Erkuden Casales

Virotherapy with a Semliki Forest Virus–Based Vector Encoding IL12 Synergizes with PD-1/PD-L1 Blockade

Quetglas and colleagues report that intratumoral injection of cytolytic nonreplicative Semliki Forest virus vector expressing IL12, along with systemic administration of anti-PD-1/PD-L1 antibodies, induced regression of both virally injected and distal tumors and synergistically prolonged survival in mouse tumor models. Virotherapy and checkpoint inhibitors can be combined for the treatment of cancer with complementarity and potential for synergistic effects. We have developed a cytolytic but nonreplicative viral vector system based on Semliki Forest virus that encodes IL12 (SFV-IL12). Following direct intratumoral injection, infected cells release transgenic IL12, die, and elicit an inflammatory response triggered by both abundantly copied viral RNA and IL12. In difficult-to-treat mouse cancer models, such as those derived from MC38 and bilateral B16-OVA, SFV-IL12 synergized with an anti–PD-1 monoclonal antibody (mAb) to induce tumor regression and prolong survival. Similar synergistic effects were attained upon PD-L1 blockade. Combined SFV-IL12 + anti–PD-1 mAb treatment only marginally increased the elicited cytotoxic T-lymphocyte response over SFV-IL12 as a single agent, at least when measured by in vivo killing assays. In contrast, we observed that SFV-IL12 treatment induced expression of PD-L1 on tumor cells in an IFNγ-dependent fashion. PD-L1–mediated adaptive resistance thereby provides a mechanistic explanation of the observed synergistic effects achieved by the SFV-IL12 + anti–PD-1 mAb combination. Cancer Immunol Res; 3(5); 449–54. ©2015 AACR.

A novel system for the production of high levels of functional human therapeutic proteins in stable cells with a Semliki Forest virus noncytopathic vector

Semliki Forest virus (SFV) vectors lead to high protein expression in mammalian cells, but expression is transient due to vector cytopathic effects, inhibition of host cell proteins and RNA-based expression. We have used a noncytopathic SFV mutant (ncSFV) RNA vector to generate stable cell lines expressing two human therapeutic proteins: insulin-like growth factor I (IGF-I) and cardiotrophin-1 (CT-1). Therapeutic genes were fused at the carboxy-terminal end of Puromycin N-acetyl-transferase gene by using as a linker the sequence coding for foot-and-mouth disease virus (FMDV) 2A autoprotease. These cassettes were cloned into the ncSFV vector. Recombinant ncSFV vectors allowed rapid and efficient selection of stable BHK cell lines with puromycin. These cells expressed IGF-I and CT-1 in supernatants at levels reaching 1.4 and 8.6 microg/10(6)cells/24 hours, respectively. Two cell lines generated with each vector were passaged ten times during 30 days, showing constant levels of protein expression. Recombinant proteins expressed at different passages were functional by in vitro signaling assays. Stability at RNA level was unexpectedly high, showing a very low mutation rate in the CT-1 sequence, which did not increase at high passages. CT-1 was efficiently purified from supernatants of ncSFV cell lines, obtaining a yield of approximately 2mg/L/24 hours. These results indicate that the ncSFV vector has a great potential for the production of recombinant proteins in mammalian cells.

Development of a new noncytopathic Semliki Forest virus vector providing high expression levels and stability

Alphavirus vectors express high levels of recombinant proteins in mammalian cells, but their cytopathic nature makes this expression transient. In order to generate a Semliki Forest virus (SFV) noncytopathic vector we introduced mutations previously described to turn Sindbis virus noncytopathic into a conserved position in an SFV vector expressing LacZ. Interestingly, mutant P718T in replicase nsp2 subunit was able to replicate in only a small percentage of BHK cells, producing beta-gal-expressing colonies without selection. Puromycin N-acetyl-transferase (pac) gene was used to replace LacZ in this mutant allowing selection of an SFV noncytopathic replicon containing a second mutation in nsp2 nuclear localization signal (R649H). This latter mutation did not confer a noncytopathic phenotype by itself and did not alter nsp2 nuclear translocation. Replicase synthesis was diminished in the SFV double mutant, leading to genomic and subgenomic RNA levels that were 125-fold and 66-fold lower than in wild-type vector, respectively. Interestingly, this mutant expressed beta-gal levels similar to parental vector. By coexpressing pac and LacZ from independent subgenomic promoters this vector was able to generate stable cell lines maintaining high expression levels during at least 10 passages, indicating that it could be used as a powerful system for protein production in mammalian cells.

Strict Requirement for Vector-Induced Type I Interferon in Efficacious Antitumor Responses to Virally Encoded IL12

Host responses are increasingly considered important for the efficacious response to experimental cancer therapies that employ viral vectors, but little is known about the specific nature of host responses required. In this study, we investigated the role of host type I interferons (IFN-I) in the efficacy of virally delivered therapeutic genes. Specifically, we used a Semliki Forest virus encoding IL12 (SFV-IL12) based on its promise as an RNA viral vector for cancer treatment. Intratumoral injection of SFV-IL12 induced production of IFN-I as detected in serum. IFN-I production was abolished in mice deficient for the IFNβ transcriptional regulator IPS-1 and partially attenuated in mice deficient for the IFNβ signaling protein TRIF. Use of bone marrow chimeric hosts established that both hematopoietic and stromal cells were involved in IFN-I production. Macrophages, plasmacytoid, and conventional dendritic cells were each implicated based on cell depletion experiments. Further, mice deficient in the IFN-I receptor (IFNAR) abolished the therapeutic activity of SFV-IL12, as did a specific antibody-mediated blockade of IFNAR signaling. Reduced efficacy was not caused by an impairment in IL12 expression, because IFNAR-deficient mice expressed the viral IL12 transgene even more strongly than wild-type (WT) hosts. Chimeric host analysis for the IFNAR involvement established a strict requirement in hematopoietic cells. Notably, although tumor-specific CD8 T lymphocytes expanded robustly after intratumoral injection of WT mice with SFV-IL12, this did not occur in mice where IFNAR was inactivated genetically or pharmacologically. Overall, our results argued that the antitumor efficacy of a virally based transgene therapeutic relied strongly on a vector-induced IFN-I response, revealing an unexpected mechanism of action that is relevant to a broad array of current translational products in cancer research.

Eradication of Liver-Implanted Tumors by Semliki Forest Virus Expressing IL-12 Requires Efficient Long-Term Immune Responses

Semliki Forest virus vectors expressing IL-12 (SFV–IL-12) were shown to induce potent antitumor responses against s.c. MC38 colon adenocarcinomas in immunocompetent mice. However, when MC38 tumors were implanted in liver, where colon tumors usually metastasize, SFV–IL-12 efficacy was significantly reduced. We reasoned that characterization of immune responses against intrahepatic tumors in responder and nonresponder animals could provide useful information for designing more potent antitumor strategies. Remarkably, SFV–IL-12 induced a high percentage of circulating tumor-specific CD8 T cells in all treated animals. Depletion studies showed that these cells were essential for SFV–IL-12 antitumor activity. However, in comparison with nonresponders, tumor-specific cells from responder mice acquired an effector-like phenotype significantly earlier, were recruited more efficiently to the liver, and, importantly, persisted for a longer period of time. All treated mice had high levels of functional specific CD8 T cells at 8 d posttreatment reflected by both in vivo killing and IFN-γ–production assays, but responder animals showed a more avid and persistent IFN-γ response. Interestingly, differences in immune responses between responders and nonresponders seemed to correlate with the immune status of the animals before treatment and were not due to the treatment itself. Mice that rejected tumors were protected against tumor rechallenge, indicating that sustained memory responses are required for an efficacious therapy. Interestingly, tumor-specific CD8 T cells of responder animals showed upregulation of IL-15Rα expression compared with nonresponders. These results suggest that SFV–IL-12 therapy could benefit from the use of strategies that could either upregulate IL-15Rα expression or activate this receptor.

Capsid-deficient alphaviruses generate propagative infectious microvesicles at the plasma membrane

Alphavirus budding is driven by interactions between nucleocapsids assembled in the cytoplasm and envelope proteins present at the plasma membrane. So far, the expression of capsid and envelope proteins in infected cells has been considered an absolute requirement for alphavirus budding and propagation. In the present study, we show that Semliki Forest virus and Sindbis virus lacking the capsid gene can propagate in mammalian and insect cells. This propagation is mediated by the release of infectious microvesicles (iMVs), which are pleomorphic and have a larger size and density than wild-type virus. iMVs, which contain viral RNA inside and viral envelope proteins on their surface, are released at the plasma membrane and infect cells using the endocytic pathway in a similar way to wild-type virus. iMVs are not pathogenic in immunocompetent mice when injected intravenously, but can infect different organs like lungs and heart. Finally, we also show that alphavirus genomes without capsid can mediate the propagation of heterologous genes, making these vectors potentially interesting for gene therapy or vaccination studies. The minimalist infectious system described in this study shows that a self-replicating RNA able to express membrane proteins with binding and fusion properties is able to propagate, providing some insights into virus evolution.

A simple and efficient method for the production of human glycosylated glial cell line-derived neurotrophic factor using a Semliki Forest virus expression system

Human glial cell line-derived neurotrophic factor (hGDNF) is a very promising protein for the treatment of Parkinsons disease and other neurodegenerative disorders. The present work describes a quick and simple method to obtain a high amount of purified hGDNF using a mammalian cell-derived system. The method is based on the high expression level provided by a Semliki Forest virus vector and its ability to induce a strong shut-off of host-cell protein synthesis in mammalian cells. As a result, hGDNF is the only protein present in the supernatant and can be efficiently purified by a single chromatographic step. Using this system it was possible to eliminate other secreted proteins from the culture medium, like insulin-like growth factor-5, which are hard to remove using other hGDNF production methods. Purified hGDNF presents a complex glycosylation pattern typical of mammalian expression systems and is biologically active. This protocol could be extended to other secreted proteins and could be easily scaled up for industrial purposes.

Recent Patents on Alphavirus Protein Expression and Vector Production

Alphaviruses contain a single-strand RNA genome that can be modified to express heterologous genes at high levels. Alphavirus vectors can be packaged within viral particles (VPs) or used as DNA/RNA layered systems. The broad tropism and high expression levels of alphavirus vectors have made them very attractive for applications like recombinant protein expression, vaccination or gene therapy. Expression mediated by alphavirus vectors is generally transient due to induction of apoptosis. However, during the last years several non-cytopathic mutations have been identified within the replicase sequence of different alphaviruses, allowing prolonged protein expression in culture cells. Some of these mutants, which have been patented, have allowed the generation of stable cell lines able to express recombinant proteins for extended periods of time in a constitutive or inducible manner. Production of alphavirus VPs usually requires cotransfection of cells with vector and helper RNAs providing viral structural proteins in trans. During this process full-length wild type (wt) genomes can be generated through recombination between different RNAs. Several new strategies to reduce wt virus generation during packaging, optimize VP production, increase packaging capacity, and provide VPs with specific targeting have been recently patented. Finally, hybrid vectors between alphavirus and other types of viruses have led to a number of patents with applications in vaccination, cancer therapy or retrovirus production.

576. Antitumoral Effect Mediated by Two SFV-IL-12 Expression Vectors in a Murine Model of Colon Adenocarcinoma

Two Semliki Forest Virus (SFV) based vectors expressing murine interleukin-12 (IL-12) have been developed and their antitumoral efficacy has been tested in the MC38 mouse model of colon carcinoma. In SFV-IL-12 vector, genes coding for IL-12 subunits were cloned under a single viral subgenomic promoter using an IRES between them. SFV-enhIL-12 vector carries each gene fused to the SFV capsid translation enhancer, under independent viral subgenomic promoters. Recombinant SFV viral particles were produced and IL-12 expression was tested in supernatants of BHK and MC38 infected cells by ELISA. Biologically active IL-12 was expressed at high levels from both vectors with a 9-fold increase in the case of SFV-enhIL-12. Single tumor nodules were implanted in the flank of C57BL/6 mice by subcutaneous injection of MC38 colon carcinoma cells and treated with a single intratumoral injection of SFV-IL-12, SFV-enhIL-12, and SFV-LacZ or saline as controls. With 108 viral particles of SFV-IL-12 or SFV-enhIL-12 more than 80% of treated mice experienced complete tumor regression with long-term tumor-free survival. However, when lower doses of vectors were used SFV-enhIL-12 was more efficient than SFV-IL-12 in inducing antitumoral responses. In order to compare the SFV vectors with other viral vectors more commonly used in gene therapy some groups of mice were treated in parallel with a first generation adenovirus vector coding IL-12 (Ad-IL-12). Both SFV vectors were more efficient inducing antitumoral responses than Ad-IL-12. Treatment with repeated intratumoral injection of suboptimal doses of SFV-enhIL-12 increased significatively the antitumoral response. This effect was also observed with Ad-IL-12 but only when 108 PFU were injected. In addition, all mice that rejected tumors showed specific protection against tumor rechallenge. CTL assays and depletion studies showed a CD8+ T cell dependent response. Intratumoral expression was measured in tumor homogenates after infection with SFV IL-12 vectors resulting in a maximum cytokine level at 24 hours postinjection. The length of expression was also determined in vivo by intratumoral injection of a SFV vector expressing luciferase (SFV-Luc) using the Xenogen IVIS system. Luciferase expression localized into the tumor mass for at least 6 days after infection. Systemic administration of SFV-Luc in tumor bearing mice by intraperitoneal or intravenous injection produced high luciferase expression in large body areas but it did not localized at the tumor nodule.