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To investigate the efficiency of encapsidation of plasmid by papillomavirus virus-like particles (PV VLPs), and the infectivity of the resultant PV pseudovirions, Cos-1 cells were transfected with an 8-kb plasmid incorporating a green fluorescent protein (GFP) reporter gene (pGSV), and infected with bovine PV (BPV-1) L1/L2 recombinant vaccinia virus to produce BPV1 pseudovirions. Approximately 1 in 1.5×104 of dense (1.35 g/ml) PV pseudovirions and 0.3 in 104 of less-dense (1.29 g/ml) pseudovirions packaged an intact pGSV plasmid. The majority (>75%) of packaged plasmids contained deletions, and the deletions affected all tested genes. After exposure of Cos-1 cells to BPV-1 pseudovirions at an MOI of 40,000:1, 6% of cells expressed GFP, giving a calculated efficiency of delivery of the pGSV plasmid, by pseudovirions which had packaged an intact plasmid, of approximately 5%. Plasmid delivery was not effected by purified pGSV plasmid, was blocked by antiserum against BPV-1, and was not blocked by DNase treatment of pseudovirions, confirming that delivery was mediated by DNA within the pseudovirion. We conclude that a major limitation to the use of PV pseudovirions as a gene delivery system is that intact plasmid DNA is not efficiently selected for packaging by VLPs in cell-based pseudovirions production systems. [ABSTRACT FROM AUTHOR]

An immunoradiometric assay for IgG class autoantibody to liver membrane antigens, based on serum binding to glutaraldehyde treated monkey hepatocytes, was used to examine sera from patients with chronic active hepatitis (CAH) and other acute and chronic liver diseases. All sera from normals and patients showed binding, up to a titre of 1/2,048. For comparison of assays, results were normalized by selecting two reference sera, one with a high degree of binding, and one from a healthy subject with a low degree of binding: at a dilution of 1/2,048, these sera were given binding values of 100% and 0%. The values for the binding of unknown sera at the same dilution were calculated from these two reference values. For 26 patients with autoimmune CAH, the mean (±s.d.) percentage binding value (70 ±33%) was significantly higher than the mean value for 26 healthy subjects (10±15%), and high binding values were significantly associated with biochemically active hepatitis. The mean percentage binding value was moderately increased for eight patients with HBsAg associated CAH (42±12%), 13 patients with alcoholic hepatitis with cirrhosis (37±25%) and 45 patients with acute viral hepatitis A (40±27%) or B (52±37%). At a cut-off binding value of 65%, the assay as a single diagnostic procedure was shown to have a 70% sensitivity and a 95% specificity for the diagnosis of autoimmune CAH. Better understanding of the pathogenetic significance of antibodies to liver membrane antigens in CAH and other liver diseases will depend upon biochemical analysis of the presumably multiple antigenic determinants on the hepatocyte membrane. [ABSTRACT FROM AUTHOR]

Cell mediated immunity (CMI) to hepatitis B viral antigens was studied in BALB/mice after immunization with purified hepatitis B surface antigen (HBsAg), or core antigen (HBcAg), with adjuvants. The two in vitro assays for cell-mediated immunity (CMI), utilizing lymph node cells, were release of interferon after exposure to antigen, and blast transformation of lymphocytes, and the in vivo assay was ear swelling at 24 h after local injection of antigen. Immunization with HBsAg or HBcAg with adjuvants induced antigen-specific cutaneous reactivity; if no adjuvants were given, immunization with HBcAg, but not HbsAg, induced cutaneous reactivity. CMI could be adoptively transferred by lymph node cells, but for only a limited period after immunization with HbsAg or MBcAg. The ability of lymph node cells from mice immunized with HBV antigens to transfer adoptively CMI correlated well with their production of interferon after challenge with antigen in vitro, but less well with blastogenesis after challenge with antigen in vitro, or with cutaneous reactivity to antigen in the donor mouse. Reliable antigen-specific lymphokine release assays, rather than blast transformation of lymphocytes or cutaneous reactivity after antigen challenge, arc required to assess CMI to HBV antigens in the mouse and, by inference, in man. [ABSTRACT FROM AUTHOR]

In 18 cases of chronic liver disease due to non-A, non-B hepatitis virus(es) in which the diagnosis was established by transmission, including chimpanzee inoculation in nine, sera were tested for the autoantibodies characteristically associated with autoimmune chronic active hepatitis. The frequency of autoantibodies to nuclear, smooth muscle, cytofilament, mitochondrial and liver membrane antigens was low, being not greater than that recorded for a normal population, and the few positive reactions obtained were at very low titre. These findings suggest that among cases of "HBsAg negative" chronic hepatitis, those due to NANB infection are distinguishable from those due to autoimmune chronic hepatitis by negative serological tests for autoantibodies. [ABSTRACT FROM AUTHOR]

ABSTRACT- The mononuclear cell infiltrate in the liver was analyzed, using a panel of monoclonal antibodies (MAbs) of known specificity, in 10 patients with auto-immune chronic active hepatitis and, for contrast, in 14 with other types of chronic parenchymal liver diseases. In all cases, the mononuclear cell (MNC) infiltrate in the liver consisted mostly of T lymphocytes. Helper (Th) cells were more frequent than suppressor/cytotoxic T cells (Tsc) in the portal tracts and cirrhotic scar tissue, while Tsc were more common in the hepatic parenchyma. The number of Tsc cells in the parenchyma was greatest in patients with histologically active CAH and least in patients with quiescent cirrhosis. 'Plasmacytoid' cells with the morphology of plasma cells, a hallmark of the MNC infiltrate in auto-immune CAH, were more frequent in histologically active CAH than in quiescent cirrhosis. These plasmacytoid cells were T200 + ve, and hence of bone marrow origin, but the majority expressed neither membrane nor cytoplasmic immunoglobulin nor any lineage-specific marker antigens, and hence did not fulfil criteria for B lymphocytes; however, these cells were positive for OKT10 and HLA DR. No difference was evident between the MNC infiltrate in the liver in auto-immune CAH and that in the other acute or chronic liver diseases studied, including HBV-associated CAH; hence immunohistological studies do not point to any pathological processes uniquely responsible for the pattern of hepatocyte damage seen in auto-immune CAH. [ABSTRACT FROM AUTHOR]

Currently available vaccines prevent HPV infection and development of HPV-associated malignancies, but do not cure existing HPV infections and dysplastic lesions. Persistence of infection(s) in immunocompetent patients may reflect induction of local immunosuppressive mechanisms by HPV, providing a target for therapeutic intervention. We have proposed that a mouse, expressing HPV16 E7 oncoprotein under a Keratin 14 promoter (K14E7 mice), and which develops epithelial hyperplasia, may assist with understanding local immune suppression mechanisms that support persistence of HPV oncogene-induced epithelial hyperplasia. K14E7 skin grafts recruit immune cells from immunocompetent hosts, but consistently fail to be rejected. Here, we review the literature on HPV-associated local immunoregulation, and compare the findings with published observations on the K14E7 transgenic murine model, including comparison of the transcriptome of human HPV-infected pre-malignancies with that of murine K14E7 transgenic skin. We argue from the similarity of i) the literature findings and ii) the transcriptome profiles that murine K14E7 transgenic skin recapitulates the cellular and secreted protein profiles of high-grade HPV-associated lesions in human subjects. We propose that the K14E7 mouse may be an appropriate model to further study the immunoregulatory effects of HPV E7 expression, and can facilitate development and testing of therapeutic vaccines., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

We have shown that the expression of human papillomavirus type 16 E7 (HPV16.E7) protein within epithelial cells results in local immune suppression and a weak and ineffective immune response to E7 similar to that occuring in HPV-associated premalignancy and cancers. However, a robust acute inflammatory stimulus can overcome this to enable immune elimination of HPV16.E7-transformed epithelial cells. 2,4-Dinitrochlorobenzene (DNCB) can elicit acute inflammation and it has been shown to initiate the regression of HPV-associated genital warts. Although the clinical use of DNCB is discouraged owing to its mutagenic potential, understanding how DNCB-induced acute inflammation alters local HPV16.E7-mediated immune suppression might lead to better treatments. Here, we show that topical DNCB application to skin expressing HPV16.E7 as a transgene induces a hyperinflammatory response, which is not seen in nontransgenic control animals. The E7-associated inflammatory response is characterized by enhanced expression of Th2 cytokines and increased infiltration of CD11b(+)Gr1(int)F4/80(+)Ly6C(hi)Ly6G(low) myeloid cells, producing arginase-1. Inhibition of arginase with an arginase-specific inhibitor, N(omega)-hydroxy-nor-L-arginine, ameliorates the DNCB-induced inflammatory response. Our results demonstrate that HPV16.E7 protein enhances DNCB-associated production of arginase-1 by myeloid cells and consequent inflammatory cellular infiltration of skin.

Papillomavirus infection is a major antecedent of anogenital malignancy. We have previously established that the L1 and L2 capsid genes of papillomavirus have suboptimal codon usage for expression in mammalian cells. We now show that the lack of immunogenicity of polynucleotide vaccines based on the L1 gene can be overcome with codon modified L1, which induces strong immune responses, including conformational virus neutralising antibody and delayed type hypersensitivity. Conjugation of a ubiquitin gene to a hybrid gene incorporating L1 and the E7 non-structural papillomavirus protein improved E7 specific CTL responses, and induced protection against an E7 expressing tumour, but induced little neutralising antibody. However, a mixture of ubiquitin conjugated and non-ubiquitin conjugated polynucleotides induced virus neutralising antibody and E7 specific CD8 T cells. An optimal combined prophylactic/therapeutic viral vaccine might therefore comprise ubiquitin conjugated and non-ubiquitinated genes, to induce prophylactic neutralising antibody and therapeutic cell mediated immune responses.

Mice transgenic for the E7 tumor Ag of human papillomavirus type 16, driven from a keratin 14 promoter, express E7 in keratinocytes but not dendritic cells. Grafted E7-transgenic skin is not rejected by E7-immunized mice that reject E7-transduced transplantable tumors. Rejection of recently transplanted E7-transgenic skin grafts, but not of control nontransgenic grafts or of established E7-transgenic grafts, is induced by systemic administration of live or killed Listeria monocytogenes or of endotoxin. Graft recipients that reject an E7 graft reject a subsequent E7 graft more rapidly and without further L. monocytogenes exposure, whereas recipients of an E7 graft given without L. monocytogenes do not reject a second graft, even if given with L. monocytogenes. Thus, cross-presentation of E7 from keratinocytes to the adaptive immune system occurs with or without a proinflammatory stimulus, but proinflammatory stimuli at the time of first cross-presentation of Ag can determine the nature of the immune response to the Ag. Furthermore, immune effector mechanisms responsible for rejection of epithelium expressing a tumor Ag in keratinocytes are different from those that reject an E7-expressing transplantable tumor. These observations have implications for immunotherapy for epithelial cancers.