cmvpp65 peptides pool  (Miltenyi Biotec)

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Monitoring cell-mediated immune responses in AAV gene therapy clinical trials using a validated IFN-γ ELISpot method

doi: 10.1016/j.omtm.2021.05.012

Figure Lengend Snippet: Distribution of screening responses for LOD, confirmatory cutpoint, and PHA minimum evaluation PBMCs from 99 normal human donors were mock stimulated or stimulated with peptide pools from AAV5, FVIII, or CMVpp65 or PHA and tested by IFN-γ ELISpot. (A) The distribution of mock responses (outliers excluded) was used to establish the LOD as the statistical 95 th percentile. (B) The pooled distribution of antigen-specific over mock response ratios (outliers excluded) was used to establish the confirmatory cutpoint as the empirical 95 th percentile. (C) The distribution of PHA responses was used to establish the PHA minimum as the empirical 1 st percentile.

Article Snippet: Aliquots of lyophilized human CMVpp65 peptide pool (JPT Peptide Technologies GmbH) containing 138 peptides with >70% purity were purchased.

Techniques: Enzyme-linked Immunospot

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Monitoring cell-mediated immune responses in AAV gene therapy clinical trials using a validated IFN-γ ELISpot method

doi: 10.1016/j.omtm.2021.05.012

Figure Lengend Snippet: Intra-triplicate, intra-assay and interassay precision

Article Snippet: Aliquots of lyophilized human CMVpp65 peptide pool (JPT Peptide Technologies GmbH) containing 138 peptides with >70% purity were purchased.

Techniques: Intra Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Monitoring cell-mediated immune responses in AAV gene therapy clinical trials using a validated IFN-γ ELISpot method

doi: 10.1016/j.omtm.2021.05.012

Figure Lengend Snippet: Linearity, ULOQ, and specificity evaluation (A) Using PBMCs from 3 medium to strong CMVpp65-responding donors, IFN-γ ELISpot was performed by stimulation to CMVpp65 in 3 different runs at 7 different cell densities (400,000–6,250 PBMCs/well). Each symbol indicates a different donor (●, ▲, and ▼). The linear range was defined as the range of cell densities with responses ≥LOD, through which a linear regression yields a coefficient of correlation r 2 ≥0.90. (B) Human PBMCs from 6 donors were stimulated at varying concentrations with PHA in 3 runs. The ULOQ was defined as the 95 th percentile of the highest SFU/well responses elicited that consistently showed an intra-triplicate CV ≤30% (total of 18 data points). (C) PBMCs from 6 donors were stimulated with 4 negative control peptide pools (HIV, AAV5 pool 1, AAV5 pool 2, and FVIII pool 1) along with positive (CMVpp65 and PHA) and mock controls across 4 runs. Each bar color indicates a different donor. Error bars represent the standard deviation from the mean for each donor across the 4 runs. Specificity met acceptance criteria if the SFU/well for the negative control peptide pools were

Article Snippet: Aliquots of lyophilized human CMVpp65 peptide pool (JPT Peptide Technologies GmbH) containing 138 peptides with >70% purity were purchased.

Techniques: Enzyme-linked Immunospot, Negative Control, Standard Deviation

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Monitoring cell-mediated immune responses in AAV gene therapy clinical trials using a validated IFN-γ ELISpot method

doi: 10.1016/j.omtm.2021.05.012

Figure Lengend Snippet: LLOQ determination

Article Snippet: Aliquots of lyophilized human CMVpp65 peptide pool (JPT Peptide Technologies GmbH) containing 138 peptides with >70% purity were purchased.

Techniques:

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Monitoring cell-mediated immune responses in AAV gene therapy clinical trials using a validated IFN-γ ELISpot method

doi: 10.1016/j.omtm.2021.05.012

Figure Lengend Snippet: Trending and stability of control donor PBMCs (A–E) PBMCs from a control donor were monitored during clinical sample testing for IFN-γ responses to (A) mock, (B) FVIII pool 2, (C) FVIII pool 3, (D) CMVpp65, and (E) PHA. Additional FVIII or AAV5 peptide pools were not tested since they did not generate a positive response in this control donor. Responses were analyzed for trends using Levey-Jennings plots. The blue line indicates the limit of detection (LOD, or 60 SFU/million PBMCs). The dashed black line indicates the lower limit of quantification (LLOQ, or 222 SFU/million PBMCs). The green line indicates the mean of each dataset. The red lines indicate the upper confidence limit (UCL) and lower confidence limit (LCL) of the dataset, representing 3 multiples of the standard deviation above and below the mean of all of the data points plotted, respectively. (F) PHA responses that were too numerous to count were not plotted since no numerical values could be assigned. PBMC viability remained >79% throughout.

Article Snippet: Aliquots of lyophilized human CMVpp65 peptide pool (JPT Peptide Technologies GmbH) containing 138 peptides with >70% purity were purchased.

Techniques: Control, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Comparative Analysis of Clinical-Scale IFN-γ-Positive T-Cell Enrichment Using Partially and Fully Integrated Platforms

doi: 10.3389/fimmu.2016.00393

Figure Lengend Snippet: Comparison of ex vivo restimulation of CMV-specific T-cells by antigenic restimulation by platform . Restimulation frequencies of CMV-specific T-cells were compared between all manufacturing processes of Plus and Prodigy. Samples were collected from the PreS fractions after incubation (4 h, 37°C, 5% CO 2 ) with the HCMVpp65 peptide pool (MACS GMP PepTivator HCMV pp65, 1 μg/ml per peptide). Flow cytometric analyses were performed to identify total IFN-γ + -releasing CD3 + T-cells (A) and (CD4 + /CD8 + ) T-cell subsets (B,C) in those PreS fractions to compare both restimulation procedures.

Article Snippet: Ex vivo restimulation (4 h, 37°C, 5% CO 2 ) with the GMP-grade CMVpp65 peptide pool (MACS ® GMP PepTivator ® HCMV pp65, 1 μg/ml per peptide, Miltenyi Biotec), labeling of WBCs with the CliniMACS CCS ® Catchmatrix Reagent (37°C, 5% CO 2 ), cooling and cooled centrifugation steps (2–6°C), and labeling were carried out manually with the Plus and autonomously within the Prodigy instrument.

Techniques: Comparison, Ex Vivo, Incubation