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Leukocyte proliferation responses to cotton-tail rabbit papillomavirus (CRPV) were measured in vitro with fresh whole blood as well as with ammonium chloride lysis-separated leukocytes. The antigens used were (1) CRPV particles produced in the athymic( (nu/nu) mouse xenograft system and (2) purified bacterial fusion proteins of the CRPV major and minor capsid proteins L1 and L2. CRPV-infected domestic rabbits with persistent papillomas or after papilloma regression, as well as uninfected controls were studied. There was a clearcut difference between infected and uninfected animals. We demonstrated antigen-specific leukocyte proliferation to at least one CRPV antigen in 12 of 21 infected rabbits but there was no positivity in 9 control animals (P = 0.004). There was whole-blood reactivity preferentially to intact CRPV particles in regressors. Specific but weak leukocyte proliferation against CRPV particles was detected in 6 of 9 regressor rabbits (66%) but only in 1 of 12 progressors (8%; P = 0.0158). This trend of greater reactivity to intact CRPV particles in regressors as compared with progressors was not seen with peripheral blood leukocytes isolated by ammonium chloride lysis. We conclude that specific leukoproliferative responses against capsid CRPV proteins exist in rabbits experimentally infected with CRPV.

This study analyzed in vivo antiviral cellular immune reactions in the Shope rabbit papilloma – carcinoma model. Antigens studied in experimentally infected domestic rabbits were cottontail rabbit papillomavirus particles produced with the athymic (nu/nu) mouse xenograft system and bacterial fusion proteins containing the major or minor capsid protein. Recall reactions to antigens were tested by classic intracutaneous tests. Positive reactions had a biphasic course. Histopathology of skin test biopsy specimens showed infiltrating polymorphonuclear cells during the early stages. Later they were replaced by predominantly perivascular infiltrates composed of mononuclear cells. Time course of swelling and infiltrates resembled a delayed-type hypersensitivity reaction. Ten of 11 regressor rabbits (p = 0.00006) and 10 of 20 progressors (p = 0.009) had positive skin tests with intact and/or denaturated virus particles and individual capsid proteins also could elicit specific skin reactions. Skin reactivity to the cottontail rabbit papillomavirus particles was also greater (p = 0.042) in regressor rabbits (8 of 11) when compared to progressors (7 of 20). Recall reactions remained detectable at post-regression times, ranging from several months up to more than 2 years. We conclude that specific skin reactions against the cottontail rabbit papillomavirus in infected domestic rabbits exist, and are strongly positive to intact particles of this papillomavirus in animals (regressors) clinically free of disease.

Spontaneous regressions of papillomavirus lesions frequently occur in both human and animal infections. The mechanism by which this occurs is currently unknown. Mononuclear infiltrates are found in regressing human and rabbit papillomas. To assess the potential functional role of these infiltrates in regression, we have characterized and quantitated the cell types present in regressing rabbit lesions. Forty New Zealand white rabbits were inoculated with cottontail rabbit papillomavirus (CRPV) at 2 sites on the dorsal skin. All tumors on 6 rabbits markedly decreased in volume within 6 to 8 weeks of inoculation. Tumors on 4 of these 6 regressor rabbits were studied by immunohistochemistry. Regressor papillomas had conspicuous leucocytic infiltrates, most concentrated at the epithelial basement membrane, and often obliterating the basal cells of the germinal layer. Infiltrating leucocytes were also concentrated in the subjacent dermis immediately beneath the basement membrane. The infiltrates gradually lessened at increased depths in the dermis. In contrast, progressor papillomas contained fewer leucocytes, which were randomly distributed in the dermis. The phenotype of the infiltrating leucocytes was examined in 4 regressing and 12 progressing papillomas. In regressing papillomas, infiltrating leucocytes were predominantly T cells (68.0%), with relatively few B cells (7.4%). Progressing papilloma dermis contained fewer T cells and B cells than regressing papillomas. Most of the infiltrating T cells in regressing papillomas were labelled with a rabbit MHC-class-II-specific monoclonal antibody (MAC) (2C4), in contrast to only a small number in progressing papillomas. In addition to the leucocytic infiltrates, keratinocytes in regressing, but not in progressing, papillomas, frequently exhibited strong 2C4 staining. These results demonstrate that infiltration with T cells expressing rabbit class II is characteristic of regressing Shope papillomas and strengthens the assertion that cell-mediated immunity is the mechanism of Shope papilloma regression.