Journal: Frontiers in Immunology

Article Title: A 10-Day-Old Murine Model of Coxsackievirus A6 Infection for the Evaluation of Vaccines and Antiviral Drugs

doi: 10.3389/fimmu.2021.665197

Figure Lengend Snippet: Cytokine expression in the coxsackievirus A6 (CVA6)-A and CVA6-W infected mice. Here, 10-day-old suckling mice were inoculated with a similar load of CVA6-A and CVA6-W. The expression levels of IL-4 (A) , IL-6 (B) , IL-10 (C) , and IFN-γ (D) were detected by using enzyme-linked immunosorbent assay from the serum of the post-infected CVA6-A and CVA6-W 4 d.p.i. (n = 3). **p < 0.01, ***P < 0.001, ****P < 0.0001, n.s., insignificant difference.

Article Snippet: Purified CVA6-W of 1×104 TCID50 were diluted with the ELISA coating buffer (pH 9.6) (Solarbio, China) and added into each well of 96-well ELISA plate (Costar, USA).

Techniques: Expressing, Infection, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: A 10-Day-Old Murine Model of Coxsackievirus A6 Infection for the Evaluation of Vaccines and Antiviral Drugs

doi: 10.3389/fimmu.2021.665197

Figure Lengend Snippet: Antibody responses of the 3-day-old mice immunized with inactivated coxsackievirus A6 (CVA6)-W, CVA6-A, or PBS. Three groups of three-day-old mice (5 per group) were immunized with inactivated CVA6-W, inactivated CVA6-A, or PBS. Their sera were collected 7 and 14 days later, respectively. The levels of IgM (A) and IgG (B) were detected by the enzyme-linked immunosorbent assay method. (C) All the sera collected 14 days post-immunization were used to detect the neutralization titer against CVA6-W by performing in vitro micro-neutralization test. The inactivated CVA6-A or CVA6-W induced the suckling mice to produce neutralizing antibodies against CVA6-W.

Article Snippet: Purified CVA6-W of 1×104 TCID50 were diluted with the ELISA coating buffer (pH 9.6) (Solarbio, China) and added into each well of 96-well ELISA plate (Costar, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Neutralization, In Vitro

Journal: bioRxiv

Article Title: Gut IgA Enhances Systemic IgG Responses to Pneumococcal Vaccines Through the Commensal Microbiota

doi: 10.1101/2021.04.29.439534

Figure Lengend Snippet: IgA Enhances Systemic IgG Responses to TI Immunizations (A) ELISA of serum IgG3 (top) and IgM (bottom) to PPS from Pneumovax23 in 7 WT (solid circles) or Igha −/− (open circles) mice prior to immunization (PI) and 3 or 7 days following i.v. immunization with Pneumovax23. (B) ELISA of serum IgG3 (top) and IgM (bottom) to TNP from 5 WT or Igha −/− mice before and 3 or 5 days following i.v. immunization with TNP-Ficoll. This experiment involved WT mice purchased from Jackson Laboratories. (C) ELISA of total serum IgG3 and IgM in 15-27 WT or 28-41 Igha −/− mice at steady state. (D) Flow cytometry of CD21 and CD23 on splenic MZ B cells from representative WT or Igha −/− mice. Numbers indicate MZ B cell frequency (%) within the CD19 + population. (E) Frequency (% of CD19 + cells; left) and absolute number (right) of splenic CD21 high CD23 low MZ B cells from 10 WT mice or 11 Igha −/− mice (E). (F, G) Flow cytometry of intracellular (ic) IgG3 and extracellular (ec) IgG3 from splenic ecIgG3 + icIgG3 − switched B cells (SBCs), ecIgG3 + icIgG3 + plasmablasts (PBs) and ecIgG3 lo icIgG3 + PCs of representative WT or Igha −/− mice (F) as well as absolute number (G) of SBCs, PBs and PCs from 5 WT or 5 Igha −/− mice 5 days following i.v. immunization with TNP-Ficoll. Numbers in (F) indicate frequency within total cells. These experiments involved WT mice purchased from Jackson Laboratories. Data show representative flow plots (D, F) or summarize results from one (A, B, E, G) or 5-6 experiments (C). Results are shown with mean; a two-tailed unpaired Student’s t-test was performed when data followed a Gaussian distribution, otherwise a Mann-Whitney test was used to determine significance. *p < 0.05, **p < 0.01, ***p <0.001. See also Figure S1.

Article Snippet: For total Ig subclass quantification, Immulon 4 HBX 96-well plates (ThermoFisher Scientific) were coated with a primary antibody diluted in UltraCoat ELISA Coating Buffer (Leinco Technologies) at 50 μ l per well overnight at 4 ° C. Antigen-specific Ig subclass quantification was determined by coating Immulon 4HBX 96-well plates overnight at 4 ° C with 50 μ l/well of the following antigens diluted in PBS at 5 μ g/mL: TNP-bovine serum antigen (BSA), CPS9 or CPS14 from Streptococcus pneumoniae , Staphylococcus aureus -derived lipoteichoic acid, Salmonella typhimurium -derived LPS, or mitomycin-C- inactivated whole Streptococcus pneumonia (4 ×10 6 bacteria/mL).

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Gut IgA Enhances Systemic IgG Responses to Pneumococcal Vaccines Through the Commensal Microbiota

doi: 10.1101/2021.04.29.439534

Figure Lengend Snippet: IgA Increases Systemic IgG Responses to TD immunizations (A) ELISA of serum IgG1 to PPS from 5 WT or 7 Igha −/− mice prior to immunization (PI) and 7 or 28 days following i.v. immunization with Prevnar13. (B) Flow cytometry of intracellular (ic) IgG1 and extracellular (ec) IgG1 from splenic ecIgG1 + icIgG1 − switched B cells (SBCs), ecIgG1 + icIgG1 + plasmablasts (PBs) and ecIgG1 lo icIgG1 + PCs of representative WT or Igha −/− mice 28 days following i.v. immunization with Prevnar13. Numbers indicate frequency (% of B220 + cells) of SBCs, PBs and PCs. (C) ELISA of serum high-affinity (NP7-BSA) IgG1 and low-affinity (NP23-BSA) IgG1 from 13 WT or 11 Igha −/− mice before immunization (PI) and 7, 14, 21, 28 or 62 days following i.p. immunization with NP15-OVA and alum. (D) Affinity maturation of IgG1 calculated as a ratio of IgG1 specific to NP7-BSA versus IgG1 specific to NP23-BSA in 13 WT or 11 Igha -/- mice 7, 14, 21, 28 or 62 days following i.p. immunization with NP15- OVA and alum. (E, F) Flow cytometry of CD21 and CD23 on splenic FO B cells from representative WT or Igha −/− mice at steady state (E) as well as frequency (% of CD19+ cells) and absolute number of CD21 low CD23 high FO B cells from 10 WT or 11 Igha −/− mice (F). (G, H) Flow cytometry of B220 and IgG1 on purified splenic B cells from representative WT or Igha −/− mice incubated with medium alone (ctrl) or anti-CD40 and IL-4 for 6 days (G) as well as frequency (% of live cells) of splenic IgG1 + B220 + B cells from 6 WT or Igha −/− mice cultured as above (H). (I, J) Flow cytometry of B220 and CD138 on purified splenic B cells from representative WT or Igha −/− mice incubated with medium alone (ctrl) or anti-CD40 and IL-4 for 6 days (I) as well as frequency (% of live cells) of splenic B220 + CD138 + PBs from 6 WT or Igha −/− mice cultured as above (J). (K) ELISA of IgG1 secreted by purified splenic B cells from representative 4 WT or Igha −/− mice incubated with medium alone (ctrl) or anti-CD40 and IL-4 for 6 days. Data show one representative result (B, E, G, I) or summarize results from either a single (F), two (A, C, D, K), or three experiments (H, J) Results are shown with mean (A, F) or mean ± s.e.m. (C, D, H, J, K); a two-tailed unpaired Student’s t-test was performed when data followed a Gaussian distribution, otherwise a Mann-Whitney test was used to determine significance. *p < 0.05, **p < 0.01, ***p <0.001. See also Figure S2.

Article Snippet: For total Ig subclass quantification, Immulon 4 HBX 96-well plates (ThermoFisher Scientific) were coated with a primary antibody diluted in UltraCoat ELISA Coating Buffer (Leinco Technologies) at 50 μ l per well overnight at 4 ° C. Antigen-specific Ig subclass quantification was determined by coating Immulon 4HBX 96-well plates overnight at 4 ° C with 50 μ l/well of the following antigens diluted in PBS at 5 μ g/mL: TNP-bovine serum antigen (BSA), CPS9 or CPS14 from Streptococcus pneumoniae , Staphylococcus aureus -derived lipoteichoic acid, Salmonella typhimurium -derived LPS, or mitomycin-C- inactivated whole Streptococcus pneumonia (4 ×10 6 bacteria/mL).

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Purification, Incubation, Cell Culture, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Gut IgA Enhances Systemic IgG Responses to Pneumococcal Vaccines Through the Commensal Microbiota

doi: 10.1101/2021.04.29.439534

Figure Lengend Snippet: IgA Helps Systemic IgG Responses to Vaccines through Gut Commensals (A) ELISA of serum IgG1 to PPS from 8 WT mice or Pigr −/− mice prior to immunization (PI) and 7, 14, 21 or 28 days following i.p. immunization with Prevnar13. (B) ELISA of serum IgG1 to PPS PI or 7, 14, 21 or 28 days following i.p. immunization with Prevnar13 of ex-GF mice obtained by reconstituting 6-7 GF recipient mice with cecal content from either an SPF WT or an SPF Igha −/− donor mouse. Recipient mice were vaccinated two weeks following reconstitution. (C) ELISA of serum IgG1 to PPS prior to immunization or 7, 14, 21 or 28 days following i.p. immunization with Prevnar13 of ex-GF mice obtained by reconstituting 12 or 18 GF recipient mice with fecal material from 4 human HCs or 4 IGAD patients, respectively. Immunization was performed four weeks following reconstitution of the gut microbiota. (D) Flow cytometry of IgA + gut bacteria from 16 or 20 ex-GF mice reconstituted with fecal material from 4 healthy controls (HC) and 4 IGAD donors, respectively. Measurements were performed four weeks following reconstitution of the gut microbiota. Results are from 1 (B) or 2 (A, C, D) independent experiments. Data are shown with mean (D) or mean ± s.e.m (A, B, C); a two-tailed unpaired Student’s t-test was performed when data followed a Gaussian distribution, otherwise a Mann-Whitney test was used. *p < 0.05, **p < 0.01. See also Figure S3.

Article Snippet: For total Ig subclass quantification, Immulon 4 HBX 96-well plates (ThermoFisher Scientific) were coated with a primary antibody diluted in UltraCoat ELISA Coating Buffer (Leinco Technologies) at 50 μ l per well overnight at 4 ° C. Antigen-specific Ig subclass quantification was determined by coating Immulon 4HBX 96-well plates overnight at 4 ° C with 50 μ l/well of the following antigens diluted in PBS at 5 μ g/mL: TNP-bovine serum antigen (BSA), CPS9 or CPS14 from Streptococcus pneumoniae , Staphylococcus aureus -derived lipoteichoic acid, Salmonella typhimurium -derived LPS, or mitomycin-C- inactivated whole Streptococcus pneumonia (4 ×10 6 bacteria/mL).

Techniques: Vaccines, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Bacteria, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Gut IgA Enhances Systemic IgG Responses to Pneumococcal Vaccines Through the Commensal Microbiota

doi: 10.1101/2021.04.29.439534

Figure Lengend Snippet: IgA Restrains Systemic IgG Responses to Translocated Gut Antigens (A) ELISA of total serum IgG1 from 20 WT or 27 Igha −/− mice at steady state. (B) ELISA of serum IL-4 from 10 WT or 10 Igha −/− mice at steady state. (C) ELISA of serum IgG1 to lipoteichoic acid (LTA) from Staphylococcus aureus , CPS14 from Streptococcus pneumoniae or LPS from Salmonella typhimurium in 8 WT or 6 Igha −/− littermate mice at steady state. (D) ELISA of serum IgG1 reactive to paired colonic microbial antigens from 15 WT or 16 Igha −/− mice at steady state (fecal and serum samples from same mouse). (E) Flow cytometry of IgG1-coated small intestinal (SI) (left) or colonic (right) bacteria from 6 WT or 5-6 Igha −/− mice with or without (ctrl) incubation with paired serum. (F) Quantification of CFUs of anaerobic bacteria from homogenates of spleen, MLNs, mesenteric adipose tissue (MAT) and SI from 8-13 WT or 10-13 Igha −/− mice at steady state. (G) Taxonomic classification of anaerobic bacterial colonies isolated in the MAT from 2 WT or 7 Igha −/− mice, as determined by mass spectrometry. (H) 16S rRNA gene sequencing analysis of microbiota (MB) from colon feces of 6 WT or 7 Igha −/− co- housed littermate mice at steady state. Bacterial families detected in (G) or potentially relevant to gut homeostasis, including persistence of intraluminal SIgA, are underlined in red. Results summarize 2-3 (A, E) or 5 (F, G) independent experiments and one experiment (B, C, D, H). Data are presented as mean ± s.e.m.; a two-tailed unpaired Student’s t-test was performed when data followed a Gaussian distribution, otherwise a Mann-Whitney test was used. *p < 0.05, **p < 0.01, ***p <0.001. See also Figure S4.

Article Snippet: For total Ig subclass quantification, Immulon 4 HBX 96-well plates (ThermoFisher Scientific) were coated with a primary antibody diluted in UltraCoat ELISA Coating Buffer (Leinco Technologies) at 50 μ l per well overnight at 4 ° C. Antigen-specific Ig subclass quantification was determined by coating Immulon 4HBX 96-well plates overnight at 4 ° C with 50 μ l/well of the following antigens diluted in PBS at 5 μ g/mL: TNP-bovine serum antigen (BSA), CPS9 or CPS14 from Streptococcus pneumoniae , Staphylococcus aureus -derived lipoteichoic acid, Salmonella typhimurium -derived LPS, or mitomycin-C- inactivated whole Streptococcus pneumonia (4 ×10 6 bacteria/mL).

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Bacteria, Incubation, Isolation, Mass Spectrometry, Sequencing, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Gut IgA Enhances Systemic IgG Responses to Pneumococcal Vaccines Through the Commensal Microbiota

doi: 10.1101/2021.04.29.439534

Figure Lengend Snippet: IgA Constrains Systemic T Cell Expression of the immune inhibitor PD-1 and Anti-PD-1 Rescues Vaccine-Specific IgG Production in IgA Deficiency (A) Gene set variation analysis (GSVA) of anergy gene signatures identified by RNA-seq and differentially expressed by splenic MZ and FO B cells from 4-5 WT or 5 Igha −/− mice at steady state. (B) Heat map shows z-score of gene-expression for top 30 differentially expressed anergy genes identified by RNA-seq from gene set differentially expressed (in A) by splenic FO but not MZ B cells from 4-5 WT or 5 Igha −/− mice at steady state. Asterisks indicate highly differentially expressed genes discussed in the text. The scale bar shows color coding (blue, white, red) for z-score. (C) Flow cytometry of PD-1 and CD4 (top) or CD8 (bottom) on splenic CD3 + T cells from representative WT or Igha −/− mice at steady state. Numbers indicate frequency (%) of PD-1-expresing cells. (D) Frequency (%) of splenic CD4 + or CD8 + T cells expressing PD-1 from 5 WT or 6 Igha −/− mice determined by flow cytometry at steady state. (E) PD-L1-expression (MFI, mean fluorescence intensity) on splenic MZ B cells, FO B cells, total B-1 cells, B-1a cells, and B-1b cells from 5 WT or 6 Igha −/− mice determined by flow cytometry at steady state. (F) ELISA of serum IgG1 to PPS 14, 21, or 28 days following i.p. immunization with Prevnar13 of 13 WT or Igha −/− mice treated with anti-PD-1 mAb and of 11 WT or 12 Igha-/- mice treated with control isotype-matched mAb. Mice received 200 μ g i.p. of either anti-PD-1 or control mAb one day prior to immunization and one day after immunization, followed by injections every third day through day 19 post-immunization. (G) Frequency (%) of circulating anergic IgM lo CD21 lo B cells within IgD + CD27 − naïve B cells from 17 healthy controls (HCs), 6 IGAD-R patients and 6 IGAD-NR patients. (H) ELISA of serum sCD14 from 18 HCs, 9 IGAD-R patients, and 6 IGAD-NR patients. Results summarize one (A, B, D, E, H), two (F), or 15 independent experiments (G), or show representative flow dot plots (C). Data are presented with mean GSVA score and 95% confidence interval (A) or mean and significance (D-H). Significance was determined using limma modeling (A), Mann- Whitney test (D, E, F) or Kruskal-Wallis test with Dunn’s correction for multiple comparisons (G, H). *p < 0.05, **p < 0.01. See also Figure S6.

Article Snippet: For total Ig subclass quantification, Immulon 4 HBX 96-well plates (ThermoFisher Scientific) were coated with a primary antibody diluted in UltraCoat ELISA Coating Buffer (Leinco Technologies) at 50 μ l per well overnight at 4 ° C. Antigen-specific Ig subclass quantification was determined by coating Immulon 4HBX 96-well plates overnight at 4 ° C with 50 μ l/well of the following antigens diluted in PBS at 5 μ g/mL: TNP-bovine serum antigen (BSA), CPS9 or CPS14 from Streptococcus pneumoniae , Staphylococcus aureus -derived lipoteichoic acid, Salmonella typhimurium -derived LPS, or mitomycin-C- inactivated whole Streptococcus pneumonia (4 ×10 6 bacteria/mL).

Techniques: Expressing, RNA Sequencing, Gene Expression, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Gut IgA Enhances Systemic IgG Responses to Pneumococcal Vaccines Through the Commensal Microbiota

doi: 10.1101/2021.04.29.439534

Figure Lengend Snippet: IgA Increases Systemic BCAAs Capable of Enhancing IgG Production (A) Heat map of significantly differentially abundant plasma metabolites from 9 HCs, 4 IGAD-R patients and 3 IGAD-NR patients. Data represent standardized peak areas and have been row mean-centered and row-normalized. Metabolites highlighted in bold are discussed in the text. SPA, standardized peak area. The positioning of the methyl group on methyl-2-oxvaleric acid was unable to be differentiated between attachment to either the third or fourth carbon of the molecule. (B) Plasma concentration of leucine, isoleucine and valine in donors described in A. Standardized peak area compared to internal standard and quantitative estimation ( μ M) was performed by normalizing relative peak areas and comparing to standard curve. (C) ELISA of IgG secreted by B cells from 7-10 HCs upon exposure of peripheral blood mononuclear cells to medium alone (ctrl) or T cell-associated stimuli CD40L and IL-21 for 6 days in the presence of decreasing amounts of BCAAs. From left to right: complete BCAA-sufficient media, (120 mg/L BCAAs at a 2:5:5 ratio of L-valine, L-leucine, and L-isoleucine), BCAA-depleted media with one tenth as much BCAAs (12 mg/L BCAAs at the same ratio as above), and BCAA-deficient media with no BCAAs. (D) ELISA of IgM secreted by B cells from 10 HCs upon exposure of peripheral blood mononuclear cells for 6 days to medium alone (ctrl) or the TI ligand CpG-DNA in the presence of decreasing amounts of BCAAs as in (C). Metabolomics (A, B) were from one experiment. In vitro BCAA experiments (C, D) summarize two independent experiments involving peripheral blood mononuclear cells from 5 HCs each. Data are presented with mean and significance was determined through Kruskal-Wallis test with Dunn’s correction for multiple comparisons. *p < 0.05, **p < 0.01, ***p <0.001. See also Figure S7.

Article Snippet: For total Ig subclass quantification, Immulon 4 HBX 96-well plates (ThermoFisher Scientific) were coated with a primary antibody diluted in UltraCoat ELISA Coating Buffer (Leinco Technologies) at 50 μ l per well overnight at 4 ° C. Antigen-specific Ig subclass quantification was determined by coating Immulon 4HBX 96-well plates overnight at 4 ° C with 50 μ l/well of the following antigens diluted in PBS at 5 μ g/mL: TNP-bovine serum antigen (BSA), CPS9 or CPS14 from Streptococcus pneumoniae , Staphylococcus aureus -derived lipoteichoic acid, Salmonella typhimurium -derived LPS, or mitomycin-C- inactivated whole Streptococcus pneumonia (4 ×10 6 bacteria/mL).

Techniques: Clinical Proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay, In Vitro