Peptide vaccination in the presence of adjuvants in patients after hematopoietic stem cell transplantation with CD4+ T cell reconstitution elicits consistent CD8+ T cell responses.

Rationale: Patients receiving an allogeneic stem cell graft from cytomegalovirus (CMV) seronegative donors are particularly prone to CMV reactivation with a high risk of disease and mortality. Therefore we developed and manufactured a novel vaccine and initiated a clinical phase I trial with a CMV phosphoprotein 65 (CMVpp65)-derived peptide. Methods: Ten patients after allogeneic stem cell transplantation received four vaccinations at a biweekly interval. All patients were monitored for CMVpp65 antigenemia. Flow cytometry for CMV-specific CD8+ and γδ T cells as well as neutralizing anti-CMV antibodies were correlated to clinical parameters. Results: The vaccination was well tolerated. Seven of nine patients cleared CMVpp65 antigenemia after four vaccinations and are still free from antigenemia to this day. Two patients with CMV reactivation showed persisting CMV antigenemia. One patient received prophylactic vaccination and did not develop antigenemia. An increase of up to six-fold in frequency of both CMV-specific CD8+ T cells and/or Vδ2negative γδ T cells was detected. Titers of neutralizing antibodies increased up to the tenfold. Humoral and cellular immune responses correlated with clearance of CMV. Conclusion: In summary, CMVpp65 peptide vaccination for patients after allogeneic stem cell transplantation at high risk for CMV reactivation was safe, well tolerated and clinically encouraging. A study in solid-organ transplant patients is ongoing.

Cytomegalovirus vaccination of leukemia and lymphoma patients after allogeneic stem cell transplantation--validation of a peptide vaccine.

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. As a paradigm for peptide vaccine preparation the immunogenic CMV peptide 495-503 NLVPMVATV lyophilisate was resolubilized in dimethyl sulfoxide, phosphate buffered saline and admixed with Montanide. Addition of different amounts of peptide (10-80 microg) to a mixed lymphocyte peptide culture (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 degrees C over 24 h did not result in a significant change of the immunogenicity of the vaccine. In contrast, cryopreservation of the vaccine at -20 degrees C resulted in a loss of immunogenicity. Quantitation of tumor/viral antigen peptides admixed with adjuvants, such as incomplete Freund's adjuvant (IFA), is feasible through bio-assays as the modified ELISPOT/FACS assay described here, meeting GMP criteria for multi-center trials.