rabbit polyclonal anti sars cov 2 orf8  (Novus Biologicals)

Journal: bioRxiv

Article Title: One and Done: A safe, adaptable single-cycle SARS-CoV-2 vaccine platform blocks XBB.1.5 infection and transmission

doi: 10.64898/2026.03.09.709481

Figure Lengend Snippet: (a) Schematic illustration of the SARS-CoV-2 genomic landscape and the deletions/substitutions in ΔE673* XBB (scVac XBB ); position of main structural and accessory proteins as indicated. Positions of the overlapping PCR fragments (A-D2) covering the whole SARS-CoV-2 genome are indicated. (b) Cell-free virus after passage of either wild-type SARS-CoV-2 expressing an Omicron XBB.1.5 Spike (rCoV2 XBB ) or scVac XBB after 0-3 virus passages is shown for three non-complementing cell lines as indicated. The level of input virus is indicated by a dotted line. (c) Staining of N, S, and ORF7a (magenta), F-actin (green), nuclei (blue), and ORF6, ORF3a, or ORF8 in Vero E6-TMPRSS2 cells infected with rCoV2 XBB and scVac XBB . (d) Immunoblot analysis of viral proteins 24h after infection of Vero E6-TMPRSS2 cells with the indicated viruses or medium only (control), probed with anti-NSP2, anti-N, anti-S, anti-ORF3a (full-length [fl] and truncated [tr] forms indicated with arrows), anti-ORF6, anti-ORF7a, and anti-beta-actin (β-ACT) antibodies. Scale bar is 50 µm and 20 µm in (c) (overview and ROI images, respectively).

Article Snippet: The following antibodies were used for immunocytochemistry and immunoblotting: mouse monoclonal anti-β-actin (Cell Signaling Technology; 3700; RRID: AB_2242334; LOT# 20), rabbit polyclonal anti-SARS-CoV-2 nsp2 (GeneTex; GTX135717; RRID: AB_2909866; LOT# B318853), mouse monoclonal anti-SARS-CoV-2 Nucleocapsid protein (4F3C4, gift from S. Reiche [ ], sheep polyclonal anti-SARS-CoV-2 ORF3a [ ], rat monoclonal anti-SARS-CoV-2 ORF6 (8B10, gift from Y. Miyamoto [ ]), mouse monoclonal anti-SARS-CoV-2 ORF7a (3C9; GeneTex; GTX632602; RRID: AB_2888320; LOT# 42219), rabbit polyclonal anti-SARS-CoV-2 ORF8 (Novus Biologicals; NBP3-07972; LOT# 25966-2102), mouse monoclonal anti-SARS-CoV-2 Spike protein (4B5C1, gift from S. Reiche).

Techniques: Virus, Expressing, Staining, Infection, Western Blot, Control

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Overview of the COVID-19 pregnancy cohort (total n = 29). Pregnant women included in the study had negative (Control, n = 6), or positive SARS-CoV-2 diagnoses (COVID-19 n = 23) during the first ( n = 2), second ( n = 5), or third ( n = 16) trimesters of pregnancy. Participants had biospecimens collected at the time of the delivery. Biospecimens collected included (i) biofluid specimens: maternal plasma, newborn blood, cord plasma, and amniotic fluid; and (ii) placental tissues: umbilical cord, chorion, and amnion. The biospecimens underwent ultrasensitive SARS-CoV-2 detection using digital droplet PCR (ddPCR), SARS-CoV-2 ORF8 ELISA, proteomics profile for 97 inflammatory biomarkers, and global transcriptomics profile (~22,000 host genes) by RNAseq. ( B ) Box and whiskers plot representing average and minimum and maximum values of copies/mL of SARS-CoV-2 N1 and N2 proteins detected maternal plasma (controls, n = 5 for N1; n = 6 for N2; COVID-19, n = 23 for N1/N2), chorion (controls, n = 5 for N1, n = 6 for N2; COVID-19, n = 21), amnion (controls, n = 2 for N1; n = 4 for N2; COVID-19, n = 21), amniotic fluid (controls, n = 3; COVID-19, n = 9), cord plasma (controls, n = 5 for N1, n = 3 for N2; COVID-19, n = 20), newborn plasma (controls, n = 0; COVID-19, n = 12) from controls and COVID-19-affected pregnancies. ( C ) Bar charts representing the percentage of samples positive (purple bar) and negative (blue bar) from SARS-CoV-2 in the biospecimens analyzed. Pie chart representing the total SARS-CoV-2 positivity considering at least one fetal-skewed specimen. Cut-off value defined as counts/mL 3 ≥ 2. ( D ) Stacked bar charts (mean with SD) representing the levels of viral RNA of samples positive for SARS-CoV-2 in the fetal compartment, according to the trimester of pregnancy that maternal SARS-CoV-2 occurred (First trimester, n = 2; Second trimester, n = 5 and Third trimester, n = 16). .

Article Snippet: Ten-micron sections were dewaxed, rehydrated and stained with hematoxylin and eosin (H&E) or via immunohistochemical staining with rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody (Cat. No. GTX135591, Genetex) and recombinant anti-C3 antibody (clone: EPR19394; Abcam) and counterstained with hematoxylin.

Techniques: Negative Control, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Graphical representation of samples analyzed for ORF8 quantitative and qualitative ELISA: maternal plasma (Control, n = 4; COVID-19, n = 23), umbilical cord plasma (Control, n = 4; COVID-19, n = 20), and amniotic fluid (Control, n = 3; COVID-19, n = 8). ORF8 levels in samples from control and COVID-19-affected pregnancies. Data are presented in violin plots showing the median (middle line) and describe numerical data distributions using density curves. Values are represented in ng/mL with cut off 1.7 ng/mL in maternal plasma, cut off of 13.4 ng/mL in umbilical cord plasma and cut off of 36.1 ng/mL in amniotic fluid. ( B ) Pie charts illustrating qualitative analysis of ORF8 ELISA. ( C ) Stacked bars representing the circulating ORF8 according to trimester of SARS-CoV-2 infection (First trimester, n = 2; Second trimester, n = 4; Third trimester, n = 10). ( D ) Boxplots representing levels of anti-Spike S1 IgM with cut off of 2720 ng/mL and anti-Nucleocapsid IgG with cut off of 6420 ng/mL in maternal plasma ( n = 22) and newborn cord plasma ( n = 15) from COVID-19-affected pregnancies. Data are presented in boxplot, middle line represents means, the bound of box represent interquartile range, and whiskers represent maximum and minimum values. ( E ) Comparative analysis of DEGs (−2 < FC > 2, p < 0.05) in COVID-19 amnion ORF8 negatives and positives. ( F ) Bar plot representing gene ontology of upregulated biological pathways in COVID-19 amnion ORF8 positive group. ( G , H ) Violin plots representing the fold change values for individual genes related with ( G ) complement (ORF8 (−), n = 5–6; ORF8 (+), n = 9–10) and ( H ) inflammation in amnion (ORF8 (−), n = 5–7; ORF8 (+), n = 8–11) transcriptomics. ( I ) Comparative analysis of differentially expressed proteins (DEPs) ( p < 0.05) in COVID-19 amniotic fluid ORF8 negatives ( n = 4) and positives ( n = 8). Bubble plot of DEPs upregulated exclusively in COVID-19 amniotic fluid ORF8 positive group. ( J ) Comparative analysis of DEGs in COVID-19 umbilical cord ORF8 negatives and positives. ( K ) Bar plot of gene ontology biological pathways associated with DEGs upregulated exclusively in COVID-19 umbilical cord ORF8 positive. ( L ) Violin plots representing the fold change values for individual genes related to complement in umbilical cord transcriptomics. ( M ) Amnion serial sections were analyzed by immunohistochemistry for detection of SARS-CoV-2 ORF8 and C3b. From left to right, tissues stained with (i) Hematoxylin and Eosin (H&E) (hematoxylin—purple, eosin—pink); (ii) Hematoxylin (purple) and the secondary antibody anti-rabbit (Motulsky and Brown, ); (iii) Hematoxylin (purple) and anti-ORF8 produced (Motulsky and Brown, ); and (iv) Hematoxylin (purple) and anti-C3b (Motulsky and Brown, ). Images were taken at 4X magnification. Representative images from three control and three COVID-19 amnions. Fold change calculated by individual expression divided by average expression of control. Violin plots data are presented as means ± SEMs pg/mL, using Mann–Whitney U test ( p < 0.05). Comparative analysis relative to controls. .

Article Snippet: Ten-micron sections were dewaxed, rehydrated and stained with hematoxylin and eosin (H&E) or via immunohistochemical staining with rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody (Cat. No. GTX135591, Genetex) and recombinant anti-C3 antibody (clone: EPR19394; Abcam) and counterstained with hematoxylin.

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Control, Infection, Immunohistochemistry, Staining, Produced, Expressing, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Chorion serial sections were analyzed by immunohistochemistry for detection of SARS-CoV-2 ORF8 and C3b. From left to right, tissues stained with (i) Hematoxylin (purple) and the secondary antibody anti-rabbit (Motulsky and Brown, ); (ii) Hematoxylin (purple) and anti-ORF8 produced (Motulsky and Brown, ); and (iii) Hematoxylin (purple) and anti-C3b (Motulsky and Brown, ). Images were taken at 20X magnification. Representative images from two control and two COVID-19 chorion specimens.

Article Snippet: Ten-micron sections were dewaxed, rehydrated and stained with hematoxylin and eosin (H&E) or via immunohistochemical staining with rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody (Cat. No. GTX135591, Genetex) and recombinant anti-C3 antibody (clone: EPR19394; Abcam) and counterstained with hematoxylin.

Techniques: Immunohistochemistry, Staining, Produced, Control

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Chorion and ( B ) amnion serial sections were stained with SARS-CoV-2 ORF8 (red) and C3b (green); or with Krt8/18 (red) and ORF8 (green). Images were taken at 40× magnification. Images are representative of one control and one COVID-19+. Scale bars: 40 μm. .

Article Snippet: Ten-micron sections were dewaxed, rehydrated and stained with hematoxylin and eosin (H&E) or via immunohistochemical staining with rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody (Cat. No. GTX135591, Genetex) and recombinant anti-C3 antibody (clone: EPR19394; Abcam) and counterstained with hematoxylin.

Techniques: Staining, Control

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Chorion and ( B ) amnion serial sections were stained with SARS-CoV-2 ORF8 (red) and C3b (green). Mander’s and Pearson’s correlations coefficient between SARS-CoV-2 ORF8 and C3b. White circles represent the colocalization areas analyzed. Images were taken at 40x magnification. Images are representative of one COVID-19+ pregnancy. Scale bars: 40 μm.

Article Snippet: Ten-micron sections were dewaxed, rehydrated and stained with hematoxylin and eosin (H&E) or via immunohistochemical staining with rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody (Cat. No. GTX135591, Genetex) and recombinant anti-C3 antibody (clone: EPR19394; Abcam) and counterstained with hematoxylin.

Techniques: Staining

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Graphical representation detailing the placental cell culture models for in vitro SARS-CoV-2 ORF8 treatment. ( B ) Boxplots representing the fold change values of gene expression for individual genes, obtained from standardization of in vitro SARS-CoV-2 ORF8 treatment with 10 ng/mL, 20 ng/mL, or 100 ng/mL for 8 h from eight independent experiments ( n = 8) and 16 h from eleven independent experiments ( n = 11) on immortalized trophoblasts (HTR8/SVneo cells). Fold change calculated by DDCt method, relative to mock. Data are presented in boxplot where the middle line represent means, the bound of box represent interquartile range, and whiskers represent maximum and minimum values in ng/mL, using one-way ANOVA Kruskal–Wallis with uncorrected Dunn’s test. ( C – E ) Relative expression of genes associated with complement activation in the placental cell culture models treated with 20 ng/mL of SARS-CoV-2 ORF8 for 8 h. The models included ( C ) HTR8/SVneo cells (mock, n = 6–8; and ORF8, n = 7–8) ( D ) primary human villous trophoblasts (HVT) (mock, n = 4–5; and ORF8, n = 4–5), and ( E ) iPSC-derived trophoblasts (mock, n = 5–6; and ORF8, n = 6). Relative expression was calculated by DCt method using GAPDH as the normalizing gene. Mann–Whitney U test was used for analysis. ( F ) Boxplots representing the percentage of C3b + HTR8/SVneo cells treated with SARS-CoV-2 ORF8 for 30 min (mock, n = 5; and ORF8, n = 6), 8 h (mock, n = 4; and ORF8, n = 4) or 16 h (mock, n = 4; and ORF8, n = 4). Data are presented in boxplot, middle line represents means, the bound of box represent interquartile range, and whiskers represent maximum and minimum values. ( G ) Violin plots representing the levels of complement proteins in the supernatant of HTR8/SVneo cells treated with SARS-CoV-2 ORF8 for 8 h (mock, n = 5–6; and ORF8, n = 6) or 16 h (mock, n = 4–5; and ORF8, n = 6). Mann–Whitney U test was used for analysis ( p < 0.05). Immortalized pluripotent stem cells (iPSC). .

Article Snippet: Ten-micron sections were dewaxed, rehydrated and stained with hematoxylin and eosin (H&E) or via immunohistochemical staining with rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody (Cat. No. GTX135591, Genetex) and recombinant anti-C3 antibody (clone: EPR19394; Abcam) and counterstained with hematoxylin.

Techniques: Cell Culture, In Vitro, Gene Expression, Expressing, Activation Assay, Derivative Assay, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Line chart representing absorbance values obtained in the SARS-CoV-2 ORF8 - C1q in vitro binding assay using different molarities of C1q (5, 10, 20, and 50 nM) and SARS-CoV-2 ORF8 (0, 20, 40, 60, 80, and 100 nM). Absorbance calculated by discounting the blank (0 nM C1q). ( B ) Co-immunoprecipitation of SARS-CoV-2 ORF8 with C1qA, C1qB and C1qC. Graphical representation of the plasmid constructs containing the expression cassettes for the subcomponents C1qA, C1qB, and C1qC contained an N-terminal signal peptide (SP), and a C-terminal FLAG tag. Western blot images for co-immunoprecipitation using an anti-ORF8 antibody. ( C ) Co-immunoprecipitation of C1qA full-length (FL), globular (G), disordered domain (D) with SARS-CoV-2 ORF8. Graphical representation of the plasmid constructs containing expression cassettes for the C1qA-FL, C1qA-G, and C1qA-D, with N-terminal SP and a C-terminal 3FLAG tag, cloned in pIRES vectors. Western blot images for co-immunoprecipitation using an anti-ORF8 antibody. Co-immunoprecipitated product (IP). Whole-cell lysate (WCL). .

Article Snippet: Ten-micron sections were dewaxed, rehydrated and stained with hematoxylin and eosin (H&E) or via immunohistochemical staining with rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody (Cat. No. GTX135591, Genetex) and recombinant anti-C3 antibody (clone: EPR19394; Abcam) and counterstained with hematoxylin.

Techniques: In Vitro, Binding Assay, Immunoprecipitation, Plasmid Preparation, Construct, Expressing, FLAG-tag, Western Blot, Clone Assay

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Co-immunoprecipitation of SARS-CoV-2 ORF8 with C1qA, C1qB, and C1qC. Graphical representation of the plasmid constructs containing the expression cassettes for the subcomponents C1qA, C1qB, and C1qC contained an N-terminal signal peptide (SP), and a C-terminal FLAG tag. Western blot images for co-immunoprecipitation using an anti-ORF8 antibody. ( B ) Co-immunoprecipitation of C1qA full-length (FL), globular (G), disordered domain (D) with SARS-CoV-2 ORF8. Graphical representation of the plasmid constructs containing expression cassettes for the C1qA-FL, C1qA-G, and C1qA-D, with N-terminal SP and a C-terminal 3FLAG tag, cloned in pIRES vectors. Western blot images for co-immunoprecipitation using an anti-Flag antibody. Co-immunoprecipitated product (IP). Whole-cell lysate (WCL).

Article Snippet: Ten-micron sections were dewaxed, rehydrated and stained with hematoxylin and eosin (H&E) or via immunohistochemical staining with rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody (Cat. No. GTX135591, Genetex) and recombinant anti-C3 antibody (clone: EPR19394; Abcam) and counterstained with hematoxylin.

Techniques: Immunoprecipitation, Plasmid Preparation, Construct, Expressing, FLAG-tag, Western Blot, Clone Assay

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Line chart of absorbance values obtained in the SARS-CoV-2 ORF8 peptides - C1q binding assay using different molarities of C1q (10, 20, and 50 nM) and 1 mM of 28 different peptides spanning SARS-CoV-2 ORF8 region. Absorbance calculated by discounting the blank (0 nM C1q). ( B ) The ORF8 peptides presenting dose-dependent binding with C1q (#4 red, #10 purple, #19 blue, and #25 yellow) in molecular binding model between SARS-CoV-2 ORF8 dimers and C1qA-Globular (model 1). ( C ) Bar charts representing docking and confidence scores for in silico docking models SARS-CoV-2 ORF8 and C1qA-G. The ORF8 peptides #4, #10, #19, and #25 represented on amino acid (aa) sequence alignment of docking models. ( D ) Representative docking poses of the globular domain of C1q (PDB ID: 1PK6) with SARS-CoV-2 ORF8 protein (PDB ID:7JTL) in 2D interaction diagrams. The ORF8 and C1q residues with significant interactions from each protein are highlighted, labeled, and properly annotated. The hydrogen bonds, pi-pi, and salt bridges formed between ORF8 and C1q are also shown by colored dash or arrow lines. The polar (turquoise), hydrophobic (green), positively charged (purple), and negatively charged (orange) residues are represented in colored spheres. Shown below each of the interaction diagrams is a table of interacting or contact residues between the SARS-CoV-2 ORF8 protein and the globular C1q domain, including their aa positions, aa distance measured in Angstrom (Å), and the corresponding specific binding interactions. ( E ) Boxplots representing the relative expression of genes analyzed in HTR8/SVneo trophoblast treated with ORF8 peptides #4, #10, #19, and #25 using 0.1 nM and 1 nM ( n = 5–6) or mock ( n = 7–10). ( F ) Boxplots representing the relative expression of genes analyzed in primary human villous trophoblast (HVT) treated with 0.1 nM and 1 nM of ORF8 peptide #10 or Mock. Relative expression was calculated by DCt method using GAPDH as the normalizing gene. ( G ) Boxplots representing the percentage of live C3b+ cells in mock ( n = 4) and ORF8 peptides #4, #10, #19, and #25 0.1 nM and 1 nM treated ( n = 4–6) HTR8/SVneo trophoblasts. Data are presented in boxplot, middle line represents means, the bound of box represents interquartile range, and whiskers represent maximum and minimum values. ( H ) Graphical representation of main results. Comparisons using one-way ANOVA Kruskal–Wallis with Fisher test ( p < 0.05). .

Article Snippet: Ten-micron sections were dewaxed, rehydrated and stained with hematoxylin and eosin (H&E) or via immunohistochemical staining with rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody (Cat. No. GTX135591, Genetex) and recombinant anti-C3 antibody (clone: EPR19394; Abcam) and counterstained with hematoxylin.

Techniques: Binding Assay, In Silico, Sequencing, Labeling, Expressing

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: Correlation analyses between the number of days post SARS-CoV-2 infection to delivery and ( A ) SARS-CoV-2 N1 RNA copies, SARS-CoV-2 N2 RNA copies, and ( B ) circulating ORF8 levels in all biospecimens analyzed, including maternal plasma, chorion, amnion, cord plasma and newborn plasma. Simples linear regression and Spearman’s rank correlation test was used for all the correlations analysis. Scatter plots with Pearson correlation coefficients (r) and dotted lines represent 95% confidence intervals.

Article Snippet: Ten-micron sections were dewaxed, rehydrated and stained with hematoxylin and eosin (H&E) or via immunohistochemical staining with rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody (Cat. No. GTX135591, Genetex) and recombinant anti-C3 antibody (clone: EPR19394; Abcam) and counterstained with hematoxylin.

Techniques: Infection, Clinical Proteomics

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Sequence alignment of SARS-CoV-2 ORF8 and human immunoglobulin variable lambda chain (IGLV) genes. Representative amino acid residues of SARS-CoV-2 ORF8-specific region located at the loop flanking β 4- β 5 strands were compared with a portion of the framework 3 (FR3) region of the variable light chain of human IGLV5-39*01, IGLV5-39*02, and IGLV1-47*01 germline gene sequences. Identical residues were highlighted in red box and marked by an asterisk (*), while semi-conserved positions due to minor structural differences are in blue boxes and are indicated by a period (.). The β strands are shown by green arrows above the ORF8 sequence while the spanning loops were represented by black lines (Flower et al, ). The FR3 region (shown in the yellow box) was defined according to the IMGT delimitations. ( B ) Comparison of the overlapping sequences of SARS-CoV-2 ORF8 and human IGLV genes highlighted in the dashed boxes using peptide properties: buried index, hydrophobicity, polarity, and turn and helix propensity. The legend for each property is shown as a gradient color scheme on the right. ( C – E ) Structures of ORF8 ( C ), IGLV5-39*01 ( D ), and superimposed images after pairwise structural alignment as viewed from the top (left) and side (right). PBD entries: 7JTL (SARS-CoV-2, blue) and 2CD0 (human IGLV5-39, green). Overlapping sequences between the two structures are highlighted in red. ( F ) Pairwise structural alignment of the human IgG-Fc region (cyan, PDB ID:1FC1) and ORF8 (in blue) showing close structural similarity at the CH2 domain (193 atoms aligned; RMSD: 5.9 Å). The CH2 and CH3 domains are highlighted, while residues in red sphere show the neonatal Fc receptor (FcRn) interaction site. ( G ) Superimposition of CH2 domain of the human IgG-Fc and ORF8 from different rotational perspectives. CH2 residues with the closest folding to ORF8 (blue) is shown in flesh while CH3 are in cyan. The glycosylation site at N297 at the CH2 interface is shown in cyan stick models.

Article Snippet: Ten-micron sections were dewaxed, rehydrated and stained with hematoxylin and eosin (H&E) or via immunohistochemical staining with rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody (Cat. No. GTX135591, Genetex) and recombinant anti-C3 antibody (clone: EPR19394; Abcam) and counterstained with hematoxylin.

Techniques: Sequencing, Comparison, Glycoproteomics

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Ten-micron sections were dewaxed, rehydrated and stained with hematoxylin and eosin (H&E) or via immunohistochemical staining with rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody (Cat. No. GTX135591, Genetex) and recombinant anti-C3 antibody (clone: EPR19394; Abcam) and counterstained with hematoxylin.

Techniques: Recombinant, Variant Assay, Generated, Plasmid Preparation, FLAG-tag, Sequencing, Digital PCR, cDNA Synthesis, SYBR Green Assay, Blocking Assay, Software, Cell Culture, Enzyme-linked Immunosorbent Assay, Transfection, Real-time Polymerase Chain Reaction, Spectrophotometry, Imaging

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Overview of the COVID-19 pregnancy cohort (total n = 29). Pregnant women included in the study had negative (Control, n = 6), or positive SARS-CoV-2 diagnoses (COVID-19 n = 23) during the first ( n = 2), second ( n = 5), or third ( n = 16) trimesters of pregnancy. Participants had biospecimens collected at the time of the delivery. Biospecimens collected included (i) biofluid specimens: maternal plasma, newborn blood, cord plasma, and amniotic fluid; and (ii) placental tissues: umbilical cord, chorion, and amnion. The biospecimens underwent ultrasensitive SARS-CoV-2 detection using digital droplet PCR (ddPCR), SARS-CoV-2 ORF8 ELISA, proteomics profile for 97 inflammatory biomarkers, and global transcriptomics profile (~22,000 host genes) by RNAseq. ( B ) Box and whiskers plot representing average and minimum and maximum values of copies/mL of SARS-CoV-2 N1 and N2 proteins detected maternal plasma (controls, n = 5 for N1; n = 6 for N2; COVID-19, n = 23 for N1/N2), chorion (controls, n = 5 for N1, n = 6 for N2; COVID-19, n = 21), amnion (controls, n = 2 for N1; n = 4 for N2; COVID-19, n = 21), amniotic fluid (controls, n = 3; COVID-19, n = 9), cord plasma (controls, n = 5 for N1, n = 3 for N2; COVID-19, n = 20), newborn plasma (controls, n = 0; COVID-19, n = 12) from controls and COVID-19-affected pregnancies. ( C ) Bar charts representing the percentage of samples positive (purple bar) and negative (blue bar) from SARS-CoV-2 in the biospecimens analyzed. Pie chart representing the total SARS-CoV-2 positivity considering at least one fetal-skewed specimen. Cut-off value defined as counts/mL 3 ≥ 2. ( D ) Stacked bar charts (mean with SD) representing the levels of viral RNA of samples positive for SARS-CoV-2 in the fetal compartment, according to the trimester of pregnancy that maternal SARS-CoV-2 occurred (First trimester, n = 2; Second trimester, n = 5 and Third trimester, n = 16). .

Article Snippet: Rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody , Genetex , Cat. No. GTX135591 Dilution WB: 1:5000 Dilution IHC/IF: 1:2000.

Techniques: Negative Control, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Graphical representation of samples analyzed for ORF8 quantitative and qualitative ELISA: maternal plasma (Control, n = 4; COVID-19, n = 23), umbilical cord plasma (Control, n = 4; COVID-19, n = 20), and amniotic fluid (Control, n = 3; COVID-19, n = 8). ORF8 levels in samples from control and COVID-19-affected pregnancies. Data are presented in violin plots showing the median (middle line) and describe numerical data distributions using density curves. Values are represented in ng/mL with cut off 1.7 ng/mL in maternal plasma, cut off of 13.4 ng/mL in umbilical cord plasma and cut off of 36.1 ng/mL in amniotic fluid. ( B ) Pie charts illustrating qualitative analysis of ORF8 ELISA. ( C ) Stacked bars representing the circulating ORF8 according to trimester of SARS-CoV-2 infection (First trimester, n = 2; Second trimester, n = 4; Third trimester, n = 10). ( D ) Boxplots representing levels of anti-Spike S1 IgM with cut off of 2720 ng/mL and anti-Nucleocapsid IgG with cut off of 6420 ng/mL in maternal plasma ( n = 22) and newborn cord plasma ( n = 15) from COVID-19-affected pregnancies. Data are presented in boxplot, middle line represents means, the bound of box represent interquartile range, and whiskers represent maximum and minimum values. ( E ) Comparative analysis of DEGs (−2 < FC > 2, p < 0.05) in COVID-19 amnion ORF8 negatives and positives. ( F ) Bar plot representing gene ontology of upregulated biological pathways in COVID-19 amnion ORF8 positive group. ( G , H ) Violin plots representing the fold change values for individual genes related with ( G ) complement (ORF8 (−), n = 5–6; ORF8 (+), n = 9–10) and ( H ) inflammation in amnion (ORF8 (−), n = 5–7; ORF8 (+), n = 8–11) transcriptomics. ( I ) Comparative analysis of differentially expressed proteins (DEPs) ( p < 0.05) in COVID-19 amniotic fluid ORF8 negatives ( n = 4) and positives ( n = 8). Bubble plot of DEPs upregulated exclusively in COVID-19 amniotic fluid ORF8 positive group. ( J ) Comparative analysis of DEGs in COVID-19 umbilical cord ORF8 negatives and positives. ( K ) Bar plot of gene ontology biological pathways associated with DEGs upregulated exclusively in COVID-19 umbilical cord ORF8 positive. ( L ) Violin plots representing the fold change values for individual genes related to complement in umbilical cord transcriptomics. ( M ) Amnion serial sections were analyzed by immunohistochemistry for detection of SARS-CoV-2 ORF8 and C3b. From left to right, tissues stained with (i) Hematoxylin and Eosin (H&E) (hematoxylin—purple, eosin—pink); (ii) Hematoxylin (purple) and the secondary antibody anti-rabbit (Motulsky and Brown, ); (iii) Hematoxylin (purple) and anti-ORF8 produced (Motulsky and Brown, ); and (iv) Hematoxylin (purple) and anti-C3b (Motulsky and Brown, ). Images were taken at 4X magnification. Representative images from three control and three COVID-19 amnions. Fold change calculated by individual expression divided by average expression of control. Violin plots data are presented as means ± SEMs pg/mL, using Mann–Whitney U test ( p < 0.05). Comparative analysis relative to controls. .

Article Snippet: Rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody , Genetex , Cat. No. GTX135591 Dilution WB: 1:5000 Dilution IHC/IF: 1:2000.

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Control, Infection, Immunohistochemistry, Staining, Produced, Expressing, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Chorion serial sections were analyzed by immunohistochemistry for detection of SARS-CoV-2 ORF8 and C3b. From left to right, tissues stained with (i) Hematoxylin (purple) and the secondary antibody anti-rabbit (Motulsky and Brown, ); (ii) Hematoxylin (purple) and anti-ORF8 produced (Motulsky and Brown, ); and (iii) Hematoxylin (purple) and anti-C3b (Motulsky and Brown, ). Images were taken at 20X magnification. Representative images from two control and two COVID-19 chorion specimens.

Article Snippet: Rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody , Genetex , Cat. No. GTX135591 Dilution WB: 1:5000 Dilution IHC/IF: 1:2000.

Techniques: Immunohistochemistry, Staining, Produced, Control

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Chorion and ( B ) amnion serial sections were stained with SARS-CoV-2 ORF8 (red) and C3b (green); or with Krt8/18 (red) and ORF8 (green). Images were taken at 40× magnification. Images are representative of one control and one COVID-19+. Scale bars: 40 μm. .

Article Snippet: Rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody , Genetex , Cat. No. GTX135591 Dilution WB: 1:5000 Dilution IHC/IF: 1:2000.

Techniques: Staining, Control

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Chorion and ( B ) amnion serial sections were stained with SARS-CoV-2 ORF8 (red) and C3b (green). Mander’s and Pearson’s correlations coefficient between SARS-CoV-2 ORF8 and C3b. White circles represent the colocalization areas analyzed. Images were taken at 40x magnification. Images are representative of one COVID-19+ pregnancy. Scale bars: 40 μm.

Article Snippet: Rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody , Genetex , Cat. No. GTX135591 Dilution WB: 1:5000 Dilution IHC/IF: 1:2000.

Techniques: Staining

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Graphical representation detailing the placental cell culture models for in vitro SARS-CoV-2 ORF8 treatment. ( B ) Boxplots representing the fold change values of gene expression for individual genes, obtained from standardization of in vitro SARS-CoV-2 ORF8 treatment with 10 ng/mL, 20 ng/mL, or 100 ng/mL for 8 h from eight independent experiments ( n = 8) and 16 h from eleven independent experiments ( n = 11) on immortalized trophoblasts (HTR8/SVneo cells). Fold change calculated by DDCt method, relative to mock. Data are presented in boxplot where the middle line represent means, the bound of box represent interquartile range, and whiskers represent maximum and minimum values in ng/mL, using one-way ANOVA Kruskal–Wallis with uncorrected Dunn’s test. ( C – E ) Relative expression of genes associated with complement activation in the placental cell culture models treated with 20 ng/mL of SARS-CoV-2 ORF8 for 8 h. The models included ( C ) HTR8/SVneo cells (mock, n = 6–8; and ORF8, n = 7–8) ( D ) primary human villous trophoblasts (HVT) (mock, n = 4–5; and ORF8, n = 4–5), and ( E ) iPSC-derived trophoblasts (mock, n = 5–6; and ORF8, n = 6). Relative expression was calculated by DCt method using GAPDH as the normalizing gene. Mann–Whitney U test was used for analysis. ( F ) Boxplots representing the percentage of C3b + HTR8/SVneo cells treated with SARS-CoV-2 ORF8 for 30 min (mock, n = 5; and ORF8, n = 6), 8 h (mock, n = 4; and ORF8, n = 4) or 16 h (mock, n = 4; and ORF8, n = 4). Data are presented in boxplot, middle line represents means, the bound of box represent interquartile range, and whiskers represent maximum and minimum values. ( G ) Violin plots representing the levels of complement proteins in the supernatant of HTR8/SVneo cells treated with SARS-CoV-2 ORF8 for 8 h (mock, n = 5–6; and ORF8, n = 6) or 16 h (mock, n = 4–5; and ORF8, n = 6). Mann–Whitney U test was used for analysis ( p < 0.05). Immortalized pluripotent stem cells (iPSC). .

Article Snippet: Rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody , Genetex , Cat. No. GTX135591 Dilution WB: 1:5000 Dilution IHC/IF: 1:2000.

Techniques: Cell Culture, In Vitro, Gene Expression, Expressing, Activation Assay, Derivative Assay, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Line chart representing absorbance values obtained in the SARS-CoV-2 ORF8 - C1q in vitro binding assay using different molarities of C1q (5, 10, 20, and 50 nM) and SARS-CoV-2 ORF8 (0, 20, 40, 60, 80, and 100 nM). Absorbance calculated by discounting the blank (0 nM C1q). ( B ) Co-immunoprecipitation of SARS-CoV-2 ORF8 with C1qA, C1qB and C1qC. Graphical representation of the plasmid constructs containing the expression cassettes for the subcomponents C1qA, C1qB, and C1qC contained an N-terminal signal peptide (SP), and a C-terminal FLAG tag. Western blot images for co-immunoprecipitation using an anti-ORF8 antibody. ( C ) Co-immunoprecipitation of C1qA full-length (FL), globular (G), disordered domain (D) with SARS-CoV-2 ORF8. Graphical representation of the plasmid constructs containing expression cassettes for the C1qA-FL, C1qA-G, and C1qA-D, with N-terminal SP and a C-terminal 3FLAG tag, cloned in pIRES vectors. Western blot images for co-immunoprecipitation using an anti-ORF8 antibody. Co-immunoprecipitated product (IP). Whole-cell lysate (WCL). .

Article Snippet: Rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody , Genetex , Cat. No. GTX135591 Dilution WB: 1:5000 Dilution IHC/IF: 1:2000.

Techniques: In Vitro, Binding Assay, Immunoprecipitation, Plasmid Preparation, Construct, Expressing, FLAG-tag, Western Blot, Clone Assay

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Co-immunoprecipitation of SARS-CoV-2 ORF8 with C1qA, C1qB, and C1qC. Graphical representation of the plasmid constructs containing the expression cassettes for the subcomponents C1qA, C1qB, and C1qC contained an N-terminal signal peptide (SP), and a C-terminal FLAG tag. Western blot images for co-immunoprecipitation using an anti-ORF8 antibody. ( B ) Co-immunoprecipitation of C1qA full-length (FL), globular (G), disordered domain (D) with SARS-CoV-2 ORF8. Graphical representation of the plasmid constructs containing expression cassettes for the C1qA-FL, C1qA-G, and C1qA-D, with N-terminal SP and a C-terminal 3FLAG tag, cloned in pIRES vectors. Western blot images for co-immunoprecipitation using an anti-Flag antibody. Co-immunoprecipitated product (IP). Whole-cell lysate (WCL).

Article Snippet: Rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody , Genetex , Cat. No. GTX135591 Dilution WB: 1:5000 Dilution IHC/IF: 1:2000.

Techniques: Immunoprecipitation, Plasmid Preparation, Construct, Expressing, FLAG-tag, Western Blot, Clone Assay

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Line chart of absorbance values obtained in the SARS-CoV-2 ORF8 peptides - C1q binding assay using different molarities of C1q (10, 20, and 50 nM) and 1 mM of 28 different peptides spanning SARS-CoV-2 ORF8 region. Absorbance calculated by discounting the blank (0 nM C1q). ( B ) The ORF8 peptides presenting dose-dependent binding with C1q (#4 red, #10 purple, #19 blue, and #25 yellow) in molecular binding model between SARS-CoV-2 ORF8 dimers and C1qA-Globular (model 1). ( C ) Bar charts representing docking and confidence scores for in silico docking models SARS-CoV-2 ORF8 and C1qA-G. The ORF8 peptides #4, #10, #19, and #25 represented on amino acid (aa) sequence alignment of docking models. ( D ) Representative docking poses of the globular domain of C1q (PDB ID: 1PK6) with SARS-CoV-2 ORF8 protein (PDB ID:7JTL) in 2D interaction diagrams. The ORF8 and C1q residues with significant interactions from each protein are highlighted, labeled, and properly annotated. The hydrogen bonds, pi-pi, and salt bridges formed between ORF8 and C1q are also shown by colored dash or arrow lines. The polar (turquoise), hydrophobic (green), positively charged (purple), and negatively charged (orange) residues are represented in colored spheres. Shown below each of the interaction diagrams is a table of interacting or contact residues between the SARS-CoV-2 ORF8 protein and the globular C1q domain, including their aa positions, aa distance measured in Angstrom (Å), and the corresponding specific binding interactions. ( E ) Boxplots representing the relative expression of genes analyzed in HTR8/SVneo trophoblast treated with ORF8 peptides #4, #10, #19, and #25 using 0.1 nM and 1 nM ( n = 5–6) or mock ( n = 7–10). ( F ) Boxplots representing the relative expression of genes analyzed in primary human villous trophoblast (HVT) treated with 0.1 nM and 1 nM of ORF8 peptide #10 or Mock. Relative expression was calculated by DCt method using GAPDH as the normalizing gene. ( G ) Boxplots representing the percentage of live C3b+ cells in mock ( n = 4) and ORF8 peptides #4, #10, #19, and #25 0.1 nM and 1 nM treated ( n = 4–6) HTR8/SVneo trophoblasts. Data are presented in boxplot, middle line represents means, the bound of box represents interquartile range, and whiskers represent maximum and minimum values. ( H ) Graphical representation of main results. Comparisons using one-way ANOVA Kruskal–Wallis with Fisher test ( p < 0.05). .

Article Snippet: Rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody , Genetex , Cat. No. GTX135591 Dilution WB: 1:5000 Dilution IHC/IF: 1:2000.

Techniques: Binding Assay, In Silico, Sequencing, Labeling, Expressing

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: Correlation analyses between the number of days post SARS-CoV-2 infection to delivery and ( A ) SARS-CoV-2 N1 RNA copies, SARS-CoV-2 N2 RNA copies, and ( B ) circulating ORF8 levels in all biospecimens analyzed, including maternal plasma, chorion, amnion, cord plasma and newborn plasma. Simples linear regression and Spearman’s rank correlation test was used for all the correlations analysis. Scatter plots with Pearson correlation coefficients (r) and dotted lines represent 95% confidence intervals.

Article Snippet: Rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody , Genetex , Cat. No. GTX135591 Dilution WB: 1:5000 Dilution IHC/IF: 1:2000.

Techniques: Infection, Clinical Proteomics

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Sequence alignment of SARS-CoV-2 ORF8 and human immunoglobulin variable lambda chain (IGLV) genes. Representative amino acid residues of SARS-CoV-2 ORF8-specific region located at the loop flanking β 4- β 5 strands were compared with a portion of the framework 3 (FR3) region of the variable light chain of human IGLV5-39*01, IGLV5-39*02, and IGLV1-47*01 germline gene sequences. Identical residues were highlighted in red box and marked by an asterisk (*), while semi-conserved positions due to minor structural differences are in blue boxes and are indicated by a period (.). The β strands are shown by green arrows above the ORF8 sequence while the spanning loops were represented by black lines (Flower et al, ). The FR3 region (shown in the yellow box) was defined according to the IMGT delimitations. ( B ) Comparison of the overlapping sequences of SARS-CoV-2 ORF8 and human IGLV genes highlighted in the dashed boxes using peptide properties: buried index, hydrophobicity, polarity, and turn and helix propensity. The legend for each property is shown as a gradient color scheme on the right. ( C – E ) Structures of ORF8 ( C ), IGLV5-39*01 ( D ), and superimposed images after pairwise structural alignment as viewed from the top (left) and side (right). PBD entries: 7JTL (SARS-CoV-2, blue) and 2CD0 (human IGLV5-39, green). Overlapping sequences between the two structures are highlighted in red. ( F ) Pairwise structural alignment of the human IgG-Fc region (cyan, PDB ID:1FC1) and ORF8 (in blue) showing close structural similarity at the CH2 domain (193 atoms aligned; RMSD: 5.9 Å). The CH2 and CH3 domains are highlighted, while residues in red sphere show the neonatal Fc receptor (FcRn) interaction site. ( G ) Superimposition of CH2 domain of the human IgG-Fc and ORF8 from different rotational perspectives. CH2 residues with the closest folding to ORF8 (blue) is shown in flesh while CH3 are in cyan. The glycosylation site at N297 at the CH2 interface is shown in cyan stick models.

Article Snippet: Rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody , Genetex , Cat. No. GTX135591 Dilution WB: 1:5000 Dilution IHC/IF: 1:2000.

Techniques: Sequencing, Comparison, Glycoproteomics

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rabbit polyclonal anti-SARS-CoV-2 ORF8 antibody , Genetex , Cat. No. GTX135591 Dilution WB: 1:5000 Dilution IHC/IF: 1:2000.

Techniques: Recombinant, Variant Assay, Generated, Plasmid Preparation, FLAG-tag, Sequencing, Digital PCR, cDNA Synthesis, SYBR Green Assay, Blocking Assay, Software, Cell Culture, Enzyme-linked Immunosorbent Assay, Transfection, Real-time Polymerase Chain Reaction, Spectrophotometry, Imaging