Recombinant Myxoma Virus Research Articles

Myxoma virus expressing a fusion protein of interleukin-15 (IL15) and IL15 receptor alpha has enhanced antitumor activity.

Myxoma virus, a rabbit poxvirus, can efficiently infect various types of mouse and human cancer cells. It is a strict rabbit-specific pathogen, and is thought to be safe as a therapeutic agent in all non-rabbit hosts tested including mice and humans. Interleukin-15 (IL15) is an immuno-modulatory cytokine with significant potential for stimulating anti-tumor T lymphocytes and NK cells. Co-expression of IL15 with the α subunit of IL15 receptor (IL15Rα) greatly enhances IL15 stability and bioavailability. Therefore, we engineered a new recombinant myxoma virus (vMyx-IL15Rα-tdTr), which expresses an IL15Rα-IL15 fusion protein plus tdTomato red fluorescent reporter protein. Permissive rabbit kidney epithelial (RK-13) cells infected with vMyx-IL15Rα-tdTr expressed and secreted the IL15Rα-IL15 fusion protein. Functional activity was confirmed by demonstrating that the secreted fusion protein stimulated proliferation of cytokine-dependent CTLL-2 cells. Multi-step growth curves showed that murine melanoma (B16-F10 and B16.SIY) cell lines were permissive to vMyx-IL15Rα-tdTr infection. In vivo experiments in RAG1-/- mice showed that subcutaneous B16-F10 tumors treated with vMyx-IL15Rα-tdTr exhibited attenuated tumor growth and a significant survival benefit for the treated group compared to the PBS control and the control viruses (vMyx-IL15-tdTr and vMyx-tdTr). Immunohistological analysis of the subcutaneous tumors showed dramatically increased infiltration of NK cells in vMyx-IL15Rα-tdTr treated tumors compared to the controls. In vivo experiments with immunocompetent C57BL/6 mice revealed a strong infiltrate of both NK cells and CD8+ T cells in response to vMyx-IL15Rα-tdTr, and prolonged survival. We conclude that delivery of IL15Rα-IL15 in a myxoma virus vector stimulates both innate and adaptive components of the immune system.

Recombinant myxoma virus lacking all poxvirus ankyrin-repeat proteins stimulates multiple cellular anti-viral pathways and exhibits a severe decrease in virulence

Although the production of single gene knockout viruses is a useful strategy to study viral gene functions, the redundancy of many host interactive genes within a complex viral genome can obscure their collective functions. In this study, a rabbit-specific poxvirus, myxoma virus (MYXV), was genetically altered to disrupt multiple members of the poxviral ankyrin-repeat (ANK-R) protein superfamily, M-T5, M148, M149 and M150. A particularly robust activation of the NF-κB pathway was observed in A549 cells following infection with the complete ANK-R knockout (vMyx-ANKsKO). Also, an increased release of IL-6 was only observed upon infection with vMyx-ANKsKO. In virus-infected rabbit studies, vMyx-ANKsKO was the most extensively attenuated and produced the smallest primary lesion of all ANK-R mutant constructs. This study provides the first insights into the shared functions of the poxviral ANK-R protein superfamily in vitro and in vivo.

An Interleukin‐15 Fusion Protein Enhances the Oncolytic Effects of Myxoma Virus in a Murine Melanoma Model

Myxoma virus (MYXV) has potential as an oncolytic virus, but is not fully effective as a single‐agent therapy. We hypothesized that intratumoral (IT) expression of interleukin‐15 (IL‐15) by MYXV would recruit cytotoxic effector cells and improve treatment efficacy. To test this, MYXV expressing a fluorescent protein (MYXV‐Tred) and MYXV‐Tred encoding murine IL‐15 (MYXV‐IL15) were evaluated in a murine melanoma model. C57/BL6 mice that received weekly IT injections of virus lived significantly longer than controls. Unexpectedly, the median survival time of mice treated with MYXV‐IL15 was similar to MYXV‐Tred. In MYXV‐IL15‐treated tumors, virus titer, IL‐15 concentration, lymphocyte grade, and CD3+ cell counts were elevated at 4 days post‐inoculation (dpi), but decreased at 7 dpi. In subsequent experiments, recombinant MYXV was injected IT every 3 days. Additionally, to further enhance IL‐15 functions, MYXV‐Tred was modified to express IL‐15 fused with IL‐15 receptor alpha (MYXV‐RLI). The median survival time and number of IT Ly‐49+ cells were significantly increased in RAG mice bearing melanomas treated with MYXV‐RLI compared with MYXV‐IL15. These findings suggest that IT expression of an IL‐15/IL‐15Rα fusion protein enhances the oncolytic effects of MYXV.

Histological evaluation of intratumoral myxoma virus treatment in an immunocompetent mouse model of melanoma.

Two recombinant myxoma viruses (MYXV expressing a fluorescent protein [MYXV-Tred] and MYXV-Tred encoding murine interleukin-15 [MYXV-IL15]) were evaluated for therapeutic effects in an aggressive B16F10 melanoma model in immunocompetent mice. It was hypothesized that continuous expression of IL-15 within a tumor would recruit cytotoxic effector cells to induce an antitumor immune response and improve treatment efficacy. Weekly intratumoral injections were given to evaluate the effect of treatment on the median survival time of C57BL/6 mice bearing established B16F10 melanomas. Mice that received MYXV-Tred or MYXV-IL15 lived significantly longer than mice given treatment controls. Unexpectedly, the median survival time of MYXV-IL15-treated mice was similar to that of MYXV-treated mice. At 1, 2, and 4 days postinoculation, viral plaque assays detected replicating MYXV-Tred and MYXV-IL15 within treated tumors. At these time points in MYXV-IL15-treated tumors, IL-15 concentration, lymphocyte grades, and cluster of differentiation-3+ cell counts were significantly increased when compared to other treatment groups. However, viral titers, recombinant protein expression, and lymphocyte numbers within the tumors diminished rapidly at 7 days postinoculation. These data indicate that treatment with recombinant MYXV should be repeated at least every 4 days to maintain recombinant protein expression within a murine tumor. Additionally, neutrophilic inflammation was significantly increased in MYXV-Tred- and MYXV-IL15-treated tumors at early time points. It is speculated that neutrophilic inflammation induced by intratumoral replication of recombinant MXYV contributes to the antitumoral effect of MYXV treatment in this melanoma model. These findings support the inclusion of neutrophil chemotaxins in recombinant poxvirus oncolytic virotherapy.

Myxomavirus as a vector for the immunisation of sheep: Protection study against challenge with bluetongue virus

Recombinant poxviruses are well suited for the development of new vaccine vectors. Our previous data supported the idea that Myxomavirus (MYXV) is efficient at priming antibody responses in sheep. To provide definitive evidence on the potential of MYXV for vaccination against infectious diseases in ruminants, we investigated the immune protection provided by recombinant MYXV against bluetongue, a devastating disease in sheep. To test this concept, sheep were injected twice with an MYXV expressing the immunodominant VP2 protein (SG33-VP2). The SG33-VP2 vector promoted the production of neutralising antibodies and partially protected sheep against disease after challenge with a highly virulent strain of serotype-8 bluetongue virus (BTV-8). In contrast, an MYXV expressing both VP2 and VP5 proteins (SG33-VP2/5) elicited very little protection. The expression levels of the VP2 and VP5 proteins suggested that, greater than the co-expression of the VP5 protein which was previously thought to favour anti-VP2 antibody response, the high expression of VP2 may be critical in the MYXV context to stimulate a protective response in sheep. This highlights the requirement for a careful examination of antigen expression before any conclusion can be drawn on the respective role of the protective antigens. As a proof of principle, our study shows that an MYXV vaccine vector is possible in ruminants.

Infection of Nonhost Species Dendritic CellsIn Vitrowith an Attenuated Myxoma Virus Induces Gene Expression That Predicts Its Efficacy as a Vaccine Vector

Recombinant myxoma virus (MYXV) can be produced without a loss of infectivity, and its highly specific host range makes it an ideal vaccine vector candidate, although careful examination of its interaction with the immune system is necessary. Similar to rabbit bone marrow-derived dendritic cells (BM-DCs), ovine dendritic cells can be infected by SG33, a MYXV vaccine strain, and support recombinant antigen expression. The frequency of infected cells in the nonhost was lower and the virus cycle was abortive in these cell types. Among BM-DC subpopulations, Langerhans cell-like DCs were preferentially infected at low multiplicities of infection. Interestingly, ovine BM-DCs remained susceptible to MYXV after maturation, although apoptosis occurred shortly after infection as a function of the virus titer. When gene expression was assessed in infected BM-DC cultures, type I interferon (IFN)-related and inflammatory genes were strongly upregulated. DC gene expression profiles were compared with the profiles produced by other poxviruses in interaction with DCs, but very few commonalities were found, although genes that were previously shown to predict vaccine efficacy were present. Collectively, these data support the idea that MYXV permits efficient priming of adaptive immune responses and should be considered a promising vaccine vector along with other poxviruses.

Safety and immunogenicity of myxoma virus as a new viral vector for small ruminants

Myxoma virus (MYXV), a leporide-specific poxvirus, represents an attractive candidate for the generation of safe and non-replicative vaccine vectors for other species. With the aim of developing new recombinant vaccines for ruminants, we evaluated the safety and the immunogenicity of recombinant MYXV in sheep. In vitro studies indicated that ovine primary fibroblasts were not permissive for MYXV and that infection of ovine peripheral blood mononuclear cells occurred at a low rate. Although non-specific activation significantly improved the susceptibility of lymphocytes, MYXV infection remained abortive. Histological and immunohistochemical examination at the inoculation sites revealed the development of an inflammatory process and allowed the detection of sparse infected cells in the dermis. In addition, inoculated sheep developed an antibody response directed against MYXV and the product of the transgene. Overall, these results provide the first line of evidence on the potential of MYXV as a viral vector for ruminants.

Myxoma Virus Oncolysis of Primary and Metastatic B16F10 Mouse Tumors In Vivo

Myxoma virus (MV) is a rabbit-specific poxvirus, whose unexpected tropism to human cancer cells has led to studies exploring its potential use in oncolytic therapy. MV infects a wide range of human cancer cells in vitro, in a manner intricately linked to the cellular activation of Akt kinase. MV has also been successfully used for treating human glioma xenografts in immunodeficient mice. This study examines the effectiveness of MV in treating primary and metastatic mouse tumors in immunocompetent C57BL6 mice. We have found that several mouse tumor cell lines, including B16 melanomas, are permissive to MV infection. B16F10 cells were used for assessing MV replication and efficacy in syngeneic primary tumor and metastatic models in vivo. Multiple intratumoral injections of MV resulted in dramatic inhibition of tumor growth. Systemic administration of MV in a lung metastasis model with B16F10LacZ cells was dramatically effective in reducing lung tumor burden. Combination therapy of MV with rapamycin reduced both size and number of lung metastases, and also reduced the induced antiviral neutralizing antibody titres, but did not affect tumor tropism. These results show MV to be a promising virotherapeutic agent in immunocompetent animal tumor models, with good efficacy in combination with rapamycin.

Prospects for fertility control in the European rabbit (Oryctolagus cuniculus) using myxoma virus-vectored immunocontraception

Research over the last 15 years has examined whether fertility control can reduce overabundant rabbit populations and whether an effective immunocontraceptive agent can be developed and delivered. The results of this research indicate that for fertility control to have an environmental impact at least 80% of females will need to be infertile and that this infertility will need to be permanent. Epidemiological studies suggest that this level of infertility may be very difficult to obtain with a recombinant myxoma virus because of competition with field strains of virus. Research with laboratory rabbits using recombinant myxoma virus to deliver an immunocontraceptive antigen demonstrated that it was possible to obtain the required level of infertility using rabbit zona pellucida C as an antigen. However, only ~50% of animals remained infertile in the medium term. Further research on delivery vector and antigen selection would be needed to produce a practical immunocontraceptive virus for laboratory testing. Such a virus would then need to be optimised for transmissibility before it would be suitable for field testing.

Immunocontraceptive Effects on Female Rabbits Infected with Recombinant Myxoma Virus Expressing Rabbit ZP2 or ZP3

Recombinant myxoma viruses expressing rabbit zona pellucida 2 (rZP2) or rabbit zona pellucida 3 (rZP3) glycoproteins were constructed and tested in domestic rabbits to assess their potential to induce autoimmune infertility. The recombinant virus expressing rZP2 had no effect on fertility or ovarian histology, despite all animals developing antibodies against the rZP2 antigen. However, recombinant viruses expressing rZP3 induced infertility in 70% of animals at the first breeding. Serum antibodies were relatively short-lived, but antibody was bound to zona pellucida of all rabbits from Day 10 onward. There was no obvious correlation between infertility and rZP3 antibody titer. There was a transient inflammatory response in the ovaries of rZP3-immunized rabbits at Day 15 but no T-cell response to rZP3 could be detected at any time. Dysfunctional follicular formation was present in ovaries from rabbits infected with rZP3-expressing viruses 15-40 days postinfection but this had disappeared at later time points. A recombinant myxoma virus expressing a modified rZP3 antigen with the C-terminal hydrophobic putative anchor sequence deleted was also tested. This virus did not induce either infertility or an antibody response against the zona pellucida. Thus, the context of antigen presentation was crucial for an autoimmune response.

Potential for broad-spectrum protection against feline calicivirus using an attenuated myxoma virus expressing a chimeric FCV capsid protein

It has previously been demonstrated that recombinant myxoma viruses expressing FCV capsid protein are capable of eliciting protective responses against virulent FCV challenge, following vaccination, in cats. An attempt was made to produce a bivalent myxoma recombinant expressing the capsid protein genes of both FCV strains F9 and LS015. The FCV capsid protein genes were inserted into the myxoma growth factor gene (MGF) locus, and the serine protease inhibitor (SERP 2) gene locus. Subsequent recombination between myxoma-FCV viruses resulted in a recombinant expressing a chimeric form of the capsid protein. Nonetheless, cats immunised with this myxoma-FCV recombinant demonstrate high levels of serum neutralising antibodies against both F9 and LS015 strains. Such a chimeric vaccine may provide effective protection against a wide range of FCV strains.

Expression of rabbit IL-4 by recombinant myxoma viruses enhances virulence and overcomes genetic resistance to myxomatosis

Rabbit IL-4 was expressed in the virulent standard laboratory strain (SLS) and the attenuated Uriarra (Ur) strain of myxoma virus with the aim of creating a Th2 cytokine environment and inhibiting the development of an antiviral cell-mediated response to myxomatosis in infected rabbits. This allowed testing of a model for genetic resistance to myxomatosis in wild rabbits that have undergone 50 years of natural selection for resistance to myxomatosis. Expression of IL-4 significantly enhanced virulence of both virulent and attenuated virus strains in susceptible (laboratory) and resistant (wild) rabbits. SLS-IL-4 completely overcame genetic resistance in wild rabbits. The pathogenesis of SLS-IL-4 was compared in susceptible and resistant rabbits. The results support a model for resistance to myxomatosis of an enhanced innate immune response controlling virus replication and allowing an effective antiviral cell-mediated immune response to develop in resistant rabbits. Expression of IL-4 did not overcome immunity to myxomatosis induced by immunization.

Immune response in rabbit ovaries following infection of a recombinant myxoma virus expressing rabbit zona pellucida protein B

In this study, we investigated the autoimmune response in rabbit ovaries following infection with a recombinant myxoma virus expressing rabbit zona pellucida protein B (MV-ZPB). A specific IgG antibody response to ZPB was elicited in the serum of infected rabbits and the antibody strongly bound to the zona pellucida of oocytes in secondary and tertiary follicles. T cell infiltration in the ovary was detected in a small proportion of the infected rabbits. In spite of this, the mean number of preovulatory and tertiary follicles in the ovary was significantly reduced at 30 days postinfection compared with that of the infected and uninfected controls. Histological analysis revealed that the cortex and medulla of these ovaries had accumulated a large number of probably luteinized cells and there were no follicles in these areas, indicating the ovaries were in a severe pathological condition. The data suggest that the delivery of ZP antigens using a recombinant myxoma virus is a prospective way to develop immunocontraceptive vaccines for rabbit population control, but that more understanding of the kinetics of the autoimmune response induced by viral delivery is needed.

Antibody response in the female rabbit reproductive tract to influenza haemagglutinin encoded by a recombinant myxoma virus

The antibody response in serum and the reproductive tract of female rabbits to a model antigen, influenza virus haemagglutinin (HA), encoded by a recombinant myxoma virus was investigated. Strong and lasting IgG antibody responses to HA were induced in serum following intradermal, intranasal, and intravaginal immunisations. HA IgG was also detected in reproductive tract fluids but was only about 1% the titer of that in serum. HA IgA was not detected in serum of any infected groups and was occasionally detected in reproductive tract fluids at a low titer only after infections through mucosal sites. HA IgM was also detected only in some of the reproductive tract fluids at very low levels. Induction of ovulation did not change these patterns and B cell homing to the reproductive tract was not profound. In contrast, HA IgG and IgM titers in ovarian follicular fluids were comparable to that in serum. These data suggest that if this virus is used to deliver an immunocontraceptive vaccine that requires a high-level antibody response, the target antigen needs to be accessible to serum antibody or in the ovary.

Vaccination of cats with an attenuated recombinant myxoma virus expressing feline calicivirus capsid protein

Myxoma virus, a member of the Poxviridae family (genus Leporipoxvirus) is the agent responsible for myxomatosis in the European rabbit. Recombinant myxoma viruses expressing the capsid gene of an F9 strain of feline calicivirus (FCV) were constructed from an apathogenic, laboratory attenuated, isolate of myxoma virus. The FCV capsid genes were recombined into the myxoma growth factor (MGF) locus of the myxoma genome and expressed from synthetic poxvirus promoters. Myxoma virus is unable to replicate productively in feline cells in vitro, however, cells infected with recombinant viruses do express the heterologous antigens from both late and early/late synthetic promoters. Cats immunised with myxoma–FCV recombinant virus generated high levels of serum neutralising antibody and were protected from disease on subsequent challenge with virulent FCV. Furthermore, there was no evidence of transmission of myxoma–FCV recombinant virus from vaccinated to non-vaccinated cats. These results demonstrate the potential of myxoma virus as a safe vaccine vector for use in non-lepori species and in particular the cat.

First field trial of a transmissible recombinant vaccine against myxomatosis and rabbit hemorrhagic disease

As a novel approach for immunisation of wild rabbits, we have recently developed a transmissible vaccine against myxomatosis and rabbit hemorrhagic disease (RHD) based on a recombinant myxoma virus (MV) expressing the RHDV capsid protein [J. Virol. 74 (2000) 1114]. The efficacy and safety of the vaccine have been extensively evaluated under laboratory conditions. In this study, we report the first limited field trial of the candidate vaccine that was undertaken in an island of 34 Has containing a population of around 300 rabbits. Following administration by the subcutaneous route to 76 rabbits, the vaccine induced specific antibody responses against both myxomatosis and RHDV in all the inoculated rabbits. Furthermore, the recombinant virus exhibited a limited horizontal transmission capacity, promoting seroconversion of around 50% of the uninoculated rabbit population. No evidence of undesirable effects due to the recombinant virus field release was detected.

Safety evaluation of a recombinant myxoma-RHDV virus inducing horizontal transmissible protection against myxomatosis and rabbit haemorrhagic disease

We have recently developed a transmissible vaccine to immunize rabbits against myxomatosis and rabbit haemorrhagic disease based on a recombinant myxoma virus (MV) expressing the rabbit haemorrhagic disease virus (RHDV) capsid protein [Bárcena et al. Horizontal transmissible protection against myxomatosis and rabbit haemorragic disease using a recombinant myxoma virus. J. Virol. 2000;74:1114–23]. Administration of the recombinant virus protects rabbits against lethal RHDV and MV challenges. Furthermore, the recombinant virus is capable of horizontal spreading promoting protection of contact animals, thus providing the opportunity to immunize wild rabbit populations. However, potential risks must be extensively evaluated before considering its field use. In this study several safety issues concerning the proposed vaccine have been evaluated under laboratory conditions. Results indicated that vaccine administration is safe even at a 100-fold overdose. No undesirable effects were detected upon administration to immunosuppressed or pregnant rabbits. The recombinant virus maintained its attenuated phenotype after 10 passages in vivo.

Effects of surgically imposed sterility on free‐ranging rabbit populations

Summary 1. Demographic changes in response to surgically imposed female sterility were monitored in 12 free‐ranging rabbit populations in south‐western Australia over a 4‐year period. This was part of a research programme aimed at examining the potential for virally vectored immunocontraception to limit the abundance of rabbits (e.g. using a recombinant myxoma virus) and other mammalian pests. Sterility levels were 0%, 40%, 60% and 80% of all females in year 1, with a similar proportion of female recruits sterilized surgically in subsequent years. 2. There was a significant decrease in rabbit productivity with increasing sterility level. This was overcome by increased survival of kittens and adults on the high‐sterility sites, such that the base‐level numbers of rabbits were maintained, and mean annual rates of increase (r) were near zero for all treatments in all years. However, in the high‐sterility populations this compensation was insufficient to overcome the effects of sterility totally, and there was a marked decrease in the seasonal peaks in rabbit abundance for these treatments. 3. Survival and recruitment were dependent upon the level of sterility, and consequently the density of rabbits, with greatest survival of adult rabbits occurring on the 80% sites. Survival of sterile females was greater than that of other adults, probably because of their increased ability to maintain body condition during times of low pasture biomass (summer drought). Thus two density‐dependent processes were identified: the first was operating through increased survival of juvenile rabbits, the second through increased adult survival, particularly sterilized females. 4. Because the proportional impact of immigration was greater (i.e. immigrants constituted a greater proportion of the population) and emigration was less, from the 80% sites, the effects of sterility may have been underestimated on these sites. 5. The abundance of European rabbit fleas, a vector of myxomatosis, was significantly lower on the 80% sites, but this did not appear to affect the transmission of myxoma. Myxomatosis occurred as an annual epizootic in three of four years, with > 90% of rabbits on site after each epizootic testing positive for myxoma antibodies. 6. To achieve a sustained long‐term reduction in rabbit abundance, 60–80% of female rabbits would need to be prevented from breeding. This could be achieved by a recombinant strain of myxoma provided the strain retained good transmissibility and all infected rabbits became sterile for life.

Horizontal transmissible protection against myxomatosis and rabbit hemorrhagic disease by using a recombinant myxoma virus.

We have developed a new strategy for immunization of wild rabbit populations against myxomatosis and rabbit hemorrhagic disease (RHD) that uses recombinant viruses based on a naturally attenuated field strain of myxoma virus (MV). The recombinant viruses expressed the RHDV major capsid protein (VP60) including a linear epitope tag from the transmissible gastroenteritis virus (TGEV) nucleoprotein. Following inoculation, the recombinant viruses induced specific antibody responses against MV, RHDV, and the TGEV tag. Immunization of wild rabbits by the subcutaneous and oral routes conferred protection against virulent RHDV and MV challenges. The recombinant viruses showed a limited horizontal transmission capacity, either by direct contact or in a flea-mediated process, promoting immunization of contact uninoculated animals.

Role of the C-Terminal RDEL Motif of the Myxoma Virus M-T4 Protein in Terms of Apoptosis Regulation and Viral Pathogenesis

The purpose of this study was to investigate the significance of the C-terminal RDEL motif of the myxoma virus M-T4 protein in terms of apoptosis regulation and role in viral virulence. To accomplish this, a recombinant myxoma virus was created in which the C-terminal RDEL motif of M-T4 was deleted and a selectable marker (Ecogpt) was inserted immediately downstream. We hypothesized that removal of the RDEL motif from M-T4 would alter the subcellular localization of the protein and provide insight into its antiapoptotic role. Surprisingly, removal of the RDEL motif from M-T4 did not affect localization of the protein within the endoplasmic reticulum (ER), but it did reduce the stability of the mutant protein. Pulse-chase immunoprecipitation and endoglycosidase H analysis coupled with confocal fluorescent light microscopy demonstrated that the M-T4 RDEL− mutant protein is retained in the ER like wildtype M-T4 and suggests that the C-terminal RDEL motif is not the sole determinant for M-T4 localization to the ER. Infection of cultured rabbit lymphocytes with the M-T4 RDEL− mutant virus results in an intermediate apoptosis phenotype compared with the wildtype and M-T4 knockout mutant viruses. A novel myxomatosis phenotype was observed in European rabbits when infected with the recombinant M-T4 RDEL− mutant virus. Rabbits infected with the M-T4 RDEL− virus on day 9 postinfection exhibited an exacerbated edematous and inflammatory response at secondary sites of infections, particularly the ears. Our results indicate that the C-terminal RDEL motif may not be solely responsible for retention of M-T4 to the ER and that M-T4 may have a dual function in protecting infected lymphocytes from apoptosis and in modulating the inflammatory response to virus infection.