Transient complement inhibition promotes a tumor-specific immune response through the implication of natural killer cells.

Although the role of the complement system in cancer development has been studied, its involvement in the development of an antitumoral immune response remains poorly understood. Using cobra venom factor (CVF) to inhibit the complement cascade via C3 molecule exhaustion in immunocompetent mice bearing B16gp33 melanoma tumors, we show that transient inhibition of the complement system allowed for the development of a more robust gp33-specific antitumoral CD8(+) T-cell response. This immune response proved to be natural killer (NK) dependent, suggesting an interaction of complement proteins with this cellular subset leading to T lymphocyte activation and enhanced cytotoxic T-cell activity against tumor cells. This study demonstrates for the first time the implication of the complement system in the development of NK-mediated cytotoxic T-cell-dependent antitumoral immune responses. The complement pathway could therefore be a potent therapeutic target to improve NK-dependent antitumoral immune responses in patients with cancer.

The strength of the T cell response against a surrogate tumor antigen induced by oncolytic VSV therapy does not correlate with tumor control.

Cancer therapy using oncolytic viruses has gained interest in the last decade. Vesicular stomatitis virus is an attractive candidate for this alternative treatment approach. The importance of the immune response against tumor antigens in virotherapy efficacy is now well recognized, however, its relative contribution versus the intrinsic oncolytic capacity of viruses has been difficult to evaluate. To start addressing this question, we compared glycoprotein and matrix mutants of vesicular stomatitis virus (VSV), showing different oncolytic potentials for B16/B16gp33 melanoma tumor cells in vitro, with the wild-type virus in their ability to induce tumor-specific CD8(+) T cell responses and control tumor progression in vivo. Despite the fact that wild-type and G mutants induced a stronger gp33-specific immune response compared to the MM51R mutant, all VSV strains showed a similar capacity to slow down tumor progression. The effectiveness of the matrix mutant treatment proved to be CD8(+) dependent and directed against tumor antigens other than gp33 since adoptive transfer of isolated CD8(+) T lymphocytes from treated B16gp33-bearing mice resulted in significant protection of naive mice against challenge with the parental tumor. Remarkably, the VSV matrix mutant induced the upregulation of major histocompatibility class-I antigen at the tumor cell surface thus favoring recognition by CD8(+) T cells. These results demonstrate that VSV mutants induce an antitumor immune response using several mechanisms. A better understanding of these mechanisms will prove useful for the rational design of viruses with improved therapeutic efficacy.

[Vesicular stomatitis virus in the fight against cancer]. / Le virus de la stomatite vésiculaire dans la lutte contre le cancer.

Cancer is a complex disease that affects more and more people around the world. Unfortunately, existing treatments are only partially efficient and often induce major side effects. Thus, the use of viruses to selectively kill cancer cells is a new promising therapeutic approach. Recently, VSV has been used in oncolytic virotherapy because of its capacity to preferentially infect most human tumor cells. However, despite the availability of good oncolytic VSV mutants, the large variability of tumor cell types and the multiple ways in which they can evade viral infection suggests that therapeutic combinations of various viruses will be necessary to efficiently treat most cancers. A better understanding of the infection mechanisms and immune system recruitment by oncolytic viruses will be of great value for the development of safe and efficient strategies for cancer treatment.

Mutations in the glycoprotein of vesicular stomatitis virus affect cytopathogenicity: potential for oncolytic virotherapy.

Vesicular stomatitis virus (VSV) has been widely used to characterize cellular processes, viral resistance, and cytopathogenicity. Recently, VSV has also been used for oncolytic virotherapy due to its capacity to selectively lyse tumor cells. Mutants of the matrix (M) protein of VSV have generally been preferred to the wild-type virus for oncolysis because of their ability to induce type I interferon (IFN) despite causing weaker cytopathic effects. However, due to the large variability of tumor types, it is quite clear that various approaches and combinations of multiple oncolytic viruses will be needed to effectively treat most cancers. With this in mind, our work focused on characterizing the cytopathogenic profiles of four replicative envelope glycoprotein (G) VSV mutants. In contrast to the prototypic M mutant, VSV G mutants are as efficient as wild-type virus at inhibiting cellular transcription and host protein translation. Despite being highly cytopathic, the mutant G(6R) triggers type I interferon secretion as efficiently as the M mutant. Importantly, most VSV G mutants are more effective at killing B16 and MC57 tumor cells in vitro than the M mutant or wild-type virus through apoptosis induction. Taken together, our results demonstrate that VSV G mutants retain the high cytopathogenicity of wild-type VSV, with G(6R) inducing type I IFN secretion at levels similar to that of the M mutant. VSV G protein mutants could therefore prove to be highly valuable for the development of novel oncolytic virotherapy strategies that are both safe and efficient for the treatment of various types of cancer.

The lectin concanavalin-A signals MT1-MMP catalytic independent induction of COX-2 through an IKKgamma/NF-kappaB-dependent pathway.

The lectin from Canavalia ensiformis (Concanavalin-A, ConA), one of the most abundant lectins known, enables one to mimic biological lectin/carbohydrate interactions that regulate extracellular matrix protein recognition. As such, ConA is known to induce membrane type-1 matrix metalloproteinase (MT1-MMP) which expression is increased in brain cancer. Given that MT1-MMP correlated to high expression of cyclooxygenase (COX)-2 in gliomas with increasing histological grade, we specifically assessed the early proinflammatory cellular signaling processes triggered by ConA in the regulation of COX-2. We found that treatment with ConA or direct overexpression of a recombinant MT1-MMP resulted in the induction of COX-2 expression. This increase in COX-2 was correlated with a concomitant decrease in phosphorylated AKT suggestive of cell death induction, and was independent of MT1-MMP's catalytic function. ConA- and MT1-MMP-mediated intracellular signaling of COX-2 was also confirmed in wild-type and in Nuclear Factor-kappaB (NF-kappaB) p65(-/-) mutant mouse embryonic fibroblasts (MEF), but was abrogated in NF-kappaB1 (p50)(-/-) and in I kappaB kinase (IKK) gamma(-/-) mutant MEF cells. Collectively, our results highlight an IKK/NF-kappaB-dependent pathway linking MT1-MMP-mediated intracellular signaling to the induction of COX-2. That signaling pathway could account for the inflammatory balance responsible for the therapy resistance phenotype of glioblastoma cells, and prompts for the design of new therapeutic strategies that target cell surface carbohydrate structures and MT1-MMP-mediated signaling. Concise summary Concanavalin-A (ConA) mimics biological lectin/carbohydrate interactions that regulate the proinflammatory phenotype of cancer cells through yet undefined signaling. Here we highlight an IKK/NF-kappaB-dependent pathway linking MT1-MMP-mediated intracellular signaling to the induction of cyclooxygenase-2, and that could be responsible for the therapy resistance phenotype of glioblastoma cells.

VSV strains with defects in their ability to shutdown innate immunity are potent systemic anti-cancer agents.

Ideally, an oncolytic virus will replicate preferentially in malignant cells, have the ability to treat disseminated metastases, and ultimately be cleared by the patient. Here we present evidence that the attenuated vesicular stomatitis strains, AV1 and AV2, embody all of these traits. We uncover the mechanism by which these mutants are selectively attenuated in interferon-responsive cells while remaining highly lytic in 80% of human tumor cell lines tested. AV1 and AV2 were tested in a xenograft model of human ovarian cancer and in an immune competent mouse model of metastatic colon cancer. While highly attenuated for growth in normal mice, both AV1 and AV2 effected complete and durable cures in the majority of treated animals when delivered systemically.

Ability of the matrix protein of vesicular stomatitis virus to suppress beta interferon gene expression is genetically correlated with the inhibition of host RNA and protein synthesis.

The vesicular stomatitis virus (VSV) matrix (M) protein plays a major role in the virus-induced inhibition of host gene expression. It has been proposed that the inhibition of host gene expression by M protein is responsible for suppressing activation of host interferon gene expression. Most wild-type (wt) strains of VSV induce little if any interferon gene expression. Interferon-inducing mutants of VSV have been isolated previously, many of which contain mutations in their M proteins. However, it was not known whether these M protein mutations were responsible for the interferon-inducing phenotype of these viruses. Alternatively, mutations in other genes besides the M gene may enhance the ability of VSV to induce interferons. These hypotheses were tested by transfecting cells with mRNA expressing wt and mutant M proteins in the absence of other viral components and determining their ability to inhibit interferon gene expression. The M protein mutations were the M51R mutation originally found in the tsO82 and T1026R1 mutant viruses, the double substitution V221F and S226R found in the TP3 mutant virus, and the triple substitution E213A, V221F, and S226R found in the TP2 mutant virus. wt M proteins suppressed expression of luciferase from the simian virus 40 promoter and from the beta interferon (IFN-beta) promoter, while M proteins of interferon-inducing viruses were unable to inhibit luciferase expression from either promoter. The M genes of the interferon-inducing mutants of VSV were incorporated into the wt background of a recombinant VSV infectious cDNA clone. The resulting recombinant viruses were tested for their ability to activate interferon gene expression and for their ability to inhibit host RNA and protein synthesis. Each of the recombinant viruses containing M protein mutations induced expression of a luciferase reporter gene driven by the IFN-beta promoter and induced production of interferon bioactivity more effectively than viruses containing wt M proteins. Furthermore, the M protein mutant viruses were defective in their ability to inhibit both host RNA synthesis and host protein synthesis. These data support the idea that wt M protein suppresses interferon gene expression through the general inhibition of host RNA and protein synthesis.

Matrix protein mutations contribute to inefficient induction of apoptosis leading to persistent infection of human neural cells by vesicular stomatitis virus.

In a model system to study factors involved in the establishment of a persistent viral infection that may lead to neurodegenerative diseases, Indiana and New Jersey variants of vesicular stomatitis virus (VSV) with different capacities to infect and persist in human neural cells were studied. Indiana matrix (M) protein mutants and the wild-type New Jersey strain persisted in the human neural cell line H4 for at least 120 days. The Indiana wild-type virus (HR) and a non-M mutant (TP6), both unable to persist, induced apoptosis more strongly than all the other variants tested, as indicated by higher levels of DNA fragmentation and caspase-3-like activity. Transfection of H4 cells with mRNA coding for the VSV M protein confirmed the importance of this protein in the induction of apoptosis. Furthermore, the pan-caspase inhibitor ZVAD-fmk maintained cell survival to about 80%, whereas inhibition of caspase-8, caspase-9, or both only partially protected the cells against death, consistent with the fact that anti-apoptotic molecules from the Bcl-2 family also protect cells from death only partially. These results suggest that VSV activates many pathways of cell death and that an inefficient induction of caspase-3-related apoptosis participates in the establishment of a persistent infection of human neural cells by less virulent VSV variants.