Hypervariable Region 1 Peptides Research Articles

Immune Reactivity of Synthetic Peptides Originated from Hypervariable Region 1 of Hepatitis C Synthetic Consensuses with Egyptian Sera Infected with Hepatitis C Virus Type 4

We have studied the immune reactivity of hypervariable region 1 (HVR1) of hepatitis C virus (HCV) against HCV immune positive and negative sera. Two published HVR1 consensus nucleotide sequences (Italian and Chinese) were synthesized, and with both of them, a splicing by overlap extension polymerase chain reaction, cloning and sequencing were performed. From the corresponding amino acid sequences, 3 Italian and 1 Chinese HVR1 peptides were selected for synthesis. The 4 peptides (MB1-MB4; GenBank No.: HQ846888-HQ846891) were used to screen 47 and 31 HCV (type 4) immune positive and negative sera, respectively, by enzyme-linked immunosorbent assay (ELISA). The 3 Italian HVR1 peptides (MB1, MB2, MB3) showed reactivities of 83, 68 and 76.6%, respectively, while the Chinese HVR1 peptide (MB4) showed a reactivity of 80.8%. Our results supported that the HVR1 is an attractive target for a peptide-based vaccine as it contains neutralizing epitopes, and all of the HCV patients' sera used in this study have anti-HVR1 antibodies. Interestingly, the amino acid sequence of peptide MB1 has a close sequence similarity with the published mimotope R9, and it shows a high ELISA reactivity. So, MB1 could be tested in combination with other HCV-related peptides as a supplemental assay for HCV diagnosis.

Chimeric monoclonal antibodies to hypervariable region 1 of hepatitis C virus

Two chimeric monoclonal antibodies (cAbs), 2P24 and 15H4, to hypervariable region 1 (HVR1) of hepatitis C virus (HCV) were constructed by grafting the variable regions of murine monoclonal antibodies (mAbs) 2P24 and 15H4 to a human IgG1 kappa constant region. Two cAb-producing cell lines were adapted to serum-free media. Both cAb 2P24 and cAb 15H4 cell lines produced 3-5 microg antibodies ml(-1) after 3-5 days culture. cAbs retained binding characteristics similar to those observed in the original mAbs. There was no clear difference in affinity between binding of cAbs and mAbs to seven HVR1 peptides. Mixtures of biotinylated cAbs or mAbs reacted with 32 (86 %) and 31 (84 %) of 37 HVR1 peptides, respectively, but not with non-HVR1 control peptides. HCV from 16 out of 18 (89 %) random HCV-containing plasmas was captured by the mixture of biotinylated cAbs. The capture from IgG-depleted plasmas suggested that cAbs captured mainly free rather than complexed HCV, irrespective of genotype. A mixture of the two cAbs inhibited HCV binding to Molt-4 cells in a dose-dependent manner. These cAbs may be useful for prevention of nosocomial HCV infection and passive immunization to prevent HCV reinfection after liver transplantation.

Sequences in the hypervariable region 1 of hepatitis C virus show only minimal variability in the presence of antibodies against hypervariable region 1 during acute infection in chimpanzees.

We analyzed sequences of hypervariable region 1 (HVR1) of hepatitis C virus (HCV) in six chimpanzees, experimentally infected with a single HCV inoculum, to clarify the correlation between HVR1 mutation and antibodies to HVR1. Two chimpanzees had been immunized with synthetic HVR1 peptides before HCV inoculation. All six animals became infected with HCV but cleared the infection within the acute phase. The major HVR1 sequences in longitudinal sera were unchanged in animals both with and without anti-HVR1 antibodies. Additionally, sequences of HVR1 variants in each chimpanzee converged after 11 to 19 weeks. The data show that anti-HVR1 antibodies are unlikely to drive variation in HVR1.

Production and characterization of monoclonal antibodies specific for a conserved epitope within hepatitis C virus hypervariable region 1.

Frequent mutations in hypervariable region 1 (HVR1) of the main envelope protein of hepatitis C virus (HCV) is a major mechanism of persistence by escaping the host immune recognition. HVR1 contains an epitope eliciting neutralizing antibodies. This study was aimed to prepare broadly cross-reacting, high-affinity, monoclonal antibodies (MAb) to the HVR1 C terminus of HCV with potential therapeutic neutralizing capacity. A conserved amino residue group of glycine (G) at position 23 and glutamic acid (Q) at position 26 in HVR1 was confirmed as a key epitope against which two MAbs were selected and characterized. MAbs 2P24 and 15H4 were immunoglobulin G1 kappa chain [IgG1(kappa)], cross-reacted with 32 and 30 of 39 random C-terminal HVR1 peptides, respectively, and did not react with other HCV peptides. The V(H) of 2P24 and 15H4 heavy chains originated from Igh germ line v gene family 1 and 8, respectively. In contrast, the V(L) kappa sequences were highly homologous. The affinity (K(d)) of 2P24 and 15H4 (10(-9) or 10(-8) M with two immunizing peptides and 10(-8) M with two nonimmunizing HVR1 peptides) paralleled the reactivity obtained with peptide enzyme immunoassay. MAbs 2P24 and 15H4 captured 25 of 31 (81%) HCV in unselected patients' plasmas. These antibodies also blocked HCV binding to Molt-4 cells in a dose-dependent fashion. The data presented suggest that broadly cross-reactive MAbs to a conserved epitope within HCV HVR1 can be produced. Clinical application for passive immunization in HCV-related chronic liver disease and after liver transplantation is considered.

Antibody responses against B‐cell epitopes of the hypervariable region 1 of hepatitis C virus in self‐limiting and chronic human hepatitis C followed‐up using consensus peptides

A rare collection of serum samples from patients with hepatitis C virus (HCV) infection followed up from the onset of clinical symptoms was acquired. RNA corresponding to the hypervariable region 1 (HVR1) of E2 protein of HCV isolated from nine patients was reverse-transcribed, amplified, sequenced, and HVR1 amino acid sequences were deduced. These sequences and a selection of HVR1 amino acid sequences of matching HCV genotypes from protein and translated DNA sequence databanks were used to create the HVR1 amino acid consensus. The degenerated peptides mimicking N- and C-termini of the consensus were synthesized. Most (76%) of 17 patients followed up for the period from 1 week to a minimum of 7 months from the onset of acute symptoms developed antibodies reacting with peptides representing N- and/or C- termini of HVR1. Antibody recognition of the consensus HVR1 peptides indicates that the variability of HVR1 sequence on the protein level is limited with certain conserved structure(s) being untouched. A tendency was observed for a slower development of anti-HVR1 antibody response in patients developing chronic HCV, as compared to those with self-limiting HCV infection.

Hypervariable region 1 of hepatitis C virus: immunological decoy or biologically relevant domain?

The hypervariable region 1 (HVR1) of the E2 protein of hepatitis C virus (HCV) is highly heterogeneous and is responsible for significant inter- and intra-individual variation of the infecting virus, which may represent an important pathogenetic mechanism leading to escape and persistent infection. Moreover, a binding site for neutralizing antibodies (Ab) has been allegedly identified in this region. Prospective studies of serological responses to synthetic oligopeptides derived from HVR1 sequences of patients with acute and chronic HCV infection showed extensive serological cross-reactivity for unrelated HVR1 peptides in the majority of the patients. A significant correlation was found between HVR1 sequence variation, and intensity, and cross-reactivity of humoral immune responses providing strong evidence in support of the contention that HCV variant selection is driven by the host immune pressure. Monoclonal Ab (mAb) generated following immunization of mice with peptides derived from natural HVR1 sequences also showed cross-reactivity for several HVR1 sequences attesting to the existence of conserved amino acid motifs among different variants. These findings suggest that it is possible to induce a broadly cross-reactive immune response to HVR1 and that this mechanism can be used to generate protective immunity for a large repertoire of HCV variants.

Prevention of hepatitis C virus infection in a chimpanzee by vaccination and epitope mapping of antiserum directed against hypervariable region 1

We previously reported on a chimpanzee immunized with both putative envelope glycoproteins (E1 and E2) of hepatitis C virus (HCV), strain HCV-N2, and synthetic peptides of hypervariable region 1 (HVR1) of a different isolate, HCV-#6. The chimpanzee showed complete protection against HCV-#6 infection only when the titer of anti-HVR1 increased, suggesting that an immune response to the HVR1 is more essential in protecting a chimpanzee from HCV infection than an immune response to E1 and E2. In this study, we immunized this chimpanzee with only synthetic HVR1 peptides after anti-E1 and anti-E2 antibody levels dropped and then rechallenged with 10 infectious chimpanzee doses of HCV. The immunized animal was protected, and neutralization of HCV with the antiserum from the protected animal was achieved by inoculating another chimpanzee with HCV preneutralized by this antiserum mixture. Epitope analysis of HVR1 by Pin-ELISA using this antiserum seemed to demonstrate that the antibody response was directed mainly against the C terminus of HVR1. Moreover, our results showed that, if a part of the sequences was conserved, a broad cross-reactivity of the antiserum could be observed, even if amino-acid sequences in this epitope were substituted for those of other HCV strains.

High Prevalence of Hypervariable Region 1-Specific and -Cross-Reactive CD4+ T Cells in HCV-Infected Individuals Responsive to IFN-α Treatment

The hypervariable region 1 (HVR1) of the putative envelope 2 protein of the hepatitis C virus (HCV) is the most variable part of the whole HCV polyprotein. Anti-HVR1 antibodies have been shown to protect against HCV infection, indicating that this region contains an important neutralization determinant. Recently we and others have demonstrated that HVR1 is also a T cell determinant able to activate helper T cell responses during HCV infection. In order to investigate the role of the immune response against HVR1 during HCV infection we have evaluated the humoral and lymphoproliferative responses to a panel of HVR1 peptides in HCV-infected patients with different outcomes of the disease following interferon-α (IFN-α) treatment. We observed that the frequency of anti-HVR1 T cell responses was significantly higher in patients who recovered after IFN-α therapy than in those who did not, while no differences in the anti-HVR1 antibody reactivities were detected. In addition, by generating HVR1-specific T cell lines and clones we identified human leukocyte-associated antigens DR4 restricted T cell epitopes in the carboxy-terminus of HVR1 and we demonstrated that broadly cross-reactive HVR1 T cells are elicited by HVR1.

Monoclonal Antibodies to the Hypervariable Region 1 of Hepatitis C Virus Capture Virus and Inhibit Virus Adsorption to Susceptible Cells in Vitro

To analyze the neutralizing-related activity of antibodies against the hypervariable region 1 (HVR1) of hepatitis C virus (HCV) in more detail, monoclonal antibodies (mAbs) against HVR1 were raised by immunizing various strains of mice with one of two synthetic HVR1 peptides that had been derived from two isolates of HCV. The epitope specificity of all six mAbs could be assigned by the use of a series of linear peptides in competitive ELISA. It seems that most subregions in the amino acid sequence of HVR1 can induce a humoral immune response in mice. All three mAbs specific to HVR1-6-1 had the ability to capture homologous HCV-6 and inhibit its absorption to susceptible cells in vitro despite the fact that the epitope of each mAb was at a different location in HVR1, whereas the other three mAbs specific to HVR1-7 could not capture HCV-6 nor inhibit the absorption of HCV-6 to susceptible cells. The data in this study suggest that mAbs against HVR1 can prevent the infectivity of HCV in an isolate-specific and epitope position-independent manner.

Antibody responses to hepatitis C virus hypervariable region 1: evidence for cross-reactivity and immune-mediated sequence variation.

Sequence heterogeneity of hepatitis C virus (HCV) is unevenly distributed along the genome, and maximal variation is confined to a short sequence of the HCV second envelope glycoprotein (E2), designated hypervariable region 1 (HVR1), whose biological function is still undefined. We prospectively studied serological responses to synthetic oligopeptides derived from HVR1 sequences of patients with acute and chronic HCV infection obtained at baseline and after a defined follow-up period. Extensive serological cross-reactivity for unrelated HVR1 peptides was observed in the majority of the patients. Antibody response was restricted to the IgG1 isotype and was focused on the carboxyterminal end of the HVR1 region. Cross-reactive antibodies could be readily elicited following immunization of mice with multiple antigenic peptides carrying HVR1 sequences derived from our patients. The vigor and heterogeneity of cross-reactive antibody responses were significantly higher in patients with chronic hepatitis compared with those with acute hepatitis and in patients infected with HCV type 2 compared with patients infected with other viral genotypes (predominantly type 1), which suggest that higher time-related HVR1 sequence diversification previously described for type 2 may result from immune selection. The finding of a statistically significant correlation between HVR1 sequence variation, and intensity, and cross-reactivity of humoral immune responses provided stronger evidence in support of the contention that HCV variant selection is driven by the host's immune pressure.

Multiple Sequence-Reactive Antibodies Induced by a Single Peptide Immunization with Hypervariable Region 1 of Hepatitis C Virus

Hypervariable region 1 (HVR1) of hepatitis C virus (HCV) is known to contain neutralizing epitopes. We previously found that murine antibodies against HVR1-#6 captured a different isolate, HCV-#7, and cross-reacted with the HVR1 peptide of HCV-#7. We investigated the inducibility and generality of cross-reaction of animal anti-HVR1 antibody responses in this study. Anti-HVR1-#7 antibodies, which were induced in mice and a chimpanzee by immunization, were found to be cross-reactive to HVR1-#6 peptide. Antibody responses against HVR1-#6-1 and HVR1-#7 peptides were detected in 11/165 (6.7%) and 26/165 (15.8%) HCV-infected individuals, respectively. Nine HVR1 sequences from six individuals, who were strongly positive for anti-HVR1-#7 antibodies, were only 50–64.5% identical to that of HVR1-#7. All nine of these HVR1 peptides were reactive to sera from the six patients and/or to antisera against HVR1-#6 and HVR1-#7 produced in mice and chimpanzees. Cross-inhibition tests of chimpanzee antisera indicated that a given species of anti-HVR1 antibodies was reactive to multiple HVR1 sequences. Fine epitope mapping of polyclonal and monoclonal anti-HVR1 antibodies showed that conserved subregions in HVR1 sequences determined the observed immunological cross-reactivity. Our data demonstrate that cross-reacting anti-HVR1 antibodies are inducible by a single peptide immunization.

Human recombinant single-chain antibody fragments, specific for the hypervariable region 1 of hepatitis C virus, from immune phage-display libraries.

The hypervariable region 1 (HVR1) of hepatitis C virus (HCV) may contain a potential neutralization site and the generation of human single-chain antibody fragments (scFv) to HVR1 may therefore provide a useful tool for the study of HCV. In this report, we have isolated and characterized three anti-HVR1 scFv clones from two patient-derived phage-displayed libraries and HCV HVR1 peptides. scFv S52/20 and S53/6 were selected with serologically cross-reactive HVR1 peptides. scFv p3f10 was obtained by screening the library from patient MH with an autologous HVR1 peptide. Nucleotide sequencing showed that the VH chains and Vkappa chains of all three scFv antibodies were derived from VH3 and Vkappa1 family germline V-genes, respectively. The specificity and affinity of the recombinant scFv antibodies were examined by enzyme-linked immunosorbent assay (ELISA) and an affinity biosensor, using HVR1 peptides. S52/20 scFv binding to S52 HVR1 peptide was blocked by preincubation with soluble peptide S52 and was partially competed by one of three HCV-infected patient sera. In addition, scFv S52/20 blocked the binding of HCV-susceptible Molt-4 cells to immobilized S52 peptide. This study demonstrates that recombinant human scFv antibodies to HCV HVR1 can be produced in vitro and directly confirms that HVR1 of HCV elicits highly specific antibodies. The very high specificity of these antibodies to HVR1 may limit their potential use in passive immunization therapy against HCV, and further engineering of the scFvs needs to be performed to generate broad-spectrum blocking scFvs.

Genetic and serological evidence for multiple instances of unrecognized transmission of hepatitis C virus in hemodialysis units.

We investigated the unrecognized patient-to-patient transmission of hepatitis C virus (HCV) in hemodialysis units by performing phylogenetic and serological analyses of hypervariable region 1 (HVR1) of HCV. Of the 62 patients in one center, 11 were positive for HCV RNA. A total of 24 HVR1 sequences, including the minor population of sequences of HCV isolates, from each patient were closely related and classified into five clusters by phylogenetic analysis. Of the 11 patients, 5 were infected with multiple clusters of HCV. Two patients were infected with HCV during an 18-month interval between examinations, and these HVR1 sequences fell into one of the five clusters. In another hemodialysis center, 5 of the 20 patients were HCV RNA positive, and two HVR1 sequences were found to be closely related and phylogenetically derived from the same cluster. The antibody responses of these patients to the HVR1 peptides representative of the genetic clusters revealed exactly the same clustering as that shown by phylogenetic analysis. These findings suggest that phylogenetic and serological analyses of HVR1 sensitively detect unrecognized and multiple transmission of HCV occurring within the same room in hemodialysis centers. Fingerprinting analyses using hypervariable regions of infectious agents are useful in identifying the precise route of transmission of infection.

Towards a solution for hepatitis C virus hypervariability: mimotopes of the hypervariable region 1 can induce antibodies cross-reacting with a large number of viral variants.

The hypervariable region 1 (HVR1) of the putative envelope protein E2 of hepatitis C virus (HCV) is the most variable antigenic fragment in the whole viral genome and is mainly responsible for the large inter-and intra-individual heterogeneity of the infecting virus. It contains a principal neutralization epitope and has been proposed as the major player in the mechanism of escape from host immune response. Since anti-HVR1 antibodies are the only species shown to possess protective activity up to date, developing an effective prevention therapy is a very difficult task. We have approached the problem of HVR1 variability by deriving a consensus profile from >200 HVR1 sequences from different viral isolates and used it as a template to generate a vast repertoire of synthetic HVR1 surrogates displayed on M13 bacteriophage. This library was affinity selected using many different sera from infected patients. Phages were identified which react very frequently with patients' sera and bind serum antibodies that cross-react with a large panel of HVR1 peptides derived from natural HCV variants. When injected into experimental animals, the 'mimotopes' with the highest cross-reactivity induced antibodies which recognized the same panel of natural HVR1 variants. In these mimotopes we identified a sequence pattern responsible for the observed cross-reactivity. These data may hold the key for future development of a prophylactic vaccine against HCV.

Virus isolate-specific antibodies against hypervariable region 1 of the hepatitis C virus second envelope protein, gp70.

Hypervariable region 1 (HVR1), located in the N‐terminal region of a putative second envelope glycoprotein (gp70) of hepatitis C virus (HCV), contains immunological B‐cell epitopes which might be neutralizing epitopes. To clarify whether B‐cell epitopes within HVR1 are common among virus isolates or specific for the homologous virus isolate, we examined the reactivities of sera from 53 patients with chronic hepatitis or hepatocellular carcinoma/liver cirrhosis against two different HVR1 peptides (HVR11‐1 and HVR1 Y‐l) derived from patient I with sporadic acute hepatitis and an asymptomatic carrier Y, respectively, using our original assay system for the detection of anti‐HVR1 antibody. All patients examined had a history of blood transfusion. Most sera showed no reactivity with either HVR1 1‐1 or HVR1 Y‐l peptide. Only seven and fourteen serum samples reacted significantly, although weakly, with HVR1 1‐1 and HVR1 Y‐l peptides, respectively, compared with the serum from patient I or asymptomatic carrier Y. The blood transfusions of most reactive cases had occurred more than thirty years earlier. Six cases reacted with both HVR1 1‐1 and HVR1 Y‐l peptides, but further analysis revealed that only three cases reacted weakly with the peptide for either epitope I or II, identified within HVR11‐1, These results indicate that the B‐cell epitopes within HVR1 are fairly specific for the homologous virus isolate, and this may represent a serious difficulty in the development of a vaccine against HCV.