ORIGINAL ARTICLE Philip Arlen á Kwong-Yok Tsang á John L. Marshall Alice Chen á Seth M. Steinberg á Diane Poole Patricia Horan Hand á Jerey Schlom J. Michael Hamilton The use of a rapid ELISPOT assay to analyze peptide-speci®c immune responses in carcinoma patients to peptide vs. recombinant poxvirus vaccines Received: 12 June 2000 /Accepted: 13 July 2000 Abstract An enzyme-linked immunosorbent spot ELI- SPOT) assay for interferon c production has been used to analyze speci®c T cell responses to a Flu 9-mer pep- tide, and a 9-mer peptide of carcinoembryonic antigen CEA). Assays were performed on peripheral blood mononuclear cells PBMC) of HLA-A2-positive patients with CEA-expressing carcinomas, both before and after vaccination with CEA-based vaccines, and from HLA- A2-positive healthy blood donors. The ELISPOT assay utilized aliquots of frozen PBMC, and assays were per- formed after 24 h in culture with peptide to rule out any artifacts due to long-term in vitro stimulation cycles. An internal standard was used for each assay to de®ne re- producibility of the assay, and all samples from a given patient pre- and post-vaccination, with both the Flu and CEA peptides) were analyzed simultaneously. The results indicated a trend towards healthy blood donors having higher levels of Flu-speci®c T cell precursors than do colon carcinoma patients, but these results were not statistically signi®cant P = 0.06). On the other hand, slightly higher CEA-speci®c T cell responses were ob- served in cancer patients with CEA-expressing carcino- mas than in healthy blood donors. PBMC from two CEA-based vaccine clinical trials were analyzed for T cell responses to the same CEA peptide and to the Flu control peptide. The ®rst trial consisted of three monthly vaccinations of CEA peptide designated PPP) in adju- vant.Thesecondtrialconsistedofcohortsreceivingthree monthly vaccinations of avipox-CEA recombinant des- ignated AAA) or cohorts receiving a primary vaccination with recombinant vaccinia-CEA followed by two monthly vaccinations with avipox-CEA designated VAA). Few, if any, CEA-speci®c T cell responses were seen in the PPP vaccinations, while the majority of patients receiving the poxvirus CEA recombinants dem- onstrated increases in CEA-speci®c T cell responses and no increases in Flu-speci®c responses. CEA-speci®c IgG responses were also demonstrated in patients following recombinant CEA poxvirus vaccinations. Statistical analyses of the T cell responses to the same CEA peptide demonstrated a P value of 0.028 for the recombinant poxvirus vaccines, as compared with the peptide vaccine. There were no dierences seen P = 0.37) in Flu-speci®c responsesafterthesetwotypesofCEAvaccination.These results thus provide the ®rst evidence that poxvirus recombinant-based vaccines are more potent in the initi- ation of tumor-antigen-speci®c T cell responses than vaccines employing peptide in adjuvant, when assays are conducted in an identical manner, and in de®ning re- sponses to the same peptide. These results also demon- strateforthe®rsttimethatanELISPOTassay,performed over a 24-h period and without in vitro sensitization, can be successfully used to monitor immune responses to a tumor-associated antigen in cancer patients. Key words Avipox-CEA á ELISPOT assay á CEA peptide á Immune responses á Cancer vaccine á Carcinoma Introduction Development of antitumor vaccines for a variety of human carcinomas is the subject of numerous ongoing studies. Recent advances in immunology and molecular biology have led to the generation of vaccines composed Cancer Immunol Immunother 2000) 49: 517±529 Ó Springer-Verlag 2000 P. Arlen á K.-Y. Tsang á D. Poole á P. H. Hand á J. Schlom &) Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, 10 Center Drive, Bethesda, MD 20892-1750, USA Tel.: +1-301-496-4343; Fax: +1-301-496-2756 J. L. Marshall Georgetown University Medical Center, Vincent T. Lombardi Cancer Center, Washington, DC 20007, USA A. Chen á J. M. Hamilton Medicine Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA S. M. Steinberg Biostatistics and Data Management Section, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-8325, USA