Journal: Vaccines

Article Title: Longitudinal Analyses after COVID-19 Recovery or Prolonged Infection Reveal Unique Immunological Signatures after Repeated Vaccinations

doi: 10.3390/vaccines10111815

Figure Lengend Snippet: Serum antibody features in COVID-19 patients during hospitalization: Graphs show serum anti-S-RBD IgM ( A ) and IgG ( B ) levels for each disease severity ( n = 9 patients/mild group; 10 patients/moderate I group; 9 patients/moderate II patients). Blue and red dots indicate admission and discharge, respectively; the duration of hospitalization is indicated at the bottom of each panel. Graphs show the correlation between anti-S-RBD IgM or IgG and neutralizing antibody titer ( C ) and age, as well as the change in the titer against the WT strain in patients infected with the WT and VOCs ( D ). Each dot denotes disease severity, and Spearman’s correlation coefficients are shown within the graph. Gray areas indicate a 95% confidence interval (CI) for the total. ( E ) Dot plot indicates the neutralizing potency against the WT strain by sex. Graphs show the serum-neutralizing antibody titer against WT ( F ), Kappa ( G ), and Delta ( H ) variants at each severity (moderate II is classified by the infected strains, n = 9 patients/mild group; 10 patients/moderate I group; 9 patients/WT-infected moderate II group; 8 patients/VOC-infected moderate II group). Statistical significances were determined using the Tukey–Kramer test and Welch’s t -test. All horizontal bars show mean values. Cross, deceased; #, long-term observed healthcare worker. * p < 0.05; *** p < 0.005; ns, not significant.

Article Snippet: Isolated PBMCs were incubated with His-tagged SARS-CoV-2 S-RBD recombinant protein (Cell Signaling Technology, Danvers, MA, USA) for 30 min on ice.

Techniques: Infection

Journal: Vaccines

Article Title: Longitudinal Analyses after COVID-19 Recovery or Prolonged Infection Reveal Unique Immunological Signatures after Repeated Vaccinations

doi: 10.3390/vaccines10111815

Figure Lengend Snippet: Longitudinal analyses of serum neutralizing antibody activities and PBMCs in a patient with high neutralizing potency: ( A ) Concentration of anti-S-RBD IgM and IgG in the serum of a healthcare worker (indicated by # in ) upon infection. ( B ) Fluctuation of each neutralizing antibody titer from onset to post-vaccination ( n = 1). ( C ) Representative FACS plots of cell population are shown in panels. The numbers indicate the positive rate of each cell population. Graphs show the absolute number of IgG + S-RBD + Bmems ( D ) and the frequency of lymphocyte ( E ), CD19 + CD27 + CD38 high , and IgG + Bmems (( F ); n = 1, 3–5 technical replicates). Black dots indicate technical replicates. Each number indicates the mean.

Article Snippet: Isolated PBMCs were incubated with His-tagged SARS-CoV-2 S-RBD recombinant protein (Cell Signaling Technology, Danvers, MA, USA) for 30 min on ice.

Techniques: Concentration Assay, Infection

Journal: Vaccines

Article Title: Longitudinal Analyses after COVID-19 Recovery or Prolonged Infection Reveal Unique Immunological Signatures after Repeated Vaccinations

doi: 10.3390/vaccines10111815

Figure Lengend Snippet: Affinity maturation of S-RBD-specific memory B-cells after vaccination: Pie charts show the distribution of heavy chain genes ( A ) and light chain genes ( B ) from onset to post-vaccination, comparing patients with the recovered individual ( n = 1) and a prolonged COVID-19 patient ( n = 1). The number in the inner circle indicates the number of sequenced clones. ( C ) Graphs show the number of somatic hypermutations in each antibody gene. Statistical significances were determined using Tukey–Kramer test. ( D ) Graphs show the distribution of CDR3 length in each antibody gene (heavy chains; n = 34–45 clones/recovered individual, 14 clones/prolonged COVID-19 patient; light chains; 18–66 clones/recovered individual, 15 clones/prolonged COVID-19 patient).

Article Snippet: Isolated PBMCs were incubated with His-tagged SARS-CoV-2 S-RBD recombinant protein (Cell Signaling Technology, Danvers, MA, USA) for 30 min on ice.

Techniques: Clone Assay

Journal: bioRxiv

Article Title: Cutaneous jet-injection of naked mRNA vaccine induces robust immune responses without systemic vaccine spillage

doi: 10.1101/2023.02.27.530188

Figure Lengend Snippet: (a-d) Balb/C mice were PYRO-injected with naked spike mRNA at both flanks in a prime-boost setting separated by a 3-week interval. 5 weeks after the prime, blood plasma and splenocytes were collected for evaluating humoral and cellular immunity. (a,b) Anti-spike IgG ELISA absorbance vs. plasma dilution curves . (c) The ability of plasma in blocking the binding between spike-RBD and ACE2 proteins. (d) Quantification of spike-specific INFγ-positive splenocytes. Data represent the mean ± SEM (n=4). **p <0.01, *p <0.05 vs. NT, non-repeated ANOVA followed by Dunnett’s test in (d) . (e-l) Vaccination in NHPs. (e) Injections and sampling schedule. (f,g) Appearance of PYRO injection site on the back of Cynomolgus monkeys (f) compared to Balb/C mice (g) . (h) Anti-spike IgG titers in NHPs PYRO-injected with buffer or mRNA solution. (i) Binding inhibition of ACE2 and SARS-CoV-2 RBD by plasma of immunized monkeys. (j-l) Toxicity evaluation in PYRO-injecting monkeys receiving either buffer or spike mRNA. (j) Body temperature measured immediately before and 24 h post dosing, after each of the 3 doses. (k) Change in body weight. (l) Plasma levels of proinflammatory cytokines before and 24 h after the first dose. n.s.: nonsignificant, **p <0.01, *p <0.05 vs. buffer treatment in unpaired Student’s t -test. BLQ: below limit of quantification.

Article Snippet: SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit was obtained from Cell Signaling Technologies.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Blocking Assay, Binding Assay, Sampling, Inhibition

Journal: Virulence

Article Title: Repertoires of SARS-CoV-2 epitopes targeted by antibodies vary according to severity of COVID-19

doi: 10.1080/21505594.2022.2073025

Figure Lengend Snippet: Prevalence of IgG specific for betacoronavirus RBDs in study cohort. Plasma was assayed by ELISA using recombinant, multimeric RBD as antigen. (a) SARS-COV-2 spike 319-591 with p values from ANOVA with Tukey HSD for all pairwise comparisons shown beneath. (b) SARS-CoV spike 384-655 with p values shown as above, (c) MERS spike 306-577. Each point indicates an individual participant. Blue bars show median values. N = 7–30. (d) Comparison of absorbance values for MERS and SARS-CoV RBD binding IgG for all participants. Each point indicates an individual participant, n = 124. The orange circle indicates participants with detectable amounts of MERS RBD-binding IgG, showing correlation with SARS-CoV-binding IgG in this subset. IgG specific to recombinant, multimeric RBD from seasonal betacoronaviruses, (e) OC43 spike 315-675, (f) HKU1 spike 307-675 assayed by ELISA as above.

Article Snippet: ELISAs were prepared using the following recombinant antigens (expressed in HEK 293 cells with 8 × His tags) coated onto 96-well plates: SARS-CoV-2 Spike RBD (multimeric, 319–591, Cell Signaling Technology #17862), SARS-CoV RBD (multimeric, 306–577), MERS-CoV RBD (multimeric, 364–655), OC43 Spike RBD (multimeric, 315–675), HKU1 Spike RBD (multimeric, 307–675).

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

Journal: Virulence

Article Title: Repertoires of SARS-CoV-2 epitopes targeted by antibodies vary according to severity of COVID-19

doi: 10.1080/21505594.2022.2073025

Figure Lengend Snippet: Prevalence of IgGs specific for SARS-CoV-2 peptides. (b) Individual peptides with significant variation between groups ( p < 0.05, ANOVA, n = 7–30). Bars denote groups with significant differences by pairwise comparison (Tukey HSD, p < 0.05).

Article Snippet: ELISAs were prepared using the following recombinant antigens (expressed in HEK 293 cells with 8 × His tags) coated onto 96-well plates: SARS-CoV-2 Spike RBD (multimeric, 319–591, Cell Signaling Technology #17862), SARS-CoV RBD (multimeric, 306–577), MERS-CoV RBD (multimeric, 364–655), OC43 Spike RBD (multimeric, 315–675), HKU1 Spike RBD (multimeric, 307–675).

Techniques:

Journal: Virulence

Article Title: Repertoires of SARS-CoV-2 epitopes targeted by antibodies vary according to severity of COVID-19

doi: 10.1080/21505594.2022.2073025

Figure Lengend Snippet: Comparison between peptide seropositivity frequencies in the primary dataset with an independent cohort. Peptides that showed >20 percentage point difference in frequency of seropositivity between participants in the Chelsea survey who had been hospitalized and those who reported exposure to SARS-CoV-2 are shown in green. The differences in frequency of seropositivity for IgG specific to those peptides between acutely hospitalized patients and people with no history of infection are shown in blue.

Article Snippet: ELISAs were prepared using the following recombinant antigens (expressed in HEK 293 cells with 8 × His tags) coated onto 96-well plates: SARS-CoV-2 Spike RBD (multimeric, 319–591, Cell Signaling Technology #17862), SARS-CoV RBD (multimeric, 306–577), MERS-CoV RBD (multimeric, 364–655), OC43 Spike RBD (multimeric, 315–675), HKU1 Spike RBD (multimeric, 307–675).

Techniques: Infection

Journal: Frontiers in Immunology

Article Title: Single-Cell Immunogenomic Approach Identified SARS-CoV-2 Protective Immune Signatures in Asymptomatic Direct Contacts of COVID-19 Cases

doi: 10.3389/fimmu.2021.733539

Figure Lengend Snippet: Study approach and distinct antibody responses to SARS-CoV-2 along with neutralization assay. (A) Outline delineating the experimental workflow. (B) Box plots showing the anti-SARS-CoV-2 total, IgA, IgM, and IgG antibody proportions in serum samples from controls, symptomatic- and asymptomatic-infected individuals and in contacts (C) . Pie chart showing the overall immunoglobulin response across subject groups. (D) Box plot depicting the percent inhibition of neutralizing antibodies in an in vitro spike RBD-ACE2 interaction-based surrogate virus neutralization assay. Statistical comparisons were performed using unpaired Wilcoxon test. p-values are written against respective comparisons, where n = number of individuals; IgA, immunoglobulin A; IgM, immunoglobulin M; IgG, immunoglobulin G; control (n = 14), infected (n = 23), symptomatic (n = 10), asymptomatic (n = 13), and contact (n = 24).

Article Snippet: For IgA and IgM quantification, receptor binding domain (RBD) (SARS-CoV-2 spike RBD recombinant protein, mFc-Tag, CST # 41701S) antigen at a concentration of 200 ng/well was coated in a 96-well high binding microtiter plate (HIMEDIA-EP1) in 1× Tris-buffered saline (TBS) pH-7.4 for 2 h at 37°C.

Techniques: Neutralization, Infection, Inhibition, In Vitro

Journal: medRxiv

Article Title: SARS-CoV-2 specific immune-signature in direct contacts of COVID-19 cases protect them from contracting disease: A Retrospective Study

doi: 10.1101/2021.03.11.21253367

Figure Lengend Snippet: A , Box plots showing the anti-SARS-CoV-2 IgA, IgM and IgG antibody proportions in serum samples from control, infected and contact along with B , Serum cytokine levels in pg/ml (Eotaxin, G-CSF, IL-7, MIF and MIP 1-□) determined by bio plex. C , The area under the receiver-operating characteristic (ROC) curve of the cytokines having more than 0.8 AUC. D , Box plot depicting the percent inhibition of neutralizing antibodies in an in -vitro spike RBD-ACE2 interaction based Surrogate Virus Neutralization assay. Statistical comparisons were performed using unpaired Wilcoxon test, p-values are written against respective comparisons. Where, n = number of individuals, IgA-Immunoglobulin A, IgM-Immunoglobulin M and IgG-Immunoglobulin G, control (n-14), infected (n-23, Symptomatic n-10 and Asymptomatic n-13) and contact (n-24).

Article Snippet: For IgA and IgM quantification, RBD (SARS-CoV-2 spike RBD recombinant protein, mFc-Tag, CST # 41701S) antigen at a concentration of 200ng/well was coated in a 96-well high binding micro titre plate (HIMEDIA-EP1) in 1x TBS pH-7.4 for 2h at 37□.

Techniques: Infection, Inhibition, In Vitro, Neutralization