human codon-optimized cdna encoding sars-cov-2 spike glycoprotein of the wa1/2020 and variants  (GenScript corporation)

Journal: Biomedicines

Article Title: Co-Expression of Niemann-Pick Type C1-Like1 (NPC1L1) with ACE2 Receptor Synergistically Enhances SARS-CoV-2 Entry and Fusion

doi: 10.3390/biomedicines12040821

Figure Lengend Snippet: Target CHO-K1 cells co-expressing human encoded ACE2 and NPC1L1 significantly enhances SARS-CoV-2 spike glycoprotein-mediated cell fusion. ( A ) Effector CHO-K1 cells expressing S glycoprotein were co-cultured with the target cells co-expressing ACE2 and NPC1L1 or pCDNA3.1 with the luciferase gene. Membrane fusion as a means of virus cell-to-cell spread was detected by monitoring relative luciferase units (RLUs). ( B ) Cell-to-cell fusion using effector CHO-K1 cells expressing S glycoprotein from three different mutants; wild type: Wuhan-Hu-1, D614G mutant, and N501Y mutant. RLU data in this experiment are expressed without TMPRSS2. Asterisks indicate a significant difference between the groups and ACE2 positive control (* p = 0.0246, ** p = 0.0044, *** p = 0.0006, and **** p < 0.0001).

Article Snippet: The mammalian expression plasmids encoding SARS-CoV-2 spike glycoprotein D614G (Cat # VG40589-UT), N501Y (Cat # VG40771-UT), and human ACE2 receptor (Cat # HG10108-UT) were purchased from Sino Biological (Wayne, PA, USA).

Techniques: Expressing, Cell Culture, Luciferase, Membrane, Virus, Mutagenesis, Positive Control

Journal: Biomedicines

Article Title: Co-Expression of Niemann-Pick Type C1-Like1 (NPC1L1) with ACE2 Receptor Synergistically Enhances SARS-CoV-2 Entry and Fusion

doi: 10.3390/biomedicines12040821

Figure Lengend Snippet: Impact of NPC1L1 expression on syncytia formation between effector and target cells using 3 different variant S glycoproteins; wild type: Wuhan-Hu-1 (panels a–c) ( A ), D614G mutant (panels d–f) ( B ), and N501Y mutant (panels g–i) ( C ). Images were captured under 10× and 40× magnification on EVOS FL auto imaging system after 24 h of co-culturing effector and target cells to highlight differences in size and abundance of syncytia in the presence and absence of NPC1L1.

Article Snippet: The mammalian expression plasmids encoding SARS-CoV-2 spike glycoprotein D614G (Cat # VG40589-UT), N501Y (Cat # VG40771-UT), and human ACE2 receptor (Cat # HG10108-UT) were purchased from Sino Biological (Wayne, PA, USA).

Techniques: Expressing, Variant Assay, Mutagenesis, Imaging

Journal: medRxiv

Article Title: Systems biological assessment of the temporal dynamics of immunity to a viral infection in the first weeks and months of life

doi: 10.1101/2023.01.28.23285133

Figure Lengend Snippet: A) Diagram depicting the experiment (n infants and young children: pre=12, acute=12, conv=21; n adults: acute=17). B-G) Spike-specific B cells were sorted using FACS, and the BCR sequence of each clone was determined. B) Frequency of SARS-CoV-2 spike-specific IgG+ memory B cells as a proportion of CD20+ B cells. Samples of the same donor a connected by a gray line. Blue line indicates average values; shaded areas indicate 5th to 95th percentiles. C,D) Frequency of SARS-CoV-2 specific IgG+ memory B cells in C) infants and young children at convalescent phase and D) at acute phase in adults and infants and young children. E) Clonality analysis of sorted SARS-CoV-2 spike-specific IgG+ memory B cells in infants and young children (n = 220). Each clone is represented as a circle. Circle size indicates the number of IGHV sequences in each clone; color represents the mean IGHV somatic hypermutation rate. F) Somatic hypermutation rates of the IGHV genes in single sorted SARS-CoV-2 spike specific IgG+ memory B cells at acute phase. G) Mean somatic hypermutation rate of all cloned IGHV genes in indicated infant samples. H-J) T cells were stimulated with overlapping peptides against WT and Omicron variants. Cytokine production was determined via flow cytometry. H) Box plot showing the fraction of multifunctional T cells (IFNg+, IL-2+, TNFa+) at different infection stages. I) Kinetics of multifunctional CD4+ T cell response. J) Comparison of multifunctional CD4+ T cell response during the acute phase of infection in infants and young children and adults. Statistical comparisons were conducted with Wilcoxon rank sum test. Solid line indicates median healthy response; dashed line indicates 3x median healthy response.

Article Snippet: Briefly, human codon-optimized cDNA encoding SARS-CoV-2 spike glycoprotein of the WA1/2020 and variants was synthesized by GenScript and cloned into eukaryotic cell expression vector pcDNA 3.1 between the BamH I and Xho I sites.

Techniques: Sequencing, Clone Assay, Flow Cytometry, Infection, Comparison

Journal: Nature Communications

Article Title: Breadth of SARS-CoV-2 neutralization and protection induced by a nanoparticle vaccine

doi: 10.1038/s41467-022-33985-4

Figure Lengend Snippet: a Schematic of the vaccination and challenge study. Cynomolgus macaques ( n = 5 per group) were immunized intramuscularly 3 times with 100 μg of RBD-scNP adjuvanted with 3M-052-Alum, Alum, 3M052-AF, or PBS control. Animals injected with adjuvant alone or PBS were set as control groups. Monkeys were then challenged with SARS-CoV-2 WA-1, subjected to blood, Bronchoalveolar lavage (BAL) and nasal swab collection, and necropsied for pathologic analysis. b Neutralization titers (ID 50 ) of plasma antibodies against pseudovirus of SARS-CoV-2 variants in 293T-ACE2-TMPRSS2. Each dot indicates one monkey ( n = 5 per group) and bars indicate geometric mean values of each group. Adjusted p -values: ns, not significant, * p < 0.05, two-sided Wilcoxon rank sum exact test. c Serum antibody neutralization against pseudoviruses of the SARS-CoV-2 Omicron (BA.1, BA.2, BA.2.12.1, BA.4/BA.5) variants, and SARS-CoV-2 PMS20 variant in 293T-ACE2 cells. The geometric mean titers and the fold reduction compared to D614G are shown. d Serum antibody neutralization against pseudoviruses of SARS-CoV in 293T-ACE2 cells. Each dot indicates one monkey ( n = 5 per group) and bars indicate geometric mean values of each group. Adjusted p-values: ns, not significant, * p < 0.05, two-sided Wilcoxon rank sum exact test. e SARS-CoV-2 N gene sgRNA in BAL and nasal swab samples collected on day 2 and 4 post-challenge. Each dot indicates one monkey ( n = 5 per group) and bars indicate geometric mean values of each group. Dashed line indicates limit of the detection. f , g Histopathological analysis. Lung sections from each animal were scored for lung inflammation by haematoxylin and eosin (H&E) staining ( f ), and for SARS-CoV-2 nucleocapsid antigen (Ag) expression by immunohistochemistry (IHC) staining ( g ). Each dot indicates one monkey ( n = 5 per group) and bars indicate geometric mean values of each group. Adjusted p -values: ns not significant, * p < 0.05, two-sided Wilcoxon rank sum exact test. Source data are provided as a Source Data file.

Article Snippet: Human codon-optimized cDNA encoding SARS-CoV-2 spike glycoproteins of various strains were synthesized by GenScript and cloned into eukaryotic cell expression vector pcDNA 3.1 between the BamHI and XhoI sites.

Techniques: Injection, Neutralization, Variant Assay, Staining, Expressing, Immunohistochemistry

Journal: Nature Communications

Article Title: Breadth of SARS-CoV-2 neutralization and protection induced by a nanoparticle vaccine

doi: 10.1038/s41467-022-33985-4

Figure Lengend Snippet: a Negative-stain electron microscopy 2D class averaging of RBD-scNP, NTD-scNP, and S2P-scNP. The 2D class averaging of 14,300 RBD-scNP particles, 10,800 NTD-scNP particles or 1034 S2P-scNP particles were generated using RELION. The size of each box: RBD-scNP and NTD-scNP, 257 Å; S2P-scNP, 1029 Å. b Schematic of the three-dose regimen. Cynomolgus macaques ( n = 5 per group) were immunized 3 times with RBD-scNP, NTD-scNP, or S2P-scNP adjuvanted with 3M-052-Alum. Monkeys were then challenged with SARS-CoV-2 WA-1, sampled for blood, BAL and nasal swabs, and necropsied for pathologic analysis. c Neutralization titers of plasma antibodies (week 0, 2, 6 and 10) against pseudotyped SARS-CoV-2 D614G strain in 293T/ACE2.MF cells. d Neutralization titers of plasma antibodies (week 10) against live SARS-CoV-2 WA-1 virus in Vero-E6 cells in microneutralization (MN) assay. Each dot indicates one monkey ( n = 5 per group) and bars indicate geometric mean values of each group. Adjusted p -values: ns not significant, * p < 0.05, two-sided Wilcoxon rank sum exact test. e Neutralization titers of plasma antibodies ( n = 5 per group) against pseudoviruses of the SARS-CoV-2 variants in 293T-ACE2-TMPRSS2 cells. Each dot indicates one monkey ( n = 5 per group) and bars indicate geometric mean values of each group. Adjusted p -values: ns, not significant, * p < 0.05, Two-sided Wilcoxon rank sum exact test. f , g Serum antibody neutralization against pseudoviruses of ( f ) the SARS-CoV-2 Omicron BA.1 and BA.2 variants, the PMS20 variant, and ( g ) SARS-CoV in 293T-ACE2 cells. Each dot indicates one monkey ( n = 5 per group) and bars indicate geometric mean values of each group. For SARS-CoV-2, the geometric mean titers and the fold reduction compared to D614G are shown. Adjusted p-values: ns, not significant, * p < 0.05, Two-sided Wilcoxon rank sum exact test. h SARS-CoV-2 N gene sgRNA in BAL and nasal swab samples collected on day 2 and 4 post-challenge. i , j Histopathological analysis. Scores of lung inflammation were determined by H&E staining ( i ) and SARS-CoV-2 nucleocapsid antigen expression were determined by IHC staining ( j ). Source data are provided as a Source Data file.

Article Snippet: Human codon-optimized cDNA encoding SARS-CoV-2 spike glycoproteins of various strains were synthesized by GenScript and cloned into eukaryotic cell expression vector pcDNA 3.1 between the BamHI and XhoI sites.

Techniques: Staining, Electron Microscopy, Generated, Neutralization, Variant Assay, Expressing, Immunohistochemistry

Journal: Nature Communications

Article Title: Breadth of SARS-CoV-2 neutralization and protection induced by a nanoparticle vaccine

doi: 10.1038/s41467-022-33985-4

Figure Lengend Snippet: a Schematic of the heterologous prime-boost regimen. Cynomolgus macaques ( n = 5 per group) were immunized 2 times with S2P mRNA-LNP, and boosted with adjuvanted RBD-scNP, NTD-scNP, or S2P-scNP vaccine. Monkeys were then challenged with SARS-CoV-2 WA-1, sampleded for blood, BAL and nasal swabs, and necropsied for pathologic analysis. b Neutralization titers of plasma antibodies against pseudoviruses of SARS-CoV-2 variants in 293T-ACE2-TMPRSS2 cells. Each dot indicates one monkey ( n = 5 per group) and bars indicate geometric mean values of each group. Adjusted p -values: ns not significant, * p < 0.05, Two-sided Wilcoxon rank sum exact test. Serum antibody neutralization titers against pseudoviruses of ( c ) the SARS-CoV-2 Omicron BA.1 and BA.2 variants, ( d ) the SARS-CoV-2 PMS20 variant, and ( e ) SARS-CoV in 293T-ACE2 cells. Each dot indicates one monkey ( n = 5 per group) and bars indicate geometric mean values of each group. For SARS-CoV-2, the geometric mean titers and the fold reduction compared to D614G are shown. Adjusted p -values: ns not significant, * p < 0.05, Two-sided Wilcoxon rank sum exact test. f SARS-CoV-2 N gene sgRNA in BAL and nasal swab samples collected on day 2 and 4 post-challenge. Histopathological analysis. Scores of lung inflammation were determined by H&E staining ( g ) and SARS-CoV-2 nucleocapsid antigen expression were determined by IHC staining ( h ). Source data are provided as a Source Data file.

Article Snippet: Human codon-optimized cDNA encoding SARS-CoV-2 spike glycoproteins of various strains were synthesized by GenScript and cloned into eukaryotic cell expression vector pcDNA 3.1 between the BamHI and XhoI sites.

Techniques: Neutralization, Variant Assay, Staining, Expressing, Immunohistochemistry

Journal: Nature Communications

Article Title: Breadth of SARS-CoV-2 neutralization and protection induced by a nanoparticle vaccine

doi: 10.1038/s41467-022-33985-4

Figure Lengend Snippet: a Schematic of the vaccination and challenge studies. Cynomolgus macaques were immunized twice with RBD-scNP adjuvanted with 3M-052-Alum, and challenged with SARS-CoV-2 WA-1 strain ( n = 5) or B.1.351 (Beta; n = 5) or B.1.617.2 (Delta; n = 5), collected for blood, BAL and nasal swab samples, and necropsied for pathologic analysis. b Neutralization titers of plasma antibodies against pseudoviruses of SARS-CoV-2 variants in 293T-ACE2-TMPRSS2 cells. Each dot indicates one monkey ( n = 10 per group) and bars indicate geometric mean values of each group. Adjusted p -values: ns not significant, * p < 0.05, Two-sided Wilcoxon rank sum exact test. Neutralization titers of serum antibodies against pseudoviruses of ( c ) the SARS-CoV-2 Omicron BA.1 and BA.2 variants, ( d ) the SARS-CoV-2 PMS20 variant, and ( e ) SARS-CoV in 293T-ACE2 cells. Each dot indicates one monkey ( n = 15 per group) and bars indicate geometric mean values of each group. Adjusted p-values: ns, not significant, * p < 0.05, Two-sided Wilcoxon rank sum exact test. For SARS-CoV-2 variants, the geometric mean ID 50 titers and the fold reduction compared to D614G are shown. SARS-CoV-2 sgRNA levels for nucleocapsid (N) gene in BAL and nasal swab samples collected on day 2 and 4 after ( f ) SARS-CoV-2 WA-1, ( g ) Beta variant or ( h ) Delta variant challenge. Dashed line indicates limit of the detection (LOD). Histopathological analysis of the SARS-CoV-2 ( i ) WA-1, ( j ) Beta variant or ( k ) Delta variant challenged monkeys. Scores of lung inflammation determined by H&E staining and SARS-CoV-2 nucleocapsid Ag expression determined by IHC staining. Source data are provided as a Source Data file.

Article Snippet: Human codon-optimized cDNA encoding SARS-CoV-2 spike glycoproteins of various strains were synthesized by GenScript and cloned into eukaryotic cell expression vector pcDNA 3.1 between the BamHI and XhoI sites.

Techniques: Neutralization, Variant Assay, Staining, Expressing, Immunohistochemistry

Journal: Nature Communications

Article Title: Breadth of SARS-CoV-2 neutralization and protection induced by a nanoparticle vaccine

doi: 10.1038/s41467-022-33985-4

Figure Lengend Snippet: a Schematic of the mouse challenge studies. 11-month-old female BALB/c mice ( n = 10 per group) were immunized intramuscularly twice with adjuvanted RBD-scNP and challenged with SARS-CoV-2 mouse-adapted 10 (MA10) WA-1, SARS-CoV-2 MA10 Beta variant, SARS-CoV-1 mouse-adapted 15 (MA15), or Bat coronavirus (CoV) RsSHC014 MA15. GLA-SE was used as adjuvant in the SARS-CoV challenge study, and 3M-052-Alum was used in the other challenge studies. b Weight loss ( n = 10 per group) and lung virus titers ( n = 10 per group) at 4 days post-infection (dpi) of the SARS-CoV-2 MA10 WA-1 challenged mice. c Weight loss ( n = 5 per group) and lung virus titers ( n = 4 per group) at 2 dpi of the SARS-CoV-2 MA10 Beta variant challenged mice. d Weight loss ( n = 10 per group) and lung virus titers ( n = 5 per group) at 2 dpi of the SARS-CoV-1 MA15 challenged mice. e Weight loss ( n = 10 per group) and lung virus titers ( n = 10 per group) at 4 dpi of the Bat CoV RsSHC014 MA15 challenged mice. For weight curves, data are presented as mean values ±SEM. For lung virus titers, each dot indicates one mouse and bars indicate geometric mean values of each group. P -values: ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, Two-sided Wilcoxon rank sum exact test. Source data are provided as a Source Data file.

Article Snippet: Human codon-optimized cDNA encoding SARS-CoV-2 spike glycoproteins of various strains were synthesized by GenScript and cloned into eukaryotic cell expression vector pcDNA 3.1 between the BamHI and XhoI sites.

Techniques: Variant Assay, Infection