Journal: bioRxiv

Article Title: Cutaneous jet-injection of naked mRNA vaccine induces robust immune responses without systemic vaccine spillage

doi: 10.1101/2023.02.27.530188

Figure Lengend Snippet: (a-d) Balb/C mice were PYRO-injected with naked spike mRNA at both flanks in a prime-boost setting separated by a 3-week interval. 5 weeks after the prime, blood plasma and splenocytes were collected for evaluating humoral and cellular immunity. (a,b) Anti-spike IgG ELISA absorbance vs. plasma dilution curves . (c) The ability of plasma in blocking the binding between spike-RBD and ACE2 proteins. (d) Quantification of spike-specific INFγ-positive splenocytes. Data represent the mean ± SEM (n=4). **p <0.01, *p <0.05 vs. NT, non-repeated ANOVA followed by Dunnett’s test in (d) . (e-l) Vaccination in NHPs. (e) Injections and sampling schedule. (f,g) Appearance of PYRO injection site on the back of Cynomolgus monkeys (f) compared to Balb/C mice (g) . (h) Anti-spike IgG titers in NHPs PYRO-injected with buffer or mRNA solution. (i) Binding inhibition of ACE2 and SARS-CoV-2 RBD by plasma of immunized monkeys. (j-l) Toxicity evaluation in PYRO-injecting monkeys receiving either buffer or spike mRNA. (j) Body temperature measured immediately before and 24 h post dosing, after each of the 3 doses. (k) Change in body weight. (l) Plasma levels of proinflammatory cytokines before and 24 h after the first dose. n.s.: nonsignificant, **p <0.01, *p <0.05 vs. buffer treatment in unpaired Student’s t -test. BLQ: below limit of quantification.

Article Snippet: SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit was obtained from Cell Signaling Technologies.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Blocking Assay, Binding Assay, Sampling, Inhibition

Journal: The Journal of Experimental Medicine

Article Title: SARS-CoV-2 Spike protein suppresses CTL-mediated killing by inhibiting immune synapse assembly

doi: 10.1084/jem.20220906

Figure Lengend Snippet: Spike suppresses TCR accumulation and phosphotyrosine signaling at the CTL IS. (A and B) RT-qPCR of human ACE2 mRNA (A) and immunoblot analysis of human ACE2 protein (B) in purified CD8 + T cells prior to stimulation (day 0) or after stimulation with anti-CD3/CD28 mAb–coated beads in the presence of IL-2 for the indicated times. The migration of molecular mass markers is indicated ( n = 3, one-way ANOVA test for RT-qPCR analysis; *, P ≤ 0.05; n = 4, paired two-tailed Student’s t test for immunoblot analysis; **, P ≤ 0.01). (C) Immunofluorescence analysis of Spike W binding to purified CD8 + T cells prior to stimulation (day 0) or 7 d after stimulation with anti-CD3/CD28 mAb–coated beads in the presence of IL-2 ( n = 2). Scale bar, 15 μm. (D) Immunofluorescence analysis of CTL binding of fluorescently labeled MiniVs (Spike W embedded in fluorescent NDP-labeled liposomes) or control liposomes. Representative images are shown ( n = 2). Scale bar, 15 μm. (E–G) Immunofluorescence analysis of CD3ζ and PTyr in CTLs (day 7) pretreated with either vehicle (PBS) or 0.05 μg/μl Spike W (cell viability after pretreatment 91.7 ± 0.2%), mixed with Raji cells (APCs) either unpulsed or pulsed with a combination of SEA, SEB, and SEE (SAgs), and incubated for 15 min at 37°C. Representative images (medial optical sections) of the T cell:APC conjugates are shown (E). Scale bar, 5 μm. (F) Left: Quantification (%) of 15-min SAg-specific conjugates harboring PTyr staining at the IS (≥50 cells/sample, n = 3, one-way ANOVA test; ****, P ≤ 0.0001; **, P ≤ 0.01). Right: Relative PTyr fluorescence intensity at the IS (recruitment index; 10 cells/sample, n = 3, Kruskal–Wallis test; ****, P ≤ 0.0001). (G) Left: Quantification (%) of 15-min SAg-specific conjugates harboring CD3ζ staining at the IS (≥50 cells/sample, n = 3, one-way ANOVA test; ***, P ≤ 0.001; *, P ≤ 0.05). Right: Relative CD3ζ fluorescence intensity at the IS (recruitment index; 10 cells/sample, n = 3, Kruskal–Wallis test; ****, P ≤ 0.0001). (H and I) Immunofluorescence analysis of CD3ζ and PTyr in CTLs (day 7) pretreated with either 1.2 × 10 9 control liposomes or MiniVs, mixed with Raji cells (APCs) either unpulsed or pulsed with a combination of SEA, SEB, and SEE (SAgs), and incubated for 15 min at 37°C. (H) Left: Quantification (%) of 15-min SAg-specific conjugates harboring PTyr staining at the IS (≥50 cells/sample, n = 3, one-way ANOVA test; ****, P ≤ 0.0001; **, P ≤ 0.01). Right: Relative PTyr fluorescence intensity at the IS (recruitment index; 10 cells/sample, n = 3, Kruskal–Wallis test; ****, P ≤ 0.0001). Side-by-side comparison of PTyr + immune synapses formed by CTLs pretreated with the same amount of soluble and MiniV-associated Spike showed a ∼2.3-fold increase in the suppressive ability of Spike when associated with MiniVs (51 vs. 22%, n = 3). (I) Left: Quantification (%) of 15-min SAg-specific conjugates harboring CD3ζ staining at the IS (≥50 cells/sample, n = 3, one-way ANOVA test; ****, P ≤ 0.0001; **, P ≤ 0.01; *, P ≤ 0.05). Right: Relative CD3ζ fluorescence intensity at the IS (recruitment index; 10 cells/sample, n = 3, Kruskal–Wallis test; ****, P ≤ 0.0001). Nonsignificant differences are not shown. Source data are available for this figure: .

Article Snippet: Immunoblotting was carried out using anti-phospho-Erk1/2 ([1:500], phosphorylated p44/42 [MAPK] Erk1/2, #9101; Cell Signaling), anti-Erk2 ([1:500], clone D2, #sc-1647; SantaCruz), anti-ACE2 ([1:500], clone SN0754, #MA5-32307; Invitrogen), and anti-actin ([1:10,000], #MAB1501; Merck Millipore) Abs and peroxidase-labeled secondary Ig, and a chemiluminescence detection kit (Pierce Rockford).

Techniques: Quantitative RT-PCR, Western Blot, Purification, Migration, Two Tailed Test, Immunofluorescence, Binding Assay, Labeling, Incubation, Staining, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: SARS-CoV-2 Spike protein suppresses CTL-mediated killing by inhibiting immune synapse assembly

doi: 10.1084/jem.20220906

Figure Lengend Snippet: The inhibitory effects of Spike on IS formation are mediated by ACE2 and can be detected in acutely infected SARS-CoV-2 patients. (A and B) Top: Quantification (%) of 15-min antigen-specific conjugates harboring PTyr (A) or CD3ζ (B) staining at the IS in CTLs (day 7) pretreated with either vehicle (PBS) or 0.05 μg/μl Spike W, Spike Omicron BA.1 (Spike BA.1), alone or in the presence of the respective neutralizing mAb (Spike W + J08; Spike BA.1 + 02M04); or Spike Omicron BA.2 (Spike BA.2). Samples pretreated with only the neutralizing mAbs (J08 and 02M04) were also included in the analysis. CTLs were mixed with Raji cells (APCs), either unpulsed or pulsed with a combination of SEA, SEB, and SEE (SAgs), and incubated for 15 min at 37°C (≥50 cells/sample, n = 3, unpaired two-tailed Student’s t test; ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05). Bottom: Relative PTyr (A) or CD3ζ (B) fluorescence intensity at the IS (recruitment index; 10 cells/sample, n = 3, Kruskal–Wallis test; ****, P ≤ 0.0001). (C) Top: Quantification (%) of 15-min antigen-specific conjugates formed as in panel A harboring PCTN1 staining at the IS (≥50 cells/sample, n = 3, one-way ANOVA test; ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05). Bottom: Measurement of the distance (μm) of the centrosome (PCNT) from the T cell–APC contact site (10 cells/sample, n = 3, Kruskal–Wallis test; ****, P ≤ 0.0001). (D and E) Immunofluorescence analysis of PTyr (D) and CD3ζ (E) in purified CD8 + T cells transfected with an ACE2-GFP–expressing construct or empty vector, pretreated with either vehicle (PBS) or 0.05 μg/μl Spike W, then mixed with Raji cells (APCs) either unpulsed or pulsed with a combination of SEA, SEB, and SEE (SAgs), and incubated for 15 min at 37°C. The histograms show (left) the quantification (%) of conjugates harboring PTyr (D) and CD3ζ (E) staining at the IS (top; ≥50 cells/sample, n = 2, one-way ANOVA test; ***, P ≤ 0.001; **, P ≤ 0.01), or (right) the relative PTyr (D) and CD3ζ (E) fluorescence intensity at the IS (recruitment index; 10 cells/sample, n = 2, Kruskal–Wallis test; ****, P ≤ 0.0001). Quantification was carried out on GFP + cells. (F) Immunofluorescence analysis of PTyr and GrzB in BALs or PBLs from patients with acute SARS-CoV-2 infection, or PBLs from healthy donors, mixed with Raji cells (APCs) pulsed with a combination of SEA, SEB, and SEE (SAgs), and incubated for 15 min at 37°C. Representative images (medial optical sections) of the T cell:APC conjugates are shown. (G) The histograms show (left) the quantification (%) of 15-min SAg-specific conjugates harboring PTyr staining at the IS (≥50 cells/sample, n = 3, one-way ANOVA test; ***, P ≤ 0.001; **, P ≤ 0.01), or (right) the relative PTyr fluorescence intensity at the IS (recruitment index; 10 cells/sample, n = 3, Kruskal–Wallis test; ****, P ≤ 0.0001). The analysis was restricted to CTLs, identified by GrzB staining. Data are expressed as mean ± SD. Nonsignificant differences are not shown. Scale bar, 5 μm.

Article Snippet: Immunoblotting was carried out using anti-phospho-Erk1/2 ([1:500], phosphorylated p44/42 [MAPK] Erk1/2, #9101; Cell Signaling), anti-Erk2 ([1:500], clone D2, #sc-1647; SantaCruz), anti-ACE2 ([1:500], clone SN0754, #MA5-32307; Invitrogen), and anti-actin ([1:10,000], #MAB1501; Merck Millipore) Abs and peroxidase-labeled secondary Ig, and a chemiluminescence detection kit (Pierce Rockford).

Techniques: Infection, Staining, Incubation, Two Tailed Test, Fluorescence, Immunofluorescence, Purification, Transfection, Expressing, Construct, Plasmid Preparation

Journal: The Journal of Experimental Medicine

Article Title: SARS-CoV-2 Spike protein suppresses CTL-mediated killing by inhibiting immune synapse assembly

doi: 10.1084/jem.20220906

Figure Lengend Snippet: ACE2 is recruited to the CTL IS and suppresses IS formation and cytotoxicity. (A) Immunofluorescence analysis of ACE2 localization in purified CD8 + T cells transfected with an ACE2-GFP–expressing construct or empty GFP vector, pretreated with either vehicle (PBS) or 0.05 μg/μl Spike W, then mixed with Raji cells (APCs) either unpulsed or pulsed with a combination of SEA, SEB, and SEE (SAgs), and incubated for 15 min at 37°C. Representative images are shown. The histograms show (left) the quantification (%) of conjugates harboring ACE2-GFP staining at the IS (≥50 cells/sample, n = 2, one-way ANOVA test; *, P ≤ 0.05), or (right) the relative ACE2-GFP fluorescence intensity at the IS (recruitment index; 10 cells/sample, n = 2, Kruskal–Wallis test; ****, P ≤ 0.0001). (B and C) Left: Quantification (%) of 15-min conjugates harboring PTyr (B) or CD3ζ (C) staining at the IS. CTLs (day 7) were conjugated with Raji cells (APCs) in the absence or presence of SAgs and either an anti-ACE2 Ab (cell viability after pretreatment 93.7 ± 0.8%), or Ang II, or the peptide Ang 1-7 (≥50 cells/sample, n = 3, one-way ANOVA test; ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05). Right: Relative PTyr (B) or CD3ζ (C) fluorescence intensity at the IS (recruitment index; 10 cells/sample, n = 3, Kruskal–Wallis test; ****, P ≤ 0.0001). (D) Left: Quantification (%) of 15-min conjugates formed as in A harboring PCTN staining at the IS (≥50 cells/sample, n = 3, one-way ANOVA test; ****, P ≤ 0.0001; **, P ≤ 0.01; *, P ≤ 0.05). Right: Measurement of the distance (μm) of the centrosome (PCNT) from the T cell–APC contact site in CTL-APC conjugates formed as in A (10 cells/sample, n = 3, Kruskal–Wallis test; ****, P ≤ 0.0001). (E) Measurement of the distance (μm) of the LGs (marked by GzmB) from the centrosome (PCNT) in 15-min CTL-APC conjugates formed as in A (10 cells/sample, n = 3, Kruskal–Wallis test; ****, P ≤ 0.0001; ***, P ≤ 0.001). (F) Fluorimetric analysis of cytotoxicity of CTLs (day 7) using the calcein release assay. CTLs were pretreated with either vehicle (PBS) or 2 μg/ml anti-ACE2 Ab (ACE2) and co-cultured with SAg-pulsed, calcein AM–loaded Raji cells at different E:T cell ratios for 4 h. The representative curves show the kinetics of target cell lysis by CTLs at the indicated E:T cell ratios. The histogram shows the target cell lysis at 4 h ( n = 3, one-way ANOVA test; **, P ≤ 0.01; *, P ≤ 0.05). (G) Flow cytometric analysis of antigen-specific target cell killing by melanoma-specific CTLs derived from three patients, using as target the melanoma cell line A375. CTLs were pretreated as in F, cocultured with A375 cells for 18 h, and processed for flow cytometry after staining with propidium iodide and anti-CD8 mAb. Analyses were carried out gating on CD8 − /PI + cells. The histograms show the percentage (%) of target cells lysed ( n = 3, one-way ANOVA test; ****, P ≤ 0.0001). The data are expressed as mean ± SD. Nonsignificant differences are not shown. Scale bar, 5 μm.

Article Snippet: Immunoblotting was carried out using anti-phospho-Erk1/2 ([1:500], phosphorylated p44/42 [MAPK] Erk1/2, #9101; Cell Signaling), anti-Erk2 ([1:500], clone D2, #sc-1647; SantaCruz), anti-ACE2 ([1:500], clone SN0754, #MA5-32307; Invitrogen), and anti-actin ([1:10,000], #MAB1501; Merck Millipore) Abs and peroxidase-labeled secondary Ig, and a chemiluminescence detection kit (Pierce Rockford).

Techniques: Immunofluorescence, Purification, Transfection, Expressing, Construct, Plasmid Preparation, Incubation, Staining, Fluorescence, Release Assay, Cell Culture, Lysis, Derivative Assay, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: SARS-CoV-2 Spike protein suppresses CTL-mediated killing by inhibiting immune synapse assembly

doi: 10.1084/jem.20220906

Figure Lengend Snippet: ACE2 suppresses IS assembly and function in CTLs but not in resting CD8 + T cells. (A) ELISA-based quantification of IFNγ in 36-h supernatants of melanoma-specific CTLs derived from three patients, pretreated with either vehicle (PBS) or 2 μg/ml anti-ACE2 Ab (ACE2), and co-cultured with irradiated autologous APCs pulsed with 2 µg/ml MAGE 3 ( n = 3, unpaired two-tailed Student’s t test; **, P ≤ 0.01). (B–D) Top: Quantification (%) of 5-min and 15-min conjugates harboring PTyr (B), CD3ζ (C), or P-ZAP-70 (D) staining at the IS (≥50 cells/sample, n = 3, one-way ANOVA test; ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05). CTLs (day 7), pretreated with vehicle (PBS) or 2 μg/ml anti-ACE2 Ab (ACE2), were conjugated with Raji cells (APCs) in the absence or presence of SAgs and either an anti-ACE2 Ab, or Ang II, or the peptide Ang 1-7 (≥50 cells/sample, n = 3, one-way ANOVA test). Bottom: Relative PTyr (B), CD3ζ (C), or ZAP-70 (D) fluorescence intensity at the IS (recruitment index; 10 cells/sample, n = 3, Kruskal–Wallis test; ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05). (E and F) Immunofluorescence analysis of PTyr and CD3ζ in freshly purified CD8 + T cells (day 0) pretreated with either vehicle (PBS) or 2 μg/ml anti-ACE2 Ab (ACE2), then mixed with Raji cells (APCs) either unpulsed or pulsed with a combination of SEA, SEB, and SEE (SAgs), and incubated for 15 min at 37°C. Left: Quantification (%) of conjugates harboring PTyr (E), CD3ζ (F; ≥50 cells/sample, n = 3, one-way ANOVA test; ***, P ≤ 0.001; **, P ≤ 0.01). Right: Relative PTyr (E) and CD3ζ (F) fluorescence intensity at the IS (recruitment index; 10 cells/sample, n = 3, Kruskal–Wallis test; ****, P ≤ 0.0001; **, P ≤ 0.01). (G) Immunofluorescence analysis of PCNT in conjugates formed as described in E and F. Left: Quantification (%) of conjugates harboring PCNT at the IS (≥50 cells/sample, n = 3, one-way ANOVA test; ****, P ≤ 0.0001). Right: Measurement of the distance (μm) of the centrosome (PCNT) from the T cell–APC contact site (10 cells/sample, n = 3, Kruskal–Wallis test; ****, P ≤ 0.0001). (H) RT-qPCR of human MAS1 mRNA in purified CD8 + T cells at days 0, 5, and 7 after stimulation with anti-CD3/CD28 mAb-coated beads in the presence of IL-2 ( n = 3). Nonsignificant differences are not shown.

Article Snippet: Immunoblotting was carried out using anti-phospho-Erk1/2 ([1:500], phosphorylated p44/42 [MAPK] Erk1/2, #9101; Cell Signaling), anti-Erk2 ([1:500], clone D2, #sc-1647; SantaCruz), anti-ACE2 ([1:500], clone SN0754, #MA5-32307; Invitrogen), and anti-actin ([1:10,000], #MAB1501; Merck Millipore) Abs and peroxidase-labeled secondary Ig, and a chemiluminescence detection kit (Pierce Rockford).

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Cell Culture, Irradiation, Two Tailed Test, Staining, Fluorescence, Immunofluorescence, Purification, Incubation, Quantitative RT-PCR

Journal: mSphere

Article Title: Mouse-Adapted SARS-CoV-2 MA10 Strain Displays Differential Pulmonary Tropism and Accelerated Viral Replication, Neurodissemination, and Pulmonary Host Responses in K18-hACE2 Mice

doi: 10.1128/msphere.00558-22

Figure Lengend Snippet: Mouse Ace2 (mAce2) expression, but not hACE2 , is downregulated in the lung of SARS-CoV-2-infected K18-hACE2, C57BL/6J, and BABL/c mice. The relative expression of mAce2 (A), hACE2 (B), and mTmprss2 (C) was evaluated in the lung of K18-hACE2 mice at 2, 4, and 6 dpi following infection with SARS-CoV-2 MA10 and USA-WA1/2020 strains. The relative expression of mAce2 (D and F) and mTmprss2 (E and G) expression were evaluated at 2, 4, and 7 dpi in the lung of mock and MA10-infected C57BL/6J and BALB/c mice, respectively. Bars represent the mean ± standard deviation. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Article Snippet: Sections were then incubated with a ready-to-use hydrogen peroxide solution (Leica Biosystems) for 5 min and subsequently a rabbit monoclonal anti-ACE2 antibody (clone E5O6J [Cell Signaling Technologies, Danvers, MA, USA] diluted at 1:100 in primary antibody diluent [Leica Biosystems]) for 30 min at room temperature.

Techniques: Expressing, Infection, Standard Deviation

Journal: mSphere

Article Title: Mouse-Adapted SARS-CoV-2 MA10 Strain Displays Differential Pulmonary Tropism and Accelerated Viral Replication, Neurodissemination, and Pulmonary Host Responses in K18-hACE2 Mice

doi: 10.1128/msphere.00558-22

Figure Lengend Snippet: Expression and distribution of mAce2 mRNA and ACE2 protein in the lungs of K18-hACE2 mice. mAce2 mRNA expression and distribution was evaluated by RNAscope ISH in the lung of mock-, MA10-, and USA-WA1/2020-infected mice at 4 dpi (A to C). Expression was predominantly observed within the bronchiolar epithelium, with expression in only rare pneumocytes (A to C, insets). mAce2 mRNA expression was semiquantitated in the bronchiolar epithelium by H-score calculation; while a reduction trend in expression level was noted, this was not statistically significant (D). Distribution of ACE2 protein in the lung of mock-, MA10-, and USA-WA1/2020-infected mice at 4 dpi (E to G). ACE2 has an apical distribution predominantly within bronchiolar epithelium and rarely in AT2 cells. Fast Red ( mAce2 mRNA) (A to C) and DAB (brown, ACE2) (E to G). ×200 total magnification.

Article Snippet: Sections were then incubated with a ready-to-use hydrogen peroxide solution (Leica Biosystems) for 5 min and subsequently a rabbit monoclonal anti-ACE2 antibody (clone E5O6J [Cell Signaling Technologies, Danvers, MA, USA] diluted at 1:100 in primary antibody diluent [Leica Biosystems]) for 30 min at room temperature.

Techniques: Expressing, Infection

Journal: mSphere

Article Title: Mouse-Adapted SARS-CoV-2 MA10 Strain Displays Differential Pulmonary Tropism and Accelerated Viral Replication, Neurodissemination, and Pulmonary Host Responses in K18-hACE2 Mice

doi: 10.1128/msphere.00558-22

Figure Lengend Snippet: Primer sequences used for gene expression analysis

Article Snippet: Sections were then incubated with a ready-to-use hydrogen peroxide solution (Leica Biosystems) for 5 min and subsequently a rabbit monoclonal anti-ACE2 antibody (clone E5O6J [Cell Signaling Technologies, Danvers, MA, USA] diluted at 1:100 in primary antibody diluent [Leica Biosystems]) for 30 min at room temperature.

Techniques: Expressing