Plasmacytoid dendritic cells (pDCs) rapidly produce type I interferon (IFN-I) in response to acute virus infections. During chronic HIV-1 infection, persistent activation of pDCs contributes to inflammatory diseases. Combination antiretroviral therapy (cART) effectively suppresses HIV-1 replication and prolongs the life span of people living with HIV-1 (PLWH). The persistence of viral reservoir cells under cART, however, is associated with suboptimal immune reconstitution, impaired anti-HIV immunity, and non–AIDS-defining inflammatory diseases through unclear mechanisms. We report here that pDC depletion in HIV-infected humanized mice with suppressive cART alleviated HIV-associated inflammation, reversed T cell immune exhaustion, enhanced HIV-specific CD8 ⁺ T cell responses, and reduced HIV-1 reservoirs in lymphoid and nonlymphoid tissues through CD8 ⁺ T cell–dependent mechanisms. Specifically, pDC depletion in the mice led to an increase in TCF-1 ⁺ PD-1 ⁺ Tim-3 ⁻ stem-like memory CD8 ⁺ T cells in HIV-infected lymphoid tissues, which correlated with a reduction in the HIV-1 reservoir. We further showed that pDCs suppressed the polyfunctional activity of anti-HIV stem-like memory CD8 ⁺ T cells isolated from PLWH in vitro. As in the HIV-infected humanized mice, pDC depletion or IFN-I blockade functionally rescued the stem-like memory CD8 ⁺ T cells, enhancing their anti-HIV responses. Combination therapy with PD-1 blockade further improved stem-like memory T cell function both in vitro and in HIV-infected humanized mice. Our findings indicate that HIV-induced inflammatory pDCs impair anti-HIV stem-like memory CD8 ⁺ T cell responses and suggest that the combination of pDC depletion with PD-1 immune checkpoint blockade represents a candidate therapeutic approach to treating HIV-1 infection and its associated inflammatory diseases.

We developed a novel hepatitis B virus (HBV) infection-monitoring system using a luminescent, 11-amino acid reporter (HiBiT). We performed high-throughput antiviral screening using this system to identify anti-HBV compounds. After the infection of primary human hepatocytes with the recombinant virus HiBiT-HBV, which contains HiBiT at its preS1, 1262 compounds were tested in a first screening using extracellular HiBiT activity as an indicator of viral infection. Following a second screening, we focused on the compound skimmianine, which showed a potent antiviral effect. When skimmianine was added at the same time as HiBiT-HBV infection, skimmianine inhibited HiBiT activity with EC₅₀ of 0.36 pM, CC₅₀ of 1.67 μM and a selectivity index (CC₅₀:EC₅₀ ratio) of 5,100,000. When skimmianine was added 72 h after HiBiT-HBV infection, the EC₅₀, CC₅₀ and selectivity index were 0.19 μM, 1.87 μM and 8.79, respectively. Time-lapse fluorescence imaging analysis using another recombinant virus, ReAsH-TC155HBV, with the insertion of tetra-cysteine within viral capsid, revealed that skimmianine inhibited the accumulation of the capsid into hepatocytes. Furthermore, skimmianine did not inhibit either attachment or internalization. These results imply that skimmianine inhibits the retrograde trafficking of the virus after internalization. This study demonstrates the usefulness of the recombinant virus, HiBiT-HBV, for high-throughput screening to identify anti-HBV compounds.

Hepatitis B virus (HBV) remains a major global health threat because covalently closed circular DNA (cccDNA) persists in hepatocytes and limits the efficacy of current antiviral therapies. Effective HBV research and drug screening require culture models that recapitulate the complete viral life cycle and allow for quantitative monitoring of replication. In this study, an 11-amino acid luminescent reporter, HiBiT, was inserted at multiple sites within the preS1 region of a genotype D HBV genome, and the C terminus of preS1 was identified as optimal for maintaining robust replication. We then established HepG2-B4 cells stably replicating HiBiT-HBV with HiBiT at the preS1 C terminus. Extracellular HiBiT activity and supernatant levels of HBV-DNA, HBsAg, and HBcAg increased continuously until day 42 and were reduced by nucleos(t)ide analog treatment, and cccDNA was confirmed by Southern blot analysis. Supernatants from HepG2-B4 cells infected naïve HepG2-NTCP cells and primary human hepatocytes, as shown by extracellular HiBiT activity. Transcriptome analysis revealed distinct gene expression changes in HepG2-B4 cells compared with parental HepG2 cells. These findings indicate that the HepG2-B4 system provides a rapid, quantitative, and scalable platform for HBV replication and infection studies and is suitable for mechanistic investigations and high-throughput antiviral screening.

Background: Chronic hepatitis B virus (HBV) infection (CHB) affects nearly 300 million individuals globally and remains incurable with current antiviral therapies, which suppress viral replication but rarely achieve a functional cure, defined by sustained loss of hepatitis B surface antigen (HBsAg). CHB is characterized by profound virus-induced immune tolerance that limits the efficacy of conventional therapeutic vaccination strategies. Objective: To evaluate the therapeutic efficacy and immunological mechanisms of HEPLISAV-B, a CpG-1018 adjuvanted HBsAg vaccine, in breaking immune tolerance and inducing functional cure-like responses in a murine model of CHB. Design: Using the adeno-associated virus-HBV (AAV-HBV) mouse model, mice with high levels of persistent HBV viremia were vaccinated with two doses of HEPLISAV-B. Virological outcomes in the blood and liver, immune responses and mechanisms were assessed. Results: HEPLISAV-B induced rapid and durable HBsAg clearance, markedly reduced circulating and intrahepatic HBV DNA and RNA, and suppressed viral replication without hepatocellular injury. Vaccination elicited robust, sustained anti-HBs IgG1 and IgA responses, enhanced HBsAg-specific T- and B-cell immunity, reduced CD4 regulatory T cells, and decreased PD-1 expression on CD4 T cells. Therapeutic efficacy was strictly dependent on CD4 T cells and the CD40/CD40L signaling pathway, but independent of CD8 T cells, indicating a CD4-driven, non-cytolytic antiviral mechanism critical for HEPLISAV-B-induced HBV control. Conclusions: HEPLISAV-B effectively breaks HBV-induced immune tolerance and restores coordinated antiviral immunity through a CD4 T cell/CD40L-dependent pathway. The findings support its potential as a therapeutic vaccine in CHB patients.