Long-term antigen reduction does not achieve durable HBV control in mice but enhances therapeutic vaccine efficacy

Chronic HBV infection has an increasing death toll, but curative therapies are lacking. The study aimed to determine whether long-term suppression of viral antigens using RNA-interference restores HBV-specific immunity and achieves durable virus control, and how long HBV antigens must be suppressed to enable therapeutic vaccination to restore immunity.

HBV-transgenic or AAV-HBV-infected, HBV-carrier mice were treated for up to 7 months with liver-directed small interfering RNAs (siRNAs) or short-hairpin RNAs (shRNAs) that target all HBV transcripts. The antiviral effect and development of B- and T-cell immunity were evaluated. A subcohort of mice received the heterologous prime/boost therapeutic vaccine, TherVacB, before cessation of siRNA.

Continuous siRNA therapy reduced HBsAg by up to 4 log₁₀, but a threshold effect was observed after 12-16 weeks. Suppression of viral antigens for up to 7 months did not lead to spontaneous reconstitution of T-cell immunity; this required stimulation by therapeutic vaccination. Without vaccination, HBV replication rebounded after discontinuation of siRNA therapy, even though HBsAg and HBeAg seroconversion had occurred. However, a longer duration of siRNA pretreatment correlated with increasing interferon type I signaling in the liver. siRNA pretreatment for 8 instead of 3.5 weeks improved the ability of therapeutic vaccination to activate functional antiviral T cells, fully control HBV replication, and significantly reduce the number of HBV-positive hepatocytes (p <0.01 to p <0.001).

Our data show that, in addition to suppressing viral antigens, immune stimulation is necessary to achieve long-lasting HBV control after treatment discontinuation. These results will help design clinical trials that can ultimately achieve HBV control.

Drug entities in development to cure chronic hepatitis B include direct-acting antivirals, nucleic acid-based therapeutics, immunotherapies, and combinations thereof. In our study, long-term suppression of HBV in HBV-carrier mice using siRNAs targeting all HBV antigens neither allowed spontaneous reconstitution of T-cell immunity nor sustained HBV control, even when accompanied by anti-HBs seroconversion. However, prolonged viral antigen suppression by siRNA enhanced the efficacy of therapeutic vaccination. Our study highlights that achieving loss of viral parameters from serum, or even anti-HBe/HBs seroconversion, should not represent the ultimate goal of curative therapy, but rather be viewed as a prerequisite that empowers immunostimulatory drugs to induce curative T-cell responses.