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: Gene Therapy

Article Title: Significant alterations of biodistribution and immune responses in Balb/c mice administered with adenovirus targeted to CD40(+) cells

doi: 10.1038/sj.gt.3303085

Figure Lengend Snippet: Determination of interferon-γ (IFN-γ) and interleukin-2 (IL-2) in mice receiving either Ad-NP or tAd-NP. The mice were administrated with either Ad-NP (open bar) or tAd-N (filled bar) and boosted 4 weeks later. The splenocytes were then harvested on days 0, 15 and 30 for the analyses of Ag-specific IFN-γ or IL-2 using ELISPOT as described in the ‘Materials and methods’ section. Data represent spots per 10 5 splenocytes stimulated by 10 μg ml −1 of NP. ( a ) IFN-γ levels in mice injected with either Ad-NP or tAd-NP (i.d. route); ( b ) IL-2 levels in mice injected with either Ad-NP or tAd-NP (i.d. route); ( c ) IFN-γ levels in mice injected with either Ad-NP or tAd-NP through the i.n. route); ( d ) IL-2 levels in mice injected with either Ad-NP or tAd-NP (i.n. route). All data represent the means±s.d. from four mice. Asterisks ( * ) indicate significant difference between Ad-NP and tAd-NP (same below). Statistical comparisons were conducted with the use of a two-tailed t -test, with P <0.05 being considered significant. Open bar depicts Ad-NP, while filled bar designates tAd-NP.

Article Snippet: Anti-CD40 mAb, rh-IL-4, recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), the ELISPOT kits with various controls, including IFN-γ (EL485) and IL-2 (EL402), were purchased from R&D systems, Minneapolis, MN, USA.

Techniques: Enzyme-linked Immunospot, Injection, Two Tailed Test

Journal: OncoImmunology

Article Title: An adjuvant-containing cDC1-targeted recombinant fusion vaccine conveys strong protection against murine melanoma growth and metastasis

doi: 10.1080/2162402x.2022.2115618

Figure Lengend Snippet: Figure 3. A CD4-biased cDC1 vaccine confers partial protection against metastasis. (a) Results show the number of lung metastases ± s.e.m. formed 17 days after the intravenous inoculation of B16F1-OVA melanoma cells in mice receiving PBS (control, open circles), the CD4-biased CTA1-p323-aCD103 vaccine (CTA1-II-aCD103, blue circles) or the CD4/CD8-biased CTA1-SIINFEKL-p323-aCD103 vaccine (CTA-I/II-aCD103, red circles) 3 and 10 days after tumor cell inoculation. Statistics by ANOVA (n = 5 mice per group). (b-d) show analyses of CD8+ T cells in single-cell suspensions of lungs from naïve mice (gray circles), control tumor-bearing mice (open circles), CTA1-II-aCD103-vaccinated mice (blue circles) and CTA1-I/II-aCD103-vaccinated mice (red circles) at the experimental endpoint. Results in (b) show the % of CD8+ T cells (flow cytometry); (c) shows the % of SIINFKEL tetramer MHC I+ CD8+ T cells (flow cytometry); and (d) shows the no. of SIINFEKL-specific IFN γ -producing CD8+ T cells (ELISPOT). Statistics by ANOVA (n = 5 mice per group). (e-h) show corresponding analyses of CD4+ T cells in lung suspensions, where (e) shows the % of CD4+ T cells (flow cytometry); (f) shows the no. of p323-specific IFN γ-producing CD4+ T cells (ELISPOT); (g) shows the no. of p323-specific IL 17-producing CD4+ T cells (ELISPOT); and (h) shows frequency of RORγt cells (Th17 cells) out of CD4+ T cells (flow cytometry). Statistics by ANOVA (n = 5 mice per group).

Article Snippet: Formation of IFN-γ and interleukin-17 (IL-17) was determined using the mouse IFN-γ/IL-17 double-color ELISPOT kit (Immunospot) according to the manufacturer’s instructions.

Techniques: Control, Flow Cytometry, Enzyme-linked Immunospot

Journal: OncoImmunology

Article Title: An adjuvant-containing cDC1-targeted recombinant fusion vaccine conveys strong protection against murine melanoma growth and metastasis

doi: 10.1080/2162402x.2022.2115618

Figure Lengend Snippet: Figure 4. The anti-metastatic efficacy of the cDC1 vaccine is mediated by CD8+ T cells. (a) Study design in mice receiving the CTA1-SIINFEKL-p323-aCD103 (CTA1-I/ II-aCD103) vaccine followed by depletion of CD4+ and CD8+ T cells prior to intravenous inoculation of B16F1-OVA melanoma cells. (b) Results show the number of lung melanoma metastases ± s.e.m. formed in mice receiving the CTA1-I/II-aCD103 vaccine without T cell depletion (red circles), followed by depletion of CD4+ T cells (light red circles) or followed by depletion of CD8+ T cells as outlined in (A). (c) shows analysis of SIINFEKL-specific IFN-γ producing CD8+ T cells (ELISPOT) in single cell suspensions of lungs from these mice (n = 5, ANOVA). (d) Study design in adoptive transfer of T cell subsets retrieved from vaccinated mice. CD8+ and CD4+ T cells were sorted from the lymph node of mice after administration of the CTA1-I/II-aCD103 vaccine. The sorted T cells were injected intraperitoneally to naïve mice followed by intravenous challenge with B16F1-OVA cells. (e) shows the number of lung melanoma metastases ± s.e.m. formed in mice receiving PBS (control, open circles), CD4+

Article Snippet: Formation of IFN-γ and interleukin-17 (IL-17) was determined using the mouse IFN-γ/IL-17 double-color ELISPOT kit (Immunospot) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunospot, Adoptive Transfer Assay, Injection, Control

Journal: OncoImmunology

Article Title: An adjuvant-containing cDC1-targeted recombinant fusion vaccine conveys strong protection against murine melanoma growth and metastasis

doi: 10.1080/2162402x.2022.2115618

Figure Lengend Snippet: Figure 5. The CD4/CD8-biased cDC1 vaccine elicits long-lasting anti-tumor memory. (a) Study design for analyses of T cell memory after administration of the CT1A-SIINFEKL-p323-aCD103 (CTA1-I/II-aCD103) vaccine followed by a three month resting period, prior to intravenous challenge with B16F1-OVA melanoma cells. Results in (b) show the mean number of lung melanoma metastases ± s.e.m. formed in mice that had received PBS (control, open circles) or the CTA1-I/II-aCD103 vaccine (red circles) three months prior to intravenous challenge with melanoma cells as outlined in (a). Statistics by Student´s t-test (n = 6 mice). (c) shows analysis of SIINFEKL-specific IFN-γ producing CD8+ T cells (ELISPOT) in single cell suspensions of lungs from these mice. (d) shows the quantification of SIINFEKL MHC-I tetramer+

Article Snippet: Formation of IFN-γ and interleukin-17 (IL-17) was determined using the mouse IFN-γ/IL-17 double-color ELISPOT kit (Immunospot) according to the manufacturer’s instructions.

Techniques: Control, Enzyme-linked Immunospot

Journal: FASEB bioAdvances

Article Title: AJP001, a novel helper T‐cell epitope, induces a humoral immune response with activation of innate immunity when included in a peptide vaccine

doi: 10.1096/fba.2019-00056

Figure Lengend Snippet: Vaccination with AJP001 induces antibody production and T‐cell activation in mice. A. Ang II, a mixture of Ang II and AJP001 or the AJP001‐Ang II conjugate vaccine was administered intracutaneously to 7‐week‐old female BALB/cA three times at 2‐week intervals. The anti‐Ang II IgG antibody titer in serum samples collected at 0, 2, 4, 6, and 8 weeks was detected by ELISA. The data represent the mean OD at 450 nm and the SD at each serum dilution fold (n = 3). B, C. The AJP001‐Ang II conjugate vaccine (AJP001‐Ang II 100 μg) was administered intracutaneously to 7‐week‐old female BALB/cA (B) or 7‐week‐old male C57BL/6 (C) mice three times at 2‐week intervals without any adjuvant cotreatment. The anti‐Ang II IgG antibody titer in serum sample collected at 0, 2, 4, 6, and 8 weeks was detected by ELISA. The data represent the mean OD at 450 nm and the SD at each serum dilution fold (n = 3). D. The AJP001‐Ang II conjugate vaccine was administered intracutaneously to 7‐week‐old female BALB/cA mice at a dose of 20, 100, or 500 μg per mouse three times at 2‐week intervals without any adjuvant cotreatment. The anti‐Ang II IgG antibody titer in serum samples collected at 0, 2, 4, 6, and 8 weeks was measured by ELISA. The titers are expressed as the dilution fold of the serum giving half‐maximal absorbance at 450 nm. All data are expressed as the mean ± SD (n = 3). * P < .05, ** P < .01 and *** P < .001 vs the saline group. E, F. Antigen‐specific activation of T cells in AJP001‐Ang II‐immunized mice was evaluated by an ELISpot assay. Splenocytes were isolated from AJP001‐Ang II‐immunized mice and stimulated with Angiotensin II or AJP001 at a concentration of 10 μg/mL. PMA and ionomycin (100 ng/m each) were added to positive control wells, and medium was added as a negative control. The number of IFN‐γ‐ (E) or IL‐4‐producing (F) cells was detected by counting spots using a stereomicroscope. The number of spots was quantified in the duplicate or triplicate wells of each mouse. The data represent the mean ± SD (n = 3). * P < .05 and ** P < .01 vs the saline group

Article Snippet: Mouse IFN‐γ ELISpot Development Module, Mouse IL‐4 ELISpot Development Module and ELISpot Blue Color Module were obtained from R&D Systems, Inc.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Adjuvant, Saline, Enzyme-linked Immunospot, Isolation, Concentration Assay, Positive Control, Negative Control

Journal: PLoS ONE

Article Title: Priming with Recombinant Auxotrophic BCG Expressing HIV-1 Gag, RT and Gp120 and Boosting with Recombinant MVA Induces a Robust T Cell Response in Mice

doi: 10.1371/journal.pone.0071601

Figure Lengend Snippet: Groups of mice were primed with the indicated rBCG vaccines (10 7 cfu or 10 5 cfu) and then boosted with rMVA (10 4 pfu) on day 28. One group of mice was left unvaccinated then vaccinated on day 28 with rMVA. On day 40 spleens were harvested and splenocytes pooled from 5 mice per group. (A) IFN-γ ELISPOT assays with Gag CD8 + T cells and CD4 + T cell peptides or peptide H CD8 + T cell peptide. Bars are the average and standard deviation of the average IFN-γ ELISPOT responses for the indicated individual peptides for 3 separate experiments. Asterisks indicate statistical significance of the mean IFN-γ ELISPOT responses for the individual peptides for a rBCG-Gag or rBCGpan-Gag vaccine prime and rMVA boost compared to that for the respective rBCG-Control or rBCGpan-Control vaccine prime and rMVA boost. (B) Splenocytes were stained with H-2K d and H-2D d MHC class I pentamers folded with the Gag CD8 + T cell peptide or peptide H CD8 + T cell peptide and flow cytometry was used to determine the frequency of Gag- and peptide H-specific CD8 + T cells in the splenocyte population. Bars are the average of triplicate values for Gag- and peptide H-specific CD8 + T cells expressed as a percentage of the total gated CD8 + T cell population for a single experiment. The coefficient of variation of all average values (standard deviation of the average expressed as a percentage of the average) was less than 0.01%. Asterisks indicate the statistical significance of the mean values for the percentage of Gag- or peptide H-specific CD8 + T cells for a rBCG-Gag or rBCGpan-Gag vaccine prime and rMVA boost compared to that for the respective rBCG-Control or BCGpan-Control vaccine prime and rMVA boost. Respective differences for peptide responses between groups are also indicated. *<0.01; **<0.05; Student's t-test for means of unpaired data.

Article Snippet: The Mouse IFN-γ and IL-2 ELISPOT sets (BD Pharmingen, The Scientific Group, Johannesburg, SA) were used as per manufacturer's instructions.

Techniques: Enzyme-linked Immunospot, Standard Deviation, Staining, Flow Cytometry

Journal: PLoS ONE

Article Title: Priming with Recombinant Auxotrophic BCG Expressing HIV-1 Gag, RT and Gp120 and Boosting with Recombinant MVA Induces a Robust T Cell Response in Mice

doi: 10.1371/journal.pone.0071601

Figure Lengend Snippet: Groups of mice were primed with the indicated rBCG vaccines (10 7 cfu or 10 5 cfu) then boosted with rMVA (10 4 pfu) on day 28. One group of mice was left unvaccinated then vaccinated on day 28 with rMVA. On day 40 spleens were harvested and splenocytes pooled from 5 mice per group. (A) IFN-γ ELISPOT assays with RT CD8 + T cell and CD4 + T cell peptides or peptide H CD8 + T cell peptide. Bars are the average and standard deviation of the average IFN-γ ELISPOT responses for the indicated individual peptides for 3 separate experiments. Asterisks indicate statistical significance of the mean IFN-γ ELISPOT responses for the individual peptides for a rBCG-RT or rBCGpan-RT vaccine prime and rMVA boost compared to that for the respective BCG- Control or rBCGpan-Control vaccine prime and rMVA boost. (B) Splenocytes were stained with H-2K d and H-2D d MHC class I pentamers folded with the RT CD8 + T cell peptide or peptide H CD8 + T cell peptide and flow cytometry was used to determine the frequency of RT- and peptide H-specific CD8 + T cells in the splenocyte population. Bars are the average of triplicate values for RT- and peptide H-specific CD8 + T cells expressed as a percentage of the total gated CD8 + T cell population for a single experiment. The coefficient of variation of all average values (standard deviation of the average expressed as a percentage of the average) was less than 0.01%. Asterisks indicate the statistical significance of the mean values for the percentage of CD8 + RT- or peptide H-specific T cells for a rBCG-RT or rBCGpan-RT vaccine prime and rMVA boost compared to that for the respective rBCG-Control or rBCGpan-Control vaccine prime and rMVA boost. Respective differences for peptide responses between groups are also indicated. *<0.01; **<0.05; Student's t-test for means of unpaired data.

Article Snippet: The Mouse IFN-γ and IL-2 ELISPOT sets (BD Pharmingen, The Scientific Group, Johannesburg, SA) were used as per manufacturer's instructions.

Techniques: Enzyme-linked Immunospot, Standard Deviation, Staining, Flow Cytometry

Journal: PLoS ONE

Article Title: Priming with Recombinant Auxotrophic BCG Expressing HIV-1 Gag, RT and Gp120 and Boosting with Recombinant MVA Induces a Robust T Cell Response in Mice

doi: 10.1371/journal.pone.0071601

Figure Lengend Snippet: Groups of mice were primed with either the individual rBCGpan-Gag, rBCGpan-RT or rBCGpan-gp120 vaccines (10 7 cfu) or a rBCG vaccine mix (3×10 7 cfu) prepared by mixing 10 7 cfu of the individual rBCGpan-Gag, rBCGpan-RT and rBCGpan-gp120 vaccines. Groups of mice vaccinated with the rBCGpan-Control vaccine (10 7 cfu to act as control for the individual vaccine vaccinations or 3×10 7 cfu to act as control for the mix of the individual vaccine vaccinations) served as controls. All groups of mice were then boosted with MVA (10 4 pfu) on day 28. On day 40 spleens were harvested and splenocytes pooled from 5 mice per group were used in an IFN-γ ELISPOT ( A ) or IL-2 ELISPOT ( B ) assay with Gag, RT, Env and peptide H CD8 + T cell and CD4 + T cell peptides. Bars are the average and standard deviation of the average IFN-γ ELISPOT (A) or IL-2 ELISPOT responses for the indicated individual peptides for 3 separate experiments. Statistical significance (Student's t-test for means of unpaired data) of the mean IFN-γ ELISPOT or IL-2 ELISPOT responses for the individual peptides compared to that for the control is indicated.

Article Snippet: The Mouse IFN-γ and IL-2 ELISPOT sets (BD Pharmingen, The Scientific Group, Johannesburg, SA) were used as per manufacturer's instructions.

Techniques: Enzyme-linked Immunospot, Standard Deviation

Journal: PLoS ONE

Article Title: Priming with Recombinant Auxotrophic BCG Expressing HIV-1 Gag, RT and Gp120 and Boosting with Recombinant MVA Induces a Robust T Cell Response in Mice

doi: 10.1371/journal.pone.0071601

Figure Lengend Snippet: Groups of mice were primed with either the individual rBCGpan-Gag, rBCGpan-RT or rBCGpan-gp120 vaccines (10 7 cfu) or a rBCG vaccine mix (3×10 7 cfu) prepared by mixing 10 7 cfu of the individual vaccines rBCGpan-Gag, rBCGpan-RT and rBCGpan-gp120. Groups of mice vaccinated with the BCGpan-Control vaccine (10 7 cfu to act as control for the individual vaccine vaccinations or 3×10 7 cfu to act as control for the mix of the individual vaccine vaccinations) served as controls. All groups of mice were then boosted with rMVA (10 4 pfu) on day 28. On day 40 spleens were harvested and splenocytes pooled from 5 mice per group were stimulated with Gag, RT, Env and peptide H CD8 + T cell and CD4 + T cell peptides. Culture supernatants collected at 48 h were analyzed for IFN-γ ( A ), TNF-α ( B ) and IL-2 ( C ) using a cytokine bead array assay and flow cytometric analysis. Data was expressed as pg cytokine/10 6 splenocytes. Bars are the average and standard deviation of the average cytokine released into the supernatant during stimulation with the indicated individual peptide for 3 separate experiments. Statistical significance (Student's t-test for means of unpaired data) of the mean cytokine response for the individual peptides compared to that for the control is indicated.

Article Snippet: The Mouse IFN-γ and IL-2 ELISPOT sets (BD Pharmingen, The Scientific Group, Johannesburg, SA) were used as per manufacturer's instructions.

Techniques: Standard Deviation