Targeted therapy for patients with hepatitis B virus (HBV) infection can lead to objective responses, although response times may be short. At the same time, the response rate to programmed cell death-1 (PD-1) treatment was more durable. It is speculated that HBV targeted therapy can synergistically enhance the antitumor activity with PD-1 blockade. To test this hypothesis, we evaluated the effect of crispr-cas9 on HBV and PD-1 in vitro and in vivo. We found that HBV targeting gRNA/cas9 induced a decrease in the expression of HBsAg, while the PD-1 gene could be knocked out by electroporation targeting gRNA / cas9 by polymerase chain reaction. In HBV transgenic mice, the immunophenotype and cytokine expression of human dendritic cells (DCS) were detected by crispr-cas9 system stimulation, flow cytometry and polymerase chain reaction. These results indicate that gRNA/cas9 treatment upregulates the expression of CD80, CD83 and CD86, and significantly increases the mRNA levels of IL-6, IL-12, IL-23 and tumor necrosis factor alpha. The combination of anti HBV and anti PD-1 therapy can inhibit HBV expression and significantly improve the survival of HBV transgenic mice. In addition, the combination therapy increased the production of interferon by T cells, and then enhanced the expression of Th1 related immunostimulatory genes, thereby reducing the transcription of regulatory / inhibitory immune genes. In general, this response can reshape the tumor microenvironment from immunosuppression to immune stimulation. Finally, anti HBV therapy can induce the expression of interferon dependent programmed cell death ligand-1 in HBV transgenic mice in vivo. To sum up, these results demonstrate that the combination of HBV targeted therapy and PD-1 immune checkpoint block has a strong synergistic effect, thus supporting the transformation potential of this combined therapy strategy in clinical treatment of HBV infection.

尽管反应时间可能很短，但针对乙型肝炎病毒（HBV）感染的患者进行的靶向治疗可导致客观反应。同时，对程序性细胞死亡1（PD-1）治疗的反应率更持久。推测HBV靶向治疗可以通过PD-1阻滞协同增强抗肿瘤活性。为了验证这一假设，我们在体外和体内评估了crisp-cas9对HBV和PD-1的作用。我们发现靶向gRNA / cas9的HBV诱导了HBsAg表达的降低，而PD-1基因可通过聚合酶链反应靶向gRNA / cas9的电穿孔而被敲除。在HBV转基因小鼠中，通过crisps-cas9系统刺激，流式细胞仪和聚合酶链反应检测人树突状细胞（DCS）的免疫表型和细胞因子表达。这些结果表明，gRNA / cas9处理可上调CD80，CD83和CD86的表达，并显着增加IL-6，IL-12，IL-23和肿瘤坏死因子α的mRNA水平。抗HBV和抗PD-1疗法的组合可以抑制HBV表达，并显着提高HBV转基因小鼠的存活率。此外，联合疗法可增加T细胞产生的干扰素，然后增强Th1相关免疫刺激基因的表达，从而减少调节性/抑制性免疫基因的转录。通常，这种反应可以使肿瘤的微环境从免疫抑制重塑为免疫刺激。最后，抗HBV疗法可诱导HBV转基因小鼠中干扰素依赖性程序性细胞死亡配体-1的表达体内。综上所述，这些结果表明，针对HBV的靶向疗法与PD-1免疫检查点阻滞剂的联合具有强大的协同作用，从而支持了该联合疗法在HBV感染临床治疗中的转化潜力。