Addition of TAT protein transduction domain and GrpE to human p53 provides soluble fusion proteins that can be transduced into dendritic cells and elicit p53-specific T-cell responses in HLA-A*0201 transgenic mice.

The protein p53 has been shown to be an efficient tumour antigen in both murine and human cancer vaccine studies and cancer vaccines targeting p53 based on major histocompatibility complex (MHC) class I binding p53-derived peptides that induce cytotoxic T lymphocytes (CTLs) without p53-specific CD4(+) T-cell help have been tested by several research groups including ours. To obtain such CD4(+) T-cell help and cover a broader repertoire of MHC haplotypes we have previously attempted to produce recombinant human p53 for vaccination purposes. However, attempts to refold a hexahis-tagged p53 protein in our laboratory were unsuccessful. Here, we show that fusion of an 11-amino-acid region of the human immunodeficiency virus TAT protein transduction domain (PTD) to human p53 increases the solubility of the otherwise insoluble p53 protein and this rTAT-p53 protein can be transduced into human monocyte-derived dendritic cells (DCs). The induction of a p53-specific HLA-A*0201 immune response was tested in HLA-A*0201/K(b) transgenic mice after immunization with rTAT-p53-transduced bone-marrow-derived DCs. In these mice, p53-specific CD4(+) and CD8(+) T-cell proliferation was observed and immunization resulted in the induction of HLA-A*0201-restricted CTLs specific for two human p53-derived HLA-A*0201-binding peptides, p53(65-73) and p53(149-157). Addition of GrpE to generate rTAT-GrpE-p53 led to a further increase in protein solubility and to a small increase in DC maturation but did not increase the observed p53-specific T-cell responses. The use of rTAT-p53 in ongoing clinical protocols should be applicable and offers advantages to current strategies omitting the use of HLA-typed patients.

SARS CTL vaccine candidates; HLA supertype-, genome-wide scanning and biochemical validation.

An effective Severe Acute Respiratory Syndrome (SARS) vaccine is likely to include components that can induce specific cytotoxic T-lymphocyte (CTL) responses. The specificities of such responses are governed by human leukocyte antigen (HLA)-restricted presentation of SARS-derived peptide epitopes. Exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information (Lauemoller et al., Rev Immunogenet 2001: 2: 477-91). The latter was recently established when a causative coronavirus (SARS-CoV) was isolated and full-length sequenced (Marra et al., Science 2003: 300: 1399-404). Here, we have combined advanced bioinformatics and high-throughput immunology to perform an HLA supertype-, genome-wide scan for SARS-specific CTL epitopes. The scan includes all nine human HLA supertypes in total covering >99% of all individuals of all major human populations (Sette & Sidney, Immunogenetics 1999: 50: 201-12). For each HLA supertype, we have selected the 15 top candidates for test in biochemical binding assays. At this time (approximately 6 months after the genome was established), we have tested the majority of the HLA supertypes and identified almost 100 potential vaccine candidates. These should be further validated in SARS survivors and used for vaccine formulation. We suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design.

Identification of a macromolecular crystal growth inhibitor in human urine as osteopontin.

Macromolecules occurring in human urine inhibit the growth and/or aggregation of calcium oxalate crystals and may prevent the formation of kidney stones. Attention has focused particularly on proteins, as these seem to be most responsible for the inhibitory activity; three proteins, nephrocalcin, an unidentified protein rich in uronic acid, and uropontin have all been described as possessing such activity. We have recently isolated an unknown inhibitor of calcium oxalate crystal growth that co-eluted with trypsin inhibitor in several separation steps, which suggested its identity. The aim of the present study was to outline a simple procedure for isolating and identifying this inhibitor. Purification was done as follows: precipitation of the major proteins (albumin and uromucoid) with trichloroacetic acid, followed by anion exchange chromatography, hydroxyapatite chromatography, anion exchange chromatography, negative affinity chromatography, and twice reversed phase chromatographies of the supernatant. By this procedure, the inhibitor was identified as being a fragment of osteopontin; urinary trypsin inhibitor and nucleic acids were excluded as being responsible for inhibitory action.

An unidentified macromolecular inhibitory constituent of calcium oxalate crystal growth in human urine.

We have detected and isolated a macromolecular constituent in normal human urine possessing calcium crystal growth inhibitory activity. The purification procedure consisted of two anion exchange chromatographies and one affinity chromatography. The crystal growth inhibitor was found to be heterogeneous in net charge as well as in size. It has not been identified. It is not an uronic acid-containing glycosaminoglycan, hitherto presumed to be responsible for the inhibitory activity. Whether an urinary fragment of inter-alpha-trypsin inhibitor is responsible has yet to be resolved.