构建溶瘤腺病毒的策略

溶瘤腺病毒是通过遗传工程改造的腺病毒,具有溶细胞复制周期的特性和特异靶向肿瘤细胞和裂解肿瘤细胞的功能。溶瘤腺病毒通过复制、释放子代病毒感染邻近肿瘤细胞。现在对腺病毒生活周期的认识水平,可以对其基因组进行改造从而使其特异性裂解肿瘤细胞而不杀伤正常细胞,并且构建了多种溶瘤腺病毒。本文主要概述了溶瘤腺病毒的三种构建策略:剔除腺病毒在正常细胞中复制所必需而在肿瘤细胞中不需要的某些基因;利用肿瘤特异性启动子控制病毒复制所必需的基因;对腺病毒衣壳蛋白进行基因修饰,达到特异性结合肿瘤细胞的目的。     

溶瘤痘苗病毒对肿瘤细胞作用机制的研究进展

溶瘤病毒治疗,是指利用具有复制能力的病毒选择性地感染和摧毁癌细胞,而对正常细胞或组织影响很少甚至没有影响的一种癌症治疗方法.痘苗病毒在肿瘤的基因治疗应用研究中以其独特的优势而备受关注.痘苗病毒感染细胞后具有产生病毒颗粒快、宿主范围广、基因组容量大、安全性高、传播高效等优点.尤为重要的是,基于溶瘤痘苗病毒的天然特性或遗传工程的改造,溶瘤痘苗病毒可以选择性地在肿瘤细胞中复制并对其产生杀伤效果.痘苗溶瘤病毒引起肿瘤细胞裂解和死亡是由多种不同的机制引起的.本文就近年来溶瘤痘苗病毒对细胞作用机制和相关信号通路的影响作一简要综述.     

一株新发现新城疫病毒体外高效杀伤肝癌细胞作用机制初探

目的:与NDV疫苗株LaSota对比研究一株NDV D817株对肝癌细胞高效特异性的杀伤效应和作用机制,进一步筛选NDV溶瘤毒株.方法:用MTT法对比病毒对三株传代肝癌细胞株SMMC-7721、Bel-7404和HepG-2及一株正常肝细胞株HL-7702的杀伤效应,并用TUNNL法及透射电镜观察病毒诱导肿瘤细胞发生凋亡作用.结果:NDV D817株对肝癌细胞株SMMC-7721、Bel-7404和HepG-2杀伤效应高达80%,显著高于疫苗LaSota株(P<0.01),而对人正常肝细胞HL-7702无明显影响;病毒在肝癌细胞中明显复制增殖,对细胞的杀伤活性与病毒作用剂量和病毒作用时间成正比;NDV D817株有效诱导肝癌细胞发生凋亡.结论:NDV D817株有效诱导肝癌细胞发生凋亡,对SMMC-7721、Bel-7404和HepG-2细胞具有高效杀伤性,而对正常肝细胞HL-7702未见明显影响.推测为溶瘤株.     

溶瘤病毒靶向治疗肿瘤的策略

近年来,随着国内外几款溶瘤病毒制剂的相继上市,溶瘤病毒疗法成为肿瘤免疫治疗的焦点。溶瘤病毒可选择性感染并裂解肿瘤细胞,同时释放肿瘤相关抗原激活机体的抗肿瘤免疫反应,达到杀伤肿瘤细胞和抑制肿瘤生长的目的。溶瘤病毒对肿瘤的靶向杀伤作用决定了其安全性和溶瘤效果。为了开发出安全高效的溶瘤病毒,目前主要采用以下策略：利用某些病毒载体对肿瘤细胞的天然靶向性,使溶瘤病毒选择性地在肿瘤细胞内复制并杀伤肿瘤细胞;或者对病毒基因组进行缺失和插入等修饰,通过靶向肿瘤细胞特异性表面受体、胞内信号通路或者肿瘤微环境等提高溶瘤病毒的肿瘤靶向性。其中,肿瘤微环境中的低氧状态、新血管生成以及免疫抑制状态等都可成为溶瘤病毒的靶点。而溶瘤病毒通过表达细胞因子和免疫检查点抑制剂,或者与CAR-T细胞联合作用,靶向调节肿瘤微环境中免疫抑制状态,成为提高溶瘤病毒肿瘤靶向性的常用方法。本文将对以上溶瘤病毒靶向治疗肿瘤策略的研究进展进行综述。     

溶瘤病毒调控肿瘤微环境及联合治疗的研究进展

溶瘤病毒是一类天然的或经改造后获得具有靶向杀伤癌细胞能力的病毒,除了能特异性杀伤肿瘤细胞外,经改造后的溶瘤病毒对肿瘤微环境的调控作用也会影响其最终疗效.通过调控肿瘤微环境中肿瘤细胞抗原的表达、免疫抑制状态、肿瘤相关成纤维细胞及肿瘤血管新生等,溶瘤病毒为肿瘤的治疗提供了更为系统的治疗策略;联合免疫检查点抑制剂的使用能使两者获得协同和互补的功效,进一步提升了肿瘤全面和有效的治疗.本文将对溶瘤病毒对肿瘤微环境调控作用及联合治疗的研究进展进行综述.     

溶瘤病毒的免疫学机制及临床研究进展

溶瘤病毒（oncolytic virus,OVs）历经百年发展,应用于当前最具潜力的肿瘤免疫疗法。它主要是天然的或基因修饰的DNA病毒和RNA病毒。近年来随着基因工程技术的飞跃发展,经基因改造的溶瘤病毒在肿瘤治疗领域取得很大进展,很多不同类型的病毒（包括单纯疱疹病毒、腺病毒、痘病毒、麻疹病毒和呼肠孤病毒等）正处于临床前研究、临床试验阶段或已批准上市,显示了良好的安全性和临床疗效。普遍认为溶瘤病毒靶向杀伤肿瘤细胞是通过选择性在肿瘤细胞内自我复制,最终裂解肿瘤细胞,同时可激发机体的免疫应答反应,进而增强抗肿瘤免疫效果,靶向杀伤肿瘤细胞而对正常细胞无明显影响。运用基因重组技术将溶瘤病毒与免疫检查点相结合以及肿瘤免疫联合疗法的兴起和不断进步,使溶瘤病毒的应用更加广泛,但仍存在病毒靶向性、安全性、给药途径等瓶颈问题。本文综述了溶瘤病毒的发展史、病毒分类、不同类型溶瘤病毒产品的临床研究进展、溶瘤病毒靶向杀伤肿瘤的免疫学机制及未来发展面临的挑战与展望等。     

溶瘤病毒联合CAR-T细胞治疗实体瘤研究进展

嵌合抗原受体修饰的T细胞(chimeric antigen receptor T cells, CAR-T)疗法属于肿瘤免疫治疗的范畴。该疗法在血液系统恶性肿瘤治疗中表现优异,但在实体瘤治疗中存在诸多挑战。近年来,溶瘤病毒(oncolytic viruses, OVs)在针对黑色素瘤、脑胶质瘤等实体瘤适应证的临床试验中展现出良好的疗效。溶瘤病毒一方面选择性地在肿瘤细胞中复制杀伤肿瘤细胞,另一方面通过激活机体自身的免疫系统发挥抗肿瘤作用。因此,溶瘤病毒与免疫检查点抑制剂、肿瘤浸润淋巴细胞疗法(tumor infiltrating lymphocytes, TIL)等免疫疗法的联合应用也在广泛开展。目前研究表明,溶瘤病毒不仅能够增加CAR-T细胞的抗肿瘤活性,而且通过传递肿瘤相关抗原或特异性抗原来增强CAR-T细胞对肿瘤的杀伤作用,同时溶瘤病毒还可以帮助CAR-T细胞克服免疫抑制性的肿瘤微环境。两种疗法的联合应用在临床前研究中展现出了良好的疗效和安全性,能够解决CAR-T在实体瘤治疗领域的瓶颈,具有广阔的临床转化前景。本文就溶瘤病毒与CAR-T细胞联合治疗实体瘤的药效及相关机制研究展开论述...     

溶瘤性流感病毒的研究进展

溶瘤病毒疗法属于免疫治疗的手段之一。其可通过病毒特异性地感染裂解肿瘤细胞和激活肿瘤免疫两种途径来达到杀伤肿瘤的目的;同传统疗法比,具有安全、高效、副作用小等优点。流感病毒自1900年代首次发现其可能作为“有益”的病毒缓解白血病病情以来,不断有研究证明流感病毒具有杀伤肿瘤细胞的能力;利用反向遗传操作技术对病毒进行改造,有望将其发展成为一种更加安全、有效的肿瘤治疗生物制剂。本文将对近年来溶瘤流感病毒利用肿瘤分泌的胰蛋白酶促进病毒感染并在RAS基因突变导致干扰素缺陷的肿瘤中复制来提高肿瘤靶向性,编码CTLA-4的单链抗体或HER-2增强流感病毒的抗癌特异性及作为外源基因IL-2、IL-15、GM-CSF和抗PD-1单克隆抗体的载体激活机体免疫几个方向进行综述。     

基于水疱性口炎病毒(VSV)的溶瘤病毒研究进展

溶瘤病毒利用肿瘤细胞抗病毒信号通路缺损或病毒受体过表达的特点,实现在其中选择性高复制进而杀伤肿瘤细胞,同时刺激机体产生特异性抗肿瘤免疫反应,是目前肿瘤治疗研究领域的热点。水疱性口炎病毒(vesicular stomatitis virus,VSV)能依赖肿瘤细胞干扰素信号通路的缺陷特异性靶向肿瘤细胞,具有复制高效、广泛组织嗜性、人群低致病性、基因组较小且易操纵等优势,是一种具有发展潜力的溶瘤病毒载体。对水疱性口炎病毒的病毒学特征以及目前基于VSV溶瘤病毒关于提高肿瘤选择性、延长半衰期、增强溶瘤效果的研究进展进行综述,为基于VSV溶瘤制剂的开发提供依据,为肿瘤治疗提供新的策略。     

靶向宿主代谢重编程的溶瘤病毒调控策略

溶瘤病毒是一类天然的或经过基因编辑后能特异性在肿瘤细胞中复制、发挥抗肿瘤效应的病毒。溶瘤病毒的抗肿瘤效应主要通过以下两个方面实现：a. 直接的溶瘤效应，例如诱导肿瘤细胞发生凋亡、促使细胞裂解等；b. 溶瘤病毒作为一种激活免疫的药物，通过诱导机体产生强烈的抗肿瘤免疫，达到清除肿瘤的目的。溶瘤病毒疗法作为免疫疗法的一个重要分支，因其具有肿瘤特异性，便于基因改造等优点，成为该领域的研究热点。截至目前，处在临床实验招募和完成阶段的溶瘤病毒疗法虽然已达100多例，但已批准上市的产品仅有4款。溶瘤疗法应用于肿瘤治疗领域还面临着诸多挑战。因此，系统性回顾溶瘤病毒的改造策略，深入了解溶瘤病毒的生物学过程显得尤为必要。病毒依赖于宿主完成复制、增殖过程，其生物学过程与宿主的代谢状态密切相关。肿瘤的标志性特征为代谢重编程，即肿瘤细胞重新构建代谢网络以满足指数生长和增殖的需求并防止氧化应激的过程。通常包括糖酵解的增强和谷氨酰胺分解，以及线粒体功能和氧化还原稳态的变化。通过靶向宿主代谢重编程增强溶瘤病毒的复制、溶瘤能力是当前极具前景的方向。本文综述溶瘤病毒的临床应用现状及与代谢相关的调控机制，为进一步开发新型溶瘤病毒以及联用方式提供新的思路。     

Incomplete but Infectious Vaccinia Virions Are Produced in the Absence of Oncolysis in Feline SCCF1 Cells

Vaccinia virus is a large, enveloped virus of the poxvirus family. It has broad tropism and typically virus replication culminates in accumulation and lytic release of intracellular mature virus (IMV), the most abundant form of infectious virus, as well as release by budding of extracellular enveloped virus (EEV). Vaccinia viruses have been modified to replicate selectively in cancer cells and clinically tested as oncolytic agents. During preclinical screening of relevant cancer targets for a recombinant Western Reserve strain deleted for both copies of the thymidine kinase and vaccinia growth factor genes, we noticed that confluent monolayers of SCCF1 cat squamous carcinoma cells were not destroyed even after prolonged infection. Interestingly, although SCCF1 cells were not killed, they continuously secreted virus into the cell culture supernatant. To investigate this finding further, we performed detailed studies by electron microscopy. Both intracellular and secreted virions showed morphological abnormalities on ultrastructural inspection, suggesting compromised maturation and morphogenesis of vaccinia virus in SCCF1 cells. Our data suggest that SCCF1 cells produce a morphologically abnormal virus which is nevertheless infective, providing new information on the virus-host cell interactions and intracellular biology of vaccinia virus.     

Enhancement of vaccinia virus based oncolysis with histone deacetylase inhibitors

Histone deacetylase inhibitors (HDI) dampen cellular innate immune response by decreasing interferon production and have been shown to increase the growth of vesicular stomatitis virus and HSV. As attenuated tumour-selective oncolytic vaccinia viruses (VV) are already undergoing clinical evaluation, the goal of this study is to determine whether HDI can also enhance the potency of these poxviruses in infection-resistant cancer cell lines. Multiple HDIs were tested and Trichostatin A (TSA) was found to potently enhance the spread and replication of a tumour selective vaccinia virus in several infection-resistant cancer cell lines. TSA significantly decreased the number of lung metastases in a syngeneic B16F10LacZ lung metastasis model yet did not increase the replication of vaccinia in normal tissues. The combination of TSA and VV increased survival of mice harbouring human HCT116 colon tumour xenografts as compared to mice treated with either agent alone. We conclude that TSA can selectively and effectively enhance the replication and spread of oncolytic vaccinia virus in cancer cells.     

Virotherapy of Canine Tumors with Oncolytic Vaccinia Virus GLV-1h109 Expressing an Anti-VEGF Single-Chain Antibody

Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for cancer therapy. We have previously reported that oncolytic vaccinia virus strains expressing an anti-VEGF (Vascular Endothelial Growth Factor) single-chain antibody (scAb) GLAF-1 exhibited significant therapeutic efficacy for treatment of human tumor xenografts. Here, we describe the use of oncolytic vaccinia virus GLV-1h109 encoding GLAF-1 for canine cancer therapy. In this study we analyzed the virus-mediated delivery and production of scAb GLAF-1 and the oncolytic and immunological effects of the GLV-1h109 vaccinia virus strain against canine soft tissue sarcoma and canine prostate carcinoma in xenograft models. Cell culture data demonstrated that the GLV-1h109 virus efficiently infect, replicate in and destroy both tested canine cancer cell lines. In addition, successful expression of GLAF-1 was demonstrated in virus-infected canine cancer cells and the antibody specifically recognized canine VEGF. In two different xenograft models, the systemic administration of the GLV-1h109 virus was found to be safe and led to anti-tumor and immunological effects resulting in the significant reduction of tumor growth in comparison to untreated control mice. Furthermore, tumor-specific virus infection led to a continued production of functional scAb GLAF-1, resulting in inhibition of angiogenesis. Overall, the GLV-1h109-mediated cancer therapy and production of immunotherapeutic anti-VEGF scAb may open the way for combination therapy concept i.e. vaccinia virus mediated oncolysis and intratumoral production of therapeutic drugs in canine cancer patients.     

Highly effective production of biologically active,secreted, human granulocyte-macrophage colony-stimulating factor by recombinant vaccinia virus

The recombinant vaccinia virus strain VV-GMCSF-S1/3, which contains an insertion of full-length DNA copy of messenger RNA of human granulocyte-macrophage colony-stimulating factor (GM-CSF) in the structural part of the viral thymidine kinase gene, was obtained. The expression of the GM-CSF gene as a part of the recombinant virus is under the control of the native vaccinia virus promoter р7.5K; this results in the production of a mature form of the secreted protein with a molecular mass of 32 kDa. The biological activity of GM-CSF was evaluated by stimulation of the proliferation of cytokine-dependent human TF-1 erythroleukemia cells. The secretion level of biologically active human GM-CSF in the system of recombinant vaccinia virus/mammalian cells was 1–40 μg/mL of culture medium. The recombinant strain VV-GMCSF-S1/3 can be used as a producer of the glycosylated mature form of human GM-CSF, as well as a vector for the construction of oncolytic viruses and multivalent vaccine preparations.     

基于痘苗病毒载体的恶性肿瘤基因治疗研究进展

基因治疗一直是肿瘤生物治疗的重要策略,而以溶瘤痘苗病毒为载体的肿瘤治疗近年来受到较多关注。该文总结了目前用于恶性肿瘤治疗的痘苗病毒和基于痘苗病毒载体的基因治疗研究进展及其在各个领域的成果。     

Evaluation of a New Recombinant Oncolytic Vaccinia Virus Strain GLV-5b451 for Feline Mammary Carcinoma Therapy

Virotherapy on the basis of oncolytic vaccinia virus (VACV) infection is a promising approach for cancer therapy. In this study we describe the establishment of a new preclinical model of feline mammary carcinoma (FMC) using a recently established cancer cell line, DT09/06. In addition, we evaluated a recombinant vaccinia virus strain, GLV-5b451, expressing the anti-vascular endothelial growth factor (VEGF) single-chain antibody (scAb) GLAF-2 as an oncolytic agent against FMC. Cell culture data demonstrate that GLV-5b451 virus efficiently infected, replicated in and destroyed DT09/06 cancer cells. In the selected xenografts of FMC, a single systemic administration of GLV-5b451 led to significant inhibition of tumor growth in comparison to untreated tumor-bearing mice. Furthermore, tumor-specific virus infection led to overproduction of functional scAb GLAF-2, which caused drastic reduction of intratumoral VEGF levels and inhibition of angiogenesis.In summary, here we have shown, for the first time, that the vaccinia virus strains and especially GLV-5b451 have great potential for effective treatment of FMC in animal model.     

Efficient colonization and therapy of human hepatocellular carcinoma (HCC) using the oncolytic vaccinia virus strain GLV-1h68

Virotherapy using oncolytic vaccinia virus strains is one of the most promising new strategies for cancer therapy. In this study, we analyzed for the first time the therapeutic efficacy of the oncolytic vaccinia virus GLV-1h68 in two human hepatocellular carcinoma cell lines HuH7 and PLC/PRF/5 (PLC) in cell culture and in tumor xenograft models. By viral proliferation assays and cell survival tests, we demonstrated that GLV-1h68 efficiently colonized, replicated in, and did lyse these cancer cells in culture. Experiments with HuH7 and PLC xenografts have revealed that a single intravenous injection (i.v.) of mice with GLV-1h68 resulted in a significant reduction of primary tumor sizes compared to uninjected controls. In addition, replication of GLV-1h68 in tumor cells led to strong inflammatory and oncolytic effects resulting in intense infiltration of MHC class II-positive cells like neutrophils, macrophages, B cells and dendritic cells and in up-regulation of 13 pro-inflammatory cytokines. Furthermore, GLV-1h68 infection of PLC tumors inhibited the formation of hemorrhagic structures which occur naturally in PLC tumors. Interestingly, we found a strongly reduced vascular density in infected PLC tumors only, but not in the non-hemorrhagic HuH7 tumor model. These data demonstrate that the GLV-1h68 vaccinia virus may have an enormous potential for treatment of human hepatocellular carcinoma in man.     

Targeting of interferon-beta to produce a specific, multi-mechanistic oncolytic vaccinia virus

Background

Oncolytic viruses hold much promise for clinical treatment of many cancers, but a lack of systemic delivery and insufficient tumor cell killing have limited their usefulness. We have previously demonstrated that vaccinia virus strains are capable of systemic delivery to tumors in mouse models, but infection of normal tissues remains an issue. We hypothesized that interferon-beta (IFN-β) expression from an oncolytic vaccinia strain incapable of responding to this cytokine would have dual benefits as a cancer therapeutic: increased anticancer effects and enhanced virus inactivation in normal tissues. We report the construction and preclinical testing of this virus.

Methods and Findings

In vitro screening of viral strains by cytotoxicity and replication assay was coupled to cellular characterization by phospho-flow cytometry in order to select a novel oncolytic vaccinia virus. This virus was then examined in vivo in mouse models by non-invasive imaging techniques. A vaccinia B18R deletion mutant was selected as the backbone for IFN-β expression, because the B18R gene product neutralizes secreted type-I IFNs. The oncolytic B18R deletion mutant demonstrated IFN-dependent cancer selectivity and efficacy in vitro, and tumor targeting and efficacy in mouse models in vivo. Both tumor cells and tumor-associated vascular endothelial cells were targeted. Complete tumor responses in preclinical models were accompanied by immune-mediated protection against tumor rechallenge. Cancer selectivity was also demonstrated in primary human tumor explant tissues and adjacent normal tissues. The IFN-β gene was then cloned into the thymidine kinase (TK) region of this virus to create JX-795 (TK⁻/B18R⁻/IFN-β⁺). JX-795 had superior tumor selectivity and systemic intravenous efficacy when compared with the TK⁻/B18R⁻ control or wild-type vaccinia in preclinical models.

Conclusions

By combining IFN-dependent cancer selectivity with IFN-β expression to optimize both anticancer effects and normal tissue antiviral effects, we were able to achieve, to our knowledge for the first time, tumor-specific replication, IFN-β gene expression, and efficacy following systemic delivery in preclinical models.     

Vaccinia virus infection during murine pregnancy: a new pathogenesis model for vaccinia fetalis

Vaccinia fetalis, the vertical transfer of vaccinia virus from mother to fetus, is a relatively rare but often fatal complication of primary vaccinia virus vaccination during pregnancy. To date there has been no attempt to develop an animal model to study the pathogenesis of this acute viral infection in vivo. Here we report that infection of gestating BALB/c mice by either intravenous or intraperitoneal routes with the Western Reserve strain of vaccinia virus results in the rapid colonization of the placenta and vertical transfer of virus to the developing fetus. Systemic maternal infections during gestation lead to the death of all offspring prior to or very shortly after birth. Using in situ hybridization for vaccinia virus mRNA to identify infected cells, we show that the virus initially colonizes cells lining maternal lacunae within the trophospongium layer of the placenta. The study of this model will significantly enhance our understanding of the pathogenesis of fetal vaccinia virus infections and aid in the development of effective treatments designed to reduce the risk of vaccinia virus-associated complications during pregnancy.