Journal: Frontiers in Immunology

Article Title: Females with obesity exhibit greater influenza vaccine-induced immunity and protection than males in a mouse model

doi: 10.3389/fimmu.2025.1699275

Figure Lengend Snippet: Vaccine-induced antibody responses in males and females, with or without obesity. Male and female C57BL/6J mice, with or without obesity were vaccinated twice with the 2009 pandemic H1N1 influenza A virus (IAV) vaccine at a 3-week interval. At 35 days post-vaccination (dpv), blood samples were collected, and antibodies were measured in plasma. (A) IgG, (B) IgG1, (C) IgG2c, (D) IgG1 to IgG2c ratio, and (E) virus-neutralizing antibody (nAb) titers are compared (dashed line – limit of detection; n=13-15/group). Subjects that had an antibody titer below the limit of detection are shown in percent in each figure. Statistical comparisons were made using two-way analysis of variance (ANOVA) (A-E) followed by Tukey’s multiple comparisons. An asterisk (*) indicates a significant difference (p<0.05) and a hash ( # ) represents a trend (0.05≤p≤0.1). NO-M, non-obese males; NO-F, non-obese females; O-M, males with obesity; O-F, females with obesity.

Article Snippet: Secondary antibodies used were IgG (#31430, Invitrogen, CA, USA), IgG1 (#PA1-74421, Invitrogen, CA, USA), and IgG2c (#56970, Cell Signaling Technologies, MA, USA), followed by the tetramethylbenzidine (TMB) substrate reagent (#555214, BD Biosciences, CA, USA) addition (50μL/well).

Techniques: Virus, Clinical Proteomics

Journal: Frontiers in Immunology

Article Title: Females with obesity exhibit greater influenza vaccine-induced immunity and protection than males in a mouse model

doi: 10.3389/fimmu.2025.1699275

Figure Lengend Snippet: Comparisons of immunity and protection between non-responder females (i.e., NR-F) and responder females with obesity (i.e., R/O-F). Following 12 weeks on HFD, nearly 2/3 rd of females developed obesity, and the remainder were non-responders. (A) Baseline body mass; (B) body mass over 12 weeks on HFD; (C) week 12 body mass; (D) body mass index (BMI); (E) glucose tolerance test (GTT) time course (0–120 min); and (F) GTT area under the curve (AUC) are shown (n=9-19/group). After prime-boost vaccination with the inactivated 2009 H1N1 influenza vaccine, at 35 days post-vaccination (dpv), (G) IgG, (H) IgG1, (I) IgG2c, (J) IgG1/IgG2c, and (K) virus-neutralizing antibody (nAb) titers were measured (dashed line – limit of detection, n=9-15/group). Following the challenge with the drift variant of H1N1 virus, (L) absolute (g) and (M) relative (%) body mass change through 21 days post-challenge (dpc); (N) lung viral titers at 3 dpc; and (O) log 10 -transformed cytokines/chemokines concentrations at 3 dpc were compared (n=3-5/group). Statistical comparisons were made using the (A, C, D, F, G-K, N) Mann-Whitney test, (B, E, L, M) two-way repeated measures analysis of variance (ANOVA) followed by Tukey’s multiple comparisons, and (O) , unpaired T-test followed by Holm-Sidak test for multiple comparisons. An asterisk (*) indicates a significant difference (p<0.05) and a hash ( # ) represents a trend (0.05≤p≤0.1).

Article Snippet: Secondary antibodies used were IgG (#31430, Invitrogen, CA, USA), IgG1 (#PA1-74421, Invitrogen, CA, USA), and IgG2c (#56970, Cell Signaling Technologies, MA, USA), followed by the tetramethylbenzidine (TMB) substrate reagent (#555214, BD Biosciences, CA, USA) addition (50μL/well).

Techniques: Virus, Variant Assay, Transformation Assay, MANN-WHITNEY

Journal: Kidney international

Article Title: TACI deletion protects against progressive murine lupus nephritis induced by BAFF overexpression

doi: 10.1016/j.kint.2018.03.012

Figure Lengend Snippet: (A, B) Representative flow plots (A) and total number (B) of splenic B220loCD138+ plasma cells/plasmablasts in WT, Taci-/-, BAFF-Tg and Taci-/-.BAFF-Tg mice sacrificed at indicated ages. (C, D) Representative flow plots (C) and percentage (D) of BM B220negCD138+ plasma cells/plasmablasts in WT, Taci-/-, BAFF-Tg and Taci-/-.BAFF-Tg mice sacrificed at indicated ages. (A, C) Flow plots are representative of indicated genotypes sacrificed at 10-12 months, gated on total splenocytes (A) and total BM cells (C), respectively. Number equals percentage within gate. (E, F) Number of isotype-specific IgM+ (E) and IgG2c+ (F) splenic plasmablasts in WT, Taci-/-, BAFF-Tg and Taci-/-.BAFF-Tg mice. (G, H) Percentage of BM plasma cells expressing IgM (G) and IgG2c (H) by intracellular staining, from indicated genotypes sacrificed at 10-12 months. (I, J) Total serum IgM, IgG and IgA titers (I), and lgG1, lgG2b, lgG2c and lgG3 subclass titers (J) in 3-month-old mice of indicated genotype. (A-H) Error bars indicate SEM; *, P<0.05; ***, P<0.001; ****, P<0.0001; by one-way ANOVA, followed by Tukey's multiple comparison test (n=9-12 mice analyzed per group).

Article Snippet: Anti-murine antibodies used in this study include: B220 (RA3-6B2), CD4 (RM4-5), CD138 (281-2), CD80 (16-10A1), from BD Biosciences; CD62L (MEL-14), CD11c (N418), Gr-1 (RB6-8C5), CD11b (M1/70), GL7 (GL-7), from eBioscience; CD19 (ID3), CD44 (IM7), CD4 (RM4-4), IgM (MHM-88), CD21 (7E9), CD24 (M1/69), from BioLegend; FITC Goat Anti-Mouse IgG2c (SouthernBiotech).

Techniques: Clinical Proteomics, Expressing, Staining, Comparison

Journal: NAR Molecular Medicine

Article Title: Self-amplifying RNAs generated with the modified nucleotides 5-methylcytidine and 5-methyluridine mediate strong expression and immunogenicity in vivo

doi: 10.1093/narmme/ugae004

Figure Lengend Snippet: Figure 5. Reduced immunogenicity of m5U-containing saRNA vaccines in vivo versus those generated with m5C or canonical nucleotides. C57BL / 6 mice ( n = 5 per group) were immunized i.m. on Days 0 and 21 with 1 or 5 μg of canonical, m5C- or m5U-modified O V A saRNA encapsulated within LNPs. Serum collected on Da y s 14, 21 and 28 was analyzed by ELISA against O V A protein to determine the antigen-specific IgG antibody titers ( A ). Grouped data are presented as geometric mean + 95% confidence interval. Splenocytes harvested on Days 14 and 28 were analyzed by IFN- γ ELISpot when stimulated by SIINFEKL peptide ( B ). Values obtained with media alone were subtracted from those measured in the presence of the peptide. Grouped data are presented as mean + SEM. Serum collected on Day 28 was further analyzed by ELISA against O V A protein to characterize the subtype (i.e. IgG1 versus IgG2c) of the antigen-specific IgG response ( C ). The statistical significance of differences when compared to the canonical saRNA at a similar saRNA dose is shown: * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0 0 01 by one-way ANO V A followed by Dunett’s multiple comparison test. Results shown have been confirmed in a separate experiment. NT, not tested.

Article Snippet: Bound IgG was detected with goat anti-mouse IgG, IgG1 or IgG2c-horseradish peroxidase (HRP; 1:4000, Southern Biotech, Birmingham, AL, USA) prior to the addition of the substrate o -phenylenediamine dihydrochloride (Sigma– Aldrich).

Techniques: Immunopeptidomics, Vaccines, In Vivo, Generated, Modification, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Comparison

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Neutral Polymer Micelle Carriers with pH-Responsive, Endosome-Releasing Activity Modulate Antigen Trafficking to Enhance CD8 T-Cell Responses

doi: 10.1016/j.jconrel.2014.03.041

Figure Lengend Snippet: Splenocytes were isolated on day 29 from mice immunized twice, 3 weeks apart, with free ova, physical mixture, and pH-responsive conjugate. a) Cells were plated and tested for IFN-γ in vitro recall responses by incubating with CD8+ T cell epitope ova257-264 (SINFEKL). CD8+ T cells were identified by ICS based on CD8 expression, and the percentage of these cells expressing IFN-γ determined. Means ± SEM (n = 7–15) shown are pooled from three independent experiments. Representative flow cytometry dot plots of CD8+ IFN-γ+ T cells from individual mice. b) Ova-specific IgG1 (black bars) and IgG2c (white bars) antibody titres were measured on day 28 in sera from immunized mice. Data shown are pooled from four independent experiments and represent the mean of the reciprocal dilution ± SEM (n = 7–15). One-way ANOVA followed by Tukey’s Multiple Comparison Test was used for all statistical analyses. *p < 0.05.

Article Snippet: Post incubation plates were washed with PBST and incubated with biotin-conjugated anti-mouse antibodies to IgG1 (BD Pharmingen) or IgG2c (Bethyl Laboratories) at 0.005 μg/mL in 0.1% BSA/PBST for 1 h at RT.

Techniques: Isolation, In Vitro, Expressing, Flow Cytometry

Journal: Journal of neuroimmunology

Article Title: Increased maternofoetal transfer of antibodies in a murine model of systemic lupus erythematosus, but no immune activation and neuroimmune sequelae in offspring.

doi: 10.1016/j.jneuroim.2022.577927

Figure Lengend Snippet: Fig. 1. Total IgG2a levels of either the IgG2A or IgG2C allotype in BXSB/MpJ, C57BL/6 J and DBA/2JRj dams. (A) Total IgG2A in BXSB/MpJ (n = 4), C57BL/6 J (n = 6), and DBA/2JRj (n = 8), controls. Significantly higher levels of IgG2A were observed in DBA/2JRj controls compared to both C57BL/6 J (p = 0.0013) and BXSB/MpJ (p = 0.0036). (B) Total IgG2C in BXSB/MpJ (n = 4), C57BL/6 (n = 6), and DBA/2JRj (n = 8) controls. IgG2C levels in BXSB/MpJ and C57BL/6 J controls were significantly greater compared to DBA/2JRj (p = 0.0292 and p = 0.0005, respectively). Statistical significance based on multiplicity- adjusted p-value determined using one-way ANOVA with Tukey's test for multiple com parisons. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. n = sample size.

Article Snippet: Wells were next incubated 2 h at RT with biotinylated Goat anti-mouse antibody, diluted in TBS/Tw to 1 μg/ml, according to the desired antibody or allotype measured: Goat anti-mouse IgG2A (Southern Biotech, Cat.: 1083–08), Goat anti-mouse IgG2C (Southern Biotech, Cat.: 1077–08) or Goat anti-mouse Ig (Southern Biotech, Cat.: 1010–08).

Techniques:

Journal: Journal of neuroimmunology

Article Title: Increased maternofoetal transfer of antibodies in a murine model of systemic lupus erythematosus, but no immune activation and neuroimmune sequelae in offspring.

doi: 10.1016/j.jneuroim.2022.577927

Figure Lengend Snippet: Fig. 5. Anti-dsDNA antibody levels in BXSB/MpJ, C57BL/6 J, and DBA/2JRj dams. (A) Anti-dsDNA IgG2A in BXSB/MpJ (n = 4), C57BL/6 J (n = 5), and DBA/2JRj (n = 5) controls. Anti-dsDNA IgG2A was hardly detectable in all groups. (B) Anti-dsDNA IgG2C in BXSB/MpJ (n = 4), C57BL/6 J (n = 5), and DBA/2JRj (n = 8) controls. Between all groups, levels of anti- dsDNA IgG2C were diminutive and compa rable to the DBA/2JRj controls. Statistical significance was tested by one- way ANOVA with Tukey's test for multiple comparisons. No significant differences were detected. AU = arbitrary units, n = sample size.

Article Snippet: Wells were next incubated 2 h at RT with biotinylated Goat anti-mouse antibody, diluted in TBS/Tw to 1 μg/ml, according to the desired antibody or allotype measured: Goat anti-mouse IgG2A (Southern Biotech, Cat.: 1083–08), Goat anti-mouse IgG2C (Southern Biotech, Cat.: 1077–08) or Goat anti-mouse Ig (Southern Biotech, Cat.: 1010–08).

Techniques:

Journal: Frontiers in Immunology

Article Title: STING Is Required in Conventional Dendritic Cells for DNA Vaccine Induction of Type I T Helper Cell- Dependent Antibody Responses

doi: 10.3389/fimmu.2022.861710

Figure Lengend Snippet: STING is required for DNA vaccine induction of antigen-specific Th1 associated IgG 2C antibody responses. Wild type (WT), cGAS -/- and STING -/- mice were IM/EP vaccinated with a DNA vaccine (pNP) expressing influenza nucleoprotein (pNP). Sera were collected prior to vaccination (D0), and at 14 (D14), 21 (D21), and 28 days post-vaccination (D28). The concentration of anti-NP (A) IgG, (B) IgG 2C and (C) IgG 1 antibody responses was measured by ELISA. Each ELISA plate used goat anti-mouse IgG and known concentrations of mouse IgG, IgG 2C , or IgG 1 to create a standard curve to back calculate antibody titers (μg/ml). (D) The Th1:Th2 ratio was calculated as IgG 2C :IgG 1 , a higher ratio is indicative of a Th1 response. Three independent experiments consisting of 3-9 mice were performed consisting of 3-9; representative data are the average ±SD 3-9 mice/genotype. At each timepoint a one-way ANOVA was used. ****p < 0.0001 and NS, not significant.

Article Snippet: Anti-NP IgG, IgG1 and IgG 2C concentrations (μg/ml) in sera were calculated using purified mouse IgG (Southern Biotech cat. no. 0107-01), IgG 1 (Southern Biotech cat. no. 0102-01), or IgG 2C (Southern Biotech cat. no. 1078-01) to produce a standard curve.

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: STING Is Required in Conventional Dendritic Cells for DNA Vaccine Induction of Type I T Helper Cell- Dependent Antibody Responses

doi: 10.3389/fimmu.2022.861710

Figure Lengend Snippet: STING is required within conventional dendritic cells for DNA vaccine induction of antigen-specific IgG 2C antibody responses. WT, STING -/- , cDC STING cKO and cDC STING LitC mice IM/EP vaccinated with pNP. Sera were collected prior to vaccination (D0), 14 days (D14), 21 (D21) and 28 days post-vaccination (D28). Anti-NP (A) IgG, (B) IgG 2C and (C) IgG 1 antibody responses was measured by ELISA as described above. (D) The Th1:Th2 ratio was calculated as the ratio of IgG 2C :IgG 1 . A Th1:Th2 score >1 indicates a predominantly Th1 responses with a higher score indicating a strong Th1 response. Antibody secreting cells (ASCs) in WT, STING -/- , cDC STING cKO and cDC STING LitC mice vaccinated with pNP were measure 21 days post-vaccination by B cell ELIspot. Shown are (E) IgG, (F) IgG 2C and (G) IgG 1 ASCs. Three independent experiments were performed consisting of 3-7 mice; representative data shown are the average± SD of 3-7 mice/genotype. A one-way ANOVA was employed to compare groups, *p < 0.05, **p < 0.01 and NS, not significant.

Article Snippet: Anti-NP IgG, IgG1 and IgG 2C concentrations (μg/ml) in sera were calculated using purified mouse IgG (Southern Biotech cat. no. 0107-01), IgG 1 (Southern Biotech cat. no. 0102-01), or IgG 2C (Southern Biotech cat. no. 1078-01) to produce a standard curve.

Techniques: Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot