Department of Pathology, Cambridge University, Cambridge CB2 1QP, UK1
Glaxo Smithkline Research and Development, Medicines Research Centre, Stevenage, Hertfordshire SG1 2NY, UK2
Author for correspondence: Richard Moore. Fax +44 1223 333730. e-mail ram36{at}cam.ac.uk
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Abstract |
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COPV infection is a valuable mucosal model of its human counterparts, including the cervical carcinoma-inducing high-risk HPV types. COPV induces warts on the oral mucosa in domestic dogs and wild canids. Typically, COPV infection has an incubation period of 48 weeks followed by the production of florid papillomatosis and spontaneous immune-mediated regression within a further 48 weeks; recurrence of disease has rarely been reported. In this study, six beagles were challenged with COPV at the oral mucosa (OM) and five of the six animals developed warts at the vast majority of challenge sites. The infection was in a well-characterized laboratory species with an immunology similar to that of humans, and the target tissue was easily observable and mucosal rather than cutaneous.
The recurrence rate of genital warts in humans is high, so any vaccine that can prevent a latent infection or induce clearance would be a significant advance. In this report, the COPV model was employed as a mucosal model of PV infection in humans. Prophylactic vaccination using particle-mediated immunotherapeutic delivery (PMID) of COPV L1 was used and the results of this vaccination have been reported elsewhere (Stanley et al., 2001 ). PMID is an intra-epithelial vaccination technique whereby plasmid DNA bound to gold beads is propelled using helium. In this study, six animals were vaccinated with hepatitis B virus antigen (HBS) DNA (animals 16) and six with COPV L1 DNA (animals 712), both encoded on cytomegalovirus (CMV)-driven plasmids. All animals were challenged at ten sites of the upper OM using purified native COPV. Five of the six HBS-vaccinated animals developed warts at the majority of challenge sites. Animal number 1 had no visible lesions but seroconverted at a similar time to the other control animals and was thought to have undergone a subclinical infection. In contrast, all of the L1-vaccinated animals were protected against subsequent virus challenge (Stanley et al., 2001
). Vaccination and challenge were as previously reported (Stanley et al., 2001
).
Post-mortem, the entire upper oral mucosa was removed from each animal and lysed as follows. The OM was finely chopped and resuspended in proteinase K buffer containing 50 µg/ml RNase A. This was incubated for 30 min at 37 °C, before addition of 50 µg/ml proteinase K. The OM was then placed in a 50 °C water bath for 30 min and incubated overnight at 37 °C.
PCR was performed using 12 µl of OM lysate as template in a 100 µl reaction. Sets of primers were used covering a short region of each open reading frame (ORF) within the COPV genome; these primer sequences have been previously published (Nicholls et al., 1999 ). PCR reaction conditions were 94 °C for 5 min, followed by 30 cycles of 1 min at 94 °C, 1 min at 51 °C and 2 min at 72 °C, with a final extension of 10 min at 72 °C. The results were then viewed directly by agarose gel electrophoresis.
The serological and biological readout has been reported elsewhere in full (Stanley et al., 2001 ). The OM of the animals was removed post-regression and analysed by PCR, as described above, for detection of latent COPV DNA. Table 1
indicates the timing of wart appearance and regression, as well as the date of OM removal.
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The data presented in this communication demonstrate that PMID is capable not only of protection from subsequent challenge but also of prevention of any significant virus entry. An alternative explanation for the lack of COPV DNA in the L1 vaccinees could be that the vaccine induced virus clearance, although this explanation seems the less probable of the two.
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References |
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Received 11 January 2002;
accepted 5 April 2002.