Studies of expression of the HBsAg: occult hepatitis B, genotypes of the HBV and chimeras of HBV and HCV.
Introduction: Hepatitis B virus (HBV) is classified into eight genotypes (A-H) that have a distinct distribution in different geographical regions. In Brazil, genotypes A, D and F are the most prevalent. HBV infection is commonly diagnosed by the presence of the surface antigen (HBsAg) in serum. Amino acid mutations may affect HBsAg secretion levels, as well as, alter HBsAg conformation, and thus, inhibit its detection by immunoassays. The detection of HBV DNA in HBsAg negative patients characterizes the occurrence of occult HBV infection. HBsAg hybrid particles, also known as chimeras, have been successfully used as vectors for presentation of other epitopes. Objectives: To conduct HBsAg expression assays in eukaryotic cells aiming to (i) associate occult HBV infection with amino acid mutations in HBsAg; (ii) analyze possible differences in the levels of HBsAg detection related to HBV genotypes; and (iii) construct hybrid particles of HBsAg and hepatitis C virus (HCV) epitopes to produce chimeric immunogenic proteins. Methods: Cloning procedures of the coding regions for the HBV envelop proteins were done by using pcDNA3 and/or pCI eukaryotic expression vectors. A natural restriction site located at the HBsAg neutralizing epitope was used for insertion of the HCV segment into the HBsAg. Transient transfection assays were carried out in CHO and/or HuH7 cells. Medium and extracts of transfected cells were analyzed for the presence of HBsAg by immunoassays. The intracellular localization of HBsAg was observed by immunofluorescence using a monoclonal anti-HBs antibody. Results: 1) Occult HBV infection was detected in 6/43 (14%) HIV-infected patients. It was not associated with alanine aminotransferase elevation but significantly correlated with the presence of HCV infection. All except one patient, showed low HBV loads. S gene sequencing revealed mutations in two patients that may inhibit HBsAg detection by commercial assays. 2) HBV genotype A displayed the highest levels of HBsAg detection in comparison with genotypes D (reduction of 37%) and F (reduction of 30%). However, the presence of two single mutations (T143M, in a genotype A clone, and T125M, in a genotype D clone) changed considerably HBsAg detection levels. 3) A unique amino acid mutation in the carboxi-terminal region of the HBsAg at residue 215 (L215Q), from a genotype F isolate, that inhibits secretion of HBsAg, was identified. By immunofluorescence microscopy, an intracellular localization of the HBsAg mutant, indicating retention in the endoplasmic reticulum, was observed. 4) The insertion of the HCV segment into the HBsAg neutralizing epitope by a natural restriction site did not alter patterns of secretion and antigenicity of the chimeric HBsAg. Conclusions: The detection of HBsAg mutants and low HBV loads confirm that multifactorial mechanisms are involved in occult HBV infection. Single mutations in HBsAg may have more influence on the patterns of HBsAg detection and secretion than the set of variations characteristic of each genotype. The natural restriction site identified in this study for insertion of HCV epitopes did not change HBsAg antigenic characteristics.
Advisor:Selma de Andrade Gomes
School:Faculdades Oswaldo Cruz
Source Type:Master's Thesis
Keywords:Transfecção Hepatitis B virus Antigens Transfection
Date of Publication:07/11/2008