Virus-like particles of bovine papillomavirus type 4 in prophylactic and therapeutic immunization.

Virus-like particles were produced in insect cells containing either the L1 and L2 capsid proteins of bovine papillomavirus type 4 (BPV-4) or only the L1 protein. Both preparations of VLPs proved to be extremely effective prophylactic vaccines. Thirteen of 15 calves immunised with either L1-L2 VLPs or L1-VLPs were refractory to experimental challenge with high doses of BPV-4 and did not develop papillomas, while 9 of 10 control animals developed multiple oral papillomas. VLPs were not efficient as therapeutic vaccine in calves with established papillomas, although VLP-vaccinated animals appeared to undergo tumour regression more rapidly than nonvaccinated control animals. Antibody responses in VLP-vaccinated calves were associated with prevention of disease but not with regression of papillomas. Thus prophylactic VLP vaccination is effective in preventing disease in this model of mucosal papillomavirus infection. VLPs and native virus share at least some conformational epitopes, as shown by the cross-reactivity of their antibodies.

Vaccination of cattle with the N-terminus of L2 is necessary and sufficient for preventing infection by bovine papillomavirus-4.

We have previously shown that cattle vaccinated with L2, the minor structural protein of bovine papillomavirus-4 (BPV-4), do not develop alimentary papillomas upon challenge with BPV-4. Analysis of the B and T cell response in L2-vaccinated animals showed that the majority of the response was directed against the N-terminus and C-terminus of L2 with little response against the middle portion. Cattle were vaccinated with the N-terminus or the C-terminus of L2. The animals vaccinated with the N-terminus were completely protected from viral challenge, whereas the animals vaccinated with the C-terminus were not. Further analysis with synthetic overlapping peptides spanning the entire N-terminus mapped a B cell immunodominant epitope at amino acid 101-120. This epitope was recognised by all vaccinated animals.

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.

Humoral immune response to the E7 protein of bovine papillomavirus type 4 and identification of B-cell epitopes.

We have previously vaccinated cattle with E7, the major transforming protein of bovine papillomavirus-4, prior to homologous virus challenge. This retarded the development of papillomas and promoted their early regression compared to control animals. To understand the mechanism for this regression, we have studied the B and T cell response in vaccinated animals and compared it to that of non-vaccinated, virus-infected animals. The B cell response is reported here. The development of E7 IgG antibodies was detected after vaccination and before viral challenge, indicating that vaccine E7 is effectively presented to the immune system. In vaccinated animals titres of E7 antibodies remained high 10 weeks after viral challenge, whereas E7 antibodies in control animals were not detectable until 13 weeks post-viral challenge. Further analysis with synthetic overlapping peptides spanning the entire E7 protein mapped major immunodominant epitopes in the N and C termini of the protein and a minor epitope in the middle of the protein.

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.

Recombinant interleukin-4 promotes expression of the CD25 (Tac) antigen at the plasma membrane of high-density human tonsillar B lymphocytes.

High-density human B lymphocytes were prepared from tonsillar mononuclear cells by depletion of adherent cells and T lymphocytes, followed by discontinuous density gradient centrifugation. The B cells were analysed for the levels of expression of the CD25 (Tac) antigen marker by flow cytometry following culture with a variety of cytokines. IL-4 could induce elevated levels of CD25 on high-density, putatively resting B lymphocytes in a dose-dependent fashion. Expression of CD25 at the B-cell surface could not be promoted by interleukin-2 (IL-2), interferon-gamma (IFN-gamma) or by a crude preparation of B-cell growth factor 2 (BCGF-2). Mitogenic challenge of the B cells with pokeweed mitogen (PWM) and a combination of phorbol ester and calcium ionophore were similarly ineffective, although a small increase in CD25 expression could be detected when the B cells were cultured with phytohaemagglutinin A (PHA). The ability of IL-4 to promote CD25 expression was abolished by the presence of IFN-gamma in the culture. Titration experiments suggested that the amount of IL-4 required to produce a half-maximal increase in CD25 expression was approximately 40 U/ml; this is considerably greater than the 8-10 U/ml required to produce the equivalent effect on CD23 expression. The ability of IL-4 to promote CD25 expression in the high-density B-lymphocyte population was apparently independent of proliferation of the cells. IL-4 could not promote Tac expression on high-density T cells prepared from the same tissue source.

Appearance of a novel plasma membrane phosphoprotein following culture of resting murine B lymphocytes with recombinant interleukin 4.

Culture of high density resting murine B cells with recombinant IL-4 caused changes in the patterns of phosphoproteins associated with the plasma membranes isolated from such cells. The principal difference was the appearance of an alkali-resistant phosphoprotein of Mr = 75,000-80,000. Culture with LPS also gave rise to a characteristic alteration in phosphoprotein profiles, but this was distinct from the cytokine-induced changes and did not include induction of a 75 kDa signal. Culture in medium plus serum only failed to produce an alteration of the phosphoprotein profile of membranes isolated from B cells cultured in this manner. Culture of high density murine splenic B cells with IL-4 in the presence of the 11B11 anti-IL-4 monoclonal antibody prevented the appearance of the 75 kDa phosphoprotein, indicating that IL-4 binding was necessary for induction of the 75 kDa phosphoprotein signal. The possible molecular identity of the 75 kDa phosphoprotein and its role in control of B lymphocyte differentiation is discussed.

The effect of recombinant interleukin 4 upon protein kinase activities associated with murine and human B lymphocyte plasma membranes.

Exposure of plasma membranes isolated from high density resting murine B cells to recombinant IL-4 in the presence of gamma-[32P]-ATP promoted phosphorylation of a protein of Mr = 42,000. The 42 Kd protein kinase substrate could be detected in membranes prepared from low density B cells following a 24 h culture with lipopolysaccharide, but not in membranes prepared from B cells exposed to LPS for 48 h. Treatment of the cells with LPS resulted in the appearance of a number of new membrane-associated phosphoproteins. Treatment with the cytokine also resulted in the disappearance of a protein kinase substrate of Mr = 30,000 from phosphoprotein profiles of membranes prepared from cells exposed to LPS for 24 h. The 42 Kd structure appears to be a protein kinase substrate rather than possessing intrinsic phosphotransferase activity as judged from experiments employing 8-azido-gamma-[32P]-ATP as a photoaffinity label. No 42 Kd species was detectable using this reagent. Experiments employing identical protocols failed to reveal any enhanced or diminished phosphorylation of membrane-associated proteins in human peripheral blood B cells or in human B lymphoma cell lines.