Supplementary Table 1: Antigenic regions/sites on Ebola-GP identified using GFPDL*

Transcription

1 Supplementary Table 1: Antigenic regions/sites on Ebola-GP identified using GFPDL* Site AA Sequence 3x10 6 3x x x x x10 6 Post-1 st Post-2 nd Post-1 st Post-2 nd Post-1 st Post-2 nd I 1-36 MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLG 0% 1% 1% 0% 0% 0% II SIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDV PSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAP DGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGT TFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQAT GFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTG KL 0% 2% 0% 0% 0% 1% II AFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLR EPVNATEDPSSGYYSTTIRY 0% 2% 18% 15% 0% 2% II SSHPLREPVNATEDPSSGYYSTTIRYQATGFG 2% 0% 11% 15% 0% 1% III ATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQF LLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKI RSEELSFTVVSNGAKNISGQSPARTSSDPGTNTTTEDHKIMASENSSA MVQVHS 1% 2% 1% 3% 2% 4% III SSGYYSTTIRY 0% 0% 13% 10% 0% 1% IV SNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSNGA KNISGQSPARTSSDPGTNTTTEDHKIMASENSSAMVQVHSQGREAAV SHLTTLATISTSPQSLTTKPGPDNSTHNTPVYKLDISEATQVEQHHRRT DNDSTASD 2% 1% 1% 1% 7% 0% IV DTTIGEWAFWETKKNLTRKIRSEE 2% 1% 1% 6% 8% 9% IV GEWAFWETKKN 1% 5% 1% 7% 10% 12% IV GEWAFWETKKNLTRKIRSEELSFTVVSNGAKNISGQSPARTSSDPGT NTTTEDHKIMASENSSAMVQVHSQGREAAVSH 2% 2% 1% 6% 3% 5% V TEDHKIMASENSSAMVQVHSQGREAAVSHLTTLATISTSPQSLTTKPG PDNSTHNTPVYKLDISEATQVEQHHRRTDNDSTASDTPSATTAAGPP KAENTNTSKSTDFLDPATTTSPQNHSETAGNNNTHHQDTGEESASSG KLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGA AIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLR ATTELRTF 0% 1% 0% 0% 1% 3% V ASENSSAMVQVHSQGREAAVSHLTTL 3% 5% 4% 8% 4% 7% V STSPQSLTTKPGPDNSTHNTPVYKLDISEATQVEQHHRRTDNDSTAS DT V TKPGPDNSTHNTPVYKLDISEATQVEQHHRRTDNDSTASDTPSATTAA GPPKAENTNTSKSTDFLDPATTTSPQNHSETAGNNNTHHQDTGEESA SSGKLGLITNTIAGVAG 22% 23% 6% 9% 10% 8% 3% 5% 4% 1% 7% 2% V TTAAGPPKAENTNTSKSTDFLDPA 5% 1% 5% 1% 1% 0% V NTSKSTDFLDPATTTSPQNHSETAGNNNTHHQDTGEESASSGKLGLIT NTIAGVAG 8% 5% 1% 2% 18% 11% V SETAGNNNTHHQDTGEESASSGKLGLITN 1% 2% 1% 0% 8% 17% V TGEESASSGKLGLITNTIAGVAGLITGGRR 3% 6% 3% 4% 2% 3% VI KNITDKIDQIIHDFVDKTLPDQGDNDNWW 22% 9% 13% 4% 1% 1% * Table showing frequency of antigenic sites for different vaccine dose groups after first and second rvsv G-ZEBOV-GP vaccination. The frequency of the clone per antigenic site is calculated by dividing the frequency of occurrence of a particular clone by the total number of phage clones for each vaccine dose.

2 Supplementary Table 2: Sequence conservation of Antigenic regions/sites among different EBOV strains Antigenic sites in the GP that are >70% conserved in diverse EBOV strains are shown in bold

3 Supplementary Table 3: Phage Titers from affinity selection of pooled sera with different doses of rvsv G-ZEBOV-GP vaccine following first vaccination using Protein A/G, IgM and IgA matrices 3 x 10 6 rvsv-ebola pfu 20 x 10 6 rvsv-ebola pfu 100 x 10 6 rvsv-ebola pfu A/G IgM IgA A/G IgM IgA A/G IgM IgA Phage Titer 7x x x x x x x10 5 2x x10 4

4 Supplementary Figure 1: Complete EBOV-Mayinga (1976) GP gene translated sequence used for construction of EBOV-GFPD library and depiction in Figure 1 3.

5 Mayinga_Strain MGVTGILQLP RDRFKRTSFF LWVIILFQRT FSIPLGVIHN STLQVSDVDK LVCRDKLSST NQLRSVGLNL EGNGVATDVP Kikwit_Strain Makona_Strain Mayinga_Strain SATKRWGFRS GVPPKVVNYE AGEWAENCYN LEIKKPDGSE CLPAAPDGIR GFPRCRYVHK VSGTGPCAGD FAFHKEGAFF Kikwit_Strain Makona_Strain.V Mayinga_Strain LYDRLASTVI YRGTTFAEGV VAFLILPQAK KDFFSSHPLR EPVNATEDPS SGYYSTTIRY QATGFGTNET EYLFEVDNLT Kikwit_Strain Makona_Strain Mayinga_Strain Kikwit_Strain Makona_Strain YVQLESRFTP QFLLQLNETI YTSGKRSNTT GKLIWKVNPE IDTTIGEWAF WETKKNLTRK IRSEELSFTV VSNGAKNISG A...R A A...P Mayinga_Strain QSPARTSSDP GTNTTTEDHK IMASENSSAM VQVHSQGREA AVSHLTTLAT ISTSPQSLTT KPGPDNSTHN TPVYKLDISE Kikwit_Strain PP Makona_Strain... E...N K PP...T Mayinga_Strain ATQVEQHHRR TDNDSTASDT PSATTAAGPP KAENTNTSKS TDFLDPATTT SPQNHSETAG NNNTHHQDTG EESASSGKLG Kikwit_Strain P...L...G..L Makona_Strain...G... A....P...L... A.S..L......Y Mayinga_Strain LITNTIAGVA GLITGGRRTR REAIVNAQPK CNPNLHYWTT QDEGAAIGLA WIPYFGPAAE GIYIEGLMHN QDGLICGLRQ Kikwit_Strain......A T Makona_Strain V T Mayinga_Strain LANETTQALQ LFLRATTELR TFSILNRKAI DFLLQRWGGT CHILGPDCCI EPHDWTKNIT DKIDQIIHDF VDKTLPDQGD Kikwit_Strain Makona_Strain Mayinga_Strain NDNWWTGWRQ WIPAGIGVTG VIIAVIALFC ICKFVF Kikwit_Strain Makona_Strain Supplementary Figure 2: Alignment of glycoprotein (GP) sequences from EBOV Mayinga (1976), Kikwit (1995) and Makona (2014) strains.

6 Supplementary Figure 3: Random distribution of size and sequence of the EBOV-GFPDL. Sequencing of GP fragments expressed by the phages of the EBOV GFPD libraries were aligned to the Zaire EBOV GP translated sequence (shown in Supplementary figure 1).

7 D8 13F D8 13F6 Known site GFPDL Known site GFPDL AA Sequence 390-NTPVYKLDISEATQVEQ TKPGPDNSTHNTPVYKLDISEATQVEQH TQVEQHHRRTDNDSTAS TQVEQHHRRTDND-414 Supplementary Figure 4: GFPDL based epitope mapping of protective MAbs 6D8 and 13F6 used in the MB-003 cocktail. Graphical distribution of representative clones with a frequency of >2, obtained after affinity selection, are shown. The common conserved minimal sequence for each MAb identified using GFPDL mapping is shown in the table compared to the sequence previously identified known site (Davidson, et al. J Virol; 2015). The reactivity of the GFPDL identified sequence to the respective MAb was confirmed by phage ELISA.

8 a b MAb 289 MAb 289 Absorbance (A490 nm) GP GP GP :5 1:25 1:125 1:625 Phage dilution c MAb 324 MAb 324 Absorbance (A490 nm) GP GP GP :5 1:25 1:125 1:625 Phage dilution Supplementary Figure 5: GFPDL based epitope mapping of cross-reactive conformation dependent neutralizing and protective human MAb 289 and MAb 324 from EBOV survivors (Flyak, et al. ;Cell; 2016). The reactivity of the GFPDL identified sequence to the respective MAb was confirmed by phage ELISA shown in panel B-C using the selected phage clones displaying the peptides shown in black and green for MAb 289, and in red and orange for Mab 324 in panel A.

9 Absorbance (A490 nm) Anti-GP IgA + IgG + IgM ELISA following GFPDL adsorption 1:1000 1:2000 1:4000 1:8000 1:16000 Sera dilution 20e6 D56 100e6 D56 20e6 D56- M13 ads 100e6 D56- M13 ads Supplementary Figure 6: Anti-GP reactivity of post-second vaccination sera for 20 million and 100 million pfu vaccine dose in ELISA before and after EBOV- GFPDL adsorption. Post second vaccination sera from individuals vaccinated with 20 million and 100 million rvsv G-ZEBOV-GP vaccine dose was adsorbed on EBOV GFPDL coated petri dishes. Binding to recombinant EBOV-GP is shown before (blue and red lines) and after (green and black) GFPDL-adsorption in ELISA using HRPconjugated goat anti-human IgA + IgG + IgM specific antibody.

10 Model 3CSY Supplementary Figure 7: Model of the complete Zaire Ebola GP monomer generated using I-TASSER (left) showing transmembrane (TM) domain in red and membrane in grey, and solved crystal GP structure (PDB Id #3CSY; includes GP residues , , and ) in monomeric form on right.

11 A 4 Anti-GP IgM OD 490 nm Serial Dilutions 20e6 D0 20e6 D28 20e6 D56 100e6 D0 100e6 D28 100e6 D56 B 5 Anti-GP IgG OD 490 nm Serial Dilutions 200e6 D0 20e6 D28" 20e6 D56 100e6 D0 100e6 D28 100e6 D56 C 2 Anti-GP IgA OD 490 nm Serial Dilutions 20e6 D0 20e6 D28 20e6 D56 100e6 D0 100e6 D28 100e6 D56 Supplementary Figure 8: Anti-GP reactivity of post-first and post-second vaccination sera for 20 million and 100 million pfu vaccine dose in ELISA. The isotype of two-fold serial dilution of serum antibodies (starting at 200-fold serum dilution) bound to EBOV-GP are shown for serum samples collected at different time points from adults vaccinated with 20 million and 100 million rvsv G-ZEBOV-GP vaccine dose administered groups as measured in ELISA using HRP conjugated anti-human IgM muchain specific antibody (A), HRP conjugated anti-human IgG Fc-chain specific antibody (B) and HRP conjugated anti-human IgA alpha-chain specific antibody (C).

12 IgG subclass of GP binding antibodies Percentage Isotyping (%) 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% D28 D42 D56 D84 D180 D28 D42 D56 D84 D180 D28 D42 D56 D84 D180 3E6 pfu 20E6 pfu 100E6 pfu IgG4 IgG3 IgG2 IgG1 Supplementary Figure 9: IgG subclass of human serum binding to EBOV-GP following rvsv G-ZEBOV-GP vaccination. The subclass of total IgG of serum antibodies bound to EBOV-GP are shown for serum samples collected at different time points from adults vaccinated with three different rvsv G-ZEBOV-GP vaccine dose administered groups.as measured in SPR experiment. The resonance unit for each anti-gp antibody IgG subclass was divided by the total resonance units for total bound IgG antibodies combined for each sera and represented as a percentage.

13 A 20 x 10 6 rvsv-ebola pfu 100 x 10 6 rvsv-ebola pfu A/G IgM IgA A/G IgM IgA Phage Titer 1x x x x x x10 4 B 20 x 10 6 DOSE GP GP C 100 x 10 6 DOSE GP2 GP IgG IgG IgM IgM IgA IgA 1 18

14 Supplementary Figure 10: Individual antibody repertoires elicited following first vaccination with rvsv G-ZEBOV-GP vaccine using IgA, IgG and IgM specific capture beads. (A) Number of captured phage clones isolated using EBOV-GFPDL affinity selection with sera obtained from individual vaccinee after first vaccination with 20 or 100 million pfu rvsv G- ZEBOV-GP vaccine dose group using IgA, IgG and IgM specific capture beads by EBOV- Kikwit GP GFPDL. (B, C) Schematic alignment of the peptides recognized by IgG, IgM and IgA antibodies following first vaccination with 20 million (B) and 100 million (C) rvsv G- ZEBOV-GP vaccine, identified by panning with GFPDL. The amino acid designation is based on the GP protein sequence encoded by the complete EBOV GP gene (Supplementary Figure 2). Bars indicate identified inserts in GP sequence. The GP receptor binding region (RBR) is depicted as yellow and the mucin like domain is shown in light green. Bars indicate identified inserts in GP sequence. Bar location indicates the homology of the displayed EBOV-GP protein sequence on the phage clones after affinity selection. Graphical distribution of representative clones with a frequency of >2, obtained after affinity selection, are shown. The thickness of each bar represents the frequency of repetitively isolated phage, with the scale shown below the alignment.