Journal: Cell host & microbe

Article Title: Longitudinal human antibody repertoire against complete viral proteome from Ebola virus survivor reveals protective sites for vaccine design

doi: 10.1016/j.chom.2020.01.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Zaire EBOV VP40 Matrix protein , Sino Biologicals , 40446-V07E.

Techniques: Recombinant

Journal: Cell host & microbe

Article Title: Longitudinal human antibody repertoire against complete viral proteome from Ebola virus survivor reveals protective sites for vaccine design

doi: 10.1016/j.chom.2020.01.001

Figure Lengend Snippet: Serial dilutions of serum samples collected at different time points from the EBOV survivor were analyzed for antibody binding to purified proteins from EBOV/Makona strain by SPR. (A-H) Total antibody binding is represented in SPR resonance units (RU) in black for binding to NP (A), VP35 (B). VP40 (B), GP (B). sGP (E), VP30 (F), VP24 (G) and L polymerase (H). Total antibody binding show is calculated RU for an undiluted serum sample. (A-H) Polyclonal antibody affinity maturation to EBOV proteins following EBOV infection in survivor was determined by SPR. Binding affinity of serially diluted post-infection serum to EBOV proteins was measured and is plotted in blue for each of the proteins as mentioned above for total antibody binding. Antibody off-rate constants that describe the fraction of antibody-antigen complexes decaying per second were determined directly from the serum sample interaction with EBOV proteins using SPR in the dissociation phase as described in Materials and Methods. All SPR experiments were performed twice and the researchers performing the assay were blinded to sample identity. The variation for each sample in duplicate SPR runs was <5%. The data shown is average value of two experimental runs. The maximum resonance units (Max RU) data shown was the calculated RU signal for the undiluted serum sample. (I - P) Antibody isotype of EBOV/Makona protein binding antibodies following EBOV infection. The isotype composition of serum antibodies bound to different proteins of EBOV/Makona isolate as measured in SPR. The resonance units for each anti-Makona protein antibody isotype (IgM in black, IgG in green, and IgA in red) was divided by the total resonance units for all antibody isotypes combined to calculate the percentage of each antibody isotype. for individual serum sample.

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Human anti-EBOV GP mAb (KZ52) IBT Bioservices 0260–001 HRP-conjugated goat anti-human IgA + IgG + IgM Jackson ImmunoResearch 109-035-064 Bacterial and Virus Strains E coli TG1 Khurana et al, 2011b In house maEBOV Bray et al, 1998 In house EBOV/Makona USAMRIID In house EBOV/Makona GFPDL This manuscript N/A M13K07 NEB N0315S Biological Samples EBOV post-infection samples Chertow et al, 2016 N/A Chemicals, Peptides, and Recombinant Proteins Zaire EBOV VP24 Protein Sino Biologicals 40454-V07E Recombinant Zaire EBOV Minor nucleotide VP30 My BioSource MBS1288327 Recombinant Zaire EBOV polymerase cofactor VP35 My BioSource MBS1308847 Zaire EBOV VP40 Matrix protein Sino Biologicals 40446-V07E Zaire EBOV RNA directed RNA polymerase L My BioSource MBS1414035 Recombinant Zaire EBOV NP Alpha Diagnostics EVNP15-R-10 EBOV H.sapiens-wt/GIN/2014/ Kissidougou-C15 GP Sino Biologicals 40442-V08H1 Recombinant EBOV Soluble GP IBT 0565–001 Recombinant Mayinga 1976 EBOV GP Sino Biologicals 40304-V08B1 Recombinant EBOV Kikwit-95 GP Acros Bio ZE5-V5221 Recombinant SUDV GP Immune Tech IT-014–007p Peptides & KLH-Peptides This manuscript N/A Protein A UltraLink™ Resin Thermo Scientific 53139 Fab Preparation Kit Thermo Scientific 44985 Emulsigen MVP Adjuvants N/A Experimental Models: Cell Lines Vero E6 cells ATCC CCL-81 293T cells ATCC CRL-3216 Experimental Models: Organisms/Strains Female New Zealand white rabbits Charles River Labs KBL(NZW)BR Female C57Bl/6 mice Charles River Labs N/A Open in a separate window KEY RESOURCES TABLE Longitudinal analysis of Ebola survivor reveals long-lasting IgM/IgG/IgA epitope diversity Despite undetectable virus, an EBOV-specific IgM suggests occult viral persistence Antibody affinity and immunodominant sites associated with EVD resolution are revealed Sites in C-terminus of GP1 and GP2 are promising therapeutic and vaccine targets

Techniques: Binding Assay, Purification, Infection, Protein Binding

Journal: Cell host & microbe

Article Title: Longitudinal human antibody repertoire against complete viral proteome from Ebola virus survivor reveals protective sites for vaccine design

doi: 10.1016/j.chom.2020.01.001

Figure Lengend Snippet: (A) IgM, IgG and IgA antibody epitope repertoire recognized in the EBOV infected sera at different days post-onset of symptoms ((D7, D13, D19, D31, D110 and D361) and their alignment to the whole proteome of EBOV showing different proteins (NP, VP35, VP40, GP, VP30, VP24 and L). Graphical distribution of representative clones with a frequency of ≥2, obtained after affinity selection, are shown. The horizontal position and the length of the bars indicate the peptide sequence displayed on the selected phage clone to its homologous sequence in the EBOV proteome on alignment. The thickness of each bar represents the frequency of repetitively isolated phage, with the scale shown below the alignment. Scale value for IgM, IgG and IgA is shown enclosed in a red box beneath the respective alignments. The GFPDL affinity selection data was performed in duplicate (two independent experiments by researcher in the lab, who was blinded to sample identity), and similar number of phage clones and epitope repertoire was observed in both phage display analysis. (B) Elucidation of antibody epitope profile against the EBOV proteome following EBOV infection. Antigenic sites within the EBOV proteins recognized by serum antibodies following EBOV infection (based on data presented in Fig. 1A). The amino acid designation is based on the EBOV protein sequence encoded by the complete EBOV/Makona genome. The antigenic regions/sites discovered in this study using the post-infection antibodies are depicted below the EBOV proteome schematic and are color coded. Epitopes of each protein are numbered in a sequential fashion indicated in black and the epitopes are color coded according to the protein color code in the proteome map.

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Human anti-EBOV GP mAb (KZ52) IBT Bioservices 0260–001 HRP-conjugated goat anti-human IgA + IgG + IgM Jackson ImmunoResearch 109-035-064 Bacterial and Virus Strains E coli TG1 Khurana et al, 2011b In house maEBOV Bray et al, 1998 In house EBOV/Makona USAMRIID In house EBOV/Makona GFPDL This manuscript N/A M13K07 NEB N0315S Biological Samples EBOV post-infection samples Chertow et al, 2016 N/A Chemicals, Peptides, and Recombinant Proteins Zaire EBOV VP24 Protein Sino Biologicals 40454-V07E Recombinant Zaire EBOV Minor nucleotide VP30 My BioSource MBS1288327 Recombinant Zaire EBOV polymerase cofactor VP35 My BioSource MBS1308847 Zaire EBOV VP40 Matrix protein Sino Biologicals 40446-V07E Zaire EBOV RNA directed RNA polymerase L My BioSource MBS1414035 Recombinant Zaire EBOV NP Alpha Diagnostics EVNP15-R-10 EBOV H.sapiens-wt/GIN/2014/ Kissidougou-C15 GP Sino Biologicals 40442-V08H1 Recombinant EBOV Soluble GP IBT 0565–001 Recombinant Mayinga 1976 EBOV GP Sino Biologicals 40304-V08B1 Recombinant EBOV Kikwit-95 GP Acros Bio ZE5-V5221 Recombinant SUDV GP Immune Tech IT-014–007p Peptides & KLH-Peptides This manuscript N/A Protein A UltraLink™ Resin Thermo Scientific 53139 Fab Preparation Kit Thermo Scientific 44985 Emulsigen MVP Adjuvants N/A Experimental Models: Cell Lines Vero E6 cells ATCC CCL-81 293T cells ATCC CRL-3216 Experimental Models: Organisms/Strains Female New Zealand white rabbits Charles River Labs KBL(NZW)BR Female C57Bl/6 mice Charles River Labs N/A Open in a separate window KEY RESOURCES TABLE Longitudinal analysis of Ebola survivor reveals long-lasting IgM/IgG/IgA epitope diversity Despite undetectable virus, an EBOV-specific IgM suggests occult viral persistence Antibody affinity and immunodominant sites associated with EVD resolution are revealed Sites in C-terminus of GP1 and GP2 are promising therapeutic and vaccine targets

Techniques: Infection, Clone Assay, Selection, Sequencing, Isolation

Journal: Cell host & microbe

Article Title: Longitudinal human antibody repertoire against complete viral proteome from Ebola virus survivor reveals protective sites for vaccine design

doi: 10.1016/j.chom.2020.01.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Human anti-EBOV GP mAb (KZ52) IBT Bioservices 0260–001 HRP-conjugated goat anti-human IgA + IgG + IgM Jackson ImmunoResearch 109-035-064 Bacterial and Virus Strains E coli TG1 Khurana et al, 2011b In house maEBOV Bray et al, 1998 In house EBOV/Makona USAMRIID In house EBOV/Makona GFPDL This manuscript N/A M13K07 NEB N0315S Biological Samples EBOV post-infection samples Chertow et al, 2016 N/A Chemicals, Peptides, and Recombinant Proteins Zaire EBOV VP24 Protein Sino Biologicals 40454-V07E Recombinant Zaire EBOV Minor nucleotide VP30 My BioSource MBS1288327 Recombinant Zaire EBOV polymerase cofactor VP35 My BioSource MBS1308847 Zaire EBOV VP40 Matrix protein Sino Biologicals 40446-V07E Zaire EBOV RNA directed RNA polymerase L My BioSource MBS1414035 Recombinant Zaire EBOV NP Alpha Diagnostics EVNP15-R-10 EBOV H.sapiens-wt/GIN/2014/ Kissidougou-C15 GP Sino Biologicals 40442-V08H1 Recombinant EBOV Soluble GP IBT 0565–001 Recombinant Mayinga 1976 EBOV GP Sino Biologicals 40304-V08B1 Recombinant EBOV Kikwit-95 GP Acros Bio ZE5-V5221 Recombinant SUDV GP Immune Tech IT-014–007p Peptides & KLH-Peptides This manuscript N/A Protein A UltraLink™ Resin Thermo Scientific 53139 Fab Preparation Kit Thermo Scientific 44985 Emulsigen MVP Adjuvants N/A Experimental Models: Cell Lines Vero E6 cells ATCC CCL-81 293T cells ATCC CRL-3216 Experimental Models: Organisms/Strains Female New Zealand white rabbits Charles River Labs KBL(NZW)BR Female C57Bl/6 mice Charles River Labs N/A Open in a separate window KEY RESOURCES TABLE Longitudinal analysis of Ebola survivor reveals long-lasting IgM/IgG/IgA epitope diversity Despite undetectable virus, an EBOV-specific IgM suggests occult viral persistence Antibody affinity and immunodominant sites associated with EVD resolution are revealed Sites in C-terminus of GP1 and GP2 are promising therapeutic and vaccine targets

Techniques: Recombinant

Journal: Cell host & microbe

Article Title: Longitudinal human antibody repertoire against complete viral proteome from Ebola virus survivor reveals protective sites for vaccine design

doi: 10.1016/j.chom.2020.01.001

Figure Lengend Snippet: Serial dilutions of serum samples collected at different time points from the EBOV survivor were analyzed for antibody binding to purified proteins from EBOV/Makona strain by SPR. (A-H) Total antibody binding is represented in SPR resonance units (RU) in black for binding to NP (A), VP35 (B). VP40 (B), GP (B). sGP (E), VP30 (F), VP24 (G) and L polymerase (H). Total antibody binding show is calculated RU for an undiluted serum sample. (A-H) Polyclonal antibody affinity maturation to EBOV proteins following EBOV infection in survivor was determined by SPR. Binding affinity of serially diluted post-infection serum to EBOV proteins was measured and is plotted in blue for each of the proteins as mentioned above for total antibody binding. Antibody off-rate constants that describe the fraction of antibody-antigen complexes decaying per second were determined directly from the serum sample interaction with EBOV proteins using SPR in the dissociation phase as described in Materials and Methods. All SPR experiments were performed twice and the researchers performing the assay were blinded to sample identity. The variation for each sample in duplicate SPR runs was <5%. The data shown is average value of two experimental runs. The maximum resonance units (Max RU) data shown was the calculated RU signal for the undiluted serum sample. (I - P) Antibody isotype of EBOV/Makona protein binding antibodies following EBOV infection. The isotype composition of serum antibodies bound to different proteins of EBOV/Makona isolate as measured in SPR. The resonance units for each anti-Makona protein antibody isotype (IgM in black, IgG in green, and IgA in red) was divided by the total resonance units for all antibody isotypes combined to calculate the percentage of each antibody isotype. for individual serum sample.

Article Snippet: Zaire EBOV VP40 Matrix protein , Sino Biologicals , 40446-V07E.

Techniques: Binding Assay, Purification, Infection, Protein Binding

Journal: Cell host & microbe

Article Title: Longitudinal human antibody repertoire against complete viral proteome from Ebola virus survivor reveals protective sites for vaccine design

doi: 10.1016/j.chom.2020.01.001

Figure Lengend Snippet: (A) IgM, IgG and IgA antibody epitope repertoire recognized in the EBOV infected sera at different days post-onset of symptoms ((D7, D13, D19, D31, D110 and D361) and their alignment to the whole proteome of EBOV showing different proteins (NP, VP35, VP40, GP, VP30, VP24 and L). Graphical distribution of representative clones with a frequency of ≥2, obtained after affinity selection, are shown. The horizontal position and the length of the bars indicate the peptide sequence displayed on the selected phage clone to its homologous sequence in the EBOV proteome on alignment. The thickness of each bar represents the frequency of repetitively isolated phage, with the scale shown below the alignment. Scale value for IgM, IgG and IgA is shown enclosed in a red box beneath the respective alignments. The GFPDL affinity selection data was performed in duplicate (two independent experiments by researcher in the lab, who was blinded to sample identity), and similar number of phage clones and epitope repertoire was observed in both phage display analysis. (B) Elucidation of antibody epitope profile against the EBOV proteome following EBOV infection. Antigenic sites within the EBOV proteins recognized by serum antibodies following EBOV infection (based on data presented in Fig. 1A). The amino acid designation is based on the EBOV protein sequence encoded by the complete EBOV/Makona genome. The antigenic regions/sites discovered in this study using the post-infection antibodies are depicted below the EBOV proteome schematic and are color coded. Epitopes of each protein are numbered in a sequential fashion indicated in black and the epitopes are color coded according to the protein color code in the proteome map.

Article Snippet: Zaire EBOV VP40 Matrix protein , Sino Biologicals , 40446-V07E.

Techniques: Infection, Clone Assay, Selection, Sequencing, Isolation

Journal: Cell host & microbe

Article Title: Longitudinal human antibody repertoire against complete viral proteome from Ebola virus survivor reveals protective sites for vaccine design

doi: 10.1016/j.chom.2020.01.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Zaire EBOV VP40 Matrix protein , Sino Biologicals , 40446-V07E.

Techniques: Recombinant