This study measured the ability of a standard smallpox vaccine, given by scarification (by bifurcated needle), to induce primary human vaccinia virus-specific cytotoxic and interferon (IFN)-gamma-producing T lymphocyte responses. Because protection against smallpox may be mediated in part by T cell memory responses induced by vaccination, an analysis of the induction of primary human cytotoxic T lymphocytes (CTL) and IFN-gamma-producing T cell responses was performed. Although smallpox is no longer an epidemic threat under natural conditions, vaccination is still recommended for persons working with vaccinia viruses in the laboratory and for those who may be at risk from the potential use of smallpox virus as a bioterrorism agent. The results demonstrate that smallpox vaccine given by bifurcated needle induces strong vaccinia virus-specific CD8(+) CTL and IFN-gamma-producing T cell responses and provide baseline information useful for planning the immunologic assessment of future smallpox vaccines.

Identification of T cell epitopes is a vital but often slow and difficult step in studying the immune response to infectious agents and autoantigens. We report a spatially addressable technique for screening large numbers of T cell epitopes for both specific antigen recognition and functional activity induced. This system uses microarrays of immobilized, recombinant MHC-peptide complexes, costimulatory molecules, and cytokine-capture antibodies. The array elements act as synthetic antigen-presenting cells and specifically elicit T cell responses, including adhesion, secretion of cytokines, and modulation of surface markers. The method allows facile identification of pertinent T cell epitopes in a large number of candidates and simultaneous determination of the functional outcome of the interaction. Using this method, we have characterized the activation of human CD4 ؉ and CD8 ؉ T cells responding to vaccinia, influenza, HIV-1, and Epstein-Barr viruses.MHC class I and class II ͉ protein array ͉ T cell epitopes T cells recognize specific antigenic peptides bound to MHC proteins on the surface of antigen-presenting cells, triggering intracellular signaling and T cell effector functions. The particular functions expressed determine how successfully the infection can be eliminated, and insufficient or inappropriate responses can result in lack of clearance (1, 2), immunopathology (3), or autoimmunity (4). An important step in studying T cell responses is to identify the relevant epitopes, usually short peptides derived from pathogenic molecules that are recognized by clonotypic T cells present in the overall repertoire. The typically low frequency of specific T cells within the overall circulating T cell population and the large number of potential epitopes present in a pathogenic or autoimmune proteome present challenges to the facile and routine identification of T cell epitopes.T cell epitopes currently are identified by testing overlapping peptides from pathogenic sequences in cellular assays such as T cell-proliferation (5), target-lysis (6), bulk or intracellularcytokine secretion (7), or ELISPOT (8) assays. Screening synthetic peptides that span the entire length of a pathogenic sequence in cellular assays is time-and reagent-intensive and, in particular, consumes large numbers of T cells, which are often a limiting factor when testing clinical samples. This normally tedious and expensive process can be simplified by using an array format that can both identify pathogen-derived epitopes from many candidates and give broad functional information about the nature of the T cell response induced. MethodsRecombinant Production of Human MHC Proteins. Recombinant MHC proteins were prepared by adaptation of standard methods for multiple, parallel, small-scale preparations. Soluble, extracellular portions of HLA-DR1 (HLA-DRB1*0101 and HLA-DRA1*0101), including a uniquely reactive cysteine engineered at the C terminus of the alpha chain, were produced in S2 Schneider cells, chemically modified with PEO-maleimidebiotin (...

Peripheral T lymphocytes can be classified into two groups: naive and memory T cells. The focus of this study was to examine the duration of T-cell memory in humans. Vaccinia virus replicates in the cytoplasm of infected cells and is not thought to persist or become latent after the acute phase of infection. We identified long-lived vaccinia virus-specific memory cytotoxic T cells in adults who had been immunized against smallpox as children. Initially, we detected vaccinia virus-specific T cells in peripheral blood mononuclear cells while screening for human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses in HIV-1-seropositive subjects. These individuals had not had contact with vaccinia virus since their primary immunization in early childhood. Several vaccinia virus-specific CD4 ؉ T-cell clones were derived from these donors and characterized. Healthy, HIV-1-seronegative donors who had been immunized against smallpox many (35 to 50) years earlier were also screened for vaccinia virus-specific T-cell immunity. We found significant CD8 ؉ and CD4 ؉ cytotoxic T-cell responses to vaccinia virus after in vitro stimulation, indicating that these memory cells are maintained in vivo for many years. The peripheral blood mononuclear cells of young adults with no history of immunization against smallpox did not develop vaccinia virus-specific T-cell responses after in vitro stimulation. Precursor frequency analysis of the vaccinia virus-specific memory CD4 ؉ T cells from a donor immunized with vaccinia virus 35 years earlier revealed a frequency of 1 in 65,920 CD4 ؉ T cells. We concluded that specific vaccinia virus T-cell immunity can persist for up to 50 years after immunization against smallpox in childhood in the presumed absence of exposure to vaccinia virus.

Vaccinia virus (W) is a potent immunogen, but the nature of VV proteins involved in the activation of the immune response of the host is not yet known. By screening a lambda gtll expression library of rabbitpox virus DNA with serum from humans vaccinated against smallpox or with serum from VV-immunized animals, we identified several VV genes that encode highly antigenic viral proteins with molecular masses of 62, 39, 32, 25, 21, and 14 kDa. It was found that VV proteins of 62, 39, 25, and 21 kDa are part of the virus core, while proteins of 32 and 14 kDa are part of the virus envelope. All of these proteins were synthesized at late times postinfection. Proteins of 62 and 25 kDa were produced by cleavage of larger precursors of 95 kDa (p4a) and 28 kDa, respectively. The 21-kDa protein was the result of a cleavage of p4a, presumably at amino acid Gly-697. DNA sequence analysis, in comparison with the known nucleotide sequence of W, provided identification of the corresponding open reading frames. Expression of the viral genes in Escherichia coli was used to monitor which of the viral antigens elicit immunodominant responses and the location of antigenic domains. Three viral antigens of 62, 39, and 32 kDa exhibited immunodominant characteristics. The most antigenic sites of 62 and 39 kDa were identified at the N terminus (amino acids 132 to 295) and C terminus (last 103 amino acids), respectively. Immunization of mice with the 62-, 39-, or 14-kDa antigenic proteins conferred different degrees of protection from VV challenge. Proteins of 32 and 14 kDa induced cellular proliferative responses in VV-infected mice. Our findings demonstrate the nature of VV proteins involved in the activation of host immune responses after vaccination, provide identification of the viral gene locus, and define structural and immunological properties of these antigenic VV proteins. * Corresponding author. MATERIALS AND METHODS Materials. Restriction endonucleases, modifying enzymes, isopropyl- ,-D-thiogalactopyranoside (IPTG), and protein A were obtained from Bethesda Research Laboratories, Inc., New England Biolabs, or Boehringer Mannheim Biochemicals. The radioisotopes [3H]thymidine, [35S]methionine, 125I, and [32P]dATP were from Amersham or ICN. Goat anti-rabbit peroxidase and goat anti-human peroxidase were from Organon Teknika, Cappel. Cloning, mapping, and DNA sequence analysis. DNA manipulations were carried out as previously described (31). DNA fragments obtained by digestion of viral and phage 386

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