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Methods. 1997 Jun;12(2):117-23.

Identification of a Kb-restricted CTL epitope of beta-galactosidase: potential use in development of immunization protocols for "self" antigens.

Author information

1
Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

Abstract

The use of recombinant and synthetic vaccines in the treatment of cancer has recently been explored using model tumor associated antigens (TAA), many of which do not model the immunological state of affairs in which the TAA is expressed by normal tissues. One potentially useful model Ag is beta-galactosidase (beta-gal). Because the activity of this enzyme is so easily detectable, this gene has been inserted into a large number of recombinant viruses and tumors useful to the cancer vaccinologist. In addition, numerous transgenic mouse colonies that have tissue-specific expression of beta-gal have been developed, enabling the modeling of tolerance to "self" Ags. Since most of these mice have an H-2b background, we generated cytotoxic T lymphocytes (CTL) capable of recognizing beta-gal-expressing tumor cells of C57BL\6 origin and have determined that their restriction element is the K(b) molecule. Using an allele-specific epitope forecast to generate a panel of candidate peptides, we have determined that the K(b)-restricted sequence is DAPIYTNV and corresponds to amino acids 96-103 of the intact beta-gal molecule. A recombinant vaccinia virus (rVV-ES beta-gal96-103) was constructed that encoded the peptide epitope preceded by an endoplasmic reticulum insertion signal sequence. Tumor cells infected with this rVV were recognized by the original CTL that had been used to identify the epitope. Furthermore, splenocytes of mice immunized with a rVV encoding the full-length beta-gal molecule and restimulated with the DAPIYTNV peptide specifically recognized tumor cells expressing beta-gal. The identification of this immunogenic beta-gal sequence enables the modeling of immunization strategies in animal models of malignant disease in which the target antigen is a "self" protein.

PMID:
9184376
DOI:
10.1006/meth.1997.0461
[Indexed for MEDLINE]

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