Ex vivo virotherapy with myxoma virus does not impair hematopoietic stem and progenitor cells.

BACKGROUND: Relapsing disease is a major challenge after hematopoietic cell transplantation for hematological malignancies. Myxoma virus (MYXV) is an oncolytic virus that can target and eliminate contaminating cancer cells from auto-transplant grafts. The aims of this study were to examine the impact of MYXV on normal hematopoietic stem and progenitor cells and define the optimal treatment conditions for ex vivo virotherapy. METHODS: Bone marrow (BM) and mobilized peripheral blood stem cells (mPBSCs) from patients with hematologic malignancies were treated with MYXV at various time, temperature and incubation media conditions. Treated BM cells from healthy normal donors were evaluated using flow cytometry for MYXV infection, long-term culture-initiating cell (LTC-IC) assay and colony-forming cell (CFC) assay. RESULTS: MYXV initiated infection in up to 45% of antigen-presenting monocytes, B cells and natural killer cells; however, these infections were uniformly aborted in >95% of all cells. Fresh graft sources showed higher levels of MYXV infection initiation than cryopreserved specimens, but in all cases less than 10% of CD34(+) cells could be infected after ex vivo MYXV treatment. MYXV did not impair LTC-IC colony numbers compared with mock treatment. CFC colony types and numbers were also not impaired by MYXV treatment. MYXV incubation time, temperature or culture media did not significantly change the percentage of infected cells, LTC-IC colony formation or CFC colony formation. CONCLUSIONS: Human hematopoietic cells are non-permissive for MYXV. Human hematopoietic stem and progenitor cells were not infected and thus unaffected by MYXV ex vivo treatment.

Oncolytic Poxviruses.

Current standard treatments of cancer can prolong survival of many cancer patients but usually do not effectively cure the disease. Oncolytic virotherapy is an emerging therapeutic for the treatment of cancer that exploits replication-competent viruses to selectively infect and destroy cancerous cells while sparing normal cells and tissues. Clinical and/or preclinical studies on oncolytic viruses have revealed that the candidate viruses being tested in trials are remarkably safe and offer potential for treating many classes of currently incurable cancers. Among these candidates are vaccinia and myxoma viruses, which belong to the family Poxviridae and possess promising oncolytic features. This article describes poxviruses that are being developed for oncolytic virotherapy and summarizes the outcomes of both clinical and preclinical studies. Additionally, studies demonstrating superior efficacy when poxvirus oncolytic virotherapy is combined with conventional therapies are described.

Myxoma virus protein M029 is a dual function immunomodulator that inhibits PKR and also conscripts RHA/DHX9 to promote expanded host tropism and viral replication.

Myxoma virus (MYXV)-encoded protein M029 is a member of the poxvirus E3 family of dsRNA-binding proteins that antagonize the cellular interferon signaling pathways. In order to investigate additional functions of M029, we have constructed a series of targeted M029-minus (vMyx-M029KO and vMyx-M029ID) and V5-tagged M029 MYXV. We found that M029 plays a pivotal role in determining the cellular tropism of MYXV in all mammalian cells tested. The M029-minus viruses were able to replicate only in engineered cell lines that stably express a complementing protein, such as vaccinia E3, but underwent abortive or abated infection in all other tested mammalian cell lines. The M029-minus viruses were dramatically attenuated in susceptible host European rabbits and caused no observable signs of myxomatosis. Using V5-tagged M029 virus, we observed that M029 expressed as an early viral protein is localized in both the nuclear and cytosolic compartments in virus-infected cells, and is also incorporated into virions. Using proteomic approaches, we have identified Protein Kinase R (PKR) and RNA helicase A (RHA)/DHX9 as two cellular binding partners of M029 protein. In virus-infected cells, M029 interacts with PKR in a dsRNA-dependent manner, while binding with DHX9 was not dependent on dsRNA. Significantly, PKR knockdown in human cells rescued the replication defect of the M029-knockout viruses. Unexpectedly, this rescue of M029-minus virus replication by PKR depletion could then be reversed by RHA/DHX9 knockdown in human monocytic THP1 cells. This indicates that M029 not only inhibits generic PKR anti-viral pathways, but also binds and conscripts RHA/DHX9 as a pro-viral effector to promote virus replication in THP1 cells. Thus, M029 is a critical host range and virulence factor for MYXV that is required for replication in all mammalian cells by antagonizing PKR-mediated anti-viral functions, and also conscripts pro-viral RHA/DHX9 to promote viral replication specifically in myeloid cells.

Oncolytic myxoma virus: the path to clinic.

Many common neoplasms are still noncurative with current standards of cancer therapy. More therapeutic modalities need to be developed to significantly prolong the lives of patients and eventually cure a wider spectrum of cancers. Oncolytic virotherapy is one of the promising new additions to clinical cancer therapeutics. Successful oncolytic virotherapy in the clinic will be those strategies that best combine tumor cell oncolysis with enhanced immune responses against tumor antigens. The current candidate oncolytic viruses all share the common property that they are relatively nonpathogenic to humans, yet they have the ability to replicate selectively in human cancer cells and induce cancer regression by direct oncolysis and/or induction of improved anti-tumor immune responses. Many candidate oncolytic viruses are in various stages of clinical and preclinical development. One such preclinical candidate is myxoma virus (MYXV), a member of the Poxviridae family that, in its natural setting, exhibits a very restricted host range and is only pathogenic to European rabbits. Despite its narrow host range in nature, MYXV has been shown to productively infect various classes of human cancer cells. Several preclinical in vivo modeling studies have demonstrated that MYXV is an attractive and safe candidate oncolytic virus, and hence, MYXV is currently being developed as a potential therapeutic for several cancers, such as pancreatic cancer, glioblastoma, ovarian cancer, melanoma, and hematologic malignancies. This review highlights the preclinical cancer models that have shown the most promise for translation of MYXV into human clinical trials.

Myxoma and vaccinia viruses bind differentially to human leukocytes.

Myxoma virus (MYXV) and vaccinia virus (VACV), two distinct members of the family Poxviridae, are both currently being developed as oncolytic virotherapeutic agents. Recent studies have demonstrated that ex vivo treatment with MYXV can selectively recognize and kill contaminating cancerous cells from autologous bone marrow transplants without perturbing the engraftment of normal CD34(+) hematopoietic stem and progenitor cells. However, the mechanism(s) by which MYXV specifically recognizes and eliminates the cancer cells in the autografts is not understood. While little is known about the cellular attachment factor(s) exploited by MYXV for entry into any target cells, VACV has been shown to utilize cell surface glycosaminoglycans such as heparan sulfate (HS), the extracellular matrix protein laminin, and/or integrin ß1. We have constructed MYXV and VACV virions tagged with the Venus fluorescent protein and compared their characteristics of binding to various human cancer cell lines as well as to primary human leukocytes. We report that the binding of MYXV or VACV to some adherent cell lines could be partially inhibited by heparin, but laminin blocked only VACV binding. In contrast to cultured fibroblasts, the binding of MYXV and VACV to a wide spectrum of primary human leukocytes could not be competed by either HS or laminin. Additionally, MYXV and VACV exhibited very different binding characteristics against certain select human leukocytes, suggesting that the two poxviruses utilize different cell surface determinants for the attachment to these cells. These results indicate that VACV and MYXV can exhibit very different oncolytic tropisms against some cancerous human leukocytes.

The A33-dependent incorporation of B5 into extracellular enveloped vaccinia virions is mediated through an interaction between their lumenal domains.

There are two mechanisms for the incorporation of B5 into the envelope of extracellular virions produced by orthopoxviruses, one that requires A33 and one that does not. We have hypothesized that the A33-dependent mechanism requires a direct interaction between A33 and B5. In this study, chimeric constructs of A33 and B5/B5-green fluorescent protein (GFP) were used to show that the two proteins interact through their lumenal domains and that the coiled-coil domain of B5 is sufficient for an interaction with A33. Furthermore, our experiments reveal that a transmembrane domain, not necessarily its own, is requisite for the lumenal domain of B5 to interact with A33. In contrast, the lumenal domain of A33 is sufficient for interaction with B5. Furthermore, the lumenal domain of A33 is sufficient to restore the proper localization of B5-GFP in infected cells. Taken together, our results demonstrate that the lumenal domains of A33 and B5 interact and that the interaction is required for the incorporation of B5-GFP into extracellular virions, whereas the incorporation of A33 is independent of B5. These results suggest that viral protein incorporation into extracellular virions is an active process requiring specific protein-protein interactions.

Increased interaction between vaccinia virus proteins A33 and B5 is detrimental to infectious extracellular enveloped virion production.

Two mechanisms exist for the incorporation of B5 into extracellular virions, one of which is dependent on A33. In the companion to this paper (W. M. Chan and B. M. Ward, J. Virol. 86:8210-8220, 2012), we show that the lumenal domain of A33 is sufficient for interaction with the coiled-coil domain of B5 and capable of directing B5-green fluorescent protein (GFP) into extracellular virions. Here, we have created a panel of charge-to-alanine mutations in the lumenal domain of A33 to map the B5 interaction site. While none of these mutations abolished the interaction with B5, a subset displayed an increased interaction with both B5 and B5-GFP. Both B5 and B5-GFP recombinant viruses expressing these mutant proteins in place of normal A33 had a small-plaque phenotype. The increased interaction of the mutant proteins was detected during infection, suggesting that normally the interaction is either weak or transient. In addition, the increased A33-B5 interaction was detected on virions produced by recombinant viruses and correlated with reduced target cell binding. Taken together, these results show that both B5 and B5-GFP interact with A33 during infection and that the duration of this interaction needs to be regulated for the production of fully infectious extracellular virions.

Selective purging of human multiple myeloma cells from autologous stem cell transplantation grafts using oncolytic myxoma virus.

Autologous stem cell transplantation and novel therapies have improved overall survival of patients with multiple myeloma; however, most patients relapse and eventually succumb to their disease. Evidence indicates that residual cancer cells contaminate autologous grafts and may contribute to early relapses after autologous stem cell transplantation. Here, we demonstrate that ex vivo treatment with an oncolytic poxvirus called myxoma virus results in specific elimination of human myeloma cells by inducing rapid cellular apoptosis while fully sparing normal hematopoietic stem and progenitor cells. The specificity of this elimination is based on strong binding of the virus to myeloma cells coupled with an inability of the virus to bind or infect CD34(+) hematopoietic stem and progenitor cells. These 2 features allow myxoma to readily identify and distinguish even low levels of myeloma cells in complex mixtures. This ex vivo rabbit-specific oncolytic poxvirus called myxoma virus treatment also effectively inhibits systemic in vivo engraftment of human myeloma cells into immunodeficient mice and results in efficient elimination of primary CD138(+) myeloma cells contaminating patient hematopoietic cell products. We conclude that ex vivo myxoma treatment represents a safe and effective method to selectively eliminate myeloma cells from hematopoietic autografts before reinfusion.

The inability of vaccinia virus A33R protein to form intermolecular disulfide-bonded homodimers does not affect the production of infectious extracellular virus.

The orthopoxvirus protein A33 forms a disulfide-bonded high molecular weight species that could be either a homodimer or a heteromultimer. The protein is a major target for neutralizing antibodies and the majority of antibodies raised against A33 only recognize the disulfide-bonded form. Here, we report that A33 is present as a disulfide-bonded homodimer during infection. Additionally, we examined the function of intermolecular disulfide bonding in A33 homodimerization during infection. We show that the cysteine at amino acid 62 is required for intermolecular disulfide bonding, but not dimerization as this mutant was still able to form homodimers. To investigate the role of disulfide-bonded homodimers during viral morphogenesis, recombinant viruses that express an A33R with cysteine 62 mutated to serine were generated. The recombinant viruses had growth characteristics similar to their parental viruses, indicating that intermolecular disulfide-bonded homodimerization of A33 is not required for its function.

There is an A33-dependent mechanism for the incorporation of B5-GFP into vaccinia virus extracellular enveloped virions.

Orthopoxviruses produce two, antigenically distinct, infectious virions, intracellular mature virions and extracellular virions (EV). A33 and B5 are found on EV but not on intracellular mature virions. To investigate the function of A33, a recombinant virus that has A33R deleted and expresses B5R-GFP (vB5R-GFP/DeltaA33R) was generated. A comparison of vB5R-GFP/DeltaA33R to an analogous virus (vDeltaA33R) revealed an additional defect in infectious EV production that was not apparent when A33R was present. Characterization of these recombinants revealed that EV produced in the absence of A33 had undetectable levels of B5-GFP. Both recombinants released similar amounts of EV but there were differences in their infectivity. Approximately equal numbers of virions produced by these recombinants were able to bind cells even though EV produced by vB5R-GFP/DeltaA33R do not contain B5. These results suggest that in the absence of A33, the cytoplasmic tail of B5 contributes to its incorporation into the envelope of progeny virions.

Omental immune aggregates and tumor metastasis within the peritoneal cavity.

The omentum, an important peritoneal tissue, is studded with a high number of immune aggregates, or "milky spots," the number, function, and phenotype of which is largely unknown. We have analyzed the immune composition on the normal omentum and also have shown that both free immune cells and tumor cells in the peritoneal fluid bind preferentially to these immune aggregates. This binding may be mediated by the network of collagen I fibers, which overlay these areas. In addition, we have shown that not only do omental vessels express vascular endothelial growth factor receptor 3 (VEGFR3), a receptor that is only found on angiogenic blood vessels, but that tumor cells co-localize with these vessels, possibly increasing the ability of tumor to induce neovascularization and therefore thrive.

The vaccinia virus B5 protein requires A34 for efficient intracellular trafficking from the endoplasmic reticulum to the site of wrapping and incorporation into progeny virions.

The glycoproteins encoded by the vaccinia virus A34R and B5R genes are involved in intracellular envelope virus formation and are highly conserved among orthopoxviruses. A recombinant virus that has the A34R gene deleted and the B5R gene replaced with a B5R gene fused to the enhanced green fluorescent protein (B5R-GFP) gene was created (vB5R-GFP/DeltaA34R) to investigate the role of A34 during virion morphogenesis. Cells infected with vB5R-GFP/DeltaA34R displayed GFP fluorescence throughout the cytoplasm, which differed markedly from that seen in cells infected with a normal B5R-GFP-expressing virus (vB5R-GFP). Immunofluorescence and subcellular fractionation demonstrated that B5-GFP localizes with the endoplasmic reticulum in the absence of A34. Expression of either full-length A34 or a construct consisting of the lumenal and transmembrane domains restored normal trafficking of B5-GFP to the site of wrapping in the juxtanuclear region. Coimmunoprecipitation studies confirmed that B5 and A34 interact through their luminal domains, and further analysis revealed that in the absence of A34, B5 is not efficiently incorporated into virions released from the cell.