One hundred and fifty-one peptides (18-aa long with 11-aa overlap) spanning the viral core, NS3, and NS4 coding regions of RHV were synthesized by Genemed Synthesis. Peptides were dissolved in sterile water containing 10% DMSO and incorporated into four testing pools (core, NS3_1, NS3_2, and NS4). The final concentration of each peptide in all functional assays was 2 µg/mL. Matrix pools for mapping responses against NS5A and NS5B were kindly provided by Charles Rice (The Rockefeller University) and tested at a concentration of 1 µg/mL per peptide. Peptides identified by matrix analysis were synthesized by Genemed Synthesis and reconstituted as described above. For testing of single peptides alone, the final concentration was 10 µg/mL. For the pool of MHC class I and II epitopes, the concentration of each peptide was 5 µg/mL. Peptides for the Ad5 hexon protein (PepTivator AdV5 Hexon) were acquired from Miltenyi Biotec and used according to manufacturer’s instructions.
Peptivator adv5 hexon
Manufactured by Miltenyi Biotec
Sourced in Germany
PepTivator AdV5 Hexon is a peptide pool specifically designed to stimulate T-cell responses against the Hexon protein of Adenovirus 5. It is intended for use in research applications.
Automatically generated - may contain errors
5 protocols using peptivator adv5 hexon
1
Synthetic Peptide Immunogenicity Assay
2
Isolation of Virus-Specific T Cells
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Unstimulated apheresis products containing at least 1x109 T cells from the stem cell donor or, in the case of ineligibility, a third-party donor (n=5), were collected. Thirdparty donors were haploidentical family donors except one unrelated third-party donor who was tested on two HLA alleles with 50% matching. VST were isolated using the IFN-γ CCS (Miltenyi Biotec) on a CliniMACS Prodigy® (Figure 1 ). Within this device, cells were stimulated with viral peptides (ADV: MACS GMP PepTivator AdV5 Hexon; CMV: MACS GMP PepTivator HCMV pp65 or EBV: MACS GMP PepTivator Select, all from Miltenyi Biotec) for 4 h. For multi-specific VST, the appropriate antigens were combined. Apheresis products were labeled with the CliniMACS CCS Catchmatrix Reagent, capturing the secreted IFN-γ on the surface of activated T cells. Labeled cells were separated using CliniMACS IFN-γ Enrichment Reagent, consisting of IFN-γ-specific antibody-conjugated superparamagnetic particles. The final product was ready for infusion within 2 days. Release criteria for infusion were: (i) sterility; (ii) absence of endotoxins; (iii) purity (>20% CD3+ T cells); and (iv) cell viability (>60% of CD3+ cells). A maximum of 25x103 and 50x103 T cells/kg were infused per donation from haploidentical donors and matched donors, respectively.
3
Adenoviral Vectors for Biomedical Research
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Adβgal is a ΔE1/E3 HAdV5 vector harboring a lacZ expression cassette [69 (link)]. AdL40Q is an HAdV5-based vector with a leucine to glutamine mutation of an amino acid in protein VI that decreases its membrane lytic activity [31 (link)]. Alexa555- and Alexa488-HAdV5 were generated from Adβgal by using an Alexa555 or Alexa488 Protein Labeling Kit (Life Technologies, Villebon-sur-Yvette, France) as previously described [70 (link)]. Ad2ts1 harbors a mutation in protease and results in several unprocessed capsid proteins and a hyper-stable capsid [71 (link)]. All HAdV viruses/vectors were produced in 293 or 911 cells and purified by double banding on CsCl density gradients as previously describe [14 (link)]. Vector purity typically reaches >99%. HAdV concentrations (physical particles/ml) were determined as previously described [72 (link)]. The hexon peptide pool (PepTivator AdV5 hexon, Miltenyi) is overlapping sequences of the HAdV5 hexon protein.
4
Adoptive Transfer of AdV-Specific T Cells
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Leukapheresis collections were performed in the initial HSC donors (n = 7) or related third party haploidentical donors (n = 6) for patients with UCB transplantation after informed consent. Briefly, recovered peripheral blood mononuclear cells (PBMCs) were stimulated for 6 h with a GMP adenovirus peptide pool: PepTivator-AdV5 Hexon (Miltenyi Biotec, Bergisch Gladbach, Germany). These cells were subsequently processed using the Cytokine Capture System (CCS, Miltenyi Biotec) based on an IFN-γ immunomagnetic technology on the CliniMACS device (Miltenyi Biotec) as previously described [35 (link), 37 (link)]. Quality controls including microbiological seeding and flow cytometric assessment were performed on the positive fraction. When enrichment reached trial specifications (minimum lymphocyte viability of 20% and minimum enrichment of 15% in CD4+IFN-γ+ or CD8+IFN-γ+ T cells), freshly isolated ADV-specific T cells were released (n = 12) and brought to the different French investigating centers within 8 h following cell isolation. The maximum number of infused CD3+ cells was defined according to the SFGM-TC guidelines [38 (link)]: 5 × 104 CD3/kg in a MUD setting, 1 × 104 CD3/kg in a (M)MUD setting, and in a third party haploidentical setting after UCB transplantation.
5
ELISPOT-based IFN-γ quantification
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IFN-γ–producing cells were enumerated by ELISPOT according to manufacturer instructions (BD-Biosciences, Franklin Lakes, NJ) as described,41 (link) by incubating fresh PBMC with or without antigens (recombinant NS3, 1 µg/ml; 20-mer NS3 peptide pool, 1 µg/ml each peptide;15 (link) adenoviral peptide pool, 1 µg/ml (Peptivator AdV5 Hexon, Miltenyi)) or PHA (1 µg/ml).
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