Biomedical subjects

Wim Jiskoot

Publications and source records attributed to Wim Jiskoot.

At least 37 records · Page 2Linked to original sources

PubMed2004

Ligand-induced changes in the conformational dynamics of a bacterial cytotoxic endonuclease.

Knowledge about the conformational dynamics of a protein is key to understanding its biochemical and biophysical properties. In the present work we investigated the dynamic properties of the enzymatic domain of DNase colicins via time-resolved fluorescence and anisotropy decay analysis in combination with steady-state acrylamide quenching experiments. The dynamic properties of the apoenzyme were compared to those of the E9 DNase ligated to the transition metal ion Zn(2+) and the natural inhibitor Im9. We further investigated the contributions of each of the two tryptophans within the E9 DNase (Trp22 and Trp58) using two single-tryptophan mutants (E9 W22F and E9 W58F). Wild-type E9 DNase, E9 W22F, and E9 W58F, as well as Im9, showed multiple lifetime decays. The time-resolved and steady-state fluorescence results indicated that complexation of E9 DNase with Zn(2+) induces compaction of the E9 DNase structure, accompanied by immobilization of Trp22 along with a reduced solvent accessibility for both tryptophans. Im9 binding resulted in immobilization of Trp22 along with a decrease in the longest lifetime component. In contrast, Trp58 experienced less restriction on complexation of E9 DNase with Im9 and showed an increase in the longest lifetime component. Furthermore, the results point out that the Im9-induced changes in the conformational dynamics of E9 DNase are predominant and occur independently of the Zn(2+)-induced conformational effects.

Bacterial Proteins↗

PubMed2004

Stability of mono- and trivalent meningococcal outer membrane vesicle vaccines.

The stability during storage of outer membrane vesicles (OMVs) of Neisseria meningitidis group B was studied. Three types of OMVs were compared for their stability, containing either one (monovalent) or three different PorA subtypes (trivalent), the latter with and without class 4 outer membrane protein (OMO, RmpM). Aqueous formulations were stored freeze-dried (4 degrees C), frozen (-70 degrees C) and in liquid form at 4, 37 and 56 degrees C. Physico-chemical properties and immunogenicity of the OMVs as well as PorA conformation and antigenicity (P1.7-2,4, the subtype present in all formulations) were monitored during 1 year. At -70 or 4 degrees C, the structure and immunogenicity of OMVs was preserved. Storage of OMVs at high temperatures (37 or 56 degrees C) induced destruction of the OMV structure and denaturation of PorA, followed by chemical degradation. Immunogenicity decreased or was lost completely. Changes observed in the fluorescence spectra of degraded OMVs were also seen in tryptophan (Trp) and tyrosine (Tyr) derivatives incubated at 56 degrees C, indicating the occurrence of chemical degradation of tryptophan and tyrosine residues in PorA. Trivalent OMVs were slightly more stable at 37 degrees C than monovalent OMVs as assessed by in vitro methods, but these differences did not result in differences in the immunogenicity. The stability of trivalent OMVs was not affected by the presence of RmpM. Both trivalent and monovalent OMVs could be freeze-dried with preservation of their immunogenicity. In conclusion, OMVs are sensitive to elevated temperatures, but are stable in the frozen or freeze-dried state or when stored at 4 degrees C in the liquid state.

Animals↗

PubMed2004

Immunogenicity of meningococcal PorA formulations encapsulated in biodegradable microspheres.

The purpose of our study was to investigate the possibility to microencapsulate liposomes and meningococcal outer membrane vesicles (OMV), both containing neisserial pore protein A (PorA), in biodegradable dextran- and mannan-based microspheres and to study the immunogenicity of the encapsulated PorA formulations. PorA-liposomes and OMV were encapsulated in dextran- or mannan-based microspheres by using an aqueous two-phase system consisting of a polyethylene glycol solution and a methacrylated dextran or mannan solution. The formulations were characterized for size distribution, PorA structure and antigen recovery after release. Calcein-containing model liposomes were used to establish the encapsulation efficiency and release profiles from both types of microspheres. The immunogenicity of the PorA-containing formulations was determined in mice after subcutaneous immunization. Liposomes were encapsulated in dextran and mannan microspheres with a high efficiency (70-90%). Calcein liposomes, after a 5-day lag period, exhibited apparent zero-order release kinetics from both types of microspheres between Days 5 and 10 of incubation in vitro. The total release was 80 and 100% from mannan and dextran microspheres, respectively. The trimeric PorA conformation was preserved in the released liposomes and OMV and the antigen was partly recovered. The immunogenicity of PorA-liposomes and OMV encapsulated in dextran or mannan microspheres was preserved. In conclusion, PorA-liposomes and OMV could be encapsulated in dextran- and mannan-based microspheres with high efficiency. The immunogenicity of encapsulated antigen was preserved.

Animals↗

PubMed2004

Virosome and ISCOM vaccines against Newcastle disease: preparation, characterization and immunogenicity.

The purpose of this study was to prepare and characterize virosomes and ISCOMs containing envelope proteins of Newcastle disease virus (NDV) and to evaluate their immunogenicity in target animals (chickens). Virosomes were prepared by solubilization of virus with either Triton X-100 or octyl glucoside (OG) followed by detergent removal. Biochemical analysis revealed that these virosomes contained both the haemagglutinin-neuraminidase protein (HN) and the fusion protein (F), with preserved biological activity. Acidic environment triggered the fusion between virosomes and chicken erythrocyte ghosts. Formation of ISCOMs was achieved by solubilizing phospholipids, cholesterol, envelope protein antigen and Quil A in Triton X-100. The ISCOM particles were formed by removal of the detergent. In each formulation the relative HN content correlated with the capability to agglutinate red blood cells. The immunogenicity of these lipid-based subunit vaccines was determined in chickens after subcutaneous immunization. The relative HN content of the subunit vaccines correlated with the haemagglutination-inhibition (HI) antibody titres. Virosomes prepared with Triton X-100 and ISCOMs offered high clinical protection (> 80%) upon challenge with virulent NDV. Virosomes prepared with OG yielded lower clinical protection despite high HI antibody titres. Virosomes with reduced antigen density showed poor immunogenicity and protection. In conclusion, ND virosomes and ISCOMs were found to be immunogenic and provided good protection.

Animals↗

PubMed2004

Physicochemical studies on the stability of influenza haemagglutinin in vaccine bulk material.

The relative unknown conformational stability of monovalent bulks of influenza virus haemagglutinin (HA) from three different strains (B/Guangdong, A/New Caledonia and A/Panama) was investigated with fluorescence and circular dichroism (CD) spectroscopy. Various stress conditions (concentration of denaturant, freeze-thawing, pH and temperature) affected the spectroscopic properties of the haemagglutinin proteins differently. Unfolding experiments revealed a poor stability of Guangdong haemagglutinin (GD-HA) in comparison with New Caledonia (NC-HA) and Panama haemagglutinin (P-HA). Freeze-thawing altered the secondary and tertiary structure of Guangdong haemagglutinin and only the tertiary structure of Panama haemagglutinin. From pH 4.6-9.2 the tertiary structures of Guangdong, New Caledonia and Panama haemagglutinin were all affected to a different extent. The secondary structure was only altered at low pH. Incubation of haemagglutinin at 60 degrees C resulted in denaturation of the protein and a dramatic change of the fluorescence spectrum, indicative of oxidised tryptophan (Trp). In conclusion, fluorescence and circular dichroism spectroscopy are highly suitable techniques to monitor the stability of haemagglutinin in a straightforward and fast way.

Chemical Phenomena↗

PubMed2004

Structure-immunogenicity relationships of therapeutic proteins.

As more recombinant human proteins become available on the market, the incidence of immunogenicity problems is rising. The antibodies formed against a therapeutic protein can result in serious clinical effects, such as loss of efficacy and neutralization of the endogenous protein with essential biological functions. Here we review the literature on the relations between the immunogenicity of the therapeutic proteins and their structural properties. The mechanisms by which protein therapeutics can induce antibodies as well as the models used to study immunogenicity are discussed. Examples of how the chemical structure (including amino acid sequence, glycosylation, and pegylation) can influence the incidence and level of antibody formation are given. Moreover, it is shown that physical degradation (especially aggregation) of the proteins as well as chemical decomposition (e.g., oxidation) may enhance the immune response. To what extent the presence of degradation products in protein formulations influences their immunogenicity still needs further investigation. Immunization of transgenic animals, tolerant for the human protein, with well-defined, artificially prepared degradation products of therapeutic proteins may shed more light on the structure-immunogenicity relationships of recombinant human proteins.

Animals↗

PubMed2004

Stabilization of proteins in dry powder formulations using supercritical fluid technology.

Therapeutic proteins have become essential in the treatment of many diseases. Their formulation in dry form is often required to improve their stability. Traditional freeze-drying or spray-drying processes are often harmful to labile proteins and could be replaced by supercritical fluid (SCF) drying to produce particles with defined physicochemical characteristics in a mild single step. A survey of the current SCF drying processes for proteins is presented to give insight into the effect of SCF drying on protein stability and to identify issues that need further investigation. Methods used for drying aqueous and organic protein solutions are described. In particular, effects of process and formulation parameters on particle formation and protein stability are discussed. Although SCF methodology for drying proteins is still in its infancy, it can provide a serious alternative to existing drying methods for stabilizing proteins.

Carbon Dioxide↗

PubMed2004

Distinct conformational stability and functional activity of four highly homologous endonuclease colicins.

The family of conserved colicin DNases E2, E7, E8, and E9 are microbial toxins that kill bacteria through random degradation of the chromosomal DNA. In the present work, we compare side by side the conformational stabilities of these four highly homologous colicin DNases. Our results indicate that the apo-forms of these colicins are at room temperature and neutral pH in a dynamic conformational equilibrium between at least two quite distinct conformers. We show that the thermal stabilities of the apo-proteins differ by up to 20 degrees C. The observed differences correlate with the observed conformational behavior, that is, the tendency of the protein to form either an open, less stable or closed, more stable conformation in solution, as deduced by both tryptophan accessibility studies and electrospray ionization mass spectrometry. Given these surprising structural differences, we next probed the catalytic activity of the four DNases and also observed a significant variation in relative activities. However, no unequivocal link between the activity of the protein and its thermal and structural stability could easily be made. The observed differences in conformational and functional properties of the four colicin DNases are surprising given that they are a closely related (> or =65% identity) family of enzymes containing a highly conserved (betabetaalpha-Me) active site motif. The different behavior of the apo-enzymes must therefore most likely depend on more subtle changes in amino acid sequences, most likely in the exosite region (residues 72-98) that is required for specific high-affinity binding of the cognate immunity protein.

Amino Acid Sequence↗

PubMed2004

Innovative therapeutics: from molecules to medicines. 27 June-7 July 2004, Parma, Italy.

The Socrates Intensive Programme offers annual courses focusing on the specific aspects of innovative therapeutics. The 2004 meeting was coordinated by the University of Parma and covered various subjects in the field of advanced drug delivery and pharmaceutical technology, including sessions on biopharmaceutics, pharmacokinetics, polymers, oral delivery, colloidal vectors, peptide and protein delivery, vaccines, oligonucleotide delivery, gene delivery, non-conventional routes of administration, and a graduate student symposium. The meeting had a highly interactive character and provided a unique opportunity for young scientists to present and discuss their work in an international setting.

Drug Administration Routes↗

PubMed2003

Physicochemical and immunochemical techniques predict the quality of diphtheria toxoid vaccines.

The most critical step in the production of diphtheria vaccines is the inactivation of the toxin by formaldehyde. Diphtheria toxoid (DTx) is produced during this inactivation process through partly unknown, chemical modifications of the toxin. Consequently, diphtheria vaccines are difficult to characterise completely and the quality of the toxoids is routinely determined with potency and safety tests. This article describes the possibility of monitoring the quality in diphtheria vaccine production with a selection of physicochemical and immunochemical tests as an alternative to established in vivo tests. To this end, diphtheria toxin was treated with increasing formaldehyde concentrations resulting in toxoid products varying in potency and residual toxicity. Differences in the quality of the experimental toxoids were also assessed with physicochemical and immunochemical techniques. The results obtained with several of these analyses, including SDS-PAGE, primary amino group determination, fluorescence spectroscopy, circular dichroism (CD) and biosensor analysis, showed a clear correlation with the potency and safety tests. A set of criteria is proposed that a diphtheria toxoid must comply with, i.e. an apparent shift of the B-fragment on SDS-PAGE, a reduction of primary amino groups in a diphtheria molecule, an increased resistance to denaturation, an increased circular dichroism signal in the near-UV region and a reduced binding to selected monoclonal antibodies. In principle, a selected set of in vitro analyses can replace the classical in vivo tests to evaluate the quality of diphtheria toxoid vaccines, provided that the validity of these tests is demonstrated in extensive validation studies and regulatory acceptance is obtained.

Animals↗

PubMed2003

Identification of formaldehyde-induced modifications in proteins: reactions with model peptides.

Formaldehyde is a well known cross-linking agent that can inactivate, stabilize, or immobilize proteins. The purpose of this study was to map the chemical modifications occurring on each natural amino acid residue caused by formaldehyde. Therefore, model peptides were treated with excess formaldehyde, and the reaction products were analyzed by liquid chromatography-mass spectrometry. Formaldehyde was shown to react with the amino group of the N-terminal amino acid residue and the side-chains of arginine, cysteine, histidine, and lysine residues. Depending on the peptide sequence, methylol groups, Schiff-bases, and methylene bridges were formed. To study intermolecular cross-linking in more detail, cyanoborohydride or glycine was added to the reaction solution. The use of cyanoborohydride could easily distinguish between peptides containing a Schiff-base or a methylene bridge. Formaldehyde and glycine formed a Schiff-base adduct, which was rapidly attached to primary N-terminal amino groups, arginine and tyrosine residues, and, to a lesser degree, asparagine, glutamine, histidine, and tryptophan residues. Unexpected modifications were found in peptides containing a free N-terminal amino group or an arginine residue. Formaldehyde-glycine adducts reacted with the N terminus by means of two steps: the N terminus formed an imidazolidinone, and then the glycine was attached via a methylene bridge. Two covalent modifications occurred on an arginine-containing peptide: (i) the attachment of one glycine molecule to the arginine residue via two methylene bridges, and (ii) the coupling of two glycine molecules via four methylene bridges. Remarkably, formaldehyde did not generate intermolecular cross-links between two primary amino groups. In conclusion, the use of model peptides enabled us to determine the reactivity of each particular cross-link reaction as a function of the reaction conditions and to identify new reaction products after incubation with formaldehyde.

Chromatography, Liquid↗

PubMed2003

Shifting paradigms: biopharmaceuticals versus low molecular weight drugs.

Biopharmaceuticals are pharmaceutical products consisting of (glyco)proteins. Nowadays a substantial part of the FDA-approved drugs belong to this class of drugs. Biopharmaceuticals deserve special attention as they have a number of characteristics that set them aside from low molecular weight drugs. Their activity depends on their complicated shape based on secondary, tertiary and (sometimes) quaternary structures. These structures cannot be fully defined with our present set of analytical techniques and approaches for potency testing. They often are the same as (or closely resemble) endogenous proteins. This means that in safety testing and clinical test programs questions have to be addressed regarding species specific responses, selection of dosing schedules and route of administration, and the possible occurrence of immunogenicity. As the conformational structure of a protein is easily disturbed, formulation and handling of biopharmaceuticals needs special attention in order to optimize the therapeutic effect and minimize adverse reaction, among which immune responses. The issue of biogenerics is gaining more and more interest and different critical elements in the development of biogenerics are touched upon. In conclusion, biopharmaceuticals cannot be characterized fully in terms of their structure like low molecular weight drugs. The performance of biopharmaceuticals relies on strict production protocols and close monitoring of their activity in the clinical situation.

Animals↗

PubMed2003

Nanotechnological approaches for the delivery of macromolecules.

In this overview, novel approaches are described for the controlled release and/or for the targeted delivery of macromolecules such as proteins and DNA. The building stones of these highly complex systems are (phospho)lipids and/or (biodegradable) polymers. They should be carefully chosen and preparation protocols should be rationally designed to maximize chances for success.

Animals↗

PubMed2003

Restored functional immunogenicity of purified meningococcal PorA by incorporation into liposomes.

The impact of the conformation, lipooligosaccharide (LOS)-depletion and the presentation form of outer membrane protein PorA from Neisseria meningitidis (PorA) subtype P1.7-2,4 on the immune response in mice was studied. Native PorA was purified from outer membrane vesicles (OMVs) derived from meningococci and reconstituted into liposomes. The conformation of PorA after purification from OMVs and reconstitution in liposomes was monitored by use of electrophoretic and spectroscopic techniques and compared with the conformation of PorA in outer membrane complexes (OMCs) and heat-denatured PorA. The antigenicity of the PorA formulations was measured by ELISA by using a bactericidal anti-P1.4 monoclonal antibody. Immunogenicity was determined in Balb/c mice. PorA-specific IgG, isotype distribution and bactericidal activity were measured after subcutaneous immunization. In all formulations except in heat-denatured OMVs, PorA was present as trimers. The lipooligosaccharide (LOS) content was reduced by 96% in the purified protein with respect to the original OMVs. The antigenicity of purified PorA (i.e. ELISA response) was substantially higher as compared to PorA in liposomes, OMVs or OMCs. The results of the immunogenicity studies showed that all formulations were able to induce comparable IgG titers. However, whereas the antibodies raised by OMVs were bactericidal, the antibodies elicited by immunization with purified PorA were unable to kill meningococci. Remarkably, the ability to induce bactericidal antibodies was fully recovered by incorporation of the purified PorA into liposomes, in the absence of other adjuvants, as compared to LOS-containing OMVs.

Animals↗

PubMed2003

Micelle-associated protein in epoetin formulations: aA risk factor for immunogenicity?

PURPOSE: An upsurge of pure red cell aplasia (PRCA) cases associated with subcutaneous treatment with epoetin alpha has been reported. A formulation change introduced in 1998 is suspected to be the reason for the induction of antibodies that also neutralize the native protein. The aim of this study was to detect the mechanism by which the new formulation may induce these antibodies. METHODS: Formulations of epoetin were subjected to gel permeation chromatography with UV detection, and the fractions were analyzed by an immunoassay for the presence of epoetin. RESULTS: The chromatograms showed that Eprex/Erypo contained micelles of Tween 80. A minute amount of epoetin (0.008-0.033% of the total epoetin content) coeluted with the micelles, as evidenced by ELISA. When 0.03% (w/v) Tween 80, corresponding to the concentration in the formulation, was added to the elution medium, the percentage of epoetin eluting before the main peak was 0.68%. CONCLUSIONS: Eprex/Erypo contains micelle-associated epoetin, which may be a risk factor for the development of antibodies against epoetin.

Chemistry, Pharmaceutical↗

PubMed2003

Liposomal meningococcal B vaccination: role of dendritic cell targeting in the development of a protective immune response.

The effect of targeting strategies for improving the interaction of liposomal PorA with dendritic cells (DC) on the immunogenicity of PorA was investigated. PorA, a major antigen of Neisseria meningitidis, was purified and reconstituted in different types of (targeted) liposomes, i.e., by using mannose or phosphatidylserine as targeting moieties, or with positively charged liposomes. We studied the efficiency of liposome uptake and its effect on the maturation of and interleukin 12 (IL-12) production by murine DC. Moreover, mice were immunized subcutaneously to study the localization and immunogenicity of PorA liposomes. Uptake of liposomes by DC was significantly increased for targeted liposomes and resulted in the maturation of DC, but to various degrees. Maturation markers (i.e., CD80, CD86, major histocompatibility complex class II, and CD40) showed enhanced expression on DC incubated with targeted PorA liposomes relative to those incubated with nontargeted PorA liposomes. Moreover, only the uptake of targeted PorA liposomes induced production of IL-12 by DC, with levels similar to those produced by lipopolysaccharide (LPS)-pulsed DC. Mannose-targeted PorA liposomes administered subcutaneously had an increased localization in draining lymph nodes compared to nontargeted PorA liposomes. Liposomes in draining lymph nodes interacted preferentially with antigen-presenting cells, an effect that was enhanced with targeted PorA liposomes. Immunization studies showed an improvement of the bactericidal antibody response (i.e., increased number of responders) generated by targeted PorA liposomes compared to that generated by nontargeted ones or LPS-containing outer membrane vesicles. In conclusion, the use of targeted PorA liposomes results in an improved uptake by and activation of DC and an increased localization in draining lymph nodes. These effects correlate with an enhanced immune response toward the vaccine.

Animals↗

PubMed2002

Reduction of animal use in human vaccine quality control: opportunities and problems.

In vivo assays play a crucial role in the assessment of the potency and safety of human vaccines. Robust vaccine production procedures, improved characterisation methods and development of well-characterised vaccines create possibilities to reduce animal use. In this paper the current status in this field is reviewed. Achievements with regard to in vivo and in vitro potency and safety testing are discussed as well as new developments and possibilities in the field of in vitro characterisation of vaccine components. Finally, validation and implementation issues will be dealt with. Although replacement of in vivo tests for batch release of existing vaccines is difficult, emerging technologies allow well-considered reduction of in vivo experiments during product and process development and improvement. Inextricably bound up with this approach is good manufacturing practice (GMP), resulting in robust, validated production processes.

Animal Testing Alternatives↗

PubMed2002

Functional characterization of an endosome-disruptive peptide and its application in cytosolic delivery of immunoliposome-entrapped proteins.

Antibody-directed liposomes (immunoliposomes) are frequently used for targeted drug delivery. However, delivery of large biotherapeutic molecules (i.e. peptides, proteins, or nucleic acids) with immunoliposomes is often hampered by an inefficient cytosolic release of entrapped macromolecules after target cell binding and subsequent endocytosis of immunoliposomes. To enhance cytosolic drug delivery from immunoliposomes present inside endosomes, a pH-dependent fusogenic peptide (diINF-7) resembling the NH(2)-terminal domain of influenza virus hemagglutinin HA-2 subunit was used. Functional characterization of this dimeric peptide showed its ability to induce fusion between liposome membranes and leakage of liposome-entrapped compounds when exposed to low pH. In a second series of experiments, diINF-7 peptides were encapsulated in immunoliposomes to enhance the endosomal escape of diphtheria toxin A chain (DTA), which inhibits protein synthesis when delivered into the cytosol of target cells. Immunoliposomes targeted to the internalizing epidermal growth factor receptor on the surface of ovarian carcinoma cells (OVCAR-3) and containing encapsulated DTA did not show any cytotoxicity toward OVCAR-3 cells. Cytotoxicity was only observed when diINF-7 peptides and DTA were co-encapsulated in the immunoliposomes. Thus, diINF-7 peptides entrapped inside liposomes can greatly enhance cytosolic delivery of liposomal macromolecules by pH-dependent destabilization of endosomal membranes after cellular uptake of liposomes.

Circular Dichroism↗