Myxoma virus M064 is a novel member of the poxvirus C7L superfamily of host range factors that controls the kinetics of myxomatosis in European rabbits.

The myxoma virus (MYXV) carries three tandem C7L-like host range genes (M062R, M063R, and M064R). However, despite the fact that the sequences of these three genes are similar, they possess very distinctive functions in vivo. The role of M064 in MYXV pathogenesis was investigated and compared to the roles of M062 and M063. We report that M064 is a virulence factor that contributes to MYXV pathogenesis but lacks the host range properties associated with M062 and M063.

Myxoma virus sensitizes cancer cells to gemcitabine and is an effective oncolytic virotherapeutic in models of disseminated pancreatic cancer.

Myxoma virus (MYXV) is a novel oncolytic virus that has been shown to replicate in pancreatic cancer cells, but its efficacy in animal models of pancreatic cancer has not been determined. The efficacy of MYXV as monotherapy or in combination with gemcitabine was evaluated in intraperitoneal dissemination (IPD) models of pancreatic cancer. The effects of an intact immune system on the efficacy of MYXV therapy was tested by comparing immunodeficient versus immunocompetent murine models and combination therapy with gemcitabine was also evaluated. In cell culture, MYXV replication was robust in a broad range of pancreatic cancer cells and also showed increased oncolysis in combination with gemcitabine. In animal models, MYXV treatment conferred survival benefits over control or gemcitabine-treated cohorts regardless of the cell line or animal model used. MYXV monotherapy was most effective in an immunocompetent IPD model, and resulted in 60% long-term survivors. In Pan02 engrafted immunocompetent IPD models, sequential treatment in which MYXV was administered first, followed by gemcitabine, was the most effective and resulted in 100% long-term survivors. MYXV is an effective oncolytic virus for pancreatic cancer and can be combined with gemcitabine to enhance survival, particularly in the presence of an intact host immune system.

M062 is a host range factor essential for myxoma virus pathogenesis and functions as an antagonist of host SAMD9 in human cells.

Myxoma virus (MYXV) M062R is a functional homolog of the C7L family of host range genes from orthopoxviruses. We constructed a targeted M062R-knockout-MYXV (vMyxM062-KO) and characterized its properties in vitro and in vivo. In European rabbits, infection by vMyxM062-KO was completely asymptomatic. The surviving rabbits did not gain full protection against the subsequent lethal-dose challenge with wild-type MYXV. We also looked for cellular tropism defects in a variety of cultured cells. In all of the rabbit cells tested, vMyxM062-KO conducts an abortive infection, although it initiates viral DNA replication. In many, but not all, human cancer cells that are permissive for wild-type MYXV, vMyxM062-KO exhibited a profound replication defect. We categorized human cells tested into two groups: (i) type A, which support productive replication for wild-type MYXV but are unable to produce significant levels of progeny virus by vMyxM062-KO, and (ii) type B, which are permissive to infections by both wild-type MYXV and vMyxM062-KO. Furthermore, using proteomic strategies, we identified sterile α motif domain containing 9 (SAMD9), an interferon-regulated cellular protein implicated in human inflammatory disorders, as a unique host binding partner of M062 in human cells. Significantly, knocking down SAMD9 in type A human cancer cells led to a substantial rescue of vMyxM062-KO infection. In summary, M062 is a novel host range factor that controls productive MYXV replication in rabbit cells and in a wide variety of human cells. M062 also binds and antagonizes cellular SAMD9 in human cells, suggesting that SAMD9 is a novel innate antiviral factor against poxviruses.

Oncolytic virotherapy for pancreatic cancer.

Within the past decade, many oncolytic viruses (OVs) have been studied as potential treatments for pancreatic cancer and some of these are currently under clinical trials. The applicability of certain OVs, such as adenoviruses, herpesviruses and reoviruses, for the treatment of pancreatic cancer has been intensively studied for several years, whereas the applicability of other more recently investigated OVs, such as poxviruses and parvoviruses, is only starting to be determined. At the same time, studies have identified key characteristics of pancreatic cancer biology that provide a better understanding of the important factors or pathways involved in this disease. This review aims to summarise the different replication-competent OVs proposed as therapeutics for pancreatic cancer. It also focuses on the unique biology of these viruses that makes them exciting candidate virotherapies for pancreatic cancer and discusses how they could be genetically manipulated or combined with other drugs to improve their efficacy based on what is currently known about the molecular biology of pancreatic cancer.

Intratumoral virotherapy with 4-1BBL armed modified vaccinia Ankara eradicates solid tumors and promotes protective immune memory.

Background Human cancers are extraordinarily heterogeneous in terms of tumor antigen expression, immune infiltration and composition. A common feature, however, is the host's inability to mount potent immune responses that prevent tumor growth effectively. Often, naturally primed CD8+ T cells against solid tumors lack adequate stimulation and efficient tumor tissue penetration due to an immune hostile tumor microenvironment.Methods To address these shortcomings, we cloned tumor-associated antigens (TAA) and the immune-stimulatory ligand 4-1BBL into the genome of modified vaccinia Ankara (MVA) for intratumoral virotherapy.Results Local treatment with MVA-TAA-4-1BBL resulted in control of established tumors. Intratumoral injection of MVA localized mainly to the tumor with minimal leakage to the tumor-draining lymph node. In situ infection by MVA-TAA-4-1BBL triggered profound changes in the tumor microenvironment, including the induction of multiple proinflammatory molecules and immunogenic cell death. These changes led to the reactivation and expansion of antigen-experienced, tumor-specific cytotoxic CD8+ T cells that were essential for the therapeutic antitumor effect. Strikingly, we report the induction of a systemic antitumor immune response including tumor antigen spread by local MVA-TAA-4-1BBL treatment which controlled tumor growth at distant, untreated lesions and protected against local and systemic tumor rechallenge. In all cases, 4-1BBL adjuvanted MVA was superior to MVA.Conclusion Intratumoral 4-1BBL-armed MVA immunotherapy induced a profound reactivation and expansion of potent tumor-specific CD8+ T cells as well as favorable proinflammatory changes in the tumor microenvironment, leading to elimination of tumors and protective immunological memory.

Myxoma virus expressing interleukin-15 fails to cause lethal myxomatosis in European rabbits.

Myxoma virus (MYXV) is a poxvirus pathogenic only for European rabbits, but its permissiveness in human cancer cells gives it potential as an oncolytic virus. A recombinant MYXV expressing both the tdTomato red fluorescent protein and interleukin-15 (IL-15) (vMyx-IL-15-tdTr) was constructed. Cells infected with vMyx-IL-15-tdTr secreted bioactive IL-15 and had in vitro replication kinetics similar to that of wild-type MYXV. To determine the safety of this virus for future oncolytic studies, we tested its pathogenesis in European rabbits. In vivo, vMyx-IL-15-tdTr no longer causes lethal myxomatosis. Thus, ectopic IL-15 functions as an antiviral cytokine in vivo, and vMyx-IL-15-tdTr is a safe candidate for animal studies of oncolytic virotherapy.

Evaluation of modified Vaccinia Ankara-based vaccines against foot-and-mouth disease serotype A24 in cattle.

To prepare foot-and-mouth disease (FMD) recombinant vaccines in response to newly emerging FMD virus (FMDV) field strains, we evaluated Modified Vaccinia virus Ankara-Bavarian Nordic (MVA-BN®) as an FMD vaccine vector platform. The MVA-BN vector has the capacity to carry and express numerous foreign genes and thereby has the potential to encode antigens from multiple FMDV strains. Moreover, this vector has an extensive safety record in humans. All MVA-BN-FMD constructs expressed the FMDV A24 Cruzeiro P1 capsid polyprotein as antigen and the FMDV 3C protease required for processing of the polyprotein. Because the FMDV wild-type 3C protease is detrimental to mammalian cells, one of four FMDV 3C protease variants were utilized: wild-type, or one of three previously reported mutants intended to dampen protease activity (C142T, C142L) or to increase specificity and thereby reduce adverse effects (L127P). These 3C coding sequences were expressed under the control of different promoters selected to reduce 3C protease expression. Four MVA-BN-FMD constructs were evaluated in vitro for acceptable vector stability, FMDV P1 polyprotein expression, processing, and the potential for vaccine scale-up production. Two MVA-BN FMD constructs met the in vitro selection criteria to qualify for clinical studies: MVA-mBN360B (carrying a C142T mutant 3C protease and an HIV frameshift for reduced expression) and MVA-mBN386B (carrying a L127P mutant 3C protease). Both vaccines were safe in cattle and elicited low to moderate serum neutralization titers to FMDV following multiple dose administrations. Following FMDV homologous challenge, both vaccines conferred 100% protection against clinical FMD and viremia using single dose or prime-boost immunization regimens. The MVA-BN FMD vaccine platform was capable of differentiating infected from vaccinated animals (DIVA). The demonstration of the successful application of MVA-BN as an FMD vaccine vector provides a platform for further FMD vaccine development against more epidemiologically relevant FMDV strains.

Aromatic amino acids in the juxtamembrane domain of severe acute respiratory syndrome coronavirus spike glycoprotein are important for receptor-dependent virus entry and cell-cell fusion.

The severe acute respiratory syndrome coronavirus (SARS-CoV) spike glycoprotein (S) is a class I viral fusion protein that binds to its receptor glycoprotein, human angiotensin converting enzyme 2 (hACE2), and mediates virus entry and cell-cell fusion. The juxtamembrane domain (JMD) of S is an aromatic amino acid-rich region proximal to the transmembrane domain that is highly conserved in all coronaviruses. Alanine substitutions for one or two of the six aromatic residues in the JMD did not alter the surface expression of the SARS-CoV S proteins with a deletion of the C-terminal 19 amino acids (S Delta19) or reduce binding to soluble human ACE2 (hACE2). However, hACE2-dependent entry of trypsin-treated retrovirus pseudotyped viruses expressing JMD mutant S Delta19 proteins was greatly reduced. Single alanine substitutions for aromatic residues reduced entry to 10 to 60% of the wild-type level. The greatest reduction was caused by residues nearest the transmembrane domain. Four double alanine substitutions reduced entry to 5 to 10% of the wild-type level. Rapid hACE2-dependent S-mediated cell-cell fusion was reduced to 60 to 70% of the wild-type level for all single alanine substitutions and the Y1188A/Y1191A protein. S Delta19 proteins with other double alanine substitutions reduced cell-cell fusion further, from 40% to less than 20% of wild-type levels. The aromatic amino acids in the JMD of the SARS-CoV S glycoprotein play critical roles in receptor-dependent virus-cell and cell-cell fusion. Because the JMD is so highly conserved in all coronavirus S proteins, it is a potential target for development of drugs that may inhibit virus entry and/or cell-cell fusion mediated by S proteins of all coronaviruses.

Synergistic cancer immunotherapy combines MVA-CD40L induced innate and adaptive immunity with tumor targeting antibodies.

Virus-based vaccines and appropriate costimulation potently enhance antigen-specific T cell immunity against cancer. Here we report the use of recombinant modified vaccinia virus Ankara (rMVA) encoding costimulatory CD40L against solid tumors. Therapeutic treatment with rMVA-CD40L-expressing tumor-associated antigens results in the control of established tumors. The expansion of tumor-specific cytotoxic CD8+ T cells is essential for the therapeutic antitumor effects. Strikingly, rMVA-CD40L also induces strong natural killer (NK) cell activation and expansion. Moreover, the combination of rMVA-CD40L and tumor-targeting antibodies results in increased therapeutic antitumor efficacy relying on the presence of Fc receptor and NK cells. We describe a translationally relevant therapeutic synergy between systemic viral vaccination and CD40L costimulation. We show strengthened antitumor immune responses when both rMVA-CD40L-induced innate and adaptive immune mechanisms are exploited by combination with tumor-targeting antibodies. This immunotherapeutic approach could translate into clinical cancer therapies where tumor-targeting antibodies are employed.

A novel naturally occurring tandem promoter in modified vaccinia virus ankara drives very early gene expression and potent immune responses.

Modified vaccinia virus Ankara (MVA) has been shown to be suitable for the generation of experimental vaccines against cancer and infectious diseases, eliciting strong humoral and cellular immune responses. In viral vectored vaccines, strong recombinant antigen expression and timing of expression influence the quantity and quality of the immune response. Screening of synthetic and native poxvirus promoters for strong protein expression in vitro and potent immune responses in vivo led to the identification of the MVA13.5L promoter, a unique and novel naturally occurring tandem promoter in MVA composed of two 44 nucleotide long repeated motifs, each containing an early promoter element. The MVA13.5L gene is highly conserved across orthopoxviruses, yet its function is unknown. The unique structure of its promoter is not found for any other gene in the MVA genome and is also conserved in other orthopoxviruses. Comparison of the MVA13.5L promoter activity with synthetic poxviral promoters revealed that the MVA13.5L promoter produced higher levels of protein early during infection in HeLa cells and particularly in MDBK cells, a cell line in which MVA replication stops at an early stage before the expression of late genes. Finally, a recombinant antigen expressed under the control of this novel promoter induced high antibody titers and increased CD8 T cell responses in homologous prime-boost immunization compared to commonly used promoters. In particular, the recombinant antigen specific CD8 T cell responses dominated over the immunodominant B8R vector-specific responses after three vaccinations and even more during the memory phase. These results have identified the native MVA13.5L promoter as a new potent promoter for use in MVA vectored preventive and therapeutic vaccines.

Bugs and drugs: oncolytic virotherapy in combination with chemotherapy.

Single agent therapies are rarely successful in treating cancer, particularly at metastatic or end stages, and survival rates with monotherapies alone are generally poor. The combination of multiple therapies to treat cancer has already driven significant improvements in the standard of care treatments for many types of cancers. The first combination treatments exploited for cancer therapy involved the use of several cytotoxic chemotherapy agents. Later, with the development of more targeted agents, the use of novel, less toxic drugs, in combination with the more classic cytotoxic drugs has proven advantageous for certain cancer types. Recently, the combination of oncolytic virotherapy with chemotherapy has shown that the use of these two therapies with very distinct anti-tumor mechanisms may also lead to synergistic interactions that ultimately result in increased therapeutic effects not achievable by either therapy alone. The mechanisms of synergy between oncolytic viruses (OVs) and chemotherapeutic agents are just starting to be elucidated. It is evident, however, that the success of these OV-drug combinations depends greatly on the particular OV, the drug(s) selected, and the cancer type targeted. This review summarizes the different OV-drug combinations investigated to date, including the use of second generation armed OVs, which have been studied with the specific purpose of generating synergistic interactions with particular chemotherapy agents. The known mechanisms of synergy between these OV-drug combinations are also summarized. The importance of further investigating these mechanisms of synergy will be critical in order to maximize the therapeutic efficacy of OV-drug combination therapies in the future.

The immunoregulatory properties of oncolytic myxoma virus and their implications in therapeutics.

Myxoma virus (MYXV) is a poxvirus with a strict rabbit-specific host-tropism for pathogenesis. The immunoregulatory factors encoded by MYXV can suppress some functions of immune effectors from other species. We review their mechanisms of action, implications in therapeutics and the potential to improve MYXV as an oncolytic agent in humans.