Diagnosis of Hepatitis C Virus Infection Using a Novel Mouse Monoclonal Antibody to Detect Serum HCV E1/E2 Antigens.

BACKGROUND: Hepatitis C virus (HCV) is a major cause of liver disease worldwide and in Egypt. The aim of this study was to detect HCV E1/E2 antigens using a novel mouse monoclonal antibody (mAb) designated (7G9) as a diagnostic and alternative approach for HCV detection. METHODS: The detection of HCV-E1/E2 antigens in 138 patients positive for HCV infection tested by RT-PCR and 25 healthy individuals negative for HCV as control group was done by an optimized in-house ELISA and DotELISA (based on the molecular mimicry of E2 to immunoglobulins). RESULTS: The mAb (7G9) was found to be IgM (heavy-chain)/kappa (light-chain) and characterization by western blot revealed two bands at 63 kDa for E2 and 31 kDa for E1. ELISA peptide mapping showed high reactivity with peptide derived from HCV E1 (a.a. 315 - 323) and low reactivity to peptides derived from HCV E2 (a.a. 517 - 531) and HCV E2 (a.a. 412 - 419). The mAb (7G9) showed no reactivity with HBV Ag, S. typhi or B. Abortus Ag proving high specificity. AUC for HCV-E1/E2 detection was 0.96 for all HCV patients with sensitivity 87% (119/137), specificity 88% (22/25) and efficiency 87%. The HCV-E1/E2 antigens detection by Dot-ELISA showed 76.8% sensitivity, 88% specificity and the efficiency of the assay was 78.5%. Furthermore, no correlation was found between serum HCV viral load and HCV E1/E2 antigens detection. CONCLUSIONS: The ELISA and Dot-ELISA based on the monoclonal antibody (7G9) are reliable, rapid, easy and economic diagnostic assays for HCV infection.

Instructive even after a decade: Complete results of initial virological diagnostics and re-evaluation of molecular data in the German rabies virus "outbreak" caused by transplantations.

In 2005, six patients in Germany received solid organs and both corneas from a donor with an unrecognized rabies infection. Initial virological diagnostics with the machinery available at the two national reference laboratories could quickly clarify the situation. Rabies virus antigen was detected in the organ donor's brain. In two of the three recipients with neurological alterations, intra vitam diagnosis was achieved by conventional RT-PCRs. Comparison of the phylogenetic relatedness of the different viral isolates proved transmission from the donor and, consequently, also established the diagnosis for the third patient. As indicated by the titre of neutralizing antibodies, the liver transplant recipient was protected from the lethal infection due to a vaccination against rabies virus, which he had received more than 15 years ago. All samples from the recipients of the corneas were invariably negative. Re-evaluation of the molecular data by real-time PCR did not lead to an improvement of intra vitam diagnosis but provided intriguing insights regarding the relative amounts of rabies virus RNA in different body fluids and peripheral organs. In saliva and skin, they were 250-200,000 times lower than in the infected patient's brains. Furthermore, in saliva samples taken serially from the same patient fluctuations by a factor of 160-500 were recorded. These findings highlight the problems of intra vitam diagnosis of rabies virus infections and make understandable why the virus can escape from all diagnostic attempts. Finally, in this context one should recall an almost trivial fact: Simple and appropriate postexposure prophylaxis could not only have saved the young organ donor's life but would also have prevented the whole transplantation-associated rabies "outbreak" in Germany.

HLA-B*27 subtype specificity determines targeting and viral evolution of a hepatitis C virus-specific CD8+ T cell epitope.

BACKGROUND & AIMS: HLA-B*27 is associated with spontaneous HCV genotype 1 clearance. HLA-B*27-restricted CD8+ T cells target three NS5B epitopes. Two of these epitopes are dominantly targeted in the majority of HLA-B*27+ patients. In chronic infection, viral escape occurs consistently in these two epitopes. The third epitope (NS5B2820) was dominantly targeted in an acutely infected patient. This was in contrast, however, to the lack of recognition and viral escape in the large majority of HLA-B*27+ patients. Here, we set out to determine the host factors contributing to selective targeting of this epitope. METHODS: Four-digit HLA class I typing and viral sequence analyses were performed in 78 HLA-B*27+ patients with chronic HCV genotype 1 infection. CD8+ T cell analyses were performed in a subset of patients. In addition, HLA/peptide affinity was compared for HLA-B*27:02 and 05. RESULTS: The NS5B2820 epitope is only restricted by the HLA-B*27 subtype HLA-B*27:02 (that is frequent in Mediterranean populations), but not by the prototype HLA-B*27 subtype B*27:05. Indeed, the epitope is very dominant in HLA-B*27:02+ patients and is associated with viral escape mutations at the anchor position for HLA-binding in 12 out of 13 HLA-B*27:02+ chronically infected patients. CONCLUSIONS: The NS5B2820 epitope is immunodominant in the context of HLA-B*27:02, but is not restricted by other HLA-B*27 subtypes. This finding suggests an important role of HLA subtypes in the restriction of HCV-specific CD8+ responses. With minor HLA subtypes covering up to 39% of specific populations, these findings may have important implications for the selection of epitopes for global vaccines.

Genetic heterogeneity of hepatitis C virus cell entry receptors seems to have no influence on selection of virus variants.

BACKGROUND: No information is available on the possible influence of the genetic heterogeneity of major hepatitis C virus (HCV) cell receptors on selection of virus variants. FINDINGS: Anti-D globulin preparations contaminated with the HCV strain AD78 caused hepatitis C infection in more than 3000 women in East Germany in 1978. Analysis of the core to NS2 gene sequences of this strain in several globulin batches revealed the presence of three closely related but distinct virus variants of the same strain. Apparently even distribution of these three virus variants was observed in 91 patients infected with the AD78 strain. None of these patients was infected with more than one virus variant, suggesting a selection mechanism of a particular virus variant in each patient. To verify the hypothesis that heterogeneity of HCV cell receptors might influence the virus variant selection, single-nucleotide polymorphisms (SNPs) in low-density lipoprotein receptor (LDLR), occludin (OCLN), and scavenger receptor B1 (SCARB1) genes in AD patients were analyzed. No evident correlation between receptor polymorphisms and presence of a particular virus variant was noted. CONCLUSION: SNPs of HCV cell entry receptors have no influence on virus selection in patients infected with an inoculum containing different virus variants.

Hepatic and serum levels of miR-122 after chronic HCV-induced fibrosis.

BACKGROUND & AIMS: The progression of liver fibrosis in patients with chronic hepatitis C (CHC) is important to decide on the treatment of the virus. As liver biopsy and liver stiffness measurement for staging of fibrosis present limitations, circulating levels of miR-122 have been suggested as a novel biomarker to predict the extent of liver injury. We evaluated the potential of miR-122 as an indicator of fibrosis progression in CHC infection and performed, for the first time, a comprehensive analysis of hepatic and circulating miR-122 levels in patients with CHC. METHODS: Patients with well-documented CHC infection were selected from the database of HepNet, the German-Competence-Network on Viral Hepatitis. All patients underwent blood sampling and liver biopsy with grading of inflammation and staging of fibrosis. RNA was extracted from 84 liver biopsies and 164 serum samples of CHC patients. miR-122 levels in liver and serum samples were quantified by real-time PCR normalized to RNU6 or spiked-in RNA, respectively. RESULTS: Hepatic levels of miR-122 decreased significantly with the severity of fibrosis (p = 0.001). In addition, circulating miR-122 levels correlated negatively with increasing stages of fibrosis, although the inverse correlation was moderate due to a two-phase miR-122 pattern during fibrosis progression. Thus, circulating miR-122 levels decreased in patients with severe fibrosis (F3, F4), while at early stages with distinct fibrotic structures (F2) and high inflammatory activity, miR-122 serum levels were elevated. CONCLUSIONS: We conclude that during progression of fibrosis less miR-122 is released into the blood stream due to the loss of liver cells and the decrease of hepatic miR-122 levels. Although the release of circulating miR-122 possibly mirrors acute liver injury, in chronic liver disease and fibrosis, the loss of liver cells and the decreased hepatocellular miR-122 expression render miR-122 an inappropriate marker, when exclusively used for interpretation of fibrosis progression.

CD8+ T-cell response promotes evolution of hepatitis C virus nonstructural proteins.

BACKGROUND & AIMS: Hepatitis C virus (HCV) acquires mutations that allow it to escape the CD8+ T-cell response, although the extent to which this process contributes to viral evolution at the population level is not clear. We studied viral adaptation using data from a large outbreak of HCV genotype 1b infection that occurred among women immunized with contaminated immunoglobulin from 1977 to 1978. METHODS: The HCV nonstructural protein coding regions NS3-NS5B were sequenced from 78 patients, and mutations were mapped according to their location inside or outside previously described CD8+ T-cell epitopes. A statistical approach was developed to identify sites/regions under reproducible selection pressure associated with HLA class I. RESULTS: The frequency of nonsynonymous mutations was significantly higher inside previously described CD8+ T-cell epitopes than outside-particularly in NS3/4A and NS5B. We identified new regions that are under selection pressure, indicating that not all CD8+ T-cell epitopes have been identified; 6 new epitopes that interact with CD8+ T cells were identified and confirmed in vitro. In some CD8+ T-cell epitopes mutations were reproducibly identified in patients that shared the relevant HLA allele, indicating immune pressure at the population level. There was statistical support for selection of mutations in 18 individual epitopes. Interestingly, 14 of these were restricted by HLA-B allele. CONCLUSIONS: HLA class I-associated selection pressure on the nonstructural proteins and here predominantly on NS3/4A and NS5B promotes evolution of HCV. HLA-B alleles have a dominant effect in this selection process. Adaptation of HCV to the CD8+ T-cell response at the population level creates challenges for vaccine design.

Degree of cross-genotype reactivity of hepatitis C virus-specific CD8+ T cells directed against NS3.

UNLABELLED: The inherent sequence diversity of the hepatitis C virus (HCV) with the existence of multiple genotypes that differ up to 20% at the amino acid level represents one of the major obstacles for immune control. Accordingly, immune control of a heterologous virus challenge, particularly across genotypes, is difficult to achieve; however, the overall role of genotype-specific sequence differences has not yet been defined at the epitope level. The aim of this study was to determine the role of genotype-specific sequence differences for the CD8+ T cell response against HCV. We analyzed a cohort of anti-HCV-positive injection drug users infected with HCV genotype 1 (n = 17) or genotype 3 (n = 22) or undetectable HCV-RNA (n = 14) with overlapping peptides covering consensus sequences of NS3 from both genotypes. Importantly, the majority of HCV-specific CD8 T cells were specific for one genotype only indicating that sequence differences between genotypes are relevant at the epitope level. Interestingly, T cells active against both genotypes were significantly more frequent in HCV-RNA-negative subjects. Of note, we identified five subjects with undetectable viremia and coexistence of two T cell populations-one for each genotype-suggesting immune control of two different genotypes. CONCLUSION: We systematically analyzed the degree of cross-genotype reactivity of HCV-specific T cells and have shown that CD8 responses targeting different HCV genotypes can be primed in the same individual and that such responses potentially characterize a subgroup among injection drug users being protected from chronic HCV infection.

Hepatitis C virus recombinants are rare even among intravenous drug users.

Systematic studies of the circulation of hepatitis C virus (HCV) recombinants in different parts of the world have been initiated only recently, and no detailed information on this subject is available. The aim of the current investigation was to determine the frequency of HCV recombinants in intravenous drug users (IVDU) from two European countries. HCV RNA from serum samples was tested by RT-PCR with primers derived from the core and NS5B regions with subsequent sequencing and genotype assignment. The 118 samples from Germany (100%) and 45 out of 47 (96%) sera from Russia demonstrated concordant genotyping results. In the two genotype discrepant sera from Russia 2k/1b recombinants were identified. In order to test the hypothesis that the individuals from the IVDU group might be multiply exposed to various genotypes, 145 out of 165 genotyped serum samples, which were found to be positive for anti-NS4 antibodies, were serotyped with the Murex HCV serotyping kit that is based on detection of antibodies to type-specific peptides derived from the NS4 proteins of different HCV genotypes. Discrepancy in genotype and serotype attributions was observed in 11% cases. Retesting of 99 type 1a or 3a samples with a set of type- and subtype-specific primers revealed the presence of a mixed infection only in one case (1a/3a). Thus, the cases of the mixed infection with different HCV genotypes as well as the recombinant forms of HCV are very rare even in such a highly exposed group as IVDU.

Acute infection with a single hepatitis C virus strain in dialysis patients: Analysis of adaptive immune response and viral variability.

BACKGROUND/AIMS: While the adaptive immune response is crucial for spontaneous resolution of acute hepatitis C virus (HCV) infection, it also constitutes the driving force for viral escape. For acutely HCV-infected dialysis patients, little is known about the host response and its impact on viral evolution. METHODS: Four haemodialysis patients accidentally infected with the same HCV strain were prospectively investigated with respect to the clinical course, CD4+ and CD8+ T-cell responses, neutralizing antibodies, viral kinetics and sequence variability. RESULTS: In one patient, a robust CD4+ T-cell response was associated with transient control of infection, while in the other patients, weak responses correlated with persistently high viremia. Despite the presence of CD8+ T-cell effectors in the first patient, no sequence differences were detected in targeted regions of the viral genome in any of the patients when viral persistence was established. Genetic stability in the envelope genes, including the hypervariable regions, correlated with low-level or absent neutralizing antibodies in all of the patients. CONCLUSIONS: The establishment of viral persistence in the special patient group of dialysis patients is due to a failure of the adaptive immune system, as shown by the absence of significant T-cell and antibody responses, as well as viral variability.

Analysis of the evolutionary forces in an immunodominant CD8 epitope in hepatitis C virus at a population level.

Failure of the adaptive immune response to control infection with the hepatitis C virus (HCV) can result from mutational escape in targeted T-cell epitopes. Recent studies suggest that T-cell immune pressure is an important factor in the evolution of the nonstructural proteins in HCV. The aim of this study was to characterize the forces that contribute to viral evolution in an HLA-A*01-restricted epitope in HCV NS3. This epitope represents a potentially attractive target for vaccination strategies since it is conserved across all genotypes. In our cohort of subjects with chronic HCV infection (genotype 1b or 3a), it is a frequently recognized CD8 epitope in HLA-A*01-positive subjects. Viral sequence data reveal that an escape variant is the dominant residue in both genotypes. The predominant Y1444F substitution seemingly impairs binding to the HLA-A*01 molecule, which may have an important impact on the ability to prime a functional CD8 response upon infection. Interestingly, a case of evolution toward the prototype sequence was observed during chronic infection, possibly because the helicase activity of the protein containing the Y1444F substitution is reduced compared to the prototype sequence. Comparison of HCV sequences from Asia and Europe suggests that the frequency of the HLA-A*01 allele in a population may influence the frequency of the escape variant in circulating strains. These data suggest a complex interaction of multiple forces shaping the evolution of HCV in which immune pressure both within the individual and also at the population level in addition to functional constraints are important contributing factors.

Escape from HLA-B*08-restricted CD8 T cells by hepatitis C virus is associated with fitness costs.

The inherent sequence diversity of the hepatitis C virus (HCV) represents a major hurdle for the adaptive immune system to control viral replication. Mutational escape within targeted CD8 epitopes during acute HCV infection has been well documented and is one possible mechanism for T-cell failure. HLA-B*08 was recently identified as one HLA class I allele associated with spontaneous clearance of HCV replication. Selection of escape mutations in the immunodominant HLA-B*08-restricted epitope HSKKKCDEL(1395-1403) was observed during acute infection. However, little is known about the impact of escape mutations in this epitope on viral replication capacity. Their previously reported reversion back toward the consensus residue in patients who do not possess the B*08 allele suggests that the consensus sequence in this epitope is advantageous for viral replication in the absence of immune pressure. The aim of this study was to determine the impact of mutational escape from this immunodominant epitope on viral replication. We analyzed it with a patient cohort with chronic HCV genotype 1b infection and in a single-source outbreak (genotype 1b). Sequence changes in this highly conserved region are rare and selected almost exclusively in the presence of the HLA-B*08 allele. When tested in the subgenomic replicon (Con1), the observed mutations reduce viral replication compared with the prototype sequence. The results provide direct evidence that escape mutations in this epitope are associated with fitness costs and that the antiviral effect of HLA-B*08-restricted T cells is sufficiently strong to force the virus to adopt a relatively unfavorable sequence.

In vitro replicative properties of replicons constructed using sequence variants of the hepatitis C virus strain AD78 that caused a single-source outbreak of hepatitis C.

For many aspects of HCV research it would be very useful to have a set of replicons in which different genome regions are swapped by corresponding fragments from isolates of the same viral strain that might demonstrate different biological characteristics or bear evolving antigenic determinants. The isolates of the same HCV strain that are necessary for generation of such hybrid replicons might be obtained from a single-source outbreak of HCV infection. One such outbreak caused by the HCV AD78 strain, occurred in Germany due to infection of women by contaminated anti-D globulin. Using a sequential substitution of different segments of the Con1 replicon with the corresponding fragments from the AD78 strain of HCV, a set of chimeric Con1/AD78 subgenomic and full-length, AD78-based genomic replicons were generated. These replicons might be used as a new experimental tool for different aspects of HCV research, including studies of the nature of isolate-specific differences in interactions of the replicon with the host cell and analysis of the mechanisms of HCV resistance to antivirals. The newly generated full-length replicon can also be used for preparation of AD78-specific target cell lines, which may be invaluable for the analysis of the evolution of HCV cellular immune responses in the cohort of patients infected with the HCV AD78 strain.

Interferon-alpha and ribavirin resistance of Huh7 cells transfected with HCV subgenomic replicon.

To model HCV resistance to a treatment with interferon-alpha (IFN-alpha) and ribavirin, Huh7 cells, bearing HCV subgenomic replicons, were treated with these compounds for several weeks. Analysis of the cell clones, which were able to support replication of HCV RNA in the presence of high concentrations of these antivirals, demonstrated that the observed resistance was due to changes in the host cell phenotype but not to the emergence of resistant variants of the replicon. No changes in the type I IFN receptor mRNA levels or sequences were found in IFN-treated cells suggesting that the observed resistance of replicon-containing cells to IFN-alpha was caused by modifications of some other cellular factors. The resistance of cells to high concentrations of ribavirin was due to a single point mutation in the NS5A gene of the HCV replicon, and was not associated with a defect in a ribavirin uptake. This mutation, however, did not change the sensitivity of the replicon itself to this antiviral.

Awareness of rabies risks and knowledge about preventive measures among experienced German travel health advisors.

BACKGROUND: Every year, millions of people travel to countries where rabies is enzootic. However, the quality of rabies-specific information provided by travel health advisors and the extent of their knowledge about pre- and postexposure prophylaxis have not been examined on a large-scale basis up to now. METHODS: 5,780 German physicians and pharmacists, who identified themselves as active travel health advisors, were chosen from a database. The selected providers were asked to complete an Internet-based questionnaire. The form requested both demographic information and the assessment of different concrete scenarios, each of which featured individuals seeking pre-travel advice on rabies or appropriate postexposure treatment after returning from abroad. RESULTS: Overall, 496 physicians and pharmacists completed the questionnaire. Almost all respondents indicated that they would mention the risk of rabies and appropriate preventive measures to long-term travelers and tourists planning to visit rural areas. However, only 35% to 60% of the advisors would provide this information to individuals on business trips, package tours, or travelers in urban centers as well. The assessment of the scenarios yielded 51% to 98% of adequate advice. Potentially harmful decisions included, for instance, the failure to recommend further prophylactic measures after exposure of already vaccinated people or the fact that the necessary postexposure prophylaxis was inappropriately withheld in cases where treatment had been initially delayed. CONCLUSIONS: Although the participants of this study were well aware of the travel-associated rabies risks and provided adequate information about this health hazard to most of their clients, evident flaws exist regarding the correct assessment of specific situations in pre- and postexposure rabies prophylaxis. Our findings therefore provide important cues on topics that should be more intensely covered during future postgraduate training in travel medicine and also underline the need for more practically orientated, readily available information on specific prophylactic treatment against rabies.

Acute GB virus B infection of marmosets is accompanied by mutations in the NS5A protein.

GBV-B, a member of the Flaviviridae family of viruses, is the virus most closely related to HCV, and GBV-B infection in tamarin monkeys might represent a valuable surrogate animal model of HCV infection. In the current study, GBV-B was successfully transmitted to two marmosets (Callithrix jaccus). The infection resulted in viremia of 14- and 17-week duration, respectively, and was accompanied by elevation of isocitrate dehydrogenase activity. These data confirm that marmosets might represent an attractive model for GBV-B infection. The sequence of GBV-B NS5A, which was previously reported to have one of the highest mutation rates during infection in tamarins, was determined for viruses recovered from the inoculum and from marmoset blood samples obtained at weeks 1, 8, and 14 post inoculation in one marmoset and at weeks 2, 8, and 17 post inoculation in the other marmoset. In both animals, we detected four substitutions (R1945K, K2052G, F2196L, and G2268E), in the virus recovered immediately before viral clearance. Interestingly, two of these mutations (F2196L and G2268E) were described recently for viruses recovered from persistently infected tamarins. Appearance of these mutations presumably reflects a mechanism of immune escape rather than adaptation of the virus to a new host.

Risk of hepatitis C virus transmission from an infected gynecologist to patients: results of a 7-year retrospective investigation.

BACKGROUND: Currently, it is not known how often hepatitis C virus (HCV) is transmitted from infected health care workers to patients during medical care. In the present investigation, we tried to determine the rate of provider-to-patient transmission of HCV among former patients of an HCV-positive gynecologist after it was proven that he infected one of his patients with HCV during a cesarean section. METHODS: All 2907 women who had been operated on by the HCV-positive gynecologist between July 1993 and March 2000 were notified about potential exposure and were offered free counseling and testing. The crucial differentiation between HCV transmissions caused by the gynecologist and infections contracted from other sources was achieved by epidemiological investigations, nucleotide sequencing, and phylogenetic analysis. RESULTS: Of the 2907 women affected, 78.6% could be screened for markers of HCV infection. Seven of these former patients were found to have HCV. Phylogenetic analysis of HCV sequences from the gynecologist and the women did not indicate that the virus strains were linked. Therefore, no further iatrogenic HCV infections caused by the gynecologist could be detected. The resulting overall HCV transmission rate was 0.04% (1 per 2286; 95% confidence interval, 0.008%-0.25%). CONCLUSION: To our knowledge, this is the largest retrospective investigation of the risk of provider-to-patient transmission of HCV conducted so far. Our findings support the notion that such transmissions are relatively rare events and might provide a basis for future recommendations on the management of HCV-infected health care workers.

New variant of the human metapneumovirus (HMPV) associated with an acute and severe exacerbation of asthma bronchiale.

Recently, an increasing number of studies demonstrated that the human metapneumovirus (HMPV) causes mild to severe respiratory infections in children and immunosuppressed adults, and may be a frequent but somewhat undervalued pathogen. Here, we report the detection of a new variant of HMPV that is not closely related to the HMPV strains described until now. The strain was detected in a 6.5-year-old girl with an acute and severe exacerbation of asthma bronchiale triggered by an infection with a newly detected HMPV variant. The presented data provide new information on genetic heterogeneity of HMPV and necessitate an optimization of diagnostic procedures for the detection of HMPV infection.

Hepatitis C virus hypervariable region 1 variants presented on hepatitis B virus capsid-like particles induce cross-neutralizing antibodies.

Hepatitis C virus (HCV) infection is still a serious global health burden. Despite improved therapeutic options, a preventative vaccine would be desirable especially in undeveloped countries. Traditionally, highly conserved epitopes are targets for antibody-based prophylactic vaccines. In HCV-infected patients, however, neutralizing antibodies are primarily directed against hypervariable region I (HVRI) in the envelope protein E2. HVRI is the most variable region of HCV, and this heterogeneity contributes to viral persistence and has thus far prevented the development of an effective HVRI-based vaccine. The primary goal of an antibody-based HCV vaccine should therefore be the induction of cross-reactive HVRI antibodies. In this study we approached this problem by presenting selected cross-reactive HVRI variants in a highly symmetric repeated array on capsid-like particles (CLPs). SplitCore CLPs, a novel particulate antigen presentation system derived from the HBV core protein, were used to deliberately manipulate the orientation of HVRI and therefore enable the presentation of conserved parts of HVRI. These HVRI-CLPs induced high titers of cross-reactive antibodies, including neutralizing antibodies. The combination of only four HVRI CLPs was sufficient to induce antibodies cross-reactive with 81 of 326 (24.8%) naturally occurring HVRI peptides. Most importantly, HVRI CLPs with AS03 as an adjuvant induced antibodies with a 10-fold increase in neutralizing capability. These antibodies were able to neutralize infectious HCVcc isolates and 4 of 19 (21%) patient-derived HCVpp isolates. Taken together, these results demonstrate that the induction of at least partially cross-neutralizing antibodies is possible. This approach might be useful for the development of a prophylactic HCV vaccine and should also be adaptable to other highly variable viruses.

Establishment of human clones producing neutralizing human monoclonal antibodies to the envelope E1/E2 protein of hepatitis C virus by EBV immortalization of immune CD22⁺ B cells.

We aimed to establish Human cell lines producing human monoclonal antibodies to the envelope E1/E2 protein of hepatitis C virus (HCV). Two protocols for EBV immortalization of CD22⁺ cells separated from HCV positive patients were used; 1) Immortalization with 100% EBV only, 2) immortalization by 30% EBV and CPG2006. Immortalization was checked microscopically and verified by screening the culture supernatant for antibody production using dot blot and ELISA analysis. ELISA plates were coated by HCV E1/E2 derived from cell lysate transfected by plasmid expressed HCV E1/E2. Also we tested the reactivity of human antibodies based on ELISA plates coating with one linear peptides derived from HCV E1 (a.a 315-319) and two peptides derived from HCV E2 (a.a 412-419) and (a.a 517-530). Neutralization activity was measured using H77C HCV retroviral pseudoparticles (HCVpp). Fifteen clones secreting human immunoglobulin G against HCV E1/E2 protein were isolated. Results of ELISA plates coated with HCV peptides showed that one antibody was binding to E2 peptide (a.a 517-530), and two antibodies binding to HCV E2 peptide (a.a 412-419). The three generated antibodies showed extremely neutralization activity against HCV pp. The three human antibodies were IgG3 and IgG2. These antibodies may be useful for passive immunotherapy of HCV infection.

Immunogenic properties of chimeric potato virus X particles displaying the hepatitis C virus hypervariable region I peptide R9.

The immunogenic properties of chimeric potato virus X (PVX) particles engineered to display the synthetic R9 peptide have been evaluated. The R9 peptide is a consensus sequence derived from diverse variants of the hypervariable region 1 from the hepatitis C virus (HCV) envelope protein E2. Two different constructs were designed, with the R9 peptide expressed either as an indirect fusion via the ribosomal skip 2A (PVX(R9-2A)CP) sequence or as a direct PVX coat protein fusion (PVX(R9)CP). Systemic infection of Nicotiana benthamiana plants was only achieved with PVX(R9-2A)CP constructs, and the presence of the R9 peptide was detected in extracts from these plants by ELISA, Western blot and electron microscopy using specific anti-R9 antibodies. The virus particles were recovered at yields of up to 125mg/kg from leaf material. BALB/c mice immunized with purified PVX(R9-2A)CP particles developed specific anti-R9 IgG titers of up to 1:50,000. Monoclonal anti-R9 antibodies were obtained from the spleen of a mouse immunized with PVX(R9-2A)CP particles and characterized by Western blot and electron microscopy. Sera from patients infected chronically with HCV were found to react specifically with PVX(R9-2A)CP particles in 35% of cases.