Journal: bioRxiv

Article Title: HIV broadly neutralizing antibodies with enhanced Fc-CD16 affinity increase NK cell ADCC even with limited envelope binding

doi: 10.64898/2026.04.27.721006

Figure Lengend Snippet: Cytokine stimulation enhances NK cell activation while downregulating CD16 expression through ADAM17-dependent shedding. ( A ) Representative flow plots of CD69 induction of NK cells stimulated with IL-15, IL-12/IL-15, or IL-12/IL-15/IL-18. ( B ) CD69 induction across donors (n=10). ( C ) Viability upon different treatments. ( D ) Changes in CD16 expression across multiple donors upon cytokine treatment. ( E ) Spearman correlation analysis between CD16 expression and CD69 induction. ( F ) Representative histogram of CD16 expression upon triple cytokine treatment in the presence of TAPI-I (right panel) or DMSO control (left panel). ( G ) CD16 expression in donors (n=13) stimulated with cytokines in the absence or presence of TAPI-1. ( H ) Effects of TAPI-I on NK cell activation. ( I ) Percentage remaining p24 + cells upon coculture of HIV-infected CD4 + T cells with autologous NK cells from donors (n=7) pre-stimulated with cytokines in the presence of TAPI-1 or DMSO control. ( J ) Percentage ADCC calculated as indicated in methods from data shown in ( I ). Statistical significance was calculated using one-way ANOVA with multiple comparison compared to the no cytokine condition for panel ( B-D ), two-way ANOVA with multiple comparison compared to the IL12/IL15/IL18 condition for panel ( I ), and two-way ANOVA for panel ( J ). Open symbols represent female donors and closed symbols male donors.

Article Snippet: Ten cytokines were measured using Quanterix SP-X Corplex Cytokine Panel (IFN-γ, IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-22, TNF-α) (cat# 85-0329).

Techniques: Activation Assay, Expressing, Control, Infection, Comparison

Journal: bioRxiv

Article Title: HIV broadly neutralizing antibodies with enhanced Fc-CD16 affinity increase NK cell ADCC even with limited envelope binding

doi: 10.64898/2026.04.27.721006

Figure Lengend Snippet: Functional activity, cytokine secretion, and Fc binding of VRC07-523-LS and Fc variants. ( A ) Table summarizing VRC07-523LS antibody mutants with optimized Fc biding including specific mutations and effects on CD16 binding. ( B ) CD16 reporter activity in Jurkat-Lucia NFAT-CD16 cells exposed to antibody variants. ( C ) CD69 induction across donors (n=7). ( D ) Viability upon different conditions. ( E-O ) Supernatants from NK cells cultured overnight with WT VRC07-523-LS or Fc mutant antibodies were analyzed for cytokine secretion (fg/mL). Levels of ( E ) IFN-γ (Limit of detection (LOD) 1fg/mL), ( F ) TNF-α (LOD 16.7fg/mL), ( G ) IL-1β (LOD 16.3fg/mL), ( H ) IL-4 (LOD 339.1fg/mL), ( I ) IL-5 (LOD 8.7fg/ml), ( J ) IL-6 (LOD 21.1fg/mL), ( K ) IL-8 (LOD 41.7fg/mL), ( M ) IL-10 (LOD 16.8fg/mL), ( N ) IL-12p70 (LOD 24.7 fg/mL), ( O ) IL-22 (LOD 4.5 fg/mL) (n=6). ( P ) Representative flow cytometry histograms showing VRC07-523LS binding to surface Env on uninfected (blue), uninfected exposed (red), or HIV-infected cells (black). A FMO control is included (grey). Quantification of Env binding for the different antibodies (n=7). ( Q ) Surface expression of CD107a upon co-culture of NK cells alone (no CD4 + ), or NK cells co-cultured with uninfected autologous CD4 + or NL4-3 infected CD4 + T cells in the presence of the indicated antibody (n=2). Statistical significance was assessed using a Friedman test with multiple comparisons compared to unstimulated condition for panels ( C-O ), and two-way ANOVA with multiple comparisons for panel ( P ). Open symbols represent female and closed symbols male donors.

Article Snippet: Ten cytokines were measured using Quanterix SP-X Corplex Cytokine Panel (IFN-γ, IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-22, TNF-α) (cat# 85-0329).

Techniques: Functional Assay, Activity Assay, Binding Assay, Cell Culture, Mutagenesis, Flow Cytometry, Infection, Control, Expressing, Co-Culture Assay

Journal: bioRxiv

Article Title: HIV broadly neutralizing antibodies with enhanced Fc-CD16 affinity increase NK cell ADCC even with limited envelope binding

doi: 10.64898/2026.04.27.721006

Figure Lengend Snippet: Enhancing antibody affinity to CD16 improves NK cell-mediated ADCC against HIV-infected cells independently of cytokine activation or ADAM17 inhibition. Percent of remaining HIV p24⁺ infected CD4⁺ T cells following co-culture with autologous NK cells from donors pre-treated with or without cytokines (IL-12/IL-15/IL-18) (n=16) ( A ) and ADAM17 inhibitor TAPI-1 (n=7) ( B ), in the presence of the indicated antibody. ( C ) Hierarchical cluster analysis of percentage of remaining p24⁺ cells in different conditions shown in A-B. ADCC activity under the same treatment conditions in the absence ( D ) or the presence ( E ) of the ADAM17 inhibitor TAPI-1. Statistical significance was determined using two-way ANOVA with multiple comparison compared to WT. Open symbols represent female and closed symbols male donors. ( F ) Hierarchical cluster analysis of ADCC in different conditions shown in D-E . Clusters were done using ClustVis.

Article Snippet: Ten cytokines were measured using Quanterix SP-X Corplex Cytokine Panel (IFN-γ, IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-22, TNF-α) (cat# 85-0329).

Techniques: Infection, Activation Assay, Inhibition, Co-Culture Assay, Activity Assay, Comparison

Journal: bioRxiv

Article Title: HIV broadly neutralizing antibodies with enhanced Fc-CD16 affinity increase NK cell ADCC even with limited envelope binding

doi: 10.64898/2026.04.27.721006

Figure Lengend Snippet: Effects of Fc-optimized VRC07-523LS mutants across HIV-1 subtypes. ( A ) Neutralization curves of VRC07-523LS against indicated HIV-1 viral strain. ( B ) Env binding of the different antibodies to HIV-infected CD4T cells with the indicated viral strain (n=3-4). ( C ) Spearman correlation between Env binding and neutralization IC 50 values. ( D ) Percentage of remaining p24 + cells following natural cytotoxicity by autologous NK cells pretreated with a combination of cytokines or no cytokine control (n= 6-16). ( E-I ) Percentage of remaining p24+ cells for each of the indicated viruses in the presence of the different antibodies (n= 7-8). Statistical significance was assessed using a one-sample t test with a hypothetical value set to 1 for panel ( B ), wilcoxon signed rank test with hypothetical value set to 100 for panel ( E ), and two -way ANOVA with multiple comparison compared to WT for panels ( E–I ). Open symbols represent female and closed symbols male donors. ( J ) Hierarchical cluster analysis of the percentage remaining p24 + cells. Clusters were done using ClustVis.

Article Snippet: Ten cytokines were measured using Quanterix SP-X Corplex Cytokine Panel (IFN-γ, IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-22, TNF-α) (cat# 85-0329).

Techniques: Neutralization, Binding Assay, Infection, Control, Comparison

Journal: bioRxiv

Article Title: HIV broadly neutralizing antibodies with enhanced Fc-CD16 affinity increase NK cell ADCC even with limited envelope binding

doi: 10.64898/2026.04.27.721006

Figure Lengend Snippet: Cytokine activation of NK cells requires higher levels of surface Env binding to promote ADCC. ( A ) Percentage of ADCC mediated by VRC07-523LS for each of the viral strains upon pretreatment of NK cells with the combination of cytokines or no cytokine control (n=6-16). Statistical significance between no cytokine and IL12/IL15/IL18 was determined by two-way ANOVA and Wilcoxon signed rank test with hypothetical value set to 0 for each condition. Open symbols represent female and closed symbols male donors. ( B ) Spearman correlation between the percentage of ADCC and surface Env binding for the indicated antibodies and NK cells pretreated with no cytokine (top) or IL12/IL15/IL18 (bottom). Each virus is indicated with a different color (n=4-6).

Article Snippet: Ten cytokines were measured using Quanterix SP-X Corplex Cytokine Panel (IFN-γ, IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-22, TNF-α) (cat# 85-0329).

Techniques: Activation Assay, Binding Assay, Control, Virus

Journal: Cells

Article Title: ‘Shared-Hook’ and ‘Changed-Hook’ Binding Activities of Herpesviral Core Nuclear Egress Complexes Identified by Random Mutagenesis

doi: 10.3390/cells11244030

Figure Lengend Snippet: Qualitative and quantitative analyses of HCMV AD169-GFP ΔUL53 virus reconstitution on the different recombinant HFF cell populations. HFFs that express pUL53, pUL53-Flag or pUL53::sHook1-Flag were transfected with the infectious bacterial artificial chromosome of HCMV ΔUL53. ( A ) Images of the GFP signals and the respective brightfield illuminations were taken at indicated time points; scale bar in panel A, picture 1 marks 500 μm. ( B ) Quantitative analysis of fluorescence-positive cells was achieved with automated counting by the CellReporterXpress ® software (Molecular Devices LLC, San Jose, CA, USA). Counting parameters for positive nuclei were set to an intensity of at least 100, a minimal width of 7 and a maximal width of 30. Measurements were performed in biological sextuplicates per cell population of 5.05% area of the well and mean values ± SD are given. ( C ) qPCR-based assay for the determination of viral genome equivalents referring to the respective recombinant HFF populations. Viral supernatants were harvested at day 24 p.t. and subjected to IE1-specific qPCR. Calculations were performed in biological sextuplicates per cell population; mean values ± SD are shown. ( B , C ) Statistical analysis was performed using an ordinary two-way ANOVA and post-hoc Tukey correction; ****, p < 0.0001; **, p < 0.01; *, p < 0.1; ns, not significant; no significant differences were noted for values between 1–20 d p.t. ( B ). ( D ) Wb-based expression analysis of the conditionally expressing HFF populations. Recombinant protein expression in the three HFF populations of HFF-UL53, HFF-UL53-Flag and HFF-UL53::sHook1-Flag cells was either uninduced (-) or induced (500 ng/mL Dox). Cells were either infected with HCMV ΔUL53 or remained mock-infected. At 24 h p.t., cells were harvested and lysed. Total lysate samples were subjected to standard Wb analysis using tag-specific or protein-specific monoclonal antibodies as indicated.

Article Snippet: For measurement of HCMV AD169-GFP ΔUL53-positive cells, signals were counted with the ImageXpress ® Pico device (Molecular Devices LLC, San Jose, CA, USA) using CellReporterXpress ® software (version 2.9.3.1183, Molecular Devices LLC) by the system-integrated cell count assay.

Techniques: Virus, Recombinant, Transfection, Fluorescence, Software, Expressing, Infection, Bioprocessing

Journal: Cells

Article Title: ‘Shared-Hook’ and ‘Changed-Hook’ Binding Activities of Herpesviral Core Nuclear Egress Complexes Identified by Random Mutagenesis

doi: 10.3390/cells11244030

Figure Lengend Snippet: Viral replication kinetics of HCMV ΔUL53 and its revertant (HCMV Rev) determined by quantitation of GFP-positive cells and HCMV-specific qPCR on the different recombinant HFF populations. 80,000 inducibly expressing HFFs in 24-well plates were infected with HCMV ΔUL53 or HCMV Rev at a viral dose of 5 × 10 6 genome copies. pUL53, pUL53-Flag or pUL53::sHook1-Flag protein expression was either Dox-induced (+Dox) or remained non-induced (−Dox). ( A ) The number of HCMV-infected cells was measured by detection of GFP signal-positive cells at indicated time points with the CellReporterXpress ® software using the ImageXpress ® Pico device. Values represent 25.04 % of the area of a well and are given as a mean value ± SD of two independently infected wells. ( B ) Viral supernatants were harvested at indicated time points and viral genome equivalents were determined by qPCR. Each value represents the mean ± SD of two independent biological replicates, each measured twice. ( A , B ) Statistical analysis was performed using an ordinary two-way ANOVA and post-hoc Sidak correction; ****, p < 0.0001; ***, p < 0.001; *, p < 0.1; ns, not significant; no significant differences were noted for values between 1–13 d p.i. ( A ) or 1–11 d p.i. ( B ), respectively.

Article Snippet: For measurement of HCMV AD169-GFP ΔUL53-positive cells, signals were counted with the ImageXpress ® Pico device (Molecular Devices LLC, San Jose, CA, USA) using CellReporterXpress ® software (version 2.9.3.1183, Molecular Devices LLC) by the system-integrated cell count assay.

Techniques: Quantitation Assay, Recombinant, Expressing, Infection, Software