RESUMO
In this study, we report the exact localization and substitutional characterization of a B-cell epitope domain at the N-terminus of the preS2 region of the hepatitis B surface antigen. A set of deletion variants containing preS2 sequences of different length was generated on the basis of frCP as a carrier. It was found after Western blot analysis that three monoclonal antibodies (MAbs) (2-11B1, 3-11C2, HB.OT10) recognized the linear preS2 sequence within the amino acid (aa) stretch 3-WNSTTFHQTLQDP-13. The importance of each aa residue of the epitope was proved by comparison of antibody binding to alanine-substituted peptides in both free-peptide and Pepscan variants.
Assuntos
Mapeamento de Epitopos , Epitopos de Linfócito B/análise , Antígenos de Superfície da Hepatite B/análise , Precursores de Proteínas/análise , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/imunologia , Camundongos , Dados de Sequência Molecular , Precursores de Proteínas/genética , Precursores de Proteínas/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologiaRESUMO
An efficient pBR327- and Ptrp-based E. coli expression system was used to generate a large-scale library of virus like particles (VLP) formed by recombinant hepatitis B virus (HBV) core (HBc) protein derivatives. To construct the library, the gene of HBc protein of the genotype D/subtype ayw2 virus was gradually truncated from the 3`-end and twenty-two HBc variants (with truncation up to 139 aa) were expressed at high levels. The proteins were purified by salt precipitation and gel filtration. Background RNA binding was observed for VLPs formed by HBc1-149, which lacked all C-terminal Arg blocks, and the addition of three Arg residues (HBc1-152) only slightly increased RNA binding. The presence of two Arg blocks (proteins HBc1-162 and HBc1-163) resulted in approximately half of the typical level of RNA binding, and the presence of three blocks (protein HBc1-171) led to approximately 85% of the typical level of binding. Only a small increase in the level of RNA binding was found for the HBc1-175 VLPs, which contained all four Arg blocks but lacked the last 8 aa of the full-length HBc protein. VLPs containing high levels of RNA had higher antigenicity according to an ELISA with anti-HBc mAbs than the VLPs formed by HBc variants without C-terminal Arg blocks and lacking RNA. The results indicate that the VLPs were stabilised by nucleic acids. The immunogenicity in BALB/c mice was comparable for VLPs formed by different HBc proteins, but a clear switch from a Th1 response to a Th2 response occurred after the loss of encapsidated RNA. We did not observe significant differences in lymphocyte proliferation in vitro for the tested VLP variants; however, the loss of RNA encapsidation correlated with a decreased level of IFN-γ induction, which is a measure of the potential CTL activity of immunogens.
Assuntos
Biblioteca Gênica , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vírion/genética , Animais , Cromatografia em Gel , Ensaio de Imunoadsorção Enzimática , Escherichia coli , Engenharia Genética/métodos , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Antígenos do Núcleo do Vírus da Hepatite B/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
A multivalent vaccine candidate against hepatitis B virus (HBV) and hepatitis C virus (HCV) infections was constructed on the basis of HBV core (HBc) virus-like particles (VLPs) as carriers. Chimeric VLPs that carried a virus-neutralizing HBV pre-S1 epitope corresponding to amino acids (aa) 20 to 47 in the major immunodominant region (MIR) and a highly conserved N-terminal HCV core epitope corresponding to aa 1 to 60 at the C terminus of the truncated HBcDelta protein (N-terminal aa 1 to 144 of full-length HBc) were produced in Escherichia coli cells and examined for their antigenicity and immunogenicity. The presence of two different foreign epitopes within the HBc molecule did not interfere with its VLP-forming ability, with the HBV pre-S1 epitope exposed on the surface and the HCV core epitope buried within the VLPs. After immunization of BALB/c mice, specific T-cell activation by both foreign epitopes and a high-titer antibody response against the pre-S1 epitope were found, whereas an antibody response against the HBc carrier was notably suppressed. Both inserted epitopes also induced a specific cytotoxic-T-lymphocyte (CTL) response, as shown by the gamma interferon (IFN-gamma) production profile.