Langerhans cell homeostasis and activation is altered in hyperplastic human papillomavirus type 16 E7 expressing epidermis.

It has previously been shown that expression of human papillomavirus type 16 (HPV) E7 in epidermis causes hyperplasia and chronic inflammation, characteristics of pre-malignant lesions. Importantly, E7-expressing epidermis is strongly immune suppressed and is not rejected when transplanted onto immune competent mice. Professional antigen presenting cells are considered essential for initiation of the adaptive immune response that results in graft rejection. Langerhans cells (LC) are the only antigen presenting cells located in normal epidermis and altered phenotype and function of these cells may contribute to the immune suppressive microenvironment. Here, we show that LC are atypically activated as a direct result of E7 expression in the epidermis, and independent of the presence of lymphocytes. The number of LC was significantly increased and the LC are functionally impaired, both in migration and in antigen uptake. However when the LC were extracted from K14E7 skin and matured in vitro they were functionally competent to present and cross-present antigen, and to activate T cells. The ability of the LC to present and cross-present antigen following maturation supports retention of full functional capacity when removed from the hyperplastic skin microenvironment. As such, opportunities are afforded for the development of therapies to restore normal LC function in hyperplastic skin.

Transcriptional repression of E-cadherin by human papillomavirus type 16 E6.

There is increasing evidence supporting DNA virus regulation of the cell adhesion and tumour suppressor protein, E-cadherin. We previously reported that loss of E-cadherin in human papillomavirus (HPV) type 16-infected epidermis is contributed to by the major viral proto-oncogene E6 and is associated with reduced Langerhans cells density, potentially regulating the immune response. The focus of this study is determining how the HPV16 E6 protein mediates E-cadherin repression. We found that the E-cadherin promoter is repressed in cells expressing E6, resulting in fewer E-cadherin transcripts. On exploring the mechanism for this, repression by increased histone deacetylase activity or by increased binding of trans-repressors to the E-cadherin promoter Epal element was discounted. In contrast, DNA methyltransferase (DNMT) activity was increased in E6 expressing cells. Upon inhibiting DNMT activity using 5-Aza-2'-deoxycytidine, E-cadherin transcription was restored in the presence of HPV16 E6. The E-cadherin promoter was not directly methylated, however a mutational analysis showed general promoter repression and reduced binding of the transactivators Sp1 and AML1 and the repressor Slug. Expression of E7 with E6 resulted in a further reduction in surface E-cadherin levels. This is the first report of HPV16 E6-mediated transcriptional repression of this adhesion molecule and tumour suppressor protein.

Screening of drugs to counteract human papillomavirus 16 E6 repression of E-cadherin expression.

Persistent infections with certain high-risk human papillomavirus (HPV) types such as 16 and 18 can result in the development of cervical cancer. Neither of the two prophylactic vaccines against HPV16 and 18 that are in current use have any therapeutic efficacy for prevalent HPV infections. Ablative therapy is widely used for the treatment of HPV cervical dysplasia however disease recurrence is a widely recognized problem. Thus there is a continuing need for therapeutic approaches for the treatment of HPV infections. The HPV16 E6 viral oncoprotein represses surface expression of the cellular adhesion molecule, E-cadherin. Reduced E-cadherin expression on HPV-infected keratinocytes is associated with lowered numbers of antigen-presenting Langerhans cells in the infected epidermis, potentially reducing immune surveillance for HPV. Four chemicals reported to up-regulate E-cadherin were screened for their ability to counteract E6 repression of surface E-cadherin. 5-Aza-2'-deoxycytidine (AzaDC), a DNA methyltransferase inhibitor, and Indole-3-carbinol (I3C), reported to increase E-cadherin through a p21(Waf1/Cip1)-dependent mechanism, had low cytotoxicity and increased or restored E-cadherin expression and adhesive function in HPV16 E6 expressing HCT116 cells. Doxorubicin, also known to induce p21(Waf1/Cip1), increased E-cadherin in E6 expressing cells but had some associated cytotoxicity. Tamoxifen, which can restore adhesive function of surface E-cadherin, was ineffective in counteracting E6 repression of E-cadherin. AzaDC and I3C both show potential to restore antigen-presenting cells to HPV infected skin by antagonizing E6 repression of E-cadherin, thereby counteracting an important immune evasion mechanism of HPV16 and reinstating immune function at the infected site.

The immune response to papillomavirus during infection persistence and regression.

Human papillomavirus (HPV) infections cause a significant global health burden, predominantly due to HPV-associated cancers. HPV infects only the epidermal cells of cutaneous and mucosal skin, without penetration into the dermal tissues. Infections may persist for months or years, contributed by an array of viral immune evasion mechanisms. However in the majority of cases immunity-based regression of HPV lesions does eventually occur. The role of the innate immune response to HPV in persistence and regression of HPV infection is not well understood. Although an initial inflammatory infiltrate may contribute to disease regression, sustained inflammation at the HPV-induced lesions, characterized by macrophage and neutrophil infiltration, has been observed in persistence. Pathogen-associated molecular patterns (PAMPs) are important in innate recognition. The double stranded DNA and an L1 and L2 capsid components of the HPV virion are potential PAMPs that can trigger signaling through cellular pattern recognition receptors, including toll-like receptors (TLR). TLR expression is increased in regressing HPV disease but is reduced in persistent lesions, suggesting a role for TLR in HPV regression. With regard to the adaptive immune response, a key indicator of regression in humans is infiltration of the lesion with both CD4 and CD8 T cells. In individuals with persistent lesions, CD8 T cell and immune suppressive regulatory T cells (Tregs) infiltrate the infection site. There is no association between persistence or regression and the presence of serum antibodies to the viral capsid antigens of HPV. There is still much to be learned about the immunological events that trigger regression of HPV disease. Understanding the viral and host factors that influence persistence and regression is important for the development of better immunotherapeutic treatments for HPV-associated disease.

Loss of epidermal Langerhans cells occurs in human papillomavirus alpha, gamma, and mu but not beta genus infections.

Human papillomaviruses (HPVs), which are contained in the alpha, beta, gamma, mu, and nu genera, differ in their oncogenic potential and their tropism for cutaneous or mucosal epidermis. Langerhans cells (LC), the only epidermal professional antigen-presenting cells, are readily detected in normal mucosal and cutaneous epithelium. The aim of this study is to determine whether LC loss, which has been reported for HPV16, occurs in other HPV genera and establish its significance in viral pathology. We found that, as for HPV16, LCs were reduced in lesions infected with high-risk mucosal (alpha7 and alpha9 species) and low-risk cutaneous (gamma and mu) types. Lesions infected with alpha10 low-risk genital types had reduced LC but contained epidermal LC patches, coincident with dermis-localized regulatory T cells (T-regs). In contrast to other genera, LCs were common in the epidermis, and T-regs occupied the dermis of the potentially high-risk cutaneous beta-HPV type infected lesions. Therefore, LC loss in the infected lesions occurred irrespective of tropism or oncogenic potential of the HPV type. LC depletion in the HPV-infected epidermis may create an environment that is permissive for viral persistence and in HPV lesions in which LCs are found, the presence of typically immunosuppressive T-regs may compensate for their continued presence.

The orf virus inhibitor of apoptosis functions in a Bcl-2-like manner, binding and neutralizing a set of BH3-only proteins and active Bax.

We have previously shown that the Orf virus protein, ORFV125, is a potent inhibitor of the mitochondrial pathway of apoptosis and displays rudimentary sequence similarities to cellular anti-apoptotic Bcl-2 proteins. Here we investigate the proposal that ORFV125 acts in a Bcl-2-like manner to inhibit apoptosis. We show that the viral protein interacted with a range of BH3-only proteins (Bik, Puma, DP5, Noxa and all 3 isoforms of Bim) and neutralized their pro-apoptotic activity. In addition, ORFV125 bound to the active, but not the inactive, form of Bax, and reduced the formation of Bax dimers. Mutation of specific amino acids in ORFV125 that are conserved and functionally important in mammalian Bcl-2 family proteins led to loss of both binding and inhibitory functions. We conclude that ORFV125's mechanism of action is Bcl-2-like and propose that the viral protein's combined ability to bind to a range of BH3-only proteins as well as the active form of Bax provides significant protection against apoptosis. Furthermore, we demonstrate that the binding profile of ORFV125 is distinct to that of other poxviral Bcl-2-like proteins.

Increased apoptosis and reduced replication efficiency of the E3 region-modified dl309 adenovirus in cancer cells.

The Adenovirus (Ad) dl309 mutant, which lacks several E3 region genes, has been used as the backbone for a number of replication selective cytopathic Ads designed to treat tumours. We report that dl309 has enhanced cytopathogenicity in a range of different cell lines when compared with Ad5. The E3 region modifications found in dl309 contributed to reduced late gene expression in both cocksackie-adenovirus receptor (CAR) positive and negative cells. We show that completion of the dl309 viral lifecycle was less efficient and apoptosis was triggered in the CAR negative K1 thyroid cancer-derived cell line. There was increased E1A expression in dl309-infected K1 cells, compared with Ad5, and significantly, whereas E1A in Ad5-infected cells was distributed both in the nuclear and cytoplasmic compartments, E1A was predominantly nuclear in dl309-infected K1 cells. From these results we conclude that the regions of dl309 that are deleted or otherwise modified can contribute to viral replication and inhibition of apoptosis, possibly indirectly by regulating E1A. These data have implications in the development of dl309-based Ads for the treatment of tumours in vivo.

A novel Bcl-2-like inhibitor of apoptosis is encoded by the parapoxvirus ORF virus.

Apoptotic cell death forms part of the host defense against virus infection. We tested orf virus, a member of the poxvirus family, for the ability to inhibit apoptosis and found that orf virus-infected cells were fully resistant to UV-induced changes in cell morphology, caspase activation, and DNA fragmentation. By using a library of vaccinia virus-orf virus recombinants, we identified an orf virus gene (ORFV125) whose presence was linked with the inhibition of apoptosis. The 173-amino-acid predicted protein had no clear homologs in public databases other than those encoded by other parapoxviruses. However, ORFV125 possessed a distinctive C-terminal domain which was necessary and sufficient to direct the protein to the mitochondria. We determined that ORFV125 alone could fully inhibit UV-induced DNA fragmentation, caspase activation, and cytochrome c release and that its mitochondrial localization was required for its antiapoptotic function. In contrast, ORFV125 did not prevent UV-induced activation of c-Jun NH2-terminal kinase, an event occurring upstream of the mitochondria. These features are comparable to the antiapoptotic properties of the mitochondrial regulator Bcl-2. Furthermore, bioinformatic analyses revealed sequence and secondary-structure similarities to Bcl-2 family members, including characteristic residues of all four Bcl-2 homology domains. Consistent with this, the viral protein inhibited the UV-induced activation of the proapoptotic Bcl-2 family members Bax and Bak. ORFV125 is the first parapoxvirus apoptosis inhibitor to be identified, and we propose that it is a new antiapoptotic member of the Bcl-2 family.

A flow cytometry-based assay for the measurement of protein regulation of E-cadherin-mediated adhesion.

Epithelial (E)-cadherin is a transmembrane protein that mediates calcium-dependent cell adhesion. E-cadherin has significant roles in tissue development, adhesion between keratinocytes and retention of Langerhans cells in the epidermis, and its loss on tumour cells is frequently associated with metastasis. Here we describe a simple, flow cytometric adhesion assay to measure the effects of potential regulators of cell surface E-cadherin expression on E-cadherin-mediated adhesion between cells. In this assay, cells that have been transiently transfected to express the protein of interest are enzymatically treated to remove cell surface E-cadherin. Cells are then incubated in low attachment plates, during which time the E-cadherin is re-expressed and E-cadherin-mediated aggregation occurs. The effect of the protein of interest on the percentage of E-cadherin-mediated aggregates that form during incubation is measured flow cytometrically, following staining with an E-cadherin specific antibody. A major advantage of this assay is that a potential regulatory protein of interest can be tested in a transient expression system by co-expression with green fluorescent protein and analysis of adhesion conducted on green fluorescent cells only. We have applied this assay to measure the regulatory effects of E6 protein from human papillomavirus type 16 on E-cadherin-mediated adhesion but this assay potentially has broad applicability for testing the effects of other proteins on E-cadherin-mediated adhesion in an accurate and highly reproducible manner.

Flow cytometric quantitation of the protective efficacy of dendritic cell based vaccines in a human papillomavirus type 16 murine challenge model.

A murine model for the assessment of protective immunity to human papillomavirus (HPV) type 16, a virus that does not naturally infect mice, is described. In this system, protection was tested following intranasal challenge of mice with a recombinant vaccinia virus expressing both the selected HPV antigen and a beta-galactosidase (beta-gal) reporter. The extent of viral infectivity was determined by measuring beta-gal positive lung cells using flow cytometry. The efficacy of this model to measure protective immunity was evaluated by priming mice with the beta-gal vaccinia virus then challenging the mice with the same virus. Vaccinia primed mice had negligible numbers of beta-gal positive cells in the lung 5 days following viral challenge indicating protection, whereas around 50% of cells were infected in immunologically naive, challenged mice. The protective efficacy of two dendritic cell vaccines for HPV16 was measured in this model. Both vaccines provided some protection to subsequent viral challenge, compared with their controls. Although this protection model was applied to HPV in this study, it may also have broad application to other viruses that do not infect mice naturally.