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B E Clarke

Publications and source records attributed to B E Clarke.

At least 37 records · Page 2Linked to original sources
PubMed1991

Foreign epitopes in immunodominant regions of hepatitis B core particles are highly immunogenic and conformationally restricted.

The presentation of heterologous amino acid sequences on the surface of hepatitis B core antigen (HBcAg) particles has been studied using a defined linear neutralization site from human rhinovirus (HRV). Previous work has shown that fusion particles, in which the HRV peptide sequence is linked to the amino terminus of the HBcAg protein, induce excellent immune responses in experimental animals. Using predictive models of HBcAg particulate structure and the approximate location of the major immunogenic regions we have designed and constructed bacterial expression vectors which direct synthesis of chimeric particles in which heterologous sequences are presented within an immunodominant area on the particle. Immunological responses to the heterologous peptide sequence are improved by at least tenfold when compared with amino terminal fusions of the same peptide sequence to HBcAg. Moreover, the restriction placed on the heterologous peptide by its linkage at both ends within the HBcAg protein results in a more constrained structure. In the case of the rhinovirus peptide sequence this results in an antigenic conformation more closely resembling that on the native virus particle. Such a system lends itself well as a general approach to the induction of high titre antibodies against defined epitopes.

Amino Acid Sequence↗
PubMed1991

In vitro translation of hepatitis A virus subgenomic RNA transcripts.

A subgenomic cDNA clone from hepatitis A virus strain HM175, composed of the last eight nucleotides of the 5' non-translated region and the first 2248 nucleotides of the coding sequence (P1 region), was inserted into a vector under the control of the T7 promoter. Restriction enzyme digestion at sites within the structural region and subsequent transcription in vitro yielded RNA products which were translated efficiently in rabbit reticulocyte lysates to produce proteins of the predicted sizes. The translation products were specifically precipitated with antipeptide antisera; these reactions were not affected by denaturation of the antigens by boiling in 1% SDS. The translated proteins were also precipitated by antivirion antisera, but recognition was totally abolished following denaturation. Thus antivirion antisera recognized conformation-dependent epitopes expressed on the translated products exclusively.

Autoradiography↗
PubMed1990

Expression in yeast of amino-terminal peptide fusions to hepatitis B core antigen and their immunological properties.

Hepatitis B core protein (HBcAg) is a potent antigen that gives both a T-cell-dependent and a T-cell-independent antibody response. It has been shown that a foreign epitope can be fused to the amino terminus of HBcAg without affecting particle integrity, and that the resulting chimaeric cores retain the immunogenicity of the foreign epitope. Here we describe the efficient expression in yeast of two different chimaeric cores, carrying epitopes of Foot and Mouth Disease Virus (FMDV) or human chorionic gonadotrophin (hCG), which are candidates for FMD and contraceptive vaccines, respectively. These cores could not be produced in E. coli in soluble form but were expressed to high levels in yeast. We constructed a yeast expression vector that allows rapid production of different chimaeric cores by cloning in cassettes encoding foreign epitopes. Both FMDV and hCG-cores were shown to present the epitopes at the surface of the particles. The FMDV-cores produced in yeast were efficient inducers of neutralising antibodies in guinea-pigs after one low dose.

Amino Acid Sequence↗
PubMed1990

Immunological properties of hepatitis B core antigen fusion proteins.

The immunogenicity of a 19 amino acid peptide from foot-and-mouth disease virus has previously been shown to approach that of the inactivated virus from which it was derived after multimeric particulate presentation as an N-terminal fusion with hepatitis B core antigen. In this report we demonstrate that rhinovirus peptide-hepatitis B core antigen fusion proteins are 10-fold more immunogenic than peptide coupled to keyhole limpet hemocyanin and 100-fold more immunogenic than uncoupled peptide with an added helper T-cell epitope. The fusion proteins can be readily administered without adjuvant or with adjuvants acceptable for human and veterinary application and can elicit a response after nasal or oral dosing. The fusion proteins can also act as T-cell-independent antigens. These properties provide further support for their suitability as presentation systems for "foreign" epitopes in the development of vaccines.

Adjuvants, Immunologic↗
PubMed1990

Presentation and immunogenicity of viral epitopes on the surface of hybrid hepatitis B virus core particles produced in bacteria.

We recently reported the enhanced immunogenicity of a peptide epitope when it was presented as a fusion protein with hepatitis B core antigen. In those experiments the fusion protein was expressed in vaccinia virus. We have now refined the system so that large amounts of highly immunogenic particles can be produced using a simple bacterial expression system. We describe the expression of three different viral epitopes as chimeric particles that induce good antibody responses to each epitope after one dose of low amounts of antigen. Finally we demonstrate that the immunogenicity is a reflection of both T helper cell sites within the core protein and also the particulate nature of the immunogens.

Amino Acid Sequence↗
PubMed1990

Neutralization epitopes of human rhinovirus type 2.

Fourteen neutralizing monoclonal antibodies recognizing human rhinovirus (HRV) type 2 have been used to select a total of 51 virus escape mutants. Cross-resistance analysis of the mutants, together with RNA sequencing and identification of amino acid substitutions, have revealed three neutralization sites on the virus surface. Two of these appear to correspond to the NIm-IA and NIm-II sites described for HRV-type 14, although there are also substantial differences. The third site has not been described previously.

Amino Acids↗
PubMed1990

Nephropathy in model combining genetic hypertension with experimental diabetes. Enalapril versus hydralazine and metoprolol therapy.

We compared the effects of the angiotensin-converting enzyme inhibitor enalapril and a conventional antihypertensive regimen (hydralazine and metoprolol) on kidney function, albuminuria, and glomerular ultrastructure in hypertensive diabetic and nondiabetic rats. Diabetes was induced with streptozocin at 8 wk of age in spontaneously hypertensive (SHR) rats. Antihypertensive drugs were administered in drinking water from the time of induction of diabetes in all groups. Blood pressure reduction was equal in the diabetic and nondiabetic SHR rats receiving either enalapril or hydralazine plus metoprolol. In diabetic SHR rats, there was a rise in serum creatinine after 32 wk, which did not occur in diabetic rats treated with either antihypertensive regimen or in nondiabetic rats. Both drug regimens reduced albuminuria in diabetic and nondiabetic SHR rats to a similar degree. Enalapril and the combination of hydralazine and metoprolol were associated with decreased glomerular basement membrane thickness and glomerular volume in diabetic and nondiabetic SHR rats without significant effect on fractional mesangial volume. Thus, antihypertensive therapy retards the development of albuminuria, glomerular basement membrane thickening, and glomerular hypertrophy in the rat in the presence or absence of diabetes. No specific benefit of angiotensin-converting enzyme inhibition was observed in these hypertensive models of nephropathy. Human studies comparing the effects of different classes of antihypertensive drugs on kidney function, proteinuria, and glomerular morphology are warranted.

Administration, Oral↗
PubMed1990

Neutralizing antibodies to all seven serotypes of foot-and-mouth disease virus elicited by synthetic peptides.

Uncoupled peptides from all seven serotypes of foot-and-mouth disease virus (FMDV) protein VP1 have been used to elicit neutralizing antibody responses in guinea-pigs. The responses were largely serotype specific, although some significant cross-neutralization was observed. Dimeric tandem peptides have also been used to simultaneously elicit neutralizing antibodies to two different FMDV serotypes. The possible existence of structural features common to the B-cell neutralization sites or the guinea-pig helper T-cell sites within all seven peptides are analysed and discussed.

Amino Acid Sequence↗
PubMed1989

The nucleotide sequence of the structural-protein-coding region of foot-and-mouth disease virus serotype SAT3.

The nucleotide sequence coding for the structural proteins and nonstructural protein P2A has been determined for a foot-and-mouth disease virus (FMDV) isolated in Africa. This virus, serotypically designated SAT3 (South African Territories type 3), shows about 60% homology at the nucleotide level to prototype viruses from the O, A and C serotypes of FMDV. The highest region of variability was shown in structural protein VP1, presumably a consequence of its position on the surface of the virus and its exposure to selection pressure by neutralising antibody. Within this region amino acids (aa) 141-160, which have been shown to represent an immunodominant region in other FMDV serotypes, showed hypervariability as well as the insertion of 5 or 9 additional aa relative to the O1 and C1 serotypes, respectively. In contrast, the sequence of nonstructural protein P2A was completely conserved indicating a probable important role in virus replication.

Amino Acid Sequence↗
PubMed1989

Enalapril retards glomerular basement membrane thickening and albuminuria in the diabetic rat.

This study has evaluated the effects of the angiotensin converting enzyme inhibitor Enalapril on glomerular ultrastructure and albuminuria in normotensive and hypertensive diabetic rats. Streptozotocin-diabetes was induced in Wistar Kyoto and spontaneously hypertensive rats. Enalapril was administered in drinking water in diabetic normotensive, control hypertensive and diabetic hypertensive rats. Enalapril therapy prevented an increase in glomerular basement membrane thickness in diabetic normotensive, control hypertensive and diabetic hypertensive rats without any significant effect on fractional mesangial volume. Enalapril decreased albuminuria in diabetic normotensive, control hypertensive and diabetic hypertensive rats. Thus, enalapril retards the development of glomerular basement membrane thickening and albuminuria in the rat, in the presence or absence of hypertension.

Albuminuria↗
PubMed1989

Host cell selection of antigenic variants of foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV) A22 Iraq 24/64 adapted to grow in BHK monolayer cells induced antibodies which neutralized many isolates belonging to the A serotype. Plaque-purified virus isolated from this stock also induced broadly reactive antibodies, showing that this property is not due to the combined response to a mixture of variants in the original stock virus. However, viruses obtained by passage in suspension BHK cells of either the monolayer cell-adapted virus or a virus cloned from this stock resulted in the selection of virus which induced antibodies with highly specific neutralizing activity. In addition to their antigenic properties the monolayer and suspension cell-adapted viruses could be distinguished by plaque morphology, tendency to aggregate and ability to attach to BHK cells. Monoclonal antibodies (MAbs) induced with the plaque-purified monolayer-adapted virus had neutralizing activity almost as broad as polyclonal serum, showing that this property can be represented by a single epitope on the virus. These neutralizing MAbs recognize a trypsin-sensitive epitope on the virus. Surprisingly, sequence analysis of the structural protein-coding regions of the genomic RNAs of monolayer and suspension cell-adapted viruses showed no amino acid differences in VP1, the protein known to contain the major neutralization epitope in FMDV and to be the only protein susceptible to cleavage by trypsin in the virus particle. Although three coding differences were found in the capsid protein these were all located in VP2.

Amino Acid Sequence↗
PubMed1989

Epitope mapping of foot-and-mouth disease virus with neutralizing monoclonal antibodies.

Epitopes of strain A22 Iraq 24/64 of foot-and-mouth disease virus have been mapped with monoclonal antibodies (MAbs). Three methods were used: (i) an indirect ELISA using an overlapping set of peptides, (ii) production of neutralization escape variants against each MAb and (iii) sequencing of neutralization escape variants. The study has shown that the virus has at least three overlapping liner neutralizing epitopes within a major antigenic site on VP1. The presence of a second, conformational site was demonstrated but its position on the virus particle was not located. Synthetic peptides with sequences representing the major site elicit antibodies which have similar broad cross-neutralizing activity to polyclonal serum or neutralizing MAbs produced with the virus against a range of field isolates.

Amino Acid Sequence↗
PubMed1989

Serological prospects for peptide vaccines against foot-and-mouth disease virus.

Antibodies to a synthetic peptide corresponding to the 141 to 160 amino acid sequence of the protein VP1 of type O foot-and-mouth disease virus (FMDV) neutralize a wider range of type O isolates than anti-virion serum. Extending this peptide at the amino terminus reduced the number of strains neutralized by the antipeptide sera. Reactions with antisera to peptides representing non-contiguous native sequences showed that it was also possible to increase the number of strains effectively neutralized. Selected substitutions of a single amino acid at position 148 markedly altered the neutralizing specificity of antibodies elicited by the 141 to 160 peptide. In particular, a peptide with an L----S substitution at this position induced antibodies which neutralized a type O and a type A virus equally, and guinea-pigs inoculated with it were protected from challenge with either virus. Attempts to isolate variant viruses resistant to neutralization with anti-peptide antibody indicated that these occurred at low frequency, and there was some evidence that resistance may be partially conferred by mutations outside the peptide sequence.

Amino Acid Sequence↗
PubMed1988

Processing and assembly of foot-and-mouth disease virus proteins using subgenomic RNA.

Recombinant DNA clones were constructed in order to study the mechanisms of proteolytic processing and assembly in foot-and-mouth disease virus (FMDV). RNA transcripts from these clones were synthesized using SP6 polymerase and translated in rabbit reticulocyte lysates. Efficient translation occurred in the absence of all 5' untranslated sequences and processing of the structural proteins occurred in the presence of functional 3C protease which can function in trans. The specificity of 3C protease activity is not limited to Glu-Gly bonds. Translation of correctly processed structural proteins leads to assembly of subviral structures resembling 'empty' particles. Further studies on the processing of the FMDV genome show that the primary cleavage (P1-P2) is mediated neither by 3C nor the second FMDV protease L. Preliminary evidence suggests that an initial very rapid cleavage occurs between 2A and 2B with subsequent cleavage of the P1/2A junction probably being carried out by 3C.

Amino Acid Sequence↗
PubMed1988

Modification of the leader protein (Lb) of foot-and-mouth disease virus.

Translation of foot-and-mouth disease virus RNA for extended periods in rabbit reticulocyte lysates results in the appearance of a previously undescribed protein. A protein with similar properties can also be detected in BHK cells at late times after virus infection. Specific immunoprecipitation has shown that this protein (Lb') is closely related to the smaller of the two leader proteins, Lb, although it migrates with an apparently higher Mr in SDS-polyacrylamide gels. The conversion of Lb to Lb' can be mimicked by treatment with carboxypeptidase B. It is suggested that C-terminal trimming of Lb to produce Lb' results in an increase in negative charge and is responsible for its slower migration in SDS-PAGE.

Aphthovirus↗
PubMed1987

Potential secondary and tertiary structure in the genomic RNA of foot and mouth disease virus.

The nucleotide sequence of the 5' untranslated region of foot and mouth disease virus (FMDV), serotype A10 has been determined. This completes the first total genomic sequence for any one serotype of FMDV. Analysis of the sequence to the 3' side of the poly (C) tract reveals the presence of a 24 nucleotide repeated motif which has homologies with a sequence located upstream of the transcriptional initiation site from several mammalian fibrinogen genes. The function of this element in FMDV is unclear. However, computer analysis of this region predicts the presence of a high degree of secondary and tertiary structure in which these repeats form an important part. The implications of these predictions are discussed.

Aphthovirus↗
PubMed1987

All foot and mouth disease virus serotypes initiate protein synthesis at two separate AUGs.

Translation of the foot and mouth disease virus genome in vitro and in vivo indicated that all seven serotypes initiate protein synthesis at two separate AUGs. Sequence analysis of the region surrounding these AUGs has shown that the efficiency with which the initiating AUG is recognized is dependent on the flanking nucleotides. However, in vitro, the major factor determining which AUG is used is the concentration of Mg2+.

Aphthovirus↗