A review of the older literature (refs 88-95) indicates that papilloma viruses in general cannot be 
produced in tissue culture systems despite many attempts. The reason for this is the fact that the 
expression of the capsid proteins LI and L2 and virus production seem to occur only in terminally 
differentiated epithelial cells that do not grow in culture. A typical quotation states, " The virus cannot 
be propagated in cell culture and studies at the molecular level require application of its DNA in 
bacteria" (89). Live virus has been generated only from the extraction from warts or fibro papillomas 
(formed in vivo). Thus, since the intact virus can not be generated in vitro, it will also be impossible 
to generate an incomplete virus lacking both major structural proteins (LI, L2). Direct assays in this 
direction are therefore futile. 
That the LI and L2 protein (missing in pBMG-Neo) are essential for viral assembly is evident from 
a recent publication (Kirnbauer et al. 1992, ref 96) demonstrating that the LI capsid protein (produced 
by recombinant DNA techniques) on its own is sufficient for assembly of virus-like particles. LI of 
BPV1 forms pentamers which assemble into 72 pentameric capsomeres, the same organization seen in 
wild type virus extracted from bovine warts. It seems evident that a viral DNA lacking LI and L2 will 
not be able to produce functional viral particles. 
The LI protein is highly immunogenic and generates a neutralizing antibody response which would 
also decrease the risk of viral spread (96). 
4.4.4 Discussion of risk assessment of the Vector System: 
Plasmid BMG-neo is derived from the bovine papilloma virus, which is a transforming virus in 
some cells. For the purpose of this project the following points are relevant: 
1. Tumor cells are being transfected with lymphokine cDNA. Tumor cells are transformed by 
definition and the risk of further transformation by the plasmid appears insignificant. Growth rate 
comparisons and morphology of untransfected and transfected tumor cells showed no difference for 
murine LLC and human SCLC. 
2. The multicopy number (20-100/cell) of the plasmid is an important advantage of pBMG-Neo (see 
preliminary results). It allows very high levels of lymphokine cDNA expression which may be important 
for the induction of an immune response. The multicopy plasmid in addition allows continued 
production of lymphokine even after lethal irradiation of the tumor cell. 
3. Tumor cells will be lethally irradiated prior to injection precluding their proliferation at the 
injection site (see chapter 4.6.2). Lymphokine production, will mediate a cellular immune response. 
This response will make it unlikely that any of the cells escaping radiation will survive. 
4. No risk of virus production is apparent since only a viral fragment is used and since helper virus 
is unnecessary for packaging (see also next chapter 4.4.5). 
5. No risk of insertional mutagenesis, since the plasmid replicates extrachromosomally as episome 
at a copy number of 20-100 per cell (depending on the cell type). 
6. Animal papilloma viruses are generally species specific (4), further reducing the risk of viral 
spread (see also 4.4.5). 
7. Stable and high levels expression of cDNAs under the murine metallothionein promotor is 
regularly achieved in human and other mammalian cells most likely due to the intermediate plasmid 
copy number. Generally, 50-100 fold more protein can be expressed than with single copy vectors (5,6). 
High level and stable expression may be important for the success of gene therapy. 
8. The plasmid contains a selectable marker for neomycin resistance and has been constructed to 
allow expression of one or two cDNAs simultaneously. The use of the vector is not limited by size 
restrictions of inserted DNA. 
Recombinant DNA Research, Volume 18 
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