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

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