Journal: BMC Immunology

Article Title: Human CD57 + germinal center-T cells are the major helpers for GC-B cells and induce class switch recombination

doi: 10.1186/1471-2172-6-3

Figure Lengend Snippet: CD57 + GC-Th cells are more efficient than other tonsil CD4 + T cell subsets in helping B cells. (A) CD4 + T cell subsets were cultured with total tonsil CD19 + B cells for 7 days in the presence of SEB. Naïve B cells (C) or GC-B cells (D) were cultured with equal numbers of CD57 + GC-Th cells or other T cell subsets (CD57 - , CD57 - CD69 + and CD57 - CD69 - T cells) for 7 days followed by ELISA for IgM, IgG, IgA and IgE. Data from 5 independent experiments were combined and the averages are shown with standard errors. Relative production levels to CD57 + GC-Th cells are shown. *Significant differences from CD57 + GC-Th cells. The absolute Ig production levels (ng/ml) in panel A (GC-Th + Total B cells) were 5737 ± 1764 (IgM), 2111 ± 1185 (IgG), 577 ± 186 (IgA), and 4.8 ± 2.1 (IgE). The absolute Ig production levels (ng/ml) in panel B (GC-Th cells + naïve B cells) were 2045 ± 697 (IgM), 63 ± 21 (IgG), 40 ± 23 (IgA), and 2.9 ± 1.2 (IgE). The average levels (ng/ml) of Ig produced in the cultures of GC-Th cells and GC B cells were 750 ± 279 (IgM), 175 ± 52 (IgG), 51 ± 13 (IgA), and 1.0 ± 0.5 (IgE). (D) Isotype composition of the Ig induced by CD57 + GC-Th cells. Naïve B cells or GC-B cells were cultured with equal numbers of CD57 + GC-Th cells or CD57 - CD69 + T cells for 7 days followed by ELISA for IgM, IgG, IgA and IgE. Data from 4 independent experiments were combined and the averages are shown with standard errors. *Significant differences between naïve and GC-B cells, but not between the two T cell subsets, were observed.

Article Snippet: Blocking antibodies for IFN-γ (25718.111) and IL-10 (23738.111), and IgG1 isotype control antibody (11711.11) were purchased from R&D systems.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Produced

Journal: BMC Immunology

Article Title: Human CD57 + germinal center-T cells are the major helpers for GC-B cells and induce class switch recombination

doi: 10.1186/1471-2172-6-3

Figure Lengend Snippet: CD57 + GC-Th cells have the capacities to induce AID expression and to support CSR in B cells. IgD + CD38 - naïve B cells were cultured with CD57 + GC-Th cells for indicated time periods followed by RT-PCR analysis for (A) AID expression and (B) CSR. The sizes of specific PCR products are 152 bp (IgM); 416 bp (IgG1, G2, G3), 904 bp (IgG4); 904 bp (IgA1); 891 bp (IgA2); and 179 bp (IgE). Shown are productive recombination products. (C) The expression kinetics of AID and productive IgG3 transcripts over an 8 day period are shown together in a graph. In this panel, normalized expression levels calculated after dividing the levels of AID amplification by β-actin levels are shown. The time gap to reach the peak levels of the expression between AID and productive IgG3 transcripts is shown by an arrow. Representative data from at least three independent experiments are shown (panels A and B). (D) Identification of extrachromosomal reciprocal DNA recombination products. Naïve B cells were cultured with CD57 + GC-Th cells for indicated time periods and were processed to isolate genomic DNA. Fresh GC-B cells were examined for positive controls. The switch circles were detected by a nested PCR method. Representative data out of three independent experiments are shown. (E) Detection of switch circles by a DC-PCR technique. Naive B cells, CD38 + GC-B cells and naïve B cells cultured with GC-Th cells for 5 days were examined for the presence of γ3 and α1/2 switch circles.

Article Snippet: Blocking antibodies for IFN-γ (25718.111) and IL-10 (23738.111), and IgG1 isotype control antibody (11711.11) were purchased from R&D systems.

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Amplification, Nested PCR

Journal: BMC Immunology

Article Title: Human CD57 + germinal center-T cells are the major helpers for GC-B cells and induce class switch recombination

doi: 10.1186/1471-2172-6-3

Figure Lengend Snippet: CD40L and cytokines in regulation of the helper activity of CD57 + GC-Th cells. (A and B) Effects of endogenous CD40L and cytokines on the helper activity of CD57 + GC-Th cells were determined. In cultures of CD57 + GC-Th cells with naïve or GC-B cells, neutralizing antibodies to IL-4, IL-10, IFN-γ or CD40L or control antibodies (mouse IgG1) were added. *Significant differences from the control group (control antibody). (C and D) Effects of exogenously added cytokines on the helper activity of CD57 + GC-Th cells were determined. To cultures of CD57 + GC-Th cells with naïve or GC-B cells, IL-4, IL-10, IFN-γ and TGF-β1 were added separately. Cells were cultured for 7 days followed by ELISA for IgM, IgG, IgA and IgE. Relative Ig secretion levels (the medium control = 1) obtained from 9 independent experiments were combined, and averages and standard errors are shown. *Significant differences from the control group (medium).

Article Snippet: Blocking antibodies for IFN-γ (25718.111) and IL-10 (23738.111), and IgG1 isotype control antibody (11711.11) were purchased from R&D systems.

Techniques: Activity Assay, Control, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: Boost immunizations with NA-derived peptide conjugates achieve induction of NA inhibition antibodies and heterologous influenza protections.

doi: 10.1016/j.celrep.2023.112766

Figure Lengend Snippet: Figure 3. Titration of rtN1- and rtN2-spe- cific serum IgG in ELISA The sera samples collected 2 weeks after (A and D) prime immunization, (B and E) first booster, and (C and F) second booster were titrated for (A–C) rtN1 and (D–F) rtN2. Each dot represents the serum antibody titer from one mouse (n = 5 per group). Bars represent the mean values of serum antibody titers. The dash line represents the limit of detection, corresponding to the initial serum dilution. The positive wells were determined as OD values above mean +2 3 SD of the OD value of the initial pre-immune sera dilution. Statistical signifi- cance was analyzed by t test for (B), (C), (E), and (F). *p < 0.05, **p < 0.01, and ****p < 0.001. All experiments were repeated twice using the same serum samples and showed similar results.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-63His tag Sino Biological Cat#105327-MM02T-H Rabbit polyclonal anti-N1 Sino Biological Cat#11058-R001 Rabbit polyclonal anti-N2 Sino Biological Cat#40017-RP01 HRP-conjugated goat anti-human IgG Proteintech Group Cat#SA00001-17 HRP-conjugated goat anti-mouse IgG Proteintech Group Cat#SA00001-1 HRP-conjugated goat anti-mouse IgG1 Proteintech Group Cat#SA00012-1 HRP-conjugated goat anti-mouse IgG2a Proteintech Group Cat#SA00012-2 HRP-conjugated goat anti-mouse IgG3 Proteintech Group Cat#SA00012-5 HRP-conjugated goat anti-mouse IgM Proteintech Group Cat#SA00012-6 HRP-conjugated peanut agglutinin Sigma-Aldrich Cat#L7759 Bacterial and virus strains E.coli DH5a competent cells Lab stock N/A E.coli DH10Bac competent cells Lab stock N/A A/Hong Kong/8/1968 (H3N2) ATCC Cat#VR544TM A/New Jersey/8/1976 (H1N1) ATCC Cat#VR897TM A/Puerto Rico/8/1934 (H1N1) Haiyan Chang, Hunan Normal University N/A A/reassortant/NYMC X179A (H1N1) Haiyan Chang, Hunan Normal University N/A A/Guizhou/54/1989 (GZ89, H3N2) Haiyan Chang, Hunan Normal University N/A A/Puerto Rico/8/1934 (H1N1) Haiyan Chang, Hunan Normal University N/A Biological samples Human convalescent sera samples Yao-Qing Chen’s laboratory stock N/A Chemicals, peptides, and recombinant proteins N1P1 Top-peptide N/A N1P2 Top-peptide N/A N1P3 Top-peptide N/A N1P4 Top-peptide N/A N2P1 Top-peptide N/A N2P2 Top-peptide N/A N2P3 Top-peptide N/A N2P4 Top-peptide N/A rtN1 This paper N/A rtN2 This paper N/A KLH Solarbio Cat#K8160 Receptor destroying enzyme Denka Seiken Cat#340122 Fetuin from fetal bovine serum Sigma-Aldrich Cat#F2379 Incomplete Freund’s adjuvant Sigma-Aldrich Cat#F5506 Critical commercial assays ReadiLinkTM KLH Conjugation Kit AAT Bioquest Cat#5502 Mouse IFNg ELISPOT Kit BD Biosciences Cat#551083 Mouse IL-4 ELISPOT Kit Dakewe Cat#DKW22-2040-500 (Continued on next page) Cell Reports 42, 112766, July 25, 2023 11

Techniques: Titration, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Interferon-armed RBD dimer enhances the immunogenicity of RBD for sterilizing immunity against SARS-CoV-2

doi: 10.1101/2021.05.12.443228

Figure Lengend Snippet: (a) Schematic illustration of the I-R-F fusion protein. The structural elements contain a mouse IFNα4a (IFNα), receptor-binding domain (RBD), immunoglobulin Fc fragment (Fc). (b) Size exclusion chromatography of I-R-F was performed on a Superdex200 Increase Column. The ultraviolet absorption at 280mm is shown. The insert photograph presents the SDS-PAGE of the eluted protein samples. (c) The real-time binding kinetics between I-R-F and hACE2 was determined by the BIAcore T100 system. (d) Evaluation of the binding ability of monoclonal antibodies to I-R-F by ELISA. To determine the known RBD epitopes in I-F-R, ELISA Plate was coated with I-R-F. Then, 2-fold serially diluted monoclonal antibodies were detected. The anti-Pres1 XY007 monoclonal antibody was given as the control. The absorbance was read at 450-630. (e)The bioactivity of IFNα contained in I-R-F was measured by an anti-viral infection biological assay. (f) Binding of mouse IFNα-RBD-Fc to FcγR on RAW264.7 cells. Cells were incubated with serial dilutions of WT IgG fusion protein, mutant IgG fusion protein, or WT IgG fusion protein with anti-FcγR, followed by a fluorophore-conjugated anti-human IgG secondary antibody. Flow cytometry measured MFI (n=3). (g) BALB/c mice (n=8/group) were immunized intramuscularly with 10μg of I-R-F or equimolar RBD protein, mixed with alum adjuvant, respectively. Mice were re-immunized with the same dose of vaccine on day 14 post the first shot. PBS containing alum adjuvant was chosen as a negative control. Sera were collected on days 7, 14, 21, 28, 35, and 42 after initial immunization, and the IgG levels were measured by ELISA. (h) Groups of BALB/c mice (n=7/groups) were intramuscularly immunized twice on day 0 and day 14 with 10μg of alum-adjuvanted I-R-F, or equimolar RBD or alum alone as a control. Serum was collected at indicated time points, and the kinetics of the RBD-specific IgG antibody titers were determined by ELISA. The dashed line indicates the limit of detection. The data shown are presented as mean ± SEM. P-values were determined by one-way ANOVA with multiple comparison tests. ns (not significant), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: To detect the Ig subclasses, goat anti-mouse IgG1 (1:5000, Proteintech), goat anti-mouse IgG2a (1:5000, Proteintech) were added.

Techniques: Binding Assay, Size-exclusion Chromatography, SDS Page, Bioprocessing, Enzyme-linked Immunosorbent Assay, Control, Infection, Incubation, Mutagenesis, Flow Cytometry, Adjuvant, Negative Control, Comparison

Journal: bioRxiv

Article Title: Interferon-armed RBD dimer enhances the immunogenicity of RBD for sterilizing immunity against SARS-CoV-2

doi: 10.1101/2021.05.12.443228

Figure Lengend Snippet: (a). BALB/c mice (n=6/group) were i.m vaccinated with 10μg of I-P-F or equimolar of RBD, RBD-dimer, and R-F protein or PBS control with a signal vaccination regimen. The levels of RBD-specific IgG in serum on day 28 after immunization were determined by ELISA (b) BALB/c Mice (n=6/group) were intramuscularly vaccinated with 1μg of I-R-F or equimolar R-F plus IFNα protein without adjuvant and boosted with the same dose at a 14-day interval. Serum samples were collected on day 14 after the second immunization to evaluate the levels of RBD-specific IgG. (c) BALB/C mice (n=8/group) were immunized intramuscularly or subcutaneously with alum-adjuvanted 10μg of I-R-F and boosted on day 14 after initial immunization with equivalent dose. Serum samples were collected every week to determine the SARS-CoV-2-specific IgG antibody titers by ELISA. (d) BALB/C mice (n=8/group) were immunized intramuscularly with alum-adjuvanted 10μg of I-R-F by using a single dose (day0), two-dose (day0/14), and three-dose (day0/14/28) immunization procedures, respectively. Sera were collected on days 7, 14, 21, 28, 35, and 42 after the initial immunization and analyzed by ELISA to determine the IgG titer. (e) The neutralization antibody titers in serum described in on day 28 were determined by SARS-CoV-2 pseudovirus neutralization assay. The dashed line indicates the limit of detection. Data are shown as mean ± SEM. P-values were calculated by one-way ANOVA with multiple comparisons tests. ns (not significant), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: To detect the Ig subclasses, goat anti-mouse IgG1 (1:5000, Proteintech), goat anti-mouse IgG2a (1:5000, Proteintech) were added.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Adjuvant, Neutralization

Journal: bioRxiv

Article Title: Interferon-armed RBD dimer enhances the immunogenicity of RBD for sterilizing immunity against SARS-CoV-2

doi: 10.1101/2021.05.12.443228

Figure Lengend Snippet: (a) The kinetics of the RBD-specific IgG antibody response. BALB/c mice (n=7/group) were immunized with a 1:10 series dilution of the vaccine, containing 10μg, 1μg, 0.1μg, 0.01μg, 0.001μg of I-R-F, respectively. Sera were collected to assess the levels of RBD-specific IgG. (b) The serum described in Fig 1e on day 28 was used to determine the neutralization activity with live SARS-CoV-2 by FRNT. The sera from different groups were serially diluted and mixed with 600 FFU of SARS-CoV-2, and the mixtures were then transferred to Vero E6 cells. The number of SARS-CoV-2 foci was counted in the next day. The FRNT 50 was defined as the reciprocal of serum dilution, which inhibits 50% of viral infection. (c) Comparison of the neutralizing antibody titers in sera between the I-R-F vaccinated mice (n=7) and the convalescent COVID-19 patients with different severity (n=6-13/group). The sera from 3 groups of COVID-19 convalescent patients and the I-P-F immunized mice as mentioned above in were serially diluted and mixed with live SARS-CoV-2. The mixtures were added to Vero E6 cells. The neutralizing titers were presented by FRNT 50 , which was the reciprocal of serum dilution neutralizing 50% of viral infection. (d) Mouse RBD-specific IgG induced by single immunization. Mice (n=8/group) were i.m. immunized once with alum-adjuvanted 10ug I-R-F (n=8), equimolar RBD protein, or alum alone, respectively. The levels of RBD-specific IgG in sera on days 7, 14, 21, 28, 35, and 42 after the first immunization were determined by ELISA. (e) Mouse RBD-specific IgG induced by vaccines without adjuvant. Mice (n=7 or 8) were vaccinated with no adjuvant 10μg I-R-F, equimolar RBD protein, or PBS control and boosted with the same dose 14-day after the initial immunization. Sera were collected every week after immunization and used to determine the IgG titers. (f) BALB/C mice (n=8/group) were immunized i.m. with alum-adjuvanted 0.1μg I-R-F, equimolar RBD protein, or alum alone, respectively, and boosted with the same dose at a 14-day interval. Sera were collected on days 7, 14, 21, 28, 35, and 42 after the initial immunization. RBD-specific IgG levels were analyzed by ELISA. The dashed line indicates the limit of detection. Data are shown as mean ± SEM. P-values were calculated by one-way ANOVA with multiple comparisons tests. ns (not significant), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: To detect the Ig subclasses, goat anti-mouse IgG1 (1:5000, Proteintech), goat anti-mouse IgG2a (1:5000, Proteintech) were added.

Techniques: Neutralization, Activity Assay, Infection, Comparison, Enzyme-linked Immunosorbent Assay, Vaccines, Adjuvant, Control

Journal: bioRxiv

Article Title: Interferon-armed RBD dimer enhances the immunogenicity of RBD for sterilizing immunity against SARS-CoV-2

doi: 10.1101/2021.05.12.443228

Figure Lengend Snippet: (a and b) C57BL/6 mice were immunized with 10 μg I-R-F, equimolar RBD, or PBS with a prime-boost vaccination regimen in a 14-day interval. Mice were sacrificed six months post the first vaccination, and splenocytes were collected. (a) ELISPOT assay was performed to determine RBD-specific B cells in the spleen. (b) The percentage of memory B cells in the spleen was analyzed. (c) BALB/c mice (n=8/group) were immunized with 10 μg of I-R-F, equimolar RBD, or PBS twice in a 14-day interval. PBS was performed as a negative control. Sera were collected on day 28 after the initial immunization and used to determine the IgG subclasses. (d-g) C57BL/6 mice were immunized with 10 μg I-R-F, equimolar RBD, or PBS with a prime-boost vaccination regimen in a 14-day interval. Mice were sacrificed, and splenocytes were collected 28 days post the first vaccination. ELISPOT assay was performed for IFN-γ (d) and IL-4 (e) secretion from mice splenocytes stimulated with RBD peptide pool. Splenocytes were incubated with an RBD peptide pool. (f) The percentages of IFN-γ + , IL-4 + , and TNF-α + CD4 + T cells were determined by ICCS. (g) The percentages of IFN-γ + , IL-2 + , and TNF-α + CD8 + T cells were determined by ICCS. The dashed line indicates the limit of detection. Data are presented as mean ± SEM. P-Values in (a-e) were calculated by one-way ANOVA with multiple comparisons tests. P-values in (f) and (g) were calculated by two-way ANOVA with multiple comparisons tests. ns (not significant), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: To detect the Ig subclasses, goat anti-mouse IgG1 (1:5000, Proteintech), goat anti-mouse IgG2a (1:5000, Proteintech) were added.

Techniques: Enzyme-linked Immunospot, Negative Control, Incubation

Journal: bioRxiv

Article Title: Interferon-armed RBD dimer enhances the immunogenicity of RBD for sterilizing immunity against SARS-CoV-2

doi: 10.1101/2021.05.12.443228

Figure Lengend Snippet: C57BL/6 mice (n=6/group) were vaccinated intramuscularly with 0.1μg of I-R-F or I-P-R-F using a two-dose immunization procedure. Sera were collected on days 7, 14, 21, 28, 35, 42 after the initial immunization. The RBD-specific IgG antibody titer was determined by ELISA. (b) The neutralization activity of vaccinated sera collected on day 28, as shown in (a), was evaluated using a pseudovirus neutralization assay. (c) BALB/C mice (n=8/group) were immunized i.m. with 1 μg of human IFNα-RBD-Fc (human I-R-F), human IFNα-Pan-RBD-Fc (human I-P-R-F), or equimolar RBD respectively, and boosted with the same dose at a 14-day interval. Sera were collected on days 0, 14, 21, 28, 35, and 42 after initial immunization and analyzed by ELISA. The dashed line indicates the limit of detection. Data are shown as mean ± SEM. P-values were calculated by one-way ANOVA with multiple comparisons tests in (a) and (c). P-values in (b) were analyzed with an unpaired t-test. P-values in (d-e) were determined using a paired t-test. ns (not significant), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: To detect the Ig subclasses, goat anti-mouse IgG1 (1:5000, Proteintech), goat anti-mouse IgG2a (1:5000, Proteintech) were added.

Techniques: Enzyme-linked Immunosorbent Assay, Neutralization, Activity Assay

Journal: bioRxiv

Article Title: Interferon-armed RBD dimer enhances the immunogenicity of RBD for sterilizing immunity against SARS-CoV-2

doi: 10.1101/2021.05.12.443228

Figure Lengend Snippet: Male and female rhesus macaques (n=8) were equally divided into four groups with a sex ratio of 1:1 and were immunized i.m. with a high dose (50ug) or a low dose (10ug) of I-P-R-F with or without alum as an adjuvant and boost with the same dose at a 14-day interval. The sera were collected every week are analyses for I-P-R-F immunogenicity and vaccine-induced neutralizing antibodies. (a) RBD-specific IgG titers in the high-dose group were determined by ELISA at indicated time points. (b) The dynamic IgG titers in sera from the low-dose vaccinated group were determined. (c-d) The kinetics of neutralization antibody titers in serum from I-P-R-F immunized animals were determined by SARS-CoV-2 pseudovirus and authentic virus neutralization assays. (e) Neutralization of SARA-CoV-2 pseudovirus by the anti-sera from I-P-R-F immunized rhesus macaques before the virus challenge. (f) Viral load in anal swabs of rhesus macaques challenged with live SARS-CoV-2. (g) Viral loads in the lungs. For each group of vaccinated macaques, 84 specimens from fourteen lung lobes were collected on day 7 post infection and subjected to determine the viral loads in the lungs. High dose/A: immunization with high does with adjuvant, High dose/A-: immunization with high does without adjuvant, Low dose/A: immunization with low does with adjuvant, Low dose/A-: immunization with low does without adjuvant. The dashed line indicates the limit of detection. Data are shown as mean ± SEM. P-values in (d) were analyzed with the unpaired t-test. P-values were calculated by one-way ANOVA with multiple comparisons tests in (e). ns (not significant), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: To detect the Ig subclasses, goat anti-mouse IgG1 (1:5000, Proteintech), goat anti-mouse IgG2a (1:5000, Proteintech) were added.

Techniques: Adjuvant, Immunopeptidomics, Enzyme-linked Immunosorbent Assay, Neutralization, Virus, Infection

Journal: bioRxiv

Article Title: Interferon-armed RBD dimer enhances the immunogenicity of RBD for sterilizing immunity against SARS-CoV-2

doi: 10.1101/2021.05.12.443228

Figure Lengend Snippet: Rhesus macaques (n=6/group) were administered twice with 10μg or 50μg of alum-adjuvanted I-P-R-F or alum alone as control via the intramuscular injection and challenged with SARS-CoV-2 intranasally on day 21 after the initial immunization. Sera were collected on days 0, 14, 21 (before virus infection), and day 28 (after infection) and subjected to antibody assays. (a) The SARS-CoV-2 specific IgG was determined by ELISA. (b) Neutralization antibody titers were analyzed. Body temperature (c) and body weight (d) were measured after the virus challenge. Viral loads in nose swabs (e) and tracheal brushes (f) following the virus challenge were determined by qPCR. (g) Viral load in various lung lobes of macaques challenged with SARS-CoV-2 on day 7 post infection. Dashed line in(a-b, e-g)indicates the limit of detection. Data are shown as mean ± SEM. P-values were calculated by one-way ANOVA with multiple comparisons tests. ns (not significant), *P<0.05, **P<0.01, ***P<0.001, ****P<0.000

Article Snippet: To detect the Ig subclasses, goat anti-mouse IgG1 (1:5000, Proteintech), goat anti-mouse IgG2a (1:5000, Proteintech) were added.

Techniques: Control, Injection, Virus, Infection, Enzyme-linked Immunosorbent Assay, Neutralization

Journal: bioRxiv

Article Title: T-cell immunoglobulin and mucin (TIM) contributes to Hantaan virus entry into human airway epithelial cells

doi: 10.1101/872317

Figure Lengend Snippet: (A) Specificity to neutralizing activity of anti-VSVG antibody. A549 cells were infected with VSV*ΔG-Luc(VSV-G), VSV*ΔG-Luc(HTNV-G), VSV*ΔG-Luc(ANDV-G), in the presence or absence of the VSV-G neutralizing antibody I1 (mAb I1). (B) Detection of hTIM-1 on A549 cells by flow cytometry. Fixed non-permeabilised cells were stained with goat anti-TIM-1 pAb. Black line represents secondary antibody only, and red line represents primary and secondary antibodies. (C) Blocking of hTIM-1-mediated infection by antibody perturbation. A549 cells were pre-treated for 1 h at 4 °C with 30 nM of goat anti-hTIM-1 pAb and control goat IgG in the absence of serum. Cells were then infected with VSV*ΔG-Luc(HTNV-G), VSV*ΔG-Luc(ANDV-G), VSV*ΔG-Luc(EBOV-G), VSV*ΔG-Luc(LASV-G) and AdV5 and shifted to 37 °C for 90 min. Cells were washed with medium supplemented with 20 mM ammonium chloride, followed by 16 h of incubation in the presence of lysosomotropic agent. Infection was quantified by counting EGFP-positive infected cells. Data are normalized to the control DMEM and displayed as percentage of infection related to the control. Data are means + SD (n=3) with p-value ***: p ≤ 0.001.

Article Snippet: Human TIMD4 was obtained from Sino Biological Inc. Purified goat anti-human Axl IgG polyclonal antibody (pAb, #AF154), purified goat anti-human TIM-1/KIM-1/HACVR IgG pAb (#AF1750), phycoerythrin (PE)-conjugated donkey anti-goat IgG pAb, purified donkey anti-goat IgG pAb (#AF109) and monoclonal mouse IgG (#MAB002) were from R&D Systems.

Techniques: Activity Assay, Infection, Flow Cytometry, Staining, Blocking Assay, Incubation

Journal: bioRxiv

Article Title: T-cell immunoglobulin and mucin (TIM) contributes to Hantaan virus entry into human airway epithelial cells

doi: 10.1101/872317

Figure Lengend Snippet: (A) Expression of αvβ3 integrin in A549 cells. Live non-permeabilised cells were stained with the mouse anti-αvβ3 integrin mAb (red line). (B) Anti-αvβ3 integrin Ab reduces infection of VSV*ΔG-Luc(HTNV-G) but not VSV*ΔG-Luc(ANDV-G) into A549 cells. A549 cells were pre-treated for 1 h at 4 °C with mouse anti-αvβ3 mAb and a control mouse at 30 nM. Cells were then infected with VSV*ΔG-Luc(HTNV-G), VSV*ΔG-Luc(ANDV-G) and VSV*ΔG-Luc(EBOV-G), and shifted to 37 °C. Infection was quantified by counting EGFP-positive infected cells per well. (C) Relative contribution of αvβ3 integrin and hTIM-1. A549 cells were pre-treated with anti-αvβ3 and anti-hTIM-1 antibodies at 30 nM. The experiment settings were identical to the antibody perturbation assay as in panel (B). (D) Heparan sulfate reduces infection of HTNV but not ANDV pseudotypes into A549 cells. VSV*ΔG-Luc(HTNV-G), VSV*ΔG-Luc(ANDV-G), VSV*ΔG-Luc(EBOV-G), VSV*ΔG-Luc(LASV-G) and AdV5-GFP were pre-incubated with soluble heparan sulfate at various concentrations and used to infect monolayers of A549 cells. Infection levels were assessed by counting EGFP-positive infected cells. (E) Heparan sulfate and anti-hTIM-1 block HTNV-G entry. A549 cells were pre-treated for 1 h at 4 °C with the polyclonal Ab goat anti-hTIM-1 at 30 nM. Meantime, VSV*ΔG-Luc(HTNV-G), was pre-incubated with heparan sulfate (HS) at 5 µg/ml. Cells were then infected with pre-treated VSV*ΔG-Luc(HTNV-G) and shifted to 37 °C. Infection was quantified 16 h post-infection by counting EGFP-positive infected cells. (F) Relative contribution of αvβ3 integrin and heparan sulfate. Pre-treatment of cells with anti-αvβ3 mAb (30 nM), viruses with 5 µg/ml of heparan sulfate (HS) and infections were assessed as in . Infection was quantified 16 h post-infection by counting EGFP-positive infected cells. (B-F) Data are means + SD (n=3) with p-value *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001.

Article Snippet: Human TIMD4 was obtained from Sino Biological Inc. Purified goat anti-human Axl IgG polyclonal antibody (pAb, #AF154), purified goat anti-human TIM-1/KIM-1/HACVR IgG pAb (#AF1750), phycoerythrin (PE)-conjugated donkey anti-goat IgG pAb, purified donkey anti-goat IgG pAb (#AF109) and monoclonal mouse IgG (#MAB002) were from R&D Systems.

Techniques: Expressing, Staining, Infection, Incubation, Blocking Assay