Intrahepatic genetic inoculation of hepatitis C virus RNA confers cross-protective immunity.

Naturally occurring hepatitis C virus (HCV) infection has long been thought to induce a weak immunity which is insufficient to protect an individual from subsequent infections and has cast doubt on the ability to develop effective vaccines. A series of intrahepatic genetic inoculations (IHGI) with type 1a HCV RNA were performed in a chimpanzee to determine whether a form of genetic immunization might stimulate protective immunity. We demonstrate that the chimpanzee not only developed protective immunity to the homologous type 1a RNA after rechallenge by IHGI but was also protected from chronic HCV infection after sequential rechallenge with 100 50% chimpanzee infectious doses of a heterologous type 1a (H77) and 1b (HC-J4) whole-virus inoculum. These results offer encouragement to pursue the development of HCV vaccines.

Hepatitis B virus e antigen specific epitopes and limitations of commercial anti-HBe immunoassays.

Current commercial hepatitis B virus (HBV) anti-HBe immunoassays are designed so that anti-HBe is detectable only in the absence of excess HBeAg. Recently, with the use of direct anti-HBe assays, anti-HBe was detected in individuals who had been seropositive for several years for HBeAg [Maruyama et al. (1993) J. Clin. Invest. 91:2586-2595]. Although anti-HBe seroconversion does not necessarily indicate subsequent HBeAg clearance, the ability to detect earlier anti-HBe seroconversion could have clinical significance for monitoring patients undergoing HBV immunotherapy (e.g., alpha interferon therapy). Because the HBeAg and the HBcAg share 149 amino acids, an anti-HBe assay must distinguish anti-HBe from anti-HBc antibodies. Although the HBV HBeAg and HBcAg display distinct immunogenic determinants, much remains unknown regarding the complete epitope spectrum specific to each antigen. The goal of this study was 3-fold. The first objective was to identify HBeAg specific linear epitopes. The second objective was to design an anti-HBe immunoassay capable of detecting anti-HBe specific antibody in the presence of excess HBeAg. The third objective was to characterize early anti-HBe seroconversion antibodies. The major linear epitope residing in the HBeAg amino acid sequence was mapped and 2 novel minor epitopes (delta, gamma) which appear to be HBeAg specific have been identified. An anti-HBe immunoassay capable of detecting anti-HBe specific antibody in the presence of excess HBeAg was designed. Finally, it was found that early anti-HBe seroconversion antibodies appear to be conformational, whereas later seroconversion, more typically associated with the clearance of HBeAg, is characterized by the presence of antibodies to the linear HBeAg epitopes.

Use of a novel hepatitis C virus (HCV) major-epitope chimeric polypeptide for diagnosis of HCV infection.

The genome of hepatitis C virus (HCV) consists of seven functional regions: the core, E1, E2/NS1, NS2, NS3, NS4, and NS5 regions. The U. S. Food and Drug Administration-licensed 2.0G immunoassay for the detection of anti-HCV uses proteins from the core, NS3, and NS4 regions (McHutchinson et al., Hepatology 15:19-25, 1992). The 3.0G enzyme-linked immunosorbent assay includes the protein from the NS5 region (Uyttendaele et al., Vox Sang. 66:122-129, 1994). The necessity of detecting antibodies to viral envelope proteins (E1 and E2) and to different genotype samples has been demonstrated previously (Chien et al., Lancet 342:933, 1993; Lok et al., Hepatology 18:497-502, 1993). In this study we have attempted to improve the sensitivity of the anti-HCV assay by developing a single multiple-epitope fusion antigen (MEFA; MEFA-6) which incorporates all of the major immunodominant epitopes from the seven functional regions of the HCV genome. A nucleic acid sequence consisting of proteins from the viral core, E1, E2, NS3, NS4, and NS5 regions and different subtype-specific regions of the NS4 region was constructed, cloned, and expressed in yeast. The epitopes present on this antigen can be detected by epitope-specific monoclonal and polyclonal antibodies. In a competition assay, the MEFA-6 protein competed with 83 to 96% of genotype-specific antibodies from HCV genotype-specific peptides. This recombinant antigen was subsequently used to design an anti-HCV chemiluminescent immunoassay. We designed our assay using a monoclonal anti-human immunoglobulin G antibody bound to the solid phase. Because MEFA-6 is fused with human superoxide dismutase (h-SOD), we used an anti-human superoxide dismutase, dimethyl acridinium ester-labeled monoclonal antibody for detection. Our results indicate that MEFA-6 exposes all of the major immunogenic epitopes. Its excellent sensitivity and specificity for the detection of clinical seroconversion are demonstrated by this assay.

Signal transduction mediated by the truncated trkB receptor isoforms, trkB.T1 and trkB.T2.

The trkB family of transmembrane proteins serves as receptors for BDNF and NT-4/5. The family is composed of a tyrosine kinase-containing isoform as well as several alternatively spliced "truncated receptors" with identical extracellular ligand-binding domains but very small intracellular domains. The two best-characterized truncated trkB receptors, designated as trkB.T1 and trkB.T2, contain intracellular domains of only 23 and 21 amino acids, respectively. Although it is known that the tyrosine kinase isoform (trkB.FL) is capable of initiating BDNF and NT-4/5-induced signal transduction, the functional role or roles of the truncated receptors remain enigmatic. At the same time, the potential importance of the truncated receptors in the development, maintenance, and regeneration of the nervous system has been highlighted by recent developmental and injury paradigm investigations. Here we have used trkB cDNA transfected cell lines to demonstrate that both trkB.T1 and trkB.T2 are capable of mediating BDNF-induced signal transduction. More specifically, BDNF activation of either trkB.T1 or trkB.T2 increases the rate of acidic metabolite release from the cell, a common physiological consequence of many signaling pathways. Further, these trkB.T1- and trkB. T2-mediated changes occur with kinetics distinct from changes mediated by trkB.FL, suggesting the participation of at least some unique rate-limiting component or components. Mutational analysis demonstrates that the isoform-specific sequences within the intracellular domains of each receptor are essential for signaling capability. Finally, inhibitor studies suggest that kinases are likely to be involved in the trkB.T1 and trkB.T2 signaling pathways.

Replacement of a labile aspartyl residue increases the stability of human epidermal growth factor.

Long-term storage of recombinant human epidermal growth factor (EGF), an important promoter of cell division, results in its conversion to a new species that elutes later than native EGF on a reverse-phase column. This new species, called EGF-X, has only 20% of the biological activity of native EGF. Peptide mapping indicated that the primary structure of EGF-X differs from that of native EGF solely within the first 13 residues. N-Terminal sequencing of EGF-X revealed that about 30% of the polypeptides have been cleaved at the Asp-3/Ser-4 bond. In addition, the yields after the His residue at position 10 were extremely low, indicating that a chemical modification occurs at residue 11 that is incompatible with Edman degradation. We hypothesized that aspartic acid 11 had been converted to an isoaspartyl residue, and this was confirmed with L-isoaspartyl/D-aspartyl methyltransferase, an enzyme that methylates the side-chain carboxyl group of L-isoaspartyl residues but does not recognize normal L-aspartyl residues. EGF-X, but not EGF, was found to be a substrate of this enzyme, and proteolytic digestion of EGF-X with thermolysin localized the site of methylation to a nine-residue peptide containing position 11. We did not observe formation of the isoaspartyl derivative in EGF that had been denatured by reduction of its disulfide bonds. In addition, replacement of the aspartyl residue at position 11 with glutamic acid resulted in a fully active EGF derivative that does not form detectable amounts of EGF-X. We propose that conversion of this aspartyl residue to isoaspartate is a significant nonenzymatic degradation reaction affecting this growth factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and processing of biologically active fibroblast growth factors in the yeast Saccharomyces cerevisiae.

Chemically synthesized genes for bovine and human fibroblast growth factors (FGFs) were expressed in heterologous microorganisms. Although the intracellular expression or secretion of acidic and basic FGFs in Escherichia coli or Saccharomyces cerevisiae yielded recombinant growth factors with high biological activity, the resulting proteins had structural microheterogeneity due to modified amino termini. Expression of amino-terminal extended forms of human acidic and basic FGFs in S. cerevisiae gave rise to soluble, but cell-associated polypeptides, with potent biological activity. These yeast-derived proteins were processed in vivo by removal of initiation codon-derived methionine residues and by amino-terminal acetylation. Both of these processes have been observed in mammalian tissues. The yeast systems described here, therefore, provide a good model system for the expression of FGFs as intracellular proteins, but more importantly they give high levels of authentically processed human FGFs with many potential medical applications. Since the recombinant proteins have all the biological activities of their native counterparts, their possible applications in wound healing, tissue grafting, nerve regeneration, and treatment of ischemia are discussed.