Journal: NPJ Vaccines

Article Title: Immunization-induced antigen archiving enhances local memory CD8+ T cell responses following an unrelated viral infection

doi: 10.1038/s41541-024-00856-6

Figure Lengend Snippet: a Experimental schematic for b – e . C57/BL6 mice were immunized subcutaneously in the footpad and/or flank with the indicated antigens and adjuvants. b Cells were stained with CD45, PDPN, CD31 and PD-L1. Cells were gated on CD45-PDPN + CD31- for FRC and CD45-PDPN + CD31+ for LECs. Shown are examples of LEC and FRC antigen-positive cells based on PD-L1 expression (floor, MARCO LEC) and ova-AF488+ from mice 2–3 weeks after immunization with ova conjugated to Alexa-Fluor 488 (AF488) and polyI:C and αCD40. c Quantification of the frequency of LEC, BEC, and FRC in the popliteal lymph node (pLN) that are positive for the indicated antigens administered with polyI:C and αCD40 at indicated time. d Same as ( b ) except for mice were immunized with SARS-CoV-2-RBD-AF488, polyI:C, and αCD40. e Same as in ( c ) except for SARS-CoV-2-RBD and CHIKV-E2 with polyI:C and αCD40. CHIKV-E2 was repeated for 9–14 days post-vaccine (~2 weeks). Statistical analysis was done using an unpaired t -test where the p -value between naïve and indicated antigen is <0.0001. In each experiment, at least n = 2–3 mice per group were evaluated and the experiment was repeated n = 2–5 times for c – e . Shown is the representative data from one of the experiments. Error bars are mean ± standard error of the mean. ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Ova (10 μg) was purchased from Sigma-Aldrich (Cat No. A5503) and Chikungunya virus envelope 2 protein (8 μg) (CHIKV-E2, strain SL-CK1) was purchased from Sino Biological (Cat. No. 40440-V08B).

Techniques: Staining, Expressing

Journal: bioRxiv

Article Title: Downregulation of a cell polarity protein potentiates Chikungunya Virus infection in host cells

doi: 10.1101/2023.07.24.550336

Figure Lengend Snippet: The CHIKV RNA genome has two open reading frames (ORFs) separated by a junctional region (J). The 5’ ORF (ORF 1) codes for four non-structural proteins (nsP1-4). The 3’ ORF (ORF 2) codes for 5 structural proteins, namely capsid (C), E3, E2, 6K, and E1. The 6K has a ribosomal slippery site which leads to the production of a transframe (TF) protein (Firth et al., 2008) (upper panel, A). Schematic of 6K and TF sequences and the ribosome slip site (F at 48th and L at 49th position) (lower panel, A). Potential PDZ binding motifs (PBMs) present in TF as predicted by different softwares (B). Predicted interactions between TF and Scribble PDZ1, Scribble PDZ4, and HTRA1 (C). RMSF plot for TF interactions with Scribble and HTRA1 (D). Binding of TF C-terminal residues with ScPDZ1.

Article Snippet: Total protein was quantified using the bicinchoninic acid (BCA) assay, followed by SDS-PAGE and Western blotting with a primary antibody against Scribble (1:500, Santa Cruz sc55543), beta-actin (1:1000, Cell signaling Technology 4967), beta-tubulin (1:200, Santa Cruz sc55529), CHIKV E2 (1:3000, The Native antigen company MAB12130-200), and EGFP (1:3000, Bio Bharati BB-AB0065).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Downregulation of a cell polarity protein potentiates Chikungunya Virus infection in host cells

doi: 10.1101/2023.07.24.550336

Figure Lengend Snippet: HEK293T cells were infected with CHIKV at 0.1 MOI. Scribble levels were analyzed at 24 and 48 hpi using western blot and quantitated using densitometry (A). Twenty-four hours post-infection, the cells were fixed, and immunostaining was performed for Scribble and CHIKV capsid and E2 proteins. Inset shows zoomed images of an E2 expressing cell. Arrowheads indicate Scribble puncta (B). Effect of TF on Scribble expression. HEK293T cells were transfected with pEGFPC1-TF and its mutants. Only vector and pEGFPC1-6K were also transfected, and Scribble levels were analyzed 48 h post-transfection using western blot and quantitated using densitometry (C). Electron microscopy (EM) imaging of CHIKV-infected HEK293T cells. HEK293T cells were infected with CHIKV at 1.0 MOI and Scribble was visualized using a gold-labeled secondary antibody (D-i). EM images of an infected cell at higher magnification (D-ii). Immunostaining for ubiquitin and Scribble in CHIKV infected HEK293T cells (E). Data represents mean ± SEM of two biological replicates.

Article Snippet: Total protein was quantified using the bicinchoninic acid (BCA) assay, followed by SDS-PAGE and Western blotting with a primary antibody against Scribble (1:500, Santa Cruz sc55543), beta-actin (1:1000, Cell signaling Technology 4967), beta-tubulin (1:200, Santa Cruz sc55529), CHIKV E2 (1:3000, The Native antigen company MAB12130-200), and EGFP (1:3000, Bio Bharati BB-AB0065).

Techniques: Infection, Western Blot, Immunostaining, Expressing, Transfection, Plasmid Preparation, Electron Microscopy, Imaging, Labeling

Journal: bioRxiv

Article Title: Chikungunya Virus Release is Reduced by TIM-1 Receptors Through Binding of Envelope Phosphatidylserine

doi: 10.1101/2024.01.25.577233

Figure Lengend Snippet: (A) Immunoblot analysis of total lysates and supernatants harvested from CHIKV-infected Vero and VeroΔTIM/AXL cells. Cells were infected with CHIKV-GFP at 0.5 (1x) or 5 (10x) MOI. Total cell lysates and purified supernatants were probed against CHIKV E1 or vinculin as a control. (B) Diagram of CHIKV-GFP-E2-NLuc virus genome used for release efficiency assays. Nano luciferase (NLuc) was inserted at the N-terminus of E2. Created in BioRender.com (C) Vero cells were infected with either CHIKV-GFP or CHIKV-GFP-E2-NLuc. Supernatants were purified through ultracentrifugation and analyzed using a stain-free gel. (D) Multi-step replication curve of CHIKV and CHIKV-GFP-E2-NLuc in Vero cells (0.01 MOI) was harvested at each indicated time point. (E) Ratio between TCID50U/mL and Relative Luminescence Units (RLU) from samples harvested in the multi-step replication curve of cells infected with CHIKV-GFP-E2-NLuc. Data represents the mean ±SEM from at least three independent trials.

Article Snippet: Samples were then analyzed through immunoblotting probing against TYRO3 (1:1000, R&D Systems, MAB859100), TIM (TIM (1:500, AF1750, R&D Systems), AXL (1:100, AF154, R&D Systems), GAPDH (1:2000, Santa Cruz Biotech, #sc-47724), Transferrin (1:1,000, PA5-27739, ThermoFisher), or CHIKV E1 (1:1,000, MAB97792, R&D systems).

Techniques: Western Blot, Infection, Purification, Control, Virus, Luciferase, Staining

Journal: bioRxiv

Article Title: Chikungunya Virus Release is Reduced by TIM-1 Receptors Through Binding of Envelope Phosphatidylserine

doi: 10.1101/2024.01.25.577233

Figure Lengend Snippet: Release efficiency assay of CHIKV-GFP-E2-NLuc in Vero, VeroΔTIM, VeroΔAXL, and VeroΔTIM/AXL cells (MOI 0.5, harvested at 18 hours) (A) and corresponding levels of luminescence present in the total cell lysates (TCL) and supernatants (sup) (B) . Release efficiency assay with VeroΔTIM/AXL cells infected with ten times more CHIKV-GFP-E2-NLuc than Vero cells (C) and corresponding luminescence levels (D). Release efficiency assay of VSV-GFP-M-NLuc in Vero, VeroΔTIM, VeroΔAXL, and VeroΔTIM/AXL cells (MOI of 1, harvested at 8hrs) (E) and corresponding luminescence levels (F). Release efficiency assay of CHIKV VLPs in Vero and VeroΔTIM/AXL cells transfected with a plasmid encoding CHIKV’s structural cassette tagged with nano-luciferase (G) and corresponding luminescence levels (H). Data represent the mean ±SEM from at least three independent trials. For each release assay, data was normalized to the parental cell line to determine the relative release efficiency. Unpaired parametric Student’s t-test with unequal variance (Welch’s correction) was performed to determine statistical significance in comparison to the parental cell line. *, p < .05.

Article Snippet: Samples were then analyzed through immunoblotting probing against TYRO3 (1:1000, R&D Systems, MAB859100), TIM (TIM (1:500, AF1750, R&D Systems), AXL (1:100, AF154, R&D Systems), GAPDH (1:2000, Santa Cruz Biotech, #sc-47724), Transferrin (1:1,000, PA5-27739, ThermoFisher), or CHIKV E1 (1:1,000, MAB97792, R&D systems).

Techniques: Infection, Transfection, Plasmid Preparation, Luciferase, Release Assay, Comparison

Journal: bioRxiv

Article Title: Chikungunya Virus Release is Reduced by TIM-1 Receptors Through Binding of Envelope Phosphatidylserine

doi: 10.1101/2024.01.25.577233

Figure Lengend Snippet: (A) Experimental design for fluorescent liposome competition during release of CHIKV-infected Vero and VeroΔTIM/AXL cells. Created in BioRender.com (B) Increasing concentrations of fluorescent PC:PE:PS liposomes were added to CHIKV-E2-NLuc infected Vero or VeroΔTIM/AXL cells 6 hpi and release efficiency was calculated 18hrs post-infection. Data was normalized to the no-liposome control of each cell line. Vero (C) and VeroΔTIM/AXL (D) cells were transfected with a plasmid encoding hTIM-1 and infected with CHIKV-E2-NLuc 24 hours following transfection. Release efficiency was calculated 18hrs post-infection. (E) 293T cells were transfected with plasmids encoding the indicated surface receptors and infected with CHIKV-E2-NLuc 24 hours following transfection. Release efficiency was calculated 18hrs post-infection. Data was normalized to the GFP-transfected control. Data represents the mean ±SEM from at least three independent trials. Unpaired parametric Student’s t-test with unequal variance (Welch’s correction) was performed to determine statistical significance in comparison to the parental cell line. *, p < .05; **, p < .01; ***, p < .001.

Article Snippet: Samples were then analyzed through immunoblotting probing against TYRO3 (1:1000, R&D Systems, MAB859100), TIM (TIM (1:500, AF1750, R&D Systems), AXL (1:100, AF154, R&D Systems), GAPDH (1:2000, Santa Cruz Biotech, #sc-47724), Transferrin (1:1,000, PA5-27739, ThermoFisher), or CHIKV E1 (1:1,000, MAB97792, R&D systems).

Techniques: Infection, Liposomes, Control, Transfection, Plasmid Preparation, Comparison

Journal: bioRxiv

Article Title: Chikungunya Virus Release is Reduced by TIM-1 Receptors Through Binding of Envelope Phosphatidylserine

doi: 10.1101/2024.01.25.577233

Figure Lengend Snippet: (A) Entry efficiency of CHIKV-GFP in HAP1 and HAP1ΔCDC50 cells. (B) Multi-cycle replication curve of CHIKV in HAP1 and HAP1ΔCDC50 cells (MOI 0.01). (C) Release efficiency of CHIKV-GFP-E2-NLuc in HAP1 and HAP1ΔCDC50. Data was normalized to the release efficiency of the parental cell line to determine the relative release efficiency. (D) Surface biotinylation analysis of uninfected HAP1 and HAP1ΔCDC50 cells. Total lysates and surface proteins were probed using a Tyro3 antibody or Actin antibody as a loading control. Data represents the mean ±SEM from at least three independent trials. Unpaired parametric Student’s t-test was performed to determine statistical significance in comparison to the parental cell line at each indicated timepoint. An unequal variance (Welch’s correction) t-test was performed for normalized data. *, p < .05; **, p < .01; ***, p < .001.

Article Snippet: Samples were then analyzed through immunoblotting probing against TYRO3 (1:1000, R&D Systems, MAB859100), TIM (TIM (1:500, AF1750, R&D Systems), AXL (1:100, AF154, R&D Systems), GAPDH (1:2000, Santa Cruz Biotech, #sc-47724), Transferrin (1:1,000, PA5-27739, ThermoFisher), or CHIKV E1 (1:1,000, MAB97792, R&D systems).

Techniques: Control, Comparison

Journal: bioRxiv

Article Title: Chikungunya Virus Release is Reduced by TIM-1 Receptors Through Binding of Envelope Phosphatidylserine

doi: 10.1101/2024.01.25.577233

Figure Lengend Snippet: (A) Entry efficiency of CHIKV-GFP in VeroS and VeroSΔCDC50 cells. (B) CHIKV-GFP spread in VeroS and VeroSΔCDC50 cells (MOI 0.1). (C) Multi-cycle replication curve CHIKV in VeroS and VeroSΔCDC50 cells (MOI 0.01). Supernatants were harvested and titrated at the indicated time points. (D) Release efficiency assay of CHIKV-GFP-E2-NLuc when five times more virus as added to VeroSΔCDC50 than Vero cells to equalize cell lysate luminescence levels. (E) VeroSΔCDC50 cells were transfected with a plasmid encoding CDC50a and infected with CHIKV-E2-NLuc, release efficiency was assessed 18 hours post-infection. Surface receptors of VeroS (green) and VeroSΔCDC50a (blue) were assessed via staining using (F) a TIM-1 antibody or (G) binding of DioC 18 (3) fluorescent PC:PE:PS liposomes and analyzed through flow cytometry. Data represents the mean ±SEM from at least three independent trials. Unpaired parametric Student’s t-test was performed to determine statistical significance in comparison to the parental cell line at each indicated timepoint. An unequal variance (Welch’s correction) t-test was performed for normalized data. *, p < .05; **, p < .01; ***, p < .001; ****, p < .0001.

Article Snippet: Samples were then analyzed through immunoblotting probing against TYRO3 (1:1000, R&D Systems, MAB859100), TIM (TIM (1:500, AF1750, R&D Systems), AXL (1:100, AF154, R&D Systems), GAPDH (1:2000, Santa Cruz Biotech, #sc-47724), Transferrin (1:1,000, PA5-27739, ThermoFisher), or CHIKV E1 (1:1,000, MAB97792, R&D systems).

Techniques: Virus, Transfection, Plasmid Preparation, Infection, Staining, Binding Assay, Liposomes, Flow Cytometry, Comparison

Journal: bioRxiv

Article Title: Chikungunya Virus Release is Reduced by TIM-1 Receptors Through Binding of Envelope Phosphatidylserine

doi: 10.1101/2024.01.25.577233

Figure Lengend Snippet: (A) Representative histograms of binding of fluorescent liposomes in Vero and VeroΔTIM/AXL cells as a measure for phospholipid binding receptors. (B) Fold binding of fluorescent PC:PE:PS liposomes in mammalian and mosquito cell lines. To remove differences in fluorescent background levels among cell lines, fold binding was determined by calculating the ratio of DioC 18 (3) mean fluorescent intensity (MFI) over no-liposome control for each cell line. The dotted line represents the threshold where DioC 18 (3) MFI was equivalent to no-liposome background levels indicating no binding occurred. (C) The release efficiency of CHIKV-GFP-E2-NLuc in a panel of mammalian and mosquito cell lines. (D) Correlation analysis between liposome binding and CHIKV release efficiency. The size of circles represents the degree of liposome binding and colors indicate levels of release efficiency. Data represents the mean ±SEM from at least three independent trials.

Article Snippet: Samples were then analyzed through immunoblotting probing against TYRO3 (1:1000, R&D Systems, MAB859100), TIM (TIM (1:500, AF1750, R&D Systems), AXL (1:100, AF154, R&D Systems), GAPDH (1:2000, Santa Cruz Biotech, #sc-47724), Transferrin (1:1,000, PA5-27739, ThermoFisher), or CHIKV E1 (1:1,000, MAB97792, R&D systems).

Techniques: Binding Assay, Liposomes, Control

Journal: bioRxiv

Article Title: Chikungunya Virus Release is Reduced by TIM-1 Receptors Through Binding of Envelope Phosphatidylserine

doi: 10.1101/2024.01.25.577233

Figure Lengend Snippet: Levels of TIM-1 in the surface of uninfected or CHIKV-infected VeroS cells were assessed via receptor staining using a TIM-1 antibody (A) or binding of fluorescently labeled liposomes (B) and analyzed through flow cytometry. (C) VeroS cells were infected with either CHIKV-GFP (top, MOI 0.5) or LCMV-GFP (bottom, MOI 1) resulting in similar levels of infection. (D) The total protein present in total cell lysates (TCL) and biotinylated surface proteins (SB) of uninfected, CHIKV, and LCMV-infected VeroS cells were compared using a stain-free gel. (E) Immunoblot analysis of samples shown in panel D.

Article Snippet: Samples were then analyzed through immunoblotting probing against TYRO3 (1:1000, R&D Systems, MAB859100), TIM (TIM (1:500, AF1750, R&D Systems), AXL (1:100, AF154, R&D Systems), GAPDH (1:2000, Santa Cruz Biotech, #sc-47724), Transferrin (1:1,000, PA5-27739, ThermoFisher), or CHIKV E1 (1:1,000, MAB97792, R&D systems).

Techniques: Infection, Staining, Binding Assay, Labeling, Liposomes, Flow Cytometry, Western Blot

Journal: bioRxiv

Article Title: Chikungunya Virus Release is Reduced by TIM-1 Receptors Through Binding of Envelope Phosphatidylserine

doi: 10.1101/2024.01.25.577233

Figure Lengend Snippet: (A) Total cell lysate and surface biotinylated proteins of uninfected, CHIKV or LCMV-infected VeroS cells were quantified using densitometry analysis.

Article Snippet: Samples were then analyzed through immunoblotting probing against TYRO3 (1:1000, R&D Systems, MAB859100), TIM (TIM (1:500, AF1750, R&D Systems), AXL (1:100, AF154, R&D Systems), GAPDH (1:2000, Santa Cruz Biotech, #sc-47724), Transferrin (1:1,000, PA5-27739, ThermoFisher), or CHIKV E1 (1:1,000, MAB97792, R&D systems).

Techniques: Infection

Journal: bioRxiv

Article Title: Chikungunya Virus Release is Reduced by TIM-1 Receptors Through Binding of Envelope Phosphatidylserine

doi: 10.1101/2024.01.25.577233

Figure Lengend Snippet: (A) CHIKV-GFP infected VeroS cells were infected at different time points, and all were harvested at the same time for surface biotinylation analysis. Infection was maintained for 0, 3, 6, 9 or 12 hours. Samples were probed using TIM-1, E1, or transferrin (Trfn) antibodies. (B) VeroS cells were infected with CHIKV at different time points, and subjected to fluorescent liposome binding simultaneously. Infection was maintained for 0, 3, 6, 9, 12, 15, or 18 hours and analyzed through flow cytometry. (C) Quantification of fluorescent PC:PE:PS liposomes. (D) Cells were transfected with plasmids encoding CHIKV proteins for 48 hours and analyzed through binding of fluorescently labeled liposomes using flow cytometry. Liposome binding was compared to control cells to determine the relative liposome binding levels. Data represents the mean ±SEM from at least three independent trials. An ordinary one-way ANOVA with multiple comparisons was used to evaluate statistical differences in comparison to control. An unequal variance (Welch’s correction) t-test was performed for normalized data. *, p < .05; **, p < .01; ****, p < .0001.

Article Snippet: Samples were then analyzed through immunoblotting probing against TYRO3 (1:1000, R&D Systems, MAB859100), TIM (TIM (1:500, AF1750, R&D Systems), AXL (1:100, AF154, R&D Systems), GAPDH (1:2000, Santa Cruz Biotech, #sc-47724), Transferrin (1:1,000, PA5-27739, ThermoFisher), or CHIKV E1 (1:1,000, MAB97792, R&D systems).

Techniques: Infection, Binding Assay, Flow Cytometry, Liposomes, Transfection, Labeling, Control, Comparison

Journal: bioRxiv

Article Title: Chikungunya Virus Release is Reduced by TIM-1 Receptors Through Binding of Envelope Phosphatidylserine

doi: 10.1101/2024.01.25.577233

Figure Lengend Snippet: Stain-free gel analysis of (A) total cell lysates or (B) surface biotinylation proteins infected with CHIKV for different periods. (C) Exogenous expression of CHIKV non-structural proteins tagged with FLAG tag was analyzed through SDS-PAGE using an antibody against FLAG or transferrin as loading control. nsP1 was quickly detected in the cell lysates of transfection cells but longer exposure (right) was needed for detection of nsP2, nsP3, and nsP4. (D) Exogenous expression of CHIKV structural proteins was analyzed using an antibody against CHIKV E1 or transferrin as a loading control.

Article Snippet: Samples were then analyzed through immunoblotting probing against TYRO3 (1:1000, R&D Systems, MAB859100), TIM (TIM (1:500, AF1750, R&D Systems), AXL (1:100, AF154, R&D Systems), GAPDH (1:2000, Santa Cruz Biotech, #sc-47724), Transferrin (1:1,000, PA5-27739, ThermoFisher), or CHIKV E1 (1:1,000, MAB97792, R&D systems).

Techniques: Staining, Infection, Expressing, FLAG-tag, SDS Page, Control, Transfection

Journal: F1000Research

Article Title: Broad-spectrum capture of clinical pathogens using engineered Fc-mannose-binding lectin enhanced by antibiotic treatment

doi: 10.12688/f1000research.17447.1

Figure Lengend Snippet: FcMBL binding profile of bacteria, fungi, viral antigens, parasites, and bacterial antigens. Multiple species of bacteria, including multiple isolates (number of isolates), were screened by FcMBL ELLecSA to determine FcMBL binding. Total number detected of both live and fragmented bacterial isolates is shown. Fungi were screened and total number detected for live isolates shown. Purified or inactivated viral antigens, parasites, and bacterial antigens were tested directly in TBST 5 mM CaCl 2 buffer, and number detected shown. Test samples were performed in duplicate. NT, not tested.

Article Snippet: Viral antigens: Chikungunya E1 (CHIKV-E1), Dengue serotype 1 VLP (DENV1-VLP), Ebola GP1 (EBOVKW95-GP1-100), Tick-borne Encephalitis NS1 (TBEV-NS1-100), and Zika lysate (ZIKV-LYS-100) were purchased from The Native Antigen Company (Oxford, UK).

Techniques: Binding Assay, Bacteria, Purification

Journal: F1000Research

Article Title: Broad-spectrum capture of clinical pathogens using engineered Fc-mannose-binding lectin enhanced by antibiotic treatment

doi: 10.12688/f1000research.17447.1

Figure Lengend Snippet: FcMBL ELLecSA screening of Chikungunya E1 (3.0 µg/ml), Cytomegalovirus (CMV) (10 7 PFU), Dengue serotype 1 VLP (0.36 µg/ml), Human immunodeficiency virus (HIV) gp120 (0.1 µg/ml), Ebola GP1 (0.2 µg/ml), Influenza H1N1 HA (0.1 µg/ml), Influenza H1N1 NA (1 µg/ml), Respiratory syncytial virus (RSV) glycoprotein g (10.0 µg/ml), Tick-borne Encephalitis NS1 (5 µg/ml), and Zika lysate (0.24 µg/ml).

Article Snippet: Viral antigens: Chikungunya E1 (CHIKV-E1), Dengue serotype 1 VLP (DENV1-VLP), Ebola GP1 (EBOVKW95-GP1-100), Tick-borne Encephalitis NS1 (TBEV-NS1-100), and Zika lysate (ZIKV-LYS-100) were purchased from The Native Antigen Company (Oxford, UK).

Techniques: Virus