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Biomedical subjects

M De Wilde

Publications and source records attributed to M De Wilde.

At least 19 recordsLinked to original sources
PubMed1994

HIV-1 envelope-elicited neutralizing antibody titres correlate with protection and virus load in chimpanzees.

In an attempt to compare the protective effect of vaccination with two forms of envelope antigens, and to define immunological correlates of protection against HIV infection, chimpanzees were vaccinated with either recombinant gp160 or gp120. Homologous HIV challenge was performed 3 weeks after the fourth immunization. The animal with the highest level of serum neutralizing antibodies (gp160 immunogen) was protected against HIV infection. All other chimpanzees became infected, but displayed various levels of infected PBMCs. The postchallenge data gave rise to the following conclusions: (1) protection correlated with the level of the serological immune response, but not with the nature of immunogen (gp120 versus gp160); (2) the virus-neutralizing titre at day of challenge correlated with protection from infection; (3) the relative magnitude of the lymphoproliferative T-cell response at day of challenge did not correlate with any protective effect; (4) the peak numbers of virus-infected PBMCs in vaccinated animals were lower than those observed in control animals, and this effect was correlated with the intensity of the antibody response at day of challenge. This raises the possibility that a beneficial effect of HIV vaccination may be achieved in a situation where sterile immunity is not consistently obtained.

AIDS Vaccines↗
PubMed1992

Immunoelectron microscopic detection of the hepatitis B virus major surface protein in dilated perinuclear membranes of yeast cells.

The major surface protein of hepatitis B virus produced in Saccharomyces cerevisiae can be recovered from cell lysates in the form of 22-mm lipoprotein particles. Immunoelectron microscopy was applied to investigate site and time of particle assembly. Thin sections of yeast cells revealed that production of the S protein provoked a dilation of the endoplasmic reticulum. Dilated areas were specifically labeled with a polyclonal antibody raised against glutaraldehyde-treated yeast-derived HBsAg particles. In contrast to previous postulates of particle formation during cell lysis and extract preparation, these results suggest that particle formation in yeast occurs in the endoplasmic reticulum and that transport of particles along the secretion pathway is blocked.

Cell Transformation, Viral↗
PubMed1991

Sequence analysis of the putative structural genes of hepatitis C virus from Japanese and European origin.

cDNA fragments encoding the putative structural genes of the hepatitis C genome were isolated from a plasma pool of Japanese non-A, non-B hepatitis patients and from sera of individual Spanish patients. From the Japanese plasma pool a series of E1 clones was obtained that showed 88-98% homology among each other, both at the nucleotide and amino acid level. Compared to the sequences published by the Chiron Corporation and Takeuchi et al., the amino acid homology was 75-79% and 91-94%, respectively. Analysis of the core and E2/NS1 genes showed a high conservation of the core sequence and a high sequence variation in the 5' end of the E2/NS1 gene. The E1 gene of one Spanish isolate showed greater homology to the Chiron than to the Japanese sequence. Another Spanish isolate was more homologous to the Japanese sequence indicating that both hepatitis C genotypes are present in Europe. Analysis of the E1 gene of an isolate derived from a single patient with a 5-year interval revealed nine nucleotide and five amino acid changes.

Amino Acid Sequence↗
PubMed1991

The large surface protein of hepatitis B virus is retained in the yeast endoplasmic reticulum and provokes its unique enlargement.

The coding sequences for each of the three envelope proteins of hepatitis B virus (HBV), the major (S), middle (M), and large (L) surface proteins, were expressed in Saccharomyces cerevisiae. Analysis by immunoelectron microscopy of thin sections of yeast cells showed that production of L protein but not of M or S protein provoked morphological changes in the yeast endoplasmic reticulum. A large accumulation of membranous structures connected with the perinuclear cysternae and specifically labeled by a monoclonal antibody directed against the amino-terminal (preS1) sequence of the L protein, was observed. The L protein was post-translationally modified by N- and O-linked glycosylation, indicative of its entry into the yeast secretory pathway and by N-myristoylation of its amino-terminal glycine residue. Deletion of this glycine residue resulted in the synthesis of a nonmyristoylated L protein. Proliferation of the endoplasmic reticulum was comparable in cells producing either the myristoylated or nonmyristoylated L protein, indicating that myristoylation alone is not responsible for the induction of the abnormal membrane morphology.

Blotting, Western↗
PubMed1989

Assembly and release of HIV-1 precursor Pr55gag virus-like particles from recombinant baculovirus-infected insect cells.

The unprocessed Gag precursor from HIV-1, when expressed in recombinant baculovirus-infected insect cells, is targeted to the plasma membrane and assembles in 100-120 nm particles budding from the cell surface. This process mimics HIV immature particle formation and is dependent on myristoylation of the N-terminal glycine, as deletion of the latter results in particle accumulation in the cytoplasm and, interestingly, in the nucleus, pointing to a potential role of this non-fatty-acid-acylated species in the viral life cycle. Inclusion of the pol gene in the construct results in efficient processing of Pr55gag and a pronounced decrease in particle formation. Deletion of the C terminus (p16) of the Gag precursor, including the finger domains, abolishes particle assembly, but membrane targeting and evagination still occur. Heterologous expression in insect cells may prove very useful for the study of the molecular events leading to retroviral particle morphogenesis.

Animals↗
PubMed1988

Antibodies to soluble CD4 in HIV-1-infected individuals.

A direct enzyme immunoassay (EIA) using the recombinant soluble form of CD4 (sCD4) produced in rodent cells as antigen was applied to detect antibodies to CD4 in sera from HIV-1- and HIV-2-infected patients. High titers of antibodies to sCD4 were found in sera from 12.6% of the HIV-1-infected persons included in this study, but not in 120 normal human sera. The reactivity of these antibodies with sCD4 was confirmed by a Western blot analysis. A possible anti-idiotypic origin of those antibodies was thought to be unlikely in view of the lack of inhibition of the binding of the biotin-labeled monoclonal antibody (mAb) anti-Leu3a by sCD4 positive sera. Attempts to correlate the evolution of the disease with the presence or absence of antibodies to sCD4 in a panel of well documented HIV-1-seropositive cases did not reveal any clear correlation. Sera from HIV-2-infected people (nine sera analysed), sera from HIV-1-infected chimpanzees (10 sera analysed) and sera from humans immunized with a recombinant vaccinia virus expressing gp160 (10 sera analysed) scored negative for antibodies to sCD4. The possible origin and biological significance of the observed antibodies to sCD4 are discussed.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Characterization of a recombinant fusion protein of the finger domain of tissue-type plasminogen activator with a truncated single chain urokinase-type plasminogen activator.

Human recombinant single chain urokinase-type plasminogen activator (recombinant scu-PA) and a hybrid between human tissue-type plasminogen activator (t-PA) and scu-PA, obtained by ligation of cDNA fragments encoding the NH2-terminal region (amino acids 1-67) of t-PA and the COOH-terminal region (amino acids 136-411) of scu-PA, were expressed in a mammalian cell system. The proteins were purified from conditioned culture media containing 2% fetal calf serum by chromatography on zinc chelate-Sepharose, immunoadsorption chromatography on an insolubilized murine monoclonal antibody directed against urokinase, benzamidine-Sepharose chromatography, and Ultrogel AcA 44 gel filtration. Between 180 and 230 micrograms of the purified proteins were obtained per liter of conditioned medium, with a yield of approximately 18% and a purification factor of 720-1900. On sodium dodecyl sulfate gel electrophoresis under reducing conditions, the proteins migrated as single bands with approximate Mr 50,000 for recombinant scu-PA and Mr 43,000 for the t-PA/scu-PA hybrid. Following conversion to urokinase with plasmin, the proteins had a specific amidolytic activity comparable to that of natural scu-PA. Both proteins activated plasminogen directly with Km = 0.53 and 1.4 microM and k2 = 0.0034 and 0.0027 s-1, respectively. Both proteins did not bind specifically to fibrin and had a comparable degree of fibrin selectivity as measured in a system composed of a whole human 125I-fibrin-labeled plasma clot suspended in human plasma. It is concluded that this chimeric protein, consisting of the NH2-terminal "finger-like" domain of t-PA and the COOH-terminal region of scu-PA, has very similar enzymatic properties as compared to scu-PA, but has not acquired the fibrin affinity of t-PA.

Adenocarcinoma↗
PubMed1987

Immunological properties of recombinant HBsAg produced in yeast.

Although currently available plasma-derived vaccines (PDV) against hepatitis B based on the surface antigen of the virus (HBsAg) are well-tolerated and effective, their supply is limited and time-consuming controls are necessary to assess their safety. It is therefore desirable that an alternative source of HBsAg be found. Recombinant DNA technology has provided the possibility of obtaining HBsAg in large quantities. However, it is important that a recombinant DNA hepatitis B vaccine be not only antigenically similar but also elicits a similar immune response in humans. Using the Ausria kit, the recombinant DNA vaccine of SmithKline Biologicals produced in yeast appears to have a higher antigenic content than a reference plasma-derived HBsAg preparation of similar purity when compared at equivalent protein concentrations. In competition experiments, however, antibodies obtained by immunization with PDV similarly recognized yeast-derived and plasma-derived antigens. Monoclonal antibodies directed to the common a determinant of the whole virus were also used to identify distinct epitopes on the recombinant DNA vaccine. The yeast-derived HBsAg is therefore antigenically similar to plasma-derived HBsAg. The yeast-derived vaccine (YDV) was highly immunogenic in mice, rabbits, goats, monkeys, chimpanzees, and humans. High titres of anti-HBs were reached in humans after three doses administered at 0, 1, and 6 months. The antibodies raised in humans after three doses of YDV were predominantly directed to the common a determinant. Competition studies using monoclonal antibodies raised against the whole virus showed that the antibodies had the same specificity as the antibodies induced by PDV. The affinity for the plasma-derived antigen of antibodies stimulated by YDV and PDV and antibodies present in the sera of convalescent subjects were also similar. Finally, competition experiments showed that the antibodies induced in humans by YDV and antibodies from convalescent subjects were directed to the same binding sites of the plasma-derived antigen. These studies indicate that the yeast-derived vaccine is immunologically similar to the plasma-derived vaccines both in vitro and in vivo and can therefore be expected to have similar protective efficacy.

Animals↗
PubMed1987

Construction and characterization of a Saccharomyces cerevisiae strain (RIT4376) expressing hepatitis B surface antigen.

A host/vector system suitable for large-scale production of HBsAg has been constructed and optimized in terms of the expression plasmid and yeast host strain in order to permit fermentation to very high cell densities. The final expression plasmid contains the coding sequence of the major HBsAg protein (P24) flanked by the promoter sequences from a glycolytic gene and by the transcription-termination region of the ARG3 gene. The host/vector system was found to be genetically stable under large-scale fermentation conditions as demonstrated by nucleotide sequencing and restriction mapping experiments. The P24 protein is recovered from yeast as particles whose physiochemical properties are very similar to those of plasma-derived HBsAg.

Base Sequence↗
PubMed1986

Isolation, physical characterization and expression analysis of the Saccharomyces cerevisiae positive regulatory gene PHO4.

The Saccharomyces cerevisiae PHO4 gene, which positively controls the expression of phosphatase genes, has been isolated by complementation of a pho4 mutation. The isolated DNA directed integration at the chromosomal PHO4 locus. The nucleotide sequence of PHO4 has a coding region of 930 nucleotides, flanked by sequences with typical transcription initiation and termination signals. The 5' region has characteristics of low-expression promoters and carries several uncommon elements, whose significance is not known. The predicted primary structure of the PHO4 protein, of 309 residues, does not show sequence elements typical of DNA-binding proteins. The transcription of PHO4 is independent of inorganic phosphate. Like other regulatory genes, PHO4 is transcribed at a very low level and the translation of its message uses preferentially several codons which are not employed for highly expressed genes.

Amino Acid Sequence↗
PubMed1985

beta-D-N-acetylglucosaminidase, a new cytochemical marker of human lymphocyte subpopulations.

beta-D-N-acetylglucosaminidase staining characteristics of rosetted or non-rosetted normal and malignant lymphoid cells were compared with those observed after nonspecific esterase and acid phosphatase staining. With the three cytochemical techniques a similar staining pattern was observed in T cells (E-rosettes), their subpopulations T mu and T gamma, B cells and the non-T, non-B cells, as well as in the T cell populations defined with the monoclonal antibodies OKT3,4 and 8. T mu cells mostly displayed a "dot-like" reaction, T gamma and the non-T, non-B cells a "fine to heavy granular" reaction, while most B cells were negative. OKT4 and OKT8 positive lymphocytes showed for the larger part a dot-like staining pattern, however, the frequency of cells with a granular pattern was distinctly higher in the OKT8, than in the OKT4 positive cells. E(+)mIg(-) and E(-)mIg(-) A.L.L. blasts stained either with a dot-like or granular pattern or failed to react when stained cytochemically for beta-D-N-acetylglucosaminidase, nonspecific esterase and acid phosphatase activity. Only in a few instances a discrepancy was observed between the types of staining for esterase and acid phosphatase on one hand and those for beta-D-N-acetylglucosaminidase on the other.

Acetylglucosaminidase↗
PubMed1985

Production in yeast of hepatitis B surface antigen by R-DNA technology.

Yeast synthesizes the surface antigen protein of Hepatitis B virus when the structural gene is fused to the promoter from the ARG3 gene. Analysis of extracts and total cells shows that the primary translation product can be identified as a poorly antigenic monomer with an estimated molecular weight of 22K. In cell extracts Y-HBsAg is in the form of 20 nm particles which, like serum derived particles, are highly immunogenic in mice and monkeys. Yeast derived surface antigen is thus a viable alternative to the present serum derived HBV vaccines.

Animals↗
PubMed1984

Expression of human alpha 1-antitrypsin cDNA in the yeast Saccharomyces cerevisiae.

Nucleotide sequences coding either for the precursor or the mature form of human alpha 1-antitrypsin have been placed under the control of the yeast ARG3 expression signals. Recombinant plasmids pRIT10782 and pRIT10787 express the precursor or the mature alpha 1-antitrypsin species, respectively, in two different yeast strains, with yields ranging between 0.3 and 1% of total soluble proteins. The alpha 1-antitrypsin synthesized in yeasts was specifically recognized by polyclonal and monoclonal antibodies raised against human alpha 1-antitrypsin. In addition, it was shown to be biologically active in its mature form only, with optimal activity in a peptidase-deficient yeast strain.

Antibodies, Monoclonal↗
PubMed1983

A new, simple and rapid method for enumerating human T lymphocytes in full blood: the E. coli (ATCC 11303) rosette test.

A new bacterial rosette technique for enumerating T lymphocytes is described. E. coli (strain B; ATCC 11303), fixed in formaldehyde after overnight growth in thioglycolate medium, are mixed with washed whole blood cells (100 microliters) and after incubation at 4 degrees C, slides are made, stained and counted. The nature of the lymphocytes forming E. coli rosettes was demonstrated by comparing their cytochemical staining characteristics with those of E rosetted lymphocytes, and by mixed E. coli and E, mouse E rosette and Fc receptor tests, and by mixed E. coli rosette tests and anti-Ig staining. E. coli and E rosette tests in controls and pediatric patients were also compared. The results show that T mu and T gamma cells rosette with E. coli.

B-Lymphocytes↗
PubMed1983

Expression of hepatitis B surface antigen in yeast.

The genomes of HBV viruses of two different serotypes were cloned in E. coli. Sequences coding for the major polypeptides of surface antigen (HBsAg) were fused with the 5' end of a cloned yeast arg3 gene. When introduced into yeast, on a suitable vector, the hybrid gene directed the synthesis of a fusion protein. Crude extracts of such strains were shown to contain HBsAg like material having physical properties characteristic of the antigen isolated from the plasma of chronic human carriers, as judged by isopycnic and rate zonal centrifugation. Furthermore, these extracts readily elicit specific anti-HBsAg antibodies in rabbits. Further manipulations of the 5' part of the arg3 gene resulted in the introduction of a unique restriction site located in the 5' non translated leader sequence. The resulting vector was used to construct a recombinant plasmid directing the synthesis of the mature (226 amino acids) HBsAg polypeptide.

Hepatitis B Surface Antigens↗