Methods for the Construction of Recombinant Oncolytic Myxoma Viruses.

Myxoma virus (MYXV) has proven to be an effective candidate for oncolytic virotherapy in many preclinical cancer models. As a nonhuman pathogen, MYXV does not need to overcome any preexisting antiviral immunity, and its DNA cannot integrate into the host genome, making it an extremely safe vector. Moreover, the large dsDNA genome of MYXV allows the insertion of multiple transgenes and the design of engineered recombinant oncolytic viruses (OVs) with enhanced immunostimulatory or other desired properties. In this chapter, we describe detailed protocols for the generation and characterization of transgene-armed recombinant MYXV vectors.

Poxvirus oncolytic virotherapy.

INTRODUCTION: Over the last decade, advances in biological therapies have resulted in remarkable clinical responses for the treatment of some previously incurable cancers. Oncolytic virotherapy is one of these promising novel strategies for cancer therapy. A successful oncolytic virus promotes tumor cell oncolysis and elicits a robust long-term anti-tumor immunity. AREAS COVERED: Oncolytic poxviruses (Vaccinia virus and Myxoma virus) demonstrated encouraging results in multiple pre-clinical tumor models and some clinical trials for the treatment of various cancers. This review summarizes the advances made on poxvirus oncolytic virotherapy in the last five years. EXPERT OPINION: Many challenges remain in poxvirus oncolytic virotherapy. Two key goals to achieve are enhancing the efficiency of viral delivery to tumor sites and overcoming local tumor immune-evasion. Additional efforts are necessary to explore the best combination of virotherapy with standard available treatments, particularly immunotherapies. By addressing these issues, this new modality will continue to improve as an adjunct biotherapy to treat malignant diseases.

CCL2 expression is mediated by type I IFN receptor and recruits NK and T cells to the lung during MVA infection.

Migration of leukocytes to the site of microbial infection is important for the development of effective host immunity. Recombinant modified vaccinia virus Ankara is frequently used as a viral vector vaccine in preclinical and clinical studies. In comparison to other vaccinia virus strains, modified vaccinia virus Ankara robustly induces chemokine expression and rapid attraction of leukocytes. In particular, chemokine (C-C motif) ligand 2 (CCL2) has been shown to be critical for leukocyte recruitment to the lung. In this study, MVA-induced CCL2 expression in murine macrophages was dependent on type I interferon receptor and not Toll-like receptor-2. The critical role of type I interferon receptor signaling for CCL2 production in the lung was confirmed in type I interferon receptor-deficient mice (Ifnar1(-/-)). In addition, comparing Ifnar1(-/-) and Ccl2(-/-) mice with wild-type mice, we observed a similar impairment in the recruitment of natural killer and T cells to the lung after intranasal infection with modified vaccinia virus Ankara. Conversely, neutrophil recruitment was not affected in Ifnar1(-/-) and Ccl2(-/-) mice. We conclude that type I interferons, besides their known antiviral properties, can initiate the recruitment and activation of leukocytes via induction of chemokine expression including CCL2.

Chemokine (C-C Motif) receptor 1 is required for efficient recruitment of neutrophils during respiratory infection with modified vaccinia virus Ankara.

UNLABELLED: Modified vaccinia virus Ankara (MVA) serves as a versatile platform in vaccine development. This highly attenuated orthopoxvirus, which cannot replicate in mammalian cells, triggers strong innate immune responses, including cell migration. Previously, we have shown that induction of chemokine (C-C motif) ligand 2 (CCL2) by MVA is necessary for the recruitment of monocytes and T cells, but not neutrophils, to the lung. Here, we identified neutrophil-attracting chemokines produced by MVA-infected primary murine lung fibroblasts and murine bone marrow-derived macrophages. We demonstrate that MVA, but not vaccinia virus (VACV) strain WR, induces chemokine expression, which is independent of Toll-like receptor 2 (TLR2) signaling. Additionally, we show that both chemokine (C-C motif) receptor 1 (CCR1) and chemokine (C-X-C motif) receptor 2 (CXCR2) are involved in MVA-induced neutrophil chemotaxis in vitro. Finally, intranasal infection of Ccr1(-/-) mice with MVA, as well as application of the CCR1 antagonist J-113863, revealed a role for CCR1 in leukocyte recruitment, including neutrophils, into the lung. IMPORTANCE: Rapid attraction of leukocytes to the site of inoculation is unique to MVA in comparison to other VACV strains. The findings here extend current knowledge about the regulation of MVA-induced leukocyte migration, particularly regarding neutrophils, which could potentially be exploited to improve other VACV strains currently in development as oncolytic viruses and viral vectors. Additionally, the data presented here indicate that the inflammatory response may vary depending on the cell type infected by MVA, highlighting the importance of the site of vaccine application. Moreover, the rapid recruitment of neutrophils and other leukocytes can directly contribute to the induction of adaptive immune responses elicited by MVA inoculation. Thus, a better understanding of leukocyte migration upon MVA infection is particularly relevant for further development and use of MVA-based vaccines and vectors.

Modified Vaccinia virus Ankara: innate immune activation and induction of cellular signalling.

Attenuated poxviruses are currently under development as vaccine vectors against a number of diseases including, influenza, HIV, malaria and tuberculosis. Modified Vaccinia virus Ankara (MVA) is an attenuated, replication deficient vaccinia virus (VACV) strain which, similar to replication competent VACV, is highly immunogenic. The lack of productive viral replication further improves the safety profile of MVA as a vector, minimizing the potential for reversion to virulent forms particularly if used in immunocompromised individuals. Despite its inability to replicate in most mammalian cells, MVA still efficiently expresses viral and recombinant genes making it a potent antigen delivery platform. Moreover, due to the loss of various immunomodulatory factors MVA infection leads to rapid local immune responses, fulfilling a requirement of an adjuvant. In this review we take a look at the immunostimulatory properties of MVA, paying particular attention to the signalling of the innate immune system in response to MVA and VACV infection. Understanding the cellular and molecular mechanisms modulated by VACV will help in the future design and engineering of new vaccines and may provide insight into previously unknown mechanisms of dominant virus-host interactions.