Marsupials are protected native animals in Australia and even viewed as national icons. Despite their intrinsic value to the nation, some species of marsupials have become locally overabundant and their numbers have to be controlled to prevent ' economic loss and environmental damage. This study was undertaken as part of a plant-based, immunocontraceptive vaccine development program by the Cooperative Research Centre for the Conservation and Management of Marsupials, to provide a humane and long-term effective alternative to lethal control of overabundant marsupial populations. The primary objective of this study was to develop a transient viral expression system that can be used for rapid production of candidate immunocontraceptive antigens in plants for assessment of the immunogenicity of the plant-derived antigens in animals.
A Queensland isolate of tomato mosaic virus (ToMV-QLD) was chosen for development as a vector to avoid the risk of an 'exotic' virus being introduced into Australia. Also, the low virulence of ToMV-QLD for mature tomato plants was of additional value allowing maximal yields of undamaged tomato fruit containing vaccine antigens for use as 'edible vaccines'. However, until the present study, no prior sequence analysis had been performed on an Australian isolate of ToMV, nor was an infectious clone available for an Australian isolate.
The genome of ToMV-QLD was amplified in two overlapping cDNA fragments that produced multiple, independent 5'- and 3'-half genomic cDNA clones. Sequence analysis was conducted to select clones without a lethal mutation and to obtain sequence information of ToMV-QLD for vector construction purposes. A full-length cDNA clone, from which infectious RNA transcripts could be synthesised, was assembled from the sequenced half genomic cDNA. The use of an infectious clone of ToMV-QLD, completely defined at a molecular level, should facilitate future registration of a ToMV-QLD based vaccine vector.
Sequence comparisons of ToMV-QLD and overseas isolates of ToMV showed only limited genetic variation. The genome of ToMV-QLD was most similar to that of a more virulent Japanese isolate of ToMV. Only a single amino acid difference was found in movement protein (MP) coding region and it is possible that this may be responsible for the difference in virulence. However, without introducing a reverse mutation, it is difficult to assess the influence of this amino acid change on virulence. The availability of the sequence and the infectious clone of the low virulent ToMV-QLD would provide an ideal platform for future investigation of the molecular basis of virulence.
As the primary focus for developing the viral vector in this study was to express immunocontraceptive antigens, the possibility that the recombinant viral vector could escape to the wild was of significant concern. To address this issue, an effort was made to develop a replication-limited, 'model' vector, MPToMV-QLD/( His)6, which had a 473 bp portion of the MP gene deleted, and the coat protein (CP) gene modified to express a model antigen, a six histidine peptide, (His)6, on the virion surface. The large deletion in the MP gene impaired the ability of the virus to infect natural host plants, presumably resulting from the loss of ability to spread from cell-to-cell. This viral vector could only replicate in transgenic host plants that expressed in trans the viral MP gene that had been deleted from the vector.
Tobacco was used as a model system for MP expression due to the ease of transformation and regeneration. Transgenic tobacco plants used for complementing infection with MPToMV-QLD/(His)6 constitutively expressed MP mRNA at levels 0.4-0.5% of those found in a wild-type ToMV-QLD infection in tomato plants.
A tomato transformation system was optimised for MP expression, resulting in a transformation rate of approximately 14%. This was achieved by using the Tanksley tomato cultivar, Agrobacterium strain EHA105, timentin as the antibiotic selectable marker, and phytagel as a gelling agent in the root-inducing medium. In an effort to provide an additional safeguard in the development of an expression system based on the MP-deletion ToMV-QLD vector, transgenic tomato plants were generated which had the MP gene placed under the control of an ethanol-inducible alcA promoter. Disappointingly, after induction by 1% ethanol, the transgenic tomato plants showed discontinuous MP mRNA expression at levels corresponding to only 0.5% of that of the transgenic tobacco plants whose MP expression was driven from the constitutive CaMV 35S promoter. The low expression levels are presumably due to non-optimal ethanol induction, and also the alcA promoter having a comparatively weaker transcriptional activity.
Attempts were made to complement infection of MPToMV-QLD/(His)6 in the transgenic tobacco and tomato plants that were shown to express the missing MP gene. However, MPToMV-QLD/(His)6 did not accumulate to a detectable level in the transgenic host plants. This failure to achieve infection with MPToMV-QLD/( His)6 may have resulted from accumulation of insufficient amounts of MP in the transgenic host plants. More research is required for increasing MP accumulation in plants, if MPToMV-QLD/(His)6 is used as the basis for future vaccine vector studies and applications.
A recombinant, 'model' vector, ToMV-QLD/(His)6, which contained the complete viral genome along with (His)6, was also constructed as part of this study. ToMV-QLD/(His)6 could replicate, form virions, and spread systemically in tobacco plants. In addition, the yield of ToMV-QLD/(His)6 was comparable to that of wild-type virus. More importantly, approximately 5% of the CP subunits displayed (His)6 that reacted with an anti-(His)6 antibody. This suggests that the model antigen could be presented on the viral surface without impairing self-assembly of viral particles or its ability to systemically spread. Given that the viral vector developed in this study has no biological vector and is not intended for use in the field, the recombinant ToMV-QLD vector containing the complete viral genome could potentially be employed as an efficient production and delivery system for assessing candidate immunocontraceptive antigens under laboratory conditions.