The use of a rapid ELISPOT assay to analyze peptide-specific immune responses in carcinoma patients to peptide vs. recombinant poxvirus vaccines

Cancer Immunol Immunother. 2000 Dec;49(10):517-29. doi: 10.1007/s002620000145.


An enzyme-linked immunosorbent spot (ELISPOT) assay for interferon gamma production has been used to analyze specific T cell responses to a Flu 9-mer peptide, 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 performed 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 define reproducibility 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-specific T cell precursors than do colon carcinoma patients, but these results were not statistically significant (P = 0.06). On the other hand, slightly higher CEA-specific T cell responses were observed in cancer patients with CEA-expressing carcinomas 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 first trial consisted of three monthly vaccinations of CEA peptide (designated PPP) in adjuvant. The second trial consisted of cohorts receiving three monthly vaccinations of avipox-CEA recombinant (designated 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-specific T cell responses were seen in the PPP vaccinations, while the majority of patients receiving the poxvirus CEA recombinants demonstrated increases in CEA-specific T cell responses and no increases in Flu-specific responses. CEA-specific 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 differences seen (P = 0.37) in Flu-specific responses after these two types of CEA vaccination. These results thus provide the first evidence that poxvirus recombinant-based vaccines are more potent in the initiation of tumor-antigen-specific T cell responses than vaccines employing peptide in adjuvant, when assays are conducted in an identical manner, and in defining responses to the same peptide. These results also demonstrate for the first time that an ELISPOT assay, 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.

Publication types

  • Comparative Study
  • Validation Study

MeSH terms

  • Adjuvants, Immunologic
  • Antibodies, Neoplasm / biosynthesis
  • Antibodies, Viral / biosynthesis
  • Antigen-Presenting Cells / immunology
  • Antigens, Neoplasm / genetics
  • Antigens, Neoplasm / immunology*
  • Antigens, Viral / genetics
  • Antigens, Viral / immunology
  • Avipoxvirus / immunology*
  • Blood Donors
  • Breast Neoplasms / immunology
  • Breast Neoplasms / therapy
  • Cancer Vaccines / immunology
  • Cancer Vaccines / therapeutic use*
  • Carcinoembryonic Antigen / genetics
  • Carcinoembryonic Antigen / immunology*
  • Carcinoma / immunology
  • Carcinoma / therapy*
  • Cell Line
  • Cohort Studies
  • Cytotoxicity, Immunologic
  • Digestive System Neoplasms / immunology
  • Digestive System Neoplasms / therapy
  • Enzyme-Linked Immunosorbent Assay*
  • Genes, Synthetic
  • HLA-A2 Antigen / analysis
  • Humans
  • Immunoglobulin G / biosynthesis
  • Immunotherapy, Active*
  • Interferon-gamma / analysis
  • Interferon-gamma / biosynthesis*
  • Lung Neoplasms / immunology
  • Lung Neoplasms / therapy
  • Lymphocyte Activation
  • Peptide Fragments / genetics
  • Peptide Fragments / immunology*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / immunology
  • T-Lymphocyte Subsets / immunology
  • T-Lymphocyte Subsets / metabolism*
  • Vaccines, Synthetic / immunology
  • Vaccines, Synthetic / therapeutic use*
  • Viral Vaccines / immunology
  • Viral Vaccines / therapeutic use*


  • Adjuvants, Immunologic
  • Antibodies, Neoplasm
  • Antibodies, Viral
  • Antigens, Neoplasm
  • Antigens, Viral
  • Cancer Vaccines
  • Carcinoembryonic Antigen
  • HLA-A2 Antigen
  • Immunoglobulin G
  • Peptide Fragments
  • Recombinant Fusion Proteins
  • Vaccines, Synthetic
  • Viral Vaccines
  • Interferon-gamma