Journal: Microbial biotechnology

Article Title: Rectal single dose immunization of mice with Escherichia coli O157:H7 bacterial ghosts induces efficient humoral and cellular immune responses and protects against the lethal heterologous challenge

doi: 10.1111/j.1751-7915.2011.00316.x

Figure Lengend Snippet: Cellular immune responses stimulated in mice after the immunizations with EHEC O157:H7 (N°CIP 105282) BGs. Mice were immunized as described in Fig. 1 . Spleen cells (1 × 10 5 per well) were incubated for 16 h in the presence of EHEC O157:H7 BGs (specific Ag); measles inactivated Ag (strain Edmonston, non‐specific Ag) or as a negative control spleen cells were left without stimulation. The numbers of IFN‐γ‐producing cells were determined by ELISPOT assays. Rectal immunization with EHEC O157:H7 BGs effectively increased the number of IFN‐γ‐producing spleen cells after recognition of specific Ag (A). The stimulatory effect of the boost immunization was detected on day 42 (14 days after the second immunization) and remained significant until the end of experiment (B). Data represent the mean of three independent experiments ± SD (three mice per time point). P ‐values < 0.05 were considered significant and are indicated with asterisks (* P < 0.05; ** P < 0.01; *** P < 0.001). a, day of the first immunization in all groups; b, day of the second immunization in groups B1 and D1; c, day of the challenge.

Article Snippet: The number of IFN‐γ‐producing cells was determined by Mouse IFN‐γ ELISpot Kit (R&D Systems, Inc, Minneapolis, USA) following the manufacturer's instructions.

Techniques: Incubation, Negative Control, Enzyme-linked Immunospot

Journal: NPJ Vaccines

Article Title: Multicomponent intranasal adjuvant for mucosal and durable systemic SARS-CoV-2 immunity in young and aged mice

doi: 10.1038/s41541-023-00691-1

Figure Lengend Snippet: Splenocytes were isolated from mice given three immunizations with the indicated adjuvant/antigen combinations, two weeks after the final immunization (week 10) and stimulated ex vivo with 5 μg of recombinant RBD for 72 h. Levels of secreted cytokines were measured in the cell supernatant by multiplex immunoassay and compared to unstimulated cells for ( a ) IFN-γ, b IL-2, c IP-10, d TNF-α, e IL-4, f IL-5, g IL-13, h IL-6, i IL-17A, and j IL-10. (data shown as mean ± SEM ( n = 5/grp)); (* p < 0.05, ** p < 0.01 by Mann–Whitney U test shown only for select groups for simplicity: NE/IVT/10 RBD compared to other treatments in the same age group, and NE/IVT/20 RBD compared only to high dose RBD groups within the young group. Full statistical analysis is shown in Supplementary Table ).

Article Snippet: ELISpot assays were performed using an alkaline phosphatase mouse IFN-γ ELISpot Plus kit (Mabtech).

Techniques: Isolation, Ex Vivo, Recombinant, Multiplex Assay, MANN-WHITNEY

Journal: NPJ Vaccines

Article Title: Multicomponent intranasal adjuvant for mucosal and durable systemic SARS-CoV-2 immunity in young and aged mice

doi: 10.1038/s41541-023-00691-1

Figure Lengend Snippet: cLNs were isolated from mice given three immunizations with the indicated adjuvant/antigen combinations, two weeks after the final immunization (week 10) and stimulated ex vivo with 5 μg of recombinant RBD for 72 h. Levels of secreted cytokines were measured in the cell supernatant by multiplex immunoassay relative to unstimulated cells for ( a ) IFN-γ, b IL-2, c IP-10, d TNF-α, e IL-4, f IL-5, g IL-13, h IL-6, i IL-17A, and j IL-10. (data shown as mean ± SEM ( n = 5/grp)); (* p < 0.05, ** p < 0.01 by Mann–Whitney U test shown only for select groups for simplicity: NE/IVT/10 RBD compared to other treatments in the same age group, and NE/IVT/20 RBD compared only to high dose RBD groups within the young group. Full statistical analysis is shown in Supplementary Table ).

Article Snippet: ELISpot assays were performed using an alkaline phosphatase mouse IFN-γ ELISpot Plus kit (Mabtech).

Techniques: Isolation, Ex Vivo, Recombinant, Multiplex Assay, MANN-WHITNEY

Journal: NPJ Vaccines

Article Title: Multicomponent intranasal adjuvant for mucosal and durable systemic SARS-CoV-2 immunity in young and aged mice

doi: 10.1038/s41541-023-00691-1

Figure Lengend Snippet: The durability of the antigen recall responses induced by immunization was measured by comparing the levels of secreted cytokines from splenocytes (SPL) or cervical lymph node isolates (cLN) from immunized mice in response to ex vivo stimulation with 5 μg RBD for 72 h. SPL and cLN were isolated from young (8-week old) mice given three IN immunizations with 10 μg RBD with either NE or NE/IVT either 2 or 25 weeks after the last immunization (week 10 or week 33, respectively). Levels of secreted cytokines were measured in the cell supernatant by multiplex immunoassay relative to unstimulated cells for ( a ) IFN-γ, b IL-2, c IP-10, d TNF-α, e IL-4, f IL-5, g IL-13, h IL-6, i IL-17A, and j IL-10. (data shown as mean ± SEM ( n = 5/grp)); (* p < 0.05, ** p < 0.01 as compared between week 10 and week 33 for each adjuvant group by Mann–Whitney U test).

Article Snippet: ELISpot assays were performed using an alkaline phosphatase mouse IFN-γ ELISpot Plus kit (Mabtech).

Techniques: Ex Vivo, Isolation, Multiplex Assay, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Novel Synthetic Lipopeptides as Potential Mucosal Adjuvants Enhanced SARS-CoV-2 rRBD-Induced Immune Response

doi: 10.3389/fimmu.2022.833418

Figure Lengend Snippet: Specific T cell response elicited by OVA plus the novel lipopeptide. Female BALB/C mice were intranasally immunized with OVA plus LP1-14, LP1-30, LP1-34 or LP2-2 at day 0, 14 and 28. After 14 days of final immunization, mice splenic lymphocytes were stimulated by OVA 257-264 peptide or OVA 323-339 peptide respectively. OVA plus Pam 2 CSK 4 group served as positive control. (A, B) Numbers of IFN-γ spot forming cells in splenic lymphocytes were detected by ELISpot (n=5). (C, D) Representative well images of ELISpot assay. Data were presented as the mean ± SD. ns, no significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: DMEM (C11995500BT), Fetal bovine serum qualified Australia (10099141C) and Trypsin-EDTA (0.25%), phenol red (25200072) were purchased from Gibco; CD11c-FITC (117306), CD86-PerCP (105026), CD80-APC (104714) and CD40-PE (124610) were purchased from Biolegend; Goat Anti-Mouse IgG Antibody (HRP) (ab98762), Goat Anti-Mouse IgA alpha chain (HRP) (ab97235), Goat Anti-Mouse IgG1 heavychain (HRP) (ab97240), Goat Anti-Mouse IgG2a heavy chain (HRP) (ab97245), TMB ELISA Substrate (ab171523) and 450nm stop solution for TMB substrate (ab171529) were purchased from Abcam; Penicillin-Streptomycin Solution100X (C0222), and BSA, FranctionV (ST023) were purchased from Beyotime; Mouse IFN-gamma ELISpot PLUS kit (HRP) (3321-4HPW-2), Mouse TNFa ELISA kit (1217202), Mouse IL-1β ELISA kit (1210122), and Mouse IL-12p70 ELISA kit (1211202) were purchased from Dakewe; GM-CSF (315-02-10ug) and IL-4 (214-14-20ug) were purchased from PEPROTECH; Quant-iTTM PicoGreendsDNA Reagent (P7581) was purchased from Thermo Scientific; 2019-nCoV Protein RBD (DRA36) was purchased from novoprotein; Albumin from chicken egg white (A5503) was purchased from sigma.

Techniques: Positive Control, Enzyme-linked Immunospot