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PubMed2006

Construction and characterization of an HCV-derived multi-epitope peptide antigen containing B-cell HVR1 mimotopes and T-cell conserved epitopes.

Hepatitis C (HCV) genome is highly variable, particularly in the hypervariable region 1 (HVR1) of its E2 envelope gene. The variability of HCV genome has been a major obstacle for developing HCV vaccines. Due to B-cell HVR1 mimotopes mimicking the antigenicity of natural HVR1 epitopes and some T-cell epitopes from the consensus sequence of HCV genes conserving among the different HCV genotypes, we synthesized an minigene of HCV-derived multi-epitope peptide antigen (CMEP), which contains 9 B-cell HVR1 mimotopes in E2, 2 conserved CTL epitopes in C, 1 conserved CTL epitope in NS3 and 1 conserved Th epitope in NS3. This minigene was cloned into a GST expression vector to generate a fusion protein GST-CMEP. The immunogenic properties of CEMP were characterized by HCV infected patients' sera, and found that the reactivity frequency reached 75%. The cross reactivity of anti-CEMP antibody with different natural HVR1 variants was up to 90%. Meanwhile, we constructed an HCV DNA vaccine candidate, plasmid pVAX1.0-st-CMEP carrying the recombinant gene (st) of a secretion signal peptide and PADRE universal Th cell epitope sequence in front of the CMEP minigene. Immunization of rabbits with pVAX1.0-st-CMEP resulted in the production of antibody, which was of the same cross reactivity as the fusion protein GST-CMEP. Our findings indicate that the HCV-derived multi-epitope peptide antigen in some degree possessed the characteristics of neutralizing HCV epitopes, and would be of the value as a candidate for the development of HCV vaccines.

Amino Acid Sequence↗
PubMedDate not supplied

Acute posttransfusion hepatitis C: identification of a common hepatitis C virus strain in donor and recipient using polymorphism analysis.

An 11-year-old Thai boy who had received multiple blood transfusions from 12 different donors for treatment of Dengue shock syndrome presented with symptoms of acute hepatitis 5 weeks thereafter. He was found positive for antibodies to hepatitis C virus (HCV) and HCV-RNA was detected by reverse transcription PCR (RT-PCR). When his alanine aminotransferase (ALT) level peaked at 1,879 U/l in the 8th week, interferon therapy (3 million units, thrice weekly for 6 months ) was initiated. After initially decreasing to tenfold the normal level, the ALT dropped to fivefold the normal level at 6 months. HCV RNA is still detectable in his serum 6 months later. Using RT-PCR and subsequent restriction fragment length polymorphism (RFLP) analysis we identified one of the donors as harboring HCV genotype 3a, identical to that found in the patient. Moreover, polymorphism analysis on the hypervariable region employing five distinct restriction endonucleases suggested this donor as the source of infection. We hence recommend thorough screening of all blood donors as the only means of prevention presently feasible.

Antibodies, Viral↗
PubMed2001

Reactivation of hepatitis C virus superinfection in a patient seropositive for hepatitis B e antigen.

During the course of chronic hepatitis B virus (HBV) infection, a patient seropositive for hepatitis B e antigen experienced four episodes of acute hepatic necroinflammation. Serum HBV-DNA concentration elevated immediately before the first and third exacerbations, whereas serum hepatitis C virus (HCV) RNA was detected during the second and fourth exacerbations. The nucleotide sequences of HCV hypervariable region derived from samples of the two exacerbations were identical. Interestingly, "de novo" seroconversion of anti-HCV antibody (Abbott HCV EIA 3.0) followed by reversion occurred in both the second and fourth exacerbations with low sample/cutoff ratios. Immunoblot analysis using a line-immunoassay (Inno-LIA HCV Ab III) revealed a single positive band (C1) developing after the second exacerbation. These data indicate that the second exacerbation in this patient was caused by newly acquired acute HCV superinfection, whereas the fourth exacerbation was likely due to reactivation of the previous HCV infection. Recognition of such a case suggests that the presence of de novo seroconversion of anti-HCV may indicate either reactivation or acute superinfection of HCV in a patient seropositive for hepatitis B e antigen.

Adult↗
PubMed2006

Evolution of hepatitis C virus quasispecies in children with chronic hepatitis C.

BACKGROUND: Hepatitis C virus (HCV) circulates as a mixture of different but closely related genomes: this quasispecies nature could be essential for virus persistence and could induce resistance to interferon therapy. Since little is known on the behavior of HCV quasispecies in children and adolescents with chronic hepatitis C, we analyzed the virus population in six untreated children during a 5-year follow-up. METHODS: Six children aged 1-8 years, infected early in life with HCV, were included in the study. From each of them, 2 or 3 sequential serum samples obtained over a 5-year follow-up period were examined. The HCV quasispecies heterogeneity and diversity in the E2 hypervariable region-1 (HVR-1) were analyzed among samples by the heteroduplex mobility assay, and the distance between variants was estimated by the heteroduplex mobility ratio (HMR). RESULTS: The HCV population was initially highly homogeneous in all six children. During follow-up, diversification of HVR-1 leading to a more complex viral population occurred in all cases, and was particularly evident in the three older children (HMR: 0.82-0.54). Changes in the HVR-1 sequence occurred without relation to the profile of ALT and HCV-RNA levels. CONCLUSIONS: HCV quasispecies diversification is a common event during chronic hepatitis C in childhood. Host and environmental pressure could be major determinants. The increasing viral heterogeneity could impair the response to antiviral therapy, thus indicating a rationale for early antiviral treatment in children with chronic hepatitis C.

Child↗
PubMed1988

A new look at the shared epitope hypothesis.

The striking correlation observed between T cell recognition and the sharing of the DR-beta-1 gene sequences (position 67-74) among patients with rheumatoid arthritis studied suggests that the third hypervariable region might be an important contribution to one restriction site for the putative causative agent(s) in rheumatoid arthritis. The fact that this sequence was found in DR1, DR4,Dw14, and DR4,Dw15 beta-1 genes lends support to the hypothesis that, in some cases, human leukocyte antigen and disease association may involve the association of discrete disease-related epitopes rather than entire human leukocyte antigen genes and that these epitopes are immunologically relevant in terms of T cell recognition. The association of these polymorphisms with susceptibility to rheumatoid arthritis would then support the hypothesis that binding and presentation of "arthritogenic peptides" by major histocompatibility complex class II molecules is one of the pathogenic events in developing rheumatoid arthritis.

Animals↗
PubMed1990

Differential diagnosis between complete mole and hydropic abortus by deoxyribonucleic acid fingerprints.

We used a new method of deoxyribonucleic acid fingerprint analysis to obtain the differential diagnosis between complete mole and hydropic abortus. This method with a deoxyribonucleic acid minisatellite probe requires only a small amount of tissue sample and peripheral blood, and presents individual specific restriction fragment length polymorphisms (deoxyribonucleic acid "fingerprints") by simultaneous detection of many hypervariable regions (minisatellite regions) widely dispersed in the human genome. Southern blot hybridization showed that in cases of complete mole, all polymorphic fragments were exclusively inherited from the father. Some of the polymorphic bands of paternal deoxyribonucleic acid were not observed in molar deoxyribonucleic acid. However, in the hydropic abortus, the polymorphic fragments could be traced back to its parent. These results indicate that deoxyribonucleic acid fingerprints could distinguish the abnormal fertilization of complete mole (androgenesis) from the normal fertilization of hydropic abortus by identifying the difference in genetic variations between complete mole and hydropic abortus at the deoxyribonucleic acid level.

Blotting, Southern↗
PubMed1991

Restriction fragment length polymorphism analysis to study the genetic origin of complete hydatidiform mole.

To determine the genetic origin of the complete hydatidiform mole, 20 abnormal pregnancies were studied with restriction fragment length polymorphism with five genomic probes: EJ 6.6, beta-globin gene, 3'alpha-hypervariable region, J-Bir, and St14. In the 12 cases of molar pregnancy, pure paternal origin was proved in 11 cases, but both maternal and paternal inheritance were shown in only one case. In the cases with pure paternal origin, all of the restriction fragment length polymorphisms were homozygous, although those of the fathers were heterozygous at 15 loci. In the four cases that mimicked hydatidiform mole but were diagnosed as hydropic change of villi, both paternal and maternal inheritance were noted. In the four pregnancies with blighted ovum, both paternal and maternal inheritance were shown in three cases; and in one case with a balanced translocation between chromosomes 13 and 14, only paternal inheritance was noted. This study showed that most of the complete hydatidiform moles were caused by fertilization of an empty egg by a duplicated haploid sperm, but rare exceptions may exist.

Adult↗
PubMed1986

Primary structure of a cationic Cu,Zn superoxide dismutase. The sheep enzyme.

The complete amino acid sequence of Cu,Zn superoxide dismutase from sheep erythrocytes has been determined. The sequence is very similar to that of the bovine enzyme, having the same number of residues (151) and only two substitutions in the 'hypervariable' region (residues 17-30). The 5 overall substitutions confer a positive charge on the sheep enzyme at neutral pH (pI approximately equal to 8). This charge is localized outside the active site region. The catalytic efficiency of the sheep enzyme is 15% less than that of the cow enzyme, confirming the hypothesis that the enzyme activity is related to the concentration of positive surface charge near the active site channel.

Amino Acid Sequence↗
PubMed1990

DNA fingerprinting of the intestinal parasite Giardia duodenalis with the M13 phage genome.

A DNA fingerprint procedure has been established for the intestinal parasite, Giardia duodenalis. This permits the identification of individual strains from both human and animal sources. Analysis of strain movement, resurgence and variation is now possible. The fingerprint probe is based on the tandem repetitive sequence found in bacteriophage M13 which hybridizes to a set of hypervariable polymorphic minisatellite sequences found in higher eukaryotes. This probe recognizes a weakly homologous set of hypervariable regions in the Giardia genome to provide a DNA fingerprint comparable to those seen in higher eukaryotes.

Animals↗
PubMed1990

DNA polymorphisms of the apolipoprotein B gene in patients with premature coronary artery disease.

Elevated plasma levels of low density cholesterol and their major apolipoprotein (apo B) are associated with an increased risk of coronary artery disease (CAD). We have examined allele frequencies of restriction fragment length polymorphisms (RFLP) of the apo B gene in 111 male Caucasians with premature CAD (mean age 49 +/- 7 years) and in 122 elderly Caucasian males (mean age, 73 +/- 5 years), free of clinical cardiovascular disease. The rare allele (R1) of the EcoR1 RFLP in exon 29, resulting in an amino acid change (Glu----Lys4154) was seen more frequently in CAD than in controls (0.270 vs 0.207, P less than 0.05). The R1 RFLP and the MspI insertion polymorphisms (MI) within the 3' hypervariable region (HVR) were observed together in 87% and are likely in linkage disequilibrium. The MI RFLP were slightly more frequent in CAD than control (0.239 vs. 0.211, P = 0.08). A second MspI RFLP in exon 26 results in an amino acid change (Arg----Glu3611); the rare allele M2 was seen more frequently in patients than in controls (0.150 vs. 0.057, P less than 0.005). No significant differences in allele frequencies were observed for the Xba1 RFLP in exon 26 (0.500 vs. 0.529, P = ns) or for the PvuII RFLP near the 5' end (P2) (0.105 vs. 0.088, P = ns). No statistically significant differences in lipid, lipoprotein cholesterol or apolipoproteins A-I and B were observed in patients or in controls. Two of the RFLPs examined (R1 and M2) result in changes in amino acid sequence and their allele frequencies are increased in CAD cases when compared with controls. Genetic variability within the apo B gene may thus contribute to cardiovascular risk. The physiological effects of individual mutations within apo B remain to be determined. It is unlikely, however that the single site polymorphisms examined in this study, will impart further information about CAD risk than conventional lipid parameters.

Adult↗
PubMed1991

Familial defective apolipoprotein B-100: haplotype analysis of the arginine(3500)----glutamine mutation.

Familial defective apolipoprotein B-100 (FDB) is a recently identified, dominantly inherited genetic disorder, which leads to an increased serum level of low density lipoprotein (LDL) cholesterol with reduced affinity for the LDL receptor. It is postulated that this disorder results from a G to A mutation at nucleotide 10,708 in exon 26 of the apo B gene creating a substitution of glutamine for arginine in the codon for amino acid 3500. To investigate whether recurrent mutation has contributed to the high frequency of FDB, we have conducted a haplotype analysis in previously reported and newly detected FDB heterozygotes in Germany. 5 FDB families and 6 unrelated FDB heterozygotes were genotypes at 4 polymorphic sites in the 3' end of the apo B gene. These sites consisted of the diallelic markers XbaI, MspI, EcoRI and the hypervariable region (3'HVR). In 5 FDB families and 1 unrelated FDB heterozygote the arginine(3500)----glutamine mutation could be unambiguously assigned to the haplotype XbaI-/MspI+/EcoRI-/3'HVR48, in the other 5 FDB unrelated heterozygotes this finding was consistent with the combination of the genotype. The existence of the arginine(3500)----glutamine mutation on the same and supposedly rare allele suggests that the mutant alleles are identical by descent in our population. The fact that the same mutant allele was identified in North America and Austria suggests a common European origin of the arginine(3500)----glutamine mutation.

Adult↗
PubMed1988

Anti-HV3 peptide antibodies as probes for conformational changes in immunoglobulins.

Recent studies have shown that monoclonal anti-alprenolol antibody 37A4 could mimic the beta-adrenergic receptor with respect to binding properties and alprenolol-induced changes in intrinsic fluorescence. We prepared and characterized polyclonal antibodies against a synthetic tetradecapeptide corresponding to residues 92-105 of the 37A4 VH domain. In ELISA and immunoblotting assays, the anti-peptide antibodies bound to the intact IgG molecule as well as to the isolated heavy chain. They failed to interact with the native IgG protein but recognized with high specificity the conformationally modified Ig molecule; as a consequence, their reactivity to the entire protein was found to vary with the conditions of the assay. This study illustrates that anti-peptide antibodies might be used as probes for the detection of conformational changes occurring in the Ig hypervariable regions.

Animals↗
PubMed1992

Unexpected interaction of some anti-TNP hybridoma antibodies with Superose HPLC gel filtration resins.

Five different hybridoma antibodies (the isotypes IgG1, IgG2a and IgG2b), reactive with TNP, showed increased elution times when gel filtered on a Superose-12 HPLC column corresponding to apparent molecular weights ranging from 54 kDa to 120 kDa compared to the normal of 150 kDa. One of the antibodies (C1901-B4) was studied in detail showing that the unusual gel filtration behaviour was localized to the Fab part of the molecule. SDS-PAGE analysis showed that the intact antibodies had normal molecular weights. Gel filtration on a Toyosoda G-3000SW HPLC column and Sephadex G-150 as well as Sepharose 6B generally showed normal elution times. These results support the hypothesis that the retardation on the Superose gel is probably due to aromatic interactions between amino acid residues supposedly exposed in the hypervariable region (i.e., the antibody combining site) of the antibody, and the gel matrix which is rich in ether O-atoms created during the manufacturing process. If this hypothesis is correct one might expect such interactions between Superose resins and antibodies of many different specificities in which aromatic amino acids are exposed in the combining sites.

Chromatography, Gel↗
PubMed1995

A direct non-competitive idiometric enzyme immunoassay for serum oestradiol.

We report a novel non-competitive enzyme immunoassay for oestradiol based on the use of two types of anti-idiotypic antibody that recognize different epitopes within the hypervariable region of the primary anti-oestradiol idiotypic antibody (Ab1). The first anti-idiotype, the betatype, competes with the analyte for an epitope of the primary antibody at the binding site. On the other hand, the second anti-idiotype, the alphatype, binds to the Ab1 in the presence of analyte but does not bind to the betatype/Ab1 complex because of steric hindrance. In the present format the biotinylated alphatype was captured onto anti-biotin IgG which was adsorbed on the surface of microtitre wells. Reaction mixtures containing the Ab1 complexed sequentially with an enzyme labelled second antibody reagent, with oestradiol standards or serum samples and with the betatype anti-idiotypic antibody were then allowed to react with the immobilized alphatype anti-idiotypic antibody. The enzyme activity of the bound fraction measured at 405 nm increased with increasing oestradiol concentrations over the range 0.06-2.5 ng/ml. The detection limit of the assay was 28 pg/ml. The intra-assay variation ranged from 3.5 to 12.4%, and inter-assay variation from 6 to 13.4%. The results obtained by the colorimetric idiometric immunoassay correlated well with those obtained by a direct radioimmunoassay (n = 85, r = 0.97). This non-competitive immunoassay, termed idiometric assay, for haptens permits the development of sensitive immunoassays with a wide working range, and a variety of end-point determinations depending on the label used (e.g., enzyme, chemiluminescent or fluorogenic compound).

Animals↗
PubMed1985

Domain association in immunoglobulin molecules. The packing of variable domains.

We have analyzed the structure of the interface between VL and VH domains in three immunoglobulin fragments: Fab KOL, Fab NEW and Fab MCPC 603. About 1800 A2 of protein surface is buried between the domains. Approximately three quarters of this interface is formed by the packing of the VL and VH beta-sheets in the conserved "framework" and one quarter from contacts between the hypervariable regions. The beta-sheets that form the interface have edge strands that are strongly twisted (coiled) by beta-bulges. As a result, the edge strands fold back over their own beta-sheet at two diagonally opposite corners. When the VL and VH domains pack together, residues from these edge strands form the central part of the interface and give what we call a three-layer packing; i.e. there is a third layer composed of side-chains inserted between the two backbone side-chain layers that are usually in contact. This three-layer packing is different from previously described beta-sheet packings. The 12 residues that form the central part of the three observed VL-VH packings are absolutely or very strongly conserved in all immunoglobulin sequences. This strongly suggests that the structure described here is a general model for the association of VL and VH domains and that the three-layer packing plays a central role in forming the antibody combining site.

Amino Acid Sequence↗
PubMed1988

One type of gamma-turn, rather than the other gives rise to chain-reversal in proteins.

Gamma-turns may be defined by a hydrogen bond between the carbonyl group of one amino acid residue and the amino group of the acid two residues ahead in the sequence. They occur as two types, inverse gamma-turns and classic gamma-turns (classic gamma-turns are usually called just gamma-turns but we prefer to add the adjective classic to distinguish them from the word gamma-turn, referring collectively to both). Of the two, classic gamma-turns are less common and are considered by all authors to be extreme rarities in proteins. However, we find that a number do occur in a sample of proteins of known three-dimensional structure. One occurs at the edge of the second hypervariable region of the light chain in some immunoglobulins. All classic gamma-turns except one are associated with a reversal in the main chain direction. In most cases, the turn lies at the loop end of a beta-hairpin. By contrast, inverse gamma-turns, although giving rise to a kink in the chain, rarely occur within beta-hairpins and are seldom situated at a position of reversal, by 180 degrees, in chain direction.

Amino Acids↗
PubMed1992

Refined structure of rat Clara cell 17 kDa protein at 3.0 A resolution.

The rat Clara cell 17 kDa protein (previously referred to as the rat Clara cell 10 kDa protein) has been reported to inhibit phospholipase A2 and papain, and to also bind progesterone. It has been isolated from rat lung lavage fluid and crystallized in the space group P6(5)22. The structure has been determined to 3.0 A resolution using the molecular replacement method. Uteroglobin, whose amino acid sequence is 55.7% identical, was used as the search model. The structure was then refined using restrained least-squares and simulated annealing methods. The R-factor is 22.5%. The protein is a covalently bound dimer. Two disulfide bonds join the monomers together in an antiparallel manner such that the dimer encloses a large internal hydrophobic cavity. The hydrophobic cavity is large enough to serve as the progesterone binding site, but access to the cavity is limited. Each monomer is composed of four alpha-helices. The main-chain structure of the Clara cell protein closely resembles that of uteroglobin, but the nature of many of the exposed side-chains differ. This is true, particularly in a hypervariable region between residues 23 and 36, and in the H1H4 pocket.

Amino Acid Sequence↗
PubMed1994

Investigation of shape variations in the antibody binding site by molecular dynamics computer simulation.

Molecular dynamics simulations have been used to investigate the flexibility and variations in the shape of the binding site of an antibody against human Rhinovirus serotype 2 (HRV2) and its complex with a 15 amino acid oligopeptide, the structure of which has been recently determined by X-ray crystallography. During the simulation of the unbound antibody the binding site, defined in terms of the hypervariable regions or complementarity determining regions (CDRs), shows significant fluctuations in shape. For the complex such variations in the shape of the binding site were reduced. The largest fluctuations in the unbound antibody occurred within the CDR-H3. The largest differences between the bound and unbound crystal structures are also associated with CDR-H3. The relative displacements of the loops have been analysed in terms of internal distortions, rigid body motions of the loops and changes with respect to the framework regions. The degree to which the motions of the loops are correlated and the variation in the volume of the binding pocket during the simulation have also been examined.

Amino Acid Sequence↗