Replication-defective recombinant Semliki Forest virus encoding GM-CSF as a vector system for rapid and facile generation of autologous human tumor cell vaccines.

This paper describes the production of recombinant Semliki Forest virus encoding murine or human granulocyte-macrophage colony-stimulating factor (GM-CSF) and the capacity of these vectors to transduce murine and human tumor cells ex vivo. High-titer stocks (up to 3 x 10(9) particles/ml) of conditionally infective, replication-defective, recombinant SFV particles were generated using the SFV Helper-2 system. It is shown that the recombinant SFV/GM-CSF virus, as well as recombinant SFV carrying the beta-galactosidase reporter gene, efficiently transduce both murine tumor cell lines as well as primary human renal carcinoma cells. Using ELISA's specific for GM-CSF, levels of GM-CSF production by the cells were determined. Levels of murine GM-CSF (mGM-CSF) produced by SFV/mGM-CSF transduced renal cell cancer cultures were equal to or higher than corresponding levels reported in the literature after transduction of similar renal carcinoma cell cultures using a retroviral vector system. The biological activity of GM-CSF was demonstrated by using cells which are dependent on GM-CSF for growth and by using primary bone marrow cells. All the transduced cell cultures (including the human renal cell carcinoma samples) produced GM-CSF for up to at least 4 days after transduction. The results imply that the recombinant SFV system can be used for rapid and facile preparation of autologous cancer cell vaccines.     

Recombinant Semliki Forest virus as a vector system for fast and selective in vivo gene delivery into balloon-injured rat aorta

Previously, we demonstrated that recombinant Semliki Forest virus (SFV) vector rapidly and selectively transfers genes into cultured vascular smooth muscle cells (VSMC), leaving endothelial cells (EC) unaffected. From this, we hypothesized that recombinant SFV in vivo only transfers genes into the media of balloon-injured but not intact vessel, that gene expression in VSMC is fast, and that the specificity of SFV for VSMC is caused by specific binding sites. To address these hypotheses, we studied the time course of in vivo SFV-LacZ and Ad-LacZ expression in balloon-injured rat aorta. In addition, the fusion characteristics of fluorescent pyrene-labeled SFV were explored in cultured VSMC and EC. In intact aorta, no LacZ expression was found in the intima or media at 24 h. In contrast, in denuded aorta, LacZ expression was detected in as early as 12 h after incubation. LacZ expression was predominantly present in the media. Ad-LacZ expression started after 12 h, but was predominantly present in the adventitia. Ad-LacZ expression in the media started after 72 h. In vitro transfection with SFV showed that fusion was higher and, moreover, saturable in VSMC as compared with EC, indicating the presence of specific SFV binding sites on VSMC, but not EC. From this we conclude that in vivo selectivity of SFV in balloon-injured vessels is based on the removal of the endothelium, which results in accessibility of VSMC in the media that carry specific binding sites for the SFV vector.     

Induction of P815 tumor immunity by recombinant Semliki Forest virus expressing the P1A gene.

The methylcholantrene-induced P815 mastocytoma tumor is derived from DBA/2 mice and expresses a weak tumor rejection antigen, P815A. The P1A gene, which encodes for the P815A antigen, is silent in most normal tissues with the exception of testis and placenta. These characteristics make P815 an interesting mouse model for the human MAGE-type tumor antigens. Recombinant Semliki Forest virus particles (rSFV) were constructed that expressed variants of the P815 antigen. Such particles, when used for vaccination, express the antigen only transiently since the viral vector is incapable of productive replication. Nevertheless, mice vaccinated with rSFV generated strong CTL responses and were protected against P815 tumor challenge.     

Inhibition of angiogenesis by a Semliki Forest virus vector expressing VEGFR-2 reduces tumour growth and metastasis in mice

Inhibition of tumour angiogenesis has been shown to restrict primary tumour growth and metastatic spread. This study examines the active induction of immune responses against tumour endothelial cells following immunization with recombinant Semliki Forest virus (rSFV) particles encoding murine vascular endothelial growth factor receptor-2 (VEGFR-2). This approach was tested in two murine tumour models, CT26 colon carcinoma and 4T1 metastasizing mammary carcinoma. Tumour growth and metastatic spread were shown to be significantly inhibited in mice that were prophylactically vaccinated or therapeutically treated with rSFV particles coding for VEGFR-2. Microvessel density analysis showed that immunization with rSFV led to significant inhibition of tumour angiogenesis. Therapeutic efficacy was found to be associated with the induction of an antibody response against VEGFR-2. Co-immunization of mice with rSFV particles encoding VEGFR-2 and interleukin (IL)-12 completely abrogated both the antibody response and the antitumour effect. However, co-immunization of mice with VEGFR-2 and IL-4 encoding particles was shown both to induce higher titres of anti-VEGFR-2 antibodies and lead to enhanced survival following tumour challenge when compared to mice vaccinated with VEGFR-2 particles alone. These findings indicate that active immunization with rSFV particles coding for VEGFR-2 can break immunological tolerance and could potentially be used as part of a novel treatment for cancer.     

CNS gene therapy applications of the Semliki Forest virus 1 vector are limited by neurotoxicity.

The Semliki Forest virus (SFV) 1 vector system is highly efficient at gene transduction in a broad range of host cells, including neurons. To determine the potential of SFV1-based vectors to mediate gene expression in substantia nigra neurons, we inoculated d1EGFP-expressing SFV virus-like particles stereotaxically into the mouse brain. This system selectively and extensively mediated gene expression in dopaminergic neurons of the substantia nigra. Continual reporter gene expression was evident in neuronal cell bodies for up to 3 weeks postinoculation and d1EGFP-positive neuronal processes were apparent for 12 weeks. There was no evidence of an apoptotic response to infection, but with time cell degeneration and an axonopathy, indicative of neuronal loss, were increasingly apparent. This system has potential for experimental studies requiring efficient transient gene transduction of mouse CNS neurons. The current SFV1 vector system is, however, limited in its potential for CNS gene therapy by neurotoxicity.     

大鼠组织激肽释放酶8重组腺病毒载体的构建及鉴定

目的构建大鼠组织激肽释放酶8（KLK8）的腺病毒表达载体,为进一步研究KLK8对心肌细胞的作用提供实验基础。方法采用AdEasy系统构建携带外源性KLK8编码基因的重组腺病毒载体pAd-KLK8,转染AD-293细胞包装产生重组腺病毒Ad-KLK8,TCID50法对收获病毒进行滴度鉴定,RT-PCR及Western-blot方法检测目的基因在Hela细胞的表达。结果 PCR、序列测定以及限制性酶切证实KLK8基因正确插入腺病毒表达载体,并在AD-293细胞中包装出重组腺病毒;收获病毒的滴度为3.16×1012 IU/mL。结论成功构建pAd-KLK8重组腺病毒表达载体,且重组腺病毒在Hela细胞能有效表达。     

Efficacy of recombinant adenovirus as vector for allergen gene therapy in a mouse model of type I allergy

DNA-based immunization represents an attractive alternative approach to the current treatment of allergic diseases by specific immunotherapy with allergen extracts. In this study, we used a replication-deficient adenovirus vector (AdCMV), to examine the in vivo efficacy of preventive and therapeutic genetic immunization in a mouse model of type I allergy. Primary immunization with a recombinant adenovirus expressing the model antigen beta-galactosidase (AdCMV-(beta)gal) induced a Th1 immune response (predominance of IgG2a antibodies, high frequency of IFN-gamma producing T cells) and large numbers of cytotoxic T lymphocytes. Prophylactic vaccination with AdCMV-(beta)gal abolished the production of specific IgE following subsequent immunization with (beta)gal-protein, and skewed the Th2-biased immune response to a Th1-orientated response. In contrast, therapeutic administration of AdCMV-(beta)gal after priming with (beta)gal-protein neither significantly inhibited ongoing IgE production nor modulated a manifest Th2 immune response. Thus, allergen gene transfer via recombinant adenovirus represents an effective method to establish protection against the development of allergic disorders, but does not qualify as a therapeutic tool to interfere with ongoing high IgE production.     

Treatment of rapidly growing K-BALB and CT26 mouse tumours using Semliki Forest virus and its derived vector

To assess the potential of immune stimulation in combination with apoptosis induction by Semliki Forest virus (SFV) and its derived vector for tumour treatment, we have utilized the poorly immunogenic and rapidly growing K-BALB and CT26 murine tumour models. Both cell lines underwent apoptosis and expressed viral antigen when infected with the SFV4 strain of SFV, or recombinant SFV (rSFV) virus-like particles (VLPs) encoding the p62-6k viral structural proteins. VLPs were used to immunize groups of BALB/c and BALB/c nu/nu mice prior to subcutaneous tumour induction and treatment. Direct intratumoral injection of VLPs or SFV4 resulted in an immediate and intense inflammatory reaction in immunized groups that was not observed in naive groups until day 5 of treatment, and was not observed in nu/nu groups. A significantly higher level of tumour growth inhibition was observed in immunocompetent groups than in athymic mice. For K-BALB tumours, SFV4 treated groups showed greater inhibition than that observed in VLP-treated groups, with immunization prior to treatment enhancing the overall antitumour effect and immune response. No significant difference was observed in CT26 tumours between VLP and SFV4-treated groups, but prior immunization considerably enhanced the antitumoural response. It is concluded that use of the inherent apoptosis-inducing capability of SFV or its vector, by perfusion in combination with immune stimulation, may have potential for the treatment of rapidly growing tumours.     

Regression of mouse tumours and inhibition of metastases following administration of a Semliki Forest virus vector with enhanced expression of IL-12

The Semliki Forest virus (SFV) vector is an RNA-based suicide expression vector that has been used experimentally for tumour therapy. Recently, a new enhanced vector pSFV10-E has been developed that expresses foreign genes at levels up to 10 times higher than the original vector. Interleukin-12 (IL-12), an immunomodulatory cytokine, plays a key role in the induction of T-helper1 responses. The two IL-12 gene subunits were cloned from mouse splenocytes and inserted into the pSFV10-E and pSFV10 (non-enhanced) vectors. Both constructs expressed and secreted biologically active murine IL-12. Administration of high titre rSFV10-E-IL12 particles intratumourally to treat implanted K-BALB tumours in BALB/c mice demonstrated complete tumour regression in comparison to control or rSFV10-IL12 treated groups. High titre rSFV10-E-IL12 particles were also effective in the CT26 tumour model. Histological and immunohistochemical studies revealed tumour necrosis in addition to aggressive influx of CD4+ and CD8+ T cells and other immune cells. Furthermore, inhibition of primary tumour growth and lung metastases of a metastatic (4T1) tumour model indicated the potential of high titres of rSFV10-E-IL12 particles as an efficient antitumour therapeutic agent.     

IL-2和(或)IL-3重组腺病毒直接体内注射对大剂量化疗后小鼠造血功能恢复的影响

目的：检测应用直接腹腔注射白细胞介素２（ＩＬ－２）重组腺病毒和（或）白细胞介素３（ＩＬ－３）重组腺病毒方法对化疗后小鼠造血功能恢复的影响。方法：正常小鼠腹腔注射大剂量环磷酰胺（２００ｍｇ／ｋｇ）２４小时后，直接腹腔注射ＩＬ－２重组腺病毒（Ａｄ－ＩＬ－２）和（或）ＩＬ－３重组腺病毒（Ａｄ－ＩＬ－３），用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）检测相应细胞内细胞因子ｍＲＮＡ，并持续观察小鼠外周血白细胞、股骨单个核细胞、骨髓ＣＦＵ－ＧＭ的恢复情况。结果：腹腔注射Ａｄ－ＩＬ－２和（或）Ａｄ－ＩＬ－３组小鼠腹腔细胞可测及相应细胞因子ｍＲＮＡ，血清中可测及相应细胞因子；腹腔注射Ａｄ－ＩＬ－３组小鼠外周血白细胞、股骨单个核细胞、骨髓ＣＦＵ－ＧＭ最低值均显著提高（Ｐ＜０．０５），但腹腔注射Ａｄ－ＩＬ－２组小鼠对化疗后小鼠造血功能无明显影响，联合应用Ａｄ－ＩＬ－２和Ａｄ－ＩＬ－３与单独应用Ａｄ－ＩＬ－３相比无显著性差异。结论：腹腔直接注射Ａｄ－ＩＬ－３能促进化疗后小鼠造血功能的恢复。     

Therapeutic potential of adenovirus as a vaccine vector for chronic virus infections

Therapeutic vaccines for chronic infections and cancer are needed. Challenges faced by therapeutic vaccines differ from those of preventative vaccines. Whereas the latter target a naive immune system, the former have to readjust an antigen-experienced immune system that is subverted due to sustained exposure to antigen. E1-deleted adenoviral vectors have succeeded preclinically as preventative vaccines and are now in clinical trials. Their potential as therapeutic vaccines for diseases caused by chronic virus infections or virus-associated malignancies remains to be explored in more depth and may require modifications to circumvent negative immunoregulatory pathways that develop following chronic infections or during tumour progression.     

Transfer of the murine interleukin-12 gene in vivo by a Semliki Forest virus vector induces B16 tumor regression through inhibition of tumor blood vessel formation monitored by Doppler ultrasonography.

To elucidate further the potential of a Semliki Forest virus (SFV) vector in vivo for gene therapy, we constructed a vector, SFV-IL12, to transfer murine IL-12 genes into tumors. A single intratumoral injection of established B16 murine melanoma with SFV-IL12 resulted in a significant inhibition of tumor growth, while injection with SFV-LacZ had no effect. This antitumoral activity correlated with an increase of IFN gamma production, MIG and IP-10 mRNA expression, both at the tumor site and at the periphery. In contrast, no increase in CTL- or NK cell-mediated cytotoxic response could be detected, ruling out the involvement of T and NK cell cytotoxicity. To determine how the transfer to IL-12 genes induced tumor regression, the antiangiogenic-activity of SFV-IL12 was investigated using Doppler ultrasonography (DUS). SFV-IL12 inhibited in situ neovascularization within the tumor, without affecting the resistance index of pre-existing intratumoral blood flows. In addition, histological analysis of SFV-IL12-treated tumors showed massive tumor necrosis induced by SFV-IL12 treatment. These data indicate that SFV-IL12 inhibits tumor growth through its antiangiogenic activity, demonstrated for the first time in vivo by DUS, and suggest that the SFV vector may be a novel valuable tool in tumor gene transfer.     

Recombinant Semliki Forest virus vaccine vectors: the route of injection determines the localization of vector RNA and subsequent T cell response

Vectors based on Semliki Forest virus (SFV) have been widely used in vitro and in vivo to express heterologous genes in animal cells. In particular, the ability of recombinant SFV (rSFV) to elicit specific, protective immune responses in animal models suggests that rSFV may be used as a vaccine vehicle. In this study, we examined the distribution of rSFV in vivo by immunohistochemistry and RT-PCR after intravenous, intramuscular and subcutaneous injection of rSFV particles and related this to the degree of cytotoxic T lymphocyte (CTL) responses and frequency of specific T cells detected by MHC-I tetramers. We found that after i.v. injection, rSFV-RNA was distributed to a variety of different tissues, whereas it was confined locally after i.m. and s.c. injections. The persistence of the rSFV vector was transient, and no viral RNA could be detected 10 days after inoculation. All tested routes of immunization generated significant levels of antigen-specific CTL responses and increased numbers of specific CD8+ T cells, as detected by tetramer binding. The distribution of antigen-specific CTLs correlated with the in vivo distribution pattern of rSFV, with a highest frequency in the spleen or local lymph node, depending on the injection route.     

Adeno-associated virus-mediated local delivery of LIGHT suppresses tumorigenesis in a murine cervical cancer model

LIGHT is a tumor necrosis factor superfamily ligand that is considered as a promising candidate for cancer therapy. It has a potent antitumor activity through establishing lymphoid-like tissues inside tumor sites and recruiting naive T cells into the tumor. In this study, we examined the possibility of antitumor activity by expressing LIGHT in cervical cancer (CC) model. A recombinant adeno-associated virus (AAV) vector was chosen for the transfer, based on its transfection efficiency and lack of detectable pathology. In vitro transfer of recombinant AAV vector expressing LIGHT (AAV-LIGHT) stimulated T-lymphocyte proliferation and activation. AAV-mediated gene transfer of LIGHT by intratumoral injection exerted a very potent antitumor effect against preexisting TC-1 cell CC in C57BL/6 mice. This study confirmed that AAV-LIGHT regressed tumor growth by activating cytotoxic T lymphocyte, enhancing infiltration of inflammatory cells in tumor and increasing stimulatory cytokine expression in tumor microenvironment. Therefore, AAV-LIGHT therapy might have potential utility for the treatment of CC.     

A therapy modality using recombinant IL-12 adenovirus plus E7 protein in a human papillomavirus 16 E6/E7-associated cervical cancer animal model

Interleukin (IL)-12 has been reported to induce cellular immune responses for protection against tumor formation. Here we investigate the utility of adenoviral delivery of IL-12 as an adjuvant for a human papillomavirus E7 subunit vaccine in a mouse tumor challenge model. Direct intratumoral injection of AdIL-12 resulted in a significant suppression of tumor growth compared to the control group. Injection of E7 protein into either a tumor site or the distance site along with AdIL-12 further enhanced antitumor effects significantly higher than either AdIL-12 or E7 injection alone. This combined injection resulted in complete regression of 9-mm-sized tumor in 40% of animals as well as lasting antitumor immunity against tumor recurrence. We also evaluated immune responses induced by these injections. AdIL-12 plus E7 enhanced E7-specific antibody responses significantly higher than AdIL-12 or E7 injection. In particular, the production level of interferon (IFN)-gamma from E7-specific CD4(+) T cells was similar between AdIL-12 group and AdIL-12 + E7 group. However, IFN-gamma production from E7-specific CD8(+) T cells was the most significant when injected with AdIL-12 + E7. This was consistent with intracellular IFN-gamma staining levels of CD8(+) T cells, suggesting that AdIL-12 + E7 injection enhances antitumor immunity in the human papillomavirus (HPV) 16 tumor model through increased expansion of the cytotoxic T-lymphocyte (CTL) subset. This enhanced protection appeared to be mediated by CD8(+) T cells, as determined by in vivo T-cell subset deletion. Thus, these studies demonstrate that E7 vaccines can induce CTL responses responsible for antitumor effects in the presence of IL-12 delivered via adenovirus vectors. This likely provides one additional approach for immune therapy against cervical cancers.     

Production of recombinant adeno-associated virus vectors using a packaging cell line and a hybrid recombinant adenovirus.

Recombinant adeno-associated virus (rAAV) vectors are under consideration for a wide variety of gene therapy applications. One of the limitations of the rAAV vector system has been the difficulty in producing the vector in sufficient quantity for adequate preclinical and clinical evaluation. A common method for vector production involves large-scale transient transfection of multiple plasmids into cultured cells. Because this approach might not be feasible for clinical scale manufacturing, we have sought approaches for rAAV vector production that avoid transient transfection procedures. In previously reported work, we generated an AAV packaging cell line that produces infectious rAAV when the vector genome is transfected into the cell line as plasmid DNA. We have now extended this approach by constructing a hybrid recombinant adenovirus (rAd) that contains a complete rAAV vector genome in the E1 region. This hybrid virus is used to deliver the rAAV genome to the packaging cell line (in the place of plasmid transfection). rAAV is produced when the packaging cell line is infected with the hybrid adenovirus and wild-type adenovirus. This method avoids the need for plasmid transfection and is adaptable to large-scale manufacturing processes.     

宫颈癌术后调强放疗与三维适形放疗的对比研究

目的比较宫颈癌术后行调强放疗(IMRT)与辅助性三维适形放疗(3DCRT)的有效性、安全性及远期疗效,优化临床治疗措施。方法 2009年1月至2010年10月收治的宫颈癌术后行辅助性放疗患者90例,据随机数字表法分为IMRT组与3DCRT组。IMRT组患者行5野6 MVX射线放疗,计划靶区(PTV)内剂量梯度<10%;3DCRT组行4野6MV X射线盒式照射。两组处方剂量、总剂量分别为1.8 Gy/次和45~48.6 Gy。对比两组PTV剂量分布、危及器官照射体积、副反应及3年复发率。结果 IMRT组PTV最小照射剂量(Dmin)、平均照射剂量(Dmean)显著低于3DCRT组,差异均有统计学意义(P<0.001,P=0.014),适形指数(CI 95%)明显高于3DCRT组(P<0.001)。经归一化处理后,IMRT组小肠、直肠、膀胱、骨髓、双侧股骨头受照体积在V45处方水平均明显低于3DCRT组(P<0.05),骨髓抑制、消化系统反应、放射性膀胱炎发生率亦明显低于3DCRT组,差异有统计学意义(P<0.05)。随访期内均无死亡病例,两组复发率、平均复发时间差异无统计学意义(P>0.05)。结论宫颈癌术后IMRT放疗相对于3DCRT放疗可以达到更好的靶区剂量分布,明显降低危及器官的受照射体积,减少放射性损伤,但患者的远期预后无明显获益。     

膀胱上皮特异性重组腺病毒的构建及其对膀胱癌细胞的抑制作用

目的构建特异作用于尿路上皮细胞的重组腺病毒并研究其对膀胱癌细胞株的抑制作用。方法 RT-PCR测定人类uroplakinⅡ（hUPⅡ）基因的表达模式以及柯萨奇病毒腺病毒联合受体（CAR）对多重细胞系的影响。瞬时转染和荧光素酶检测分析技术测定hUPⅡ启动子的组织特异性。构建重组腺病毒Ad-UPⅡ-E1A和Ad-UPⅡ-Null,限制性内切酶酶切分析和PCR技术检测其构建的正确性。Western blot分析细胞感染重组腺病毒后腺病毒E1A蛋白在膀胱癌细胞株BIU-87中的表达。测定重组腺病毒Ad-UPⅡ-E1A对于膀胱癌细胞系BIU-87细胞的抑制作用。结果膀胱癌细胞系BIU-87细胞表达hUPⅡ和CAR,其中hUPⅡ启动子具有高活性。在细菌技术上使用同源重组,将hUPⅡ启动子和E1A基因插入到5型的重组腺病毒的基因组中。在BIU-87细胞感染重组腺病毒Ad-UPⅡ-E1A后,E1A蛋白的表达呈强阳性。MTT分析证实重组腺病毒Ad-UPⅡ-E1A抑制了膀胱癌BIU-87细胞的生长。结论 hUPⅡ启动子有高组织特异性。重组腺病毒Ad-UPⅡ-E1A对膀胱癌细胞系BIU-87细胞有抑制作用。     

小鼠白细胞介素10重组腺病毒载体构建及对树突状细胞的基因修饰

背景:对于抗原递呈细胞树突状细胞及其分泌的细胞因子白细胞介素10在气道高反应性和炎症中的作用国内外文献报道较少.目的:构建小鼠白细胞介素10腺病毒重组体Ad-mIL-10,获得小鼠白细胞介素10基因修饰的树突状细胞,以期为下一步基因治疗的动物实验打下基础.方法:通过人工合成获得小鼠白细胞介素10基因,根据白细胞介素10基因序列及腺病毒载体的多克隆位点,合成包括酶切位点的基因序列,连接到pMD18-T载体并测序鉴定.用基因工程的方法将小鼠白细胞介素10基因克隆到BDAdeno-X~(TM)腺病毒载体,于人胚肾293细胞中包装、扩增病毒并测定白细胞介素10蛋白表达,转染到体外培养的小鼠骨髓来源的树突状细胞.结果与结论:成功构建了小鼠Ad-mIL-10重组腺病毒载体并高包装、扩增成功,测定高表达白细胞介素10蛋白,体外成功培养小鼠骨髓来源的小鼠树突状细胞并顺利转染Ad-mIL-10.提示用基因工程方法构建小鼠Ad-mIL-10重组腺病毒载体并转染小鼠骨髓来源的树突状细胞是可行的,可为进行相关基因治疗的可能性提供更充足的理论依据.