Jansons Juris, Skrastina Dace, Kurlanda Alisa, Petkov Stefan, Avdoshina Darya, Kuzmenko Yulia, Krotova Olga, Trofimova Olga, Gordeychuk Ilya, Sominskaya Irina, Isaguliants Maria
Institute of Microbiology and Virology, Riga Stradins University, LV-1007 Riga, Latvia.
Latvian Biomedical Research and Study Centre, LV-1067 Riga, Latvia.
Microorganisms. 2021 May 17;9(5):1073. doi: 10.3390/microorganisms9051073.
Chronic HCV infection and associated liver cancer impose a heavy burden on the healthcare system. Direct acting antivirals eliminate HCV, unless it is drug resistant, and partially reverse liver disease, but they cannot cure HCV-related cancer. A possible remedy could be a multi-component immunotherapeutic vaccine targeting both HCV-infected and malignant cells, but also those not infected with HCV. To meet this need we developed a two-component DNA vaccine based on the highly conserved core protein of HCV to target HCV-infected cells, and a renowned tumor-associated antigen telomerase reverse transcriptase (TERT) based on the rat TERT, to target malignant cells. Their synthetic genes were expression-optimized, and HCV core was truncated after aa 152 (Core152opt) to delete the domain interfering with immunogenicity. Core152opt and TERT DNA were highly immunogenic in BALB/c mice, inducing IFN-γ/IL-2/TNF-α response of CD4+ and CD8+ T cells. Additionally, DNA-immunization with TERT enhanced cellular immune response against luciferase encoded by a co-delivered plasmid (Luc DNA). However, DNA-immunization with Core152opt and TERT mix resulted in abrogation of immune response against both components. A loss of bioluminescence signal after co-delivery of TERT and Luc DNA into mice indicated that TERT affects the in vivo expression of luciferase directed by the immediate early cytomegalovirus and interferon-β promoters. Panel of mutant TERT variants was created and tested for their expression effects. TERT with deleted N-terminal nucleoli localization signal and mutations abrogating telomerase activity still suppressed the IFN-β driven Luc expression, while the inactivated reverse transcriptase domain of TERT and its analogue, enzymatically active HIV-1 reverse transcriptase, exerted only weak suppressive effects, implying that suppression relied on the presence of the full-length/nearly full-length TERT, but not its enzymatic activity. The effect(s) could be due to interference of the ectopically expressed xenogeneic rat TERT with biogenesis of mRNA, ribosomes and protein translation in murine cells, affecting the expression of immunogens. HCV core can aggravate this effect, leading to early apoptosis of co-expressing cells, preventing the induction of immune response.
慢性丙型肝炎病毒（HCV）感染及相关肝癌给医疗系统带来了沉重负担。直接作用抗病毒药物可清除HCV（除非其具有耐药性），并部分逆转肝脏疾病，但无法治愈HCV相关癌症。一种可能的补救方法是开发一种多组分免疫治疗疫苗，该疫苗既能靶向HCV感染细胞和恶性细胞，也能靶向未感染HCV的细胞。为满足这一需求，我们基于HCV高度保守的核心蛋白开发了一种双组分DNA疫苗，用于靶向HCV感染细胞；同时基于大鼠端粒酶逆转录酶（TERT）开发了一种著名的肿瘤相关抗原，用于靶向恶性细胞。对它们的合成基因进行了表达优化，并且HCV核心蛋白在第152个氨基酸后被截短（Core152opt），以删除干扰免疫原性的结构域。Core152opt和TERT DNA在BALB/c小鼠中具有高度免疫原性，可诱导CD4+和CD8+ T细胞产生IFN-γ/IL-2/TNF-α应答。此外，用TERT进行DNA免疫增强了针对共递送质粒（Luc DNA）编码的荧光素酶的细胞免疫应答。然而，用Core152opt和TERT混合物进行DNA免疫导致对两种组分的免疫应答均被消除。将TERT和Luc DNA共递送至小鼠体内后，生物发光信号的丧失表明TERT影响由立即早期巨细胞病毒和干扰素-β启动子驱动的荧光素酶在体内的表达。构建了一组突变TERT变体，并测试了它们的表达效果。删除N端核仁定位信号且突变消除端粒酶活性的TERT仍能抑制IFN-β驱动的Luc表达，而TERT的失活逆转录酶结构域及其类似物（具有酶活性的HIV-1逆转录酶）仅产生微弱的抑制作用，这意味着抑制作用依赖于全长/近全长TERT的存在，而不是其酶活性。这种效应可能是由于异位表达的异种大鼠TERT干扰了小鼠细胞中mRNA、核糖体的生物合成以及蛋白质翻译，从而影响了免疫原的表达。HCV核心蛋白可加重这种效应，导致共表达细胞早期凋亡，从而阻止免疫应答的诱导。
