WO2001095725A1

WO2001095725A1 - Biological control of pest animals

Info

Publication number: WO2001095725A1
Application number: PCT/AU2001/000698
Authority: WO
Inventors: Ronald James Jackson; Alistair J. Ramsay; Ian Ramshaw
Original assignee: The Executive Director Of The Department Of Conservation And Land Management; Commonwealth Scientific And Industrial Research Organisation; The Australian National University; Agriculture Protection Board Of Western Australia; The University Of Sydney; The University Of Western Australia; The University Of Adelaide
Priority date: 2000-06-13
Filing date: 2001-06-13
Publication date: 2001-12-20
Also published as: AUPQ811700A0

Abstract

The present invention provides a biological control agent for the control of pest animals, and methods of use therefor. Generally, the present invention relies on exposing the pest animal to a pathogen(s) and an exogenous source of at least one type-2 cytokine and/or at least one type-2 polarising chemokine, wherein the combined action of these factors causes substantial mortality of the pest animals.

Description

BIOLOGICAL CONTROL OF PEST ANIMALS

FIELD OF THE INVENTION

The present invention relates to a biological control agent for pest animals such as rodents and rabbits.

BACKGROUND OF THE INVENTION

Ectromelia virus (ECTV; family Poxviήdae, genus Orthopoxvirus) is a natural, specific and highly infective pathogen of mice of the species Mus and Nannomys subgenera of the genus Mus, that causes a generalized disease termed mousepox (Fenner and Buller, 1997). All house mice are equally and highly, susceptible to infection by footpad inoculation however development of clinical mousepox among inbred and outbred mouse strains differs greatly (Wallace and Buller, 1985). In mousepox sensitive mice (e.g. BALB/c) the disease is an acute systemic infection with high viral titers in the liver and spleen with resultant necrosis and high mortality. In contrast, infection of mousepox resistant mice (e.g. C57BL/6) is usually subclinical with lower levels of viral replication in the visceral organs and nonfatal lesions. Genetic resistance has been found to act through the combined activity of innate host defenses including natural killer (NK) cells, interferon-α (IFN-α), IFN-β and IFN-γ, activated macrophages and inducible nitric oxide (iNO) production (Jacoby et al, 1989; Karupiah et al, 1998; Karupiah et al, 1993a; Karupiah et al, 1993b; Rams haw et al, 1997). Mousepox resistant mice also display the early activation of a strong viral-specific cytotoxic T lymphocyte (CTL) response (Karupiah et al, 1996; O'Neil and Brenan, 1987) and produce high levels of type-1 cytokines interleukin-2 (IL-2), IL-12, IFN-γ, and tumor necrosis factor-α (TNFα) in response to ECTV infection, whereas these factors are absent or produced at low levels in susceptible mice (Karupiah, 1998; Ramshaw et al, 1997). Due to its high specificity, infectivity and mortality (in susceptible mice), ECTV provides a promising candidate for use as a biological control agent for pest mice. However, widespread use of a particular pathogen (such as a virus) for biological control of pests typically results in the selection of individuals which are genetically resistant, as well as producing acquired immunity within potentially susceptible individuals, allowing reproduction and recovery of the population. Accordingly, to optimise (or prolong) the biological control effect of a pathogen on a pest species, it would be desirable to take steps to overcome any genetic resistance or acquired immunity that may exist or develop in the target population.

Previous studies using a variety of viral infection models have shown that over-expression or systemic administration of IL-4 impedes the development of virus-specific CTL activity causing a delay in viral clearance (Andrew and Coupar, 1992; Bembridge et al, 1998; Fischer et al, 1997; Moran et al, 1996; Sharma et al, 1996). It has also been suggested that over- expression of IL-4 down regulates NK cell activity, leading to failure of early macrophage activation and exacerbation of vaccinia virus infection (Cheers et al., 1999). The present applicants have investigated what effect IL-4 over- expression may have on the virulence of ECTV and have found, surprisingly, that not only does it increase the frequency and rate of mortality in susceptible (i.e. genetically sensitive) mice, it also produces a high rate of mortality in mice which are genetically resistant to ECTV or which have acquired immunity to ECTV. Accordingly, the present applicants have discovered a means for controlling pest animal species which is capable of circumventing resistance mechanisms which can be acquired by the pest animal.

DISCLOSURE OF THE INVENTION

Thus, in a first aspect, the present invention provides a biological control agent for the control of pest animals, said agent comprising a recombinant pathogen which includes an introduced nucleic acid sequence encoding at least one type-2 cytokine and/or at least one type-2 polarising chemokine that is/are expressed upon infection of a pest animal by said recombinant pathogen, wherein said infection causes substantial mortality of said pest animals.

In a second aspect, the present invention provides a biological control agent for the control of pest animals, said agent comprising a recombinant pathogen which has been produced from a pathogen that causes substantial mortality in said pest animals, wherein said recombinant pathogen includes an introduced nucleic acid sequence which encodes at least one type-2 cytokine and/or at least one type-2 polarising chemokine that is/are expressed upon infection of a pest animal by said recombinant pathogen. In a preferred embodiment of the first and second aspects, the pest animal is selected from the group consisting of rabbits, hares, cats, foxes, kangaroos, possums, and a species of Muridae. Preferably, the species of Muήdae is selected from the group consisting of Rattus species and Mus species. More preferably, the Mus species is selected from the group consisting of Mus domesticus and Mus musculus. Further, it is preferred that the Rattus species is selected from the group consisting of Rattus rattus and Rattus norvegicus.

In another preferred embodiment, the pathogen is selected from the group consisting of fungi, protozoans, bacteria and viruses. More preferably, the virus is selected from the group consisting of ECTV, murine cytomegalo virus, the murine hepatitis virus, and myxoma virus.

The introduced nucleic acid sequence may encode any type-2 cytokine(s) and/or any type-2 polarising chemokine(s), so that expression in pest animals infected with the recombinant pathogen brings about the downregulation of type 1 cytokines such as LL-2, IL-12 and IFNγ which are pivotal to NK, CD8+ CTL and CD4+ Thl immune responses.

Preferably, the type-2 cytokine is IL-4 or IL-10. Most preferably, the type-2 cytokine is IL-4. Preferably, the type-2 polarising chemokine is monocyte chemoattractant protein-1.

In a third aspect, the present invention provides a biological control agent for the control of pest mice, said agent comprising a recombinant ectromelia virus (ECTV) which includes an introduced nucleic acid sequence encoding at least one type-2 cytokine and/or at least one type-2 polarising chemokine that is/are expressed upon infection of a pest mouse by said recombinant ECTN.

In a preferred embodiment of the third aspect, the type-2 cytokine is murine IL-4. In a fourth aspect, the present invention provides a biological control agent for the control of pest rabbits, said agent comprising a recombinant myxoma virus which includes an introduced nucleic acid sequence encoding at least one type-2 cytokine and/or at least one type-2 polarising chemokine that is/are expressed upon infection of a pest rabbit by said recombinant myxoma virus. In a preferred embodiment of the fourth aspect, the type-2 cytokine is rabbit IL-4.

In a fifth aspect, the present invention provides a biological control agent for the control of pest animals, said agent comprising a first pathogen, and a recombinant second pathogen which includes an introduced nucleic acid sequence encoding at least one type-2 cytokine and/or at least one type-2 polarising chemokine that is/are expressed upon infection of a pest animal by said second pathogen, wherein infection of the pest animal with each pathogen causes substantial mortality of said pest animals. In a sixth aspect, the present invention provides a biological control agent for the control of pest animals, said agent comprising a first pathogen that causes substantial mortality in said pest animals, and a recombinant second pathogen which includes an introduced nucleic acid sequence encoding at least one type-2 cytokine and/or at least one type-2 polarising chemokine that is/are expressed upon infection of a pest animal by said second pathogen.

The second pathogen may or may not be a pathogen that normally causes substantial mortality of said pest animals.

In a preferred embodiment of the fifth and sixth aspects, the pest animal is selected from the group consisting of rabbits, hares, cats, foxes, kangaroos, possums, and a species of Muridae. Preferably, the species of Muridae is selected from the group consisting of Rattus species and Mus species. More preferably, the Mus species is selected from the group consisting of Mus domesticus and Mus musculus. Further, it is preferred that the Rattus species is selected from the group consisting of Rattus rattus and Rattus norvegicus.

The first and second pathogens may be selected from any pathogen of a pest animal species and, therefore, may be selected from the group consisting of: fungi, protozoans, bacteria and viruses. Preferably, the first and second pathogens are viruses such as ECTV, murine cytomegalo virus, the murine hepatitis virus, and myxoma virus.

The introduced nucleic acid sequence may encode any type-2 cytokine(s) and/or any type-2 polarising chemokine(s), so that expression in pest animals infected with the recombinant pathogen brings about the downregulation of type 1 cytokines such as IL-2, LL-12 and IFNγ which are pivotal to NK, CD8+ CTL and CD4+ Thl immune responses. Preferably, the type-2 cytokine is IL-4 or IL-10. Most preferably, the type-2 cytokine is IL-4.

Preferably, the type-2 polarising chemokine is monocyte chemoattractant protein-1. In a seventh aspect the present invention provides a biological control agent for the control of pest mice, said agent comprising a first pathogen which is an ectromelia virus (ECTV), and a recombinant second pathogen which includes an introduced nucleic acid sequence encoding at least one type-2 cytokine and/or at least one type-2 polarising chemokine that is/are expressed upon infection of a pest mouse by said second pathogen.

In a preferred embodiment of the seventh aspect, the type-2 cytokine is murine IL-4.

In an eighth aspect the present invention provides a biological control agent for the control of pest rabbits, said agent comprising a first pathogen which is an myxoma virus, and a recombinant second pathogen which includes an introduced nucleic acid sequence encoding at least one type-2 cytokine and/or at least one type-2 polarising chemokine that is/are expressed upon infection of a pest rabbit by said second pathogen.

In a preferred embodiment of the eighth aspect, the type-2 cytokine is rabbit IL-4.

The biological control agent may be formulated with an appropriate acceptable carrier and applied by, for example, spraying an area inhabited by a population of pest animals or, alternatively, may be formulated with an appropriate solid carrier (such as a foodstuff) and applied to an area by scattering or more selective placement (i.e. "baiting"). In addition, distribution of the biological control agent could be achieved by the release of infected animals which could initiate an epidemic among the local population. Transmission of the pathogen can occur in numerous ways, for example, by mating or via abrasions through contaminated bedding, grooming, fighting and cannibalism. In addition, the transmission of some pathogens, for example the myxoma virus, could occur via biting insects such as mosquitoes, fleas, mites, ticks and the like.

Accordingly, in a ninth aspect, the present invention provides a method for controlling a population of pest animals, said method comprising applying a biological control agent according to the present invention to an area inhabited by said population of pest animals. Furthermore, in a tenth aspect, the present invention provides a composition comprising a biological control agent according to the present invention and an acceptable carrier.

In an eleventh aspect, the present invention provides a method for controlling a population of pest animals, said method comprising applying a composition according to the present invention to an area inhabited by said population of pest animals.

In an twelfth aspect, the present invention provides a bait for controlling pest animals, said bait comprising a biological control agent according to the present invention in admixture with a foodstuff.

In a thirteenth aspect, the present invention provides a bait for controlling pest animals, said bait comprising a pathogen in admixture with a foodstuff including at least one type-2 cytokine and/or at least one type-2 polarising chemokine, wherein infection of a pest animal by said pathogen and ingestion of the foodstuff by said pest animal causes substantial mortality of said pest animal.

In a fourteenth aspect, the present invention provides a bait for controlling pest animals, said bait comprising a pathogen that causes substantial mortality in said pest animals in admixture with a foodstuff including a type-2 cytokine and/or type-2 polarising chemokine.

In a preferred embodiment of the thirteenth and fourteenth aspects, the type-2 cytokine is IL-4 or IL-10. Most preferably, the type-2 cytokine is IL-4.

In a further preferred embodiment of the thirteenth and fourteenth aspects, the type-2 polarising chemokine is monocyte chemoattractant protein-1.

In a fifteenth aspect, the present invention provides a method for controlling a population of pest animals, said method comprising applying a bait according to the present invention to an area inhabited by said population of pest animals. Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. The invention will hereinafter be described by way of the following non-limiting Examples and accompanying Figures. BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES

FIG. 1. Anti-Orthopoxvirus CTL responses in C57BL/6 mice following footpad inoculation with varying doses of ECTV-IL4(TK+) or ECTN- 602(TK+). Lytic activity of splenocytes taken 7 days p.i. at different effector- to-target ratios. Assays were carried out in triplicate with four mice per group, and standard errors of the mean at all points were <5%.

FIG. 2. ΝK cell response in C57BL/6 mice following footpad inoculation with 10⁴ PFU of ECTV-IL4(TK+) or ECTV-602(TK+) 3 days p.i. Lytic activity at different effector-to-target ratios are shown. Assays were carried out in triplicate with four mice per group, and standard errors of the means at all points were <5%.

DETAILED DECRIPTIOΝ OF THE INVENTION

General Recombinant Techniques Unless otherwise indicated, the recombinant DNA techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (Editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present) and are incorporated herein by reference.

Substantial Mortality

As used herein, "substantial mortality" refers to mortality of >50%, more preferably > 75% and most preferably >90%, of pest animals infected with said recombinant pathogen.

As used herein, "a pathogen that causes substantial mortality in said pest animals" refers to a pathogen which causes mortality in >50%, more preferably > 75% and most preferably >90%, of susceptible animals of said pest animals. Recombinant Pathogens

As used herein the term "pathogen" refers to any organism that can infect the pest animal, typically causing at least one disease symptom associated with the infection. Preferred pathogens include fungi, protozoans, bacteria and viruses.

The term "recombinant pathogen" refers to pathogens which have been engineered to incorporate and express a foreign nucleic acid sequence. As outlined above, the foreign nucleic acid sequence at least encodes a type-2 cytokine and/or a type-2 polarising chemokine. Methods of introducing and expressing a foreign nucleic acid sequence in an organism are well known in the art. The introduced nucleic acid sequence may be inserted into the genomic DNA or RNA of a pathogen or, and particularly where the pathogen is a bacteria, may be borne on a extra- chromosomal nucleic acid such as a plasmid. The foreign nucleic acid sequence will typically be introduced into the pathogen via an expression vector. As used herein, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and effecting expression of a type-2 cytokine and/or a type-2 polarising chemokine. Preferably, the expression vector is also capable of replicating within the host cell. The vector is typically a virus or a plasmid. Expression vectors suitable for use in the present invention include any vectors that function (i.e., direct gene expression) when introduced into a pathogen.

In particular, the expression vector, or an expression cassette to be introduced into the genome of the pathogen, contains regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of the introduced nucleic acid sequence. In particular, the expression vector, or an expression cassette to be introduced into the genome of the pathogen, suitable for use in the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one pathogen as defined herein. The transcriptional control sequences can be constitutively expressed or expressed in a spatial and/or temporal specific manner. Preferably, the transcriptional control sequences direct the constitutive expression of the introduced nucleic acid sequence encoding the type-2 cytokine and/or the type-2 polarising chemokine. A variety of such transcription control sequences are well known to those skilled in the art. Transformation of a nucleic acid sequences into a cell can be accomplished by any method known in the art. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. Transformed nucleic acid sequences suitable for use in the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained.

Recombinant DNA techniques can be used to improve expression of a transformed nucleic acid sequence by manipulating, for example, the number of copies of the nucleic acid sequence within a host cell, the efficiency with which those nucleic acid sequence are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications. Recombinant techniques useful for increasing the expression of nucleic acid sequence suitable for use in the present invention include, but are not limited to, operatively linking the nucleic acid sequence to high-copy number plasmids, integration of the nucleic acid sequence into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of a nucleic acid sequence suitable for use in the present invention to correspond to the codon usage of the host cell, and the deletion of sequences that destabilize transcripts.

Compositions and Acceptable Carriers

Compositions of the present invention include acceptable carriers, also referred to herein as "excipients". An acceptable carrier can be any material that an area to which the composition is applied can tolerate, for example carriers which are toxic to non-pest animal species or other organisms would probably not be suitable. Examples of suitable acceptable carriers include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran. The compositions of the present invention can also contain minor amounts of additives, such as substances that enhance isotonicity and pathogen stability. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer. Acceptable carriers can also be used to increase the half-life of the pathogen, for example, but are not limited to, polymeric controlled release vehicles, liposomes, bacteria, viruses, other cells, oils, esters, and glycols.

One embodiment of the present invention is a controlled release formulation that is capable of slowly releasing a composition of the present invention into an area. As used herein, a controlled release formulation comprises a composition of the present invention in a controlled release vehicle. Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems. Preferred controlled release formulations are biodegradable (i.e., bioerodible).

A preferred controlled release formulation of the present invention is capable of releasing a composition of the present invention to an area inhabited by the target population of pest animals. The formulation is preferably released over a period of time ranging from about 1 to about 12 months. A preferred controlled release formulation of the present invention is capable of effecting a treatment preferably for at least about 1 month, more preferably for at least about 3 months, even more preferably for at least about 6 months, still more preferably for at least about 9 months, and most preferably for at least about 12 months.

Baits

The type-2 cytokine and/or type-2 polarising chemokine may be incorporated into the foodstuff merely by, for example, mixing a solution of the type-2 cytokine and/or type-2 polarising chemokine with the foodstuff or by mixing solid dosage forms (e.g. tablets) of the type-2 cytokine and/or type- 2 polarising chemokine. Such solutions and tablets may include acceptable carriers, stabilisers, etc. as is well known in the agricultural and veterinary fields. Solid dosage forms may use protective enteric coats to ensure delivery of the type-2 cytokine and/or type-2 polarising chemokine to the intestine for absorption. The amount of type-2 cytokine and/or type-2 polarising chemokine incorporated into the foodstuff should be sufficient such that a pest animal will ingest, in a normally consumed amount of the foodstuff, an amount of the type-2 cytokine and/or type-2 polarising chemokine which downregulates endogenous type-1 cytokine expression.

The bait may also include agents which enhance the attractiveness of the bait to the target pest animal. For example, US 5,720,951 describes baits optimized for consumption by rodents. Furthermore, the bait may include agents which make the bait unattractive to non-target animals such as pet dogs and cats, domestic animals such as cows, horses and sheep, as well as non-pest native animals whose population is endangered or whose population is not at a size to be considered a pest.

EXAMPLE 1: Materials and Methods Cells and viruses.

L-M(TK-) (mouse [Mus musculus) ATCC CCL-1.3) and B-SC-1 (African green monkey (Cercopithecus aethiops) ATCC CCL-26) cells were maintained in minimal essential medium (MEM) supplemented with 5% fetal bovine serum at 37°C, 5% C0₂. Ectromelia virus (ECTV)s were grown on the above cells in MEM at 35°C, 5% C0₂. Recombinant ECTVs were constructed by infection of L-M(TK-) cells at a multiplicity of infection of 0.1 PFU/cell with the TK- virus ECTV-602, which contains the insertion of the E. coli lacZ (β- galactosidase) gene inactivating the Orthopoxvirus TK gene (Jackson et al, 1998). The virus infected cells were transfected with either plasmids pTK- 7.5A (Couper et al, 1988) or pFB-TK-D.,4 (Andrew and Coupar, 1992) using LipofectAMINE Reagent (Gibco-BRL Life Technologies Inc., Gaithersburg, MD). Using these vectors DNA recombination should occur between the Vaccinia virus (VACV) Hzz. dlll-F sequences contained in the vectors and the "homologous" sequences within ECTV HindlH-E fragment. Recombinant ECTVs expressing the herpes simplex virus (HSV) TK gene were selected by growth on L-M(TK-) cells in MEM containing HAT supplement (Gibco-BRL Life Technologies Inc.). Recombinant virus ECTV-602(TK+) which was constructed using pTK-7.5A contains a copy of the HSV TK gene inserted into the natural BamLHL site located immediately downstream of the VACV F7 ORF early promoter. Virus ECTV-IL4(TK+) which was constructed using pFB-TK- IL4 is similar to ECTv⁷-602(TK+) except that it also contains a copy of the mouse IL-4 cDNA under the transcriptional control of the vaccinia virus P7.5 early/late promoter inserted immediately downstream of the HSV TK gene. IL-4 expression was confirmed in vitro by bioassay of supernatants overlying ECTv⁷-IL4(TK+) infected L-M(TK-) cells (Hogan et al, 1998; Sharma et al, 1996).

Virus titration.

Titration of recombinant ECTVs recovered from mouse tissues was performed on B-SC-1 cells grown in 6 well culture dishes overlaid with 2ml MEM containing 1% (w/v) low melting point agarose (SeaPlaque GTG, FMC

BioProducts, Rockland, ME) and grown at 35°C for 72 hours. Plaques produced by viruses expressing β-galactosidase were visualized by overlaying the infected cells with a further 2ml MEM, 1% agarose containing 300μg/ml

X-gal.

Mice and inoculation.

Animal studies were conducted in accordance with the Australian

Code of Practice for the Care and Use of Animals for Scientific Purposes.

Specific pathogen free 6- to 8-wk-old female mice were obtained from the Australian National University Animals Services Division.

Virulence studies.

BALB/c and C57BL/6 mice were inoculated with varying doses of virus into the right hind footpad and disease symptoms observed for two weeks post infection when surviving animals were euthanized.

Delayed Type Hypersensitivity (DTH) studies.

BALB/c and C57BL/6 mice were immunized by inoculation into the right hind footpad with 10³ PFU of highly attenuated ECTV-602. The immune mice were challenged four weeks post immunization by inoculation into the same footpad with 10⁴PFU of either ECTV-602(TK+) or ECTV- IL4(TK+). DTH responses were measured 24 and 48 hours post challenge by measuring the dorsal-lateral thickness of the inoculated right foot using calipers and compared to the thickness of the uninoculated left foot. Mice were monitored for a further two weeks to observed signs of disease and mortality.

Assessment of antiviral cytolytic responses and IFN-γ production.

Female C57BL/6 mice were infected by footpad inoculation with 10², 10³, or 10⁴ PFU for CTL assays, or 10⁴ PFU for NK cell assays or assessment of IFN-γ production, of either ECTV-602(TK-I-) or ECTV-LL4(TK+). For NK cell assays spleens were removed on day 1, 2 and 3 post infection (p.i.), whilst for assays of CTL activity or IFN-γ expression spleens were removed on day 7 p.i.. NK cell cytolytic activity was measured on YAC-1 cells, while CTL activity was measured on VACV infected MC57G targets (a C57BL/6 derived fibroblast line, H-2^b, MHC class I⁺ and MHC class II^") using the standard 6- hour ⁵¹Cr release assay (Karupiah et al, 1990). IFN-γ production was measured using microcultures set up in parallel with those used in CTL assays. Splenic effector cells were cultured with VACV infected or uninfected MC57G targets at a ration of 20:1, supernatants were collected after 6 hours, and IFN-γ levels were assayed by ELISA (Hogan et al, 1998).

Rabbits and myxoma virus.

Recombinant myxoma virulent Standard Laboratory Strain (SLS) derivative of Moses strain (ATCC VR-116) expressing rabbit IL-4 was constructed by an intergenic insertion at a site located between the viral thymidine kinase (TK: ORF M061R) and ORF M062R to maintain virulence phenotype.

Six wild rabbits naturally selected for genetic resistance to myxoma virus were infected with 1000 plaque forming units of recombinant myxoma virus expressing rabbit IL-4.

Results

Disease symptoms following infection of mice with recombinant ECTVs. Control virus: Footpad inoculation of mousepox resistant C57BL/6 mice with 10³ PFU

ECιN-602(TK+) causes symptoms similar to the virulent wild-type Moscow strain of ECTV, however recovery is not normally associated with necrosis and sloughing of the infected limb. The ECTV-602(TK+) virus was clearly less virulent than the Moscow strain since it did not cause mortality in mousepox sensitive BALB/c mice which behave similarly to infected C57BL/6 mice. However, footpad inoculation of the highly susceptible A/J strain mice with ECTV-602(TK+) is generally lethal (data not shown). TK+ IL-4 expressing virus:

To assess the effects of IL-4 expression by a recombinant ECTV upon virulence, mice were infected with 10³ PFU of ECTV-IL4(TK+) and disease symptoms and mortality monitored. Infection of BALB/c or C57BL/6 mice with ECTV-IL4(TK+) proved to be uniformly lethal, mean survival time (days) 7.4±0.7 (n=10/10); 8.6±1.2 (n=10/10), respectively. In both strains, swelling of the inoculated foot was clearly visible by 6 day p.i., and continued to increase in size until the mice succumbed to the infection. Shortly before death the infected mice became lethargic with ruffled fur and hunched posture. At autopsy, the mice contained enlarged pallid spleens and livers with both organs containing numerous necrotic lesions. These symptoms are typical of acute mousepox as seen in genetically susceptible mice infected with the virulent Moscow strain (Fenner and Buller, 1997). C57BL/6 mice infected with ECTV-IL4(TK+) contained high levels of virus in the spleen just prior to displaying early symptoms of acute mousepox (Table 1). In contrast, virus titers in the spleens of mice infected with equivalent doses of ECTV- 602(TK+) were reduced suggestive of immune mediated clearance. Increasing the infectious dose of the IL-4 expressing virus exacerbated the onset of symptoms and decreased survival times (data not shown). All control mice infected with equivalent doses of ECTV-602(TK+) survived infection.

Assessment of cytolytic lymphocyte responses and IFN-γ production following infection.

Due to the enhanced virulence of the recombinant ECTV, the effects of IL-4 expression on the development of antiviral CD8+ CTL and NK activity and LFN-γ expression (responses which are crucial for controlling ECTV infection) was assessed. Increasing doses of ECTV-IL4(TK-r-) virus were used to infect C57BL/6 mice and spleens isolated 7 days post infection and assayed Table 1. Virus titer in spleens of naϊve C57BL/6 mice following infection with recombinant control ECTV or ECTV expressing IL-4.

Average virus titer in spleen (PFU/g) at specified inocula 7 days p.i. Virus 10² PFU 10³ PFU 10⁴ PFU

ECTV-602(TK+) 3.0xl0⁴ 2.8xl0⁴ 1.5xl0²

ECTV-IL4(TK+) 2.5xl0⁵ 3.5xl0⁷ 6.6xl0⁷

for CTL activity using the standard ⁵¹Cr release assay (Figure 1). Splenocytes isolated from mice infected with the control virus ECTV-602(TK+) displayed significant specific cytolytic activity towards Orthopoxvirus (VACV) infected MC57G cells. In contrast, splenocytes isolated from C57BL/6 mice infected with ECTV-IL4(TK-I-) contained no detectable virus-specific cytolytic activity. IL-4 expression in ECTV also markedly suppressed LFN-γ secretion by antiviral CD8+ T cells. Mean levels of virus-specific IFN-γ produced by splenocytes from mice infected with 10⁴ PFU ECTV-602(TK+) were 7-fold higher than background levels (733±139 compared to 129±29 Units/ml). In contrast, CD8+ T cells from mice given ECTV-LL4(TK+) failed to produce IFN-γ at levels above background (130 ±47 Units/ml). Viral replication in these mice appeared to be uncontrolled with the mice dying shortly thereafter. To assay for induction of innate NK cell activity, spleen cells were isolated from ECTV-IL4(TK+) infected mice on days 1, 2 and 3 post infection and assayed for lytic activity on YAC-1 cells. Cytolytic activity was undetectable at day 1 p.i. following infection with either control or IL-4 expressing viruses. At day 2 post infection, both groups of mice expressed approximately equivalent levels of NK activity (20% lysis of YAC-1 targets at 20:1 effector:target ratio). However, by day 3 p.i., when NK cell activity is usually near maximal during ectromelia virus infection (Mtillbacher et al, 1996), ECTV-rL4(TK+) infected C57BL/6 mice displayed approximately a three fold reduction in splenic NK cell mediated lysis of YAC-1 targets compared to similarly infected controls (compare % specific lysis of ECTV- IL4(TK+) infected mice 20:1 effector:target ratio and control virus infected mice 6:1 effector:target ratio) (Figure 2).

Reinfection of mice immunized against ectromelia virus. Due to the observed suppression of NK and CTL activity and IFN-γ expression during the primary immune response to ECTV-IL4(TK+) and the known inhibitory effect of IL-4 upon -the Thl mediated delayed type hypersensitivity (DTH) response (Powrie et al, 1993), the effects of ECTV IL-4 expression upon the memory response to ECTV in immune mice were investigated. Immunization of both C57BL/6 and BALB/c mice with the attenuated thymidine kinase negative virus ECTV-602 caused a mild swelling at the inoculation site which resolved within 14 day p.i., with all mice recovering from infection (Jackson et al, 1998). Twenty-eight days post immunization the mice were challenged with either control ECTV-602 (TK+) or ECTV-IL4(TK-)-) viruses. These immune mice displayed greatly differing DTH responses to infection (Table 2). Mice challenged with the control virus displayed a mild DTH response that appeared within 24 hours and was resolving 48 hours post challenge. The mice infected with the control virus showed no further evidence of viral infection indicating that they were immune to infection with ECTV. Similarly immunized mice were also immune to reinfection with the virulent Moscow strain (data not shown). In contrast, mice challenged with ECTV-IL4 (TK+) displayed an exacerbated "DTH response" characterized by extreme swelling of the inoculated foot 24 and 48 hours p.i. (Table 2). The inoculated feet of challenged immune mice continued to increase in size until 60% of the mice died between days 6 to 8 p.i. At death, the livers and spleens of these mice where enlarged and contained numerous pock lesions, suggestive of death due to acute mousepox. The mice which survived challenge with ECTV-IL4(TK+) were autopsied 21 day p.i., at which time they still displayed a marked swelling of the inoculated foot. Viral titration of tissues isolated from the surviving mice indicated they were controlling systemic infection since none contained detectable virus in the spleen. However, at the site of inoculation, virus clearance was considerably delayed with average titers of virus in the inoculated feet of the surviving C57BL/6 and BALB/c mice 21 days p.i. being 6xl0³ and 1.3xl0⁵ PFU/g tissue, respectively. Table 2. Susceptibility of ECTV immune mice to re-infection with ECTN-IL4(TK+)

DTH response difference in thickness of the inoculated footpad

(10⁴PFU virus) compared to the uninoculatec foot. (0.1 mm units ± SEM)

Mouse 24 hr p.i. 24 hr p.i. 48 hr p.i. 48 hr p.i. Mortality 6-8 days p.i.

Strain

ECTN- ECTN- ECTN- ECTN- ECTN- ECTN-

^• i

602 (TK+) IL4(TK+) 602(TK+) IL4(TK+) 602(TK+) IL4(TK+)

C57BL/6 4.4 + 0.4 9.4 + 0.5 2.2 ± 0.4 12.0 ± 0.5 0% (0/5) 60% (3/5)

BALB/c 4.8 + 0.7 12.0 ± 0.3 2.0 ± 0.3 12.1 + 0.8 0% (0/5) 60% (3/5)

Infection of rabbits with myxoma virus expressing rabbit IL-4.

Infection of genetically resistant wild rabbits with the recombinant virus expressing rabbit IL-4 resulted in 100% mortality with an average survival time of 8 days post-infection.

Discussion

The original Moscow strain of ECTV is highly virulent and normally lethal to mousepox sensitive mice. In constructing the recombinant ectromelia virus disclosed in the Example, the thymidine kinase gene was initially inactivated by insertion of the Escherichia coli lacZ (β-galactosidase) gene generating a highly attenuated recombinant virus. To partially restore the virulent phenotype, the herpes simplex virus thymidine kinase (TK) gene was introduced. The HSV TK gene is actually a deoxypyrimidine kinase with a partial thymidine kinase activity, approximately one sixth of the activity of the natural poxvirus thymidine kinase gene. As disclosed herein, the ECTV-

602 (TK+) contains both the lacZ gene inactivating the ECTV TK gene and the HSV TK gene inserted complementing the genetic deletion. This virus although reasonably virulent is not lethal to BALB/c mice which are considered genetically sensitive and would normally die of acute mousepox if infected with the wild-type Moscow strain.

The above results show that when an IL-4 gene is introduced into ECTV-602(TK-I-), the virus is capable of systemic infection and suppression of splenic NK and CTL cytolytic activity and IFN-γ expression. The expression of E -4 thereby enhances the virulence of ECTV and, indeed, renders the virus lethal to mice that are normally genetically resistant. Further, the expression of IL-4 also inhibits memory responses in mice previously immunized with ECTV leading to uncontrolled viral replication in the visceral organs resulting in classic symptoms of acute mousepox.

Accordingly, recombinant ECTV in accordance with the invention can be used as a biological control agent for pest mice. Similarly, recombinant myxoma virus expressing rabbit IL-4 in accordance with the invention can be used as a biological control agent for pest rabbits.

With regard to the ectromelia virus, it has been found experimentally that this virus only affects mice of the species Mus and Nannomys subgenera of the genus Mus. Accordingly, there is no evidence that the genetically engineered ectromelia virus of the present invention would be harmful to other species. Furthermore, at present, it is believed that the biological activity of mouse IL-4 is limited to the binding of the mouse IL-4 receptor and not the homologous receptors of other species, further increasing the attractiveness of the genetically engineered ectromelia virus for controlling mice species as disclosed herein.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

All publications discussed above are incorporated herein in their entirety.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed, particularly in Australia, before the priority date of each claim of this application.

REFERENCES

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Fenner, F. and R. M. L. Buller. 1997. Mousepox, p. 535-553. In N. Nathanson, R. Ahmed, F. Gonzalez-Scarano, D. E. Griffin, K. V. Holmes, F. A. Murphy, and H. L. Robinson (eds.), Viral Pathogenesis. Lippincott-Raven Publishers, Philadelphia Fischer, J. E., J. E. Johnson, R. K. Kuli-Zade, T. R. Johnson, S. Aung, R.

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Jackson, R. J., D. J. Maguire, L. A. Hinds, and I. A. Ramshaw. 1998. Infertility in mice induced by a recombinant ectromelia virus expressing mouse zona pellucida glycoprotein 3. Biol. Reprod. 58:152-159 Jacoby, R. O., P. N. Bhatt, and D. G. Brownstein. 1989. Evidence that

NK cells and interferon are required for genetic resistance to lethal infection with ectromelia virus. Arch. Virol. 108:49-58

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Karupiah, G., B. E. H. Coupar, A. E. Andrew, D. B. Boyle, S. M. Phillips, A. Mtillbacher, R. V. Blanden, and I. A. Ramshaw. 1990. Elevated NK cell responses in mice infected with recombinant vaccinia virus encoding murine IL-2. J. Immunol. 144:290-298

Karupiah, G, T. N. Fredrickson, K. L. Holmes, L. H. Khairallah, and R. M. Buller. 1993a. Importance of interferons in recovery from mousepox. J. Virol. 67:4214-4226 Karupiah, G, Q. W. Xie, R. M. Buller, C. Nathan, C. Duarte, and J. D.

MacMicking. 1993b. Inhibition of viral replication by interferon-γ-induced nitric oxide synthase. Science 261:1445-1447

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Mtillbacher, A., K. Ebnet, R. V. Blanden, R. Tha Hla, T. Stehle, C. Museteanu, and M. M. Simon. 1996. Granzyme A is critical for recovery of mice from infection with the natural cytopathic viral pathogen, ectromelia. Proc. Natl. Acad. Sci. USA 93:5783-5787 O'Neil, H. C. and M. Brenan. 1987. A role for early cytotoxic T cells in resistance to ectromelia virus infection in mice. J. Gen. Virol. 68:2669-2673

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Claims

1. A biological control agent for the control of pest animals, said agent comprising a recombinant pathogen which includes an introduced nucleic acid sequence encoding at least one type-2 cytokine and/or at least one type-2 polarising chemokine that is/are expressed upon infection of a pest animal by said recombinant pathogen, wherein said infection causes substantial mortality of said pest animals.

2. A biological control agent for the control of pest animals, said agent comprising a recombinant pathogen which has been produced from a pathogen that causes substantial mortality in said pest animals, wherein said recombinant pathogen includes an introduced nucleic acid sequence which encodes at least one type-2 cytokine and/or at least one type-2 polarising chemokine that is/are expressed upon infection of a pest animal by said recombinant pathogen.

3. The biological control agent of claim 1 or claim 2, wherein the pest animal is selected from the group consisting of rabbits, hares, cats, foxes, kangaroos, possums, and a species of Muridae.

4. The biological control agent of claim 3, wherein the species of Muridae is selected from the group consisting of Rattus species and Mus species.

5. The biological control agent of claim 4, wherein the Mus species is selected from the group consisting of Mus domesticus and Mus musculus.

6. The biological control agent of claim 4, wherein the Rattus species is selected from the group consisting of Rattus rattus and Rattus norvegicus.

7. The biological control agent according to any one of claims 1 to 6, wherein the pathogen is selected from the group consisting of fungi, protozoans, bacteria and viruses.

8. The biological control agent of claim 7, wherein the virus is selected from the group consisting of ECTV, murine cytomegalo virus, the murine hepatitis virus, and myxoma virus.

9. The biological control agent according to any one of claims 1 to 8, wherein the type-2 cytokine is IL-4 or IL-10.

10. The biological control agent according to claim 9, wherein the type-2 cytokine is IL-4.

11. The biological control agent according to any one of claims 1 to 8, wherein the type-2 polarising chemokine is monocyte chemoattractant protein-1.

12. A biological control agent for the control of pest mice, said agent comprising a recombinant ectromelia virus (ECTV) which includes an introduced nucleic acid sequence encoding at least one type-2 cytokine and/or at least one type-2 polarising chemokine that is/are expressed upon infection of a pest mouse by said recombinant ECTV.

13. The biological control agent according to claim 12, wherein the type-2 cytokine is murine IL-4.

14. A biological control agent for the control of pest rabbits, said agent comprising a recombinant myxoma virus which includes an introduced nucleic acid sequence encoding at least one type-2 cytokine and/or at least one type-2 polarising chemokine that is/are expressed upon infection of a pest rabbit by said recombinant myxoma virus.

15. The biological control agent according to claim 14, wherein the type-2 cytokine is rabbit IL-4.

16. A biological control agent for the control of pest animals, said agent comprising a first pathogen, and a recombinant second pathogen which includes an introduced nucleic acid sequence encoding at least one type-2 cytokine and/or at least one type-2 polarising chemokine that is/are expressed upon infection of a pest animal by said second pathogen, wherein infection of the pest animal with each pathogen causes substantial mortality of said pest animals.

17. A biological control agent for the control of pest animals, said agent comprising a first pathogen that causes substantial mortality in said pest animals, and a recombinant second pathogen which includes an introduced nucleic acid sequence encoding at least one type-2 cytokine and/or at least one type-2 polarising chemokine that is/are expressed upon infection of a pest animal by said second pathogen.

18. The biological control agent of claim 16 or claim 17, wherein the pest animal is selected from the group consisting of rabbits, hares, cats, foxes, kangaroos, possums, and a species of Muridae.

19. The biological control agent of claim 18, wherein the species of Muridae is selected from the group consisting of Rattus species and Mus species.

20. The biological control agent of claim 19, wherein the Mus species is selected from the group consisting of Mus domesticus and Mus musculus.

21. The biological control agent of claim 19, wherein the Rattus species is selected from the group consisting of Rattus rattus and Rattus norvegicus.

22. The biological control agent according to any one of claims 16 to 21, wherein the first and second pathogens are selected from the group consisting of fungi, protozoans, bacteria and viruses.

23. The biological control agent of claim 22, wherein the virus is selected from the group consisting of ECTV, murine cytomegalovirus, the murine hepatitis virus, and myxoma virus.

24. The biological control agent according to any one of claims 16 to 23, wherein the type-2 cytokine is IL-4 or IL-10.

25. The biological control agent according to claim 24, wherein the type-2 cytokine is IL-4.

26. The biological control agent according to any one of claims 16 to 23, wherein the type-2 polarising chemokine is monocyte chemoattractant protein-1.

27. A biological control agent for the control of pest mice, said agent comprising a first pathogen which is an ectromelia virus (ECTV), and a recombinant second pathogen which includes an introduced nucleic acid sequence encoding at least one type-2 cytokine and/or at least one type-2 polarising chemokine that is/are expressed upon infection of a pest mouse by said second pathogen.

28. The biological control agent according to claim 27, wherein the type-2 cytokine is murine IL-4.

29. A biological control agent for the control of pest rabbits, said agent comprising a first pathogen which is an myxoma virus, and a recombinant second pathogen which includes an introduced nucleic acid sequence encoding at least one type-2 cytokine and/or at least one type-2 polarising chemokine that is/are expressed upon infection of a pest rabbit by said ^• second pathogen.

30. The biological control agent according to claim 29, wherein the type-2 cytokine is rabbit IL-4.

31. A method for controlling a population of pest animals, said method comprising applying a biological control agent according to any one of claims 1 to 30 to an area inhabited by said population of pest animals.

32. A composition comprising a biological control agent according to any one of claims 1 to 30 and an acceptable carrier.

33. A method for controlling a population of pest animals, said method comprising applying a composition according to claim 32 to an area inhabited by said population of pest animals.

34. A bait for controlling pest animals, said bait comprising a biological control agent according to any one of claims 1 to 30 in admixture with a foodstuff.

35. A bait for controlling pest animals, said bait comprising a pathogen in admixture with a foodstuff including at least one type-2 cytokine and/or at least one type-2 polarising chemokine, wherein infection of a pest animal by said pathogen and ingestion of the foodstuff by said pest animal causes substantial mortality of said pest animal.

36. A bait for controlling pest animals, said bait comprising a pathogen that causes substantial mortality in said pest animals in admixture with a foodstuff including a type-2 cytokine and/or type-2 polarising chemokine.

37. The bait according to claim 35 or claim 36, wherein the type-2 cytokine is IL-4 or IL-10.

38. The bait according to claim 37, wherein the type-2 cytokine is IL-4.

39. The bait according to claim 35 or claim 36, wherein the type-2- polarising chemokine is monocyte chemoattractant protein-1.

40. A method for controlling a population of pest animals, said method comprising applying a bait according to any one of claims 34 to 39 to an area inhabited by said population of pest animals.