Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , Mutations on the spike glycoprotein of SARS-CoV-2 Omicron subvariants. Residues that are not identical among Omicron subvariants are colored red. b , Workflow to generate cryo-EM structure of BA.2, BA.3, BA.2.13, BA.2.12.1, BA.4/5 spike glycoprotein trimer with S6P and R683A, R685A substitutions. c , Binding affinities of Omicron variants spike trimers to hACE2 measured by SPR. SPR analyses were conducted in biological duplicates. d , MD simulated interactions between hACE2 and RBD of Omicron variants. Structures of the RBD from Omicron variants and hACE2 are shown as ribbons.

Article Snippet: WT RBD used here was SARS-CoV-2 (2019-nCoV) Spike RBD-His Recombinant Protein (Sino Biological, 40592-V08H).

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , FACS analysis of pooled memory B cells (IgM − CD27 + ) from plasma of individuals who have recovered from BA.1 breakthrough infection after vaccination, vaccinated individuals and unvaccinated individuals who have recovered from BA.1 breakthrough infection. The percentage of cells recognizing WT or BA.1 RBD are shown. b , The heavy chain V domain somatic hypermutation (SHM) rate of BA.1-specific ( n = 968) and BA.1–WT cross-reactive ( n = 4,782) BCRs obtained from 10X scVDJ-seq from individuals who have recovered from BA.1 breakthrough infection after vaccination. Two-tailed Wilcoxon rank-sum test. Boxes show 25th percentile, median and 75th percentile, and violin plots show kernel density estimation curves of the distribution. c , t -SNE and unsupervised clustering of antibodies that bind WT SARS-CoV-2 RBD. Twelve epitope groups were identified on the basis of DMS of 1,538 antibodies. d , e , Epitope distribution and projection of antibodies from plasma of individuals who had recovered from infection with the WT virus, individuals who have recovered from BA.1 breakthrough infection after vaccination, and vaccinated individuals who had recovered from SARS. f , ACE2 competition level determined by competition ELISA ( n = 1,286) were projected onto the t -SNE. g , Neutralizing activity against SARS-CoV-2 D614G ( n = 1,509) and SARS-CoV-1 (HKU-39849; n = 1,457). h , Average mutational escape score projection of each epitope group on SARS-CoV-2 RBD (Protein Data Bank (PDB): 6M0J). All neutralization assays were performed as biological duplicates.

Article Snippet: WT RBD used here was SARS-CoV-2 (2019-nCoV) Spike RBD-His Recombinant Protein (Sino Biological, 40592-V08H).

Techniques: Infection, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Activity Assay, Neutralization

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a – c , Neutralizing activity against SARS-CoV-1 and SARS-CoV-2 subvariants by NAbs in group E1 ( a ; n = 70), F2 ( b ; n = 171) and F3 ( c ; n = 69). The geometric mean of the fold change in IC 50 relative to BA.2 is shown above each plot. P -values were calculated using a two-tailed Wilcoxon signed-rank test of paired samples, compared with the IC 50 for BA.2. d , The epitope of Group E1 antibody BD55-3152 on the BA.1 RBD. e , Overlay of BD55-5840 in the complex with BA.1 or BA.2 RBD. f , g , The epitope and interactions on the binding interface of BD55-1239 (group F2) ( f ) and BD55-3372 (group F3) ( g ). Antibody residues are shown in blue, and RBD residues are in black or red. Residues highlighted in red indicate sites that are mutated in Omicron variants. h , Average escape maps of antibodies in epitope groups E1, F2 and F3, and the corresponding multiple sequence alignment of various sarbecovirus RBDs. The height of each amino acid in the escape map represents its mutation escape score. Sites that are mutated in Omicron subvariants are marked in bold. All neutralization assays were performed as biological duplicates.

Article Snippet: WT RBD used here was SARS-CoV-2 (2019-nCoV) Spike RBD-His Recombinant Protein (Sino Biological, 40592-V08H).

Techniques: Activity Assay, Two Tailed Test, Binding Assay, Sequencing, Mutagenesis, Neutralization

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , Cartoon models of Cryo-EM structures of BD55-3152 in complex of BA.1 RBD, BD55-1239 in complex of BA.1 RBD, and BD55-3372 in complex of Delta RBD. b , Workflow to generate refined structural model of BD55-3152 and BD55-1239 in complex of BA.1 RBD, BD55-3372 in complex of Delta RBD, and BD55-5840 in complex of BA.2 RBD. c , Neutralizing activity of representative NAbs in group E1 (n = 68), F2 (n = 139) and F3 (n = 61) against SARS-CoV-2 D614G, in addition to D614G+D405N and D614G+R408S. Geometric mean of IC50 fold changes compared to IC50 against D614G are annotated above the bars. P-values were calculated using a two-tailed Wilcoxon signed-rank test of paired samples. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant, p > 0.05. All neutralization assays were conducted in biological duplicates. d , Conformational comparison between BA.1 and BA.2 RBD regarding the 366-377 hairpin. e , Biolayer interferometry analysis of Group E1 antibodies S309 and BD55-5840 binding to Omicron BA.1 and BA.2 Spike trimer. Biolayer interferometry analyses were conducted in biological duplicates.

Article Snippet: WT RBD used here was SARS-CoV-2 (2019-nCoV) Spike RBD-His Recombinant Protein (Sino Biological, 40592-V08H).

Techniques: Cryo-EM Sample Prep, Activity Assay, Two Tailed Test, Neutralization, Binding Assay

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , Four epitope groups were identified among 102 BA.1-specific NAbs via k -means clustering and t -SNE of BA.1 RBD-based DMS profiles. b , c , Distribution of ACE2 competition level ( b ) and neutralizing activities ( c ) against BA.1. d , Neutralizing activities of BA.1-specific antibodies against pseudovirus with SARS-CoV-1 and SARS-CoV-2 spike variants (A Omi , n = 18; B Omi , n = 30; D Omi , n = 22; F3 Omi , n = 32). The geometric mean of the fold change in IC 50 relative to BA.1 is shown above each plot. e , Average mutational escape score projection of each BA.1-specific epitope group on SARS-CoV-2 RBD (PDB: 7WPB). f , Averaged escape maps at escape hotspots of the 102 NAbs in the four epitope groups, and corresponding multiple sequence alignment of various sarbecovirus RBDs. The height of each amino acid in the escape map represents its mutation escape score. Sites that are mutated in Omicron variants are marked in bold. WT-related escaping mutations are highlighted. g , Neutralizing activities of BA.1-specific NAbs against BA.1- or BA.2-based pseudoviruses carrying single substitutions (A Omi , n = 18; B Omi , n = 30; D Omi , n = 22; F3 Omi , n = 32). The geometric mean of the fold change in IC 50 relative to BA.1 is shown above each plot. Wilcoxon signed-rank test of paired samples, compared with IC 50 for BA.1. All neutralization assays were performed as biological duplicates.

Article Snippet: WT RBD used here was SARS-CoV-2 (2019-nCoV) Spike RBD-His Recombinant Protein (Sino Biological, 40592-V08H).

Techniques: Sequencing, Mutagenesis, Neutralization

Journal: Kidney International Reports

Article Title: Hybrid Immunity Overcomes Defective Immune Response to COVID-19 Vaccination in Kidney Transplant Recipients

doi: 10.1016/j.ekir.2023.12.008

Figure Lengend Snippet: Binding IgG, RBD IgG avidity, and neutralizing antibody responses to SARS-CoV-2 mRNA vaccination in naïve and experienced KTRs. Serum levels of SARS-CoV-2 RBD specific binding IgG (BAU: binding antibody units), RBD specific IgG avidity (koff: dissociation rate constant) and titers of neutralizing antibodies (NT50: 50% neutralization titer) were measured before vaccination (D0) and 1 month after 2 doses of mRNA vaccine (D49) in naïve KTRs (N-KTRs, light pink), experienced KTRs (E-KTRs, dark pink), naïve HCs (N-HCs, light blue) and experienced HCs (E-HCs, dark blue). Bars indicate median values. Horizontal grid lines indicate a technical negative signal (blank). Groups were compared using the analysis of variance Kruskal-Wallis test with Dunn’s correction. For within HC or KTR comparisons, ns; aP < 0.05; bP < 0.01; cP < 0.001. For comparisons between HC and KTR, ns; dP < 0.05; eP < 0.01; fP < 0.001. HC, healthy control; KTR, kidney transplant recipient; ns, not significant; RBD, receptor binding domain.

Article Snippet: 42 , 46 , 47 Antigens used for multiplex assays included Wuhan SARS-CoV-2 S1 protein (Sanyou Biopharmaceuticals #PNA002), SARS-CoV-2 S2 protein (Sinobiological #40590-V08H1), SARS-CoV-2 RBD (Proteogenix #PX-COV-P046) and SARS-CoV-2 N protein (Sanyou Biopharmaceuticals #PNA006).

Techniques: Binding Assay, Neutralization

Journal: Kidney International Reports

Article Title: Hybrid Immunity Overcomes Defective Immune Response to COVID-19 Vaccination in Kidney Transplant Recipients

doi: 10.1016/j.ekir.2023.12.008

Figure Lengend Snippet: IgG Subclasses and IgA responses to SARS-CoV-2 mRNA vaccination in naïve and experienced KTRs. Serum levels of SARS-CoV-2 RBD specific, spike S1 subunit specific, and spike S2 subunit specific IgG1, IgG2, IgG3, IgG4 and IgA were measured before vaccination (D0, panel a) and 1 month after vaccination (D49, panel b) in naïve KTRs (N-KTRs, light pink), experienced KTRs (E-KTRs, dark pink), naïve HCs (N-HCs, light blue) and experienced HCs (E-HC, dark blue). Bars indicate median values. Horizontal grid lines indicate a technical negative signal (blank). Groups were compared using the analysis of variance Kruskal-Wallis test with Dunn’s correction. For within HC or KTR comparisons, aP < 0.05; bP < 0.01; cP < 0.001. For comparisons between HC and KTR, ns; dP < 0.05; eP < 0.01; fP < 0.001. HC, healthy control; KTR, kidney transplant recipient; MFI, median fluorescent intensity; ns, not significant; RBD, receptor binding domain.

Article Snippet: 42 , 46 , 47 Antigens used for multiplex assays included Wuhan SARS-CoV-2 S1 protein (Sanyou Biopharmaceuticals #PNA002), SARS-CoV-2 S2 protein (Sinobiological #40590-V08H1), SARS-CoV-2 RBD (Proteogenix #PX-COV-P046) and SARS-CoV-2 N protein (Sanyou Biopharmaceuticals #PNA006).

Techniques: Binding Assay

Journal: Kidney International Reports

Article Title: Hybrid Immunity Overcomes Defective Immune Response to COVID-19 Vaccination in Kidney Transplant Recipients

doi: 10.1016/j.ekir.2023.12.008

Figure Lengend Snippet: Fcγ receptors binding antibody responses to SARS-CoV-2 mRNA vaccination in naïve and experienced KTRs. Serum levels of SARS-CoV-2 RBD specific, spike S1 subunit specific, and spike S2 subunit specific antibodies binding the Fcγ receptors, FcγRIIa and FcγRIIIa were measured before vaccination (D0, panel a) and 1 month after vaccination (D49, panel b) in naïve KTRs (N-KTRs, light pink), experienced KTRs (E-KTRs, dark pink), naïve HCs (N-HCs, light blue) and experienced HCs (E-HC, dark blue). Bars indicate median values. Horizontal grid lines indicate a technical negative signal (blank). Groups were compared using the analysis of variance Kruskal-Wallis test with Dunn’s correction. For within HC or KTR comparisons, ns; aP < 0.05; bP < 0.01; cP < 0.001. For comparisons between HC and KTR, ns; dP < 0.05; eP < 0.01; fP < 0.001. HC, healthy control; KTR, kidney transplant recipient; MFI, median fluorescent intensity; ns, not significant; RBD, receptor binding domain

Article Snippet: 42 , 46 , 47 Antigens used for multiplex assays included Wuhan SARS-CoV-2 S1 protein (Sanyou Biopharmaceuticals #PNA002), SARS-CoV-2 S2 protein (Sinobiological #40590-V08H1), SARS-CoV-2 RBD (Proteogenix #PX-COV-P046) and SARS-CoV-2 N protein (Sanyou Biopharmaceuticals #PNA006).

Techniques: Binding Assay

Journal: Kidney International Reports

Article Title: Hybrid Immunity Overcomes Defective Immune Response to COVID-19 Vaccination in Kidney Transplant Recipients

doi: 10.1016/j.ekir.2023.12.008

Figure Lengend Snippet: IgG-dependent complement deposition and phagocytosis responses to SARS-CoV-2 mRNA vaccination in naïve and experienced KTRs. Serum levels of SARS-CoV-2 RBD specific, spike S1 subunit specific, and spike S2 subunit specific IgG promoting complement deposition (ADCD) and cellular phagocytosis (ADCP) were measured before vaccination (D0, panel a) and 1 month after vaccination (D49, panel b) in naïve KTRs (N-KTRs, light pink), experienced KTRs (E-KTRs, dark pink) naïve HCs (N-HCs, light blue) and experienced HCs (E-HCs, dark blue). Levels of ADCD are expressed as MFI. Levels of ADCP are expressed as phagocytic score (see methods). Bars indicate median values. Horizontal grid lines indicate a technical negative signal (blank). Groups were compared using the analysis of variance Kruskal-Wallis test with Dunn’s correction. For within HC or KTR comparisons, ns; aP <0.05; bP < 0.01; cP < 0.001. For comparisons between HC and KTR, ns; dP < 0.05; eP < 0.01; fP < 0.001. HC, healthy control; KTR, kidney transplant recipient; MFI, median fluorescent intensity; ns, not significant; RBD, receptor binding domain.

Article Snippet: 42 , 46 , 47 Antigens used for multiplex assays included Wuhan SARS-CoV-2 S1 protein (Sanyou Biopharmaceuticals #PNA002), SARS-CoV-2 S2 protein (Sinobiological #40590-V08H1), SARS-CoV-2 RBD (Proteogenix #PX-COV-P046) and SARS-CoV-2 N protein (Sanyou Biopharmaceuticals #PNA006).

Techniques: Binding Assay

Journal: Kidney International Reports

Article Title: Hybrid Immunity Overcomes Defective Immune Response to COVID-19 Vaccination in Kidney Transplant Recipients

doi: 10.1016/j.ekir.2023.12.008

Figure Lengend Snippet: CD4 and CD8 T cell responses to SARS-CoV-2 mRNA vaccination in naïve and experienced KTRs. Percentage of SARS-CoV-1 spike S1 subunit and spike S2 subunit specific CD4 T cells expressing CD154, IFNγ and IL2, and of CD8 T cells expressing IFNγ and IL-2 were measured in peripheral blood before vaccination (D0, panel a) and 1 month after vaccination (D49, panel b) in naïve KTRs (N-KTRs, light pink), experienced KTRs (E-KTRs, dark pink), naïve HCs (N-HCs, light blue) and experienced HCs (E-HC, dark blue). Bars indicate median values. Groups were compared using the analysis of variance Kruskal-Wallis test with Dunn’s correction. HC, healthy control; KTR, kidney transplant recipient; ns, not significant. For within HC or KTR comparisons, ns; aP < 0.05; bP < 0.01; cP < 0.001. For comparisons between HC and KTR, ns; dP < 0.05; eP < 0.01; fP < 0.001.

Article Snippet: 42 , 46 , 47 Antigens used for multiplex assays included Wuhan SARS-CoV-2 S1 protein (Sanyou Biopharmaceuticals #PNA002), SARS-CoV-2 S2 protein (Sinobiological #40590-V08H1), SARS-CoV-2 RBD (Proteogenix #PX-COV-P046) and SARS-CoV-2 N protein (Sanyou Biopharmaceuticals #PNA006).

Techniques: Expressing

Journal: Kidney International Reports

Article Title: Hybrid Immunity Overcomes Defective Immune Response to COVID-19 Vaccination in Kidney Transplant Recipients

doi: 10.1016/j.ekir.2023.12.008

Figure Lengend Snippet: Principal component analysis of immune responses to SARS-CoV-2 mRNA vaccination in naïve and experienced KTRs. (a) Scatter plot of PCA including all immune response parameters except IgG2, IgG4 and IgA measured 1 month after vaccination (D49) in naïve KTRs (N-KTRs, light pink), experienced KTRs (E-KTRs, dark pink), naïve HCs (N-HCs, light blue) and experienced HCs (E-HC, dark blue). (b) Comparison of PC1 and PC2 values between groups by analysis of variance Kruskal-Wallis test with Dunn’s correction with P < 0.1. (c) Relative weighting of individual immune response parameters in PC1 and PC2. HC, healthy control; KTR, kidney transplant recipient, PC, principal component; PCA, principal component analysis.

Article Snippet: 42 , 46 , 47 Antigens used for multiplex assays included Wuhan SARS-CoV-2 S1 protein (Sanyou Biopharmaceuticals #PNA002), SARS-CoV-2 S2 protein (Sinobiological #40590-V08H1), SARS-CoV-2 RBD (Proteogenix #PX-COV-P046) and SARS-CoV-2 N protein (Sanyou Biopharmaceuticals #PNA006).

Techniques: Comparison

Journal: Virus Research

Article Title: MERS-CoV RBD-mRNA vaccine induces potent and broadly neutralizing antibodies with protection against MERS-CoV infection

doi: 10.1016/j.virusres.2023.199156

Figure Lengend Snippet: Design and characterization of MERS-CoV RBD-mRNA. (A) Schematic map of MERS-CoV spike (S) protein and design of MERS-CoV RBD-mRNA vaccines. Each RBD-mRNA consists of genes encoding signal peptide (tPA) and open reading frame of RBD flanked by the 5′ and 3′ untranslated regions (UTRs), a 5′ cap and a 3′ poly(A) tail. RBD-mRNA-wild-type (WT) was synthesized in the presence of nucleosides (UTP, CTP, ATP, and GTP), and RBD-mRNA-PseudoU was constructed by changing the UTP to modified Pseudo-UTP during mRNA synthesis. SP, signal peptide. NTD, N-terminal domain. RBD, receptor-binding domain. TM, transmembrane; CT, cytoplasmic tail. The synthesized mRNA was encapsulated with lipid nanoparticles (LNPs) and used for the subsequent experiments. (B) Characterization of the stability of each RBD-mRNA or empty-LNP (control) at different temperatures (4 °C, 25 °C, and 37 °C). Each LNP-encapsulated RBD-mRNA or LNP control was stored for 0, 24, 48, and 72 h, respectively, at the above temperatures, and the particle size (hydrodynamic diameter: nm) was measured using a Dynamic Light Scattering. (C) Representative images of flow cytometry for expression of MERS-CoV RBD protein. 293T cells transfected with each RBD-mRNA were tested for expression of RBD protein using a MERS-CoV RBD-specific monoclonal antibody (mAb, Mersmab1). 293T cells transfected with the empty-LNP were used as control. MFI, median fluorescence intensity. The data are presented as mean ± standard deviation of the mean (s.e.m.) of triple wells.

Article Snippet: The RBD-mRNA-WT (without the nucleoside modification) was synthesized in the presence of ATP, GTP, CTP, and UTP nucleosides, and the RBD-mRNA-PseudoU (with the nucleoside modification) was synthesized in the presence of ATP, GTP, and CTP nucleosides, as well as modified nucleoside pseudouridine-5′-O-triphosphate (Pseudo-UTP; APExBIO).

Techniques: Synthesized, Construct, Modification, Binding Assay, Flow Cytometry, Expressing, Transfection, Fluorescence, Standard Deviation

Journal: Virus Research

Article Title: MERS-CoV RBD-mRNA vaccine induces potent and broadly neutralizing antibodies with protection against MERS-CoV infection

doi: 10.1016/j.virusres.2023.199156

Figure Lengend Snippet: Immunization and challenge schedules. Mice were immunized with MERS-CoV RBD-mRNA-PseudoU, RBD-mRNA-WT, or empty-LNP (control) according to the three procedures described in Protocols 1–3 (A-C). Sera were collected to detect specific IgG antibodies (Abs), subtype (IgG1 and IgG2a) Abs, or neutralizing Abs against single or multiple strains of pseudotyped MERS-CoV. Splenocytes were collected to test specific T-cell or B-cell responses. Immunized mice were also challenged with MERS-CoV to evaluate protective efficacy of RBD-mRNA-PseudoU vaccine. I.M., intramuscular; I.N., intranasal; I.V., intravenous; S.C., subcutaneous; I.D., intradermal.

Article Snippet: The RBD-mRNA-WT (without the nucleoside modification) was synthesized in the presence of ATP, GTP, CTP, and UTP nucleosides, and the RBD-mRNA-PseudoU (with the nucleoside modification) was synthesized in the presence of ATP, GTP, and CTP nucleosides, as well as modified nucleoside pseudouridine-5′-O-triphosphate (Pseudo-UTP; APExBIO).

Techniques:

Journal: Virus Research

Article Title: MERS-CoV RBD-mRNA vaccine induces potent and broadly neutralizing antibodies with protection against MERS-CoV infection

doi: 10.1016/j.virusres.2023.199156

Figure Lengend Snippet: Nucleoside-modified RBD-mRNA induced potent and durable antibody responses with broadly neutralizing activity. Mice were immunized I.M. with LNP-encapsulated RBD-mRNA with or without the nucleoside modification (30 μg/mouse), or empty-LNP control, boosted twice at 4 weeks and around 7 months, respectively, and sera were collected for the tests described below. (A) Mouse sera from different time points were detected for IgG antibodies (Abs) by ELISA. ELISA plates were coated with MERS-CoV S1 protein, and the Ab titer was determined based on the endpoint serum dilution that remained positively detectable. (B) Mouse sera from different time points were measured for neutralizing Abs against infection of pseudotyped MERS-CoV encoding S protein of the original virus strain (EMC2012). (C) Mouse sera from the last immunization were measured for cross-neutralizing antibodies against multiple variants of pseudotyped MERS-CoV-2. NT 50 was calculated as 50% neutralizing Ab titer against infection of the respective MERS-CoV pseudovirus in Huh-7 cells. The data are presented as mean ± s . e.m. of duplicate wells from five mice in each group. *** ( P < 0.001) indicates significant difference between RBD-mRNA-PseudoU and other groups. The experiments were repeated twice, resulting in similar results.

Article Snippet: The RBD-mRNA-WT (without the nucleoside modification) was synthesized in the presence of ATP, GTP, CTP, and UTP nucleosides, and the RBD-mRNA-PseudoU (with the nucleoside modification) was synthesized in the presence of ATP, GTP, and CTP nucleosides, as well as modified nucleoside pseudouridine-5′-O-triphosphate (Pseudo-UTP; APExBIO).

Techniques: Modification, Activity Assay, Enzyme-linked Immunosorbent Assay, Infection

Journal: Virus Research

Article Title: MERS-CoV RBD-mRNA vaccine induces potent and broadly neutralizing antibodies with protection against MERS-CoV infection

doi: 10.1016/j.virusres.2023.199156

Figure Lengend Snippet: Nucleoside-modified RBD-mRNA induced effective T-cell responses. Mice were immunized I.M. with LNP-encapsulated RBD-mRNA with or without the nucleoside modification (30 μg/mouse), or empty-LNP control, and boosted twice at 4 weeks and around 7 months, respectively. Splenocytes from the last immunization were analyzed for SARS-CoV-2 RBD-specific T-cell responses by flow cytometry. IFN-γ, TNF-α, and IL-4-secreting CD4 + (A) or CD8 + (B) T cells were stained for each cell surface marker or cytokine. The data are presented as mean ± s . e.m. of duplicate wells from five mice in each group. * ( P < 0.05), ** ( P < 0.01), and *** ( P < 0.001) indicate significant difference between RBD-mRNA-PseudoU and other groups. The experiments were repeated twice, resulting in similar results.

Article Snippet: The RBD-mRNA-WT (without the nucleoside modification) was synthesized in the presence of ATP, GTP, CTP, and UTP nucleosides, and the RBD-mRNA-PseudoU (with the nucleoside modification) was synthesized in the presence of ATP, GTP, and CTP nucleosides, as well as modified nucleoside pseudouridine-5′-O-triphosphate (Pseudo-UTP; APExBIO).

Techniques: Modification, Flow Cytometry, Staining, Marker

Journal: Virus Research

Article Title: MERS-CoV RBD-mRNA vaccine induces potent and broadly neutralizing antibodies with protection against MERS-CoV infection

doi: 10.1016/j.virusres.2023.199156

Figure Lengend Snippet: An effective immunization route and antigen dose for nucleoside-modified RBD-mRNA to induce strong B-cell responses. Mice were immunized with LNP-encapsulated nucleoside modified RBD-mRNA (10 μg/mouse) or empty-LNP control at different routes, and boosted at 4 weeks. Splenocytes from the last immunization were analyzed for B-cell responses by flow cytometry. The cells were stained for B220 + -CD27 + -CD138 + -IgG + cells. The data are presented as mean ± s.e.m. of duplicate wells from five mice in each group. ** ( P < 0.01) indicates significant difference between RBD-mRNA-PseudoU and other groups. The experiments were repeated twice, resulting in similar results.

Article Snippet: The RBD-mRNA-WT (without the nucleoside modification) was synthesized in the presence of ATP, GTP, CTP, and UTP nucleosides, and the RBD-mRNA-PseudoU (with the nucleoside modification) was synthesized in the presence of ATP, GTP, and CTP nucleosides, as well as modified nucleoside pseudouridine-5′-O-triphosphate (Pseudo-UTP; APExBIO).

Techniques: Modification, Flow Cytometry, Staining

Journal: Virus Research

Article Title: MERS-CoV RBD-mRNA vaccine induces potent and broadly neutralizing antibodies with protection against MERS-CoV infection

doi: 10.1016/j.virusres.2023.199156

Figure Lengend Snippet: An effective immunization route and antigen dose for nucleoside-modified RBD-mRNA to induce potent antibodies with neutralizing activity . Mice were immunized with LNP-encapsulated nucleoside modified RBD-mRNA (10 μg/mouse) or empty-LNP control at different routes, including I.M., I.N., I.V., S.C., and I.D., and boosted as described in Fig. 5 . Sera from the first and second immunizations were detected for antibodies (Abs) and neutralizing Abs against MERS-CoV. MERS-CoV S1-specific IgG (A), IgG1 (B), IgG2a (C) Abs, and anti-MERS-CoV neutralizing Abs (D) 10 days after the first immunization, as well as MERS-CoV S1-specific IgG (E), IgG1 (F), IgG2a (G) Abs, and anti-MERS-CoV neutralizing Abs (H) 10 days after the second immunization, were evaluated by ELISA and pseudovirus neutralization assays, respectively. MERS-CoV S1-specific IgM Abs were tested by ELISA in the sera of I.D.-immunized mice from the 1st and 2nd doses (I-J). ELISA plates were coated with MERS-CoV S1 protein, and the Ab titer was determined based on the endpoint serum dilution that remained positively detectable. Neutralizing antibody activity was reported as 50% neutralizing Ab titer (NT 50 ) against infection of pseudotyped MERS-CoV (EMC2012 strain) in Huh-7 cells. The data are presented as mean ± s . e.m. of duplicate wells from five mice in each group. ** ( P < 0.01) and *** ( P < 0.001) indicate significant difference of RBD-mRNA-PseudoU injected via the I.D. route and other routes. The experiments were repeated twice, resulting in similar results.

Article Snippet: The RBD-mRNA-WT (without the nucleoside modification) was synthesized in the presence of ATP, GTP, CTP, and UTP nucleosides, and the RBD-mRNA-PseudoU (with the nucleoside modification) was synthesized in the presence of ATP, GTP, and CTP nucleosides, as well as modified nucleoside pseudouridine-5′-O-triphosphate (Pseudo-UTP; APExBIO).

Techniques: Modification, Activity Assay, Enzyme-linked Immunosorbent Assay, Neutralization, Infection, Injection

Journal: Virus Research

Article Title: MERS-CoV RBD-mRNA vaccine induces potent and broadly neutralizing antibodies with protection against MERS-CoV infection

doi: 10.1016/j.virusres.2023.199156

Figure Lengend Snippet: Nucleoside-modified RBD-mRNA protected mice from MERS-CoV infection and the protection was correlated with neutralizing antibodies. Mice were immunized I.D. with LNP-encapsulated nucleoside modified RBD-mRNA (10 μg/mouse) or empty-LNP control, and boosted twice at 4 weeks. Sera collected 10 days after the last immunization were tested for MERS-CoV S1-specific IgG antibodies (Abs) by ELISA (A) and neutralizing Abs (NT 50 ) against infection of pseudotyped MERS-CoV (EMC2012 strain) in Huh-7 cells (B). One month after the last immunization, mice were transduced with Ad5/hDPP4, and then challenged with MERS-CoV (EMC2012 strain) (5 × 10 3 PFU/mouse). Lungs were collected three days after challenge, and viral titer was detected from the challenged mice, which are reported as PFU/g of tissues (C). The data are presented as mean ± s . e.m. of five mice in each group. *** ( P < 0.001) indicates significant difference between RBD-mRNA-PseudoU and the control group. The experiments were repeated twice, resulting in similar results.

Article Snippet: The RBD-mRNA-WT (without the nucleoside modification) was synthesized in the presence of ATP, GTP, CTP, and UTP nucleosides, and the RBD-mRNA-PseudoU (with the nucleoside modification) was synthesized in the presence of ATP, GTP, and CTP nucleosides, as well as modified nucleoside pseudouridine-5′-O-triphosphate (Pseudo-UTP; APExBIO).

Techniques: Modification, Infection, Enzyme-linked Immunosorbent Assay, Transduction