Journal: Cell Reports Methods

Article Title: Direct capture of neutralized RBD enables rapid point-of-care assessment of SARS-CoV-2 neutralizing antibody titer

doi: 10.1016/j.crmeth.2022.100273

Figure Lengend Snippet:

Article Snippet: SARS-CoV-2 Neutralizing Antibody , ABclonal , Cat# A19215; RRID: AB_2862633.

Techniques: Purification, Recombinant, Virus, Luciferase, Clinical Proteomics, Generated, Suspension, Cloning, Transfection, Western Blot, Expressing, Software, Membrane, Chromatography

Journal: NPJ Vaccines

Article Title: Mucosal administration of a live attenuated recombinant COVID-19 vaccine protects nonhuman primates from SARS-CoV-2

doi: 10.1038/s41541-022-00509-6

Figure Lengend Snippet: a Syncytia formed by MV-014-212 and MVK-014-212. Micrographs were taken at a total amplification of 100× under phase contrast or using tetramethylrhodamine filter. Yellow arrows point to syncytia. Scale bars are 100 µm. b Western blot showing full-length purified SARS-CoV-2 spike protein lacking the furin cleavage site (lane 1), MVK-014-212 (lane 2), MV-014-212 (lane 3), mock-infected Vero cell lysate (lane 4), blank (lane 5). The molecular weight markers correspond to the migration of the BIO-RAD Precision Plus Protein Dual Color Standards. The blue arrows indicate the expected size of the full-length spike and the cleaved protein (S1 + S2). The blots were derived from the same experiment and processed in parallel. c Multicycle replication kinetics of MV-014-212 compared to respiratory syncytial virus (RSV) A2 in serum-free Vero cells. Cells were infected at an MOI of 0.01 and incubated at 32 °C. Cells and supernatants were collected at 0, 12, 24, 48, 72, 96, and 120 h post infection. Titers of the samples were determined by plaque assay in Vero cells. Data points represent the means of two replicate wells and error bars represent the standard deviation. d Multicycle replication kinetics of MV-014-212 compared with MVK-014-212 in serum-free Vero cells. Cells were infected at an MOI of 0.01 and incubated at 32 °C. Cells and supernatants were collected at 0, 3, 24, and 72 h post infection. Titers of the samples were determined by plaque assay in Vero cells. Data points represent the means of three replicate wells and error bars represent the standard deviation. e Short-term thermal stability assay. Virus stocks of MV-014-212 prepared in Williams E medium supplemented with sucrose phosphate glutamate (SPG) buffer or prepared in SPG alone were incubated for 6 h at −80 °C, −20 °C, 4 °C, and room temperature and the titer determined by plaque assay. The bars show the mean of 2 technical replicates of 3 different samples, and the error bars denote SD. f Two-week thermal stability assay. Virus stocks of MV-014-212 in Williams E medium supplemented with SPG buffer were incubated for 14 days at 4 °C and room temperature (18 °C to 22 °C ± 2 °C) and the titer determined by plaque assay. The bars show the mean of 2 technical replicates of 3 different samples, and the error bars denote SD.

Article Snippet: Rabbit anti-SARS-CoV-2 spike (Sino Biological Inc., Beijing, China) was diluted in iBind solution (Invitrogen) at 1:1000.

Techniques: Amplification, Western Blot, Purification, Infection, Molecular Weight, Migration, Derivative Assay, Incubation, Plaque Assay, Standard Deviation, Stability Assay

Journal: NPJ Vaccines

Article Title: Mucosal administration of a live attenuated recombinant COVID-19 vaccine protects nonhuman primates from SARS-CoV-2

doi: 10.1038/s41541-022-00509-6

Figure Lengend Snippet: a AGM study design. Nasal swabs (NS) were obtained on days 1 through 12 after vaccination. Bronchoalveolar lavages (BALs) were collected on days 2, 4, 6, 8, 10, and 12. Viral shedding of the vaccine or control virus in NS and BAL samples was determined by plaque assay using fresh samples. On day 28 post-inoculation, AGMs were challenged with wild type ( wt ) SARS-CoV-2. Nasal swabs were obtained on days 1, 2, 4, 6, and 7 post challenge. BAL was obtained on days 2, 4, 7, and 10 post challenge. b Attenuation of MV-014-212 in the upper and lower respiratory tract of AGM. Viral titer in NS (left) or BAL (right) from AGMs following inoculation with MV-014-212 or wt respiratory syncytial virus (RSV) A2 were measured by plaque assay on Vero cells. Each data point is the mean of 2 technical replicates. The box represents the range of the data and the horizontal line is the mean value for each time point. The dotted line represents the limit of detection (50 PFU/mL). N = 4 AGMs for each group. For shedding kinetics of individual animals see Supplementary Fig. . Protection of MV-014-212-vaccinated AGMs against wt SARS-CoV-2 challenge in nasal swabs ( c ) and BAL ( d ). SARS-CoV-2 sgRNA levels were measured by RT-qPCR and expressed as genome equivalents (GE)/mL. The horizontal line in the box is the mean of all measurements at each time point. The dashed line represents the limit of detection of 50 GE/mL. Each data point corresponds to one animal ( N = 3 AGMs for MV-014-212 and mock groups and N = 4 AGMs for RSV A2 group). For shedding kinetics of individual animals see Supplementary Fig. . Statistical analysis was one-way ANOVA with Tukey’s multiple comparisons. P -values are shown. Bars are mean and error bars denote SD.

Article Snippet: Rabbit anti-SARS-CoV-2 spike (Sino Biological Inc., Beijing, China) was diluted in iBind solution (Invitrogen) at 1:1000.

Techniques: Plaque Assay, Quantitative RT-PCR

Journal: NPJ Vaccines

Article Title: Mucosal administration of a live attenuated recombinant COVID-19 vaccine protects nonhuman primates from SARS-CoV-2

doi: 10.1038/s41541-022-00509-6

Figure Lengend Snippet: Neutralization assays using day 25 sera from MV-014-212-vaccinated AGMs (1) 09720, (2) C8959, and (3) C6672. a Pseudovirus neutralization assay (PNA) showing NT 50 values and a convalescent sera control at 1859 IU/mL. The data shown is the average of two technical replicates, bars are geometric mean and error bars indicate geometric standard deviation. b Surrogate virus neutralization assay (sVNA) results using the USA-WA1/2020 strain RBD. The data shown is the average of two technical replicates, bars are mean and error bars indicate standard deviation. c Pre-immune and post-vaccination surrogate virus neutralization titers of individual AGMs. Each data point represents the mean of two technical replicates, except for post-vaccination D3356, D3254, D4339, and C8546. For these animals, only one technical replicate was tested due to insufficient sample volume. Error bars denote SD. d Plaque reduction neutralization assay (PRNA). Serum from the AGM O9720, showing the highest titers in ( a ) and ( b ) was used in a 50% plaque-reduction neutralization test performed in a biosafety level 3 facility. The neutralization titers of this AGM against wild-type SARS-CoV-2 or two variants of concern (Beta/B.1.351 and Alpha/B.1.1.7) are shown. The convalescent serum used in this assay is the same as in ( a ). The limit of detection is 20 and is indicated as a dashed line. e Surrogate virus neutralization results using the Delta/B.1.617.2 strain RBD or the USA-WA1/2020 (WA-1) RBD on serum from the AGM O9720. For ( d ) and ( e ), the data shown is the average of two technical replicates and bars indicate standard deviation. Statistical analysis was one-way ANOVA using Tukey’s multiple comparisons test. p -values are shown. LOD: limit of detection. LLOQ: lower limit of quantification.

Article Snippet: Rabbit anti-SARS-CoV-2 spike (Sino Biological Inc., Beijing, China) was diluted in iBind solution (Invitrogen) at 1:1000.

Techniques: Neutralization, Standard Deviation, Plaque Reduction Neutralization Test

Journal: NPJ Vaccines

Article Title: Mucosal administration of a live attenuated recombinant COVID-19 vaccine protects nonhuman primates from SARS-CoV-2

doi: 10.1038/s41541-022-00509-6

Figure Lengend Snippet: a Spike-specific serum immunoglobulin (Ig) G antibodies were measured by ELISA using serum collected on day 25 from AGMs inoculated with MV-014-212, wild-type respiratory syncytial virus (RSV) A2, or phosphate-buffered saline (mock). The titer was expressed as ELISA units (ELU/mL) calculated by comparison against a standard curve generated with a pool of human COVID-19 convalescent serum (Std/012020, Nexelis). b IgA antibodies specific to SARS-CoV-2 spike protein were measured by ELISA using nasal swabs collected on day 25 post-inoculation. The Log 2 of the ratio of the values obtained at day 25 over day 1 are shown. The calculated ELU/mL concentration was obtained from a standard curve generated using total purified human IgA by a capture ELISA. For ( a ) and ( b ), N = 3 AGMs for the MV-014-212 and mock groups and N = 4 AGMs for the RSV A2 group. Statistical analysis was one-way ANOVA using Tukey’s multiple comparisons test. p- values are shown. Vaccine responses for individual AGMs are indicated as (1) O9720, (2) C8959, and (3) C6672 in panel ( a ) and ( b ). Bars are mean and error bars denote SD.

Article Snippet: Rabbit anti-SARS-CoV-2 spike (Sino Biological Inc., Beijing, China) was diluted in iBind solution (Invitrogen) at 1:1000.

Techniques: Enzyme-linked Immunosorbent Assay, Generated, Concentration Assay, Purification

Journal: NPJ Vaccines

Article Title: Mucosal administration of a live attenuated recombinant COVID-19 vaccine protects nonhuman primates from SARS-CoV-2

doi: 10.1038/s41541-022-00509-6

Figure Lengend Snippet: a Interferon (IFN)-γ and b interleukin (IL)-5 expressing T cells were quantified by ELISpot. Splenocytes isolated from mice expressing human ACE2 ( N = 5 mice per group) were collected on day 28 post-inoculation with MV-014-212 or phosphate-buffered saline (PBS) and stimulated with a peptide pool that spanned the SARS-CoV-2 spike protein (pool), media, or the mitogen concanavalin A (Con A). Control mice were vaccinated with purified SARS-CoV-2 spike protein adjuvanted with alum by intramuscular injection at day −20 and day 0. c Log of the ratio of IFN-γ to IL-5–expressing cells. Immunoglobulin levels of d IgG2a and e IgG1 from day 28 serum were determined by Spike-specific ELISA. The concentration of each immunoglobulin isotype was determined from standard curves generated with purified SARS-CoV-2 spike specific monoclonal IgG2a or IgG1 antibodies. f Log of the ratio of IgG2a/IgG1. Each data point corresponds to one animal. For ( a ), ( b ), ( c ), and ( f ), the horizontal bar marks the mean and error bars are SD. For ( d ) and ( e ), the horizontal bars are the geometric mean and error bars are geometric SD. Statistical analysis is an unpaired t test.

Article Snippet: Rabbit anti-SARS-CoV-2 spike (Sino Biological Inc., Beijing, China) was diluted in iBind solution (Invitrogen) at 1:1000.

Techniques: Expressing, Enzyme-linked Immunospot, Isolation, Purification, Injection, Enzyme-linked Immunosorbent Assay, Concentration Assay, Generated

Journal: Microbiology Spectrum

Article Title: Comprehensive Comparison of Seven SARS-CoV-2-Specific Surrogate Virus Neutralization and Anti-Spike IgG Antibody Assays Using a Live-Virus Neutralization Assay as a Reference

doi: 10.1128/spectrum.02314-22

Figure Lengend Snippet: Correlation analyses of sVNTs and nAb titers measured by in-house live-virus NT. Spearman correlation coefficients ( r ) and coefficients of determination ( R 2 ) are indicated. (A) cPass (GenScript); (B) SARS-CoV-2-NeutraLISA (Euroimmun); (C) ACE2-RBD neutralization assay (DiaPro); (D) TECO SARS-CoV-2 neutralization antibody assay (TECOmedical). Symbols represent samples from different cohorts, as indicated at the top. The dotted lines indicate the cutoffs of the specific assays.

Article Snippet: The four antibody assays with the highest AUC included three sVNTs: the SARS-CoV-2-NeutraLISA (Euroimmun) with an AUC of 0.92, cPass (GenScript) with an AUC of 0.92, and the TECO SARS-CoV-2 neutralization antibody assay (TECOmedical) with an AUC of 0.90. shows the specific cutoffs calculated using ROC analyses for the commercial antibody assays that would be optimal to detect a nAb titer of ≥80 with a specificity of >90%, >95%, and >99%.

Techniques: Neutralization

Journal: Microbiology Spectrum

Article Title: Comprehensive Comparison of Seven SARS-CoV-2-Specific Surrogate Virus Neutralization and Anti-Spike IgG Antibody Assays Using a Live-Virus Neutralization Assay as a Reference

doi: 10.1128/spectrum.02314-22

Figure Lengend Snippet: Correlation analyses of the IgG antibody binding tests and nAb titers measured with an in-house live-virus NT. Spearman correlation coefficients ( r ) and coefficients of determination ( R 2 ) are indicated. (A) Anti-SARS-CoV-2 QuantiVac ELISA (Euroimmun); (B) Liaison SARS-CoV-2 TrimericS IgG CLIA (DiaSorin); (C and D) SARS-CoV-2 ViraChip IgG microarray (Viramed Biotech) RBD (C) and S1-directed IgG (D). Symbols represent samples from different cohorts, as indicated at the top. The dotted lines indicate the cutoffs of the specific assays.

Article Snippet: The four antibody assays with the highest AUC included three sVNTs: the SARS-CoV-2-NeutraLISA (Euroimmun) with an AUC of 0.92, cPass (GenScript) with an AUC of 0.92, and the TECO SARS-CoV-2 neutralization antibody assay (TECOmedical) with an AUC of 0.90. shows the specific cutoffs calculated using ROC analyses for the commercial antibody assays that would be optimal to detect a nAb titer of ≥80 with a specificity of >90%, >95%, and >99%.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Microarray

Journal: Microbiology Spectrum

Article Title: Comprehensive Comparison of Seven SARS-CoV-2-Specific Surrogate Virus Neutralization and Anti-Spike IgG Antibody Assays Using a Live-Virus Neutralization Assay as a Reference

doi: 10.1128/spectrum.02314-22

Figure Lengend Snippet: Longitudinal changes in neutralizing antibodies measured by live-virus NT and sVNTs. Samples from 34 convalescent individuals for whom the first sample was taken <90 days postonset (dpo) and an additional sample ≥90 days postonset were tested with an NT and all sVNTs. (A) cPass (GenScript); (B) SARS-CoV-2-NeutraLISA (Euroimmun); (C) ACE2-RBD neutralization assay (DiaPro); (D) TECO SARS-CoV-2 neutralization antibody assay (TECOmedical). The dotted lines indicate the cutoffs of the specific assays. Connected lines show medians. Comparison of paired values was performed by the Wilcoxon test. Gray brackets show the results for NT values, and black brackets show those for the respective commercial antibody assay. ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, not significant. IH, inhibition.

Article Snippet: The four antibody assays with the highest AUC included three sVNTs: the SARS-CoV-2-NeutraLISA (Euroimmun) with an AUC of 0.92, cPass (GenScript) with an AUC of 0.92, and the TECO SARS-CoV-2 neutralization antibody assay (TECOmedical) with an AUC of 0.90. shows the specific cutoffs calculated using ROC analyses for the commercial antibody assays that would be optimal to detect a nAb titer of ≥80 with a specificity of >90%, >95%, and >99%.

Techniques: Neutralization, Inhibition

Journal: Microbiology Spectrum

Article Title: Comprehensive Comparison of Seven SARS-CoV-2-Specific Surrogate Virus Neutralization and Anti-Spike IgG Antibody Assays Using a Live-Virus Neutralization Assay as a Reference

doi: 10.1128/spectrum.02314-22

Figure Lengend Snippet: Rise in nAb values between pre- and post-COVID-19 vaccination samples measured by live-virus NT and sVNTs for 20 convalescent individuals. (A) cPass (GenScript); (B) SARS-CoV-2-NeutraLISA (Euroimmun); (C) ACE2-RBD neutralization assay (DiaPro); (D) TECO SARS-CoV-2 neutralization antibody assay (TECOmedical). The dotted lines indicate the cutoffs of the specific assays. Connected lines show medians. Comparison of paired values was performed by the Wilcoxon test. Gray brackets show the results for NT values, and black brackets show those for the respective commercial antibody assay. ****, P ≤ 0.0001.

Article Snippet: The four antibody assays with the highest AUC included three sVNTs: the SARS-CoV-2-NeutraLISA (Euroimmun) with an AUC of 0.92, cPass (GenScript) with an AUC of 0.92, and the TECO SARS-CoV-2 neutralization antibody assay (TECOmedical) with an AUC of 0.90. shows the specific cutoffs calculated using ROC analyses for the commercial antibody assays that would be optimal to detect a nAb titer of ≥80 with a specificity of >90%, >95%, and >99%.

Techniques: Neutralization