Efficient cellular and humoral immune response and production of virus-neutralizing antibodies by the Hepatitis B Virus S/preS1 16-42 antigen

Despite the availability of improved antiviral therapies, infection with Hepatitis B virus (HBV) remains a3 significant health issue, as a curable treatment is yet to be discovered. Current HBV vaccines relaying on the efficient expression of the small (S) envelope protein in yeast and the implementation of mass vaccination programs have clearly contributed to containment of the disease. However, the lack of an efficient immune response in up to 10% of vaccinated adults, the controversies regarding the seroprotection persistence in vaccine responders and the emergence of vaccine escape virus mutations urge for the development of better HBV immunogens. Due to the critical role played by the preS1 domain of the large (L) envelope protein in HBV infection and its ability to trigger virus neutralizing antibodies, including this protein in novel vaccine formulations has been considered a promising strategy to overcome the limitations of S only-based vaccines. In this work we aimed to combine relevant L and S epitopes in chimeric antigens, by inserting preS1 sequences within the external antigenic loop of S, followed by production in mammalian cells and detailed analysis of their antigenic and immunogenic properties. Of the newly designed antigens, the S/preS116–42 protein assembled in subviral particles (SVP) showed the highest expression and secretion levels, therefore, it was selected for further studies in vivo. Analysis of the immune response induced in mice vaccinated with S/preS116–42- and S-SVPs, respectively, demonstrated enhanced immunogenicity of the former and its ability to activate both humoral and cellular immune responses. This combined activation resulted in production of neutralizing antibodies against both wild-type and vaccine-escape HBV variants. Our results validate the design of chimeric HBV antigens and promote the novel S/preS1 protein as a potential vaccine candidate for administration in poor-responders to current HBV vaccines.publishedVersio

An enrichment protocol and analysis pipeline for long read sequencing of the hepatitis B virus transcriptome

Hepatitis B virus (HBV) is one of the smallest human DNA viruses and its 3.2 Kb genome encodes multiple overlapping open reading frames, making its viral transcriptome challenging to dissect. Previous studies have combined quantitative PCR and Next Generation Sequencing to identify viral transcripts and splice junctions, however the fragmentation and selective amplification used in short read sequencing precludes the resolution of full length RNAs. Our study coupled an oligonucleotide enrichment protocol with state-of-the-art long read sequencing (PacBio) to identify the repertoire of HBV RNAs. This methodology provides sequencing libraries where up to 25 % of reads are of viral origin and enable the identification of canonical (unspliced), non-canonical (spliced) and chimeric viral-human transcripts. Sequencing RNA isolated from de novo HBV infected cells or those transfected with 1.3 × overlength HBV genomes allowed us to assess the viral transcriptome and to annotate 5' truncations and polyadenylation profiles. The two HBV model systems showed an excellent agreement in the pattern of major viral RNAs, however differences were noted in the abundance of spliced transcripts. Viral-host chimeric transcripts were identified and more commonly found in the transfected cells. Enrichment capture and PacBio sequencing allows the assignment of canonical and non-canonical HBV RNAs using an open-source analysis pipeline that enables the accurate mapping of the HBV transcriptome

Efficient cellular and humoral immune response and production of virus-neutralizing antibodies by the Hepatitis B Virus S/preS116-42 antigen

Despite the availability of improved antiviral therapies, infection with Hepatitis B virus (HBV) remains a3 significant health issue, as a curable treatment is yet to be discovered. Current HBV vaccines relaying on the efficient expression of the small (S) envelope protein in yeast and the implementation of mass vaccination programs have clearly contributed to containment of the disease. However, the lack of an efficient immune response in up to 10% of vaccinated adults, the controversies regarding the seroprotection persistence in vaccine responders and the emergence of vaccine escape virus mutations urge for the development of better HBV immunogens. Due to the critical role played by the preS1 domain of the large (L) envelope protein in HBV infection and its ability to trigger virus neutralizing antibodies, including this protein in novel vaccine formulations has been considered a promising strategy to overcome the limitations of S only-based vaccines. In this work we aimed to combine relevant L and S epitopes in chimeric antigens, by inserting preS1 sequences within the external antigenic loop of S, followed by production in mammalian cells and detailed analysis of their antigenic and immunogenic properties. Of the newly designed antigens, the S/preS116–42 protein assembled in subviral particles (SVP) showed the highest expression and secretion levels, therefore, it was selected for further studies in vivo. Analysis of the immune response induced in mice vaccinated with S/preS116–42- and S-SVPs, respectively, demonstrated enhanced immunogenicity of the former and its ability to activate both humoral and cellular immune responses. This combined activation resulted in production of neutralizing antibodies against both wild-type and vaccine-escape HBV variants. Our results validate the design of chimeric HBV antigens and promote the novel S/preS1 protein as a potential vaccine candidate for administration in poor-responders to current HBV vaccines

N-Glycosylation and N-Glycan Processing in HBV Biology and Pathogenesis

Hepatitis B Virus (HBV) glycobiology has been an area of intensive research in the last decades and continues to be an attractive topic due to the multiple roles that N-glycosylation in particular plays in the virus life-cycle and its interaction with the host that are still being discovered. The three HBV envelope glycoproteins, small (S), medium (M) and large (L) share a very peculiar N-glycosylation pattern, which distinctly regulates their folding, degradation, assembly, intracellular trafficking and antigenic properties. In addition, recent findings indicate important roles of N-linked oligosaccharides in viral pathogenesis and evasion of the immune system surveillance. This review focuses on N-glycosylation’s contribution to HBV infection and disease, with implications for development of improved vaccines and antiviral therapies