Production of Bioactive Soluble Interleukin-15 in Complex with Interleukin-15 Receptor Alpha from a Conditionally-Replicating Oncolytic HSV-1

Oncolytic type-1 herpes simplex viruses (oHSVs) lacking the γ134.5 neurovirulence gene are being evaluated for treatment of a variety of malignancies. oHSVs replicate within and directly kill permissive cancer cells. To augment their anti-tumor activity, oHSVs have been engineered to express immunostimulatory molecules, including cytokines, to elicit tumor-specific immune responses. Interleukin-15 (IL-15) holds potential as an immunotherapeutic cytokine because it has been demonstrated to promote both natural killer (NK) cell-mediated and CD8+ T cell-mediated cytotoxicity against cancer cells. The purpose of these studies was to engineer an oHSV producing bioactive IL-15. Two oHSVs were constructed encoding murine (m)IL-15 alone (J100) or with the mIL-15 receptor α (mIL-15Rα, J100D) to determine whether co-expression of these proteins is required for production of bioactive mIL-15 from oHSV. The following were demonstrated: i) both oHSVs retain replication competence and cytotoxicity in permissive tumor cell lines. ii) Enhanced production of mIL-15 was detected in cell lysates of neuro-2a cells following J100D infection as compared to J100 infection, suggesting that mIL-15Rα improved mIL-15 production. iii) Soluble mIL-15 in complex with mIL-15Rα was detected in supernates from J100D-infected, but not J100-infected, neuro-2a, GL261, and CT-2A cells. These cell lines vary in permissiveness to oHSV replication and cytotoxicity, demonstrating soluble mIL-15/IL-15Rα complex production from J100D was independent of direct oHSV effects. iv) The soluble mIL-15/IL-15Rα complex produced by J100D was bioactive, stimulating NK cells to proliferate and reduce the viability of syngeneic GL261 and CT-2A cells. v) J100 and J100D were aneurovirulent inasmuch as no neuropathologic effects were documented following direct inoculation into brains of CBA/J mice at up to 1x107 plaque forming units. The production of mIL-15/mIL-15Rα from multiple tumor lines, as well as the lack of neurovirulence, renders J100D suitable for investigating the combined effects of oHSV and mIL-15/IL-15Rα in various cancer models.缺乏γ134.5神经毒力基因的溶瘤1型单纯疱疹病毒（oncolytic type-1 herpes simplex viruses，缩写oHSVs）目前正被评估用于治疗多种恶性肿瘤。oHSVs可在易感肿瘤细胞内复制并直接裂解肿瘤细胞。为增强其抗肿瘤活性，研究人员已对oHSVs进行基因工程改造，使其表达包括细胞因子在内的免疫刺激分子，以诱导肿瘤特异性免疫应答。白细胞介素-15（Interleukin-15，以下简称IL-15）作为一种免疫治疗细胞因子具有广阔应用潜力，已有研究证实其可促进自然杀伤（natural killer，以下简称NK）细胞与CD8+T细胞介导的抗肿瘤细胞毒性作用。本研究的目的在于构建可产生活性IL-15的oHSV。本研究构建了两种oHSVs：一种仅编码鼠源IL-15（murine IL-15，以下简称mIL-15），命名为J100；另一种同时编码mIL-15与鼠源IL-15受体α（murine IL-15 receptor α，以下简称mIL-15Rα），命名为J100D，以探究从oHSV产生活性mIL-15是否需要这两种蛋白的共表达。实验结果如下：① 两种oHSVs在易感肿瘤细胞系中均保留复制能力与细胞毒性活性；② 相较于J100感染组，J100D感染后的Neuro-2a细胞裂解液中检测到的mIL-15表达量显著升高，提示mIL-15Rα可提升mIL-15的产量；③ 在J100D感染的Neuro-2a、GL261及CT-2A细胞的培养上清中，可检测到与mIL-15Rα形成复合物的可溶性mIL-15，而J100感染组未检测到该复合物。上述细胞系对oHSV复制与细胞毒性的易感程度存在差异，这表明J100D产生的可溶性mIL-15/mIL-15Rα复合物的生成不受oHSV直接作用的影响；④ J100D产生的可溶性mIL-15/mIL-15Rα复合物具有生物活性，可刺激NK细胞增殖，并降低同基因GL261与CT-2A细胞的存活率；⑤ J100与J100D均无神经毒力：当以最高1×10^7噬斑形成单位（plaque forming units，以下简称PFU）的剂量直接接种至CBA/J小鼠脑内后，未观察到任何神经病理损伤。J100D可在多种肿瘤细胞系中产生mIL-15/mIL-15Rα复合物，且无神经毒力，因此适用于探究oHSV与mIL-15/IL-15Rα联合应用在多种肿瘤模型中的综合效应。