Antibodies in human sera specific to hypervariable region 1 of hepatitis C virus can block viral attachment

It has been postulated that antibodies specific to the hypervariable region 1 (HVR1) within the putative envelop protein E2 of hepatitis C virus (HCV) can neutralize virus. We studied such antibodies in sera of patients who were infected in a single-source outbreak by a contaminated anti-D immunoglobulin preparation (HCV-AD78). The nucleotide sequences of cDNAs encoding HVR1 of HCV-AD78 were determined. The four major variants (HVR1.A, B, C, and D) were expressed as fusion proteins in Escherichia coli. Sixty-seven percent of sera contained antibodies to HVR1.A. Sera unrelated to infection of the outbreak also recognized HVR1.A but to a lesser extent (15%), suggesting that not all HVR1-specific antibodies are absolutely isolate-specific. Antibodies directed against individual variants of HVR1 were found in sera obtained early postinfection (p.i.) (< or = 1 year) but also in sera obtained several years later. An in vitro binding assay of HCV to tissue culture cells was employed to further characterize these sera. Five of seven sera that were obtained early p.i. prevented binding of HCV to cells. Preincubation of such sera with HVR1-specific fusion proteins restored binding of HCV to cells in four of five sera. These findings suggest that the majority of neutralizing antibodies are directed against HVR1.     

Murine antibodies against E2 and hypervariable region 1 cross-reactively capture hepatitis C virus

The purpose of this study was to determine whether supplemental estrogen influences cardiovascular hemodynamics at peak exercise in endurance trained and sedentary postmenopausal women. Subjects were 22 women between 3 and 10 yr past menopause who had engaged in endurance exercise at least three times per week for one or more years. Twelve of the women had taken estrogen replacement for at least 1 yr (ER) while the other 10 had never taken supplemental estrogen (NOER). Peak cardiac output (Qpeak) and, subsequently, peak cardiac index (QIpeak) were calculated by regressing submaximal cardiac output values on corresponding oxygen consumptions and extrapolating to peak exercise. Peak oxygen consumption in the two groups were almost identical; however, the ER group demonstrated a higher QIpeak in conjunction with a lower arteriovenous oxygen difference and a lower peripheral resistance. It was concluded that estrogen supplementation may be associated with higher peak cardiac outputs in exercise trained postmenopausal women via alterations in the peripheral vascular and oxygen kinetic responses to maximal exercise.     

Variation of hepatitis C virus hypervariable region 1 in immunocompromised patients

The viral variability of 5 hepatitis C virus (HCV)-infected immunocompromised patients was analyzed and compared with that in isolates from immunocompetent subjects. The patients were followed longitudinally with regard to changes in hypervariable region 1 (HVR1) of HCV using a direct DNA sequencing approach. For the immunocompromised patients, viral nucleotide sequence variability was markedly lower than in immunocompetent HCV-positive patients. For 1 agammaglobulinemic patient and 1 AIDS patient, no variation in the major amino acid sequence of HCV HVR1 could be observed, while another agammaglobulinemic patient exhibited transient variations and amino acid substitutions despite the lack of functioning humoral immune response. The study supports the general hypothesis of humoral immune selection as the main force of sequence variation in the HVR1 region but suggests that other selection mechanisms may contribute to modulation of the composition of the viral population.     

Evolution of hypervariable region 1 of hepatitis C virus in primary infection

The hypervariable region 1 (HVR-1) of the putative envelope encoding E2 region of hepatitis C virus (HCV) RNA was analyzed in sequential samples from three patients with acute type C hepatitis infected from different sources to address (i) the dynamics of intrahost HCV variability during the primary infection and (ii) the role of host selective pressure in driving viral genetic evolution. HVR-1 sequences from 20 clones per each point in time were analyzed after amplification, cloning, and purification of plasmid DNA from single colonies of transformed cells. The intrasample evolutionary analysis (nonsynonymous mutations per nonsynonymous site [Ka], synonymous mutations per synonymous site [Ks], Ka/Ks ratio, and genetic distances [gd]) documented low gd in early samples (ranging from 2. 11 to 7.79%) and a further decrease after seroconversion (from 0 to 4.80%), suggesting that primary HCV infection is an oligoclonal event, and found different levels and dynamics of host pressure in the three cases. The intersample analysis (pairwise comparisons of intrapatient sequences; rKa, rKs, rKa/rKs ratio, and gd) confirmed the individual features of HCV genetic evolution in the three subjects and pointed to the relative contribution of either neutral evolution or selective forces in driving viral variability, documenting that adaptation of HCV for persistence in vivo follows different routes, probably representing the molecular counterpart of the viral fitness for individual environments.     

Long-term evolution of the hypervariable region of hepatitis C virus in a common-source-infected cohort

The long-term evolution of the hepatitis C virus hypervariable region (HVR) and flanking regions of the E1 and E2 envelope proteins have been studied in a cohort of women infected from a common source of anti-D immunoglobulin. Whereas virus sequences in the infectious source were relatively homogeneous, distinct HVR variants were observed in each anti-D recipient, indicating that this region can evolve in multiple directions from the same point. Where HVR variants with dissimilar sequences were present in a single individual, the frequency of synonymous substitution in the flanking regions suggested that the lineages diverged more than a decade previously. Even where a single major HVR variant was present in an infected individual, this lineage was usually several years old. Multiple lineages can therefore coexist during long periods of chronic infection without replacement. The characteristics of amino acid substitution in the HVR were not consistent with the random accumulation of mutations and imply that amino acid replacement in the HVR was strongly constrained. Another variable region of E2 centered on codon 60 shows similar constraints, while HVR2 was relatively unconstrained. Several of these features are difficult to explain if a neutralizing immune response against the HVR is the only selective force operating on E2. The impact of PCR artifacts such as nucleotide misincorporation and the shuffling of dissimilar templates is discussed.     

T cell recognition of hypervariable region-1 from hepatitis C virus envelope protein with multiple class II MHC molecules in mice and humans: preferential help for induction of antibodies to the hypervariable region

Hypervariable region-1 (HVR1) from the hepatitis C virus (HCV) envelope protein is thought to be a target for neutralizing Abs. To explore HVR1 recognition by helper T cells, and their role in Ab responses, we attempted to generate helper T cells specific for HVR1 in mice of three MHC types, and with PBMC from HCV-infected HLA-diverse humans. In both species, HVR1 was presented by >1 class II MHC molecule to CD4+ helper T cells and showed surprising interisolate cross-reactivity. The epitope for two DR4+ patients was mapped to a more conserved C-terminal sequence containing a DR4 binding motif, possibly accounting for cross-reactivity. Strikingly, Abs to patients' own HVR1 sequences were found only in patients with T cell responses to HVR1, even though all had Abs to envelope protein, suggesting that induction of Abs to HVR1 depends on helper T cells specific for a sequence proximal to the Ab epitope. Thus, helper T cells specific for HVR1 may be functionally important in inducing neutralizing Abs to HCV. These results may be the first example of "T-B reciprocity," in which proximity of a helper T cell epitope determines Ab epitope specificity, in a human disease setting.     

Hepatitis C virus (HCV) hypervariable region 1 complexity does not correlate with severity of liver disease, HCV type, viral load or duration of infection

OBJECTIVE: To determine whether increased sympathetic vasoconstrictor drive to the calf in heart failure is associated with reduced symptom-limited submaximal exercise performance. DESIGN: Blood pressure, heart rate and peroneal muscle sympathetic nerve activity (MSNA) were recorded during rest and before symptom-limited treadmill exercise at 70% of resting heart rate reserve for up to 45 mins. PATIENTS: Thirteen young patients with dilated cardiomyopathy (age 36 +/- 2 years). RESULTS: MSNA (45 +/- 6 bursts/min) was more than double the level documented in an age-, sex- and weight-matched group of normal subjects studied under identical baseline conditions. Patients with ventricular dysfunction exercised for 30 +/- 3 mins on average, but exercise distance and duration were independent of resting MSNA. CONCLUSIONS: In young patients with dilated cardiomyopathy, sympathetic nerve traffic to the calf vascular bed, measured at rest, does not predict the distance or duration of symptom-limited submaximal treadmill exercise.     

Mutations in NS5A region of hepatitis C virus genome correlate with presence of NS5A antibodies and response to interferon therapy for most common European hepatitis C virus genotypes

A part of the hepatitis C virus (HCV) nonstructural protein 5A (NS5A) amino acid sequence, designated as an interferon (IFN)-sensitive determining region (ISDR), has been shown to be correlated with a response to IFN in Japanese patients. We have shown previously that the presence of NS5A antibodies (Abs) detected by the INNOLIA test (IL-NS5A Ab) is also correlated with a response to IFN. The aim of this study was to investigate, in a wide range of patients, the possible relationship within the NS5A protein between the sequence of ISDR and that used in the INNOLIA test designated as IL3R. Serum samples from 52 patients infected by HCV genotypes 1, 2, and 3 were analyzed before and after treatment. The patients were classified as nonresponders (NRs), responder-relapsers (RRs), or long-term responders (LTRs). We amplified the NS5A region for 42 patients using polymerase chain reaction (PCR), and these amplicons were sequenced directly. The 10 remaining patients were analyzed using PCR with mutation-specific primers. No correlation was found between the IL3R sequence of the HCV strains and the presence of the IL-NS5A Ab for all genotypes. However, for the subtype 1b, only 2 of 11 NR patients tested had an arginin in position 2218 within the ISDR versus 3 of 3 LTR and 10 of 13 RR patients. All patients with R-2218 had IL-NS5A Ab. For the genotype 1a, 2 of 2 LTR and 1 of 3 RR were mutated in position 2216-2218 in comparison to three NR sequences. For the genotype 3, no mutations were found in the region homologous to 1b-ISDR, but 4 of 5 LTR and RR patients had a mutation T-2161 to A or V versus 0 of 3 NR patients. A close correlation was found between arginin in position 2218 in ISDR, the presence of IL-NS5A Ab, and the response to IFN therapy for genotype 1b, but this association did not predict a long-term response. For genotype 3, a potential ISD mutation could be located at the codon 2161.     

Gradual loss of IgG antibodies against GB virus C/hepatitis G virus in a patient with AIDS

GB virus C/hepatitis G virus (GBV-C/HGV) is a positive-sense, single-stranded RNA virus belonging to the family Flaviviridae and is distantly related to hepatitis C virus (HCV). GBV-C/HGV can be transmitted by the parenteral and the sexual route. Among individuals infected with human immunodeficiency virus type 1 (HIV-1) by the sexual route, we and others have demonstrated a high prevalence of GBV-C/HGV infection. Recently, Woolley and colleagues reported that AIDS patients co-infected with GBV-C/HGV had a significantly lower mean CD4 cell count than AIDS patients without GBV-C/HGV infection, suggesting that GBV-C/HGV antibody may be lost with progression to AIDS. To our knowledge no data are available on the loss of antibody against GBV-C/HGV in AIDS patients. We now report on an HIV-infected patient who exhibited gradual loss of IgG antibodies against GBV-C/HGV, as well as HCV, with progression of HIV disease.     

Suppression of host immune response by the core protein of hepatitis C virus: possible implications for hepatitis C virus persistence

Hepatitis C virus (HCV) is a major human pathogen causing mild to severe liver disease worldwide. This positive strand RNA virus is remarkably efficient at establishing chronic infections. Although a high rate of genetic variability may facilitate viral escape and persistence in the face of Ag-specific immune responses, HCV may also encode proteins that facilitate evasion of immunological surveillance. To address the latter possibility, we examined the influence of specific HCV gene products on the host immune response to vaccinia virus in a murine model. Various vaccinia/HCV recombinants expressing different regions of the HCV polyprotein were used for i.p. inoculation of BALB/c mice. Surprisingly, a recombinant expressing the N-terminal half of the polyprotein (including the structural proteins, p7, NS2, and a portion of NS3; vHCV-S) led to a dose-dependent increase in mortality. Increased mortality was not observed for a recombinant expressing the majority of the nonstructural region or for a negative control virus expressing the beta-galactosidase protein. Examination of T cell responses in these mice revealed a marked suppression of vaccinia-specific CTL responses and a depressed production of IFN-gamma and IL-2. By using a series of vaccinia/HCV recombinants, we found that the HCV core protein was sufficient for immunosuppression, prolonged viremia, and increased mortality. These results suggest that the HCV core protein plays an important role in the establishment and maintenance of HCV infection by suppressing host immune responses, in particular the generation of virus-specific CTLs.     

Involvement of the 5'' proximal coding sequences of hepatitis C virus with internal initiation of viral translation

To examine patterns of identity development for late adolescents raised in the Norwegian mixed liberal welfare-state economic system compared with late adolescents raised in the free-market economic system of the United States, ego identity status scores and distributions were examined for 56 (37 women, 19 men). Norwegian and 1498 (814 women, 684 men) United States undergraduate university students using the Extended Objective Measure of Ego Identity Status-2. The United States sample was drawn from four geographic regions and comprised of those who had participated in prior studies performed by Adams. Significant differences were found between the two nations on all identity status subscales in the ideological and interpersonal domains for each sex. The more moderate identity status scale scores evidenced by the Norwegian sample may reflect a cultural trend toward greater moderation in the exploration and commitment process.     

Truncating the putative membrane association region circumvents the difficulty of expressing hepatitis C virus protein E1 in Escherichia coli

The putative E1 of hepatitis C virus (HCV) was expressed in Escherichia coli using a glutathione-S-transferase (GST) fusion protein system. The full length E1 protein is difficult to express. A series of E1 DNA fragments was generated and used for expression vector construction. Fusion proteins containing the E1 C-terminal region could not be expressed. When this region was truncated, the fusion proteins were synthesized to high levels. The possibility of this C-terminal region hampering the production of fusion protein was further explored. A construct with this segment directly fused to the C-terminus of GST indeed generated no detectable recombinant protein. According to the predicted structure of E1, this region may have membrane-associating properties. The expression results suggest a general approach to facilitate the production of viral membrane proteins in prokaryotes. Furthermore, these recombinant E1 proteins generated as antigens were used for Western blotting with sera from HCV-infected individuals. It was found that E1 is antigenic during HCV natural infection.     

Seroprevalence survey of Egyptian tourism workers for hepatitis B virus, hepatitis C virus, human immunodeficiency virus, and Treponema pallidum infections: association of hepatitis C virus infections with specific regions of Egypt

Blood samples from 740 Egyptian Nationals working in the tourism industry at two sites in the South Sinai governorate were screened for markers of infection with hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and Treponema pallidum. Study subjects included 467 individuals from a rural seashore tourist village and 273 persons at two hotels in a well-established resort town. Subjects' ages ranged from 15 to 70 years; 99.3% were male. The prevalence of serologic markers for currently asymptomatic or past HBV infection alone was 20.7% (n = 153), of markers for past or chronic HCV infection alone was 7.4% (n = 55), and of markers for both HBV and HCV was 6.9% (n = 51). Of the 204 individuals positive for anti-HBV core antibody, 12 (5.9%) were also positive for hepatitis B surface antigen. Two individuals (0.3%) had a serologic market suggestive of an active syphilitic infection. No subject was found to be HIV-seropositive. History of prior injections and number of injections were associated with infection with HCV. Primary residence in the Nile delta and valley areas where schistosomiasis is highly endemic, was also a statistically significant risk factor for HCV, but not HBV infection.     

Serological determination of hepatitis C virus genotype: comparison with a standardized genotyping assay

In patients with chronic hepatitis C, determination of hepatitis C virus (HCV) genotype could be routinely run in the future to tailor treatment schedules. The suitabilities of two versions of a serological, so-called serotyping assay (Murex HCV Serotyping Assay version 1-3 [SA1-3] and Murex HCV Serotyping Assay version 1-6 [SA1-6]; Murex Diagnostics Ltd.), based on the detection of genotype-specific antibodies directed to epitopes encoded by the NS4 region of the genome, for the routine determination of HCV genotypes were studied. The results were compared with those of a molecular biology-based genotyping method (HCV Line Probe Assay [INNO-LiPA HCV]; Innogenetics S.A.), based on hybridization of PCR products onto genotype-specific probes designed in the 5' noncoding region of the genome, obtained with pretreatment serum samples from 88 patients with chronic hepatitis C eligible for interferon therapy. Definitive genotyping was performed by sequence analysis of three regions of the viral genome in all samples with discrepant typing results found among at least two of the three assays studied. In all instances, sequence analysis confirmed the result of the INNO-LiPA HCV test. The sensitivity of SA1-3 was 75% relative to the results obtained by the genotyping assay. The results were concordant with those of genotyping for 92% of the samples typeable by SA1-3. The sensitivity of SA1-6 was 89% relative to the results obtained by the genotyping assay. The results were concordant with those of genotyping for 94% of the samples typeable by SA1-6. Overall, SA1-6 had increased sensitivity relative to SA1-3 but remained less sensitive than the genotyping assay on the basis of PCR amplification of HCV RNA. Cross-reactivities between different HCV genotypes could be responsible for the mistyping of 8 (SA1-3) and 6% (SA1-6) of the samples. Subtyping of 1a and 1b is still not possible with the existing peptides, but discriminating between subtypes may not be necessary for routine use.     

Evaluation of a novel serotyping system for hepatitis C virus: strong correlation with standard genotyping methodologies

Direct sequencing and analysis of viral genomes are definitive methods for identifying various hepatitis C virus (HCV) genotypes. However, HCV genome sequencing methods are cumbersome and unsuitable for analyzing large numbers of clinical samples. We have developed a convenient, reliable, and reproducible RIBA strip immunoblot assay system for determining HCV serotype. Briefly, the assay consists of an immunoblot strip on which there are five lanes of immobilized serotype-specific HCV peptides from the nonstructural (NS-4) and core regions of the genomes of HCV types 1,2, and 3. HCV serotype is deduced by determining the greatest intensity of reactivity to the NS-4 serotype-specific HCV peptide band in relation to the intensity of the human immunoglobulin G internal control bands on each strip. HCV core peptide reactivity is used only in the absence of NS-4 reactivity. We used this assay to successfully serotype a high percentage of sera from well-documented HCV-infected patients. Our serotyping results correlated 99% with the findings from the standard restriction fragment length polymorphism genotyping methods. Less than 5% of the serum samples were untypeable. For a selected group of alpha interferon-treated patients we observed that the nonresponders (76.2%) and a majority of the responders who relapsed (72.2%) had type 2 HCV infection. A small population (n= 8) of complete responders was split 3:4:1 as type 1, type 2, and type 3, respectively. Our data indicate that this new serotyping assay has the potential to be a highly specific and reliable method for typing of HCV infection in patients.     

Evolutionary variants of the human immunodeficiency virus type 1 V3 region characterized by using a heteroduplex tracking assay

Syncytium-inducing (SI) variants of human immunodeficiency virus type 1 (HIV-1) are evolutionary variants that are associated with rapid CD4+ cell loss and rapid disease progression. The heteroduplex tracking assay (HTA) was used to detect evolutionary V3 variants by amplifying the V3 sequences from viral RNA derived from 50 samples of patient plasma. For this V3-specific HTA (V3-HTA), heteroduplexes were formed between the patient V3 sequences and a probe with the subtype B consensus V3 sequence. Evolution was then measured by divergence from the consensus. The presence of evolutionary variants was correlated with SI detection data on the same samples from the MT-2 cell culture assay. Evolutionary variants were correlated with the SI phenotype in 88% of the samples, and 96% of the SI samples contained evolutionary variants. In most cases the evolutionary V3 variants represented discrete clonal outgrowths of virus. Sequence analysis of the six discordant samples that did not show this correlation indicated that three non-syncytium-inducing (NSI) samples had V3 sequences that had evolved away from the consensus sequence but not toward an SI genotype. A fourth sample showed little evolution away from the consensus but was SI, which indicates that not all SI variants require basic substitutions in V3. The other two samples had SI-like genotypes and NSI phenotypes, suggesting that V3-HTA was able to detect SI emergence in these samples in the absence of their detection in vitro. V3-HTA was also used to confirm SI variant selection in MT-2 cells and to examine the possibility of variant selection during virus culture in peripheral blood cells.     

Search for hepatitis C virus extrahepatic replication sites in patients with acquired immunodeficiency syndrome: specific detection of negative-strand viral RNA in various tissues

Systemically administered interleukin-1 (IL-1) has been shown to preferentially bind to IL-1 receptors (IL-1Rs) in inflammation. Using radiolabeled IL-1alpha and molecular methods to assess gene expression for these receptors, the in vivo behavior of these receptors was investigated in a number of experimental inflammatory conditions. The uptake of 125I-labeled IL-1alpha in inflammatory foci significantly correlated with the mRNA expression for the type I and type II IL-1Rs (P < .05). Type II IL-1R mRNA showed a greater increase in expression than type I IL-1R mRNA. In neutropenic mice, inflammatory lesions, which are devoid of granulocytes, significantly lower 125I-labeled IL-1alpha uptake (P < .001), and type II IL-1R mRNA expression (P < .005) was found. Thus, there is strong up-regulation of IL-1Rs at sites of focal inflammation. Of interest, this mainly involved the type II IL-1R on granulocytes, which is not involved in signal transduction.