Additional file 1 of Development of a highly effective combination monoclonal antibody therapy against Herpes simplex virus
Creators: name: Seyfizadeh, Narges
name: Kalbermatter, David
name: Imhof, Thomas
name: Ries, Moritz
name: Müller, Christian
name: Jenner, Leonie
name: Blumenschein, Elisabeth
name: Yendrzheyevskiy, Alexandra
name: Grün, Frank
name: Moog, Kevin
name: Eckert, Daniel
name: Engel, Ronja
name: Diebolder, Philipp
name: Chami, Mohamed
name: Krauss, Jürgen
name: Schaller, Torsten
name: Arndt, Michaela
Publisher: name: figshare
Year: 2024
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Supplementary Material 1. Fig. S1. HDIT102 IgG does not cross-react with other herpesviruses than HSV-1/2 and has a very low dissociation rate (kdis). (A) Binding of a dilution series of HDIT102 at several concentrations to HSV-1/2, VZV, HCMV and EBV antigens was analyzed by ELISA using microplates coated with respective viral antigens (Enzygnost, Siemens). Absorbance at 450 nm was measured. Anti-HSV-ELISA does not discriminate between detection of anti-HSV-1 and anti-HSV-2 IgGs. Binding was detected with an HRP-conjugated anti-human gamma Fc-specific IgG. Cytotect (anti-CMV polyclonal antibody preparation) was used as a positive control for all. (B) HDIT101 IgG was tested in biolayer-interferometry against immobilized HSV-1F gB. (C) HDIT102 IgG was tested in biolayer-interferometry against immobilized HSV-1F gB. Fig. S2. HDIT102 efficiently inhibits cell-to-cell spread of HSV-1F in Vero cells. Inhibition of HSV-1F cell-to-cell spread by HDIT102. Fluorescence microscopy images of Vero cells infected with HSV-1F and subsequently treated with either HDIT102, HDIT101, human polyclonal anti-HSV antibody or left untreated. Plaque formation was visualised by anti-HSV immuno- and Hoechst staining. Representative images are shown. Arrows show plaques or initially infected cells. Fig. S3. Cryo-EM data analysis of co-structures of trimeric HSV-1F and HSV-2G gB. (A) - (D) The plots show the Fourier shell correlation (FSC) curves of the final calculated density map (black) and the FSC curve calculated between the final map and the atomic model (grey). The reported resolutions for the maps is based on the “gold-standard” FSC = 0.143 criterion and FSC = 0.5 for the FSC between map and model. (E) - (H) The final 3D reconstructions are shown in three different views (top, side and bottom) and colored according to the local resolution calculated using the ResMap [53] implementation in RELION 4.0 [20]. Fig. S4. HSV-1 gB cryo-EM co-structure with HDIT101 or HDIT102 Fab. (A) Side, bottom and top view of co-structure of trimeric HSV-1F gB in post-fusion conformation (green) with three HDIT101 Fab molecules (blue) solved by cryo-EM at a resolution of 3.27 Å are shown. (B) The co-structure of HDIT102 Fab (magenta) bound to trimeric HSV-1F gB in post-fusion conformation (green) was determined at a resolution of 3.44 Å and side, bottom and top views are shown. (C) Overlay of the co-structures indicating overlapping epitopes of HDIT101 and HDIT102 Fabs on HSV-1F gB indicating perpendicular orientation of both Fabs to another. (D) Overlay of published x-ray crystal structure (PDB:2GUM) with HSV-1 cryoEM gB structure derived in complex with HDIT101 Fab. (E) Overlay of published x-ray crystal structure (PDB:2GUM) with HSV-1 cryoEM gB structure derived in complex with HDIT102 Fab. (F) Overlay of HSV-1 cryoEM gB structure derived in complex with HDIT101 Fab and HDIT102 Fab. (G) Overlay of HSV-1 and HSV-2 cryoEM gB structures derived in complex with HDIT101 Fab. (H) Overlay of HSV-1 and HSV-2 cryoEM gB structures derived in complex with HDIT102 Fab. (I) Overlay of HSV-1 and HSV-2 cryoEM gB structures derived in complex with HDIT102 Fab with differing amino acid residues marked in magenta. (J) Detailed image of the HSV-1 gB residues interacting with HDIT101 (compare with Fig. 3D). (K) Detailed image of the HSV-1 gB residues interacting with HDIT102 (compare with Fig. 3E). Fig. S5. HSV-2 gB interactions with HDIT101 or HDIT102 CDR amino acid residues. HSV-2 gB residues within 4Å distance of residues in the HDIT101 or HDIT102 CDRs were determined by Pymol and subsequently analyzed individually for polar (red) or non-polar (yellow) interactions. Oxygen groups are highlighted in red, nitrogen groups in blue. Carbon atoms involved in non-polar interactions were not coloured. (A)-(C) Interaction of HDIT101 CDR residues with HSV-2 gB residues in the binding interface. (D)-(G) Interaction of HDIT102 CDR residues with HSV-2 gB residues in the binding interface. Fig. S6. Analysis of epitope residues conferring resistance to HDIT101 or HDIT102 induced fusion inhibition. (A) Amino acid alignment of HSV-1F and HSV-2G gB with indicated key residues conferring resistance to HDIT101 (HSV-1 gB R304Q/ HSV-2 gB R296Q) or HDIT102 (HSV-1 gB R335Q/ HSV-2 gB R327Q) evolving in vitro. (B) Cell-cell fusion assay and inhibition of selected HSV-1 gB mutants by HDIT102 IgG. Changes in fusion inhibition by HDIT102 were calculated comparing HDIT102 treated with untreated cells for each mutant and normalized to wild type gB (WT gB) mean values are shown for at least three independent biological replicates (n=3). Statistical analyses were performed using unpaired t-tests.  (C) Relative fusion activity of selected gB mutants compared to wild type protein. Fusion activity as a combined indicator of cell surface expression and fusogenicity of gB mutants was analyzed by comparison to wild type gB (WT gB) in the absence of any antibody. Mean values of three independent biological replicates are shown with error bars indicating standard deviation (n=3). Statistical analyses were performed using unpaired t-tests. (D) Cell-cell fusion assay and inhibition of selected HSV-1 gB mutants by HDIT101. Changes in fusion inhibition by HDIT102 were calculated comparing HDIT101 treated with untreated cells for each mutant and normalized to wild type gB (WT gB) mean values are shown for at least three independent biological replicates (n=3). Statistical analyses were performed using unpaired t-tests. (E) Relative fusion activity of selected gB mutants compare to wild type protein. Fusion activity as combined indicator of cell surface expression and fusogenicity of gB mutants was analyzed by comparison to wild type gB (WT gB) in the absence of any antibody. Mean values of three independent biological replicates are shown with error bars indicating standard deviation (n=3). Statistical analyses were performed using unpaired t-tests. 451 HSV-1 (F) and 368 HSV-2 (G) gB DNA sequences were retrieved from the NCBI sequence database by using nucleotide BLAST against HSV-1F or HSV-2G gB DNA sequences, aligned and analyzed for variations in the HDIT101 or HDIT102 epitope regions to study signatures of evolutionary pressure from natural HDIT102-like antibodies. The graphs illustrate the conservation at each epitope amino acid position in the gB proteins. The residue number is indicated in the x-axis and the percentage of conservation is indicated in the y-axis. (H) Single-likelihood ancestor counting analysis was performed on alignments of 185 full-length HSV-1 gB and 201 full-length HSV-2 gB sequences to identify positively selected residues. The difference in non-synonymous and synonymous substitution rates are shown on the y-axis and gB codon position in the alignment on the x-axis. (I) Phylogentic tree demonstrating HSV-2 gB isolates containing R327 or Q327. Fig. S7. HDIT102 does not elicit ADCC on HSV-1F or HSV-2G infected Vero cells or HEK293T cells ectopically expressing gB. Different dilutions of HDIT102 IgG or human polyclonal serum containing anti-HSV IgGs were added to HSV-1F or HSV-2G infected target cells, followed by incubation with effector Jurkat cells (Promega #G7010) stably expressing FcγRIIIA receptor, V158 (high affinity) variant and the NFAT-luciferase reporter at an effector-to-target cell ratio of 6:1. Infected Vero cells or HEK293T cells expressing either HSV-1 gB or HSV-2 gB were used as target cells. ADCC was quantified by luminescence readout from luciferase activity upon NFAT pathway activation. (A) ADCC activity of HDIT102 IgG or isotype control on Vero cells infected with either HSV-1F or HSV-2G or uninfected. Mean of three technical replicates is shown. (B) Same as in A, however using human polyclonal antibody as control. (C) Summary of A and B showing fold ADCC induction at highest antibody concentration. (D) ADCC activity of HDIT102 IgG on parental HEK293T or HEK293T stably expressing either HSV-1F gB or HSV-2G gB. Mean of three technical replicates is shown. (E) Same as in D, however using human polyclonal antibody as control. (F) Summary of D and E showing fold ADCC induction at highest antibody concentration. (G) and (H) Analysis of HDIT102-mediated complement dependent cytotoxicity by quantitative evaluation of the terminal C5b-9 complement complex. An ELISA was employed to measure supernatant levels of C5b-9 complex to quantify complement activation by HDIT102. HSV-1F or HSV-2G infected or uninfected Vero cells were incubated with heat-inactivated or with not heat-inactivated IgG-depleted human serum in the presence or absence of test antibodies (either HDIT102, human polyclonal IgG, or Cytotect as positive controls, or unrelated isotype IgG as negative control). Statistical analysis was done using two-way ANOVA, error bars represent standard deviation of the mean for two technical replicates, ****p&lt; 0.0001)