Peptide Vaccine Preparation and Validation with a Bio-Assay

Introduction: Peptide vaccination constitutes a novel immunotherapeutical approach for the treatment of patients with solid tumors, lymphoma and leukemia. Moreover it might be of use in hematooncological patients for the prevention and therapy of infections like cytomegalovirus (CMV) reactivation due to immunosuppression. To meet good manufacturing practice (GMP) criteria, we introduce here a bio-assay to validate peptide vaccines for peptide content and bio-activity.

Methods: As a paradigm for peptide vaccine preparation the immunogenic CMV peptide (pp65, pos.: 495–503 NLVPMVATV) lyophilisate was resolubilized in dimethyl sulfoxide (DMSO), phosphate buffered saline (PBS) and admixed with Montanide^™ (ISA51: incomplete Freund’s adjuvant [IFA]). This mixture freshly prepared, stored at +4°C and cryopreserved at −20°C was subjected to lymphocyte peptide cultures (MLPC) followed by enzyme linked immunospot (ELISPOT) assays and flow cytometry (FACS) as bio-assay.

Results: Addition of different amounts of peptide (10–80 μg) to a MLPC resulted in the generation of interferon (IFN) gamma and granzyme B releasing CD8⁺ CMV tetramer+ T cells in a dose dependent manner. The combination of FACS and ELISPOT results allowed the definition of the peptide amount in a vaccine preparation. Storage at ±4°C over 24 hrs did not result in a significant change of the immunogenicity of the vaccine, as assessed by ELISPOT and FACS. In contrast, cryopreservation of the vaccine at −20°C resulted in a loss of immunogenicity.

Conclusion: Quantitation of tumor/viral antigen peptides admixed with adjuvants, such as IFA, is feasible through bio-assays as the modified ELISPOT/FACS assay described here, meeting GMP criteria for multi-center trials.