Journal: Frontiers in Immunology

Article Title: Immunogenicity and protective efficacy of the HC009 mRNA vaccine against SARS-CoV-2

doi: 10.3389/fimmu.2024.1416375

Figure Lengend Snippet: Vaccine design and expression of the mRNA vaccine encoding COVID-19 spike. (A) Structure of HC009 RNA. UTR, untranslated region; CDS, coding domain sequence. (B) Lipid nanoparticle–mRNA formulations used as COVID-19 vaccines. (C) Spike protein expression by flow cytometry using biotinylated hACE2 protein. (D) Spike protein expression analyzed by Western blot using anti-spike as the primary antibody. The asterisk indicates a non-specific band. All data are representative of three independent experiments.

Article Snippet: Human embryonic kidney 293 cells (HEK293 cells) (ATCC CRL-3216) and HEK293T/hACE2-TMPRSS2 cells (SinoBiological, OEC003, China) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, 11965092, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, 11965092, USA) and penicillin (100 U/mL)–streptomycin (100 mg/mL) (Gibco, 15140148, USA).

Techniques: Expressing, Sequencing, Vaccines, Flow Cytometry, Western Blot

Journal: Frontiers in Immunology

Article Title: Immunogenicity and protective efficacy of the HC009 mRNA vaccine against SARS-CoV-2

doi: 10.3389/fimmu.2024.1416375

Figure Lengend Snippet: Evaluation of the immune protection provided by HC009 during in vivo challenge. (A) Immunization and challenge procedures for the 0.5-, 2-, and 10-μg dose of HC009 in mice. Six- to 8-week-old female hACE2 transgenic mice were immunized with two doses of the vaccines via the intramuscular route at 3-week intervals ( n = 12). Subsequently, they were challenged with live SARS-CoV-2 at 50 days post-vaccination, and the lung and nasal turbinate tissues were collected at the indicated time points after immunization. (B) The body weights of the mice were monitored and recorded for six consecutive days after the challenge ( n = 6). The mice were euthanized after observation. (C) Average clinical scores for disease signs, including lethargy, ruffled fur, hunched back posture, and rapid breathing. A score of 1 was given to each of these clinical signs ( n = 12). (D) Viral RNA in the lungs and nasal turbinate tissues of challenged mice were measured with qRT-PCR at 1, 3, 5, and 6 dpi, respectively ( n = 6). (E) H&E staining was performed to assess pathological changes in the lungs of mice at 1, 3, 5, and 6 dpi ( n = 3). The data are shown as the mean ± SEM. Horizontal dashed line indicates the lower limit of quantification. All the data are representative of three independent experiments. A two-way ANOVA with Tukey’s multiple comparisons test was performed, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Human embryonic kidney 293 cells (HEK293 cells) (ATCC CRL-3216) and HEK293T/hACE2-TMPRSS2 cells (SinoBiological, OEC003, China) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, 11965092, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, 11965092, USA) and penicillin (100 U/mL)–streptomycin (100 mg/mL) (Gibco, 15140148, USA).

Techniques: In Vivo, Transgenic Assay, Vaccines, Quantitative RT-PCR, Staining

Journal: Biomedicines

Article Title: Herbal Compounds Dauricine and Isoliensinine Impede SARS-CoV-2 Viral Entry

doi: 10.3390/biomedicines11112914

Figure Lengend Snippet: Screening of Chinese Herbal Medicine compounds reveals a reduction in SARS-CoV-2 wild-type (WT) Viral pseudoparticle (VPP) infection in the 293T-ACE2 cell line. ( A ) Cell viability was assessed following 24 h treatment with 10 µM of indicated compound or vehicle control (DMSO) in 293T-ACE2 cells by MTT assay. Values are normalized to vehicle control (100%) and shown as mean ± SD (n = 3). All data are shown as mean ± SD ( n = 3). ( B ) 293T-ACE2 cells were pretreated with 10 μM of indicated compound or vehicle control (DMSO) for one hour and infected with SARS-CoV-2 WT-VPP. After 24 h of infection, the infection efficiency rate was measured according to luciferase activities. Values are normalized to vehicle control (100%) and shown as mean ± SD ( n = 3). * p ≤ 0.05; ** p ≤ 0.01 compared to vehicle control.

Article Snippet: 293T-ACE2 cell line was constructed by transfection of human ACE2 expressing plasmids (HG10108-UT; Sino Biological, Beijing, China) into 293T cells, followed by stable cell selection with hygromycin, and can be used for in vitro screening and characterization of drug candidates against SARS-CoV [ ] and SARS-CoV-2 [ ].

Techniques: Infection, Control, MTT Assay, Luciferase

Journal: Biomedicines

Article Title: Herbal Compounds Dauricine and Isoliensinine Impede SARS-CoV-2 Viral Entry

doi: 10.3390/biomedicines11112914

Figure Lengend Snippet: Cytotoxic activity of Dauricine and Isoliensinine in 293T-ACE2 and VeroE6 cells. ( A , B ) 293T-ACE2 cells were treated with different concentrations (3.125, 6.25 12.5, 25, and 50 µM) of Dauricine ( A ) or Isoliensinine ( B ), and cell viability was detected using MTT assay. Values are normalized to vehicle control (100%) and shown as mean ± SD ( n = 3). ( C , D ) VeroE6 cells were treated with different concentrations (3.125, 6.25, 12.5, 25, and 50 µM) of Dauricine ( C ) or Isoliensinine ( D ), and cell viability was detected using MTT assay. Values are normalized to vehicle control (100%) and shown as mean ± SD ( n = 3).

Article Snippet: 293T-ACE2 cell line was constructed by transfection of human ACE2 expressing plasmids (HG10108-UT; Sino Biological, Beijing, China) into 293T cells, followed by stable cell selection with hygromycin, and can be used for in vitro screening and characterization of drug candidates against SARS-CoV [ ] and SARS-CoV-2 [ ].

Techniques: Activity Assay, MTT Assay, Control

Journal: Biomedicines

Article Title: Herbal Compounds Dauricine and Isoliensinine Impede SARS-CoV-2 Viral Entry

doi: 10.3390/biomedicines11112914

Figure Lengend Snippet: Trend in infection rates post-treatment with Dauricine and Isoliensinine. ( A , B ) 293T-ACE2 cells were pretreated with varying concentrations (0.08, 0.4, 2, and 50 µM) of Dauricine ( A ) or Isoliensinine ( B ) for one hour and infected with SARS-CoV-2 WT-VPP. After 24 h of infection, the infection efficiency rate was measured according to luciferase activities. Values are normalized to vehicle control (100%) and shown as mean ± SD ( n = 3). ( C , D ) VeroE6 cells were pretreated with varying concentrations (0.08, 0.4, 2, and 50 µM) of Dauricine ( C ) or Isoliensinine ( D ) for one hour and infected with SARS-CoV-2 WT-VPP. After 24 h of infection, the infection efficiency rate was measured according to luciferase activities. Values are normalized to vehicle control (100%) and shown as mean ± SD ( n = 3).

Article Snippet: 293T-ACE2 cell line was constructed by transfection of human ACE2 expressing plasmids (HG10108-UT; Sino Biological, Beijing, China) into 293T cells, followed by stable cell selection with hygromycin, and can be used for in vitro screening and characterization of drug candidates against SARS-CoV [ ] and SARS-CoV-2 [ ].

Techniques: Infection, Luciferase, Control

Journal: Biomedicines

Article Title: Herbal Compounds Dauricine and Isoliensinine Impede SARS-CoV-2 Viral Entry

doi: 10.3390/biomedicines11112914

Figure Lengend Snippet: Cytotoxicity and antiviral activity of Dauricine and Isoliensinine against SARS-CoV-2 VPP based on Figure 2 and Figure 3 . a IC50 is the half-maximal effective concentration such as the concentration of a compound required to inhibit SARS-CoV-2 infection by 50%. Values are shown as mean ± SD ( n = 3). b CC50 is the median cytotoxic concentration, such as the dose causing 50% cell death. Values are shown as mean ± SD ( n = 3). c SI is the selectivity index, such as the ratio of CC50 to EC50 (SI = CC50/IC50).

Article Snippet: 293T-ACE2 cell line was constructed by transfection of human ACE2 expressing plasmids (HG10108-UT; Sino Biological, Beijing, China) into 293T cells, followed by stable cell selection with hygromycin, and can be used for in vitro screening and characterization of drug candidates against SARS-CoV [ ] and SARS-CoV-2 [ ].

Techniques: Activity Assay, Concentration Assay, Infection

Journal: Biomedicines

Article Title: Herbal Compounds Dauricine and Isoliensinine Impede SARS-CoV-2 Viral Entry

doi: 10.3390/biomedicines11112914

Figure Lengend Snippet: Trend in infection rates among mutant forms of SARS-CoV-2. ( A , B ) 293T-ACE2 cells were pretreated with 2 µM Dauricine ( A ) or 0.2 µM Isoliensinine ( B ) for one hour and infected with SARS-CoV-2 VPP of different variants. After 24 h of infection, the infection efficiency rate was measured according to luciferase activities. Values are normalized to vehicle control (100%) and shown as mean ± SD ( n = 3). * p ≤ 0.05; ** p ≤ 0.01 compared to vehicle control. ( C , D ) VeroE6 cells were pretreated with 1.5 µM Dauricine ( C ) or 0.5 µM Isoliensinine ( D ) for one hour and infected with SARS-CoV-2 VPP of different variants. After 24 h of infection, the infection efficiency rate was measured according to luciferase activities. Values are normalized to vehicle control (100%) and shown as mean ± SD ( n = 3). ** p ≤ 0.01; *** p ≤ 0.001 compared to vehicle control.

Article Snippet: 293T-ACE2 cell line was constructed by transfection of human ACE2 expressing plasmids (HG10108-UT; Sino Biological, Beijing, China) into 293T cells, followed by stable cell selection with hygromycin, and can be used for in vitro screening and characterization of drug candidates against SARS-CoV [ ] and SARS-CoV-2 [ ].

Techniques: Infection, Mutagenesis, Luciferase, Control

Journal: Biomedicines

Article Title: Herbal Compounds Dauricine and Isoliensinine Impede SARS-CoV-2 Viral Entry

doi: 10.3390/biomedicines11112914

Figure Lengend Snippet: Inhibitory Effect of Dauricine and Isoliensinine on ACE2-Spike protein interaction and TMPRSS2 activity. ( A , B ) The percentage of Spike-ACE2 interaction from the FRET-base assay was shown with the indicated concentration of Dauricine ( A ) or Isoliensinine ( B ). Values are normalized to vehicle control (100%) and shown as mean ± SD ( n = 3). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001 compared to vehicle control. ( C , D ) The TMPRSS2 enzymatic activity in vivo was measured by using a FRET-base assay with an increasing amount of Dauricine ( C ) or Isoliensinine ( D ). Values are normalized to vehicle control (100%) and shown as mean ± SD ( n = 3).

Article Snippet: 293T-ACE2 cell line was constructed by transfection of human ACE2 expressing plasmids (HG10108-UT; Sino Biological, Beijing, China) into 293T cells, followed by stable cell selection with hygromycin, and can be used for in vitro screening and characterization of drug candidates against SARS-CoV [ ] and SARS-CoV-2 [ ].

Techniques: Activity Assay, Concentration Assay, Control, In Vivo

Journal: Biomedicines

Article Title: Herbal Compounds Dauricine and Isoliensinine Impede SARS-CoV-2 Viral Entry

doi: 10.3390/biomedicines11112914

Figure Lengend Snippet: Impaired attenuation of infection rates in TMPRSS2 overexpressed cells. ( A , B ) 293T-ACE2 cells with and without TMPRSS2 expression were pretreated with the indicated concentrations (0.08, 0.4, 2, and 10 µM) of Dauricine ( A ) or Isoliensinine ( B ) and then infected with SARS-CoV-2 WT-VPP. After 24 h of infection, the infection efficiency rate was measured according to luciferase activities. Blue line (cont): empty vector control group; Orange line (TMPRSS2): TMPRSS2 overexpressing group. Values are normalized to vehicle control (100%) and shown as mean ± SD ( n = 3). ( C , D ) VeroE6 cells with and without TMPRSS2 expression were pretreated with the indicated concentrations (0.08, 0.4, 2, and 10 µM) of Dauricine ( C ) or Isoliensinine ( D ), and then infected with SARS-CoV-2 WT-VPP. After 24 h of infection, the infection efficiency rate was measured according to luciferase activities. Blue line (cont): empty vector control group; Orange line (TMPRSS2): TMPRSS2 overexpressing group. Values are normalized to vehicle control (100%) and shown as mean ± SD ( n = 3).

Article Snippet: 293T-ACE2 cell line was constructed by transfection of human ACE2 expressing plasmids (HG10108-UT; Sino Biological, Beijing, China) into 293T cells, followed by stable cell selection with hygromycin, and can be used for in vitro screening and characterization of drug candidates against SARS-CoV [ ] and SARS-CoV-2 [ ].

Techniques: Infection, Expressing, Luciferase, Plasmid Preparation, Control

Journal: Biomedicines

Article Title: Herbal Compounds Dauricine and Isoliensinine Impede SARS-CoV-2 Viral Entry

doi: 10.3390/biomedicines11112914

Figure Lengend Snippet: Molecular docking interaction of Dauricine and Isoliensinine in the binding pocket of SARS-CoV-2_RDB-hACE2 complex. The blue colors represent different variants of SARS-CoV-2_RDB residues, whereas green colors represent human ACE2. The orange colors represent Dauricine, whereas the gray colors represent Isoliensinine. ( A ) The binding mode between Dauricine and B.1.1.529 variant of SARS-CoV-2_RDB-hACE2 complex. ( B ) The binding mode between Isoliensinine and B.1.1.529 variant of SARS-CoV-2_RDB-hACE2 complex. ( C ) The binding mode between Isoliensinine and B.1.617 variant of SARS-CoV-2_RDB-hACE2 complex. ( D ) The surface electrostatic potential map with the best docking pose. Dauricine and Isoliensinine are colored orange and gray, respectively. Electrostatic surface potentials are colored red and blue for negative and positive charges, respectively.

Article Snippet: 293T-ACE2 cell line was constructed by transfection of human ACE2 expressing plasmids (HG10108-UT; Sino Biological, Beijing, China) into 293T cells, followed by stable cell selection with hygromycin, and can be used for in vitro screening and characterization of drug candidates against SARS-CoV [ ] and SARS-CoV-2 [ ].

Techniques: Binding Assay, Variant Assay