193 results on '"Frazer, I. H."'
Search Results
52. Immunological Abnormalities in Asymptomatic Homosexual Men: Correlation with Antibody to HTLV-III and Sequential Changes over Two Years
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FRAZER, I. H., MACKAY, I. R., CRAPPER, R. M., JONES, B., GUST, I. D., SARNGADHARAN, M. G., CAMPBELL, D. C., and UNGAR, B.
- Abstract
A prospective study on 100 homosexual male volunteers was designed to examine immunological function in relation to sexual activity and infection with the human T cell lymphotropic virus Type III (HTLV-III). Complete data were available for 71 men. In a comparison with 100 age matched heterosexual men, the study group of 100 men had a significantly higher mean serum IgG level (12.1±SD 2.7 g/l vs. 10.9±2.4 g/l, p<0.01) and a significantly lower mean number of CD4 (T4) cells (845±310×10−6/ lvs. 1128±375;p<0.01). For the study group, seropositivity for anti-HTLV-III was present initially in 22 per cent and was associated with a higher mean level of serum IgG and lower mean number of CD4 cells. Among seropositive homosexual men a low CD4/8 ratio was attributable to low numbers of CD4 cells in those without lymphadenopathy and to high numbers of CD8 cells in those with lymphadenopathy. For the seronegative homosexual men, a low CD4/8 ratio as a result of an increased CD8 cell count was present in 12 of 60, and was associated with numerous sexual partners and semen culture positive for cytomegalovirus. In two seropositive subjects a low CD4/8 ratio due to a decrease in the CD4 cell count was predictive of the development of AIDS by some two years. For the 71 men with complete data over two years, indices of cell-mediated immunity, including mean counts of CD4 cells, the CD4/8 ratio, and score for recall of cutaneous delayed type hypersensitivity increased during the first year but not during the second year in both seropositive and seronegative subjects. These increases occurred in association with changes in sexual practices and activity, but could not be attributed to any one particular factor.
- Published
- 1986
53. T-helper epitopes of the E7 transforming protein of cervical cancer associated human papillomavirus type 18 (HPV18)
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Fernando, G. J. P., Tindle, R. W., and Frazer, I. H.
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- 1995
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54. Murine HPV16 E7-expressing transgenic skin effectively emulates the cellular and molecular features of human high-grade squamous intraepithelial lesions.
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Tuong ZK, Noske K, Kuo P, Bashaw AA, Teoh SM, and Frazer IH
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- Animals, Disease Models, Animal, Female, Gene Expression Profiling, Human papillomavirus 16 immunology, Humans, Hyperplasia immunology, Hyperplasia pathology, Immunosuppression Therapy, Keratin-14 immunology, Mice, Mice, Transgenic, Papillomavirus E7 Proteins immunology, Papillomavirus Infections immunology, Papillomavirus Infections pathology, Papillomavirus Infections virology, Skin immunology, Skin virology, Skin Transplantation, Squamous Intraepithelial Lesions of the Cervix genetics, Human papillomavirus 16 genetics, Keratin-14 genetics, Papillomavirus E7 Proteins genetics, Skin pathology, Squamous Intraepithelial Lesions of the Cervix immunology, Squamous Intraepithelial Lesions of the Cervix virology
- Abstract
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.)
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- 2018
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55. Human papillomavirus e7 oncoprotein transgenic skin develops an enhanced inflammatory response to 2,4-dinitrochlorobenzene by an arginase-1-dependent mechanism.
- Author
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Tran LS, Bergot AS, Mattarollo SR, Mittal D, and Frazer IH
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- Animals, Drug Eruptions pathology, Ear, External immunology, Ear, External pathology, Female, Human papillomavirus 16 immunology, Immunity, Innate drug effects, Male, Mice, Inbred C57BL, Mice, Knockout, Myeloid Cells drug effects, Myeloid Cells immunology, Papillomavirus E7 Proteins metabolism, Papillomavirus Infections immunology, Skin drug effects, Skin pathology, Th2 Cells drug effects, Th2 Cells immunology, Arginase metabolism, Dinitrofluorobenzene toxicity, Drug Eruptions immunology, Papillomavirus E7 Proteins immunology, Skin immunology
- Abstract
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.
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- 2014
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56. Polynucleotide viral vaccines: codon optimisation and ubiquitin conjugation enhances prophylactic and therapeutic efficacy.
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Liu WJ, Zhao KN, Gao FG, Leggatt GR, Fernando GJ, and Frazer IH
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- Animals, Antibodies, Viral blood, Codon genetics, Female, Genes, Viral, Humans, Hypersensitivity, Delayed, Immunity, Cellular, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Neutralization Tests, Papillomaviridae pathogenicity, Papillomavirus Infections immunology, Papillomavirus Infections prevention & control, Papillomavirus Infections therapy, T-Lymphocytes, Cytotoxic immunology, Tumor Virus Infections immunology, Tumor Virus Infections prevention & control, Tumor Virus Infections therapy, Ubiquitin immunology, Vaccines, Conjugate genetics, Vaccines, Conjugate pharmacology, Vaccines, Conjugate therapeutic use, Vaccines, DNA genetics, Vaccines, DNA pharmacology, Vaccines, DNA therapeutic use, Viral Vaccines genetics, Viral Vaccines therapeutic use, Papillomaviridae genetics, Papillomaviridae immunology, Viral Vaccines pharmacology
- Abstract
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.
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- 2001
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57. Tolerance or immunity to a tumor antigen expressed in somatic cells can be determined by systemic proinflammatory signals at the time of first antigen exposure.
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Frazer IH, De Kluyver R, Leggatt GR, Guo HY, Dunn L, White O, Harris C, Liem A, and Lambert P
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- Animals, Antigens, Bacterial administration & dosage, Antigens, Bacterial immunology, Antigens, Neoplasm biosynthesis, Antigens, Neoplasm genetics, Antigens, Neoplasm metabolism, Cells, Cultured, Female, Graft Rejection genetics, Graft Rejection immunology, Inflammation genetics, Inflammation immunology, Injections, Intravenous, Keratinocytes immunology, Keratinocytes metabolism, Listeriosis genetics, Listeriosis immunology, Mice, Mice, Inbred C57BL, Mice, Transgenic, Oncogene Proteins, Viral biosynthesis, Oncogene Proteins, Viral genetics, Oncogene Proteins, Viral immunology, Papillomaviridae genetics, Papillomaviridae immunology, Papillomavirus E7 Proteins, Signal Transduction genetics, Skin Transplantation immunology, Skin Transplantation methods, Time Factors, Tumor Cells, Cultured, Antigen Presentation genetics, Antigens, Neoplasm immunology, Immune Tolerance genetics, Signal Transduction immunology
- Abstract
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.
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- 2001
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58. Re: Cervical carcinoma and human papillomavirus: on the road to preventing a major human cancer.
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Bosch FX, Muñoz N, de Sanjosé S, Franco EL, Lowy DR, Schiffman M, Franceschi S, Kjaer SK, Meijer CJ, Frazer IH, and Cuzick J
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- DNA, Viral isolation & purification, Female, Humans, Papillomaviridae genetics, Papillomavirus Infections complications, Tumor Virus Infections complications, Uterine Cervical Neoplasms virology, Papillomaviridae immunology, Papillomaviridae isolation & purification, Papillomavirus Infections prevention & control, Tumor Virus Infections prevention & control, Uterine Cervical Neoplasms prevention & control, Viral Vaccines therapeutic use
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- 2001
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59. Nonspecific down-regulation of CD8+ T-cell responses in mice expressing human papillomavirus type 16 E7 oncoprotein from the keratin-14 promoter.
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Tindle RW, Herd K, Doan T, Bryson G, Leggatt GR, Lambert P, Frazer IH, and Street M
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- Animals, Down-Regulation, Female, Humans, Immunization, Immunologic Memory, Keratin-14, Mice, Papillomavirus E7 Proteins, Receptors, Antigen, T-Cell physiology, Receptors, Antigen, T-Cell, alpha-beta physiology, T-Lymphocytes, Cytotoxic immunology, Uterine Cervical Neoplasms virology, CD8-Positive T-Lymphocytes immunology, Keratins genetics, Oncogene Proteins, Viral physiology, Promoter Regions, Genetic
- Abstract
The E7 oncoprotein of human papillomavirus 16 (HPV16) transforms basal and suprabasal cervical epithelial cells and is a tumor-specific antigen in cervical carcinoma, to which immunotherapeutic strategies aimed at cytotoxic T-lymphocyte (CTL) induction are currently directed. By quantifying major histocompatibility complex class I tetramer-binding T cells and CTL in mice expressing an HPV16 E7 transgene from the keratin-14 (K14) promoter in basal and suprabasal keratinocytes and in thymic cortical epithelium, we show that antigen responsiveness of both E7- and non-E7-specific CD8+ cells is down-regulated compared to non-E7 transgenic control mice. We show that the effect is specific for E7, and not another transgene, expressed from the K14 promoter. Down-regulation did not involve deletion of CD8+ T cells of high affinity or high avidity, and T-cell receptor (TCR) Vbeta-chain usage and TCR receptor density were similar in antigen-responsive cells from E7 transgenic and non-E7 transgenic mice. These data indicate that E7 expressed chronically from the K14 promoter nonspecifically down-regulates CD8+ T-cell responses. The in vitro data correlated with the failure of immunized E7 transgenic mice to control the growth of an E7-expressing tumor challenge. We have previously shown that E7-directed CTL down-regulation correlates with E7 expression in peripheral but not thymic epithelium (T. Doan et al., J. Virol. 73:6166-6170, 1999). The findings have implications for the immunological consequences of E7-expressing tumor development and E7-directed immunization strategies. Generically, the findings illustrate a T-cell immunomodulatory function for a virally encoded human oncoprotein.
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- 2001
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60. Association of bovine papillomavirus type 1 with microtubules.
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Liu WJ, Qi YM, Zhao KN, Liu YH, Liu XS, and Frazer IH
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- Active Transport, Cell Nucleus drug effects, Animals, Blotting, Western, Bovine papillomavirus 1 drug effects, Bovine papillomavirus 1 physiology, Bovine papillomavirus 1 ultrastructure, Capsid metabolism, Cattle, Cell Line, Cell Membrane drug effects, Cell Membrane metabolism, Cell Membrane virology, Cell Nucleus drug effects, Cell Nucleus metabolism, Cell Nucleus virology, Cytoplasmic Vesicles drug effects, Cytoplasmic Vesicles metabolism, Cytoplasmic Vesicles virology, Fluorescent Antibody Technique, Indirect, Microscopy, Electron, Microtubules chemistry, Microtubules drug effects, Microtubules ultrastructure, Nocodazole pharmacology, Paclitaxel pharmacology, Protein Binding, Temperature, Tubulin metabolism, Virion drug effects, Virion metabolism, Virion physiology, Virion ultrastructure, Warts virology, Bovine papillomavirus 1 metabolism, Microtubules metabolism
- Abstract
Transport of BPV-1 virus from the cell membrane to the nucleus was studied in vitro in CV-1 cells. At reduced temperature (4 degrees C), BPV-1 binding to CV-1 cells was unaffected but there was no transport of virions across the cytosol. Electron microscopy showed BPV-1 virions in association with microtubules in the cytoplasm, a finding confirmed by co-immunoprecipitation of L1 protein and tubulin. Internalization of virus was unimpaired in cells treated with the microtubule-depolymerizing drug nocodazole but virions were retained in cytoplasmic vesicles and not transported to the nucleus. We conclude that a microtubule transport mechanism in CV-1 cells moves intact BPV-1 virions from the cell surface to the nuclear membrane., (Copyright 2001 Academic Press.)
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- 2001
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61. Papillomavirus virus-like particles for the delivery of multiple cytotoxic T cell epitopes.
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Liu WJ, Liu XS, Zhao KN, Leggatt GR, and Frazer IH
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- Amino Acid Sequence, Animals, Cells, Cultured, Chimera, Enzyme-Linked Immunosorbent Assay, Epitopes, T-Lymphocyte immunology, Female, Genetic Vectors, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Mice, Transgenic, Molecular Sequence Data, Spodoptera, Virion genetics, Bovine papillomavirus 1 genetics, Epitopes, T-Lymphocyte administration & dosage, T-Lymphocytes, Cytotoxic immunology
- Abstract
Chimeric papillomavirus (PV) virus-like particles (VLPs) based on the bovine papillomavirus type 1 (BPV-1) L1 protein were constructed by replacing the 23-carboxyl-terminal amino acids of the BPV1 major protein L1 with an artificial "polytope" minigene, containing known CTL epitopes of human PV16 E7 protein, HIV IIIB gp120 P18, Nef, and reverse transcriptase (RT) proteins, and an HPV16 E7 linear B epitope. The CTL epitopes were restricted by three different MHC class I alleles (H-2(b), H-2(d), HLA-A*0201). The chimeric L1 protein assembled into VLPs when expressed in SF-9 cells by recombinant baculovirus. After immunization of mice with polytope VLPs in the absence of adjuvant, serum antibodies were detected which reacted with both polytope VLPs and wild-type BPV1L1 VLPs, in addition to the HPV16E7 linear B cell epitope. CTL precursors specific for the HPV16 E7, HIV P18, and RT CTL epitopes were also detected in the spleen of immunized mice. Polytope VLPs can thus deliver multiple B and T epitopes as immunogens to the MHC class I and class II pathways, extending the utility of VLPs as self-adjuvanting immunogen delivery systems., (Copyright 2000 Academic Press.)
- Published
- 2000
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62. Differences in the post-translational modifications of human papillomavirus type 6b major capsid protein expressed from a baculovirus system compared with a vaccinia virus system.
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Fang NX, Frazer IH, and Fernando GJ
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- Animals, Capsid genetics, Electrophoresis, Gel, Two-Dimensional, Fatty Acids metabolism, Glycosylation, Phosphorylation, Protein Engineering methods, Recombinant Proteins genetics, Recombinant Proteins metabolism, Serine, Threonine, Baculoviridae genetics, Capsid metabolism, Papillomaviridae chemistry, Protein Processing, Post-Translational, Vaccinia virus genetics
- Abstract
Virus-like particles (VLPs) are being currently investigated in vaccines against viral infections in humans. There are different recombinant-protein-expression systems available for obtaining the necessary VLP preparation for vaccination. However, the differences in post-translational modifications of the recombinant proteins obtained and their differences in efficacy in eliciting an anti-viral response in vaccines are not well established. In this study we have compared the post-translational modifications of human papillomavirus type-6b major capsid protein L1 (HPV 6bL1) expressed using recombinant baculovirus (rBV) in Sf9 (Spodoptera frugiperda) insect cells, with the protein expressed using recombinant vaccinia virus (rVV) in CV-1 kidney epithelial cells. Two-dimensional gel electrophoresis of biosynthetically labelled rBV-expressed HPV 6bL1 showed several post-translationally modified variants of the protein, whereas rVV-expressed HPV 6bL1 showed only a few variants. Phosphorylations were detected at threonine and serine residues for the L1 expressed from rBV compared with phosphorylation at serine residues only for the L1 expressed from rVV. HPV 6bL1 expressed using rBV incorporated [(3)H]mannose and [(3)H]galactose, whereas HPV 6bL1 expressed using rVV incorporated only [(3)H]galactose. We conclude that post-translational modification of recombinant HPV 6bL1 can differ according to the system used for its expression. Since recombinant L1 protein is a potential human-vaccine candidate, the implication of the observed differences in post-translational modifications on immunogenicity of L1 VLPs warrants investigation.
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- 2000
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63. BPV1 E2 protein enhances packaging of full-length plasmid DNA in BPV1 pseudovirions.
- Author
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Zhao KN, Hengst K, Liu WJ, Liu YH, Liu XS, McMillan NA, and Frazer IH
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- Animals, Bovine papillomavirus 1 genetics, COS Cells, Capsid metabolism, Cell Line, DNA, Circular metabolism, Neutralization Tests, Sequence Deletion, Virion genetics, Bovine papillomavirus 1 physiology, Capsid Proteins, DNA, Viral metabolism, DNA-Binding Proteins physiology, Enhancer Elements, Genetic, Plasmids metabolism, Viral Proteins physiology, Virion metabolism, Virus Assembly genetics
- Abstract
We studied determinants of efficient encapsidation of circular DNA, incorporating a PV early region DNA sequence (nt 584-1978) previously shown to enhance packaging of DNA within papillomavirus (PV)-like particles (VLPs). Insect coelomic cells (Sf-9) and cultured monkey kidney cells (Cos-1) were transfected with an 8-kb reporter plasmid incorporating the putative BPV packaging sequence and infected with BPV1 L1 and L2 recombinant baculovirus or vaccinia virus. Heavy (1.34 g/ml) and light (1.30 g/ml) VLPs were produced, and each packaged some of the input plasmid. In light VLPs, truncated plasmids, which nevertheless incorporated the PV-derived DNA packaging sequence, were more common than full-length plasmids. Packaging efficiency of the plasmid was estimated at 1 plasmid per 10(4) VLPs in both Cos-1 and Sf-9 cells. In each cell type, expression of the BPV1 early region protein E2 in trans doubled the quantity of heavy but not light VLPs and also increased the packaging efficiency of full-length circular plasmids by threefold in heavy VLPs. The resultant pseudovirions incorporated significant amounts of E2 protein. Pseudovirions, comprising plasmids packaged within heavy VLPs, mediated the delivery of packaged plasmid into Cos-1 cells, whereby "infectivity" was blocked by antisera to BPV1 L1, but not antisera to BPV1 E4. We conclude that (a) packaging of DNA within PV L1+L2 pseudovirions is enhanced by BPV1 E2 acting in trans, (b) E2 may be packaged with the pseudovirion, and (c) E2-mediated enhancement of packaging favors 8-kb plasmid incorporation over incorporation of shorter DNA sequences., (Copyright 2000 Academic Press.)
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- 2000
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64. E2F-1 induces proliferation-specific genes and suppresses squamous differentiation-specific genes in human epidermal keratinocytes.
- Author
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Dicker AJ, Popa C, Dahler AL, Serewko MM, Hilditch-Maguire PA, Frazer IH, and Saunders NA
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- Base Sequence, Biomarkers, Cells, Cultured, DNA Primers, DNA-Binding Proteins genetics, E2F Transcription Factors, E2F1 Transcription Factor, Epidermal Cells, Humans, Retinoblastoma-Binding Protein 1, Transcription Factor DP1, Transcription Factors genetics, Carrier Proteins, Cell Cycle Proteins, Cell Differentiation genetics, Cell Division genetics, DNA-Binding Proteins physiology, Epidermis metabolism, Keratinocytes metabolism, Transcription Factors physiology
- Abstract
Squamous differentiation of keratinocytes is associated with decreases in E2F-1 mRNA expression and E2F activity, and these processes are disrupted in squamous cell carcinoma cell lines. We now show that E2F-1 mRNA expression is increased in primary squamous cell carcinomas of the skin relative to normal epidermis. To explore the relationship between E2F-1 and squamous differentiation further, we examined the effect of altering E2F activity in primary human keratinocytes induced to differentiate. Promoter activity for the proliferation-associated genes, cdc2 and keratin 14, are inhibited during squamous differentiation. This inhibition can be inhibited by overexpression of E2F-1 in keratinocytes. Overexpression of E2F-1 also suppressed the expression of differentiation markers (transglutaminase type 1 and keratin 10) in differentiated keratinocytes. Blocking E2F activity by transfecting proliferating keratinocytes with dominant negative E2F-1 constructs inhibited the expression of cdc2 and E2F-1, but did not induce differentiation. Furthermore, expression of the dominant negative construct in epithelial carcinoma cell lines and normal keratinocytes decreased expression from the cdc2 promoter. These data indicate that E2F-1 promotes keratinocyte proliferation-specific marker genes and suppresses squamous differentiation-specific marker genes. Moreover, these data indicate that targeted disruption of E2F-1 activity may have therapeutic potential for the treatment of squamous carcinomas. Oncogene (2000).
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- 2000
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65. Immune responses induced by BCG recombinant for human papillomavirus L1 and E7 proteins.
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Jabbar IA, Fernando GJ, Saunders N, Aldovini A, Young R, Malcolm K, and Frazer IH
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- Adjuvants, Immunologic administration & dosage, Animals, Antibodies, Viral biosynthesis, BCG Vaccine administration & dosage, BCG Vaccine genetics, Base Sequence, Cloning, Molecular, DNA Primers genetics, Humans, Hypersensitivity, Delayed, Lymphocyte Activation, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Oncogene Proteins, Viral genetics, Papillomaviridae genetics, Papillomavirus E7 Proteins, Plasmids genetics, T-Lymphocytes immunology, T-Lymphocytes, Cytotoxic immunology, Vaccines, Synthetic administration & dosage, Vaccines, Synthetic genetics, Viral Proteins, BCG Vaccine immunology, Capsid Proteins, Oncogene Proteins, Viral immunology, Papillomaviridae immunology, Vaccines, Synthetic immunology
- Abstract
Recombinant bacille Calmette-Guerin (BCG) based vaccine delivery systems could potentially share the safety and effectiveness of BCG. We therefore prepared recombinant BCG vaccines which expressed the L1 late protein of the human papillomavirus (HPV) 6b or the E7 early protein of the HPV 16. The two recombinants were evaluated as immunogens in C57BL/6J and BALB/c mice, and compared with a conventional protein/adjuvant system using E7 or L1 mixed with Quil-A adjuvant. rBCG6bL1 and rBCG16E7 primed specific immune responses, represented by DTH, T-proliferation and antibody, and rBCG16E7 induced cytotoxic immune response to E7 protein. The magnitude of the observed responses were less than those elicited by protein/adjuvant vaccine. As recombinant BCG vaccines expressing HPV6bL1 or HPV16E7 persist at low levels in the immunised host, they may be beneficial to prime or retain memory responses to antigens, but are unlikely to be useful as a single component vaccine strategy.
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- 2000
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66. HPV6b virus like particles are potent immunogens without adjuvant in man.
- Author
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Zhang LF, Zhou J, Chen S, Cai LL, Bao QY, Zheng FY, Lu JQ, Padmanabha J, Hengst K, Malcolm K, and Frazer IH
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- Adolescent, Adult, Antibodies, Viral blood, Humans, Hypersensitivity, Delayed etiology, Immunization, Middle Aged, Viral Vaccines adverse effects, Capsid immunology, Condylomata Acuminata therapy, Papillomaviridae immunology, Viral Vaccines immunology
- Abstract
Subjects with genital warts were immunized three times or more with HPV6b VLPs without adjuvant. All immunized subjects had DTH to HPV6b L1 protein. Of 32 subjects, nine had HPV6b specific antibody prior to immunization and 22 acquired antibody with immunization. VLP specific antibody increased following a single immunization in 6 of 8 subjects with low level antibody at recruitment. Complete regression of genital warts was observed in 25 of 33 evaluable subjects over the 20-week observation period. We conclude that immunization with HPV6b L1 VLPs without adjuvant induces immunity to the L1 protein epitopes recognised during natural infection, and may accelerate regression of warts.
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- 2000
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67. Multiple conformational epitopes are recognized by natural and induced immunity to the E7 protein of human papilloma virus type 16 in man.
- Author
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Malcolm K, Meschede W, Pawlita M, Koutsky LA, and Frazer IH
- Subjects
- Adult, Antigens, Viral, Tumor blood, Antigens, Viral, Tumor chemistry, Blotting, Western, Enzyme-Linked Immunosorbent Assay, Female, Humans, Immunity, Innate, Oncogene Proteins, Viral blood, Oncogene Proteins, Viral chemistry, Papillomavirus E7 Proteins, Protein Conformation, Radioimmunoprecipitation Assay, Antigens, Viral, Tumor immunology, Epitopes chemistry, Oncogene Proteins, Viral immunology, Papillomaviridae immunology, Papillomavirus Infections immunology, Tumor Virus Infections immunology, Uterine Cervical Neoplasms immunology
- Abstract
The reactivity of sera from patients with cervical cancer with the E7 protein of human papilloma virus type 16 (HPV16) was estimated using a novel non-radioactive immunoprecipitation assay and four established protein- and peptide-based immunoassays. Six of 14 sera from patients with cervical cancer and 1 of 10 sera from healthy laboratory staff showed repeated reactivity with E7 in at least one assay. Four of the 7 reactive sera were consistently reactive in more than one assay, but only one was reactive in all four assays. Following immunization with E7, 2 of 5 patients with cervical cancer had increased E7-specific reactivity, measurable in one or more assays. No single assay was particularly sensitive for E7 reactivity, or predictive of cervical cancer. Mapping of E7 reactivity to specific E7 peptides was unsuccessful, suggesting that natural or induced E7 reactivity in human serum is commonly directed to conformational epitopes of E7. These results suggest that each assay employed in this study measures a different aspect of E7 reactivity, and that various reactivities to E7 may manifest following HPV infection or immunization. This finding is of significance for monitoring of E7 immunotherapy and for serological screening for cervical cancer., (Copyright 2000 S. Karger AG, Basel)
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- 2000
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68. Genetic and environmental causes of variation in basal levels of blood cells.
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Evans DM, Frazer IH, and Martin NG
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- Antigens, CD genetics, Child, Female, Genetics, Medical, Humans, Lymphocyte Subsets, Male, Twins, Dizygotic, Twins, Monozygotic, Blood Cell Count, Erythrocyte Indices genetics
- Abstract
The genetic and environmental determinants of variation in blood cell size and number were investigated in 392 pairs of 12-year-old twins. The following blood cell indices were measured: haemoglobin, red blood cell count, haematocrit, mean corpuscular volume, platelet number, total white cell count, level of neutrophils, monocytes, eosinophils, total lymphocytes, CD3+ lymphocytes, CD4+ lymphocytes, CD8+ lymphocytes, CD19+ lymphocytes, CD56+ lymphocytes and CD4+/CD8+ ratio. Genetic factors contributed significantly to all blood cell measures accounting for between 61 and 96% of variance. Heritability estimates did not differ significantly between males and females, although the sample size of the present study was not large enough to exclude the possibility of sex-limited gene expression. Common environmental factors were important in determining red blood cell count and haematocrit, but were not important in determining basal levels of any white blood cell type.
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- 1999
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69. Capture ElISA and in vitro cell binding assay for the detection of antibodies to human papillomavirus type 6b virus-like particles in patients with anogenital warts.
- Author
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Peng S, Qi Y, Christensen N, Hengst K, Kennedy L, Frazer IH, and Tindle RW
- Subjects
- Adolescent, Adult, Aged, DNA, Viral analysis, Female, Humans, Immunoglobulin G blood, Male, Middle Aged, Neutralization Tests, Oncogene Proteins, Viral genetics, Papillomaviridae genetics, Papillomaviridae isolation & purification, Polymerase Chain Reaction, Protein Binding, Reproducibility of Results, Sensitivity and Specificity, Antibodies, Viral analysis, Condylomata Acuminata virology, Enzyme-Linked Immunosorbent Assay methods, Oncogene Proteins, Viral immunology, Papillomaviridae immunology, Skin Diseases, Infectious virology, Virion immunology
- Abstract
To investigate human papillomavirus (HPV) virus-like particle (VLP)-specific antibody responses among anogenital warts patients, a VLP-based capture ELISA was established. Twenty-six percent (35/134) of control subjects and 50.0% (39/78) of patients with current anogenital warts showed IgG seropositivity to HPV 6b VLPs. HPV 6b VLP-specific antibody responses recognised native VLPs only, and had no cross-reaction with HPV type 16 VLPs. No differences in reactivity were observed between L1 and L1 + L2 VLPs, suggesting that L2 contributes little to the total immunogenicity of the papillomavirus virion. A VLP-cell binding assay was also established. Some sera from patients with anogenital warts specifically inhibited VLP binding to the surface of epithelial cells, suggesting that these antibodies might be functionally neutralising. These data show that serological responses to HPV 6b VLPs were induced among some but not all patients with anogenital warts, and give a proportional estimate of infection in the community.
- Published
- 1999
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70. Expression of the alpha6 integrin confers papillomavirus binding upon receptor-negative B-cells.
- Author
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McMillan NA, Payne E, Frazer IH, and Evander M
- Subjects
- Animals, B-Lymphocytes metabolism, Binding Sites, COS Cells, Flow Cytometry, Humans, Integrin alpha6, Virus Replication, Antigens, CD physiology, B-Lymphocytes virology, Papillomaviridae physiology, Receptors, Virus physiology
- Abstract
Papillomaviruses (PV) bind to a wide range of cell lines in a specific and saturable manner. We have recently identified a candidate receptor for papillomavirus as the alpha6 integrin (Evander et al., J. Virol. 71, 2449-2456, 1997). We have further investigated the role the alpha6 integrin plays in PV binding. Here we show that the cells expressing the alpha6 integrin, partnered with either the beta4 integrin or the beta1 integrin, are equally able to bind PV HPV6b L1 virus-like particles, indicating that the beta partner does not play a major role in virus binding. In order to provide definitive evidence that the alpha6 integrin is required for PV binding we undertook to genetically complement the receptor-negative B-cell line DG75 by expressing the human alpha6A gene. The transduction of the alpha6 integrin gene into DG75 cells results in the cell surface expression of the alpha6 protein and this expression confers upon DG75 cells the ability to bind laminin, a normal ligand for alpha6 integrin. Furthermore, the alpha6 protein is partnered with the beta1 integrin in DG75 cells. Finally, we show that the DG75-alpha6 cells were able to bind papillomavirus VLPs and this binding was inhibited by a functionally blocking anti-alpha6 antibody. Together these data indicate that the alpha6 integrin is a primary cell receptor for papillomaviruses and is both necessary and sufficient for PV binding., (Copyright 1999 Academic Press.)
- Published
- 1999
- Full Text
- View/download PDF
71. Nucleotides 1506-1625 of bovine papillomavirus type 1 genome can enhance DNA packaging by L1/L2 capsids.
- Author
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Zhao KN, Frazer IH, Jun Liu W, Williams M, and Zhou J
- Subjects
- Animals, Cattle, DNA, Viral chemistry, Nucleic Acid Conformation, Sequence Analysis, DNA, Bovine papillomavirus 1 genetics, Capsid genetics, Capsid Proteins, DNA, Viral genetics, Genome, Viral
- Abstract
We have previously described a DNA-packaging assay using bovine papillomavirus type 1 (BPV-1) virus-like particles (VLPs) and have identified a region of the BPV genome that assists in packaging. In this study, we identify a specific BPV sequence involved in DNA packaging by BPV-1 VLPs. In the initial screening of BPV-1 genomic sequences essential for DNA packaging, we observed that a plasmid with deletions between nucleotides (nt) 948 and 2113 failed to be packaged into BPV-1 VLPs. However, plasmids containing nt 948 to 2113 were efficiently packaged, suggesting that this 1.2-kb fragment contains a packaging enhancement sequence (PES). Further mapping of the BPV-1 genome showed that this packaging sequence lies between nt 1506 and 1625. Furthermore, this packaging sequence is also recognized by HPV6b VLPs, suggesting that a common packaging mechanism may be used by the two papillomavirus types. Given the phylogenetic difference between these two viral types, it is likely that other papillomavirus types may also use the same packaging mechanism. Identification of the PES has allowed a minimal viral genome sequence to be used in the packaging assay, improving the usefulness of the assay in studying the process of papillomavirus DNA encapsidation., (Copyright 1999 Academic Press.)
- Published
- 1999
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72. Potential strategies utilised by papillomavirus to evade host immunity.
- Author
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Frazer IH, Thomas R, Zhou J, Leggatt GR, Dunn L, McMillan N, Tindle RW, Filgueira L, Manders P, Barnard P, and Sharkey M
- Subjects
- Animals, Epithelial Cells immunology, Epithelial Cells virology, Hematopoietic Stem Cells immunology, Humans, Interferon-alpha immunology, Keratinocytes immunology, Keratinocytes virology, Oncogene Proteins, Viral immunology, Papillomavirus E7 Proteins, T-Lymphocytes, Cytotoxic immunology, Viral Proteins immunology, Papillomaviridae immunology, Papillomavirus Infections immunology, Tumor Virus Infections immunology
- Abstract
The co-evolution of papillomaviruses (PV) and their mammalian hosts has produced mechanisms by which PV might avoid specific and non-specific host immune responses. Low level expression of PV proteins in infected basal epithelial cells, together with an absence of inflammation and of virus-induced cell lysis, restricts the opportunity for effective PV protein presentation to immunocytes by dendritic cells. Additionally, PV early proteins, by a range of mechanisms, may restrict the efficacy of antigen presentation by these cells. Should an immune response be induced to PV antigens, resting keratinocytes (KC) appear resistant to interferon-gamma-enhanced mechanisms of cytotoxic T-lymphocyte (CTL)-mediated lysis, and expression of PV antigens by resting KC can tolerise PV-specific CTL. Thus, KC, in the absence of inflammation, may represent an immunologically privileged site for PV infection. Together, these mechanisms play a part in allowing persistence of PV-induced proliferative skin lesions for months to years, even in immunocompetent hosts.
- Published
- 1999
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73. Post translational modifications of recombinant human papillomavirus type 6b major capsid protein.
- Author
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Fang NX, Frazer IH, Zhou J, and Fernando GJ
- Subjects
- Animals, Capsid metabolism, Cell Line, Glycosylation, Humans, Myristic Acid metabolism, Palmitic Acid metabolism, Phosphorylation, Protein Processing, Post-Translational, Recombinant Proteins genetics, Recombinant Proteins metabolism, Vaccinia virus genetics, Virion metabolism, Capsid genetics, Papillomaviridae genetics
- Abstract
We have determined the post-translational modifications of the major capsid protein, L1 of human papillomavirus (HPV) type 6b. Since this virus cannot be cultured in the laboratory to obtain sufficient material for a study, a recombinant L1 protein produced in a vaccinia virus expression system was used in this investigation. Our results show that this protein is phosphorylated at serine residues and is also glycosylated. No myristoylation or palmitoylation was detected. The fraction of L1 protein incorporated into virus-like particles was not glycosylated. Since recombinant L1 protein is a potential human vaccine candidate, knowledge of the post-translation modifications of this protein may prove useful for the design of anti-HPV vaccines.
- Published
- 1999
- Full Text
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74. T-helper epitopes identified within the E6 transforming protein of cervical cancer-associated human papillomavirus type 16.
- Author
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Azoury-Ziadeh R, Herd K, Fernando GJ, Frazer IH, and Tindle RW
- Subjects
- Amino Acid Sequence, Animals, B-Lymphocytes immunology, Epitope Mapping, Female, Histocompatibility Antigens Class II immunology, Humans, Immunization, Lymph Nodes cytology, Lymph Nodes immunology, Lymphocyte Activation, Mice, Mice, Inbred C57BL, Molecular Sequence Data, Oncogene Proteins, Viral chemistry, Oncogene Proteins, Viral genetics, Papillomavirus E7 Proteins, Papillomavirus Infections immunology, Papillomavirus Infections virology, Peptides chemical synthesis, Peptides immunology, Tumor Virus Infections immunology, Tumor Virus Infections virology, Uterine Cervical Neoplasms immunology, Viral Vaccines immunology, Epitopes, T-Lymphocyte immunology, Oncogene Proteins, Viral immunology, Papillomaviridae immunology, Repressor Proteins, T-Lymphocytes, Helper-Inducer immunology, Uterine Cervical Neoplasms virology
- Abstract
The E6 oncoprotein of human papillomavirus type 16 (HPV16 E6) produced by tumor cells of HPV16-associated cervical carcinoma is poorly immunogenic in patients, but nonetheless is a tumor-specific antigen to which therapeutic vaccine strategies may be directed. To investigate the subunit immunogenicity of E6 protein at the T-helper cell level, we immunized mice with overlapping peptides spanning the entire 158 amino acid sequence. Two peptides recalled a proliferative response in lymph node cells (LNC) from C57BL/6 (H-2b)-immunized mice. One of these peptides also recalled proliferative responses in the context of 5/5 other major histocompatibility complex (MHC) class II haplotypes, indicating a "promiscuous" T-epitope. Minimal consensus motif analysis identified the epitopes as 60VYRDGNPYA68 and 98GYNKPLCDLL107. LNC from mice immunized with T-epitope proliferated in response to challenge with whole E6 protein. Immunization with E6 T-epitopes linked to B-epitopes of HPV16 E7 protein elicited specific antibody indicating that T-cells recognizing the T-epitopes provided cognate "help" for B-cells. LNC from mice co-immunized with E6 T-epitope and the major T-helper epitope of HPV16 E7 (48DRAHYNI54) proliferated comparably when challenged with the peptides individually indicating co-dominance of the two T-epitopes. The findings have implications for incorporation of E6 into a therapeutic vaccine.
- Published
- 1999
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75. T cell-mediated and non-specific inflammatory mechanisms contribute to the skin pathology of HPV 16 E6E7 transgenic mice.
- Author
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Hilditch-Maguire PA, Lieppe DM, West D, Lambert PF, and Frazer IH
- Subjects
- Age Factors, Animals, Cyclosporine therapeutic use, Dinitrochlorobenzene, Female, Humans, Hyperplasia chemically induced, Hyperplasia immunology, Hyperplasia pathology, Immunity, Cellular, Immunosuppressive Agents therapeutic use, Irritants, Mice, Mice, Inbred BALB C, Mice, Nude, Mice, SCID, Oncogene Proteins, Viral genetics, Papillomaviridae genetics, Papillomavirus E7 Proteins, Skin drug effects, Skin immunology, Skin virology, Skin Diseases chemically induced, Skin Diseases drug therapy, Specific Pathogen-Free Organisms, Mice, Transgenic genetics, Repressor Proteins, Skin pathology, Skin Diseases immunology, T-Lymphocytes immunology
- Abstract
One of three lines of mice transgenic for the E6 and E7 genes of human papillomavirus type 16 (HPV16) expressed from an alphaA-crystallin promoter also expresses the transgene ectopically in the skin. This line, designated alphaACE6E7#19, develops skin disease from 3 months of age, characterised by epidermal hyperplasia and eventual skin loss. Administration of complete Freund's adjuvant (CFA) to alphaACE6E7#19 mice, but not to non-transgenic littermate controls, induced local epidermal hyperplasia which was histologically similar to the spontaneously arising skin pathology. Local application of 2,4-dinitrochlorobenzene (DNCB) to DNCB-sensitised alphaACE6E7#19 mice, but not DNCB-sensitised controls, also induced hyperplasia. Treatment with cyclosporin A (CsA) or systemic depletion of CD4+ cells significantly reduced the incidence of skin disease. These data suggest that local inflammation, and cytokines produced by T helper cells, contribute to the induction of hyperplastic skin disease in alphaACE6E7#19 mice. Spontaneous skin disease with similar histological appearance, frequency, age of onset and severity in alphaACE6E7#19 mice was observed in scid-/- alphaACE6E7#19 mice, despite immune paresis. Antigen-specific immune responses and T-cell cytokines are therefore not necessary for the induction of skin disease. We propose that epidermal hyperplasia associated with HPV16 E6 and E7 expression in skin is accelerated by local secretion of pro-inflammatory cytokines, whose production can be enhanced by activated CD4+ T cells.
- Published
- 1999
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76. Construction and production of fluorescent papillomavirus-like particles.
- Author
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Peng S, Zhou J, and Frazer IH
- Subjects
- Baculoviridae genetics, Bovine papillomavirus 1 growth & development, Capsid Proteins physiology, Cell Line, Green Fluorescent Proteins, Luminescent Proteins physiology, Recombinant Fusion Proteins biosynthesis, Recombinant Fusion Proteins genetics, Virion genetics, Virus Assembly, Bovine papillomavirus 1 physiology, Luminescent Proteins genetics, Virion physiology
- Abstract
The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has attracted widespread interest since it was demonstrated to be fluorescent in vivo when expressed in other organisms. In order to investigate papillomavirus life cycle which hampered by the unavailability of conventional cell culture system, we constructed a chimeric bovine papillomavirus (BPV) type 1 virus-like particles (VLPs) containing GFP. It was found that fluorescent VLPs could be assembled from L2 protein in which GFP is inserted into the N-terminal region of L2 (aa 88). The fluorescent VLPs could also be assembled from a GFP/L2 fusion protein in which part of the L2 sequence had been deleted. In vitro, fluorescent VLPs could bind to CV-1 cells, and this VLP/cell interaction could be analyzed by FACS assay. These results demonstrated that GFP could incorporate into BPV1 VLPs without disruption of the VLP structure. Fluorescent VLPs might be a useful tool for study of papillomavirus virus/cell interaction.
- Published
- 1999
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77. Mucosal immunisation with papillomavirus virus-like particles elicits systemic and mucosal immunity in mice.
- Author
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Liu XS, Abdul-Jabbar I, Qi YM, Frazer IH, and Zhou J
- Subjects
- Administration, Intranasal, Animals, Antibodies, Viral biosynthesis, Cattle, Cell Division immunology, Female, HIV Envelope Protein gp120 immunology, Injections, Intramuscular, Mice, Mice, Inbred C57BL, Oncogene Proteins, Viral administration & dosage, Oncogene Proteins, Viral immunology, Papillomavirus E7 Proteins, Protein Conformation, Recombinant Fusion Proteins immunology, T-Lymphocytes, Cytotoxic cytology, T-Lymphocytes, Cytotoxic immunology, Viral Proteins administration & dosage, Viral Proteins immunology, Bovine papillomavirus 1 immunology, Immunity, Mucosal, Virion immunology
- Abstract
It has been shown previously that recombinant virus-like particles (VLPs) of papillomavirus can induce VLP-specific humoral and cellular immune responses following parenteral administration. To test whether mucosal administration of bovine papillomavirus type 1 (BPV1) VLPs could produce mucosal as well as systemic immune responses to VLPs, 50 micrograms chimeric BPV1 VLPs containing an HPV16 E7 CTL epitope (BPVL1/E7 VLP) was administered intranasally to mice. After two immunisations, L1-specific serum IgG and IgA were observed. L1-specific IgG and IgA were also found in respiratory and vaginal secretions. Both serum and mucosal antibody inhibited papillomavirus VLP-induced agglutination of RBC, indicating that the antibody induced by mucosal immunisation may recognize conformational determinants associated with virus neutralisation. For comparison, VLPs were given intramuscularly, and systemic and mucosal immune responses were generally comparable following systemic or mucosal delivery. However, intranasal administration of VLP induced significantly higher local IgA response in lung, suggesting that mucosally delivered HPV VLP may be more effective for mediating local mucosal immune responses. Intranasal immunisation with HPV6b L1 VLP produced VLP-specific T proliferative responses in splenocytes, and immunisation with BPVL1 VLP containing an HPV16 E7 CTL epitope induced E7-specific CTL responses. We conclude that immunisation with papillomavirus VLPs via mucosal and intramuscular routes, without adjuvant, can elicit specific antibody at mucosal surfaces and also systemic VLP epitope specific T cell responses. These findings suggest that mucosally delivered VLPs may offer an alternative HPV VLP vaccine strategy for inducing protective humoral immunity to anogenital HPV infection, together with cell-mediated immune responses to eliminate any cells which become infected.
- Published
- 1998
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78. The role of the immune system in anogenital human papillomavirus.
- Author
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Frazer IH
- Subjects
- Antibody Formation, Antigen-Presenting Cells immunology, Antigens, Viral immunology, Cell Division, Condylomata Acuminata virology, Cytokines immunology, Humans, Immunity, Cellular, Immunocompetence, Inflammation Mediators immunology, T-Lymphocytes immunology, Viral Proteins immunology, Virus Replication, Condylomata Acuminata immunology, Papillomaviridae immunology, Papillomavirus Infections immunology, Tumor Virus Infections immunology
- Abstract
There is substantial evidence from experiments of nature that immune competence plays a major part in determining the outcome of anogenital human papillomavirus (HPV) infection. Cellular rather than humoral immunity would appear to be the key to the control and eradication of HPV-induced warts. It seems likely that the HPV early proteins, which are responsible for viral replication (E1 and E2) and for promoting tissue proliferation (E6 and E7) will be the target antigens recognized by antigen-specific T cells. Natural infection is slow to produce an appropriate therapeutic immune response to these proteins, probably because HPV has adopted a strategy to prevent the effective presentation of viral antigens to the host immune system. Human papillomavirus infection is non-lytic so there is little release of viral antigen to professional antigen-presenting cells. Additionally no local proinflammatory cytokine production and no local inflammation is induced by HPV infection. An optimal therapeutic strategy for anogenital HPV infection would be, therefore, to accelerate the induction of a strong virus-specific immune response by inducing local inflammation and the cytokines necessary to invoke HPV-specific immunity.
- Published
- 1998
79. Simplifying the molecular mechanisms of human papillomavirus.
- Author
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Saunders NA and Frazer IH
- Subjects
- Antigens, Viral genetics, Capsid genetics, Cell Transformation, Viral genetics, Condylomata Acuminata virology, DNA-Binding Proteins genetics, Female, Genes, Tumor Suppressor genetics, Genes, Viral genetics, Humans, Keratinocytes cytology, Keratinocytes metabolism, Molecular Biology, Nuclear Proteins genetics, Oncogene Proteins, Viral genetics, Papillomaviridae classification, Papillomaviridae physiology, Papillomavirus Infections physiopathology, Transcription, Genetic, Tumor Virus Infections physiopathology, Uterine Cervical Neoplasms virology, Viral Proteins genetics, Virus Replication genetics, Papillomaviridae genetics
- Abstract
Human papillomaviruses are a common human pathogen responsible for diseases varying in severity from warts to cervical cancer. This article examines the functions of the viral gene products and how they interact with cellular factors to replicate themselves and cause disease.
- Published
- 1998
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80. Split tolerance to a viral antigen expressed in thymic epithelium and keratinocytes.
- Author
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Frazer IH, Fernando GJ, Fowler N, Leggatt GR, Lambert PF, Liem A, Malcolm K, and Tindle RW
- Subjects
- Animals, Epithelium immunology, Histocompatibility Antigens Class II immunology, Humans, Mice, Mice, Transgenic, Papillomavirus E7 Proteins, Thymus Gland cytology, Antigens, Viral immunology, Immune Tolerance, Keratinocytes immunology, Oncogene Proteins, Viral immunology, Papillomaviridae immunology, Thymus Gland immunology
- Abstract
When expressed as a transgene from the keratin 14 (K14) promoter in an MHC class II-deficient mouse, I-Ab expressed in thymic cortical epithelium promotes positive but not negative selection of I-Ab-restricted CD4+ T cells (Laufer, T. M. et al., Nature 1996. 383:81-85). Transgenic mice expressing the E7 protein of human papilloma virus 16 from the K14 promoter were studied to determine the consequence of expression of a cytoplasmic/ nuclear protein from the K14 promoter. K14E7-transgenic mice express E7 in the thymus and skin without evidence for autoimmunity to E7. Repeated immunization of FVB(H-2q) or F1(C57BL/6JxFVB) mice with E7 elicited similar antibody responses to the defined B cell epitopes of E7 in K14E7-transgenic and non-transgenic animals. In contrast, for each genetic background, a single immunization with E7 elicited demonstrable T cell proliferative responses to the major promiscuous T helper epitope of E7 in the transgenic but not the non-transgenic animals. Further, E7-immunized non-transgenic F1 (FVBxC57BL/6J) animals developed strong E7-specific cytotoxic T lymphocyte (CTL) responses and were protected against challenge with E7+ tumors, whereas similarly immunized K14E7-transgenic animals had a markedly reduced CTL response to E7 and no E7-specific tumor protection was observed, although the antibody and CTL response to ovalbumin was normal. Expression of E7 protein as a transgene from the K14 promoter in the skin and thymus thus induces E7-specific tolerance in the cytotoxic T effector repertoire, together with expansion of the E7-specific T helper repertoire. These findings demonstrate that limited tissue distribution of an autoantigen may result in "split" tolerance to that autoantigen.
- Published
- 1998
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81. Th2-type CD4+ cells neither enhance nor suppress antitumor CTL activity in a mouse tumor model.
- Author
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Fernando GJ, Stewart TJ, Tindle RW, and Frazer IH
- Subjects
- Adjuvants, Immunologic pharmacology, Alum Compounds, Amino Acid Sequence, Animals, Antigens, Neoplasm biosynthesis, Antigens, Neoplasm genetics, Antigens, Neoplasm immunology, CD8-Positive T-Lymphocytes immunology, Disease Models, Animal, Drug Combinations, Epitopes, T-Lymphocyte genetics, Epitopes, T-Lymphocyte immunology, Female, Immunity, Active, Inulin immunology, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Mice, Nude, Molecular Sequence Data, Mutagenesis, Site-Directed, Oncogene Proteins, Viral biosynthesis, Oncogene Proteins, Viral genetics, Oncogene Proteins, Viral immunology, Papillomaviridae immunology, Papillomavirus E7 Proteins, Peptide Fragments immunology, Quillaja Saponins, Saponins immunology, Skin Neoplasms genetics, Skin Neoplasms immunology, Skin Neoplasms prevention & control, Thymoma genetics, Tumor Cells, Cultured, Cytotoxicity, Immunologic immunology, T-Lymphocytes, Cytotoxic immunology, Th2 Cells immunology, Thymoma immunology, Thymoma prevention & control
- Abstract
Many cervical cancers express the E7 protein of human papillomavirus 16 as a tumor-specific Ag (TSA). To establish the role of E7-specific T cell help in CD8+ CTL-mediated tumor regression, C57BL/6J mice were immunized with E7 protein or with a peptide (GF001) comprising a minimal CTL epitope of E7, together with different adjuvants. Immunized mice were challenged with an E7-expressing tumor cell line, EL4.E7. Growth of EL4.E7 was reduced following immunization with E7 and Quil-A (an adjuvant that induced a Th1-type response to E7) or with GF001 and Quil-A. Depletion of CD8+ cells, but not CD4+ cells, from an immunized animal abrogated protection, confirming that E7-specific CTL are necessary and sufficient for TSA-specific protection in this model. Immunization with E7 and Algammulin (an alum-based adjuvant) induced a Th2-like response and provided no tumor protection. To investigate whether a Th2 T helper response to E7 could prevent the development of an E7-specific CTL-mediated protection, mice were simultaneously immunized with E7/Algammulin and GF001/Quil-A or, alternatively, were immunized with GF001/Quil-A 8 wk after immunization with E7/Algammulin. Tumor protection was observed in each case. We conclude that an established Th2 response to a TSA does not prevent the development of TSA-specific tumor protective CTL.
- Published
- 1998
82. DNA packaging by L1 and L2 capsid proteins of bovine papillomavirus type 1.
- Author
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Zhao KN, Sun XY, Frazer IH, and Zhou J
- Subjects
- Animals, Bovine papillomavirus 1 physiology, Cattle, Nucleic Acid Conformation, Recombinant Fusion Proteins metabolism, Structure-Activity Relationship, Virion, Bovine papillomavirus 1 metabolism, Capsid metabolism, Capsid Proteins, DNA metabolism, Virus Assembly
- Abstract
Encapsidation of circular DNA by papillomavirus capsid protein was investigated in Cos-1 cells. Plasmids carrying both an SV40 origin of replication (ori) and an E. coli ori were introduced into Cos-1 cells by DNA transfection PV capsid proteins were supplied in trans by recombinant vaccinia viruses. Pseudovirions were purified from infected cells and their packaged DNA was extracted and used to transform E. coli as an indication of packaging efficacy. VLPs assembled from BPV-1 L1 alone packaged little plasmid DNA, whereas VLPs assembled from BPV-1 L1 + L2 packaged plasmid DNA at least 50 times more effectively. BPV-1 L1 + L2 VLPs packaged a plasmid containing BPV-1 sequence 8.2 +/- 3.1 times more effectively than a plasmid without BPV sequences. Using a series of plasmid constructs comprising a core BPV-1 sequence and spacer DNA it was demonstrated that BPV VLPs could accommodate a maximum of about 10.2 kb of plasmid DNA, and that longer closed circular DNA was truncated to produce less dense virions with shorter plasmid sequences. The present study suggests that packaging of genome within PV virions involves interaction of L2 protein with specific DNA sequences, and demonstrates that PV pseudovirions have the potential to be used as DNA delivery vectors for plasmids of up to 10.2 kb.
- Published
- 1998
- Full Text
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83. Type specific and genotype cross reactive B epitopes of the L1 protein of HPV16 defined by a panel of monoclonal antibodies.
- Author
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Kulski JK, Sadleir JW, Kelsall SR, Cicchini MS, Shellam G, Peng SW, Qi YM, Galloway DA, Zhou J, and Frazer IH
- Subjects
- Amino Acid Sequence, Animals, Antibodies, Monoclonal immunology, Cattle, Cell Line, Chlorocebus aethiops, Cross Reactions, Epitopes, B-Lymphocyte chemistry, Epitopes, B-Lymphocyte genetics, Genotype, Humans, Mice, Mice, Inbred BALB C, Molecular Sequence Data, Oncogene Proteins, Viral chemistry, Oncogene Proteins, Viral genetics, Papillomaviridae chemistry, Papillomaviridae genetics, Recombinant Fusion Proteins immunology, Species Specificity, Antibodies, Viral immunology, Capsid Proteins, Epitopes, B-Lymphocyte immunology, Oncogene Proteins, Viral immunology, Papillomaviridae immunology
- Abstract
Mouse monoclonal antibodies (mAbs) were raised against the major capsid protein, L1, of human papillomavirus type 16 (HPV16), produced in Escherichia coli with the expression plasmid pTrcL1. Epitope specificity could be assigned to 11 of these 12 antibodies using a series of linear peptides and fusion proteins from HPV16. One mAb (MC53) recognized a novel linear epitope that appears to be unique to the HPV16 genotype. A further 11 mAbs were characterized as recognizing novel and previously defined linear and conformational epitopes shared among more than one HPV genotype. The apparently genotype specific mAb could be useful for the development of diagnostic tests for vegetative virus infection in clinical specimens.
- Published
- 1998
- Full Text
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84. Papillomavirus virus-like particles can deliver defined CTL epitopes to the MHC class I pathway.
- Author
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Peng S, Frazer IH, Fernando GJ, and Zhou J
- Subjects
- Animals, Antibody Formation, Cattle, Cell Line, Transformed, Cytotoxicity, Immunologic, Drug Design, Epitopes, Female, HIV immunology, HIV Envelope Protein gp160 immunology, Humans, Mice, Mice, Inbred C57BL, Papillomavirus E7 Proteins, Papillomavirus Infections prevention & control, T-Lymphocytes, Cytotoxic virology, Tumor Cells, Cultured, Tumor Virus Infections prevention & control, Viral Vaccines, Bovine papillomavirus 1 immunology, Histocompatibility Antigens Class I immunology, Oncogene Proteins, Viral immunology, Papillomaviridae immunology, Papillomavirus Infections immunology, T-Lymphocytes, Cytotoxic immunology, Tumor Virus Infections immunology
- Abstract
To evaluate an antigen delivery system in which exogenous antigen can target the major histocompatibility complex (MHC) class I pathway, a single human papillomavirus (HPV) 16 E7 cytotoxic T lymphocyte (CTL) epitope and a single HIV gp160 CTL epitope were separately fused to the C-terminus of bovine papillomavirus 1 (BPV1) L1 sequence to form hybrid BPV1L1 VLPs. Mice immunized with these hybrid VLPs mounted strong CTL responses against the relevant target cells in the absence of any adjuvants. In addition, the CTL responses induced by immunization with BPV1L1/HPV16E7CTL VLPs protected mice against challenge with E7-transformed tumor cells. Furthermore, a high titer-specific antibody response against BPV1L1 VLPs was also induced, and this antiserum could inhibit papillomavirus-induced agglutination of mouse erythrocytes, suggesting that the antibody may recognize conformational determinates relevant to virus neutralization. These data demonstrate that hybrid BPV1L1 VLPs can be used as carriers to target antigenic epitopes to both the MHC class I and class II pathways, providing a promising strategy for the design of vaccines to prevent virus infection, with the potential to elicit therapeutic virus-specific CTL responses.
- Published
- 1998
- Full Text
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85. Presentation of the HPV16E7 protein by skin grafts is insufficient to allow graft rejection in an E7-primed animal.
- Author
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Dunn LA, Evander M, Tindle RW, Bulloch AL, de Kluyver RL, Fernando GJ, Lambert PF, and Frazer IH
- Subjects
- Animals, DNA Primers, Dinitrochlorobenzene immunology, Graft Rejection pathology, Homozygote, Humans, Hypersensitivity, Delayed, Lymphoma pathology, Mice, Mice, Inbred C57BL, Mice, Inbred Strains, Mice, Transgenic, Neoplasm Transplantation, Oncogene Proteins, Viral biosynthesis, Oncogene Proteins, Viral genetics, Papillomavirus E7 Proteins, Polymerase Chain Reaction, Recombinant Fusion Proteins biosynthesis, Recombinant Fusion Proteins immunology, Skin Transplantation pathology, Graft Rejection immunology, Lymphoma immunology, Oncogene Proteins, Viral immunology, Papillomaviridae genetics, Skin Transplantation immunology
- Abstract
The E7 transforming protein of Human Papillomavirus type 16 (HPV16) is expressed in the skin of a line of FVB mice transgenic for the E6 and E7 open reading frames of HPV16 driven from the alpha A crystallin promoter (FVB alpha AcryHPV16E6E7). We have transferred skin from FVB alpha AcryHPV16E6E7 mice to naive or E7-primed syngeneic FVB recipients to assess whether the E7 protein of HPV16 can function as a minor transplantation antigen (MTA) and promote skin graft rejection. FVB mice did not reject E7 expressing tail or flank skin grafts. E7 immunized FVB x C57BL/6J mice recipients of FVB alpha-AcryHPV16E6E7 x C57BL/6J skin generated humoral and DTH responses to E7 in vivo and E7-specific CTL precursors in the spleen, but failed to reject E7 expressing tail skin grafts by 100 days posttransfer. Thus although HPV16 E7 + ve mesenchymal and endodermal tumors can be eliminated by an E7-specific immune response, the same protein is unable to act as a MTA and promote graft rejection when expressed in skin cells. Lack of rejection of grafts expressing MTAs such as E7 may be relevant to the immunology of epithelial tumors expressing tumor-specific antigens and to our understanding of the immunology of diseases of the skin.
- Published
- 1997
- Full Text
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86. E7 oncoprotein of human papillomavirus type 16 expressed constitutively in the epidermis has no effect on E7-specific B- or Th-repertoires or on the immune response induced or sustained after immunization with E7 protein.
- Author
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Herd K, Fernando GJ, Dunn LA, Frazer IH, Lambert P, and Tindle RW
- Subjects
- Amino Acid Sequence, Animals, Antibodies, Viral blood, Cell Division, Cells, Cultured, Epidermis, Humans, Hypersensitivity, Delayed, Immunization, Mice, Mice, Transgenic, Molecular Sequence Data, Oncogene Proteins, Viral biosynthesis, Oncogene Proteins, Viral genetics, Papillomavirus E7 Proteins, Papillomavirus Infections immunology, Papillomavirus Infections prevention & control, T-Lymphocytes, Cytotoxic immunology, Tumor Virus Infections immunology, Tumor Virus Infections prevention & control, B-Lymphocytes immunology, Oncogene Proteins, Viral immunology, Papillomaviridae immunology, Repressor Proteins, Skin immunology, T-Lymphocytes, Helper-Inducer immunology
- Abstract
A line of FVB (H-2q) mice transgenic for the E6/E7 open reading frames of Human Papillomavirus type 16 driven from the alpha-A crystallin promoter expresses E7 mRNA in lens and skin epithelium. E7 protein is detectable in adult skin, coinciding with the development of inflammatory skin disease, which progresses to papillomata and squamous carcinomata in some mice. By examining the outcome of parenteral immunization with E7 protein, we sought to determine whether endogenous expression of E7 in skin had induced a preexisting immune outcome, i.e., specific immunity or tolerance, or whether the mice remain naive ("ignorant") to E7. Our data show that the antibody response to defined E7 B-epitopes, the proliferative response to Th epitopes, and the delayed-type hypersensitivity (DTH) response to whole E7 did not differ between groups of young and old E6/E7 transgenic mice (likely having different degrees of lifetime exposure to E7 protein) or between E6/E7-transgenic and nontransgenic parental strain control mice. Although an E7-specific CTL response could not be induced in the H-2q background of these mice, incorporation of a Db allele into the genome allowed comparison of Db-restricted CTL responses in E6/E7 transgenic and nontransgenic mice. Experiments indicated that the E7-immunization-induced CTL response did not differ significantly between E6/E7 transgenic and nontransgenic mice. We interpret these results to indicate that in spite of expression of E7 protein in adult skin, E6/E7 transgenic mice remain immunologically naive (ignorant) of E7 epitopes presented by immunization.
- Published
- 1997
- Full Text
- View/download PDF
87. Human aortic valve allografts elicit a donor-specific immune response.
- Author
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Hogan P, Duplock L, Green M, Smith S, Gall KL, Frazer IH, and O'Brien MF
- Subjects
- Adult, Aged, Aortic Valve immunology, Cryopreservation, Female, Flow Cytometry, Heart Valve Diseases immunology, Heart Valve Diseases surgery, Humans, Isoantibodies analysis, Lymphocyte Culture Test, Mixed, Male, Middle Aged, T-Lymphocytes, Cytotoxic, Transplantation, Homologous immunology, Aortic Valve transplantation, Transplantation Immunology
- Abstract
Objective: The nature and magnitude of the immunologic response to implantation of human cryopreserved aortic valve allografts was investigated., Methods: Twenty aortic valve allograft recipients were investigated for donor-specific antibody and T-cell-mediated responses with serial flow cytometric and microlymphocytotoxic crossmatch assays and one-way mixed lymphocyte cultures., Results: Donor-specific immunoglobulin G antibodies to class I and II human leukocyte antigens were first detected in the serum of all aortic valve allograft recipients at 30 days after implantation and persisted in substantial amounts in all but one of the recipients at day 365. Recipient T-cell alloreactivity toward donor lymphocytes was significantly increased at day 30 compared with levels before and 10 days after operation., Conclusions: Cryopreserved aortic valve allografts elicit a substantial allogeneic response in recipients. This alloreactivity may contribute to the observed morphologic changes in aortic valve allografts and eventual long-term deterioration of allograft function.
- Published
- 1996
- Full Text
- View/download PDF
88. Immunology of papillomavirus infection.
- Author
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Frazer IH
- Subjects
- Animals, Humans, Papillomaviridae immunology, Papillomavirus Infections immunology, Tumor Virus Infections immunology
- Abstract
Studies of the immunology of papillomavirus infection have come of age. Synthetic virus-like particles have been validated as vaccines for several animal papillomaviruses, and have been used to map the sero-epidemiology of human papillomavirus infection and to define papillomavirus neutralizing antibodies. Induction of cell-mediated immunity to papillomavirus early proteins is poised to become a therapeutic approach to papillomavirus infection. Studies on the immune response to papillomavirus proteins in keratinocytes are shedding light on the immunological consequences of antigen presentation by epithelial cells.
- Published
- 1996
- Full Text
- View/download PDF
89. Familial primary antiphospholipid antibody syndrome.
- Author
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Bansal AS, Hogan PG, Gibbs H, and Frazer IH
- Subjects
- Adult, Antiphospholipid Syndrome blood, Antiphospholipid Syndrome immunology, Child, Child, Preschool, Female, Humans, Male, Thrombosis blood, Antibodies, Anticardiolipin blood, Antiphospholipid Syndrome genetics, Femoral Vein, Thrombosis immunology
- Published
- 1996
- Full Text
- View/download PDF
90. Managing HIV. Part 3: Mechanisms of disease. 3.6 How HIV promotes malignancies.
- Author
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Boyle MJ, Goldstein DA, Frazer IH, and Sculley TB
- Subjects
- Cytokines immunology, HIV Infections immunology, Humans, HIV Infections complications, Neoplasms immunology, Neoplasms virology
- Published
- 1996
91. Epithelial cells display separate receptors for papillomavirus VLPs and for soluble L1 capsid protein.
- Author
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Qi YM, Peng SW, Hengst K, Evander M, Park DS, Zhou J, and Frazer IH
- Subjects
- Animals, Cell Line, Epithelium virology, Escherichia coli, Glycosylation, Humans, Isotope Labeling, Protein Binding drug effects, Recombinant Fusion Proteins metabolism, Spodoptera cytology, Trypsin, Tunicamycin pharmacology, Viral Proteins, Virion metabolism, Capsid Proteins, Oncogene Proteins, Viral metabolism, Papillomaviridae metabolism, Receptors, Virus metabolism
- Abstract
We examined the distribution of putative receptors for papillomavirus (PV) capsid proteins on various cell types, using either Hexahis HPV6b L1 fusion protein or synthetic HPV6b virus-like particles (VLPs). Specific, saturable binding of VLPs to CV-1 cells was demonstrated using 35S-labeled VLPs, with an average receptor number of 1 x 10(4)/cell and a binding affinity constant (Ka) of 4 x 10(7) M. VLP binding was quantitated by flow cytometry using a monoclonal antibody to the L1 capsid protein. Intense staining of epithelial and mesenchymal cells was observed. Some immature bone marrow-derived cells bound VLPs weakly, while the majority of B lymphoma cells demonstrated no binding. Binding to 12 of 16 VLP receptor positive cell lines was abolished by trypsin pretreatment of cells. Removal of cellular sialic acid or O-linked oligosaccharides separately did not affect VLP binding, which was enhanced about 25% when cells were pretreated with both neuraminidase and O-glycosidase. Culture of cells with sufficient tunicamycin to inhibit Concanavalin A binding did not diminish the binding of VLPs. Denatured L1 protein, either from VLPs or expressed from Escherichia coli as a Hexahis fusion protein, bound to a trypsin-resistant structure on a range of cell types and did not block the binding of VLPs to cells. Dual-fluorescence assay with a Burkitt lymphoma line BL72 demonstrated that Hexahis L1 protein and VLPs bind to separate cell surface molecules on BL72 cells. We conclude that the first binding of PV virus to cells is via a widely distributed membrane protein receptor(s) and that subsequent processing of particles may involve other non-trypsin-sensitive structure(s) also displayed on the cell membrane.
- Published
- 1996
- Full Text
- View/download PDF
92. Immunological responses in human papillomavirus 16 E6/E7-transgenic mice to E7 protein correlate with the presence of skin disease.
- Author
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Frazer IH, Leippe DM, Dunn LA, Liem A, Tindle RW, Fernando GJ, Phelps WC, and Lambert PF
- Subjects
- Amino Acid Sequence, Animals, Antibody Formation, Enzyme-Linked Immunosorbent Assay, Epitopes analysis, Eye Neoplasms immunology, Eye Neoplasms virology, Female, Genes, Viral, Genotype, Humans, Immune Tolerance, Mice, Mice, Inbred Strains, Mice, Transgenic, Molecular Sequence Data, Oncogene Proteins, Viral biosynthesis, Papillomaviridae pathogenicity, Papillomavirus E7 Proteins, Peptides chemical synthesis, Peptides immunology, Skin Diseases virology, Skin Neoplasms virology, Uterine Cervical Neoplasms virology, Hypersensitivity, Delayed, Oncogene Proteins, Viral immunology, Papillomaviridae genetics, Repressor Proteins, Skin Diseases immunology, Skin Neoplasms immunology
- Abstract
The human papillomavirus (HPV) oncogenes, E6 and E7, are believed to contribute to the development of cervical cancers in women infected with certain HPV genotypes, most notably HPV-16 and HPV-18. Given their expression in tumor tissue, E6 and E7 have been implicated as potential tumor-specific antigens. We have examined an HPV-16 E6- and E7-transgenic mouse lineage for immune responses to these viral oncoproteins. Mice in this lineage express the HPV-16 E6 and E7 genes in their skin and eyes, and on aging, these mice frequently develop squamous cell carcinomas and lenticular tumors. Young transgenic mice, which had measurable E7 protein in the eye but not in the skin, were immunologically naive to E7 protein. They mounted an immune response to E7 on immunization comparable to that of nontransgenic controls, suggesting a lack of immune tolerance to this protein. Older line 19 mice, which are susceptible to skin disease associated with transcription of the E6 and E7 open reading frames, had measurable E7 protein in their skin. These older transgenic mice spontaneously developed antibody responses to endogenous E7 protein, particularly in association with skin disease. Also detected in older mice were delayed-type hypersensitivity responses to E7. These finding parallel the humoral immune response to E7 protein in patients with HPV-associated cervical cancer and suggest that line 19 mice may provide a model for studying the immunobiology of HPV-associated cancers.
- Published
- 1995
93. Routine HIV testing before surgery.
- Author
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Selvey L, Hogan P, Frazer IH, Smithers M, and Robinson D
- Subjects
- Australia epidemiology, Enzyme-Linked Immunosorbent Assay, HIV Seroprevalence, Humans, AIDS Serodiagnosis, Diagnostic Tests, Routine, HIV Infections prevention & control, HIV-1, Preoperative Care
- Published
- 1995
94. Peptide polymerisation facilitates incorporation into ISCOMs and increases antigen-specific IgG2a production.
- Author
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Fernando GJ, Stenzel DJ, Tindle RW, Merza MS, Morein B, and Frazer IH
- Subjects
- Adjuvants, Immunologic pharmacology, Amino Acid Sequence, Animals, Antibodies, Viral biosynthesis, Antibodies, Viral classification, Antibody Specificity, Biopolymers immunology, Female, Humans, ISCOMs chemistry, Immunoglobulin G therapeutic use, Mice, Mice, Inbred CBA, Molecular Sequence Data, Oncogene Proteins, Viral chemistry, Oncogene Proteins, Viral immunology, Papillomaviridae chemistry, Papillomaviridae immunology, Peptides chemistry, Adjuvants, Immunologic chemistry, DNA-Binding Proteins, Epitopes immunology, ISCOMs immunology, Immunoglobulin G biosynthesis, Peptides immunology
- Abstract
Synthetic peptides can be tailor-made to include any B or T epitopes desired from a single or multiple antigens or organisms. However, peptides in general are not very immunogenic and have not proven easy to incorporate into immunogenic vaccines. ISCOMs is an adjuvant system that has the capability not only to enhance the humoral immunogenicity of a protein but has also been shown to induce cell-mediated immune responses in animals. Synthetic peptide ISCOM vaccines are few because of the difficulty in incorporation of these peptides into ISCOMs. We have shown in this study that non-immunogenic peptides could be made immunogenic by polymerisation, and these polymers could be incorporated into ISCOMs to give highly immunogenic vaccines. Synthetic 20mer peptides containing known B and T-helper epitopes from the E7 protein of the cervical cancer associated human papillomavirus type 16 (HPV16 E7) have been used here as model immunogens. We have compared the humoral immunity induced by these peptides as polymers or as copolymers with a lipid binding 20mer peptide (LAP 20), with or without incorporation into ISCOMs. Unpolymerised peptide elicited no measurable antibody. When polymerised peptide was administered with CFA, or in phosphate-buffered saline (PBS) without adjuvant, or incorporated into ISCOMs, antibodies recognising both the immunising peptide and HPV16 E7 protein were produced. For equal quantities of administered peptide (5 micrograms), ISCOMs gave higher titres of antibody than CFA or PBS. Polymerised peptides induced high antigen-specific IgG2a:IgG1 ratios, which increased with multiple immunisations. These data indicate that polymerised peptides could be incorporated into ISCOMs to form efficient immunogens which may elicit a Th1 type response.
- Published
- 1995
- Full Text
- View/download PDF
95. Donor-specific immune response after aortic valve allografting in the rat.
- Author
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Zhao XM, Green M, Frazer IH, Hogan P, and O'Brien MF
- Subjects
- Animals, Aortic Valve immunology, Female, Flow Cytometry, Isoantibodies analysis, Lymphocyte Culture Test, Mixed, Rats, Rats, Inbred BUF, Rats, Inbred Lew, Rats, Inbred Strains, Skin Transplantation immunology, T-Lymphocyte Subsets, T-Lymphocytes, Cytotoxic, Transplantation, Homologous immunology, Transplantation, Isogeneic immunology, Aortic Valve transplantation, Transplantation Immunology
- Abstract
The allospecific immune response in rats to a major histocompatibility complex-disparate aortic valve allograft was investigated using three in vitro assays. In each assay, DA strain (RT-1a) rats served as allograft recipient and syngeneic donor, Lewis strain (RT-1l) rats were allogeneic donors, and Buffalo (RT-1b) rats provided third-party control cells. Mixed lymphocyte cultures using spleen cells demonstrated donor-specific stimulation indices of 3.04 +/- 0.44, 4.14 +/- 0.62, and 6.32 +/- 0.60 at 7, 14, and 28 days, respectively, after aortic valve allografting; 8.19 +/- 2.91, 8.51 +/- 1.25, and 10.80 +/- 0.53 after skin allografting; and 1.84 +/- 0.56, 1.82 +/- 0.38, and 1.82 +/- 0.53 after aortic valve isografting. Limiting dilution analysis of splenocytes showed a donor-specific cytotoxic T lymphocyte precursor frequency at 7, 14, and 28 days of 1:6,853, 1:4,714, and 1:1,964 after aortic valve allografting; 1:4,181, 1:1,611, and 1:1,018 after skin allografting; and 1:14,517, 1:11,882, and 1:10,995 after aortic valve isografting. Flow cytometry detected an increase in the level of donor-specific anti-T cell antibodies in both valve and skin allograft recipients but not in isografted animals. Aortic valve allografting from Lewis into DA rats elicits allospecific cellular and humoral immune responses similar in magnitude to skin allografting but somewhat slower in onset. Investigation of the immune response to aortic allografts in humans is warranted, as donor-specific T cells, antibodies, or both may damage the allograft.
- Published
- 1994
- Full Text
- View/download PDF
96. Glycosylation of human papillomavirus type 16 L1 protein.
- Author
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Zhou J, Sun XY, and Frazer IH
- Subjects
- Acetylglucosaminidase metabolism, Amino Acid Sequence, Base Sequence, Biological Transport, Cell Compartmentation, DNA Mutational Analysis, Fluorescent Antibody Technique, Glycosylation drug effects, Lectins, Mannose, Molecular Sequence Data, Oligosaccharides chemistry, Oncogene Proteins, Viral genetics, Oncogene Proteins, Viral isolation & purification, Plasmids genetics, Protein Sorting Signals, Recombinant Proteins metabolism, Tunicamycin pharmacology, Vaccinia virus genetics, Capsid Proteins, Oncogene Proteins, Viral metabolism, Papillomaviridae metabolism, Protein Processing, Post-Translational drug effects
- Abstract
We examined glycosylation of the L1 capsid protein of human papillomavirus type 16, using HPV16 L1 protein expressed from various recombinant vaccinia viruses in CV-1 and HaCaT cells. A minority of L1 protein was N-glycosylated, and all four potential N-glycosylation sites appeared to be used. Glycosylation was of the high-mannose type, as shown by reactivity with biotin-labeled Concanavalin A and by exoglycosidase digestions. A series of mutant L1 proteins were used to establish that an N-terminal hydrophobic sequence, common to all sequenced papillomavirus L1 capsid proteins, was a major determinant of the proportion of L1 protein glycosylated, whereas C-terminus nuclear localization signal sequences were unimportant. Subcellular localization studies showed that whereas the majority of L1 protein was found in the cell nucleus, glycosylated L1 was retained in the endoplasmic reticulum and was neither exported from the cell nor translocated to the cell membrane or the cell nucleus. We conclude that glycosylated L1 is unlikely to be an important component of the papillomavirus virion, a finding of importance for the design of papillomavirus-specific vaccines.
- Published
- 1993
- Full Text
- View/download PDF
97. The vaccinia virus K2L gene encodes a serine protease inhibitor which inhibits cell-cell fusion.
- Author
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Zhou J, Sun XY, Fernando GJ, and Frazer IH
- Subjects
- Animals, Base Sequence, Cells, Cultured, Chlorocebus aethiops, DNA, Viral genetics, Fluorescent Antibody Technique, In Vitro Techniques, Molecular Sequence Data, Mutagenesis, Site-Directed, Oligodeoxyribonucleotides chemistry, Restriction Mapping, Serpins immunology, Serpins metabolism, Cell Fusion, Genes, Viral, Serpins genetics, Vaccinia virus genetics, Viral Structural Proteins genetics
- Abstract
In certain circumstances, cells infected with vaccinia virus (VV) undergo fusion, but this does not occur in tissue cultures infected with wild-type VV. The VV genome includes three genes (B24R, B13R, and K2L) encoding polypeptides that are structurally related to members of the plasma serine proteases inhibitor (SPI) superfamily. In this study, we demonstrate by deleting these genes singly or in combination that the K2L gene encoding SPI-3, but not the B24R or B13R genes encoding SPI-1 and SPI-2, inhibits cell-cell fusion in VV-infected cells. A VV-encoded hemagglutinin (HA) has previously been demonstrated to inhibit cell-cell fusion, but fusion-promoting VVs with K2L gene deletions had normal expression and cellular location of the VV HA. As both HA and SPI-3 independently inhibit cell-cell fusion in VV-infected cells, there must be at least two fusion-promoting mechanisms encoded by VV. These may play different roles in virus-cell fusion and in cell-cell fusion after VV infection.
- Published
- 1992
- Full Text
- View/download PDF
98. Definition of linear antigenic regions of the HPV16 L1 capsid protein using synthetic virion-like particles.
- Author
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Zhou J, Sun XY, Davies H, Crawford L, Park D, and Frazer IH
- Subjects
- Amino Acid Sequence, Antibodies, Viral immunology, B-Lymphocytes immunology, Blotting, Western, Capsid chemistry, Enzyme-Linked Immunosorbent Assay, Molecular Sequence Data, Recombinant Proteins chemistry, Recombinant Proteins immunology, Antigens, Viral chemistry, Capsid immunology, Epitopes, Papillomaviridae immunology
- Abstract
Mice of three haplotypes (H-2d, H-2b, and H-2d/b) were immunized with synthetic HPV16 virus-like particles (VLPs), produced using a vaccinia virus doubly recombinant for the L1 and L2 proteins of HPV16. The resultant anti-VLP antisera recognized HPV16 capsids by ELISA assay and baculovirus recombinant HPV16 L1 and L2 protein on immunoblot. Overlapping peptides corresponding to the HPV16 L1 amino acid sequence were used to define the immunoreactive regions of the L1 protein. The majority of the L1 peptides were reactive with IgG from the mice immunized with the synthetic HPV16 capsids. A computer algorithm predicted seven B epitopes in HPV16 L1, five of which lay within peptides strongly reactive with the murine antisera. The murine anti-VLP antisera failed to react with the two peptides recognized by anti-HPV16L1 monoclonal antibodies raised by others against recombinant L1 fusion protein. We conclude that the immunoreactive epitopes of HPV16 defined using virus-like particles differ significantly from those defined using recombinant HPV16 L1 fusion proteins, which implies that such fusion proteins may not be the antigens to look for HPV16L1 specific immune responses in HPV-infected patients.
- Published
- 1992
- Full Text
- View/download PDF
99. An ELISA capture assay for the E7 transforming proteins of HPV16 and HPV18.
- Author
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Selvey LA, Dunn LA, Murray B, Tindle RW, and Frazer IH
- Subjects
- Animals, Antibodies, Monoclonal, Cell Line, Transformed, Cell Transformation, Viral, Female, HeLa Cells, Humans, Oncogene Proteins, Viral standards, Papillomavirus E7 Proteins, Rabbits, Tumor Virus Infections diagnosis, Uterine Cervical Neoplasms diagnosis, DNA-Binding Proteins, Enzyme-Linked Immunosorbent Assay standards, Oncogene Proteins, Viral analysis, Papillomaviridae chemistry
- Abstract
ELISA capture assays were established for the E7 transforming proteins of HPV16 and HPV18, based on a range of previously characterised polyclonal and monoclonal antibodies. No cross-reactivity was observed in the ELISAs between HPV18 E7 and HPV16 E7. Immunoreactive E7 protein (iE7) was measured in a series of HPV-transformed cell lines, and ranged from 0.6 to 17.7 ng iE7/mg cell protein. iE7 was labile at 22 degrees C (t1/2 = 37 min) but relatively more stable at 4 degrees C (t1/2 = 210 min). HPV16 E7 protein at concentrations from 0.10 to 0.69 ng iE7/mg cell protein was detected in 5 of 13 smears from women with abnormal cervical cytology. Assay of E7 protein may play a role in the detection of HPV-induced cervical lesions with malignant potential.
- Published
- 1992
- Full Text
- View/download PDF
100. Identification of the nuclear localization signal of human papillomavirus type 16 L1 protein.
- Author
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Zhou J, Doorbar J, Sun XY, Crawford LV, McLean CS, and Frazer IH
- Subjects
- Amino Acid Sequence, Base Sequence, DNA Mutational Analysis, Fluorescent Antibody Technique, Humans, Membrane Proteins genetics, Molecular Sequence Data, Oncogene Proteins, Viral chemistry, Plasmids genetics, Polymerase Chain Reaction, Protein Sorting Signals chemistry, Recombinant Fusion Proteins genetics, Sequence Homology, Nucleic Acid, Vaccinia virus genetics, Capsid Proteins, Oncogene Proteins, Viral genetics, Papillomaviridae genetics, Protein Sorting Signals genetics
- Abstract
Human papillomavirus type 16(HPV16) L1 and L2 capsid proteins can be detected only in the nucleus of infected cells. For other nuclear proteins, specific sequences of basic amino acids(aa) termed nuclear localization signals (NLS) direct the protein from the cytoplasm to the nucleus. We used a series of deletion and substitution mutations of the HPV16 L1 protein, produced by recombinant vaccinia virus (rVV), to identify NLS within HPV16 L1 and showed that HPV16 L1 contains two NLS sequences, each containing basic aa clusters. One NLS consisted of 6 basic amino acids (KRKKRK from aa 525 to 530) at the carboxy terminal end of L1. The other NLS contained 2 basic aa clusters(KRK from aa 510 to 512 and KR at aa 525, 526) separated by 12 amino acids. Mutations in either NLS did not alter nuclear localization of L1 when the other remained intact, but mutations to both prevented nuclear localization of L1. The L1 NLS could be overridden by introduction of a membrane binding sequence at the amino terminal end of the protein. A databases search showed that all sequenced papillomaviruses are predicted to have L1 and L2 capsid proteins with sequences of basic amino acids homologous with one or both NLS of HPV16 L1.
- Published
- 1991
- Full Text
- View/download PDF
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