**6. Future Possibilities for Enhanced DNA Vaccine or Expression Vectors Based on Circoviruses**

Our group has recently published an investigation of the possibility of using circovirus-derived replication control elements to create replicons, or replicating dsDNA plasmid-like molecules, in plants and in mammalian cells [34]. This followed our extensive success with use of a plant ssDNA geminivirus-derived expression vector in plants as an enhanced expression vector [35]: Geminiviruses are very similar to circoviruses in having small circular ssDNA genomes that replicate via a Rep-mediated rolling circle mechanism, and very similar sequences for their non-nucleotide origins of replication (TAATATT/AC vs. TAGTATT/AC). In this study, we used a synthetic, partially dimeric clone of the genome of beak and feather disease virus (BFDV), a circovirus generically related to PCV, to investigate cross-potentiation of replication between the plant and animal viruses in plants and replication of the BFDV genome alone in HEK293TT cells.

Initial experiments where both the geminivirus-derived vector bean yellow dwarf virus (BeYDV) and BFDV genome were introduced into *Nicotiana benthamiana* plants via *Agrobacterium tumefaciens*-mediated DNA transfer showed that replication of BeYDV facilitated the co-replication of BFDV, albeit to levels only 100× less than for BeYDV replicons. More importantly, however, transfection of HEK293TT cells with the BFDV construct resulted in a ten-fold increase in genome copy number after three days. This was the first time that BFDV genomes had been shown to replicate in any animal-derived cell culture, in contrast to PCVs which readily infect a variety of cells [36]. Improvement in replicon copy number could be achieved by expressing BFDV in trans from another co-transfected vector with a strong promoter: This is not surprising, considering the native *rep* promoter is quite weak and is probably not well recognized in mammalian cells, meaning expression in trans could mean a far higher availability of Rep.

These results open up a number of fascinating possibilities for using BFDV-derived sequences as replication-competent DNA expression and vaccine vectors, several of which we are currently investigating (W. de Moor, G. Regnard, A.-L. Williamson, E.P. Rybicki, unpublished results and ongoing work). There are currently no small DNA virus-derived vectors in use in vaccinology, other than recombinant adeno-associated viruses (rAAV), and AAV2 has recently been implicated in insertional mutagenesis in human hepatocellular carcinomas [37]. Papilloma- and polyomaviruses are also known to be associated with cancers, which may preclude their use as replicating vectors. The essentially ubiquitous ssDNA torque teno viruses are potentially associated with some human disease conditions, although causation is not proven [38,39].

Circoviruses have never been implicated in any human disease: Although PCV-1 and PCV-2 were famously discovered in live rotavirus vaccines given to millions of children [40], and PCV-1 was shown to be able to infect a human hepatocellular carcinoma cell line [41], there was no evidence that PCV-1 infected the infants given the contaminated rotavirus vaccine [42]. There have been concerns, however, over ssDNA viruses of pigs associated with xenotransplantation in humans [43], and swine–human contacts are frequent and worldwide in agriculture. Thus, use of a circovirus such as BFDV as the source of elements for a replicating DNA expression vector, when the virus is host-restricted to one type of birds and has never been associated with human disease, is probably more likely to be regarded as safe. Our preliminary investigations have revealed considerable promise in this regard; however, these will be reported elsewhere (W. de Moor, G. Regnard, A.-L. Williamson, E.P. Rybicki, unpublished results).
