zikv vlps  (Native Antigen Inc)

Journal: Journal of Virology

Article Title: Somatic Hypermutation and Framework Mutations of Variable Region Contribute to Anti-Zika Virus-Specific Monoclonal Antibody Binding and Function

doi: 10.1128/jvi.00071-22

Figure Lengend Snippet: Reactivity of anti-ZIKV MAbs. (A) Reactivity of anti-ZIKV MAbs with ZIKV and DENV virus-like particles (VLP) using Luminex assay. ZIKV- and DENV-VLP were conjugated to MagPlex beads (Luminex) and 10,000 beads/mL of these beads mixed with 10 μg/mL to 0.0002 ng/mL anti-ZIKV MAb 102-1, 242-3, 270-12, 289-3, 306-2, 11-3, 278-11, 78-2, and 4G2 for 90 min at room temperature. (B) Western blot analysis of anti-ZIKV MAbs. ZIKV-VLP were heat denatured under nonreduced conditions at 70°C for 5 min. Samples (left, 27 ng; right, 240 ng) were electrophoresed by Wes capillary and detected by 10 μg/mL anti-ZIKV MAb clones. Left, MAb 102-1, 242-3, 270-12, 289-3, 306-2, 78-2 and 11-3. Right, 278-11. M, molecular weight marker. The estimated molecular mass of ZIKV E protein, 55 kDa; prM protein, 23 kDa.

Article Snippet: DENV-1-VLP (Nauru/Western Pacific/1974), DENV-2-VLP (Thailand/16681/84), DENV-3-VLP (Sri Lanka D3/H/IMTSSA-SRI/2000/1266), DENV-4-VLP (Dominica/814669/1981), ZIKV-VLP (Suriname Z1106033), and ZIKV E protein (Suriname Z1106033) were purchased from The Native Antigen Company (Oxford, UK).

Techniques: Luminex, Western Blot, Clone Assay, Molecular Weight, Marker

Journal: Journal of Virology

Article Title: Somatic Hypermutation and Framework Mutations of Variable Region Contribute to Anti-Zika Virus-Specific Monoclonal Antibody Binding and Function

doi: 10.1128/jvi.00071-22

Figure Lengend Snippet: Summary of characterization of anti-ZIKV MAbs

Article Snippet: DENV-1-VLP (Nauru/Western Pacific/1974), DENV-2-VLP (Thailand/16681/84), DENV-3-VLP (Sri Lanka D3/H/IMTSSA-SRI/2000/1266), DENV-4-VLP (Dominica/814669/1981), ZIKV-VLP (Suriname Z1106033), and ZIKV E protein (Suriname Z1106033) were purchased from The Native Antigen Company (Oxford, UK).

Techniques: Luminex, Western Blot

Journal: Journal of Virology

Article Title: Somatic Hypermutation and Framework Mutations of Variable Region Contribute to Anti-Zika Virus-Specific Monoclonal Antibody Binding and Function

doi: 10.1128/jvi.00071-22

Figure Lengend Snippet: Kinetic analysis of anti-ZIKV MAbs. Kinetic analysis was conducted by Octet HTX (Sartorius). Anti-ZIKV MAbs were conjugated to an amine-reactive 2nd generation (AR2G) biosensor at 0.1 to 0.3 μg/mL, and the association constant ( k a ) was measured over 0 to 900 s and dissociation constant ( k dis ) for 1,200 s for ZIKV-VLP or E proteins at various concentrations. (A) MAb 289-3 and 102-1 to ZIKV-VLP, 3.3 to 16.7 nM. (B) MAb 289-3 and 102-1 to ZIKV E proteins, 6.67 to 33.3 nM, red line, fitting pattern. (C) k a / k dis plot for anti-ZIKV MAbs to ZIKV-VLP and ZIKV E proteins. Blue, MAb 78-2, 289-3, 306-2 and 278-11; red, MAb 102-1, 242-3. 270-12, and 11-3. k a and k dis values were calculated by two runs, and the average values are shown. Values (nM) for equilibrium dissociation constants ( K D ) are shown for each dotted line. K D = K dis / K a .

Article Snippet: DENV-1-VLP (Nauru/Western Pacific/1974), DENV-2-VLP (Thailand/16681/84), DENV-3-VLP (Sri Lanka D3/H/IMTSSA-SRI/2000/1266), DENV-4-VLP (Dominica/814669/1981), ZIKV-VLP (Suriname Z1106033), and ZIKV E protein (Suriname Z1106033) were purchased from The Native Antigen Company (Oxford, UK).

Techniques:

Journal: Journal of Virology

Article Title: Somatic Hypermutation and Framework Mutations of Variable Region Contribute to Anti-Zika Virus-Specific Monoclonal Antibody Binding and Function

doi: 10.1128/jvi.00071-22

Figure Lengend Snippet: Binding activity of framework region (FWR) amino acid reverted anti-ZIKV MAb. (A to D) Association and dissociation analysis of FWR amino acid reverted anti-ZIKV MAbs by Octet HTX (Sartorius); 2 μg/mL MAbs were captured to protein G biosensor (Sartorius), and 3 μg/mL ZIKV E protein was associated for 600 s and dissociated for 900 s. (E to H) Reactivity of anti-ZIKV MAbs of ZIKV-VLP using Luminex assay. Blue, anti-ZIKV MAbs with matured amino acid; red, anti-ZIKV MAbs with amino acid reverted to allele. (A and E) MAb 102-1. (B and F) MAb 270-12. (C and G) MAb 289-3. (D and H) MAb 306-2.

Article Snippet: DENV-1-VLP (Nauru/Western Pacific/1974), DENV-2-VLP (Thailand/16681/84), DENV-3-VLP (Sri Lanka D3/H/IMTSSA-SRI/2000/1266), DENV-4-VLP (Dominica/814669/1981), ZIKV-VLP (Suriname Z1106033), and ZIKV E protein (Suriname Z1106033) were purchased from The Native Antigen Company (Oxford, UK).

Techniques: Binding Assay, Activity Assay, Luminex

Journal: Journal of Virology

Article Title: Somatic Hypermutation and Framework Mutations of Variable Region Contribute to Anti-Zika Virus-Specific Monoclonal Antibody Binding and Function

doi: 10.1128/jvi.00071-22

Figure Lengend Snippet: Reactivity of anti-ZIKV MAbs. (A) Reactivity of anti-ZIKV MAbs with ZIKV and DENV virus-like particles (VLP) using Luminex assay. ZIKV- and DENV-VLP were conjugated to MagPlex beads (Luminex) and 10,000 beads/mL of these beads mixed with 10 μg/mL to 0.0002 ng/mL anti-ZIKV MAb 102-1, 242-3, 270-12, 289-3, 306-2, 11-3, 278-11, 78-2, and 4G2 for 90 min at room temperature. (B) Western blot analysis of anti-ZIKV MAbs. ZIKV-VLP were heat denatured under nonreduced conditions at 70°C for 5 min. Samples (left, 27 ng; right, 240 ng) were electrophoresed by Wes capillary and detected by 10 μg/mL anti-ZIKV MAb clones. Left, MAb 102-1, 242-3, 270-12, 289-3, 306-2, 78-2 and 11-3. Right, 278-11. M, molecular weight marker. The estimated molecular mass of ZIKV E protein, 55 kDa; prM protein, 23 kDa.

Article Snippet: A volume of 0.1 to 1.0 μg/mL ZIKV-VLP or 0.2 to 2 μg/mL ZIKV E protein in 1× kinetic buffer (Sartorius, Fremont, CA, USA) was associated with anti-ZIKV MAb for 900 s and dissociated for 1,200 s. Kinetic parameters, association constant ( k a ), and dissociation constant ( k dis ) were analyzed by Octet Data Analysis Software HT (ver.

Techniques: Virus, Luminex, Western Blot, Clone Assay, Molecular Weight, Marker

Journal: Journal of Virology

Article Title: Somatic Hypermutation and Framework Mutations of Variable Region Contribute to Anti-Zika Virus-Specific Monoclonal Antibody Binding and Function

doi: 10.1128/jvi.00071-22

Figure Lengend Snippet: Summary of characterization of anti-ZIKV MAbs

Article Snippet: A volume of 0.1 to 1.0 μg/mL ZIKV-VLP or 0.2 to 2 μg/mL ZIKV E protein in 1× kinetic buffer (Sartorius, Fremont, CA, USA) was associated with anti-ZIKV MAb for 900 s and dissociated for 1,200 s. Kinetic parameters, association constant ( k a ), and dissociation constant ( k dis ) were analyzed by Octet Data Analysis Software HT (ver.

Techniques: Luminex, Western Blot

Journal: Journal of Virology

Article Title: Somatic Hypermutation and Framework Mutations of Variable Region Contribute to Anti-Zika Virus-Specific Monoclonal Antibody Binding and Function

doi: 10.1128/jvi.00071-22

Figure Lengend Snippet: Kinetic analysis of anti-ZIKV MAbs. Kinetic analysis was conducted by Octet HTX (Sartorius). Anti-ZIKV MAbs were conjugated to an amine-reactive 2nd generation (AR2G) biosensor at 0.1 to 0.3 μg/mL, and the association constant ( k a ) was measured over 0 to 900 s and dissociation constant ( k dis ) for 1,200 s for ZIKV-VLP or E proteins at various concentrations. (A) MAb 289-3 and 102-1 to ZIKV-VLP, 3.3 to 16.7 nM. (B) MAb 289-3 and 102-1 to ZIKV E proteins, 6.67 to 33.3 nM, red line, fitting pattern. (C) k a / k dis plot for anti-ZIKV MAbs to ZIKV-VLP and ZIKV E proteins. Blue, MAb 78-2, 289-3, 306-2 and 278-11; red, MAb 102-1, 242-3. 270-12, and 11-3. k a and k dis values were calculated by two runs, and the average values are shown. Values (nM) for equilibrium dissociation constants ( K D ) are shown for each dotted line. K D = K dis / K a .

Article Snippet: A volume of 0.1 to 1.0 μg/mL ZIKV-VLP or 0.2 to 2 μg/mL ZIKV E protein in 1× kinetic buffer (Sartorius, Fremont, CA, USA) was associated with anti-ZIKV MAb for 900 s and dissociated for 1,200 s. Kinetic parameters, association constant ( k a ), and dissociation constant ( k dis ) were analyzed by Octet Data Analysis Software HT (ver.

Techniques:

Journal: Journal of Virology

Article Title: Somatic Hypermutation and Framework Mutations of Variable Region Contribute to Anti-Zika Virus-Specific Monoclonal Antibody Binding and Function

doi: 10.1128/jvi.00071-22

Figure Lengend Snippet: Binding activity of framework region (FWR) amino acid reverted anti-ZIKV MAb. (A to D) Association and dissociation analysis of FWR amino acid reverted anti-ZIKV MAbs by Octet HTX (Sartorius); 2 μg/mL MAbs were captured to protein G biosensor (Sartorius), and 3 μg/mL ZIKV E protein was associated for 600 s and dissociated for 900 s. (E to H) Reactivity of anti-ZIKV MAbs of ZIKV-VLP using Luminex assay. Blue, anti-ZIKV MAbs with matured amino acid; red, anti-ZIKV MAbs with amino acid reverted to allele. (A and E) MAb 102-1. (B and F) MAb 270-12. (C and G) MAb 289-3. (D and H) MAb 306-2.

Article Snippet: A volume of 0.1 to 1.0 μg/mL ZIKV-VLP or 0.2 to 2 μg/mL ZIKV E protein in 1× kinetic buffer (Sartorius, Fremont, CA, USA) was associated with anti-ZIKV MAb for 900 s and dissociated for 1,200 s. Kinetic parameters, association constant ( k a ), and dissociation constant ( k dis ) were analyzed by Octet Data Analysis Software HT (ver.

Techniques: Binding Assay, Activity Assay, Luminex

Journal: Immunology

Article Title: Non-structural protein 1-specific antibodies directed against Zika virus in humans mediate antibody-dependent cellular cytotoxicity.

doi: 10.1111/imm.13380

Figure Lengend Snippet: FIGURE 1 Isotype specificity of ZIKV-immune sera. ELISA plates were coated with ZIKV NS1 (a, c) or ZIKV VLP (b, d), and a 1:100 dilution (n = 28) (a, b) or serial dilutions (n = 11) (c, d) of ZIKV-immune sera were added to plates. Following incubation and washes, optimized concentrations of polyclonal secondary IgG, and monoclonal Abs against the hinge or Fc portion of IgG1, IgG2, IgG3 and IgG4 Abs were added to wells (a) and (b) and IgG1 hinge Abs to (c) and (d). OD values shown at 450 nm. OD values to ZIKV VLP and ZIKV NS1 using a 1:100 dilution of ZIKV-naïve sera (n = 4), <0·2 with polyclonal secondary IgG and <0·1 with IgG1 hinge, IgG1 Fc, IgG2, IgG3 and IgG4 secondary Abs. Bars represent mean with standard error of the mean of the OD values. *P < 0·05, ***P < 0·001 and ****P < 0·0001 were obtained using the non- parametric Friedman test (three or more matched groups) with Dunn's multiple comparisons test

Article Snippet: Briefly, 96- well plates were coated overnight with 20 ng/well of ZIKV virus- like particles (Suriname Z1106033) or 50 ng/ml ZIKV NS1 (Uganda MR766) (Native Antigen Company, Oxfordshire, UK).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation

Journal: Immunology

Article Title: Non-structural protein 1-specific antibodies directed against Zika virus in humans mediate antibody-dependent cellular cytotoxicity.

doi: 10.1111/imm.13380

Figure Lengend Snippet: FIGURE 2 Frequencies of ZIKV-specific IgG and IgG1 B-cell responses using fluorescently labelled ZIKV or ZIKV NS1. PBMCs from ZIKV-immune and ZIKV-naïve donors were stimulated in vitro for seven days with r848 + rIL-2. FluoroSpot plates were coated with IgG and IgG1 capture Abs. (a) Images of wells containing media, total IgG, ZIKV and ZIKV NS1 IgG- or IgG1-specific MBCs from one representative donor. (b) Frequencies of ZIKV (green) and NS1-specific (red) ASCs per 106 input cells in MBC cultures from 12 ZIKV-immune and three ZIKV- naïve donors. Values are the mean of duplicate wells for each condition. The supernatants were evaluated for isotype-specific Abs to (c) ZIKV VLPs and (D) ZIKV NS1. *P < 0·05, **P < 0·01 and ****P < 0·0001 were obtained using the non-parametric Mann-Whitney or Friedman test (three or more matched groups) with Dunn's multiple comparisons test

Article Snippet: Briefly, 96- well plates were coated overnight with 20 ng/well of ZIKV virus- like particles (Suriname Z1106033) or 50 ng/ml ZIKV NS1 (Uganda MR766) (Native Antigen Company, Oxfordshire, UK).

Techniques: In Vitro, MANN-WHITNEY

Journal: Immunology

Article Title: Non-structural protein 1-specific antibodies directed against Zika virus in humans mediate antibody-dependent cellular cytotoxicity.

doi: 10.1111/imm.13380

Figure Lengend Snippet: FIGURE 3 Opsonization and ADCC activity of a ZIKV NS1 monoclonal Ab. (a) K562 cells transfected with DC-SIGN were infected with ZIKV (moi = 0·1 and 1), and 24 h later, the expression of E and NS1 was measured with monoclonal Abs 4G2 and B4 respectively. (b) Opsonization of ZIKV NS1-transfected CEM-NKR cells with a mAb to ZIKV NS1 from Native Antigen Company. CEM-NKR cells transfected with ZIKV NS1 (c and d) or control CEM-NKR cells (e and f) were opsonized with a ZIKV NS1 mAb (d and f), added to PBMC. A standard lymphocyte gate based upon light scatter properties followed by selection of singlet viable cells and dump channel exclusion (CD3− T cells) defined the non-T-cell lymphocyte population, and Abs to CD16 and CD56 identified NK cells

Article Snippet: Briefly, 96- well plates were coated overnight with 20 ng/well of ZIKV virus- like particles (Suriname Z1106033) or 50 ng/ml ZIKV NS1 (Uganda MR766) (Native Antigen Company, Oxfordshire, UK).

Techniques: Activity Assay, Transfection, Infection, Expressing, Control, Selection

Journal: Immunology

Article Title: Non-structural protein 1-specific antibodies directed against Zika virus in humans mediate antibody-dependent cellular cytotoxicity.

doi: 10.1111/imm.13380

Figure Lengend Snippet: FIGURE 4 NK cell activation and degranulation in the presence of ZIKV- immune sera. 1:100 dilution and 1:1600 dilution of polyclonal sera from ZIKV- immune individuals were evaluated for their ability to mediate ADCC of ZIKV NS1 CEM-NKR cells using intracellular cytokine assays. Shown are the frequencies of NK cells that (a) secrete IFN-γ, (B) express CD107 or (C) express both CD107 and IFN-γ in the presence of ZIKV-immune sera. Background levels were subtracted

Article Snippet: Briefly, 96- well plates were coated overnight with 20 ng/well of ZIKV virus- like particles (Suriname Z1106033) or 50 ng/ml ZIKV NS1 (Uganda MR766) (Native Antigen Company, Oxfordshire, UK).

Techniques: Activation Assay

Journal: Immunology

Article Title: Non-structural protein 1-specific antibodies directed against Zika virus in humans mediate antibody-dependent cellular cytotoxicity.

doi: 10.1111/imm.13380

Figure Lengend Snippet: FIGURE 5 Opsonization and target cell lysis using an NK-TVA image cytometry assay. (a) Opsonization of ZIKV NS1 CEM-NKR cells by ZIKV convalescent sera. (b) Representative images of wells acquired by an ImmunoSpot S5 Analyser of an NK-TVA assay where ethanol, ZIKV NS1 or IgG Ab was added to the wells containing effector and target cells. (c) Using an optimized number of target cells, effector PBMC as a source of NK cells and different dilutions of naïve sera (n = 5), positive control sera that opsonized ZIKV NS1 CEM-NKR targets (n = 5) and experimental sera (n = 12), the loss of target cells at the single-cell level was measured by the NK-TVA image cytometry assay and calculated as a percentage of lysed target cells. *P < 0·05 and **P < 0·01 were obtained using a Kruskal–Wallis test with Dunn's multiple comparisons test

Article Snippet: Briefly, 96- well plates were coated overnight with 20 ng/well of ZIKV virus- like particles (Suriname Z1106033) or 50 ng/ml ZIKV NS1 (Uganda MR766) (Native Antigen Company, Oxfordshire, UK).

Techniques: Lysis, Cytometry, Positive Control