ABSTRACT
Induction of CD8+ cytotoxic T lymphocytes (CTLs) specific for human papillomavirus (HPV) antigens provides an attractive strategy for immunotherapy of HPV-related cancers in humans. In this study, we investigated the potential of utilizing soluble E7 protein of HPV 16 in an adjuvant formulation, PROVAX as a vaccine against a progressively growing E7 transfected K1735-X21 (H-2k) metastatic melanoma cells (HOPE2) in a mouse model. Vaccination of HOPE2 tumor bearing mice (C3H) with E7 protein in PROVAX resulted in significant inhibition of tumor growth, compared to mice vaccinated with E7 in Alum or saline. In vivo depletion of CD8+ or CD4+ cells indicated that CD8+ cells are the major effector cells in mediating the anti-tumor activity in this model. Furthermore, E7-specific CTL activity in vitro was detected in tumor bearing mice vaccinated with E7-PROVAX. Our studies suggest that recombinant HPV antigens in combination with PROVAX could serve as an effective subunit vaccine to stimulate tumor specific CD8+ T cell mediated immunity against HPV-related cancers.
Subject(s)
Adjuvants, Immunologic/administration & dosage , Melanoma, Experimental/prevention & control , Oncogene Proteins, Viral/immunology , Vaccination , Animals , CD8-Positive T-Lymphocytes/immunology , Cytotoxicity, Immunologic , Female , Gene Expression/genetics , Immunity, Cellular/immunology , Melanoma, Experimental/immunology , Mice , Mice, Inbred C3H , Oncogene Proteins, Viral/genetics , Papillomaviridae/immunology , Papillomavirus E7 Proteins , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Remission Induction , T-Lymphocytes, Cytotoxic/immunology , Transfection/genetics , Tumor Cells, Cultured/cytology , Tumor Cells, Cultured/metabolism , Viral Vaccines/chemistry , Viral Vaccines/therapeutic useABSTRACT
The mammalian CAD gene codes for a 240-kDa multifunctional protein that catalyzes the first three steps of de novo pyrimidine biosynthesis. Previously, the longest cDNA construct available was missing approximately 500 bp of coding sequence at the 5' end, thereby lacking the sequence to encode the entire carbamylphosphate synthetase (CPSase) domain. Here, a complete CAD hamster cDNA is constructed, placed into a mammalian expression vector, and transfected into hamster cells deficient in CAD. Transfectants show coordinately restored levels of all three enzyme activities and the presence of full-length CAD protein. A derivative construct of the CAD cDNA was generated that should encode only the CPSase domain. When transfected into mammalian cells, a protein was synthesized that had significant CPSase activity both in vivo and in vitro. The two constructs generated in this study will facilitate the study of CAD structure, function, and allosteric regulation.
Subject(s)
Aspartate Carbamoyltransferase/genetics , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics , Dihydroorotase/genetics , Multienzyme Complexes/genetics , Pyrimidines/biosynthesis , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Cloning, Molecular , Cricetinae , Genetic Complementation Test , Genetic Vectors/genetics , Molecular Sequence Data , Protein ConformationABSTRACT
The ability of cAMP to inhibit isoleucyl-tRNA synthetase (IRS) formation has been demonstrated in wild type K-12 Escherichia coli and two adenyl-cyclase (cya) mutants. cAMP appeared not to have any effect on either the valyl- or arginyl-tRNA synthetase (VRS and ARS respectively). Addition of cAMP led to a reduction in rate of IRS synthesis but not VRS or ARS. Furthermore, derepression of IRS and VRS by isoleucine limitation was completely prevented by cAMP.
