enzyme linked immunosorbent assay elisa kits  (Thermo Fisher)

Journal: PLoS Pathogens

Article Title: Exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to the vertebrate neuronal cells

doi: 10.1371/journal.ppat.1006764

Figure Lengend Snippet: Endothelial cell-derived exosomes mediate transmission of LGTV RNA from bEnd.3 cells to N2a neuronal cells. The bEnd.3 cells (1 x 10 5 ) were infected with 6 MOI of LGTV. QRT-PCR analysis showing total LGTV loads (A) or levels of LGTV positive-sense strand or negative-sense strand (B) in exosomes isolated from bEnd.3 cells at different time points (24, 48, 72, 96 and 120 h p.i.). (C) Viral loads were determined after 24 h p.i. by the presence of LGTV in N2a cells (1 x 10 5 ) treated with LGTV containing exosomes (20 μl) or supernatant (400 μl) fractions prepared from 24 or 48 h p.i. of bEnd.3 cells. (D) Viral loads at 48 h p.i. of N2a cells (plated in lower chamber) as determined by the presence of LGTV in a transwell assay performed with 1x 10 5 of bEnd.3 cells treated with 20 μl brain endothelial cell-derived exosomes (in upper chamber) for 4 h in the presence or absence of exosome inhibitor (5 μM) (in upper chamber) or infected with LGTV laboratory virus stocks is shown. LGTV transcript levels were normalized to mouse beta-actin. P value determined by Student’s two-tail t test is shown. Representative data is shown from three independent experiments.

Article Snippet: We also isolated exosomes from N2a cells using the total exosome isolation reagent and extracted total RNA and proteins using total exosome RNA and Protein Isolation kit (Invitrogen/ThermoScientific) as per the manufacturer’s instruction.

Techniques: Derivative Assay, Transmission Assay, Infection, Quantitative RT-PCR, Isolation, Transwell Assay

Journal: PLoS Pathogens

Article Title: Exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to the vertebrate neuronal cells

doi: 10.1371/journal.ppat.1006764

Figure Lengend Snippet: Proposed model showing arthropod-borne flaviviruses transmission to human host via arthropod exosomes. Tick bite or injection/spit of saliva from infected ticks deposits exosomes containing viral infectious RNA or proteins securely to humans. Exosomes derived from tick cells/saliva infect human skin (keratinocytes) or may directly deposit saliva enriched with exosome containing tick-borne viruses into the blood capillaries/vessels. Exosomes derived from vascular endothelial cells containing flaviviruses infect neighboring endothelial cells leading into infection of the peripheral system. Higher viral loads in peripheral tissues increase viremia in blood and eventually allow entry and replication of these arthropod-borne viruses in brain microvascular endothelial cells that line the BBB. Early production and higher loads of flaviviruses in brain endothelial cells would allow these viruses to cross BBB and infect neurons. Infected neurons produce high numbers of exosomes containing infectious RNA and proteins that fuse with cell membranes of naïve neuronal cells thereby infecting neighboring neurons and leading to spread of infection and severe neuronal loss.

Article Snippet: We also isolated exosomes from N2a cells using the total exosome isolation reagent and extracted total RNA and proteins using total exosome RNA and Protein Isolation kit (Invitrogen/ThermoScientific) as per the manufacturer’s instruction.

Techniques: Transmission Assay, Injection, Infection, Derivative Assay

Journal: PLoS Pathogens

Article Title: Exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to the vertebrate neuronal cells

doi: 10.1371/journal.ppat.1006764

Figure Lengend Snippet: Detection of LGTV RNA and proteins in exosomes isolated from N2a neuronal cell line. (A) Cryo-EM images showing exosomes isolated from uninfected and LGTV-infected (MOI 6; 72 h p.i.), N2a cells (1 x 10 7 ). Scale indicates 100nm. Quantification of diameter or sizes for heterogenous population of exosomes from uninfected (B) or LGTV- infected (C) N2a cell-derived exosomes. Number of exosomes analyzed were n = 32 (uninfected) and n = 131 (infected) groups. (D) Comparison of exosome numbers per image from uninfected (n = 9) and infected (n = 13) groups is shown. (E) DG-Exos showing presence of enhanced LGTV envelope [E]-protein loads in fractions 1–6. Fractions 3–5 showed enriched amounts of exosomal markers CD9 and HSP70. E-protein detection in fraction 5 in 0.1 μm filtered samples processed for OptiPrep DG-isolation is shown. QRT-PCR analysis showing levels of total LGTV loads (F), copy numbers (G) and LGTV positive-sense strand or negative-sense strand (H) in exosomes isolated from N2a cells at different time points. N2a (1 x 10 5 ) cells were infected with 6 MOI of LGTV, and LGTV loads were analyzed at 72 h p.i. (I) Treatment of LGTV-infected (72 h p.i.) N2a cell-derived exosomes with RNase A is shown. The uninfected samples treated with RNase serve as control. LGTV transcript levels were normalized to mouse beta-actin. (J) Immunoblotting analysis showing detection of LGTV E glycoprotein and mammalian exosomal marker CD9 in exosome fractions and total lysates from whole cells prepared at 48 h p.i. from 2 x 10 6 uninfected (UI) or infected (I) N2a cells. Stain-free gels showing total protein profiles serve as the loading control. (K) Native-PAGE followed by immunoblotting analysis showing presence of LGTV E- and NS1 proteins from LGTV-infected (MOI 1; 72 h p.i.) or uninfected N2a cell-derived exosomes treated with Triton-X-100 (0.03%), or freeze-thaw cycles (3 times freezing at -80°C for 1 h each cycle) or untreated, held on ice. Coomassie stained gel image showing total protein profiles serve as a loading control. (L) ELISA performed on uninfected or LGTV-infected (MOI 6; 72 h p.i.) N2a cell-derived exosomes either untreated or treated with Triton-X-100 (0.1%). (M) Antibody-beads binding assay performed on LGTV infected (MOI 1, 72 h p.i.) N2a cell-derived exosomes (collected from N2a cells either untreated or treated with GW4869; 5 μM, exosome inhibitor) showing no differences in LGTV-E protein loads between 4G2 or isotype and untreated samples.

Article Snippet: We also isolated exosomes from N2a cells using the total exosome isolation reagent and extracted total RNA and proteins using total exosome RNA and Protein Isolation kit (Invitrogen/ThermoScientific) as per the manufacturer’s instruction.

Techniques: Isolation, Infection, Derivative Assay, Quantitative RT-PCR, Marker, Staining, Clear Native PAGE, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: PLoS Pathogens

Article Title: Exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to the vertebrate neuronal cells

doi: 10.1371/journal.ppat.1006764

Figure Lengend Snippet: Detection of LGTV RNA and proteins in exosomes isolated from primary cultures of mouse cortical neurons. LGTV (4 MOI) was used to infect 1 x 10 5 murine cortical neuronal cells. UI indicates uninfected and I indicates LGTV-infected. (A) QRT-PCR analysis showing LGTV infection kinetics in primary cultures of mouse cortical neurons at different time points (24, 48, 72, 96 h p.i.). Total LGTV loads in exosomes isolated from cortical neurons (B), copy numbers (C) and levels of LGTV positive-sense strand or negative-sense strand (D) in exosomes isolated from cortical neuronal cells at different time points (24, 48 and 72 h p.i.). (E) Immunoblotting analysis showing detection of LGTV E-protein and mammalian exosomal marker CD9 in exosome fraction and total lysates from whole cells prepared from uninfected (UI) or infected (I) cortical neuronal cells at 48 h p.i. Stain-free gel showing total protein profile serves as the loading control. (F) Immunoblotting analysis showing NS1 levels in total neuronal lysates and exosomes derived from LGTV-infected (MOI 4; 72 h p.i.) cortical neurons. Uninfected (UI) cells and exosomes derived from these cells serve as controls in addition to total protein profiles. For immunoblotting assays, 2 x 10 7 cortical neuronal cells were infected with 4 MOI of LGTV. (G) Quantitative assessment of number of plaques from exosomal and supernatant fractions is shown. (H) QRT-PCR analysis of viral loads in 1x 10 5 naïve cortical neuronal cells at 24 h p.i., infected through treatment with exosomes (20 μl) or supernatant fractions (400 μl) prepared from 24, 48 and 72 h p.i., LGTV-infected neurons show presence of LGTV. LGTV transcript levels were normalized to mouse beta-actin. P value determined by Student’s two-tailed t test is shown. Representative data is shown from at least three independent experiments.

Article Snippet: We also isolated exosomes from N2a cells using the total exosome isolation reagent and extracted total RNA and proteins using total exosome RNA and Protein Isolation kit (Invitrogen/ThermoScientific) as per the manufacturer’s instruction.

Techniques: Isolation, Infection, Quantitative RT-PCR, Marker, Staining, Derivative Assay, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to the vertebrate neuronal cells

doi: 10.1371/journal.ppat.1006764

Figure Lengend Snippet: Arthropod exosomes mediate transmission of infectious LGTV RNA and proteins from tick to human cells. (A) Cryo-EM images showing exosomes isolated from uninfected and LGTV-infected (MOI 1; 72 h p.i.), ISE6 tick cells. Scale bar indicates 100nm. Quantification of diameter or sizes for heterogenous population of exosomes from uninfected (B) or LGTV- infected (C) tick cell-derived exosomes. Number of exosomes analyzed were n = 138 (uninfected) and n = 200 (infected) groups. (D) Comparison of exosome numbers per cryo-EM image from uninfected (n = 27) and infected (n = 14) groups is shown. (E) DG-Exos showing presence of LGTV envelope [E]-protein in fractions 1–6. QRT-PCR analysis showing total LGTV loads (F) and levels of LGTV positive-sense strand or negative-sense strand (G) in exosomes isolated from tick cells at 72 h (p.i.), Uninfected cells serve as control. LGTV transcript levels were normalized to tick beta-actin. (H) Immunoblotting analysis showing detection of LGTV E- glycoprotein in exosome fraction and total lysates from whole cells prepared from uninfected (UI) or infected (I) tick cells at 72 h (p.i.). Immunoblot detecting NS1 in both tick-cell derived exosomes and total cell lysates using monoclonal anti-Langat virus NS1 protein from LGTV-infected (MOI 1; 72 h p.i.) is also shown. HSP70 levels indicate enrichment of arthropod exosomal marker in exosome fractions. Uninfected samples and total protein profiles serve as controls. Tick cells (5 x 10 6 ) were infected with 1 MOI of LGTV in both QRT-PCR and immunoblotting analysis. I) Native-PAGE followed by immunoblotting analysis showing presence of E-protein from LGTV-infected (MOI 1; 72 h p.i.) or uninfected tick cell-derived exosomes treated with Triton-X-100 (0.03%), or freeze-thaw cycles (3 times freezing at -80°C for 1 h each cycle) or untreated samples held on ice. Coomassie stained gel showing total protein profiles serve as loading control (I). (J) Antibody-beads binding assay performed on LGTV infected (MOI 1) tick cell-derived exosomes (collected from tick cells either untreated or treated with GW4869; 5 μM, exosome inhibitor) showing no differences in Langat-E protein loads between 4G2 or isotype and untreated samples. (K) Quantitative assessment of number of plaques in exosomal and supernatant fractions is shown. TMTC indicates “too many to count”. (L) Viral re-infection kinetics as determined by the presence of total LGTV loads in HaCaT cells (1 x 10 5 cells) at different time points treated with exosomes (20 μl) or supernatant fractions (400 μl) prepared from 72 h p.i. LGTV-infected tick cells are shown. (M) Viral loads at 48 h p.i. was determined by the presence of LGTV in a transwell assay performed with 1 x 10 5 tick cells (in upper chamber) or 1 x 10 5 HaCaT cells (in lower chamber) treated with tick exosome fraction (20 μl, in upper chamber) for 4 h in the presence or absence of 5 μM exosome inhibitor GW4869. Tick cells infected with LGTV laboratory stocks were used as controls. LGTV transcript levels were normalized to human beta-actin in (L) and (M). P value determined by Student’s two-tail t test is shown. Representative data is shown from three independent experiments.

Article Snippet: We also isolated exosomes from N2a cells using the total exosome isolation reagent and extracted total RNA and proteins using total exosome RNA and Protein Isolation kit (Invitrogen/ThermoScientific) as per the manufacturer’s instruction.

Techniques: Transmission Assay, Isolation, Infection, Derivative Assay, Quantitative RT-PCR, Marker, Clear Native PAGE, Staining, Binding Assay, Transwell Assay

Journal: bioRxiv

Article Title: Evidence for a role for BK channels in the regulation of ADAM17 activity

doi: 10.1101/811000

Figure Lengend Snippet: BK channel silencing increased ADAM17 activity and enhanced secretion of TNF-α and IL-6Rα.. ( A ) ( B ) Western blot analysis of whole cell and plasma membrane proteins from siRNA treated macrophages. The cells were non-treated (ctrl), received non-silencing siRNA (Ns) or silencing siRNA for BKα genes (sBK) for 72 hours. BKα silencing effect in ( A ) whole cell (WC) and ( B ) plasma membrane (PM) lysate. Densitometry analysis of BKα expression in BKα silenced cells relative to non-silencing siRNA. Mean+/−SEM (n=3). ( C )-( F )After treatment with siRNAs, macrophages were activated with 150 ng/ml LPS for 4 hours. ( C ) TNF-α and ( E ) IL-6Rα release was analyzed by ELISA. Mean+/−SEM (n=4). ( D ) Western blot analysis of mTNF-α expression. Densitometry of mTNF-α expression in BKα silenced cells relative to non-silencing siRNA. Mean+/−SEM (n=3). ( F ) ADAM17 activity in BKα silenced (sBK) macrophages. Mean+/−SEM (n=3). *p

Article Snippet: A general inhibitor for membrane metalloproteases, GM6001, and a selective inhibitor for ADAM17, TAPI-0, were used to validate that the fluorescence signal was due to the ADAM17 activity.

Techniques: Activity Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Evidence for a role for BK channels in the regulation of ADAM17 activity

doi: 10.1101/811000

Figure Lengend Snippet: Macrophages express plasma membrane BK channels after LPS activation. Plasma membrane BK channels expression appear after LPS treatment. ( A ) TNF-α release measured by ELISA and ( B ) ADAM17 activity in macrophages received 150 ng/ml LPS for 4 hours with/without IbTX. Mean+/−SEM (n=3). Western blot analysis of BKα expression in ( C ) whole cell (WC) and ( D ) plasma membrane (PM) lysate from macrophages treated with 10 ng/ml LPS for up to 24 hours. ( E ) Densitometry of BKα expression after 24 hours of LPS treatment relative to 0 hours. Mean+/− SEM (n=3). ( F ) Immunolocalization of BKα. Staining for DAPI, anti-BKα antibody (green), merged image of DAPI and anti-BKα staining and secondary antibody only control with DAPI. Scale bar, 5μm. Arrows indicate BKα staining on the plasma membrane. *p

Article Snippet: A general inhibitor for membrane metalloproteases, GM6001, and a selective inhibitor for ADAM17, TAPI-0, were used to validate that the fluorescence signal was due to the ADAM17 activity.

Techniques: Activation Assay, Expressing, Enzyme-linked Immunosorbent Assay, Activity Assay, Western Blot, Staining

Journal: bioRxiv

Article Title: Evidence for a role for BK channels in the regulation of ADAM17 activity

doi: 10.1101/811000

Figure Lengend Snippet: Pharmacological inhibition of BK channels increased ADAM17 activity and the release of TNF-α, and IL-6Rα. ( A ) Conditioned macrophages were prepared by stimulating cells with 10 ng/ml LPS for 24 hours. Subsequently the media was replaced and the conditioned cells were activated with 150 ng/ml LPS for 4 hours with/without 30 nM IbTX. ( B ) TNF-α, ( C ) IL-6Rα and ( D ) IL-6 release in were analyzed by ELISA. Mean+/−SEM (n=3). ( E ) Western blot analysis of mTNF-α expression and densitometry of mTNF-α expression relative to LPS only control group. ( F ) TNF-α mRNA was quantified by RT-qPCR. Mean+/−SEM (n=3). ( H ) ADAM17 activity. Mean+/−SEM (n=3). (G) Effect of 100 nM NS11021 on TNF-α release. *p

Article Snippet: A general inhibitor for membrane metalloproteases, GM6001, and a selective inhibitor for ADAM17, TAPI-0, were used to validate that the fluorescence signal was due to the ADAM17 activity.

Techniques: Inhibition, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Quantitative RT-PCR

Journal: Nature Communications

Article Title: An adenovirus-vectored COVID-19 vaccine confers protection from SARS-COV-2 challenge in rhesus macaques

doi: 10.1038/s41467-020-18077-5

Figure Lengend Snippet: The immunogenicity of Ad5-S-nb2 in mice. a Schematic diagram of the schedule, dosage, and delivery routes of mouse experiment. Eight-week-old BALB/c mice ( n = 5 per group) were vaccinated with Ad5-S-nb2 using the indicated dosage through an IM or IN route. The gray blood drop symbols indicate the time points at which the serum samples were collected. Twenty-eight days after the initial vaccination, the mice were sacrificed. b The IgG antibody response in the sera of mice that received IM vaccination. c The IgA antibody response in the BALFs of mice that received IM vaccination. d The IgG antibody response in the sera of mice that received IN vaccination. e The IgA antibody response in the BALFs of mice that received IN vaccination. The endpoint titers of IgG antibodies ( b and d ) and IgA antibodies ( c and e ) against the RBD, S2, and S protein are shown. f The titers of neutralizing antibodies in mice sera. g The titers of neutralizing antibodies in mice BALFs. The sera ( f ) or BALFs ( g ) from each group of mice ( n = 5) were pooled and assessed by S-pseudotyped reporter lentivirus. h , j IFN-γ-secreting cells in the spleens of mice that received IM ( h ) or IN vaccination ( j ). Splenic lymphocytes from each group of mice ( n = 5) were pooled and stimulated with two peptide pools corresponding to the S1 and S2 region. Shown are the number of spot-forming cells (SFCs) in one million splenic lymphocytes. For b – e , each circle represents an individual mice, and the error bars indicate the means ± standard derivations (SD). For f – i , each bar reflects the mean value of three technical replicates. Data are one representative result of two independent experiments. Comparisons were performed by Student’s t-test (unpaired, two-tailed). Source data are provided as a Source Data file.

Article Snippet: Finally, Ad5-S-nb2 and an empty Ad5 vector, Ad5-empty, were propagated, purified by cesium chloride density gradient centrifugation, titrated, and stored at −80 °C.

Techniques: Mouse Assay, Two Tailed Test

Journal: Nature Communications

Article Title: An adenovirus-vectored COVID-19 vaccine confers protection from SARS-COV-2 challenge in rhesus macaques

doi: 10.1038/s41467-020-18077-5

Figure Lengend Snippet: Ad5-S-nb2 protects against SARS-CoV-2 infection in rhesus macaques. a The viral loads in the pharyngeal swabs of non-vaccinated macaques. b The viral loads in the pharyngeal swabs in macaques at 4 weeks after IM vaccination with 1 × 10 11 vp Ad5-S-nb2. c The viral loads in the pharyngeal swabs in macaques at 4 weeks after IN vaccination with 5 × 10 10 vp Ad5-S-nb2. d The viral loads in the pharyngeal swabs in macaques at 8 weeks after IM vaccination with 1 × 10 10 vp Ad5-S-nb2. The genome copy numbers in the elution of the pharyngeal swabs were determined by quantitative reverse transcription PCR (qRT-PCR). The limit of detection was 200 copies ml −1 and marked by the dot line. The data points were expressed as the mean of two technical replicates. Macaques C3, 116004, 134018, 063585, and 080066 were euthanatized on day 7 after challenge and were marked by hash symbol. Other macaques were euthanatized on day 10 after challenge. Macaques C1, C2, D1 and D2 were subsequently utilized for another study. e The viral loads calculated based on AUC in the pharyngeal swabs in macaques after challenge. Blue circles, macaques that received 1 × 10 11 vp Ad5-S-nb2 (IM); Dark cyan circles, macaques that received 1 × 10 10 vp Ad5-S-nb2 (IM); Red circles, macaques that received mucosal vaccination (IN). Black circles, non-vaccinated macaques. Black lines reflect the mean AUC. Comparison between vaccinated ( n = 9) and non-vaccinated ( n = 5) macaques was conducted using Student’s t -test (unpaired, two-tailed). f – i The neutralizing antibody titers against SARS-CoV-2 before and after challenge in non-vaccinated macaques ( f ), high-dose IM vaccinated macaques ( g ), IN vaccinated macaques ( h ), and low-dose IM vaccinated macaques ( i ). The sera were examined by PRNT using SARS-CoV-2 (strain 2019-nCoV-WIV04). Source data are provided as a Source Data file.

Article Snippet: Finally, Ad5-S-nb2 and an empty Ad5 vector, Ad5-empty, were propagated, purified by cesium chloride density gradient centrifugation, titrated, and stored at −80 °C.

Techniques: Infection, Polymerase Chain Reaction, Quantitative RT-PCR, Two Tailed Test, Plaque Reduction Neutralization Test

Journal: Nature Communications

Article Title: An adenovirus-vectored COVID-19 vaccine confers protection from SARS-COV-2 challenge in rhesus macaques

doi: 10.1038/s41467-020-18077-5

Figure Lengend Snippet: The immunogenicity of Ad5-S-nb2 in Chinese rhesus macaques. a Schematic diagram of the vaccination and challenge studies in rhesus macaques. Three groups of macaques ( n = 4 per group) were vaccinated via an IM injection of 1 × 10 10 vp or 1 × 10 11 vp Ad5-S-nb2 or via an IN and oral inoculation of 5 × 10 10 vp Ad5-S-nb2 each. The control groups include macaques ( n = 2) IM injected with 1 × 10 11 vp Ad5-empty and non-vaccinated macaques ( n = 6). On days 0, 12, 18, and 24 after vaccination, the serum samples and PBMCs were collected. On day 30 (Group 1 and 2) or 56 (Group 3) after vaccination, macaques were challenged. On day 7 or 10 after challenge, macaques were euthanatized. The gray inverted triangles indicate the time points of vaccination or challenge, whereas the gray blood drop symbols indicate the time points at which the serum samples were collected. b The kinetics of IgG response to the S protein in the sera. The endpoint IgG titers are shown. The overlapped data points represent the same values. Comparisons between different time points were performed by Student’s t -test (paired, one-tailed). c The antigen recognition profiles of macaque immune sera. Macaque immune sera collected on day 24 were assessed for the IgG antibodies against the RBD, S2, and S protein by enzyme-linked immunosorbent assay (ELISA). d IFN-γ-secreting cells in the PBMCs of rhesus macaques. PBMCs isolated on day 18 were stimulated with two peptide pools corresponding to S1 and S2, respectively. Shown are the number of SFCs in one million PBMCs. All the data points represent the mean values of two technical replicates. Source data are provided as a Source Data file.

Article Snippet: Finally, Ad5-S-nb2 and an empty Ad5 vector, Ad5-empty, were propagated, purified by cesium chloride density gradient centrifugation, titrated, and stored at −80 °C.

Techniques: Injection, One-tailed Test, Enzyme-linked Immunosorbent Assay, Isolation

Journal: Nature Communications

Article Title: An adenovirus-vectored COVID-19 vaccine confers protection from SARS-COV-2 challenge in rhesus macaques

doi: 10.1038/s41467-020-18077-5

Figure Lengend Snippet: Construction and characterization of Ad5-S-nb2. a Schematic diagram of the genome of Ad5-S-nb2 and the coding sequence for SARS-CoV-2 S protein. b Western blot analysis of the expression of S protein in HEK293 cells transfected with plasmids encoding an original S sequence (pGA1-S-nb1, 4 μg per well) or a codon-modified S sequence (pGA1- S-nb2 1#: 4 μg per well; pGA1- S-nb2 2#: 2 μg per well). A pGA1-empty plasmid was used as the negative control. A purified S protein with the transmembrane domain truncated (S ΔTM ) was used as the positive control. c Western blot analysis of the expression of S protein in HEK293 cells infected with Ad5-S-nb2. Ad5-S-nb2 1#, 0.2 TCID50 per cell; Ad5-S-nb2 2#, 0.05 TCID50 per cell. S ΔTM protein and HEK293 cells infected with Ad5-empty were examined in parallel as the positive and negative controls, respectively. The samples were derived from the same experiment and the blots were processed in parallel. d Immunofluorescence analysis of the expression of S protein in A549 cells mediated by Ad5-S-nb2. A549 cells were infected with Ad5-S-nb2 or Ad5-empty at 0.2 TCID50 per cell. Twenty-four hours later, cells were labeled with a human monoclonal antibody against S protein and then with an Alexa Fluor 488-conjugated mouse anti-human antibody. The cells were observed under a fluorescence microscope. Scale bar = 50 μm. For b and c , two independent experiments were carried out with similar results. For d , one representative result from three independent experiments is shown. Source data are provided as a Source Data file.

Article Snippet: Finally, Ad5-S-nb2 and an empty Ad5 vector, Ad5-empty, were propagated, purified by cesium chloride density gradient centrifugation, titrated, and stored at −80 °C.

Techniques: Sequencing, Western Blot, Expressing, Transfection, Modification, Plasmid Preparation, Negative Control, Purification, Positive Control, Infection, Derivative Assay, Immunofluorescence, Labeling, Fluorescence, Microscopy

Journal: Nature Communications

Article Title: An adenovirus-vectored COVID-19 vaccine confers protection from SARS-COV-2 challenge in rhesus macaques

doi: 10.1038/s41467-020-18077-5

Figure Lengend Snippet: Viological and histopathological analysis of macaque airway tissues. a The viral loads in the nine anatomic locations of macaque airway. The tissues of trachea, left and right bronchus, and the upper, middle, and lower locations of the left and right lung were collected at euthanasia. The viral genomes in the tissue homogenates were assessed by qRT-PCR. All the data points represent the mean values of two technical replicates. The dot line indicates the limit of detection (1000 copies per g tissue). b – f Histopathological changes in the lungs of SARS-CoC-2 challenged non-vaccinated macaques ( b ) and vaccinated macaques ( d – f ), and an unchallenged macaque IM vaccinated with 1 × 10 10 vp Ad5-S-nb2 ( c ). The lung tissue sections were stained with hematoxylin and eosin (H E). One representative graph from each macaque is shown. Scale bars are 50 μm and indicated in each panel. Source data are provided as a Source Data file.

Article Snippet: Finally, Ad5-S-nb2 and an empty Ad5 vector, Ad5-empty, were propagated, purified by cesium chloride density gradient centrifugation, titrated, and stored at −80 °C.

Techniques: Quantitative RT-PCR, Staining