recombinant ag85a  (BEI Resources)

Journal: International journal of molecular sciences

Article Title: Preclinical Evaluation of TB/FLU-04L-An Intranasal Influenza Vector-Based Boost Vaccine against Tuberculosis.

doi: 10.3390/ijms24087439

Figure Lengend Snippet: Figure 1. Construction and molecular characterization of the vaccine strain. (a) Schematic representa- tion of the recombinant NS gene segment expressing antigens ESAT-6 and Ag85A of M. tuberculosis. The gray box (27–57) represents 12 aa that are shared between the NS1 and NEP proteins; the gray box (339–375) represents nucleotides encoding a random 12aa segment, fused to nucleotides encoding the ESAT-6 (green box) antigen followed by an autoproteolytic 2A cleavage site (orange box). The yellow

Article Snippet: Lymphocyte stimulation assay For specific stimulation of monkey PBMCs, recombinant Mtb proteins Ag85A and ESAT-6 (Novus Biologicals, Littleton, CO, USA) at a final concentration of 5μg/mL were used.

Techniques: Recombinant, Expressing

Journal: International journal of molecular sciences

Article Title: Preclinical Evaluation of TB/FLU-04L-An Intranasal Influenza Vector-Based Boost Vaccine against Tuberculosis.

doi: 10.3390/ijms24087439

Figure Lengend Snippet: Figure 4. Immunogenicity of TB/FLU-04L in Macaca fascicularis. (a) Study timeline and sampling schedule. (b) Vector-specific antibody response in HAI and (c) antigen-specific IFNγ response (fold change [FC] log10) after the i.n. vaccination with TB/FLU-04L: seven adult male Macaca fascicularis were immunized twice with TB/FLU-04 (7.5 log10TCID50/animal), with a three-week interval between vaccinations. Blood samples for HAI assay and PBMCs were collected before each vaccination (day 0, day 21) and three weeks after the second vaccination (day 42). To assess the antigen-specific IFNγ response, PBMCs were stimulated in vitro by M. tuberculosis antigens (ESAT-6 or Ag85A 5µg/mL) for 72 h. Medium alone or Concanavalin A were used as a negative or positive control, respectively. Following incubation, cell supernatants were collected and IFNγ levels were determined by using the BD OptEIA™Monkey ELISA Set. The data were considered statistically significant when p < 0.05 in the two-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: Lymphocyte stimulation assay For specific stimulation of monkey PBMCs, recombinant Mtb proteins Ag85A and ESAT-6 (Novus Biologicals, Littleton, CO, USA) at a final concentration of 5μg/mL were used.

Techniques: Immunopeptidomics, Sampling, Plasmid Preparation, HAI Assay, In Vitro, Positive Control, Incubation, Enzyme-linked Immunosorbent Assay, Comparison

Journal: International journal of molecular sciences

Article Title: Preclinical Evaluation of TB/FLU-04L-An Intranasal Influenza Vector-Based Boost Vaccine against Tuberculosis.

doi: 10.3390/ijms24087439

Figure Lengend Snippet: Figure 6. Protective efficacy of the BCG prime with TB/FLU-04L boost immunization against the i.v. M. tuberculosis challenge in C57BL/6 mice. (a) Study timeline and sampling schedule. C57BL/6 mice (40 animals per group) were immunized with a single s.c. dose of BCG (105 CFU; BCG group) or with one dose of BCG administered s.c. followed by one intranasal immunization with the influenza vector expressing ESAT-6 and Ag85A proteins (106 TCID50, BCG prime/TB/FLU-04 boost group) four months apart. The control group was immunized with PBS only at the time of the prime and boost immunizations (non-vaccinated control group). Three weeks after the boost immunization, mice were challenged intravenously (i.v.) with the virulent Erdman strain of M. tuberculosis (1 × 106 CFU). Four weeks after the challenge, the level of protection was measured by enumerating bacterial loads

Article Snippet: Lymphocyte stimulation assay For specific stimulation of monkey PBMCs, recombinant Mtb proteins Ag85A and ESAT-6 (Novus Biologicals, Littleton, CO, USA) at a final concentration of 5μg/mL were used.

Techniques: Sampling, Plasmid Preparation, Expressing, Control

Journal: Frontiers in Immunology

Article Title: A Recombinant BCG Vaccine Is Safe and Immunogenic in Neonatal Calves and Reduces the Clinical Disease Caused by the Respiratory Syncytial Virus

doi: 10.3389/fimmu.2021.664212

Figure Lengend Snippet: Vaccination with rBCG-N-hRSV elicits antigen-specific CD4 and CD8 T cell proliferative responses. (A, C) Study 1 PBMCs and (B, D) Study 2 Tracheobronchial lymph node cells (TBLNs) were isolated on day 7 after infection, labeled with Cell Trace Violet, and restimulated in vitro with PPD-B, Ag85A/TB10.4, N-hRSV or bRSV strain 375. Mock stimulated cultures were used as negative controls. ConA stimulated cultures were used as positive controls (not shown). Six days later, (A, B) CD4 + T and (C, D) CD8 + T cell proliferation, as measured by dilution of the Cell Trace dye, was analyzed by flow cytometry. Data represented as mean ± SEM *p<0.05 **p<0.01 as determined by 2-way ANOVA and Sidak’s multiple comparisons test.

Article Snippet: For all samples, 5x10 6 cells/mL were plated in round-bottom 96-well plates with 10 μg/mL Purified Protein Derivative from M. bovis (PPD-B); a cocktail of 10 μg/mL each recombinant Ag85A and TB10.4 (Lionex GmbH); 10 μg/mL each recombinant N from human RSV (N-hRSV); or a 0.1 MOI of bRSV strain 375.

Techniques: Isolation, Infection, Labeling, In Vitro, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: A Recombinant BCG Vaccine Is Safe and Immunogenic in Neonatal Calves and Reduces the Clinical Disease Caused by the Respiratory Syncytial Virus

doi: 10.3389/fimmu.2021.664212

Figure Lengend Snippet: Vaccination with rBCG-N-hRSV increases virus and BCG-specific IFN-γ and IL-17 secretion in PBMC cultures. (A, C) Study 1 PBMCs and (B, D) Study 2 Tracheobronchial lymph node cells (TBLNs) were isolated on day 7 post-infection, labeled with Cell Trace Violet, and restimulated in vitro with PPD-B, Ag85A/TB10.4, N-hRSV or bRSV strain 375. Mock stimulated cultures were used as negative controls. ConA stimulated cultures were used as positive controls (not shown). Six days later, cell culture supernatants were analyzed for bovine IFN-γ and IL-17A secretion using commercial ELISA kits. *p < 0.05 **p < 0.01 ***p < 0.001 ****p < 0.0001 as determined by Two-way ANOVA and Sidak’s multiple comparisons test. Data represented as mean ± SEM.

Article Snippet: For all samples, 5x10 6 cells/mL were plated in round-bottom 96-well plates with 10 μg/mL Purified Protein Derivative from M. bovis (PPD-B); a cocktail of 10 μg/mL each recombinant Ag85A and TB10.4 (Lionex GmbH); 10 μg/mL each recombinant N from human RSV (N-hRSV); or a 0.1 MOI of bRSV strain 375.

Techniques: Virus, Isolation, Infection, Labeling, In Vitro, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: IL-12 DNA Displays Efficient Adjuvant Effects Improving Immunogenicity of Ag85A in DNA Prime/MVA Boost Immunizations

doi: 10.3389/fcimb.2020.581812

Figure Lengend Snippet: Experimental design. ROA, Route of Administration; i.m., intramuscular; i.p., intraperitoneally; pCI, pCI empty; pCI-IL12, pCI expressing IL-12; pCI-Ag85A, pCI expressing Ag85A; MVA, recombinant MVA expressing an irrelevant protein; MVA85A, recombinant MVA expressing Ag85A.

Article Snippet: In vitro stimulation of cells was performed with recombinant Ag85A (BEI Resources, NIAID, NIH: Ag85A, Recombinant Protein Reference Standard, NR-49427) resuspended in phosphate-buffered saline (PBS; 1.37 mM NaCl, 27 mM KCl, 2 mM KH 2 PO 4 , 80 mM Na 2 H 2 PO 4 anhydrous in water).

Techniques: Expressing, Recombinant

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: IL-12 DNA Displays Efficient Adjuvant Effects Improving Immunogenicity of Ag85A in DNA Prime/MVA Boost Immunizations

doi: 10.3389/fcimb.2020.581812

Figure Lengend Snippet: IL-12 DNA administration enhances the immune response of the host to Ag85A. (A,B) Splenocytes were stimulated in vitro with Ag85A (5 μg/mL), ConA or PBS for 72 h. IFN-γ production was then determined by ELISA. Each bar represents the mean of IFN-γ production (PBS subtracted) ± SEM. (C,D) Anti-Ag85A IgG levels in mice serum were determined by ELISA. The samples were diluted (C) 1/100 and (D) 1/10,000 in PBS-10% fetal bovine serum. Each bar represents the mean of OD 450nm ± SEM. Comparisons among groups were determined by one-way ANOVA followed by Holm-Sidak's multiple comparison test. A value of p < 0.05 was considered statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Results are representative of two independent experiments.

Article Snippet: In vitro stimulation of cells was performed with recombinant Ag85A (BEI Resources, NIAID, NIH: Ag85A, Recombinant Protein Reference Standard, NR-49427) resuspended in phosphate-buffered saline (PBS; 1.37 mM NaCl, 27 mM KCl, 2 mM KH 2 PO 4 , 80 mM Na 2 H 2 PO 4 anhydrous in water).

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

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: IL-12 DNA Displays Efficient Adjuvant Effects Improving Immunogenicity of Ag85A in DNA Prime/MVA Boost Immunizations

doi: 10.3389/fcimb.2020.581812

Figure Lengend Snippet: The co-administration of IL-12 DNA induced a strong cellular immune response against the Ag85A in mice spleen. Splenocytes from immunized mice were stimulated in vitro with PBS or recombinant Ag85A for 16 h. Then, IFN-γ production and CD107a/b surface expression were determined by flow cytometry. (A) Gate strategy. CD4 + or CD8 + IFN-γ + T cells and CD8 + CD107a/b + T lymphocytes were identified by flow cytometry first gating on lymphocytes by light scatter; then, gating on CD4 + and CD8 + T cells; and finally, gating according to IFN-γ + and CD107a/b + expression using the AND boolean gate tool from FlowJo v10 software. Each bar represents the mean (PBS subtracted) of the (B) %CD4 + IFN-γ + cells ± SEM, (C) %CD8 + IFN-γ + cells ± SEM, (D) %CD8 + CD107a/b + cells ± SEM, (E) %CD8 + CD107a/b + IFN-γ + cells ± SEM. Comparisons among groups were determined by one-way ANOVA followed by Holm-Sidak's multiple comparison test. A value of p < 0.05 was considered statistically significant. n.s., non-significant difference, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Results are representative of two independent experiments. Representative dot plots are shown for (F) % CD4 + IFN-γ + T cells, (G) % CD8 + IFN-γ + T cells, and (H) % CD8 + CD107a/b + T lymphocytes (large quadrants). Controls (small quadrants) are shown.

Article Snippet: In vitro stimulation of cells was performed with recombinant Ag85A (BEI Resources, NIAID, NIH: Ag85A, Recombinant Protein Reference Standard, NR-49427) resuspended in phosphate-buffered saline (PBS; 1.37 mM NaCl, 27 mM KCl, 2 mM KH 2 PO 4 , 80 mM Na 2 H 2 PO 4 anhydrous in water).

Techniques: In Vitro, Recombinant, Expressing, Flow Cytometry, Software, Comparison

Journal: Vaccine

Article Title: A phase I trial evaluating the safety and immunogenicity of a candidate tuberculosis vaccination regimen, ChAdOx1 85A prime – MVA85A boost in healthy UK adults

doi: 10.1016/j.vaccine.2019.10.102

Figure Lengend Snippet: Ex vivo PBMC IFN- γ ELISpot responses (Spot Forming cells (SFC)/1 × 10 6 PBMC) to Ag85A pool of 66 peptides (I) and ChAdOx1 (II) in BCG-vaccinated healthy UK adults vaccinated with 5 × 10 9 vp ChAdOx1 85A (Starter Group (D0)) and 2.5 × 10 10 vp (Groups A (D0), B (D0) and C (D0 and D28)). Group B and C were vaccinated with MVA85A (1 × 10 8 pfu) at D56 and D119 respectively. Results for IFN- γ responses to MVA peptides for CD4 (III), and CD8 epitopes (IV) are presented for study groups B and C. Individual values are shown for each volunteer. Red dots indicate median values. The Wilcoxon matched pairs signed rank test was used for comparing responses to baseline in each group. Significant differences between groups are as follows: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Article Snippet: Recombinant Ag85A (Lionex, Germany) was used to measure Ag85A-specific IgG responses.

Techniques: Ex Vivo, Enzyme-linked Immunospot

Journal: Vaccine

Article Title: A phase I trial evaluating the safety and immunogenicity of a candidate tuberculosis vaccination regimen, ChAdOx1 85A prime – MVA85A boost in healthy UK adults

doi: 10.1016/j.vaccine.2019.10.102

Figure Lengend Snippet: Whole blood ICS Ag85A-specific responses in volunteers vaccinated with ChadOx1 85A (2.5 × 10 10 vp) (Groups A (D0), B (D0) and C (D0 and D28)). Group B and C were vaccinated with MVA85A (1 × 10 8 pfu) at D56 and D119 respectively. Percentages of CD4+ T cells producing IFN- γ , TNF- α , IL-2 and IL-17 (summed responses) are shown in figure (I) and Percentages of CD8+ T cells producing IFN- γ and TNF- α (summed responses) are shown in figure (II). Individual values are shown for each volunteer. Red dots indicate median values in each group. The Wilcoxon matched pairs signed rank test was used to detect differences between time points in the same group. Significant differences between groups are as follows: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Article Snippet: Recombinant Ag85A (Lionex, Germany) was used to measure Ag85A-specific IgG responses.

Techniques:

Journal: Vaccine

Article Title: A phase I trial evaluating the safety and immunogenicity of a candidate tuberculosis vaccination regimen, ChAdOx1 85A prime – MVA85A boost in healthy UK adults

doi: 10.1016/j.vaccine.2019.10.102

Figure Lengend Snippet: Polyfunctionality of Ag85A-specific CD4+ (I-III) and CD8+ (IV) T cells in volunteers vaccinated with ChadOx1 85A (2.5 × 10 10 vp) (Groups A (D0), B (D0) and C (D0 and D28)). Group B and C were vaccinated with MVA85A (1 × 10 8 pfu) at D56 and D119 respectively. Percentages of CD4+ T cells simultaneously producing IFN- γ (g+), TNF- α (t+) and IL-2 (2+), positive for IFN- γ and TNF- α and positive for IFN- γ and IL-2 are shown in figures (I), (II) and (III) respectively. Percentages of CD8+ T cells producing both IFN-γ and TNF-α are shown in figure (IV). Individual values are shown for each volunteer. Red dots indicate median values in each group. The Wilcoxon matched pairs signed rank test was used to detect differences between time points in the same group. Significant differences between groups are as follows: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Results of the detected cytokines combinations are presented.

Article Snippet: Recombinant Ag85A (Lionex, Germany) was used to measure Ag85A-specific IgG responses.

Techniques:

Journal: Vaccine

Article Title: A phase I trial evaluating the safety and immunogenicity of a candidate tuberculosis vaccination regimen, ChAdOx1 85A prime – MVA85A boost in healthy UK adults

doi: 10.1016/j.vaccine.2019.10.102

Figure Lengend Snippet: Serum antibody IgG responses in volunteers vaccinated with ChAdOx1 85A (Starter Group with 2.5 × 10 9 vp at D0), (A, B and C with 5 × 10 10 vp at D0 for all group in addition to D28 in Group C). Groups A and B volunteers received MVA85A (1 × 10 8 pfu) at D56 and D119 respectively. Figure (I) shows responses to recombinant Ag85A, Figure (II) shows ChAdOx1-specific IgG responses and figure (III) presents anti-MVA IgG responses. Data is presented as fold change responses calculated by dividing each time point's antibody response (measured in optical density) by its corresponding D0 response. Individual values are shown for each volunteer. Red dots indicate median values in each group. The Wilcoxon matched pairs signed rank test was used to detect differences between time points in the same group. Significant differences between groups are as follows: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Article Snippet: Recombinant Ag85A (Lionex, Germany) was used to measure Ag85A-specific IgG responses.

Techniques: Recombinant

Journal: Nature Medicine

Article Title: IFN-γ-independent immune markers of Mycobacterium tuberculosis exposure

doi: 10.1038/s41591-019-0441-3

Figure Lengend Snippet: a – c , Plasma levels of IgM ( a ), IgG ( b ) and IgA1 ( c ) reactive to PPD, Ag85A, ESAT6 and CFP10, α-crystalline (HspX), GroES and LAM were quantified in ‘resisters’ ( n = 40) and LTBI individuals ( n = 39) with AUCs determined from MFIs generated using a customized Luminex assay, generated with three dilutions and plotted for each individual with medians and interquartile ranges depicted for each group. The statistical significance was calculated using the Mann–Whitney U test, and two-tailed P values are indicated. Dotted lines represent the median level detected in HIV-negative, healthy North American volunteers.

Article Snippet: Mtb antigens used were: PPD (Statens Serum Institute), recombinant Ag85A and -B in a 1:1 ratio (BEI Resources, NR-14871 and NR 14870), recombinant ESAT6 (BEI Resources, NR-14868) and CFP10 in a 1:1 ratio (BEI Resources, NR-49425), HspX (provided by T. Ottenhoff), GroES (provided by T. Ottenhoff) and LAM (BEI Resources, NR-14848).

Techniques: Clinical Proteomics, Generated, Luminex, MANN-WHITNEY, Two Tailed Test

Journal: Nature Medicine

Article Title: IFN-γ-independent immune markers of Mycobacterium tuberculosis exposure

doi: 10.1038/s41591-019-0441-3

Figure Lengend Snippet: a , COMPASS analysis identified five functionally relevant CD4 T cell subset responses to Peptide Pool 2 (Ag85A, Ag85B and TB10.4), which are summarized in the heatmap. Rows represent study subjects and columns CD4 T cell functional subsets. The depth of shading within the heatmap represents the probability of detecting a response above background. IFN-γ-containing subsets are noted in red. b , Subject-specific COMPASS results in response to stimulation with Peptide Pool 2 were summarized using the polyfunctionality score, which weights T cell subsets that include more than one function. The total number of subjects analyzed was 41. Boxplots show median and interquartile ranges. The statistical significance was calculated using the Mann–Whitney U test, and the two-tailed P value is indicated. c , Representative flow cytometry plots from a ‘resister’ and an LTBI subject show frequencies of IFN-γ + CD40L/CD154 + IL-2 + TNF + T cells (red dots) in response to stimulation with Peptide Pool 2 or DMSO, with each experiment performed once. d , The absolute magnitude of Ag85/TB10.4-specific polyfunctional CD40L/CD154+ IL-2+ TNF+ CD4 T cells after background correction is displayed stratified by the expression of IFN-γ. These functional subsets represent the two right columns of the COMPASS plot in a . To facilitate visualization, we have not displayed a single LTBI outlier with the value of 3.41%. The total number of subjects analyzed was 41. Lines identify medians. The statistical significance was calculated using the Mann–Whitney U test, and two-tailed P values are indicated. e , COMPASS analysis identified 19 functionally relevant CD4 T cell subset responses to Mtb lysate, which are summarized in the heatmap. IFN-γ-containing subsets are noted in red. f , Polyfunctionality scores for the 41 subjects in response to Mtb lysate stimulation are shown, with boxplots representing median and interquartile ranges. The statistical significance was calculated using the Mann–Whitney U test, and the two-tailed P value is shown. g , Representative flow cytometry plots from a ‘resister’ and TST/IGRA-positive subject showing frequencies of IFN-γ + CD40L + IL-2 + TNF + T cells (red dots) in response to stimulation with Mtb lysate or DMSO are shown with each experiment performed once. h , The absolute magnitude of Mtb lysate-specific polyfunctional CD154 + IL-2 + TNF + CD4 T cells after background correction is displayed, stratified by the expression of IFN-γ for 40 subjects, with lines representing medians. These functional subsets represent the 9th and 17th columns of the COMPASS plot in e . To facilitate visualization, we have not displayed a single LTBI outlier with a value of 12.61%. The statistical significance was calculated using the Mann–Whitney U test, and unadjusted two-tailed P values are shown.

Article Snippet: Mtb antigens used were: PPD (Statens Serum Institute), recombinant Ag85A and -B in a 1:1 ratio (BEI Resources, NR-14871 and NR 14870), recombinant ESAT6 (BEI Resources, NR-14868) and CFP10 in a 1:1 ratio (BEI Resources, NR-49425), HspX (provided by T. Ottenhoff), GroES (provided by T. Ottenhoff) and LAM (BEI Resources, NR-14848).

Techniques: Functional Assay, MANN-WHITNEY, Two Tailed Test, Flow Cytometry, Expressing

Journal: PLoS ONE

Article Title: Characterising antibody avidity in individuals of varied Mycobacterium tuberculosis infection status using surface plasmon resonance

doi: 10.1371/journal.pone.0205102

Figure Lengend Snippet: Factors associated with anti-Ag85A antibody titre as measured by ELISA ^b .

Article Snippet: Recombinant protein, Ag85A (NR-14871) was obtained through BEI Resources, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Techniques: Enzyme-linked Immunosorbent Assay, Infection

Journal: PLoS ONE

Article Title: Characterising antibody avidity in individuals of varied Mycobacterium tuberculosis infection status using surface plasmon resonance

doi: 10.1371/journal.pone.0205102

Figure Lengend Snippet: Factors associated with chaotrope-based anti-Ag85A antibody avidity ^e .

Article Snippet: Recombinant protein, Ag85A (NR-14871) was obtained through BEI Resources, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Techniques: Infection

Journal: PLoS ONE

Article Title: Characterising antibody avidity in individuals of varied Mycobacterium tuberculosis infection status using surface plasmon resonance

doi: 10.1371/journal.pone.0205102

Figure Lengend Snippet: Factors associated with anti-Ag85A antibody responses determined by SPR ^h .

Article Snippet: Recombinant protein, Ag85A (NR-14871) was obtained through BEI Resources, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Techniques: Infection

Journal: PLoS ONE

Article Title: Characterising antibody avidity in individuals of varied Mycobacterium tuberculosis infection status using surface plasmon resonance

doi: 10.1371/journal.pone.0205102

Figure Lengend Snippet: Factors associated with anti-Ag85A antibody dissociation rates ^k .

Article Snippet: Recombinant protein, Ag85A (NR-14871) was obtained through BEI Resources, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Techniques: Infection