Mimotopes of the hyper variable region 1 of the hepatitis C virus induce cross-reactive antibodies directed against discontinuous epitopes.

Hepatitis C virus (HCV) is a major cause worldwide of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, and the development of an effective vaccine represents a high priority goal. The hyper variable region 1 (HVR1) of the second envelope protein (E2) of HCV contains a principal neutralizing determinant, but it is highly variable among different isolates and it is involved in the escape from host immune response. To be effective, a vaccine should elicit a cross-reacting humoral response against the majority of viral variants. We show that it is possible to achieve a broadly cross-reactive immune response in rabbits by immunization with mimotopes of the HVR1, selected from a specialized phage library using HCV patients' sera. Some of the cross-reacting anti-mimotope antibodies elicited in rabbits, recognize discontinuous epitopes in a manner similar to those induced by the virus in infected patients.

Mimotopes of the hepatitis C virus hypervariable region 1, but not the natural sequences, induce cross-reactive antibody response by genetic immunization.

The hypervariable region 1 (HVR1) of the putative envelope protein E2 of hepatitis C virus (HCV) contains a principal neutralization epitope, and anti-HVR1 antibodies have been shown to possess protective activity in ex vivo neutralization experiments. However, the high rate of variability of this antigenic fragment may play a major role in the mechanism of escape from host immune response and might represent a major obstacle to developing an HCV vaccine. Thus, even if direct experimental evidence of the neutralizing potential of anti-HVR1 antibodies by active immunization is still missing, the generation of a vaccine candidate with a cross-reactive potential would be highly desirable. To overcome the problem of HVR1 variability, we have engineered cross-reactive HVR1 peptide mimics (mimotopes) at the N terminus of the E2 ectodomain in plasmid vectors suitable for genetic immunization. High levels of secreted and biologically active mimotope/E2 chimeras were obtained by transient transfection of these plasmids in cultured cells. All plasmids elicited anti-HVR1 antibodies in mice and rabbits with some of them leading to a cross-reacting response against many HVR1 variants from natural isolates. Epitope mapping revealed a pattern of reactivity similar to that induced by HCV infection. In contrast, plasmids encoding naturally occurring HVR1 sequences displayed either on full-length E2 in the context of the whole HCV structural region, or on a soluble, secreted E2 ectodomain, did not induce a cross-reacting anti-HVR1 response.

Induction of cross-reactive humoral immune response by immunization with mimotopes of the hypervariable region 1 of the hepatitis C virus.

Hepatitis C Virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, worldwide, and the development of an effective vaccine represents a high priority goal. The Hyper Variable Region 1 (HVR1) of the second Envelope protein (E2) of HCV contains a principal neutralizing determinant, but it is highly variable among different isolates and it is involved in the escape from host immune response. Thus, to be effective, a vaccine should elicit a cross-reacting humoral response against the majority of viral variants. We show that it is possible to achieve a broadly cross-reactive immune response in rabbits by immunization with mimotopes of the HVR1. selected from a specialized phage library using HCV patients' sera. At least some of the cross-reacting anti-mimotope antibodies, elicited in rabbits, recognize discontinuous epitopes in a manner similar to those induced by the virus in infected patients.

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.

Identification of peptides binding to IgG in the CSF of multiple sclerosis patients.

Phage displayed random peptide libraries were screened in order to identify phagotopes reacting with the IgG present in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients. Six families of phagotopes each composed of different isolates were identified. These phagotopes were used as reagents, to characterise IgG present in the CSF of MS patients. The following results were obtained: (a) CSF antibodies from different patients display different specificities; (b) anti-phagotope antibodies are also present in the serum of MS patients; (c) Anti-phagotope antibodies are equally frequent in the serum of MS patients and of healthy individuals; (d) some of the anti-phagotope antibodies are enriched in the CSF of MS patients. These data show that the natural antigen(s) recognised by CSF antibodies is rather common in the general population. By using the selected phagotopes as immunogens in rabbits, we have derived large quantities of anti-phagotope antisera which can provide for useful tools toward the identification of the natural antigen(s) recognised by MS CSF antibodies.

Identification of HCV core mimotopes: improved methods for the selection and use of disease-related phage-displayed peptides.

Disease-specific epitope discovery from random peptide libraries displayed on phage using sera from patients involves a number of screening steps with many immune and non-immune sera. To rapidly identify mimotopes of the human hepatitis C virus (HCV) core protein, we have used an anti-core human monoclonal antibody (mAb; B12.F8) as a probe in screening phage that were affinity-selected using a serum from an HCV infected patient. Three different positive phage were isolated displaying low or no homology with the natural antigen, but which still efficiently bound to the antigen binding site of the B12.F8 antibody. Testing the reactivity of these phage with forty-five sera from HCV infected patients showed that antibodies recognizing them are present in more than 80% of this population. These antibodies showed distinct fine specificity, as they bound the selected phage in a mutually exclusive fashion. Co-expression of two mimotopes in the same cells led to chimeric particles which were recognized by antibodies of different specificity. These data provide novel information on the potential use of the phage display technology for the characterization of antibody specificity as well as disease diagnosis and prevention.

Nonrheumatoid IgM in human hepatitis C virus-associated type II cryoglobulinemia recognize mimotopes of the CD4-like LAG-3 protein.

Type II mixed cryoglobulinemia (CryoII) is an autoimmune disorder frequently associated to human hepatitis C virus (HCV) infection and characterized by the presence of cold-insoluble immunocomplexes containing IgM with rheumatoid activity. To identify disease-related epitopes, we screened a phage-displayed random peptide library using purified IgM from patients with HCV-associated CryoII(CryoII/HCV). A dominant population of phage isolates bearing the HPLAP pentapeptide consensus motif was identified and shown to be recognized by a nonrheumatoid IgM species strongly associated to CryoII/HCV. The phage-borne mimotopes (phagotopes) displayed a strong homology with an exposed extra-loop region of human lymphocyte activation 3 gene (LAG-3) product. Consistently, rabbit sera raised against a synthetic LAG-3 peptide efficiently recognized the selected phagotopes. Furthermore, one such phagotope was revealed to be a good immunogenic mimic of LAG-3 when injected into rabbits. IgM purified from CryoII/HCV patients' sera specifically reacted with the LAG-3 peptide in ELISA, and this binding was inhibited by the selected phagotopes. These results provide experimental support for a general strategy to identify novel autoantigens.

Identification of peptides specific for cerebrospinal fluid antibodies in multiple sclerosis by using phage libraries.

The study of the origin and pathogenetic relevance of the oligoclonal antibodies present in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients has been hampered by a lack of specific ligands. We recently reported a general strategy, based on phage-displayed random peptide libraries, to identify ligands for disease-specific antibodies even in the absence of any information on the nature of the pathologic antigen. With this procedure, we identified several peptides specifically recognized by antibodies present in the CSF of MS patients. Using these peptides as reagents, we demonstrated that they mimic different natural epitopes and react with antibodies enriched in the CSF of MS patients. Antibodies recognizing the selected peptides are commonly found with equal frequency in the sera of MS patients and of normal individuals. In contrast, the repertoire of CSF antibodies appears to be individual-specific and is probably the result of a nonspecific immunodysregulation rather than a stereotyped response to a single antigen/agent.

A general strategy to identify mimotopes of pathological antigens using only random peptide libraries and human sera.

A strategy to identify disease-specific epitopes from phage-displayed random peptide libraries using human sera is described. Peptides on phage (phagotopes) that react with antibodies present in patient sera are purified from > 10(7) different sequences by affinity selection and immunological screening of plaques. Disease-specific phagotopes can be identified out of this pool through an 'antigen independent' procedure which avails itself only of patient and normal human sera. Using this strategy, we have selected antigenic mimics (mimotopes) of two different epitopes from the human hepatitis B virus envelope protein (HBsAg). We could show that a humoral response to these mimotopes is widespread in the immunized population, suggesting that the strategy identifies phagotopes that have a potential role as diagnostic reagents. Immunization of mice with the selected phagotopes elicited a strong specific response against the HBsAg. These results open new inroads into disease-related epitope discovery and provide the potential for vaccine development without a requirement for the use of, or even information about, the aetiological agent or its antigens.

Trans-dominant inhibition of transcription activator LFB1.

Liver-enriched factor LFB1 (also named HNF1) is a dimeric transcription activator which is essential for the expression of many hepatocyte-specific genes. Here we demonstrate that LFB1 mutants in the POU A-like or in the homeo domains inhibit wild-type DNA binding by forming inactive heterodimeric complexes. Co-transfection of one of these mutants with wild-type LFB1 in HeLa cells eliminated LFB1 DNA binding and transcriptional activities through a trans-dominant mechanism. Expression of the same dominant negative mutant in human hepatoma HepG2 cells only partially inhibited endogenous LFB1 activity, due to stabilization of LFB1 dimers in these cells. Dimer stabilization in hepatoma cells is mediated by a heat-labile association with an 11kD polypeptide, analogous to the DCoH cofactor identified in rat liver by Mendel et al. (1). The property of stabilizing LFB1 dimers is also shared by HeLa cells which produce a HeLa homolog of DCoH. These results demonstrate that LFB1 dimer stabilization as well as the synthesis of 'stabilizing factors' are not restricted to cells expressing LFB1 or other members of its family.