Stalking influenza by vaccination with pre-fusion headless HA mini-stem

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1 Stalking influenza by vaccination with pre-fusion headless HA mini-stem Sophie A Valkenburg a,b, V Vamsee Aditya Mallajosyula c, Olive TW Li b, Alex WH hin b, George arnell d, Nigel Temperton d, Raghavan Varadarajan c *, Leo LM Poon b* a HKU-Pasteur Research Pole, School of Public Health, HKU Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong b enter of Influenza Research and School of Public Health, The University of Hong Kong, Hong Kong c Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India d Viral Pseudotype Unit, School of Pharmacy, University of Kent, Kent, United Kingdom. * orrespondence: Raghavan Varadarajan Molecular Biophysics Unit, Indian Institute of Science (IISc), Bangalore India varadar@mbu.iisc.ernet.in Leo LM Poon School of Public Health, The University of Hong Kong Hong Kong llmpoon@hku.hk

2 Table S: binds stem-directed bnabs with similar affinity as full length HA. Kinetic parameters (mean±sd) for the binding of and H5 HA (VN/4) to conformation specific bnabs were determined by surface plasmon resonance (SPR). Immunogen Ligand a k on (M - s - ) k off (s - ) K D (nm) R66-IgG 3.4±. 4.4± ±.7 F-scFv.8±.8 5.8±.5-3.6±.4 FI6v3-scFv.4±. 5.9±.3-3.±3.8 R66-IgG.4±. 5 9.± ±.8 H5 HA (VN/4) F-scFv 3.7±. 5.3± ±.5 FI6v3-scFv.9±.5 5.4± ±. a 75RU of each ligand was immobilized on an independent activated surface channel of a Biacore M5 sensor chip.

3 Table S: retained binding to the stem-directed bnab R66 after diverse stress condition(s) treatment. Kinetic parameters (mean±sd) for the binding of to R66 were determined by biolayer interferometry (BLI). Stress condition K D (nm), R66 a binding Temperature ( ) b ±3.3 4.± ± ±. ph c d 36.9±5. 37.± ±.4 6.7±.8 Temporal T=days stability at ± ± ± ±4.4 different storage 5 36.±3. 4.8±.8 43.±.7 5.±.7 temperature ±.9 4.± ± ±8. Freeze-thaw cycles ± ±4.4 4.± ±.7 a R66 (ligand) was captured on amine reactive biosensor tips. b (analyte) was incubated at the indicated temperatures for h. The samples were then cooled and K D was determined at 5. c was incubated in buffered solutions of indicated ph for 3-45 mins and binding to R66 was subsequently evaluated. The sample ph was measured before the binding experiments. d H5 HA (VN/4) showed no detectable binding after incubation at ph 4..

4 Table S3: Antibodies elicited by the engineered HA stem-fragment immunogens showed broad cross-reactivity. The equilibrium dissociation constant (K D ) for the binding of sera (total IgG) to HA proteins was determined by BLI using the Octet RED96 instrument. The sera from mice immunized with the HA stem-fragment immunogens bound heterologous HAs with high-affinity (K D values 5nM are highlighted in red). The convalescent sera exhibited weak cross-reactivity. Analyte Primary sera Apparent K D (nm) a Secondary H5N+ sera b sera Primary HF sera Secondary HF sera HN+ sera c Group HAs (mini-stem) HF 36.4±3.6.± ± ±.3 7.7±.4 9.7± ±6.8 6.± ± ± ± ±7.7 Group HAs (full-length) H A/Puerto Rico/8/934 HA - d 74.± ±5.7 - H A/alifornia/4/9 HA 655.± ± ± ±9. 9.7± ±9. H A/Brevig Mission//98 HA - 8.7± ±.8 - H5 A/Viet Nam/94/4 HA 68.4±.6.±.9 8.± ± ±4.4 n.b e H5 A/Indonesia/5/5 HA ± ±4.9 - H A/Japan/35/957 HA ± ±. - Group HAs (full-length) H3 A/Hong Kong//968 HA 36.4± ±8.6 n.b 5.± ± ±7.8 H3 A/Brisbane//7 HA ± ±4. - H7 A/Netherlands/9/3 HA ± ±4.6 - H7 A/Anhui//3 HA 39.5± ± ±9.8 4.± ±8.7 n.b Influenza B HAs (full-length) B/Brisbane/6/8 HA (Victoria lineage) B/Florida/4/8 HA (Yamagata lineage) ± ± ± ±9.9 - a The KD values are the Mean ± SD of triplicate experiments. b anti-h5n+ sera : pooled sera of mice recovered from H5N (Wigeon/7) infection. c anti-hn+ sera : pooled sera of mice recovered from pandemic HN (a/4) infection. d - : not determined because of sera limitation. e n.b : no detectable binding.

5 Table S4: The kinetic parameters obtained for the binding of sera (primary and secondary) and H5N convalescent sera against HA proteins determined by BLI using the Octet RED96 instrument. The convalescent serum (total IgG) binds HA stem-fragment proteins with low affinity and exhibits limited cross-reactivity. Analyte Serum k on (M - s - ) k off (s - ) K D (nm) a Group HAs (mini-stem) HF Primary ±3.6 Secondary ±7.9 H5N ±9.6 Primary ±6.8 Secondary ±.9 H5N ±3.3 Group HAs (full-length) H A/Puerto Rico/8/934 HA Secondary ±7.8 Primary ±9.8 H A/alifornia/4/9 HA Secondary ±9.6 H5N ±6.9 H A/Brevig Mission//98 HA ±9.3 Primary ±.6 H5 A/Viet Nam/94/4 HA Secondary ±.9 H5N ±9.9 H5 A/Indonesia/5/5 HA Secondary ±. H A/Japan/35/957 HA Secondary ±3.6 Group HAs (full-length) Primary ±63.6 H3 A/Hong Kong//968 HA Secondary ±8.6 H5N+ n.b b n.b n.b H3 A/Brisbane//7 HA ±4.4 H7 A/Netherlands/9/3 HA ±3.4 Primary ±4.5 H7 A/Anhui//3 HA Secondary ±9.9 H5N ±9.8 Influenza B HAs (full-length) B/Brisbane/6/8 HA (Victoria lineage) Secondary ±6.8 B/Florida/4/8 HA (Yamagata lineage) Secondary ±.7 a The equilibrium dissociation constant (apparent KD ) values are the Mean ± SD of triplicate experiments. b n.b : no detectable binding.

6 Table S5: The kinetic parameters obtained for the binding of HF sera (primary and secondary) and anti-hn convalescent sera (highlighted in green) against HA proteins determined by BLI using the Octet RED96 instrument. HF elicited cross-reactive, anti-ha stem antibodies. In contrast, the convalescent serum (total IgG) displayed limited cross-reactivity. Analyte Serum k on (M - s - ) k off (s - ) K D (nm) a Group HAs (mini-stem) HF Primary ±.3 Secondary ±.4 H5N ±39.3 Primary ±3.4 Secondary ±8.6 H5N ±7.7 Group HAs (full-length) H A/Puerto Rico/8/934 HA Secondary ±5.7 Primary ±9. H A/alifornia/4/9 HA Secondary ±8.3 H5N ±9. H A/Brevig Mission//98 HA Secondary ±.8 Primary ±5.5 H5 A/Viet Nam/94/4 HA Secondary ±4.4 H5N+ n.b b n.b n.b H5 A/Indonesia/5/5 HA Secondary ±4.9 H A/Japan/35/957 HA Secondary ±. Group HAs (full-length) Primary ±9.9 H3 A/Hong Kong//968 HA Secondary ±6.9 H5N ±7.8 H3 A/Brisbane//7 HA Secondary ±4. H7 A/Netherlands/9/3 HA Secondary ±4.6 Primary ±38.9 H7 A/Anhui//3 HA Secondary ±8.7 H5N+ n.b n.b n.b Influenza B HAs (full-length) B/Brisbane/6/8 HA (Victoria lineage) B/Florida/4/8 HA (Yamagata lineage) Secondary ±.3 Secondary ±9.9 a The equilibrium dissociation constant (apparent KD ) values are the Mean ± SD of triplicate experiments. b n.b : no detectable binding.

7 A B (native) (denatured) D E Figure S: Biophysical and biochemical characterization of. (A) The far-uv D spectra of indicated that the protein was well folded and -helical, consistent with an extensive presence of helical HA subunit segments (external A-helix and the centrally located coiled-coil). (B) The thermal denaturation of (~μm) was monitored by D at 8nm. The thermal unfolding of was co-operative and reversible with an apparent thermal transition mid-point (T m ) of 38K (45 ). The consecutive unfolding traces of overlapped well with each other indicating that protein unfolding was reversible. () The redshift of the fluorescence emission maximum of upon denaturation with Gdml indicated the burial of aromatic, hydrophobic residues in the native, folded state. (D) The oligomeric state of in solution was probed by analytical gel-filtration chromatography. The protein eluted as a homogeneous trimer. The molecular weight of ( ) was calculated (67.6kDa) from the calibration curve (inset) of the S- column obtained using a broad range of markers (x). The theoretical molecular weight of is 64.kDa (.4kDa 3). The oligomeric state of the protein was also confirmed by SE-MALS. The calculated molecular weight ( ( 8.97%) Da) was in good agreement with the theoretical molecular weight of a trimer. (E) Purified was stored at different temperatures (as indicated). μl of protein was aliquoted at different time points (as indicated), flash frozen and stored at -7 until further processing. All the samples were analyzed simultaneously on SDS-PAGE under non-reducing conditions and stained with oomassie. was stable for up to 3 weeks at 4 and 5. Even at a higher storage temperature (37 ), only slight degradation of the protein is observed after 3 weeks.

8 A B D E F Figure S: binds HA stem-directed, conformation specific bnabs with high affinity. SPR binding sensograms for and H5 (VN/4) HA with (A-B) R66, (-D) F-scFv and (E-F) FI6v3-scFv. The test antibody (ligand) was immobilized (75RU) on an activated surface of a Biacore M5 sensor chip. A concentration series of the analyte was used to obtain the kinetic parameters of binding (Supplementary Table S). (A, and E) (Trace -5: 5nM, 5nM, 5nM, 75nM and 5nM). (B, D and F) H5 HA (VN/4) (Trace -5: nm, nm, 5nM, 5nM and nm). bound the bnabs with high affinity (K D ; nM). The kinetic parameters were obtained by fitting the data globally to a : Langmuir interaction model using BIA EVALUATION 3. software. The data points are in open circles, while the fits are shown as solid lines.

9 Figure S3: forms a stable complex with the bnab R66. Lane :, lane : R66 (IgG), lane 3: pre-stained broad range SDS-PAGE marker (BioRad), lanes 4-7: unbound fractions, and lanes 8-: elution fractions. was incubated with R66 (IgG) for h (at 4 ) at different molar ratios (as indicated, n.a=not applicable). The protein complex ( with R66) was then pulled down using Protein G beads that bind specifically to the Fc region of a human-igg. does not bind non-specifically to Protein G beads (lane 8). The protein complex bound to the beads was eluted with mm glycine.hl (ph 3) and neutralized with M Tris.Hl (ph 9) before SDS- PAGE analysis. All the samples were analyzed under non-reducing conditions. The gel was stained with oomassie.

10 Figure S4: A representative plot demonstrating the determination of kinetic parameters for the binding of sera to HA proteins determined by BLI. The binding of sera harvested from mice vaccinated with mini-ha stem () to H5 HA (VN/4) at different sera dilutions (as indicated) has been shown. IgG was captured from the pooled mice sera using Protein G (ProG) biosensors. The biosensors loaded with the ligand were subsequently dipped in the analyte (H5 HA) wells to monitor binding. The traces were processed using the ForteBio Data Analysis Software (v8.) and fit globally using a simple : Langmuir interaction model. The residual view (inset) indicates acceptable fits. The kinetic parameters [k on (M - s - ): 4.5 3, k off (s - ): , and K D (nm):..9] obtained for the binding of different serum with HA protein(s) are reported in Tables S3, S4 and S5. The kinetic parameters of binding determined by capturing the HA protein(s) on amine reactive biosensors for probing analyte interaction (serial dilutions of the sera) were comparable with the values obtained with the aforementioned experimental setup.

11 H HA YSD A HN sera HF sera sera Naïve sera IgG B HF sera sera lone no o. lone no o HA nt position H3 HA HA YSD D HN sera HF sera sera Naïve sera IgG F HF sera G sera HA nt position E % YSD binding HN +ve H YSD H3 YSD HFS S NMS ve Serum lone no. lone no. H % of alig gnment HA nt position % A4 HA_HF H HA YSD with HFS 8% A4 HA_ H HA YSD with S 6% HK68 HA_HF H3 HA YSD with HFS HK68 HA_HA H3 HA YSD with S 4% % HA nt position % HA nt position Figure S5: Mini-HA stem immunization elicits a robust antibody response against the subdominant HA subunit. The heat inactivated t pooled sera collected from mice (n = ) at day after the secondary immunization i with mini-ha i stem(s) were used for screening against the YSD fragment library of unmatched influenza A (A-) H HA (pdm a/9) and (D-G) H3 HA (HK/68). Representative FAS plots of sorted cells that were positive for HA fragment expression and sera binding (detected by anti-mouse IgG). The HA fragment of sorted yeast cells were sequenced (from 3 clones) and aligned to the consensus full length HA sequence for the H-HA YSD for HF sera (B), sera () and the H3-HA YSD for HF sera (F), sera (G). The % binding to the total YSD cells (E) (from A, D). Alignment of all HA YSD sequences (H) (from B,, F, G). (number of clones sequenced HFS H-HA n=8, S H-HA YSD n=3, HFS H3-HA YSD n=8, S H3-HA YSD n=6).

12 A % of DA API Day 3 PBS HF B PI+ % of DA Day 7 PBS HF % aa Macro of DAPI E In nflammation Day 7 BAL ell type PBS HF Vaccination group Lung histopathology D Protein (ug/ml) 4 3 Parenchyma Vascular Airway BALF BA PBS HF day 3 day 7 Time point ell type PBS HF PBS HF Day 7 Day Figure S6: mini-ha stem vaccination does not alter inflammation profile after HN infection despite increased survival. (A-) The cell influx to the lung and (D) local lymph node of vaccinated mice after HN (pdm a/4) infection was assessed with a panel of antibodies by flow cytometry analysis. The cell profile at (A) day 3 and (B) day 7 for innate and adaptive cell types was determined. () Alternative activated macrophages from the BAL were also determined. (D) The protein concentration from the BAL fluid was determined in a standard BA protein assay at days 3 and 7 post HN (pdm a/9) infection. (E) Histopathology by H&E staining of lung sections was assessed under a light microscope and vascular, airway and parenchyma inflammation scored. Data represents the mean SD, (n=3). PBS with Addavax vaccinated mice (PBS) were used as a negative control.

13 Ratio IgG:Ig gga A.5.5 HFS S H3N positive Serum B Day IgA HFS S H3N pos Protein Day IgM HFS S H3N pos Protein F D 4 3 Day 7 H3N serum HFS S PBS+Addavax Serum G E Day 8 H3N serum HFS S H3N positive Serum d d6 d Serum Dilution H5HA H Serum Dilution..5. H3HA Serum Dilution Figure S7: Mini-HA stem immunization does not skew the Th/Th balance, elicits an early response after infection and is stable long term. (A) The balance between Th/Th responses was evaluated by determining the ratio of IgG/IgGa titers to various antigens in the pooled mice sera (n=5-) harvested at day after the secondary immunization. Sera from mice recovered after a sub- lethal H3N (HK/68) virus challenge was used as a positive control. The isotype specific (B) IgA and () IgM cross-reactive Ab response(s) of mini-ha stem vaccinated mice sera were determined by ELISA (with serum diluted :). After H3N infection of vaccinated mice, ELISA with recovered sera at (D) day 7 and (E) day 8 serum. (F-H) The longevity of mini-ha stem induced antibodies was determined in sera harvested after, 6 and days after the second vaccine dose (n=5). Antigens used in the ELISA assay were shown as indicated. 4 48