Biomedical subjects

I H Frazer

Publications and source records attributed to I H Frazer.

At least 73 records · Page 4Linked to original sources

PubMed1994

Interaction of human papillomavirus (HPV) type 16 capsid proteins with HPV DNA requires an intact L2 N-terminal sequence.

Encapsidation of papillomavirus DNA involves DNA-protein and protein-protein interactions. We sought to define the role of each human papillomavirus (HPV) capsid protein in HPV DNA encapsidation. HPV16 major (L1) and minor (L2) capsid proteins purified from recombinant vaccinia virus-infected cells were compared for their ability to bind nucleic acids. L2 protein, but not L1 protein, could bind HPV DNA. To map the DNA-binding region of L2, a series of truncated or point-mutated L2 protein open reading frames were used to show that only the N terminal of L2 was required for L2-DNA binding. This interaction depends critically on charged amino acids (Lys or Arg) in the first 12 amino acids of the N terminal of the protein. Several techniques were used to show that L2 interaction with DNA did not require specific DNA sequences. We propose that HPV L2 protein may play a major role in papillomavirus capsid assembly by introducing HPV DNA to the virus particles formed by the self assembly of the L1 major structural protein.

Amino Acid Sequence↗

PubMed1993

Human papillomavirus type 16 E6, E7 and L1 and type 18 E7 proteins produced by recombinant baculoviruses.

Proteins derived from the E6, E7 and L1 ORFs of HPV16 and the E7 ORF of HPV18 were produced in insect cells using a baculovirus expression system. HPV ORFs were inserted into baculovirus transfer vectors pAcYM1 or pVL1393/2, and recombinant baculoviruses isolated using a combination of limiting dilution and plaque assay. Using HPV-specific antisera and monoclonal antibodies HPV proteins were identified in lysates of Spodoptera frugiperda (Sf-21) cells infected with HPV-recombinant baculovirus. Immunoreactive HPV16 E7 protein produced in Sf-21 cells had an apparent M(r) of 19 kDa, larger than that predicted from the amino acid sequence, and similar to that of native HPV16 E7 protein in HeLa and CaSki cells. The apparent M(r) of recombinant HPV18-E7, HPV16-L1 and HPV16-E6 proteins was equivalent to the M(r) values predicted from the amino acid sequence. Thermostability studies revealed that the half-life of HPV16-E7 protein in Sf-21 cell lysate was approx. 20 h at 4 degrees C, 2 h at 22 degrees C, and less than 30 min at 37 degrees C. HPV16 L1, HPV16 E7 and HPV18 E7 proteins were predominantly localised in the nucleus of recombinant baculovirus-infected Sf-21 cells, whereas recombinant HPV 16 E6 protein was localised in both the cytoplasm and nucleus of infected insect cells. Northern blot analysis of RNA derived from insect cells infected with vAc16E6E7, a recombinant baculovirus containing both HPV16 E6 and E7 ORF's, revealed the presence of only E6 ORF transcripts, suggesting that the splicing of RNA products derived from the E6 and E7 ORF's, as observed in cervical cancer-derived cell lines, is not performed in insect cells. Baculovirus-derived HPV proteins have similar biological properties to the native proteins and should be suitable for studies on the immunology of HPV.

Animals↗

PubMed1993

Glycosylation of human papillomavirus type 16 L1 protein.

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.

Acetylglucosaminidase↗

PubMed1993

Synthesis and assembly of infectious bovine papillomavirus particles in vitro.

Bovine papillomavirus type 1 (BPV-1) virions were produced in vitro using vaccinia virus (VV) recombinants expressing the BPV-1 L1 and L2 capsid proteins. Particles morphologically resembling papillomaviruses were observed in the nucleus of cells infected with a VV recombinant for the BPV-1 L1 protein, and greater numbers of similar particles were seen in the nuclei of cells infected with a VV double recombinant for L1 and L2. Virus-like particles (VLPs) assembled in cells infected with the VV double recombinant for BPV-1 L1 and L2, and not those assembled in cells infected with the VV recombinant for BPV-1 L1 alone, were able to package BPV-1 DNA. Transcription of the BPV-1 E1 viral open reading frame was observed after a mouse fibroblast cell line was exposed to VLPs produced using a BPV-1 L1/L2 VV recombinant in a cell line containing episomal BPV-1 DNA. E1 transcription was not observed when the VLPs were pre-incubated with antibodies to the capsid protein of BPV-1. This system should allow an in vitro approach to the definition of the BPV-1 cellular receptor.

Animals↗

PubMed1993

Transmission of human papillomaviruses from mother to child.

Exfoliated cervical epithelial cells from women 6 weeks postpartum were analyzed for human papillomavirus (HPV) DNA using the polymerase chain reaction, and results were compared with those from buccal mucosal smears from their babies. Eleven mothers had genital genotypes of HPV in their cervical smears, and the children of 8 of these had HPV of the same genotype in buccal mucosal cell samples. Nineteen mothers had no HPV DNA detected in their cervical smears, and 1 of the buccal mucosal cell samples from their children was positive for HPV DNA (p < 0.0001). Contamination of a child's mouth with 'genital' HPV from a mother's cervix appears to occur commonly at birth or in the perinatal period, and to persist for at least 6 weeks. This observation has implications for the epidemiology and management of HPV associated cancer and precancerous conditions in the cervix and the mouth.

Cervix Uteri↗

PubMed1993

HPV DNA in oropharyngeal squamous cell cancers: comparison of results from four DNA detection methods.

Oropharyngeal squamous cell cancers (SCCs) were examined for human papillomavirus (HPV) related DNA sequences. The techniques employed were Southern blotting under stringent and non stringent conditions, dot blotting, primer directed gene amplification using the polymerase chain reaction (PCR), and in-situ hybridization. HPV 16 DNA was found in 4 of 30 tumor samples using PCR. HPV 16 DNA was found in 2 further tumors using in-situ hybridization. No HPV DNA could be found by Southern blot or dot blot in any tumor sample. The Southern blot assays were sensitive enough to detect clonally integrated HPV 16 DNA of length greater than 250 bp in the tumors. While HPV DNA is present in some oropharyngeal SCCs, there is no molecular evidence to support a causal association of HPV 16 gene products with continued tumor growth in oropharyngeal cancer.

Adult↗

PubMed1992

Definition of linear antigenic regions of the HPV16 L1 capsid protein using synthetic virion-like particles.

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.

Amino Acid Sequence↗

PubMed1992

The vaccinia virus K2L gene encodes a serine protease inhibitor which inhibits cell-cell fusion.

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.

Animals↗

PubMed1992

An ELISA capture assay for the E7 transforming proteins of HPV16 and HPV18.

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.

Animals↗

PubMed1991

The hygromycin-resistance-encoding gene as a selection marker for vaccinia virus recombinants.

Hygromycin B (Hy), an inhibitor of RNA translation, was shown to block the replication of vaccinia virus (VV) in cultured cell lines. Insertion of the Escherichia coli Hy resistance-encoding gene (hph) into the VV genome under control of early or late synthetic VV promoters could overcome inhibition of viral replication. When hph was inserted into VV in tandem with the human papillomavirus type 16 (HPV16) L1 open reading frame, hph recombinant viruses could be selected which expressed HPV16 L1.

Base Sequence↗

PubMed1991

A "public" T-helper epitope of the E7 transforming protein of human papillomavirus 16 provides cognate help for several E7 B-cell epitopes from cervical cancer-associated human papillomavirus genotypes.

We have identified a major T-cell epitope, amino acids 48-54 (DRAHYNI, in one-letter code) in the E7 open reading frame protein of human papillomavirus (HPV) type 16. Lymph node cells from mice immunized with synthetic peptides containing DRAHYNI proliferated and produced interleukin when challenged in vitro with peptide or whole HPV-16 E7 fusion protein. The T epitope was recognized in association with all five major histocompatibility complex class II I-A and I-E alleles tested. Synthetic peptides consisting of DRAHYNI linked to major B-cell epitopes on the E7 molecule formed immunogens capable of eliciting strong antibody responses to HPV-16 E7. The T epitope could provide help for the production of antibody to several B epitopes simultaneously, including a B epitope of HPV-18 E7 protein. Mice immunized with a peptide containing DRAHYNI and B epitope and, at a later date, infected with recombinant vaccinia E7 virus, displayed secondary antibody responses to E7. Because E7 has a role in cell transformation and is the most abundant viral protein in HPV-associated neoplastic cervical epithelial cells, the data have implications for vaccine strategies.

Amino Acid Sequence↗

PubMed1991

Identification of the nuclear localization signal of human papillomavirus type 16 L1 protein.

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.

Amino Acid Sequence↗

PubMed1991

Expression of vaccinia recombinant HPV 16 L1 and L2 ORF proteins in epithelial cells is sufficient for assembly of HPV virion-like particles.

A recombinant vaccinia virus termed pLC201VV was designed to coexpress the L1 and L2 late genes of human papillomavirus type 16 (HPV16). Synthesis of the L1 and L2 proteins occurred in cells infected with pLC201VV, and 40-nm virus-like particles with a density of 1.31 g/ml were produced in the nuclei of cells synthesizing both L1 and L2, but not in cells synthesizing either protein alone. Virus-like particles were partially purified from infected cells by sucrose gradient sedimentation and shown to consist of capsomeres similar to HPV and contain glycosylated L1 viral capsid protein. The production of HPV-like particles using recombinant vaccinia virus should be useful for biochemical studies and could provide a safe source of material for the development of a vaccine.

Animals↗

PubMed1990

Identification of B-epitopes in the human papillomavirus 18 E7 open reading frame protein.

A panel of murine mAb raised against a MS2 replicase/HPV 18 E7 fusion protein included 23 reactive by ELISA with HPV 18 E7 determinants. A total of 19 of the 23 recognized linear epitopes in the N-terminal region of the E7 molecule, while the other four were deduced by binding inhibition assays to recognize conformational determinants in this region. All tested antibodies precipitated a 14-kDa peptide doublet that corresponded with the predicted size of the E7 protein, from HeLa cells, but not from HPV 16 E7 containing CaSki cells. HPV 18 E7 protein was detected by immunolabeling with electron microscopy in both the nucleus and the cytoplasm of HeLa cells with the greater proportion occurring in the cytoplasm. No antibody reacted specifically by indirect immunofluorescence with HeLa cells. Weak cross-reactivity of some mAb with the E6 MS2-replicase fusion protein of HPV 16 was detected by ELISA, but no protein of the appropriate size was immunoprecipitated from CaSki cells. It is concluded that the B cell epitopes on the HPV 18 E7 transforming protein are located in the N-terminal region of the molecule and that some are weakly cross-reactive with HPV 16 E6 protein. E7 protein is either present in HeLa cells at a concentration too low to be detected by indirect immunofluorescence, or the N-terminal epitopes are masked by protein conformation or interaction with cellular or other viral components.

Amino Acid Sequence↗

PubMed1990

A neonatally tolerant mouse model to assess pathogenicity of human autoantibodies.

Since certain autoimmune diseases, including myasthenia gravis and pemphigus vulgaris can be reproduced in mice by passive transfer of immunoglobulins from affected patients, we assessed whether this procedure could be optimised. Repeated injections of human IgG into mice during pregnancy induced tolerance to human IgG in the litter, and this persisted for at least 9 months. We show that three different human autoantibodies, to mitochondria, centromere and collagen, were retained in the serum of neonatally tolerized mice, but pathogenic effects of these particular autoantibodies were not demonstrable over the four week time scale of our experiments. However, our model should be applicable to studies on human autoantibodies which might damage the appropriate tissue in a heterologous species.

Animals↗

PubMed1990

Lymphocyte ectoenzyme activity compared in healthy persons and patients seropositive to or at high risk of HIV infection.

We measured two ectoenzymes, ecto-5'-nucleotidase (NT) and dipeptidyl peptidase IV (DP) in the peripheral blood lymphocytes of various groups of HIV-infected patients because of the previous implied relationship of these enzymes to immune function. NT expressed as mean nmol/h per mg protein (+/- s.d.) was significantly depressed in the HIV-seropositive asymptomatic (42 +/- 32; P less than 0.01) and AIDS groups (14 +/- 7; P less than 0.002) when compared with a healthy HIV-seronegative male population (83 +/- 27). The NT activities in asymptomatic HIV-seropositive and HIV-seronegative high risk groups (53 +/- 30) were not significantly different from one another but both groups had significantly higher enzyme activities than the AIDS group (P = 0.01 and less than 0.002, respectively). The seronegative high risk and normal healthy group had similar NT activities. DP activities expressed as mean nmol/h per mg protein (+/- s.d.) in both seropositive asymptomatic (0.188 +/- 0.038) and high risk seronegative (0.180 +/- 0.05) groups had higher enzyme activities than the healthy seronegative (0.117 +/- 0.015; P = 0.02 and 0.05, respectively) and AIDS group (0.096 +/- 0.036; P = 0.002 and 0.02, respectively). The healthy seronegative group had DP activities not significantly different to the AIDS groups. Similarly the high risk seronegative and healthy seropositive group had similar DP activities. These results taken together indicate that measurement of both DP and NT should be evaluated prospectively as a monitor of the clinical progression of HIV infection.

5'-Nucleotidase↗

PubMed1990

Major population differences in T cell response to a malaria sporozoite vaccine candidate.

Using a complete series of overlapping peptides, we have identified the T cell epitopes of a malaria vaccine candidate, the circumsporozoite (CS) protein, that are recognized by sporozoite-exposed residents of a non-endemic country. This protein and subunits from it are being considered as malaria sporozoite vaccine candidates, as CS-specific antibodies and cytotoxic T lymphocytes have been shown to have a role in protection. The rationale for developing an antibody-based vaccine is that in Plasmodium falciparum the immunodominant B cell epitope of the protein, (Asn-Ala-Asn-Pro)n [(NANP)n], is invariant. However, the ideal vaccine must contain CS protein-derived T cell antigenic epitopes to allow natural boosting of the antibody response following sporozoite exposure. Here, we show that major differences occur between the CS-specific T cell responses of non-endemic Caucasians and an endemic African population. HLA differences between the populations are, in part, responsible. Subunit malaria vaccines for one population may be ineffective in a different population.

Animals↗

PubMed1990

Identification of B epitopes in human papillomavirus type 16 E7 open reading frame protein.

Human papillomavirus (HPV) type 16 is implicated in the aetiology of anogenital dysplasia which may progress to malignancy. HPV-16 DNA is actively transcribed in cervical carcinomas, the most abundant transcripts being from the E6 and E7 early open reading frames. The E7 protein has been shown to have transforming activity in vitro. In this report we define four immunodominant B epitopes within the protein corresponding to the E7 gene, using a panel of murine monoclonal antibodies. Three epitopes are linear and lie within the N-terminal region of the molecule, and are unique to the HPV-16 E7 protein. One epitope is non-linear and presumed to be conformational. At least three of the four epitopes of the E7 protein are detectable by immunoprecipitation from an HPV-16-infected cervical carcinoma cell line. The demonstrated immunogenicity of the E7 protein allows us to deduce that this molecule may be a potential candidate for incorporation in a vaccine against cervical cancer.

Amino Acid Sequence↗