Journal: bioRxiv

Article Title: Dengue virus structural proteins are expressed on the surface of DENV-infected cells and are a target for antibody-dependent cellular phagocytosis

doi: 10.1101/2024.07.21.604479

Figure Lengend Snippet: A) Model for the depletion of NS1-reactive antibodies from whole DENV-immune serum as described in the methods B) Opsonization quantification on DENV3 NS1 expressing CEM.NK R cells (left) and DENV3-infected CEM.NK R cells (right) using either Naïve serum (orange), whole immune serum (blue) or whole serum that was depleted of NS1-reactive antibodies (red). Plots gated on cells positive for intracellular E antigen (4G2). C) Staining of control (grey) and DENV-3 infected (red) cells with the indicated monoclonal antibodies. DENV-3 infected samples gated on infected cells by intracellular anti-prM FITC staining.

Article Snippet: CEM.NK R and DENV3 infected DC-SIGN-expressing CEM.NK R cells were stained with PKH26 at a final concentration of 3.0×10 −6 M resuspended in Diluent C for 5 minutes at room temperature.

Techniques: Expressing, Infection, Staining, Control, Bioprocessing

Journal: bioRxiv

Article Title: Dengue virus structural proteins are expressed on the surface of DENV-infected cells and are a target for antibody-dependent cellular phagocytosis

doi: 10.1101/2024.07.21.604479

Figure Lengend Snippet: A) Assessment of DENV E protein binding activity of 4E11 IgG and 4E11 IgA mAbs utilized in this study by ELISA, B) Opsonization of parental uninfected CEM.NK R cells (left) and DENV3-infected CEM.NK R cells (right), dashed lines indicating incubation without the primary 4E11 antibody, C) Percentage of monocytes that were positive for bead fluorescence, separated by antibody used to opsonize target. This data is expressed as background subtracted from a no serum condition, n=7 individual experiments. Error bars are mean ± SEM. * p < 0.05, paired one-way ANOVA, D) Percentage of monocytes that were positive for the target cell membrane dye, separated by antibody used to opsonize target. This data is expressed as background subtracted from a no serum condition, n=4 individual experiments. Error bars are mean ± SEM. * p < 0.05, paired one-way ANOVA

Article Snippet: CEM.NK R and DENV3 infected DC-SIGN-expressing CEM.NK R cells were stained with PKH26 at a final concentration of 3.0×10 −6 M resuspended in Diluent C for 5 minutes at room temperature.

Techniques: Protein Binding, Activity Assay, Enzyme-linked Immunosorbent Assay, Infection, Incubation, Fluorescence, Membrane

Journal: bioRxiv

Article Title: Dengue virus structural proteins are expressed on the surface of DENV-infected cells and are a target for antibody-dependent cellular phagocytosis

doi: 10.1101/2024.07.21.604479

Figure Lengend Snippet: A) Representative images of microscopy of monocytes that were incubated alone (top) or with DENV3 infected cells opsonized with human 4E11 IgG (bottom) at 63x magnification and each image was created using 3D projections with Z-stacks taken every 0.5um with nearest neighbors’ deconvolution. Monocyte cytosol was stained with CFSE (green), lysosomes were stained with Lysotracker Deep Red (cyan), and DENV3 infected cell membranes stained with PKH26 (red). Scale bar is 10 microns. Images were merged to show a visual representation of the colocalization, B) Quantification of colocalization between monocyte lysosomes and DENV3-infected cell membrane with each individual cell calculated separately, calculated using the Pearsons Coefficient from JACoP in FIJI, n=52

Article Snippet: CEM.NK R and DENV3 infected DC-SIGN-expressing CEM.NK R cells were stained with PKH26 at a final concentration of 3.0×10 −6 M resuspended in Diluent C for 5 minutes at room temperature.

Techniques: Microscopy, Incubation, Infection, Staining, Membrane

Journal: bioRxiv

Article Title: Dengue virus structural proteins are expressed on the surface of DENV-infected cells and are a target for antibody-dependent cellular phagocytosis

doi: 10.1101/2024.07.21.604479

Figure Lengend Snippet: A) Example flow plot used to isolate out each cell type for use in the ddPCR. PKH-monocytes were taken from the single positive CD14+ cells, DENV3-infected target cells were taken from the single positive PKH26 membrane dye cells, and the PKH+ monocytes were taken from the double positive cells, B) Copies of DENV RNA per cell found using droplet digital PCR in DENV3-infected CEM.NK R cells, PKH26(-) monocytes, and PKH26(+) monocytes that were incubated together and sorted separately using the Aria cell sorter. Error bars are mean ± SEM, paired one-way ANOVA, n=3

Article Snippet: CEM.NK R and DENV3 infected DC-SIGN-expressing CEM.NK R cells were stained with PKH26 at a final concentration of 3.0×10 −6 M resuspended in Diluent C for 5 minutes at room temperature.

Techniques: Infection, Membrane, Digital PCR, Incubation

Journal: NPJ Vaccines

Article Title: The dengue-specific immune response and antibody identification with machine learning

doi: 10.1038/s41541-023-00788-7

Figure Lengend Snippet: a Experimental steps. Five cohorts of three mice each were immunized with dengue antigens Whole protein E DENV-2 (mice 4, 5, 6), EDIII from DENV-1 (mice 9, 10, 11), EDIII from DENV-2 (mice 12, 13, 14), EDIII from DENV-3 (mice 15, 16, 17), EDIII from DENV-4 (mice 18, 19, 20), while one cohort of three mice was treated with adjuvant only (mice 1, 2, 3). Mice were sacrificed and bone marrow was collected. Plasma cells were FACS-sorted. Antibody repertoires were sequenced, and high-throughput sequencing datasets were annotated and preprocessed. b Computational analysis performed. On the left, repertoire-level: CDR3 overlaps (repertoire similarity), germline gene usage, uniform manifold approximation and projection (UMAP) clustering, network analysis, CDR3 length distributions, gene expression. On the right, sequence-level: sequence conservation, an example of clone similarity degree distribution of an antibody repertoire, machine learning benchmarking, amino acid frequency.

Article Snippet: For testing binding of DenAb X to DENV, ELISA Maxisorp plates (Nunc, Rochester, United States) were coated with DENV Whole protein E from serotypes DENV-1, DENV-2 and DENV-3 produced by the Native Antigen Company (Native Antigen Company, Oxfordshire, UK).

Techniques: Adjuvant, Next-Generation Sequencing, Expressing, Sequencing

Journal: Viruses

Article Title: NS1 Antigenemia and Viraemia Load: Potential Markers of Progression to Dengue Fatal Outcome?

doi: 10.3390/v10060326

Figure Lengend Snippet: Non-structural protein 1 (NS1) antigenemia ( A ) according to the disease outcome (non-fatal: n = 40; fatal: n = 40), * p < 0.01 using t test; ( B ) in the overall analysis of the type of infection (primary: n = 24 versus secondary: n = 45) using t test; ( C ) in the analysis of primary non-fatal ( n = 8) versus fatal ( n = 16) cases by t test; ( D ) in the secondary non-fatal ( n = 23) and fatal ( n = 22) cases, * p < 0.01 using t test; ( E ) in the analysis of DENV serotype regardless of the disease outcome ( n = 80) by ANOVA test * p < 0.01; ( F ) in the analysis of the distinct DENV serotypes according to the disease outcome (fatal: n = 10 and non-fatal/serotype) using t test. For DENV-2, fatal versus non-fatal cases, * p < 0.01, and for DENV-4 fatal versus non-fatal cases, * p < 0.01.

Article Snippet: Briefly, a standard NS1 antigen curve (ng/mL) based on an equation (y = 1.321x + 0.1271) with R 2 = 0.9542 was used and established using synthetic NS1 proteins (Native Antigen Company, Oxforshire, UK) corresponding to the NS1 of DENV-1 (Nauru/Western Pacific/1974), DENV-2 (Thailand/16681/84), DENV-3 (Sri Lanka D3/H/IMTSSA-SRI/2000/1266) and DENV-4 (Dominica/814669/1981) with 10-fold dilution.

Techniques: Infection

Journal: Viruses

Article Title: NS1 Antigenemia and Viraemia Load: Potential Markers of Progression to Dengue Fatal Outcome?

doi: 10.3390/v10060326

Figure Lengend Snippet: Overall NS1 antigenemia and RNA viral load according to the distinct DENV serotypes, independent of the disease outcome.

Article Snippet: Briefly, a standard NS1 antigen curve (ng/mL) based on an equation (y = 1.321x + 0.1271) with R 2 = 0.9542 was used and established using synthetic NS1 proteins (Native Antigen Company, Oxforshire, UK) corresponding to the NS1 of DENV-1 (Nauru/Western Pacific/1974), DENV-2 (Thailand/16681/84), DENV-3 (Sri Lanka D3/H/IMTSSA-SRI/2000/1266) and DENV-4 (Dominica/814669/1981) with 10-fold dilution.

Techniques:

Journal: Viruses

Article Title: NS1 Antigenemia and Viraemia Load: Potential Markers of Progression to Dengue Fatal Outcome?

doi: 10.3390/v10060326

Figure Lengend Snippet: NS1 antigenemia and RNA viral load according to the distinct DENV serotype and disease outcome.

Article Snippet: Briefly, a standard NS1 antigen curve (ng/mL) based on an equation (y = 1.321x + 0.1271) with R 2 = 0.9542 was used and established using synthetic NS1 proteins (Native Antigen Company, Oxforshire, UK) corresponding to the NS1 of DENV-1 (Nauru/Western Pacific/1974), DENV-2 (Thailand/16681/84), DENV-3 (Sri Lanka D3/H/IMTSSA-SRI/2000/1266) and DENV-4 (Dominica/814669/1981) with 10-fold dilution.

Techniques:

Journal: Bulletin of the World Health Organization

Article Title: Measuring Haitian children's exposure to chikungunya, dengue and malaria

doi: 10.2471/BLT.16.173252

Figure Lengend Snippet: Reference values and positivity thresholds for a multiplex bead assay based on antigens from chikungunya and dengue viruses and Plasmodium falciparum

Article Snippet: We used one recombinant chikungunya virus antigen – that is, mutant A226V envelope 1 antigen (CTK Biotech, San Diego, USA) – two dengue virus antigens – propagated using a eukaryotic plasmid vector that expressed the premembrane/membrane and envelope proteins that self-assemble into two different non-infectious virus-like particles known as DENV-2 and DENV-3 , – and three recombinant antigens based on merozoite surface protein 1 of P. falciparum .

Techniques: Multiplex Assay, Fluorescence, Virus, Mutagenesis