A modular platform to display multiple hemagglutinin subtypes on a single immunogen
- Ragon Institute of Mass General, MIT, and Harvard, United States
- Department of Microbiology, Harvard Medical School, United States
Figures
Figure 1 with 2 supplements
Design of beads-on-a-string (BOAS) immunogens.
(A) Phylogenetic tree of influenza hemagglutinin (HA) subtypes. Subtypes are further categorized as group 1, group 2, or influenza B, and stars indicate subtypes included in the BOAS. (B) Native trimeric HA structure (A/H3/Hong Kong/1968; PDB: 4FNK) is shown on the left, with the HA1 domain in light gray, and HA2 in dark gray. A construct diagram of full-length soluble HA is shown at the top with the locations of head domain boundaries within HA1. Head domains of H1, H3, and H7 span residues 57–261, and head domains of H2, H3, H4, H9, and B span 37–316 of the HA1 region of HA. (C) Construct diagram of HA BOAS. BOAS are assembled by linking HA head domains (light gray) into a single polypeptide chain where HA head domains are separated by a flexible linker with four glycine-serine-serine (GSS) repeats. The C-terminus consists of an HRV-3C protease cleavable His-tag and streptavidin-binding-protein (SBP) tag for purification and other affinity tags. (D) BOAS used in this study.
Figure 1—figure supplement 1
Adjusting flexible linker lengths on beads-on-a-string (BOAS).
(A) Construct a diagram of H3-B-H1 3mer BOAS with 2 X, 3 X, and 4 X Gly-Ser-Ser linkers. (B) SDS-PAGE of 3mer BOAS with 2 X, 3 X, and 4 X GSS linkers from left to right. (C) ELISA with subtype-specific antibodies detecting H1 (green), H3 (orange), and B (salmon) subtypes on 2 X, 3 X, and 4 X glycine-serine-serine (GSS) linker BOAS.
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Figure 1—figure supplement 1—source data 1
Uncropped and labeled gel for Figure 1B.
- https://cdn.elifesciences.org/articles/97364/elife-97364-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
Raw unedited gel for Figure 1B.
- https://cdn.elifesciences.org/articles/97364/elife-97364-fig1-figsupp1-data2-v1.zip
Figure 1—figure supplement 2
Swapping 3mer order on beads-on-a-string (BOAS).
Determining effect on order of BOAS heads on 3mer expression and ELISA. (A, B) ELISA with H5 (yellow) and H7 (pink) subtype-specific monoclonal antibodies (mAbs) on BOAS with H5-H9-H7 (A) or H9-H5-H7 (B) in order from N- to C-terminus. (C) SDS-PAGE of H9-H5-H7 and H5-H9-H7 BOAS.
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Figure 1—figure supplement 2—source data 1
Uncropped and labeled gel for Figure Supplement 2C.
- https://cdn.elifesciences.org/articles/97364/elife-97364-fig1-figsupp2-data1-v1.zip
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Figure 1—figure supplement 2—source data 2
Raw unedited gel for Figure supplement 2C.
- https://cdn.elifesciences.org/articles/97364/elife-97364-fig1-figsupp2-data2-v1.zip
Figure 2 with 2 supplements
Biochemical characterization of beads-on-a-string (BOAS).
(A) SDS-PAGE analysis of 3mer-8mer BOAS under non-reducing conditions. (B) SDS-PAGE analysis of 3mer-8mer BOAS under non-reducing conditions following glycan digestion with PNGase-F. (C) Size exclusion chromatography traces of 3mer-8mer BOAS on a Superdex 200 column in PBS. (D) Negative stain electron microscopy (NS-EM) images of the 3mer. Arrows point to individual HA monomers. Inset image is 2.5 X zoomed, enlarged image of boxed area. (E) NS-EM images of the 8mer. Arrows point to individual HA monomers. Inset image is 2.5 X zoomed, enlarged image of boxed area. (F) Characterization via ELISA of BOAS components with subtype-specific antibodies. Apparent KD values in µM are reported for each antibody for each BOAS immunogen. Antibodies corresponding to each component are as follows: S5V2-29 (Watanabe et al., 2019) (interface, all components except B/Mal/04), 5J8 (Hong et al., 2013; Krause et al., 2011) (RBS, H1), 2G1 (Krause et al., 2012) (RBS, H2), K03.12 (McCarthy et al., 2018) (RBS, H3), P2-D955 (H4), H5.3 (Winarski et al., 2015) (RBS, H5), H7.167 (Thornburg et al., 2016) (RBS periphery, H7), H1209 (Bajic et al., 2020) (RBS, B).
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Figure 2—source data 1
Uncropped and labeled gel for Figure 2A and B.
- https://cdn.elifesciences.org/articles/97364/elife-97364-fig2-data1-v1.zip
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Figure 2—source data 2
Raw unedited gel for Figure 2A and B.
- https://cdn.elifesciences.org/articles/97364/elife-97364-fig2-data2-v1.zip
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Figure 2—source data 3
Uncropped images for Figure 2D.
- https://cdn.elifesciences.org/articles/97364/elife-97364-fig2-data3-v1.pdf
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Figure 2—source data 4
Uncropped images for Figure 2E.
- https://cdn.elifesciences.org/articles/97364/elife-97364-fig2-data4-v1.pdf
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Figure 2—source data 5
Raw ELISA data for Figure 2F.
- https://cdn.elifesciences.org/articles/97364/elife-97364-fig2-data5-v1.xlsx
Figure 2—figure supplement 1
Size exclusion chromatography traces of purified beads-on-a-string (BOAS) immunogens over time.
Traces of purified 3-, 4-, 5-, 6-, 7-, and 8mer BOAS following cobalt purification (top), immediately thawed from –80° C storage (middle), and post-thaw following 1 week of storage at 4° C. Approximately 100 µg of protein was injected onto a Superose 6 10/300 column in PBS, and each trace shows a normalized 280 nm signal.
Figure 2—figure supplement 2
P2-D9 mAb specificity for H4.
ELISA assay to determine binding of P2-D9 mAb to H1, H2, H3, H4, H5, H7, H9, and B influenza hemagglutinin (HA) trimers. P2-D9 only detectably engages H4/New Brunswick/2010 HA.
Figure 3 with 1 supplement
Murine immunizations with 3mer-8mer beads-on-a-string (BOAS).
(A) Schematic of immunization regimen with 3mer-8mer BOAS and a Mix control containing an equal molar of the eight individual hemagglutinin (HA) heads not connected with a linker. Mice were primed with 20 µg of immunogen, followed by two homologous boosts two weeks apart. (B) Immunogenicity of BOAS over time as determined by serum ELISA. Serum reactivity to each immunogen is reported as the endpoint titer (EPT) dilution factor for each group (n=8) and d14, d28, and d42. Data points are an average of n=8 mice and error bars are +/-1 s.d. (C) Serum titers to each BOAS at the final time point, d42. Data points for single time point antigen titers are from individual mice n=8, bars represent mean titers and error bars are +/-1 s.d., *=p <0.05 as determined by Kruskal-Wallis test with Dunn’s multiple comparison post hoc test relative to the 3mer. (D) Serum titers to full-length HA trimers elicited by 3mer-8mer BOAS. Solid bars below each plot indicate a matched subtype, and striped bars indicate a mismatched subtype (i.e. not present in the BOAS). (E) Side-by-side comparison of 8mer (solid bars) and Mix (dotted bars) serum titers to individual HA components. Data points are serum EPTs from individual mice (n=8) and error bars are +/-1 s.d., *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001 as determined by a Kruskal-Wallis test with Dunn’s multiple comparison post hoc test.
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Figure 3—source data 1
Raw ELISA for Figure 3B–E.
- https://cdn.elifesciences.org/articles/97364/elife-97364-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Day 28 serum reactivity from beads-on-a-string (BOAS) and BOAS NP Cohorts.
(A) Serum titers to full-length soluble hemagglutinin (HA) trimers elicited by 3mer-8mer BOAS and BOAS NP at d28. Solid bars below each plot indicate a matched subtype, and striped bars indicate a mismatched subtype (i.e. not present in the BOAS). Data points are serum endpoint titers (EPTs) from individual mice (n=8 for BOAS; n=5 for BOAS NP and Mix) and error bars are +/-1 s.d. (B) Serum reactivity to each matched and mis-matched individual full-length HA component of BOAS (H1/MI/15 light green, H2/JP/57 blue, H3/KS/17 light orange, H4/NB/10 purple, H5/VN/04 yellow, H7/SH/13 pink, H9/GD/16 maroon, B/Mal/04 salmon, H3/HK/68 dark orange, and H1/SI/06 dark green) by 3mer-8mer BOAS, Mix, and BOAS NP cohorts. Solid bars below each plot indicate a matched subtype, and striped bars indicate a mismatched subtype (i.e. not present in the BOAS or NP). *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001 as determined by the Kruskal-Wallis test with multiple comparisons relative to the mix control group. *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001 as determined by a Kruskal-Wallis test with Dunn’s multiple comparison post hoc test.
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Figure 3—figure supplement 1—source data 1
Raw ELISA data for Figure 3—figure supplement 1A and B.
- https://cdn.elifesciences.org/articles/97364/elife-97364-fig3-figsupp1-data1-v1.xlsx
Figure 4 with 1 supplement
Beads-on-a-string (BOAS) component epitope homology and serum competition.
(A) Amino acid conservation of each BOAS projected on the A/H2/Japan/1957 head domain structure (PDB: 2WRE). Variability and conservation scores were determined as the % conservation of each residue at each position for each BOAS composition. Cut-outs are RBS and Trimer interface (TI) epitopes with their respective conservation. (B) Homology score as determined by the average % conservation of each residue across the entire protein sequence (overall, circle), and the RBS (square) and TI (triangle) epitopes. (C) Serum competition to RBS and TI epitopes as determined via competition with a cocktail of RBS-directed antibodies, TI-directed antibodies, or a stem-directed negative control antibody. % competition is determined by the relative decrease in serum binding to each BOAS normalized to an untreated control. Each data point represents a mean from n=3 mice and error bars are +/-1 s.d. (D) Trend overlay of % serum competition with RBS-directed antibodies and homology score of the RBS epitope as a function of BOAS length. (E) Trend overlay of % serum competition with TI-directed antibodies and homology score of the TI epitope as a function of BOAS length.
Figure 4—figure supplement 1
Sequence identity matrices of beads-on-a-string (BOAS) components.
Comparing sequence identity of eight BOAS components H1/Michigan/45/2015, H2/Japan/305/1957, H3/Kansas/14/2017, H4/New Brunswick/00464/2010, H5/Vietnam/1203/2004, H7/Shanghai/01/2014, H9/Guangdong/MZ058/2016, B/Malaysia/2506/2004. Blosum45 (A) similarity (B) or amino acid identity matrices of total head domain amino acid sequence (left), receptor binding site (RBS) contacts (center), or interface contacts (right).
Figure 5 with 1 supplement
Design of beads-on-a-string (BOAS) nanoparticle (NP) immunogens.
(A) Schematic of design of BOAS nanoparticles (NP). The eight original 8mer components were split into two 4mers, the first containing H1, H2, H3, and H4 (H1–H2–H3–H4), and the second containing H5, H7, H9, and B (H5–H7–H9–B). Each were expressed with an N-terminal SpyTag, then attached at equimolar ratios to an N-terminally fused SpyCatcher ferritin nanoparticle (SpyCatcher NP). This yields a NP with both 4mer BOAS conjugated to its surface (2x4 mer BOAS NP). (B) Dynamic light scattering (DLS) distributions of SpyCatcher NP and 2x4 mer BOAS NP. Curves represent an average of three technical replicate measurements and error bars are +/-1 s.d. (C) Size exclusion traces of SpyCatcher NP and 2x4 mer BOAS NP on an S400 column. (D) Representative negative stain electron microscopy (NS-EM) image of SpyCatcher NP. Arrows indicate individual nanoparticles, and the inset is a 2 X zoom of the boxed area. Representative NS-EM image of BOAS NP. Arrows indicate individual nanoparticles, and the inset is a 2 X zoom of the boxed area. (E) ELISA of BOAS components, SpyCatcher NP, and 2x4 mer BOAS NP with structure-dependent subtype-specific mAbs and a negative control stem-directed mAb, MEDI8852 (Kallewaard et al., 2016). Antibodies corresponding to each component are as follows: 5J8 (Hong et al., 2013; Krause et al., 2011) (RBS, H1), 2G1 (Krause et al., 2012) (RBS, H2), K03.12 (McCarthy et al., 2018) (RBS, H3), P2-D9 (Caradonna et al., 2022) (H4), H5.3 (Winarski et al., 2015) (RBS, H5), H7.167 (Thornburg et al., 2016) (RBS periphery, H7), H1209 (Bajic and Harrison, 2021) (RBS, B). ELISA data points are technical duplicates and error bars are +/-1 s.d.
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Figure 5—source data 1
Uncropped images of Figure 5D.
- https://cdn.elifesciences.org/articles/97364/elife-97364-fig5-data1-v1.zip
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Figure 5—source data 2
Raw ELISA data for Figure 5E.
- https://cdn.elifesciences.org/articles/97364/elife-97364-fig5-data2-v1.xlsx
Figure 5—figure supplement 1
Nanoparticle conjugation efficiency SDS-PAGE.
SDS-PAGE gel of N-terminally fused SpyCatcher ferritin (middle lane), and beads-on-a-string (BOAS)-conjugated ferritin NP (right lane).
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Figure 5—figure supplement 1—source data 1
Uncropped and labeled gel for Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/97364/elife-97364-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
Raw unedited gel for Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/97364/elife-97364-fig5-figsupp1-data2-v1.zip
Figure 6
Murine immunizations with beads-on-a-string (BOAS) nanoparticles (NP).
(A) Schematic of immunization regimen with 2x4 mer BOAS NP and SpyCatcher NP control. Mice were primed with 20 µg of NP, followed by two homologous boosts two weeks apart. (B) Serum reactivity to each immunizing antigen at d42. **p<0.01 as determined by the Mann-Whitney test. (C) Serum reactivity to Control NP scaffold by both groups. **p<0.01 as determined by the Mann-Whitney test. (D) Serum reactivity elicited by BOAS NP group (n=5) at d42 to both individual BOAS components (H1–H2–H3–H4 and H5–H7–H9–B), Control NP, and BOAS NP. *** p<0.001 as determined by the Kruskal-Wallis test with Dunn’s multiple comparison post hoc test. (E) Serum reactivity elicited by Control NP group (n=5) at d42 to both individual BOAS components (H1–H2–H3–H4 and H5–H7–H9–B), Control NP, and BOAS NP. ** P<0.01 as determined by the Kruskal-Wallis test with Dunn’s multiple comparison post hoc test. (F) Serum reactivity elicited by BOAS NP group (n=5) to individual full-length HA components of BOAS at d42. *=p<0.05, **=p < 0.01, ***=p < 0.001 as determined by the Kruskal-Wallis test with Dunn’s multiple comparison post hoc test. (G) Serum reactivity to each matched and mis-matched individual full-length HA component of BOAS (H1/MI/15 light green, H2/JP/57 blue, H3/KS/17 light orange, H4/NB/10 purple, H5/VN/04 yellow, H7/SH/13 pink, H9/GD/16 maroon, B/Mal/04 salmon, H3/HK/68 dark orange, and H1/SI/06 dark green) by 3mer-8mer BOAS, Mix control, and BOAS NP. Solid bars below each plot indicate a matched subtype, and striped bars indicate a mismatched subtype (i.e. not present in the BOAS, Mix, or NP). *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001 as determined by the Kruskal-Wallis test with multiple comparisons relative to the mix control group. In all plots, data points for single time point antigen titers are from individual mice (n=5 for BOAS NP and Mix cohorts, n=8 for 3mer-8mer BOAS studies), bars represent mean titers and error bars are +/-1 s.d.
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Figure 6—source data 1
Raw ELISA data for Figure 6B–F.
- https://cdn.elifesciences.org/articles/97364/elife-97364-fig6-data1-v1.xlsx
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Figure 6—source data 2
Raw ELISA data for Figure 6G.
- https://cdn.elifesciences.org/articles/97364/elife-97364-fig6-data2-v1.xlsx
Figure 7
Microneutralization titers to matched and mismatched virus.
Microneutralization of matched and mis-matched pseudo viruses: H1N1 (green, top left), H3N2 (orange, top right), H5N1 (yellow, bottom left), and H7N9 viruses (pink, bottom right) with d42 serum. Solid bars below each plot indicate a matched subtype, and striped bars indicate a mismatched subtype (i.e. not present in the BOAS). Nanoparticles (NP negative controls) were used to determine the threshold for neutralization. Upper and lower dashed lines represent the first dilution (1:32) (for H1N1, H3N2, and H5N1) or neutralization average with negative control NP serum (H7N9), and the last serum dilution (1:32,768), respectively, and points at the dashed lines indicate IC50s at or outside the limit of detection. Individual points indicate IC50 values from individual mice from each cohort (n=5). The mean is denoted by a bar and error bars are +/-1 s.d., *=p<0.05 as determined by a Kruskal-Wallis test with Dunn’s multiple comparison post hoc test relative to the mix group.
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Figure 7—source data 1
Raw microneutralization data for Figure 7.
- https://cdn.elifesciences.org/articles/97364/elife-97364-fig7-data1-v1.xlsx
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A modular platform to display multiple hemagglutinin subtypes on a single immunogen
eLife 13:RP97364.
https://doi.org/10.7554/eLife.97364.3
