Use of a lentiviral vector encoding a HCMV-chimeric IE1-pp65 protein for epitope identification in HLA-Transgenic mice and for ex vivo stimulation and expansion of CD8(+) cytotoxic T cells from human peripheral blood cells

Hum Immunol. 2004 May;65(5):514-22. doi: 10.1016/j.humimm.2004.02.018.

Abstract

H2-deleted, HLA-A2, or HLA-B7 transgenic mice were used to identify new human cytomegalovirus (HCMV)-derived major histocompatibility complex class I-restricted epitopes. Three different approaches for mice immunization were compared for their ability to induce a cytotoxic CD8(+) T cell (CTL) response: (1). inoculation of infectious HCMV, (2). injection of immunogenic synthetic peptides, and (3). infection with a newly designed HIV-derived central DNA flap positive lentiviral vector encoding the chimeric IE1-pp65 protein (Trip-IE1-pp65). Targets pulsed with either known immunogenic peptides or computer predicted ones were used to characterize CTL. Most of the mice immunized with the pp65 (495-NLVPMVATV-503) immunodominant peptide responded after one injection whereas only two of six mice responded to two successive inoculations with HCMV. Infection of mice with Trip-IE1-pp65 induced activation and expansion of CTL directed against peptides from both pp65 and IE1 and allowed identification of new epitopes. We further demonstrated the high capacity of monocyte-macrophage cells transduced with Trip-IE1-pp65 to activate and expand CTL directed against pp65 from peripheral blood mononuclear cells of HCMV-seropositive donors. Altogether these results suggest that Trip-IE1-pp65 is a powerful construct both to characterize new epitopes in combination with human leukocyte antigen-transgenic mice immunization and to provide an alternative to the use of known infectious and noninfectious approaches to expand effector T cells for adoptive immunotherapy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antigen Presentation / immunology
  • Antigens, Ly / genetics
  • CD8-Positive T-Lymphocytes / immunology
  • Cell Line
  • Coculture Techniques
  • Cytomegalovirus / immunology
  • Epitopes, T-Lymphocyte / analysis*
  • Epitopes, T-Lymphocyte / immunology
  • Flow Cytometry
  • Genetic Vectors / genetics
  • Genetic Vectors / immunology*
  • Genetic Vectors / pharmacology
  • Histocompatibility Antigens Class I / genetics
  • Histocompatibility Antigens Class I / immunology
  • Humans
  • Immediate-Early Proteins / genetics
  • Immediate-Early Proteins / immunology*
  • Interferon-gamma / metabolism
  • Lentivirus / genetics
  • Leukocytes, Mononuclear / drug effects
  • Leukocytes, Mononuclear / immunology*
  • Lymphocyte Activation / immunology
  • Membrane Proteins / genetics
  • Mice
  • Mice, Transgenic
  • Microscopy, Fluorescence
  • Monocytes / chemistry
  • Monocytes / drug effects
  • Monocytes / immunology
  • Mutation
  • Peptide Fragments / immunology
  • Peptide Fragments / pharmacology
  • Phosphoproteins / genetics
  • Phosphoproteins / immunology*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / immunology
  • Spleen / cytology
  • Spleen / immunology
  • T-Lymphocytes, Cytotoxic / immunology*
  • Transfection
  • Viral Matrix Proteins / genetics
  • Viral Matrix Proteins / immunology*
  • Viral Proteins / genetics
  • Viral Proteins / immunology*
  • beta 2-Microglobulin / genetics

Substances

  • Antigens, Ly
  • Epitopes, T-Lymphocyte
  • Histocompatibility Antigens Class I
  • IE1 protein, cytomegalovirus
  • Immediate-Early Proteins
  • Ly6a protein, mouse
  • Membrane Proteins
  • Peptide Fragments
  • Phosphoproteins
  • Recombinant Fusion Proteins
  • Viral Matrix Proteins
  • Viral Proteins
  • beta 2-Microglobulin
  • cytomegalovirus matrix protein 65kDa
  • Interferon-gamma