The present vaccine against influenza virus has the inevitable risk of antigenic discordance between the vaccine and the circulating strains, which diminishes vaccine efficacy. This necessitates new approaches that provide broader protection against influenza. Here we designed a vaccine using the hypervariable receptor-binding domain (RBD) of viral hemagglutinin displayed on a nanoparticle (np) able to elicit antibody responses that neutralize H1N1 influenza viruses spanning over 90 years. Co-display of RBDs from multiple strains across time, so that the adjacent RBDs are heterotypic, provides an avidity advantage to cross-reactive B cells. Immunization with the mosaic RBD–np elicited broader antibody responses than those induced by an admixture of nanoparticles encompassing the same set of RBDs as separate homotypic arrays. Furthermore, we identified a broadly neutralizing monoclonal antibody in a mouse immunized with mosaic RBD–np. The mosaic antigen array signifies a unique approach that subverts monotypic immunodominance and allows otherwise subdominant cross-reactive B cell responses to emerge.
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All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary information. Coordinates for 441D6 Fab structure and cryo-electron microscopy density map have been deposited in the Protein Data Bank (PDB) under accession code 5TR8 and electron microscopy Data Bank (EMDB) under accession code EMD-7021. Hemagglutinin RBD–np and antibody sequences are deposited in the GenBank under accession codes MK273069–MK273076 and MK283705–MK283710. Requests for materials should be addressed to M.K. and B.S.G.
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We thank J. Weir (Food and Drug Administration) for providing influenza viruses; R. Webby (St Jude Children’s Research Hospital) for influenza reverse genetics plasmids; M. Dillon, K. Wuddie, G. Sarbador, C. Chiedi, A. Taylor, H. Bao and D. Scorpio (VRC) for help with animal studies; A. Kumar (VRC) for help with protein production; V. Nair and E. Fischer (NIAID) for cryo-electron microscopy data collection; M. Chen (VRC), D. Angeletti and J. Yewdell (NIAID) for help with initial epitope mapping. This work used the computational resources of the NIH HPC Biowulf cluster (http://hpc.nih.gov) and also the Office of Cyber Infrastructure and Computational Biology (OCICB) High Performance Computing (HPC) cluster at the NIAID, NIH. Use of insertion device 22 (SER-CAT) at the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Basic Energy Sciences (under contract W-31-109-Eng-38). Support for this work was provided by the NIAID Intramural Research Program to the VRC and Division of Intramural Research. This work was also supported in part with federal funds from the Frederick National Laboratory for Cancer Research, NIH (under contract HHSN261200800001E).
The US National Institutes of Health has filed a patent application on the basis of the studies presented in this paper; M.K., H.M.Y. and B.S.G. are listed as inventors on the patent application.
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Integrated supplementary information
Chromatograms of homogeneously assembled and heterogeneously co-assembled hemagglutinin RBD–np on anion-exchange column (a) and size exclusion column (b). NC99 and CA09 hemagglutinin RBD–np were produced in Expi293 cells by transient transfection and the culture supernatants were purified. Anion-exchange chromatography was carried out using a HiTrap Q HP column eluted with linear gradient of NaCl (a). Fractions containing RBD–np corresponding to the shaded area were collected and subjected to size exclusion chromatography (SEC). SEC was carried out using a Superose 6 matrix to confirm particle formation (b). Experiments were independently performed three times with similar results. Purified RBD–np expressing various hemagglutinin RBD from indicated H1N1 strains homogeneously (c) or heterogeneously co-assembled (mosaic) (d) were analyzed on SDS–PAGE and blue staining. For comparison, mechanical mixtures of 2, 4, 6 or 8 different RBD–np were prepared and analyzed on the same gel (admix) (d). Experiments were independently performed two times with similar results. Size exclusion chromatography profiles of hemagglutinin RBD–np shown in panel c and d (e and f, respectively). Experiments were performed two times with similar results.
Supplementary Fig. 2 Antibody responses elicited by hemagglutinin RBD–np and commercial vaccines to influenza virus.
Serum HAI and neutralization antibody titers to CA09 (a) and BR07 (b). BALB/c mice were immunized with NC99 RBD–np (valence 1) or various preparations of various RBD–np either as admixtures (admix) or as co-assembled particles (Mosaic). Represented data were generated using sera collected 2 weeks after the second immunization with adjuvant (2 µg of RBD–np per immunization). Shown results were obtained from a representative experiment (n = 5). Each dot indicates individual mouse with mean of each group indicated as lines. Animal experiments were independently performed two times with similar results. Statistical analyses are done with one-way ANOVA with Tukey’s multiple comparisons post-hoc test. Non-statistical significance between groups is not shown for clarity. (c) Microneutralization (MN) titers against WS33, KW86, and CC03 viruses of pooled immune sera. Mice (n = 5) were immunized with 2 µg of mosaic RBD–np (8-valent), or 1.5 µg of commercial TIV (from indicated season) formulated with SAS and delivered at weeks 0 and 4. Immune sera were collected at week 6, RDE-treated, heat-inactivated, and pooled for microneutralization assays. Animal experiment was performed once.
(a) Hemagglutinin-binding of monoclonal antibodies measured by biolayer interferometry. Recombinant hemagglutinin trimer proteins were immobilized on HIS1K biosensors through hexa-histidine tag and used to assess binding of monoclonal antibodies. All assays were performed in the Octet HTX instrument (fortéBio) at 30ºC with agitation at 1,000 rpm. PBS with 1% BSA was used as assay buffer for all steps. Plotted data indicate the response value during the mAb association step at 300 s. Control monoclonal antibodies 3u-u, 2D1 and CH65 were shown as gray bars. Experiments were performed two times with similar results. (b) Neutralization capacity of monoclonal antibodies. Pseudotype neutralization assays were used to assess neutralization potency of isolated monoclonal antibodies. Homologous NC99 and heterologous BJ95 strains were selected for screening. Experiments were performed two times with similar results.
Binding kinetics measurements were carried out using biolayer interferometry with Octet HTX instrument (fortéBio). Recombinant hemagglutinin trimers were immobilized either on HIS1K or SA sensors through hexa-histidine tag or conjugated biotin, respectively. Fab 441D6 was prepared by introducing HRV3C cleavage site in the heavy chain hinge and digesting with HRV3C enzyme followed by Protein A affinity purification to remove Fc and uncleaved antibodies as described elsewhere7. Dilution series of Fab 441D6 was made in assay buffer (PBS with 1% BSA). All assays were performed in the Octet HTX instrument (fortéBio) at 30ºC with agitation at 1,000 rpm. Represented data contain measured sensograms (red) and global fitting with 1:1 binding model (black) generated by Octet Analysis software (version 9.0, fortéBio). Experiments were independently performed two times with similar results.
(a) Micrographs indicated the HA–441D6 complex partitioned equally between the carbon surface and carbon holes. (b) Picked particles contributing to the determined structure are indicated by green circles, visualized on the same micrograph shown in a. (c). Model-free 2D class averages reveal 3-fold symmetry of the complex. Experiment was performed once. (d) Class occupancy from 2D classification is plotted corresponding to classes in c. (e) Fourier-shell correlation analysis of ‘gold-standard’ refinement using a cutoff of 0.143 estimates the model resolution as 8.0 Å.
Supplementary Fig. 6 electron microscopy analysis of mosaic RBD–np particle and its complex with 441D6 Fab.
(a) Raw electron microscopy images and representative 2D classes of mosaic RBD–np. Individual particles were visualized by PTA negative staining, revealing the ferritin core particle as either a filled or hollow white circle, depending on stochastic penetration of the ferritin core by the negative stain. RBD domains were seen as points immediately surrounding the ferritin core. The inset shows 4 representative class averages from 60,842 picked particles, after initial picking of 65,517 particles in 174 micrographs. (b) Mosaic RBD–np in complex with 441D6 Fab was similarly analyzed by PTA negative staining electron microscopy. The extra density surrounding the ferritin core is consistent with the presence of a coat of bound 441D6 Fab. The inset shows representative class averages of the complex, but unlike the RBD–np alone, the particles that stained hollow or filled required separate processing as they did not naturally partition into distinct classes during classification. From a total pool of 9,187 particles in 134 micrographs, final class averages of filled particles were made from 6,926 particles, and hollow particles were made from 957 particles. Experiment was performed once. (c) Rotationally averaged images of RBD–np particles (bottom) and 441D6 Fab complexes (top) were synthesized from class averages, and then juxtaposed to illustrate the bulk effect of 441D6 binding. The inner sphere represents the ferritin core, while the middle density of lesser intensity is either RBD or RBD+Fab. The outermost layer resulted from lighter staining of the electron microscopy grid carbon surface, since the negative stain accumulated in higher amounts proximal to particles. (d) Relative mean intensity of the rotational averages was plotted as a function of particle radius. High intensity corresponds to white in images in c. The outer radius of the RBD–np is 13 nm, while mosaic RBD–np–441D6 Fab complexes extend to 15.5 nm from the particle center. We interpret the first minima near 7 nm for the 441D6 complex as accumulation of negative stain between the ferritin core and Fabs that are tethered via the RBD domains at varying orientations.
Therapeutic administration of control VRC01 (anti-HIV) and 441D6 monoclonal antibodies in mice infected with A/California/07/2009 (H1N1) (a) or A/Puerto Rico/8/1934 (H1N1) (b). BALB/c mice were intranasally infected with a 10 × LD50 of either H1N1 viruses 24 hours before receiving monoclonal antibodies via intraperitoneal injection. Clinical symptoms and body weight were monitored throughout the study. Lines in plots showing body weight change indicate mean of group with standard deviations including only live animals at indicated time points. Experiment was performed once (n = 10).
(a) Generation and characterization of hemagglutinin with 441D6KO mutation. Two point mutations, E116K and K172E were introduced in the NC99 hemagglutinin (highlighted in black on the hemagglutinin molecular surface). Antigenicity of 441D6KO hemagglutinin was assessed by ELISA using 441D6, CH65, and anti-His (Covance) antibodies. Binding curves of antibodies to WT and 441D6KO hemagglutinin were depicted. Assays were independently performed two times with similar results. (b) Serum binding antibody titers to WT and 441D6KO hemagglutinin. The endpoint titers in immune sera raised by NC99 HA-, admix (8-valent)-, mosaic (8-valent)-, and sequential RBD–np immunization were measured by ELISA (n = 10, biologically independent samples). Assays were independently performed two times with similar results. (c) Serum binding antibody titers to WT and 441D6KO hemagglutinin. The endpoint titers were determined as above (n = 26, biologically independent samples). Assays were independently performed two times with similar results. Statistical analyses were done with nonparametric t-test using Wilcoxon matched-paired signed rank test (two-tailed).
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Kanekiyo, M., Joyce, M.G., Gillespie, R.A. et al. Mosaic nanoparticle display of diverse influenza virus hemagglutinins elicits broad B cell responses. Nat Immunol 20, 362–372 (2019). https://doi.org/10.1038/s41590-018-0305-x
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