The preS antigen of hepatitis B virus (HBV) corresponds to the N-terminal polypeptide in the large (L) antigen in addition to the small (S) antigen. The virus-like particle (VLP) of the S antigen is widely used as a vaccine to protect the population from HBV infection. The presence of the S antigen and its antibodies in patient blood has been used as markers to monitor hepatitis B. However, there is very limited knowledge about the preS antigen. We generated a preS VLP that is formed by a chimeric protein between preS and hemagglutinin (HA), and the matrix protein M1 of influenza virus. The HBV preS antigen is displayed on the surface of preS VLP. Asn112 and Ser98 of preS in VLP were found to be glycosylated and O-glycosylation of Ser98 has not been reported previously. The preS VLP shows a significantly higher immunogenicity than recombinant preS, eliciting robust anti-preS neutralizing antibodies. In addition, preS VLP is also capable of stimulating preS-specific CD8⁺ and CD4⁺ T cell responses in Balb/c mice and HBV transgenic mice. Furthermore, preS VLP immunization provided protection against hydrodynamic transfection of HBV DNA in mice. The data clearly suggest that this novel preS VLP could elicit robust immune responses to the HBV antigen, and can be potentially developed into prophylactic and therapeutic vaccines.

Hepatitis B virus (HBV) X protein (HBx) plays diverse roles in both viral life cycle and HBV-related carcinogenesis. Its interaction with DNA damage-binding protein 1 (DDB1) was shown to be essential for engendering cellular conditions favorable for optimal viral transcription and replication. Previously, we described a mouse monoclonal antibody against HBx (anti-HBx 2A7) recognizing HBx encoded by representative strains from 7 of 8 known HBV genotypes. In this work, we further characterized 2A7 in order to explore its potential usefulness in HBx-targeting applications. We demonstrated that 2A7 recognizes a linear epitope mapped to L⁸⁹PKVLHKR⁹⁶ on HBx, a segment that is highly conserved across genotypes and coincidentally overlaps with the DDB1-interacting segment. HBx-DDB1 binding could be inhibited by 2A7 in vitro, suggesting therapeutic potential. Nucleic acid and amino acid sequences of 2A7 were then obtained, which allowed construction of recombinant antibody and single chain variable fragments (scFv). 2A7-derived recombinant antibody and scFv recapitulate 2A7’s HBx-binding capacity and epitope specificity. We also reported preliminary results using cell-penetrating peptide for delivering 2A7 antibody across cell membrane to target intracellular HBx. Anti-HBx 2A7 and 2A7-derived scFv characterized here may give rise to novel HBx-targeting diagnostics and therapeutics for HBV- and HBx-related pathologies.

Non-Hodgkin lymphoma (NHL), a heterogeneous lymphoid malignancy, demonstrates molecular diversity linked to genetic and immune factors, with emerging roles for viral infections in pathogenesis. Yet, the blood virome's composition and dynamics in NHL remain poorly characterized. This study characterizes the blood virome in NHL subtypes using viral metagenomic sequencing of serum from 217 patients (B-cell: BCL, T-cell: TCL, NK-cell: NKCL) and 40 healthy controls. Bioinformatic analysis identified 45 viral families, revealing subtype-specific viromic signatures. BCL exhibited a dominance of Anelloviridae, which accounted for 86% of eukaryotic viruses, compared with only 3% in controls, correlating with immunosuppression. Additionally, picobirnavirus, an opportunistic pathogen particularly in hosts with compromised immune systems, also showed a significant difference compared to controls. NKCL showed Flaviviridae enrichment, accounting for 82% of eukaryotic viruses, with nearly all of them being human pegivirus-1 (HPgV-1). Compared with healthy controls, patients with NHL exhibited significantly lower blood virome α-diversity at the genus level, and T-cell lymphomas showed the lowest species-level richness (140 vs. 332 in controls). Beta diversity highlighted BCL-specific viral heterogeneity, contrasting conserved T/NKCL viral profiles. Anelloviridae and Picobirnavirus expansion aligns with immune dysfunction, whereas NKCL-restricted HPgV-1 prevalence underscores biomarker potential. These findings implicate blood virome alterations marked by viral family predominance and diversity loss in NHL pathogenesis via immune modulation or oncogenesis. This first comprehensive NHL virome profile identifies subtype-specific signatures (Anelloviridae/Picobirnavirus/HPgV-1) for potential diagnostic and therapeutic targeting. Validation of these biomarkers may refine NHL subtyping and elucidate virome-lymphomagenesis mechanisms.