A parallel assay for the quantification of single-molecule binding forces was developed based on differential unbinding force measurements where ligand-receptor interactions are compared with the unzipping forces of DNA hybrids. Using the DNA zippers as molecular force sensors, the efficient discrimination between specific and nonspecific interactions was demonstrated for small molecules binding to specific receptors, as well as for proteinprotein interactions on protein arrays. Finally, an antibody sandwich assay with different capture antibodies on one chip surface and with the detection antibodies linked to a congruent surface via the DNA zippers was used to capture and quantify a recombinant hepatitis C antigen from solution. In this case, the DNA zippers enable not only discrimination between specific and nonspecific binding, but also allow for the local application of detection antibodies, thereby eliminating false-positive results caused by cross-reactive antibodies and nonspecific binding.

Duck HBV recombinant vaccinia virus has also been used to attempt the termination of chronic infection with this virus in ducks. 6 A temporary drop in surface antigen titer, but no permanent resolution, was observed. Recently, Rollier et al. 7 reported that immunization of chronically infected ducks with DNA encoding HBsAg and PreS determinants resulted in a lowering of viremia and a decrease in hepatic DNA levels.Based on our previous studies on the protective efficacy of DNA-based immunization against HBV in newborn chimpanzees, 8 in the present study we attempted to terminate the HBV chronic carrier state in a chimpanzee. Chronic HBV carriers are rare, as most chimpanzees infected after birth spontaneously resolve HBV infection. One chronically HBV-infected chimpanzee, which had remained chronic after infection with HBV for 12 years, was available to us for this study. We show that immunization with HBsAg-encoding plasmids, followed by boosting with recombinant canarypox encoding HBsAg, PreS-1, and PreS-2, resulted in disappearance of HBV DNA from blood detectable by a quantitative polymerase chain reaction (PCR) assay beginning 1 week after the canarypox booster and persisting for at least 202 weeks after the onset of immunization. A more sensitive nested PCR assay revealed borderline quantities of HBV DNA during the time when HBV DNA was not detectable by the quantitative PCR. Viral clearance in the liver was also established by the decline of covalently closed circular (ccc) HBV DNA that serves as a template for viral transcription. 9 Down-regulation of HBV replication appeared to be mediated by a noncytolytic mechanism that correlated with interferon gamma (IFN-␥) production. MATERIALS AND METHODS Plasmid ConstructsPlasmid pJW-So, which encodes HBsAg, but not Pre-S1 and Pre-S2, was used for the first DNA-based immunization. The recombinant plasmids used in this study were constructed by standard cloning techniques. The pJW-So clone was constructed by PCR from the HBsAg gene of HBV (ayw subtype) corresponding to 2 to 226 amino acids (aa) of S antigen as a Nhe I/Bgl II fragment. The fragment was

Posttranslational processing and subcellular localization of the HCV core protein are critical steps involved in the assembly of hepatitis C virus (HCV). In this study, both of these events were investigated by in vitro translation and transient COS-1 cell transfection of core protein expression constructs. Mutations at amino acid residues 173 to 174 and 191 to 192 disrupted processing events at the two putative cleavage sites in the C-terminal hydrophobic region of the core protein, indicating that these residues are implicated in the pathway of core protein maturation. As a result, two forms of core protein, C173 and C191, were detected by immunoblotting. Indirect immunofluorescence experiments showed that core proteins C173 and C191, when produced from HCV full-length protein or various polyprotein precursors, displayed a cytoplasmic localization. The C173 species, however, was translocated to the nucleus when expressed in the absence of C191. These findings indicate that preferential cleavage may occur during core protein maturation and that the association of the C191 with the C173 species may contribute to the distinct subcellular distribution of core protein. This may provide a possible mechanism for the control of the diverse biological functions of core protein during HCV replication and assembly.

The ability of the total hepatitis C virus (HCV) core antigen assay was evaluated for monitoring the therapeutic responses of HCV-infected patients treated with interferon. The ability to detect and quantitate an independent structural protein component of HCV, in the presence of circulating antibodies, makes this assay a valuable new tool in diagnosis and treatment monitoring. Measurement of total core antigen showed a strong dynamic correlation with HCV RNA data and may serve as an alternative direct marker of viral infection. In addition, with the advent of additional treatment protocols, a rapid, reliable assay for changes in HCV load may permit more frequent patient assessment and tailoring of the therapeutic regimen.

DNA-based immunizations have been used to elicit cellular immunity to hepatitis C virus (HCV) proteins in mice. Mice were immunized by intramuscular or intradermal injections of plasmid DNA derived from a near-full-length HCV genotype 1b genomic clone (pRC/B2) or individual genomic clones. These immunizations induced cytotoxic T lymphocytes (CTLs), as revealed in standard chromium-release assays that used syngeneic peptide-pulsed or transfected target cells. These assays identified four CTL epitopes within the capsid, E1, and E2 regions of the polyprotein sequence of HCV genotype 1a that were cross-reactive with HCV genotype 1b. Additionally, CTLs derived from mice immunized with either NS3 or NS5 specifically lysed target cells sensitized to either the genotype 1a or 1b gene products. Nucleic acid immunizations also generated humoral immunity to HCV proteins, as detected by anti-HCV reactivity to NS3 and capsid in ELISAs and immunoblot assays.

No abstract

The Chinese hamster ovary adenine phosphoribosyl transferase gene (aprt) was reengineered to be flanked by sequences from the thymidine kinase (tk) gene of herpes simplex virus. This construct was cotransfected with DNA from herpes simplex virus type 1, and after 3 days, virus was harvested and Tkplaques were selected after the virus was plated on Tkcells in the presence of bromodeoxycytosine. Recombinant viruses were identified by dot-blot hybridization, and the arrangement of aprt and tk sequences were determined by Southern blot hybridization. Analysis of the recombinants revealed that acquisition of aprt sequences resulted from insertional inactivation of the tk locus as a consequence of homology-based recombination. Recombination was precise, as evidenced by the failure to detect plasmid sequences or the synthetic restriction endonuclease sites that bounded the mutant tk gene in the aprt-tk construct. Infection of Aprtmouse or Chinese hamster ovary cells with UV-irradiated virus and selection in medium containing azaserine and adenine resulted in the survival of numerous colonies that stably express the aprt gene. Transformed cells synthesized an aprt mRNA that is identical to wild-type mRNA as determined by Northern blot and S1 nuclease analyses. Cells lytically infected with the recombinant virus do not appear to transcribe the aprt gene. Thus, infected cells differentiate between virus and foreign promoters even when a cellular gene is cis to the virus chromosome. * Corresponding author. the adenine phosphoribosyl transferase wild-type (Aprtt) phenotype. These transformed cells transcribe an mRNA that is indistinguishable from that synthesized by Aprt+ CHO cells. Parenthetically, we note that when the aprt gene is placed cis to the virus chromosome, it is not expressed at any time during the infectious cycle of the virus. MATERIALS AND METHODS Cells and viruses. Murine Ltk-Aprtcells were maintained in Dulbecco modified Eagle medium supplemented

The trpE (or anthranilate synthetase) gene product has been used extensively as a fusion protein for the expression of a myriad of biologically active proteins. A trpE construct can be produced in high yield, is relatively resistant to proteolysis, and separates from the bulk of E. coli proteins because of its insolubility. We have isolated and characterized a monoclonal antibody against the TrpE protein for use as a detection and immunoaffinity reagent. The MAb, TRP 7.4, is highly specific for the TrpE protein and has a relative affinity of 1.0 ng. The antibody can also be used to detect TrpE constructs on Western blots. In addition, TRP 7.4 has been used to purify a TrpE-IL-6 fusion protein. These studies show the utility of this MAb as a tool for both research and protein purification.