Inactivation of hepatitis A and other enteroviruses during heat treatment (pasteurization).
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Animals↗
Biomedical subjects
S M Feinstone
Publications and source records attributed to S M Feinstone.
Use of intrinsic and extrinsic helper epitopes for in vivo induction of anti-hepatitis C virus cytotoxic T lymphocytes (CTL) with CTL epitope peptide vaccines.
The induction of virus-specific cytotoxic T lymphocytes (CTL) is an important part of vaccine strategy. CTL induction in vivo by two hepatitis C virus (HCV) peptides containing CTL epitopes, one from the NS5 region (P17) and one from the core (C7), was compared. P17 required covalent attachment of a helper peptide (PCLUS3 containing a cluster of epitopes from the human immunodeficiency virus envelope protein), whereas C7 did not. However, the minimal decapeptide of C7, C7A10, alone did not induce CTL. The helper cells induced by PCLUS3-17 or by C7 were shown to be CD4+ and to produce interleukin-2 (IL-2). Thus, help can be supplied by a natural helper epitope intrinsic to the CTL peptide, as in C7, or by attaching a helper epitope from another protein, as in the case of P17. The cluster peptides may be useful promiscuous helper peptides for a variety of CTL epitopes from diverse pathogens.
Amino Acid Sequence↗
Use of recombinant protein to identify a motif-negative human cytotoxic T-cell epitope presented by HLA-A2 in the hepatitis C virus NS3 region.
To define cytotoxic T-cell (CTL) epitopes, the common approach involving the use of a series of overlapping synthetic peptides covering the whole protein sequence is impractical for large proteins. Motifs identify only a fraction of epitopes. To identify human CTL epitopes in the NS3 region of hepatitis C virus (HCV), we modified an approach using recombinant protein and the ability of short peptides to bind to class I major histocompatibility complex (MHC) molecules. Peripheral blood mononuclear cells from an HCV-infected patient were stimulated with a proteolytic digest of the recombinant NS3 protein to expand CTL to any active peptides in the digest. The digest was fractionated by reverse-phase high-performance liquid chromatography, and fractions were assessed for the ability to sensitize targets for lysis by CTL. The most active fraction was sequenced, identifying a 15-residue peptide (NS3-1J; TITTGAPVTYSTYGK). This sequence was confirmed to be the source of the activity by synthesis of the corresponding peptide. CTL lines specific for NS3-1J were established from two HCV-infected patients (both HLA-A2 and -B7 positive) by stimulation with the synthetic peptide in vitro. The CTL were HLA-A2 restricted, and the minimal epitope was mapped to a decapeptide NS3-1J (10.4). As this minimal epitope lacks the common HLA-A2-binding motif, this technique is useful for mapping CTL epitopes independent of known motifs and without the requirement for enormous numbers of overlapping peptides. Because this peptide is presented by the most common HLA class I molecule, present in almost half the population, it might be a useful component of a vaccine against HCV.
Amino Acid Sequence↗
Identification of a highly conserved sequence element at the 3' terminus of hepatitis C virus genome RNA.
Previous reports suggest that the hepatitis C virus (HCV) genome RNA terminates with homopolymer tracts of either poly(U) or poly(A). By ligation of synthetic oligonucleotides followed by reverse transcription-PCR, cDNA cloning, and sequence analysis, we determined the 3'-terminal sequence of HCV genome RNA. Our results show that the HCV 3' nontranslated region consists of four elements (positive sense, 5' to 3'): (i) a short sequence with significant variability among genotypes, (ii) a homopolymeric poly(U) tract, (iii) a polypyrimidine stretch consisting of mainly U with interspersed C residues, (iv) a novel sequence of 98 bases. This latter nucleotide sequence is not present in human genomic DNA and is highly conserved among HCV genotypes. The 3'-terminal 46 bases are predicted to form a stable stem-loop structure. Using a quantitative-competitive reverse transcription-PCR assay, we show that a substantial fraction of HCV genome RNAs from a high- specific-infectivity inoculum contain this 3'-terminal sequence element. These results indicate that the HCV genome RNA terminates with a highly conserved RNA element which is likely to be required for authentic HCV replication and recovery of infectious RNA from cDNA.
Base Sequence↗
Hepatitis A: epidemiology and prevention.
Hepatitis A is a disease with worldwide distribution. Conditions of sanitation and hygiene determine the overall epidemiological situation in any area, but risk behaviour determines an individual's chance of exposure. The recent licensing of formalin-inactivated vaccines for the prevention of hepatitis A requires an understanding of the epidemiology in order to devise how best to use this the vaccine in individuals or populations. Although higher-risk groups can be identified in developed countries, and vaccine will be useful to those groups, it is not likely that the vaccine will have a large effect on the prevalence of hepatitis A until it becomes a part of the routine childhood immunization schedule.
Hepatitis A↗
CTL responses of HLA-A2.1-transgenic mice specific for hepatitis C viral peptides predict epitopes for CTL of humans carrying HLA-A2.1.
Vaccine development in animal models depends on ability to recognize epitopes seen by human T cells. In this work, we show that CTL responses in transgenic mice expressing human HLA-A2.1 prospectively predict the same four of 11 hepatitis C virus (HCV) structural protein-derived peptides, expressing a sequence motif for HLA-A2.1 binding, that are actually recognized by human A2.1-restricted CTLs. The CTLs also recognized targets endogenously expressing these proteins. Human CTLs from HCV-infected patients, tested by using the same peptides, revealed a virtually identical response repertoire. A highly conserved HCV core peptide was the most immunogenic, and may be a valuable component of a vaccine against a broad range of HCV isolates in HLA-A2-positive patients. These results suggest that, in spite of species differences, the T cell repertoire is plastic enough to allow a similar response when the same class I MHC molecule is presenting the peptide. Thus, the HLA molecule plays the primary role in determining which peptides are recognized by CTLs. This transgenic mouse model is important for the study of HLA-restricted CTL determinants and for an approach to design a potential HCV vaccine.
Adult↗
The physical state of the negative strand of hepatitis C virus RNA in serum of patients with chronic hepatitis C.
Negative strands of the hepatitis C virus (HCV) genome (a positive-stranded RNA virus) have been found in a nuclease-resistant form in the serum of patients with HCV infections. We determined whether a complete negative-strand copy is present in the serum, whether the negative strand is particle-associated, and finally, whether it is virion-associated and encapsidated like the positive (genomic) strand. Isopyknic sucrose and cesium chloride density ultracentrifugation followed by a strand-specific reverse transcription-polymerase chain reaction on the collected fractions was performed to determine whether both positive and negative strands were associated with similar particles. Both strands comigrated to approximately the same density (1.11-1.16 g/cm3) in sucrose. After treatment of the plasma with detergent (0.1% Nonidet P-40) to remove the viral envelope and centrifugation on cesium chloride gradients, the positive strands shifted to a density of 1.35 g/cm3, and the negative strands were not detected. By using antibodies specific for the HCV core or envelope glycoproteins E1 or E2 coated onto the wells of a microtiter plate, it was possible to specifically bind HCV or viral cores to the solid phase. Pelleted virus particles were resuspended in either PBS or PBS with 0.1% Nonidet P-40 to expose the core. These pellets were then incubated in antibody-coated microtiter wells. RNA extracted from the bound and unbound fractions was tested for HCV RNA. The anti-core antibody was able to bind positive strands but not negative strands only in detergent-treated samples. In the nondetergent-treated pellets, the anti-E1 and -E2 bound the positive strand, but only anti-E1 bound the negative strands. These findings indicate that while both strands of HCV RNA can be detected in serum, the positive strand is encapsidated within the enveloped core, and the negative strand appears to be in a membrane particle associated with the viral envelope protein E1 but does not appear to be within the HCV core of circulating virions.
Base Sequence↗
Mutational events in consecutive passages of hepatitis A virus strain GBM during cell culture adaptation.
In order to study the adaptation of hepatitis A virus (HAV) in cell culture, we examined the mutational events of the genome in early passages of HAV strain GBM propagated either in FRhK-4 cells (fetal rhesus monkey kidney-derived) or in human embryonic kidney (HEK) and human embryonic fibroblast cells (HFS) in relation to their growth characteristics. Sequence analysis of the nucleotide region encoding 2B, 2C, and the beginning of 3A as well as the nucleotide region encompassing the 5' noncoding region (5'NCR) of the genome was performed on consecutive virus passages after amplification of the viral RNA from the cell culture supernatant by antigen-capture PCR. By the 2nd passage of the GBM variants cultured in FRhK-4 or in HEK cells we found a mutation at nucleotide position 3889 (2B coding region) which results in an amino acid substitution from alanine to valine. Further mutations present in the 2B/2C region of the cell culture-adapted GBM variants differ from each other and occur after the 10th or even the 40th virus passage. Another early change, an in frame deletion of nine nucleotides in the 3A region, appeared in the 5th virus passage only in GBM cultured on FRhK-4 cells. This genome region showed different mutations in the virus passages on HEK and HFS cells. The 5'NCR of the cell culture-adapted GBM variants, in contrast, did not show any mutations before the 8th virus passage. The faster and more efficient growth of the HAV strain GBM during successive propagation on cell cultures seems to correlate with the appearance of mutations in the investigated genome regions.
Adaptation, Physiological↗
Detection and quantitation of hepatitis C virus RNA in serum using the polymerase chain reaction and a colorimetric enzymatic detection system.
A sensitive, non-isotopic method for detecting and quantifying hepatitis C virus (HCV) RNA in serum using the reverse transcriptase-polymerase chain reaction (RT-PCR) and a hybridization specific, colorimetric biotin-avidin peroxidase detection system has been developed. The sensitivity of the PCR-colorimetric system was determined using RNA synthesized from cloned HCV cDNA. The assay could detect as few as 10 molecules of HCV RNA, comparable to the sensitivity achieved with double PCR using nested primers. Thus, this colorimetric assay can detect low levels of HCV RNA in serum and appears to be quantitative, suggesting that this technique may be applied to rapid screening of large numbers of samples and to monitor the effect of antiviral therapy.
Animals↗
Nucleotide sequence of wild-type hepatitis A virus GBM in comparison with two cell culture-adapted variants.
In order to study cell tropism and attenuation of hepatitis A virus (HAV), the genome of HAV wild-type GBM and two cell culture-adapted variants, GBM/FRhK and GBM/HFS, were cloned and sequenced after amplification by reverse transcriptase-PCR. During virus cultivation, the HAV variant GBM/FRhK had a strict host range for FRhK-4 cells, in contrast to GBM/HFS, which can be grown in HFS and FRhK-4 cells. The HAV variant GBM/HFS was shown to be attenuated when inoculated into chimpanzees (B. Flehmig, R. F. Mauler, G. Noll, E. Weinmann, and J. P. Gregerson, p. 87-90, in A. Zuckerman, ed., Viral Hepatitis and Liver Disease, 1988). On the basis of this biological background, the comparison of the nucleotide sequences of these three HAV GBM variants should elucidate differences which may be of importance for cell tropism and attenuation. The comparison of the genome between the GBM wild type and HAV wild types HM175 (J. I. Cohen, J. R. Ticehurst, R. H. Purcell, A. Buckler-White, and B. M. Baroudy, J. Virol. 61:50-59, 1987) and HAV-LA (R. Najarian, O. Caput, W. Gee, S. J. Potter, A. Renard, J. Merryweather, G. Van Nest, and D. Dina, Proc. Natl. Acad. Sci. USA 82:2627-2631, 1985) showed a 92 to 96.3% identity, whereas the identity was 99.3 to 99.6% between the GBM variants. Nucleotide differences between the wild-type and the cell culture-adapted variants, which were identical in both cell culture-adapted GBM variants, were localized in the 5' noncoding region; in 2B, 3B, and 3D; and in the 3' noncoding region. Our result concerning the 2B/2C region confirms a mutation at position 3889 (C-->T, alanine to valine), which had been shown to be of importance for cell culture adaptation (S. U. Emerson, C. McRill, B. Rosenblum, S. M. Feinstone, and R. H. Purcell, J. Virol. 65:4882-4886, 1991; S. U. Emerson, Y. K. Huang, C. McRill, M. Lewis, and R. H. Purcell, J. Virol. 66:650-654, 1992), whereas other mutations differ from published HAV sequence data and may be cell specific. Further comparison of the two cell culture-adapted GBM variants showed cell-specific mutations resulting in deletions of six amino acids in the VP1 region and three amino acids in the 3A region of the GBM variant GBM/FRhK.
Adaptation, Biological↗
An epitope in hepatitis C virus core region recognized by cytotoxic T cells in mice and humans.
Several cytotoxic T-lymphocyte (CTL) epitopes have been defined in hepatitis C virus (HCV) proteins. CTL may play an important role in the control of infection by HCV. Here, we identify a highly conserved antigenic site in the HCV core recognized by both murine and human CTL. Spleen cells from mice immunized with a recombinant vaccinia virus expressing the HCV core gene were restimulated in vitro with 11 peptides from the core protein. CTL from H-2d mice responded to a single 16-residue synthetic peptide (HCV 129-144). This conserved epitope was presented by a murine class I major histocompatibility molecule (H-2Dd) to conventional CD4- CD8+ CTL mapped by using transfectants expressing Dd, Ld, or Kd, but was not seen by CTL restricted by H-2b. The murine epitope was mapped to the decapeptide LMGYIPLVGA. The same 16-residue peptide was recognized by CTL from two HCV-seropositive patients but not by CTL from any seronegative donors. CTL from two HLA-A2-positive patients with acute and chronic hepatitides C recognized a 9-residue fragment (DLMGYIPLV) of the peptide presented by HLA-A2 and containing an HLA-A2-binding motif, extending only 1 residue beyond the murine epitope. Therefore, this conserved peptide, seen with murine CTL and human CTL with a very prevalent HLA class I molecule, may be a valuable component of an HCV vaccine against a broad range of HCV isolates. This study demonstrates that the screening for CTL epitopes in mice prior to human study may be useful.
Amino Acid Sequence↗
Susceptibility of nonprimate cell lines to hepatitis A virus infection.
Hepatitis A virus (HAV) has been adapted to grow in primate cell cultures. We investigated replication of HAV in nonprimate cells by inoculating 20 cell lines from different species with the tissue culture-adapted HM175 strain. Slot blot hybridization and immunofluorescence analysis revealed that HAV replicated in GPE, SP 1K, and IB-RS-2 D10 cells of guinea pig, dolphin, and pig origin, respectively. Studies in IB-RS-2 D10 cells were discontinued because cultures were contaminated with classical swine fever virus. A growth curve showed that HAV grew poorly in GPE cells and intermediately in SP 1K cells compared with growth in FRhK-4 cells. Therefore, the cell surface receptor(s) and other host factor(s) required for HAV replication are present in nonprimate as well as primate cells.
Animals↗
Antibody response to hepatitis C virus infection after liver transplantation.
OBJECTIVE: To determine whether liver transplantation and the subsequent immunosuppression affect the antibody response to hepatitis C virus (HCV) infection. METHODS: Sera from 46 patients were compared before and after liver transplantation for markers of HCV infection. Serum HCV RNA was determined by polymerase chain reaction (PCR). Anti-HCV antibody was determined by first- and second-generation immunoassays as well as a quantitative assay of the titer of anti-HCV core antibody. RESULTS: Among individuals who acquired hepatitis C infection in association with liver transplantation, only 15% (3/12) developed antibody to the core antigen and only 25% (3/12) reacted to any antigen present on the second-generation recombinant immunoblot assay after a mean follow-up period of 18 months. Thirty-eight percent (5/13) were positive, by the second-generation enzyme immunoassay (EIA-2) Whereas 94% (16/17) of the individuals who had detectable anti-HCV core antibodies pretransplant continued to have such antibodies after transplant, the titer of these antibodies declined an average of 4-fold. No significant change was seen in the antibody titer toward rotavirus, a common viral pathogen. Patients who acquired HCV infection or in whom the allograft became reinfected had a significantly increased incidence of posttransplant hepatitis (61% vs. 33%, respectively). CONCLUSIONS: Liver transplantation and posttransplant immunosuppression lead to an attenuated antibody response to hepatitis C viral infection. Currently available assays for anti-HCV antibodies may be unreliable in the posttransplant setting.
Adult↗
A second hepatitis C virus-encoded proteinase.
Host and viral proteinases are believed to be required for the production of at least nine hepatitis C virus (HCV)-specific polyprotein cleavage products. Although several cleavages appear to be catalyzed by host signal peptidase or the HCV NS3 serine proteinase, the enzyme responsible for cleavage at the 2/3 site has not been identified. In this report, we have defined the 2/3 cleavage site and obtained evidence which suggests that this cleavage is mediated by a second HCV-encoded proteinase, located between aa 827 and 1207. This region encompasses the C-terminal portion of the 23-kDa NS2 protein, the 2/3 cleavage site, and the serine proteinase domain of NS3. Efficient processing at the 2/3 site was observed in mammalian cells, Escherichia coli, and in plant or animal cell-free translation systems in the absence of microsomal membranes. Cleavage at the 2/3 site was abolished by alanine substitutions for NS2 residues His-952 or Cys-993 but was unaffected by several other substitution mutations, including those that inactivate NS3 serine proteinase function. Mutations abolishing cleavage at the 2/3 site did not block cleavage at other sites in the HCV polyprotein. Cotransfection experiments indicate that the 2/3 site can be cleaved in trans, which should facilitate purification and further characterization of this enzyme.
Amino Acid Sequence↗
Hepatitis C virus NS3 protein polynucleotide-stimulated nucleoside triphosphatase and comparison with the related pestivirus and flavivirus enzymes.
Sequence motifs within the nonstructural protein NS3 of members of the Flaviviridae family suggest that this protein possesses nucleoside triphosphatase (NTPase) and RNA helicase activity. The RNA-stimulated NTPase activity of this protein from prototypic members of the Pestivirus and Flavivirus genera has recently been established and enzymologically characterized. Here, we experimentally demonstrate that the NS3 protein from a member of the third genus of Flaviviridae, human hepatitis C virus (HCV), also possesses a polynucleotide-stimulated NTPase activity. Characterization of the purified HCV NTPase activity showed that it exhibited reaction condition optima with respect to pH, MgCl2, and salt identical to those of the representative pestivirus and flavivirus enzymes. However, each NTPase also possessed several unique properties when compared with one another. Notably, the profile of polynucleotide stimulation of the NTPase activity was distinct for the three enzymes. The HCV NTPase was the only one whose activity was significantly enhanced by a deoxyribopolynucleotide. Additional distinguishing features among the three enzymes relating to the kinetic properties of their NTPase activities are discussed. These studies provide a foundation for investigation of the putative RNA helicase activity of these proteins and for further study of the role of the NS3 proteins of members of the Flaviviridae in the replication cycle of these viruses.
Adenosine Triphosphatases↗
Expression and identification of hepatitis C virus polyprotein cleavage products.
Hepatitis C virus (HCV) is the major cause of transfusion-acquired non-A, non-B hepatitis. HCV is an enveloped positive-sense RNA virus which has been classified as a new genus in the flavivirus family. Like the other two genera in this family, the flaviviruses and the pestiviruses, HCV polypeptides appear to be produced by translation of a long open reading frame and subsequent proteolytic processing of this polyprotein. In this study, a cDNA clone encompassing the long open reading frame of the HCV H strain (3,011 amino acid residues) has been assembled and sequenced. This clone and various truncated derivatives were used in vaccinia virus transient-expression assays to map HCV-encoded polypeptides and to study HCV polyprotein processing. HCV polyproteins and cleavage products were identified by using convalescent human sera and a panel of region-specific polyclonal rabbit antisera. Similar results were obtained for several mammalian cell lines examined, including the human HepG2 hepatoma line. The data indicate that at least nine polypeptides are produced by cleavage of the HCV H strain polyprotein. Putative structural proteins, located in the N-terminal one-fourth of the polyprotein, include the capsid protein C (21 kDa) followed by two possible virion envelope proteins, E1 (31 kDa) and E2 (70 kDa), which are heavily modified by N-linked glycosylation. The remainder of the polyprotein probably encodes nonstructural proteins including NS2 (23 kDa), NS3 (70 kDa), NS4A (8 kDa), NS4B (27 kDa), NS5A (58 kDa), and NS5B (68 kDa). An 82- to 88-kDa glycoprotein which reacted with both E2 and NS2-specific HCV antisera was also identified (called E2-NS2). Preliminary results suggest that a fraction of E1 is associated with E2 and E2-NS2 via disulfide linkages.
Antigens, Viral↗
Characterization of the hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites.
Processing of the hepatitis C virus (HCV) H strain polyprotein yields at least nine distinct cleavage products: NH2-C-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B-CO OH. As described in this report, site-directed mutagenesis and transient expression analyses were used to study the role of a putative serine proteinase domain, located in the N-terminal one-third of the NS3 protein, in proteolytic processing of HCV polyproteins. All four cleavages which occur C terminal to the proteinase domain (3/4A, 4A/4B, 4B/5A, and 5A/5B) were abolished by substitution of alanine for either of two predicted residues (His-1083 and Ser-1165) in the proteinase catalytic triad. However, such substitutions have no observable effect on cleavages in the structural region or at the 2/3 site. Deletion analyses suggest that the structural and NS2 regions of the polyprotein are not required for the HCV NS3 proteinase activity. NS3 proteinase-dependent cleavage sites were localized by N-terminal sequence analysis of NS4A, NS4B, NS5A, and NS5B. Sequence comparison of the residues flanking these cleavage sites for all sequenced HCV strains reveals conserved residues which may play a role in determining HCV NS3 proteinase substrate specificity. These features include an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position.
Amino Acid Sequence↗
B-cell epitopes on the hepatitis C virus nucleocapsid protein determined by human monospecific antibodies.
Four monospecific antibodies against the hepatitis C virus nucleocapsid protein, which was expressed by recombinant baculovirus, were obtained by Epstein-Barr virus transformation of B cells from three patients with chronic hepatitis C virus infection. One of these antibodies was IgG and the other three were IgM. Their specificities were characterized initially by enzyme-linked immunosorbent assay and immunoblotting against hepatitis C virus proteins expressed by six recombinant baculoviruses with different hepatitis C virus sequence insertions. These specificities were confirmed, and their epitopes were more precisely determined with a series of overlapping decapeptides made by solid-phase pin technology. Two antibodies (1F4 and 2G6) reacted with the same peptides located near the amino(N)-terminus of nucleocapsid protein (amino acids 33-50). The third antibody (3B5) recognized the peptide consisting of amino acids 133-142, and the fourth antibody (3B9) was mapped to the carboxy(C)-terminus and reacted with a peptide consisting of amino acids 165-174. This epitope has not previously been reported. Two antibodies, 1F4 and 3B9, which are specific to the N-terminus and C-terminus of nucleocapsid protein, respectively, have been stably produced for more than 6 mo and are being subcloned to establish monoclonality. These antibodies should be useful reagents for the study of hepatitis C virus.
Antibodies, Monoclonal↗