antibody titers are expressed as endpoint dilutions. Individual NHPs are identified by unique symbols. The immunization study was performed once. e, Antibody responses against vaccine-matched antigens and viruses elicited by unadjuvanted vaccines in immunized mice. Immunization scheme is shown. All immunizations were given intramuscularly. Groups of BALB/cJ mice (N = 10) were used. HA-specific ELISA binding antibody (top), hemagglutination inhibition (HAI) (middle), and microneutralization titers (bottom) in immune sera are shown. Microneutralization titers are reported as half maximal inhibitory dilution (IC50). Each symbol represents an individual animal and the horizontal bar indicates the geometric mean of the group. Statistical analysis was performed using nonparametric Kruskal–Wallis test with Dunn’s multiple comparisons. All animal experiments except for NHP were performed at least twice and representative data are shown.
Extended Data Fig. 5 |
Extended Data Fig. 5 |. Antibody responses against vaccine-matched antigens and viruses elicited by 2018–2019 vaccines.
a, Immunization scheme. The commercial QIV, qsCocktail-I53_dn5, and qsMosaic-I53_dn5 vaccines used in this study comprised the WHO-recommended 2018–2019 vaccine strains. Sequences for the HA-I53_dn5B fusion proteins—H1-I53_dn5, SG16-I53_dn5 (updated H3), B/Yam-I53_dn5, and CO17-I53_dn5 (updated B/Vic)—are provided in Supplementary Table 1. All immunizations were given intramuscularly with AddaVax. Groups of BALB/cJ mice (N = 10) were used. b, HA-specific antibody, c, hemagglutination inhibition (HAI), and d, microneutralization titers in immune sera. Microneutralization titers are reported as half maximal inhibitory dilution (IC50). e, Heterosubtypic HA-specific antibody titers in immune sera. Each symbol represents an individual animal and the horizontal bar indicates the geometric mean of the group. Statistical analysis was performed using nonparametric Kruskal–Wallis test with Dunn’s multiple comparisons. The animal experiment was performed once.
Extended Data Fig. 6 |
Extended Data Fig. 6 |. Neutralization of historical H1N1 and H3N2 viruses.
Immunization scheme for ferret study is shown. Groups of Finch ferrets (N = 9) were used. Phylogenetic trees of HA sequences of human H1N1 (left) and H3N2 (right) viruses are shown (see Supplementary Table 4). Each symbol represents an individual animal and the horizontal bar indicates the geometric mean of the group. Statistical analysis was performed using nonparametric Kruskal–Wallis test with Dunn’s multiple comparisons. The ferret experiment was performed twice and representative data are shown.
Extended Data Fig. 7 |
Extended Data Fig. 7 |. Antibody responses elicited by a non-assembling immunogen.
a, Model of the I53_dn5B trimer, with the computationally designed interface that drives nanoparticle assembly indicated by the solid line (top), and the 1na0C3_int2 trimer, in which the interface mutations were reverted to their original identities (bottom). The dotted line indicates the inability of this molecule to drive nanoparticle assembly. b, Analytical SEC of the non-assembling immunogen (a mixture of four HA-1na0C3_int2 trimers with pentameric I53_dn5A) using a Superose 6 Increase 10/300 GL column. Only unassembled oligomeric components were observed. c, Reducing and non-reducing SDS-PAGE analysis of the non-assembling immunogen before and after analytical SEC. d, Negative stain EM of the non-assembling immunogen, which confirmed the absence of higher-order structures indicated by analytical SEC. Scale bar, 100 nm. e, Immunization scheme in mice. All immunizations were given intramuscularly with AddaVax. Groups of BALB/cJ mice (N = 10) were used in the experiment. f, Microneutralization titers in immune sera against vaccine-matched or slightly mismatched viruses. Microneutralization titers are reported as half maximal inhibitory dilution (IC50). g, Cross-reactive antibody titers in immune sera. Each symbol represents an individual animal and the horizontal bar indicates the geometric mean of the group. Statistical analysis was performed using nonparametric Kruskal–Wallis test with Dunn’s multiple comparisons. All experiments were performed once.
Extended Data Fig. 8 |
Extended Data Fig. 8 |. NS-EM analysis of H1 HA complexed with polyclonal antibody Fabs prepared from NHPs immunized with qsCocktail-I53_dn5 or qsMosaic-I53_dn5.
a, NS-EM analysis of Fabs obtained from qsCocktail-I53_dn5-immunized NHPs in complex with recombinant H1 MI15 HA trimers. Two-dimensional classifications were generated using 847,873 particles collected from 4,112 micrographs. The frequencies of complexes containing Fab fragments bound to RBD (81%), VE (18%), or stem (1%) domains are presented as pie charts in Fig. 5c. b, NS-EM analysis of Fabs obtained from qsMosaic-I53_dn5-immunized NHPs in complex with recombinant H1 MI15 HA trimers. Two-dimensional classifications were generated using 997,557 particles collected from 3,237 micrographs. The frequencies of complexes containing Fab fragments bound to RBD (69%), VE (24%), or stem (7%) domains are presented as pie charts in Fig. 5c. Upper part of each panel shows representative reference-free 2D class averages. Scale bars, 20 nm. Lower part of each panel shows seven representative 3D reconstructions of HA–Fab complexes. Single complexes containing Fabs of multiple specificities were counted once against each specificity. The coordinates of an H1 HA crystal structure (PDB 1RUZ) and a Fab fragment (PDB 3GBN) were fitted into the EM densities. Light blue ribbons, H1 HA; cyan or magenta ribbons, Fabs. All experiments were performed once.
Extended Data Fig. 9 |
Extended Data Fig. 9 |. CryoEM analysis of heterosubtypic H5 HA in complex with polyclonal antibody Fab fragments prepared from NHP immunized with qsMosaic-I53_dn5.
a, Representative cryo-electron micrograph. Scale bar, 100 nm. b, Reference-free 2D class averages. Scale bar, 20 nm. c, Gold-standard Fourier shell correlation (FSC) curve for the asymmetric reconstruction shown in d. d, Asymmetric cryo-EM reconstruction of H5 HA–Fab complexes with Fab fragments bound to all three HA subunits at 3.6 Å resolution. The reconstruction is the same as that shown in the right panel in Fig. 5d, but here is colored by local resolution. e, FSC curve for the asymmetric reconstruction shown in f. f, Two orthogonal orientations of an asymmetric cryo-EM reconstruction of H5 HA–Fab complexes with Fab fragments bound to two HA subunits at 4.1 Å resolution. g, FSC curve for the asymmetric reconstruction shown in h. h, Two orthogonal orientations of an asymmetric cryo-EM reconstruction of H5 HA–Fab complexes with Fab fragments bound to one HA subunit at 4.0 Å resolution. The reconstruction is the same as that shown in the left panel in Fig. 5d. All experiments were performed once.
Extended Data Fig. 10 |
Extended Data Fig. 10 |. Vaccine-elicited antibody responses against vaccine-matched viruses in NHPs with pre-existing influenza immunity.
Immunization scheme for NHP study shown on the left. NHPs (N = 3) that had been immunized three times with either QIV 2017–2018 or qsMosaic-I53_dn5 2017–2018 were boosted 63 weeks later with a single dose (60 μg) of updated qsMosaic-I53_dn5 2018–2019. All immunizations were given intramuscularly with AddaVax. Microneutralization titers are reported as half maximal inhibitory dilution (IC50). Each symbol represents an individual animal and the horizontal bar indicates the geometric mean of the group. Individual NHPs are identified by unique symbols. Statistical analysis was performed using paired t test.
Figure 1 |
Figure 1 |. Design and characterization of HA nanoparticle immunogens.
a, Schematic of in vitro assembly. B/Yam, B/Yamagata/16/1988-like; B/Vic, B/Victoria/2/1987-like. b, Negative stain electron micrographs of purified nanoparticle immunogens and I53_dn5 (scale bar, 200 nm). c, 3D reconstruction of the H1-I53_dn5 nanoparticle and localized reconstruction of H1 HA obtained by single-particle cryo-EM. Scale bars, 23.5 (black) and 50 (pink) nm. d, Immunoprecipitation using RSV F-specific (MPE8) and H1-specific (5J8) mAbs. IP, immunoprecipitated; UB, unbound. All experiments except for cryo-EM were performed at least twice.
Figure 2 |
Figure 2 |. Vaccine-elicited antibody responses against vaccine-matched viruses in mice, ferrets, and NHPs.
a, Hemagglutination inhibition (HAI) and b, microneutralization (MN) titers in immune sera. Groups of BALB/cJ mice (N = 10), ferrets (N = 9), and rhesus macaques (N = 4) were used in each experiment. Each symbol represents an individual animal and the horizontal bar indicates the geometric mean of the group. Individual NHPs are identified by unique symbols. Statistical analysis was performed using one-sided nonparametric Kruskal–Wallis test with Dunn’s multiple comparisons. All animal experiments except for NHPs were performed at least twice and representative data are shown.
Figure 3 |
Figure 3 |. Neutralization of and protection against historical H1N1 and H3N2 viruses.
a, Neutralization breadth of ferret immune sera, presented as geometric mean IC50 titers ± geometric s.d. for each group. Statistical analysis was performed using one-sided parametric two-way ANOVA with Tukey’s post-hoc multiple comparisons. Global GMT calculated as the means of geometric mean IC50 titers across 10 H1N1 or 9 H3N2 viruses for each individual animal. Statistical analysis was performed using one-sided nonparametric Kruskal–Wallis test with Dunn’s multiple comparisons. The experiment was performed twice using groups of ferrets (N = 9) and representative data are shown. Heterologous H1N1 (b) and mismatched H3N2 (c) virus challenges in immunized mice. Kaplan–Meier curves were compared using Mantel-Cox log-rank test with Bonferroni correction. Pseudovirus neutralizing (PN) IC50 titers were grouped based on survival outcomes; each symbol represents an individual animal. Statistical analysis was performed using Mann-Whitney test. The challenge experiments were performed once using groups of BALB/cJ mice (N = 10, or N = 9 for qsCocktail-I53_dn5 groups in b and unadjuvanted qsCocktail-I53_dn5 group in c).
Figure 4 |
Figure 4 |. Vaccine-elicited heterosubtypic antibody responses and protective immunity.
Cross-reactive antibody responses to heterosubtypic HA antigens in a, BALB/cJ mice (N = 10), b, ferrets (N = 9), and c, rhesus macaques (N = 4). Each symbol represents the endpoint titer (log10) of an individual animal and the horizontal bar indicates the geometric mean of each group. Individual NHPs are identified by unique symbols. All animal immunization experiments except for NHPs were performed at least twice and representative data are shown. Statistical analysis was performed using one-sided nonparametric Kruskal–Wallis test with Dunn’s multiple comparisons. Heterosubtypic influenza virus challenge in immunized mice (d) and ferrets (e). Three ferrets from each group were euthanized 4 days post challenge to measure lung viral RNA (right). Individual ferrets are identified by unique symbols. Right and left caudal lung lobes are indicated as closed and open symbols, respectively. f, Heterosubtypic influenza virus challenge after passive transfer of purified NHP immune Ig in mice. The multiple Kaplan–Meier curves were compared using Mantel-Cox log-rank test with Bonferroni correction. Mouse challenge experiments were performed twice; ferret experiments and passive transfer experiments were performed once.
Figure 5 |
Figure 5 |. Molecular basis for nanoparticle-induced protection against heterosubtypic influenza viruses.
a, Serum antibody titers to H1 and H3 stem-only antigens in BALB/cJ mice (N = 10), ferrets (N = 9), and rhesus macaques (N = 4). Each symbol represents the endpoint titer (log10) of an individual animal and the horizontal bar indicates the geometric mean of each group. Individual NHPs are identified by unique symbols. All animal immunization experiments except for NHPs were performed at least twice. b, Serum MN activity against H1N1 and H5N1 viruses in the presence of competitor proteins. Statistical analysis was performed using one-sided nonparametric Kruskal–Wallis test with Dunn’s multiple comparisons. c, Selected NS-EM reconstructions of H1 HA in complex with polyclonal antibody Fab fragments elicited by qsCocktail-I53_dn5 (left) and qsMosaic-I53_dn5 (right). Frequency of complexes observed by EM containing Fab fragments bound to the RBD, VE, and stem are shown as pie charts. d, Two independent cryo-EM reconstructions of H5 HA in complex with polyclonal antibody Fab fragments elicited by qsMosaic-I53_dn5. e, Close-up of one of the dominant antibody classes that resembles MEDI8852 recognition. f, Serum antibody titers to H1 and H3 stem-only antigens in NHPs (N = 6) with pre-existing influenza immunity. Each unique symbol represents the endpoint titer (log10) of an individual animal. Statistical analysis between the two timepoints was performed using paired t test.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Wei C-J et al. Next-generation influenza vaccines: opportunities and challenges. Nat. Rev. Drug Discov. 19, 239–252 (2020). - PMC - PubMed
    1. Ueda G et al. Tailored design of protein nanoparticle scaffolds for multivalent presentation of viral glycoprotein antigens. Elife 9, e57659 (2020). - PMC - PubMed
    1. Kanekiyo M & Graham BS Next-Generation Influenza Vaccines. Cold Spring Harb. Perspect. Med. (2020) doi:10.1101/cshperspect.a038448. - DOI - PMC - PubMed
    1. Iuliano AD et al. Estimates of global seasonal influenza-associated respiratory mortality: a modelling study. Lancet 391, 1285–1300 (2018). - PMC - PubMed
    1. Flannery B et al. Interim Estimates of 2017–18 Seasonal Influenza Vaccine Effectiveness - United States, February 2018. MMWR Morb. Mortal. Wkly. Rep. 67, 180–185 (2018). - PMC - PubMed

Additional References

    1. Throsby M et al. Heterosubtypic neutralizing monoclonal antibodies cross-protective against H5N1 and H1N1 recovered from human IgM+ memory B cells. PLoS One 3, e3942 (2008). - PMC - PubMed
    1. Hong M et al. Antibody recognition of the pandemic H1N1 Influenza virus hemagglutinin receptor binding site. J. Virol. 87, 12471–12480 (2013). - PMC - PubMed
    1. Ekiert DC et al. A highly conserved neutralizing epitope on group 2 influenza A viruses. Science 333, 843–850 (2011). - PMC - PubMed
    1. Iba Y et al. Conserved neutralizing epitope at globular head of hemagglutinin in H3N2 influenza viruses. J. Virol. 88, 7130–7144 (2014). - PMC - PubMed
    1. Lee PS et al. Receptor mimicry by antibody F045–092 facilitates universal binding to the H3 subtype of influenza virus. Nat. Commun. 5, 3614 (2014). - PMC - PubMed

Publication types

MeSH terms

Substances