Ex vivo detection of adenovirus specific CD4+ T-cell responses to HLA-DR-epitopes of the Hexon protein show a contracted specificity of T(HELPER) cells following stem cell transplantation.

Human adenovirus (HAdV) is a cause of significant morbidity and mortality in immunocompromised patients, especially after stem cell transplantation (SCT). Viral clearance has been attributed to CD4(+) T-cell responses against the Hexon-protein, but the frequency of specific T(HELPER) cells is extremely low or not detectable ex vivo and preference for different CD4(+) T-cell epitopes is variable among individuals. We therefore analyzed 44 healthy donors and 6 SCT-recipients for Hexon-specific CD4(+)-responses ex vivo, to identify epitopes which would be broadly applicable. We selected 19 candidate epitopes with predicted restriction to HLA-DR1/DR3/DR4/DR7; 16 were located within the highly conserved regions, indicating cross-reactivity of T cells among HAdV-subspecies. Ten epitopes induced CD4(+)-proliferation in >50% of individuals, confirmed by intracellular IFN-gamma detection. Three SCT recipients who recovered from an infection with HAdV displayed reactivity towards only a single hexon epitope, whereas healthy individuals were responsive to two to eight epitopes (median 3). The ex vivo detection of Hexon-specific CD4(+) T-cells, without any long-term culture in vitro, enables the detection and generation of HAdV-specific CD4(+) T cells for adoptive T-cell transfer against HAdV-infection post SCT.

Clinical grade generation of hexon-specific T cells for adoptive T-cell transfer as a treatment of adenovirus infection after allogeneic stem cell transplantation.

Adenovirus infection after allogeneic hematopoietic stem cell transplantation is still causing significant morbidity and mortality, especially in children. It has been demonstrated that a sufficient host T-cell response is essential to clear the virus. Adoptive transfer of specific T-cell immunity from the donor to the recipient has become a new treatment option for patients with systemic adenoviral infection who lack specific T-cell responses. The adenoviral hexon protein was shown to be an immunodominant T-cell target. We describe here a Good Manufacturing Practice-compatible generation of hexon-specific T cells developed by isolating interferon-gamma-secreting T cells after stimulation of mononuclear cells ex vivo with hexon protein. Phenotypical and functional characterization of the generated, specific T-cell product resulted in a mixed population of CD4 and CD8-positive T cells with an intermediate effector memory phenotype. Isolated hexon-specific T cells showed high expansion potential in vitro and specific cytotoxicity. T-cell lines, directed against type 5 hexon protein showed good crossreactivity against viral strains from other adenovirus species. The availability for isolation of hexon-specific T cells among 76 hematopoietic stem cell transplantation donors showed in > 72% a sufficient T-cell response (0.05% of T cells). In conclusion, Good Manufacturing Practice-grade selection of adenovirus-specific T cells for adoptive immunotherapy by hexon-induced secretion of interferon-gamma has been established. Adoptive T-cell transfer could potentially restore T-cell immunity against adenovirus after allogeneic stem cell transplantation.

The E7 protein of the cottontail rabbit papillomavirus immortalizes normal rabbit keratinocytes and reduces pRb levels, while E6 cooperates in immortalization but neither degrades p53 nor binds E6AP.

Human papillomaviruses (HPVs) cause cervical cancer and are associated with the development of non-melanoma skin cancer. A suitable animal model for papillomavirus-associated skin carcinogenesis is the infection of domestic rabbits with the cottontail rabbit papillomavirus (CRPV). As the immortalizing activity of CRPV genes in the natural target cells remains unknown, we investigated the properties of CRPV E6 and E7 in rabbit keratinocytes (RK) and their influence on the cell cycle. Interestingly, CRPV E7 immortalized RK after a cellular crisis but showed no such activity in human keratinocytes. Co-expressed CRPV E6 prevented cellular crisis. The HPV16 or CRPV E7 protein reduced rabbit pRb levels thereby causing rabbit p19(ARF) induction and accumulation of p53 without affecting cellular proliferation. Both CRPV E6 proteins failed to degrade rabbit p53 in vitro or to bind E6AP; however, p53 was still inducible by mitomycin C. In summary, CRPV E7 immortalizes rabbit keratinocytes in a species-specific manner and E6 contributes to immortalization without directly affecting p53.