William Fleming Hoggan Jarrett

Prophylactic and therapeutic vaccination against a mucosal papillomavirus

Papillomaviruses are ubiquitous DNA viruses affecting humans and animals and causing a variety of tumours of mucosal and cutaneous epithelia. Some of these lesions, particularly those affecting mucosal epithelia, can progress to squamous cell carcinomas. Prevention or cure of viral infection would ultimately lead to a decrease in the incidence of papillomavirus-associated cancers. Using recombinant proteins, we have developed prophylactic and therapeutic vaccines against bovine papillomavirus type 4, a mucosal papillomavirus implicated in cancer of the alimentary canal in cattle; similar possibilities exist for the human mucosal papillomaviruses.

Studies on vaccination against papillomaviruses: Prophylactic and therapeutic vaccination with recombinant structural proteins

The L1 and L2 proteins of BPV-2 have been produced in Escherichia coli as beta-galactosidase fusion proteins. The fusion proteins have been used to vaccinate calves both prophylactically and therapeutically. The L1 fusion protein prevented tumor formation when administered before challenge with BPV-2, while the L2 fusion protein was very effective in promoting tumor rejection, independently from whether it was administered before or after challenge. Animals vaccinated with L1, but not with L2, responded rapidly with production of serum neutralizing antibodies, showing that this peptide contains B-cell-specific epitopes. The massive infiltration of lymphocytes in the tumors of L2-vaccinated animals suggests that the peptide contains epitopes specific for T-cells. The two structural proteins of BPV-2 therefore interact with both efferent arms of the immune system, and this observation allows the choice between two different types of antiviral vaccination.

Latent papillomavirus infection in cattle

During a long term experiment designed to identify the contribution of bovine papillomavirus type 4 (BPV-4), environmental mutagens and immunosuppressants to the development of carcinomas of the upper alimentary tract of cattle, there was evidence of latent papillomavirus infection. Papillomatosis-free animals, when immunosuppressed either by feeding bracken fern or by azathioprine treatment, developed skin warts containing either BPV-1 or BPV-2. Skin warts appeared also in an immunocompetent animal at sites of damaged skin. It was concluded that the animals harboured latent papillomavirus which was reactivated by immunosuppression and/or physical trauma, causing skin warts. Papillomavirus DNA was also detected in lymphocytes of both experimental and control animals, suggesting that one of the sites of latency may be the circulating lymphocyte.

A Cutaneous Fibropapilloma from a Red Deer (Cervus elaphus) Associated with a Papillomavirus

A cutaneous fibropapilloma was found on a Scottish red deer (Cervus elaphus), and a papillomavirus was isolated from it. The virus appeared to be related to bovine papillomavirus type 1 (BPV1) or type 2 (BPV2) because: (i) it cross-reacted in peroxidase-antiperoxidase tests with antisera raised against these virions; (ii) BPV1 and BPV2 DNAs cross-hybridized to the red deer papillomavirus in situ; and (iii) BPV1 and/or BPV2 DNA cross-hybridized to the red deer papillomavirus DNA on Southern blots under conditions of high stringency. These tests also revealed a unique restriction enzyme cleavage pattern for the red deer papillomavirus DNA.

T cell responses to BPV-4 E7 during infection and mapping of T cell epitopes.

Vaccination of cattle with the recombinant E7 protein of bovine papillomavirus type 4 (BPV-4) prior to BPV-4 infection has been shown to retard development of papillomas and accelerate their regression. To understand the mechanism of regression we have measured proliferation of peripheral blood mononuclear cells (PBM) to E7 in vitro during the course of BPV-4 infection in both vaccinated and nonvaccinated cattle. In vaccinated cattle, T cells specific for E7 could be detected at high levels shortly after challenge, whereas in nonvaccinated cattle low responses of E7-specific T cells could be detected in only a few animals at the late stages of papilloma development. Using short overlapping synthetic peptides corresponding to the E7 protein, three T cell epitopes have been identified. T1 (aa 31-59) was immunodominant and T2 (aa 70-88) and T3 (aa21-40) were minor epitopes.

The Leeuwenhoek lecture, 1986. Environmental carcinogens and papillomaviruses in the pathogenesis of cancer.

In many areas of the world there is a geographically localized high incidence of alimentary and bladder cancer in cattle. Studies in western Scotland have demonstrated that this phenomenon is associated with ingestion of bracken fern. However, the affected animals and herds were shown also to have an unusually high infection rate of alimentary papillomas caused by a previously unrecognised bovine papillomavirus (BPV) and that these tumours could undergo malignant transformation. Long-term field and experimental studies were started and indicate that the pathogenesis of the tumours and their relationship to virus infection and food-derived mutagens is complex. Results from these studies, and from cellular and molecular biology experiments, are presented and discussed in the context of recent papillomavirus findings in the human subject.

Malignant transformation of a papilloma induced by bovine papillomavirus type 4 in the nude mouse renal capsule

A papillomatous cyst was induced by implanting bovine foetal palate epithelium, infected in vitro with bovine papillomavirus type 4 (BPV-4), beneath the renal capsule of a nude mouse. The benign tumour underwent malignant progression, developing into a squamous cell carcinoma with metastatic deposits in the spleen. The bovine origin of both the renal and splenic cancers was confirmed by the presence of bovine major histocompatibility complex class I antigens in the cancer cells and by sequencing the Harvey-ras 1 gene, which was shown to be of bovine origin. BPV-4 DNA was present in the residual papillomatous fronds of the renal cancer, but was absent from the carcinoma proper and for the splenic metastasis. These results confirm that BPV-4 is a carcinogenic agent and that its genetic information is not necessary for the maintenance of the malignant phenotype. Moreover the system provides the opportunity to investigate the role of viral and chemical carcinogens in an experimental system.

Vaccination of Cattle with L2 Protein Prevents BPV-4 Infection

Papillomaviruses can infect and cause tumours in both cutaneous and mucousal. epithelia. The virus induced tumours can progress to squamous cell carcinoma, as in the case of HPV-16 associated cervical. carcinoma (zur Hausen, 1991) and BPV-4 associated alimentary cancer in cattle (Campo & Jarrett, 1986). There is a requirement for prophylactic and therapeutic vaccines which would protect against infection and accelerate rejection of established tumours. Potential. vaccines are now possible with the use of recombinant proteins. Vaccination studies are difficult in humans for several. reasons and in addition little is known about the immune response to papillomavirus. We have used BPV-4 infection in cattle as a model system allowing the opportunity for vaccination studies using the early and late proteins of BPV-4 and analyses of the immune response. We have previously shown that vaccination with the E7 protein inhibits the development of papillomas and causes early rejection of papillomas (Campo et al., 1993). This is accompanied by an increase in E7 specific antibodies and T cells. We have now shown that immunisation with the late protein, L2, of BPV-4 gives virtually complete protection against BPV-4 infection (Campo et al, 1993). In this report we investigate the immune response to the L2 protein in vaccinated and unvaccinated cattle in order to understand the reason for the protective effect of L2.

Interaction between bovine papillomavirus type 4 and cocarcinogens in the production of malignant tumours.

Bovine fetal palatine tissue infected with bovine papillomavirus type 4 (BPV-4) was implanted subcutaneously in athymic nude mice. The implants developed into cysts containing papillomas essentially the same as those in the natural host. In order to investigate the interaction of cocarcinogens with BPV-4 in cell transformation, the virus-infected implants were exposed in vivo to either the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) or the tumour initiator 7,12-dimethylbenz[a]-anthracene (DMBA). Papillomas were detected in a greater number of infected implants in the presence of either TPA or DMBA than in the absence of either of these chemicals indicating interaction between the virus and these two agents. Moreover, malignantly transformed cells arose with high frequency from infected implants that had been exposed to either chemical. In the presence of chemical and absence of virus or vice versa no neoplastic changes were seen histologically, indicating that cooperation between virus and cocarcinogen is required for transformation.

Vaccination Against Cutaneous and Mucosal Papillomavirus in Cattle

Viruses are responsible for approximately 15% of human cancer worldwide. Human papillomavirus and hepatitis B virus are the recognized agents of cervical and liver cancer, respectively, which together constitute 80% of all virally induced cancers. If measures could be found to bring viral infection under control, a great proportion of human cancer would be greatly reduced. Experimental vaccines are being developed against papillomavirus. In principle two different types of vaccine can be envisaged: prophylactic vaccines that would elicit virus-neutralizing antibodies and would prevent infection and therapeutic vaccines that would induce regression of established lesions before progression to malignancy took place. The research on vaccines against human papillomavirus is hampered by the difficulties encountered in growing the virus in tissue culture and by the unacceptable nature of experimentation in humans. Effective vaccines, both natural and genetically engineered, have been developed against bovine papillomavirus and cottontail rabbit papillomavirus. The success obtained with the animal models supports the optimistic prediction that in the relatively near future vaccines will be available against the most problematic or potentially dangerous forms of papillomatosis in humans.