Covalent linkage to beta2-microglobulin enhances the MHC stability and antigenicity of suboptimal CTL epitopes.
(9/2156)
Many CTL epitopes of clinical importance, particularly those derived from tumor Ags, display relatively poor MHC binding affinity and stability. Because in vivo immunogenicity, and thus the efficacy of peptide-based vaccines, is thought to be determined by MHC/peptide complex stability, there is a need to develop a simple strategy for enhancing the binding of suboptimal epitopes. Toward this goal, the ability to enhance suboptimal peptides through covalent linkage to beta2-microglobulin (beta2m) was explored. Two suboptimal variants of a high-affinity Db-restricted influenza nucleoprotein peptide were covalently linked, via a polypeptide spacer, to the amino terminus of human beta2m and the recombinant fusion proteins expressed in Escherichia coli. When compared with their uncoupled counterparts, the beta2m-linked epitopes display enhanced MHC stabilization and antigenicity. Thus, tethering epitopes to beta2m provides a simple method for augmenting the biological activity of suboptimal peptides and could be useful in the design of peptide-based vaccines or immunotherapeutics. (+info)
Hepatitis C virus core protein inhibits Fas- and tumor necrosis factor alpha-mediated apoptosis via NF-kappaB activation.
(10/2156)
The effects of hepatitis C virus (HCV) proteins on anti-Fas (CD95/APO-1) antibody- and tumor necrosis factor alpha (TNF-alpha)-mediated apoptosis in different human cell lines were investigated by magnetic concentration of cells which transiently produced the exogenous protein. HepG2 cells, which produced whole HCV proteins, became resistant to anti-Fas-induced apoptotic cell death. Furthermore, the core protein among HCV proteins had a key role in protecting the various cells from apoptosis mediated by not only anti-Fas but also TNF-alpha. We also found that the core functioned in the activation of nuclear factor kappaB (NF-kappaB) in all cells examined. Deletion analysis of the core revealed that the region required for NF-kappaB activation was closely correlated with that for its antiapoptotic function. In addition, we revealed in some cases that the antiapoptotic effect of the core was restrained by coproduction of the inhibitor of NF-kappaB, IkappaB-alpha protein. These results demonstrated that the core inhibits Fas- and TNF-alpha-mediated apoptotic cell death via a mechanism dependent on the activation of NF-kappaB in particular cell lines. (+info)
High- and low-affinity single-peptide/MHC ligands have distinct effects on the development of mucosal CD8alphaalpha and CD8alphabeta T lymphocytes.
(11/2156)
In this study, we compared the influence of two peptides on the selection of CD8alphaalpha and CD8alphabeta intraepithelial lymphocytes (IELs) of the intestine, which develop by a unique and partially thymus-independent process. Mice were used in which all T cells carried one transgenic T cell antigen receptor (TCR) (F5), and in which only well defined transgenic peptides were presented by H-2Db. The first peptide, for which the F5 TCR has a high affinity, derives from the influenza virus nucleoprotein (NP68). The second peptide, NP34, is an antagonistic variant of NP68 and is recognized by the F5 TCR with low affinity. To avoid presentation of endogenous peptides or production of T cells carrying alternative TCRs, F5 TCR transgenic mice were generated that were deficient for Tap-1 and Rag-1. In these mice, no CD3(+)CD8(+) cells were found in lymph nodes, spleen, or intestine. Introduction of transgenes encoding either NP34 or NP68 along with an endoplasmic reticulum signal sequence enabled Tap-1-independent expression of each peptide in these mice. Positive selection of F5TCR+CD8(+) thymocytes was not rescued by these transgenic peptides. However, the high-affinity NP68 peptide induced maturation of CD8alphaalpha IEL, whereas the low-affinity NP34 peptide stimulated development of both CD8alphabeta and CD8alphaalpha IEL, but in smaller numbers. When both peptides were present, CD8alphabeta T cells failed to develop and the number of CD8alphaalpha IELs was lower than in mice carrying the NP68 transgene alone. These data demonstrate that single ligands with a high or low affinity for TCR are capable of inducing or inhibiting the maturation of alternative subsets of IELs. (+info)
Development of a simple and highly sensitive enzyme immunoassay for hepatitis C virus core antigen.
(12/2156)
A highly sensitive enzyme immunoassay (EIA) for the hepatitis C virus (HCV) core antigen (HCVcAg) was developed, and its performance was compared with that of the AMPLICOR HCV test (Roche Molecular Systems). The developed one-step pretreatment method, 30-min incubation of the specimen with a solution containing three different types of detergents (Triton X-100, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate [CHAPS], and sodium dodecyl sulfate), does not require any special device. Because the interfering anti-core antibody in the sample was sufficiently inactivated by the pretreatment, HCVcAg in the sample could be detected. The immunoreactivity on gel filtration was shifted from void fractions to those corresponding to the molecular mass range from 20 to 25 kDa, which is equal to the estimated molecular mass of HCVcAg, after the pretreatment. By the recovery test with HCVcAg-positive serum, the recovery rate was 93.5 to 106. 5%. There was no interference with the EIA by anticoagulants or blood components in the serum. When the cutoff value was tentatively set at 0.5 mU/ml based on the distribution of healthy subjects' sera, the sera of all healthy subjects (n = 125) and patients with hepatitis B (n = 50) were negative. HCVcAg was detected in sera from 57 of 73 individuals (78.1%) with anti-HCV antibody. Similarly, HCV RNA was detected in sera from 59 individuals (80.8%) with the AMPLICOR HCV as the qualitative test (AMPLICOR HCV test) and in sera from 54 individuals (74.0%) by the AMPLICOR HCV Monitor as the quantitative test (AMPLICOR Monitor test). Concentrations of HCVcAg and HCV RNA (measured by the AMPLICOR Monitor test) correlated significantly (r = 0.8, P < 0.001). On seroconversion panels, HCVcAg was detected during the early stage of infection, when anti-HCV antibodies had not been produced. This assay for HCVcAg is simpler than assays for HCV RNA based on gene technology and shows specificity and sensitivity equivalent to those of the AMPLICOR HCV test. (+info)
Hepatitis C virus core protein interacts with a human DEAD box protein DDX3.
(13/2156)
Several studies have implicated hepatitis C virus (HCV) core in influencing the expression of host genes. To identify cellular factors with a possible role in HCV replication and pathogenesis, we looked for cellular proteins that interact with the viral core protein. A human liver cDNA library was screened in a yeast two-hybrid assay to identify cellular proteins that bind to core. Several positive clones were isolated, one of which encoded the C-terminal 253 amino acids of a putative RNA helicase, a DEAD box protein designated DDX3. Bacterially expressed glutathione-S-transferase-DDX3 fusion protein specifically pulled down in vitro translated and radiolabeled HCV core, confirming a direct interaction. Immunofluorescent staining of HeLa cells with a polyclonal antiserum showed that DDX3 is located predominantly in nuclear speckles and at low levels throughout the cytoplasm. In cells infected with a recombinant vaccinia virus expressing HCV structural proteins (core, E1, and E2), DDX3 and core colocalized in distinct spots in the perinuclear region of the cytoplasm. The regions of the proteins involved in binding were found by deletion analysis to be the N-terminal 59 amino acid residues of core and a C-terminal RS-like domain of DDX3. The human DDX3 is a putative RNA helicase and a member of a highly conserved DEAD box subclass that includes murine PL10, Xenopus An3, and yeast Ded1 proteins. Their role in RNA metabolism or gene expression is unknown. The significance of core-helicase interaction in HCV replication and pathogenesis is discussed. (+info)
Hepatitis C virus core protein regulates cell growth and signal transduction pathway transmitting growth stimuli.
(14/2156)
To investigate the transforming potential of hepatitis C virus (HCV), HCV core protein was produced in BALB/3T3 A31-I-1 cells. The cells expressing HCV core gene cooperatively with the v-H-ras gene showed loss of contact inhibition, morphological alterations, and anchorage-independent and serum-independent growth. The cells producing HCV core protein showed enhanced growth against stimulus of growth factor. In addition, antisense oligodeoxynucleotides against mRNA encoding HCV core protein suppressed the growth of HCV core-producing cells. Furthermore, HCV core protein activated mitogen-activated protein kinase and serum response element, which respond to growth stimuli. From these results, we concluded that HCV core protein is involved in the acquisition of cell growth advantage. (+info)
Monitoring antibody titers to recombinant Core-NS3 fusion polypeptide is useful for evaluating hepatitis C virus infection and responses to interferon-alpha therapy.
(15/2156)
To evaluate the clinical feasibility of the antibody titer against a chimeric polypeptide (named Core 518), in which a domain of Core and NS3 of hepatitis C virus (HCV) was fused, ELISA was performed in a total of 76 serum samples. Each serum was serially diluted using two-fold dilution method with distilled water into 10 concentrations. They were all positive for second generation anti-HCV assay (HCV EIA II; Abbott Laboratories). Genotyping RT-PCR, quantitative competitive RT-PCR, and RIBA (Lucky Confirm; LG Biotech) were also assayed. Anti-Core 518 antibody was detected in x 12800 or higher dilutions of sera from 35 of 43 chronic hepatitis C (81.4%) and nine of 16 hepatocellular carcinoma sera (56.3%), one of four cirrhosis (25%), 0 of four acute hepatitis C, and one of nine healthy isolated anti-HCV-positive subjects (p=0.0000). The anti-Core 518 antibody titers were well correlated with the presence of HCV RNA in serum (p=0.002). The anti-Core 518 antibody titers decreased significantly in nine of ten responders to IFN-alpha treatment. Monitoring anti-Core 518 titers may be helpful not only for differentiating the status of HCV infection among patients with various type C viral liver diseases, but also for predicting responses to IFN-alpha treatment. (+info)
Hepatitis C virus core protein binds to a DEAD box RNA helicase.
(16/2156)
Approximately 4 million Americans are infected with the hepatitis C virus (HCV), making it a major cause of chronic liver disease. Because of the lack of an efficient cell culture system, little is known about the interaction between HCV and host cells. We performed a yeast two-hybrid screen of a human liver cell cDNA library with HCV core protein as bait and isolated the DEAD box protein DBX. DBX has significant amino acid sequence identity to mouse PL10, an ATP-dependent RNA helicase. The binding of DBX to HCV core protein occurred in an in vitro binding assay in the presence of 1 M NaCl or detergent. When expressed in mammalian cells, HCV core protein and DBX were co-localized at the endoplasmic reticulum. In a mutant strain of Saccharomyces cerevisiae, DBX complemented the function of Ded1p, an essential DEAD box RNA helicase. HCV core protein inhibited the growth of DBX-complemented mutant yeast but not Ded1p-expressing yeast. HCV core protein also inhibited the in vitro translation of capped but not uncapped RNA. These findings demonstrate an interaction between HCV core protein and a host cell protein involved in RNA translation and suggest a mechanism by which HCV may inhibit host cell mRNA translation. (+info)