Biomedical subjects

I H Frazer

Publications and source records attributed to I H Frazer.

At least 37 records · Page 2Linked to original sources

PubMed1999

Nucleotides 1506-1625 of bovine papillomavirus type 1 genome can enhance DNA packaging by L1/L2 capsids.

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.

Animals↗

PubMed1999

Construction and production of fluorescent papillomavirus-like particles.

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.

Baculoviridae↗

PubMed1999

Post translational modifications of recombinant human papillomavirus type 6b major capsid protein.

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.

Animals↗

PubMed1999

Expression, purification and immunological characterization of the transforming protein E7, from cervical cancer-associated human papillomavirus type 16.

E7 is the major oncogenic protein produced in cervical cancer-associated human papillomavirus type 16 (HPV16). This protein was expressed in Escherichia coli as a glutathione-S-transferase (GST) fusion protein. E7-enriched inclusion bodies were collected from bacterial lysates, were solubilized in 10 M urea, and the protein was purified using anion exchange column chromatography. After removal of endotoxin with serial Triton X-114 extractions, material of high purity (about 90%) was obtained, which is suitable for use in a human clinical trial. This material was immunogenic, and when used as a vaccine, protected mice against challenge with an HPV16 E7 DNA transfected tumour cell line. Based on this observation, the E7GST fusion protein is currently being used in a human clinical trial of a vaccine against HPV16-induced cervical cancer. This fusion protein could be cleaved with thrombin to remove the GST fusion part and further purified by preparative SDS gel electrophoresis to obtain free E7 with > 98% purity.

Animals↗

PubMed1999

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.

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.

Adolescent↗

PubMed1999

T-helper epitopes identified within the E6 transforming protein of cervical cancer-associated human papillomavirus type 16.

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.

Amino Acid Sequence↗

PubMed1999

Potential strategies utilised by papillomavirus to evade host immunity.

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.

Animals↗

PubMed1999

T cell-mediated and non-specific inflammatory mechanisms contribute to the skin pathology of HPV 16 E6E7 transgenic mice.

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.

Age Factors↗

PubMed1999

Genetic and environmental causes of variation in basal levels of blood cells.

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.

Antigens, CD↗

PubMed1998

Mucosal immunisation with papillomavirus virus-like particles elicits systemic and mucosal immunity in mice.

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.

Administration, Intranasal↗

PubMed1998

Th2-type CD4+ cells neither enhance nor suppress antitumor CTL activity in a mouse tumor model.

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.

Adjuvants, Immunologic↗

PubMed1998

Type specific and genotype cross reactive B epitopes of the L1 protein of HPV16 defined by a panel of monoclonal antibodies.

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.

Amino Acid Sequence↗

PubMed1998

DNA packaging by L1 and L2 capsid proteins of bovine papillomavirus type 1.

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.

Animals↗

PubMed1998

Papillomavirus virus-like particles can deliver defined CTL epitopes to the MHC class I pathway.

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.

Animals↗

PubMed1998

Split tolerance to a viral antigen expressed in thymic epithelium and keratinocytes.

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.

Animals↗

PubMed1998

Histologic and immunohistochemical responses after aortic valve allografts in the rat.

BACKGROUND: Human aortic valve allografts elicit a cellular and humoral immune response. It is not clear whether this is important in promoting valve damage. We investigated the changes in morphology, cell populations, and major histocompatibility complex antigen distribution in the rat aortic valve allograft. METHODS: Fresh heart valves from Lewis rats were transplanted into the abdominal aorta of DA rats. Valves from allografted, isografted, and presensitized recipient rats were examined serially with standard morphologic and immunohistochemical techniques. RESULTS: In comparison with isografts, the allografts were infiltrated and thickened by increased numbers of CD4+ and CD8+ lymphocytes, macrophages, and fibroblasts. Thickening of the valve wall and leaflet and the density of the cellular infiltrate was particularly evident after presensitization. Endothelial cells were frequently absent in presensitized allografts whereas isografts had intact endothelium. Cellular major histocompatibility complex class I and II antigens in the allograft were substantially increased. A long-term allograft showed dense fibrosis and disruption of the media with scattered persisting donor cells. CONCLUSIONS: The changes in these aortic valve allograft experiments are consistent with an allograft immune response and confirm that the response can damage aortic valve allograft tissue.

Animals↗

PubMed1998

Simplifying the molecular mechanisms of human papillomavirus.

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.

Antigens, Viral↗

PubMed1998

Vaccine-induced Th1-type responses are dominant over Th2-type responses in the short term whereas pre-existing Th2 responses are dominant in the longer term.

The effect of adjuvant on induction of human papillomavirus type 16 E7 protein-specific cytotoxic T lymphocytes (CTL) and immunoglobulin G (IgG)2a antibody was studied in C57BL/6 J mice immunized with various adjuvants and E7 protein. Quil-A adjuvant, but not complete Freund's adjuvant (CFA) or Algammulin, induced a T-helper 1 (Th1)-type response to E7, which was characterized by CTL activity against a tumour cell line transfected with E7 protein and by E7-specific IgG2a. All tested adjuvants elicited comparable levels of E7-specific IgG1. The longest duration and greatest magnitude of CTL response was seen following two immunizations with the highest dose of E7 and Quil-A. Simultaneous immunization with a Th1 and a T helper 2 (Th2)-promoting adjuvant gave a Th1-type response. However, E7 and Quil-A were unable to induce a Th1-type response (as measured by the inability to generate anti-E7 IgG2a antibody) in animals with a pre-existing Th2-type response to E7. These results suggest that saponin adjuvants may be suitable for immunotherapy in humans where a Th1-type response is sought, provided that there is no pre-existing Th2-type response to the antigen.

Adjuvants, Immunologic↗