The Experts below are selected from a list of 501 Experts worldwide ranked by ideXlab platform
Rinaldo Zurbriggen - One of the best experts on this subject based on the ideXlab platform.
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A Randomized Placebo-Controlled Phase Ia Malaria Vaccine Trial of Two Virosome-Formulated Synthetic Peptides in Healthy Adult Volunteers
2013Co-Authors: Blaise Genton, Gerd Pluschke, Lukas Degen, Andreas R. Kammer, Nicole Westerfeld, Shinji L. Okitsu, Ro Schroller, Markus M. Mueller, Marcel Tanner, Rinaldo ZurbriggenAbstract:Background and Objectives. Influenza Virosomes represent an innovative human-compatible antigen delivery system that has already proven its suitability for subunit vaccine design. The aim of the study was to proof the concept that Virosomes can also be used to elicit high titers of antibodies against synthetic peptides. The specific objective was to demonstrate the safety and immunogenicity of two Virosome-formulated P. falciparum protein derived synthetic peptide antigens given in two different doses alone or in combination. Methodology/Principal Findings. The design was a single blind, randomized, placebo controlled, dose-escalating study involving 46 healthy Caucasian volunteers aged 18–45 years. Five groups of 8 subjects received virosomal formulations containing 10 mg or50mg of AMA 49-CPE, an apical membrane antigen-1 (AMA-1) derived synthetic phospatidylethanolamine (PE)-peptide conjugate or 10 ug or 50 ug of UK39, a circumsporozoite protein (CSP) derived synthetic PE-peptide conjugate or 50 ug of both antigens each. A control group of 6 subjects received unmodified Virosomes. Virosomal formulations of the antigens (designated PEV301 and PEV302 for the AMA-1 and the CSP virosomal vaccine, respectively) or unmodified Virosomes were injected i. m. on days 0, 60 and 180. In terms of safety, no serious or severe adverse events (AEs) related to the vaccine were observed. 11/46 study participants reported 16 vaccine related local AEs. Of these 16 events, all being pain, 4 occurred after the 1 st, 7 after the 2 nd and 5 after the 3 rd vaccination. 6 systemic AEs probably related to the study vaccine were reported after the 1 st injection, 10 after the 2 nd and 6 after the 3 rd. Generally, no difference in the distribution of the systemic AEs between either the doses applied (10 respectively 50 mg) or th
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Enhancement of the immunogenicity of synthetic carbohydrates by conjugation to Virosomes: a leishmaniasis vaccine candidate.
ACS chemical biology, 2006Co-Authors: Xinyu Liu, Rinaldo Zurbriggen, Gerd Pluschke, Sibylle Siegrist, Mario Amacker, Peter H. SeebergerAbstract:Novel virosomal formulations of a synthetic oligosaccharide were prepared and evaluated as vaccine candidates against leishmaniasis. A lipophosphoglycan-related synthetic tetrasaccharide antigen was conjugated to a phospholipid and to the influenza virus coat protein hemagglutinin. These glycan conjugates were embedded into the lipid membrane of reconstituted influenza virus Virosomes. The virosomal formulations elicited both IgM and IgG anti-glycan antibodies in mice, indicating an antibody isotype class switch to IgG. The antisera cross-reacted in vitro with the corresponding natural carbohydrate antigens expressed by leishmania cells. These findings support the concept of using Virosomes as universal antigen delivery platform for synthetic carbohydrate vaccines.
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mucosal antibody response induced with a nasal Virosome based influenza vaccine
Vaccine, 2003Co-Authors: P Durrer, U Gluck, Christian Spyr, Alois B Lang, Rinaldo Zurbriggen, C Herzog, Reinhard GlückAbstract:A vaccination against influenza that elicits both a systemic antibody and a mucosal IgA response would improve on the protective efficacy of currently available vaccines. Previous studies have shown the safety and efficacy of Virosomes as delivery systems in vaccination. This study was a controlled, randomised, double-blind, single centre, phase II trial assessing an intranasal Virosome vaccine, adjuvanted with heat-labile toxin (HLT) from enterotoxigenic Escherichia coli, versus an intranasal without HLT and comparing it open to an intramuscular vaccine in a total of 88 healthy adults. The development of a new technique enabled for the first time the detection of neutralising IgA antibodies in very dilute nasal wash samples. It was demonstrated that intranasally administered inactivated influenza vaccine, adjuvanted with HLT, not only elicits a spectrum of humoral and cell-mediated responses in healthy adults, critical for the protection and recovery from influenza virus infection, but is also highly effective in eliciting IgA neutralising antibodies at the mucosa. Intranasal Virosome-formulated, HLT-adjuvanted, influenza vaccine was effective and well tolerated in this study. Its potential to offer a high level of mucosal protection, not provided by conventional parenteral vaccination, could play a significant role in preventing morbidity and mortality associated with influenza.
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induction of parasite growth inhibitory antibodies by a virosomal formulation of a peptidomimetic of loop i from domain iii of plasmodium falciparum apical membrane antigen 1
Infection and Immunity, 2003Co-Authors: Markus S Mueller, Rinaldo Zurbriggen, Annabelle Renard, Francesca Boato, Denise Vogel, Martin Naegeli, John A Robinson, Gerd PluschkeAbstract:Apical membrane antigen 1 (AMA-1) of Plasmodium falciparum is a leading candidate antigen for inclusion in a malaria subunit vaccine. Its ectodomain can be divided into three subdomains, each with disulfide bond-stabilized structures. Since the majority of antibodies raised against the ectodomain appear to recognize strain-specific epitopes in domain I, we attempted to develop a vaccine formulation which directs the immune response to a region that contains more conserved epitopes. Here we demonstrate that a virosomal formulation of a peptide that mimics the semiconserved loop I of domain III elicits parasite growth-inhibitory antibodies. A synthetic peptide comprising residues 446 to 490 of AMA-1 (AMA-1(446-490)) was conjugated through the N terminus to a derivative of phosphatidylethanolamine and the phosphatidylethanolamine-peptide conjugate was incorporated into immunopotentiating reconstituted influenza Virosomes as a human-compatible antigen delivery system. Both cyclized and linear versions of the peptide antigen elicited antibodies which specifically bound to parasite-expressed AMA-1 in Western blotting with parasite lysates as well as in immunofluorescence assays with blood stage parasites. All 11 peptidomimetic-specific monoclonal antibodies generated were cross-reactive with parasite-expressed AMA-1. Antigen binding assays with a library of overlapping cyclic peptides covering the target sequence revealed differences in the fine specificity of these monoclonal antibodies and provided evidence that at least some of them recognized discontinuous epitopes. The two immunodominant epitopes comprised the conserved linear sequences K(459)RIKLN(464) and D(467)DEGNKKII(475). A key feature of the synthetic vaccine formulation proposed here is the display of the peptide antigen in a native-like state on the surface of the Virosome.
Gerd Pluschke - One of the best experts on this subject based on the ideXlab platform.
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A Randomized Placebo-Controlled Phase Ia Malaria Vaccine Trial of Two Virosome-Formulated Synthetic Peptides in Healthy Adult Volunteers
2013Co-Authors: Blaise Genton, Gerd Pluschke, Lukas Degen, Andreas R. Kammer, Nicole Westerfeld, Shinji L. Okitsu, Ro Schroller, Markus M. Mueller, Marcel Tanner, Rinaldo ZurbriggenAbstract:Background and Objectives. Influenza Virosomes represent an innovative human-compatible antigen delivery system that has already proven its suitability for subunit vaccine design. The aim of the study was to proof the concept that Virosomes can also be used to elicit high titers of antibodies against synthetic peptides. The specific objective was to demonstrate the safety and immunogenicity of two Virosome-formulated P. falciparum protein derived synthetic peptide antigens given in two different doses alone or in combination. Methodology/Principal Findings. The design was a single blind, randomized, placebo controlled, dose-escalating study involving 46 healthy Caucasian volunteers aged 18–45 years. Five groups of 8 subjects received virosomal formulations containing 10 mg or50mg of AMA 49-CPE, an apical membrane antigen-1 (AMA-1) derived synthetic phospatidylethanolamine (PE)-peptide conjugate or 10 ug or 50 ug of UK39, a circumsporozoite protein (CSP) derived synthetic PE-peptide conjugate or 50 ug of both antigens each. A control group of 6 subjects received unmodified Virosomes. Virosomal formulations of the antigens (designated PEV301 and PEV302 for the AMA-1 and the CSP virosomal vaccine, respectively) or unmodified Virosomes were injected i. m. on days 0, 60 and 180. In terms of safety, no serious or severe adverse events (AEs) related to the vaccine were observed. 11/46 study participants reported 16 vaccine related local AEs. Of these 16 events, all being pain, 4 occurred after the 1 st, 7 after the 2 nd and 5 after the 3 rd vaccination. 6 systemic AEs probably related to the study vaccine were reported after the 1 st injection, 10 after the 2 nd and 6 after the 3 rd. Generally, no difference in the distribution of the systemic AEs between either the doses applied (10 respectively 50 mg) or th
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A randomized placebo-controlled phase Ia malaria vaccine trial of two Virosome-formulated synthetic peptides in healthy adult volunteers.
Public Library of Science (PLoS), 2007Co-Authors: Blaise Genton, Gerd Pluschke, Lukas Degen, Andreas R. Kammer, Nicole Westerfeld, Shinji L. Okitsu, Markus M. Mueller, Sandro Schroller, Penelope Vounatsou, Marcel TannerAbstract:Influenza Virosomes represent an innovative human-compatible antigen delivery system that has already proven its suitability for subunit vaccine design. The aim of the study was to proof the concept that Virosomes can also be used to elicit high titers of antibodies against synthetic peptides. The specific objective was to demonstrate the safety and immunogenicity of two Virosome-formulated P. falciparum protein derived synthetic peptide antigens given in two different doses alone or in combination.The design was a single blind, randomized, placebo controlled, dose-escalating study involving 46 healthy Caucasian volunteers aged 18-45 years. Five groups of 8 subjects received virosomal formulations containing 10 microg or 50 microg of AMA 49-CPE, an apical membrane antigen-1 (AMA-1) derived synthetic phospatidylethanolamine (PE)-peptide conjugate or 10 ug or 50 ug of UK39, a circumsporozoite protein (CSP) derived synthetic PE-peptide conjugate or 50 ug of both antigens each. A control group of 6 subjects received unmodified Virosomes. Virosomal formulations of the antigens (designated PEV301 and PEV302 for the AMA-1 and the CSP virosomal vaccine, respectively) or unmodified Virosomes were injected i. m. on days 0, 60 and 180. In terms of safety, no serious or severe adverse events (AEs) related to the vaccine were observed. 11/46 study participants reported 16 vaccine related local AEs. Of these 16 events, all being pain, 4 occurred after the 1(st), 7 after the 2(nd) and 5 after the 3(rd) vaccination. 6 systemic AEs probably related to the study vaccine were reported after the 1(st) injection, 10 after the 2(nd) and 6 after the 3(rd). Generally, no difference in the distribution of the systemic AEs between either the doses applied (10 respectively 50 microg) or the synthetic antigen vaccines (PEV301 and PEV302) used for immunization was found. In terms of immunogenicity, both PEV301 and PEV302 elicited already after two injections a synthetic peptide-specific antibody response in all volunteers immunized with the appropriate dose. In the case of PEV301 the 50 microg antigen dose was associated with a higher mean antibody titer and seroconversion rate than the 10 microg dose. In contrast, for PEV302 mean titer and seroconversion rate were higher with the lower dose. Combined delivery of PEV301 and PEV302 did not interfere with the development of an antibody response to either of the two antigens. No relevant antibody responses against the two malaria antigens were observed in the control group receiving unmodified Virosomes.The present study demonstrates that three immunizations with the virosomal malaria vaccine components PEV301 or/and PEV302 (containing 10 microg or 50 microg of antigen) are safe and well tolerated. At appropriate antigen doses seroconversion rates of 100% were achieved. Two injections may be sufficient for eliciting an appropriate immune response, at least in individuals with pre-existing anti-malarial immunity. These results justify further development of a final multi-stage virosomal vaccine formulation incorporating additional malaria antigens.ClinicalTrials.gov NCT00400101
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Enhancement of the immunogenicity of synthetic carbohydrates by conjugation to Virosomes: a leishmaniasis vaccine candidate.
ACS chemical biology, 2006Co-Authors: Xinyu Liu, Rinaldo Zurbriggen, Gerd Pluschke, Sibylle Siegrist, Mario Amacker, Peter H. SeebergerAbstract:Novel virosomal formulations of a synthetic oligosaccharide were prepared and evaluated as vaccine candidates against leishmaniasis. A lipophosphoglycan-related synthetic tetrasaccharide antigen was conjugated to a phospholipid and to the influenza virus coat protein hemagglutinin. These glycan conjugates were embedded into the lipid membrane of reconstituted influenza virus Virosomes. The virosomal formulations elicited both IgM and IgG anti-glycan antibodies in mice, indicating an antibody isotype class switch to IgG. The antisera cross-reacted in vitro with the corresponding natural carbohydrate antigens expressed by leishmania cells. These findings support the concept of using Virosomes as universal antigen delivery platform for synthetic carbohydrate vaccines.
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induction of parasite growth inhibitory antibodies by a virosomal formulation of a peptidomimetic of loop i from domain iii of plasmodium falciparum apical membrane antigen 1
Infection and Immunity, 2003Co-Authors: Markus S Mueller, Rinaldo Zurbriggen, Annabelle Renard, Francesca Boato, Denise Vogel, Martin Naegeli, John A Robinson, Gerd PluschkeAbstract:Apical membrane antigen 1 (AMA-1) of Plasmodium falciparum is a leading candidate antigen for inclusion in a malaria subunit vaccine. Its ectodomain can be divided into three subdomains, each with disulfide bond-stabilized structures. Since the majority of antibodies raised against the ectodomain appear to recognize strain-specific epitopes in domain I, we attempted to develop a vaccine formulation which directs the immune response to a region that contains more conserved epitopes. Here we demonstrate that a virosomal formulation of a peptide that mimics the semiconserved loop I of domain III elicits parasite growth-inhibitory antibodies. A synthetic peptide comprising residues 446 to 490 of AMA-1 (AMA-1(446-490)) was conjugated through the N terminus to a derivative of phosphatidylethanolamine and the phosphatidylethanolamine-peptide conjugate was incorporated into immunopotentiating reconstituted influenza Virosomes as a human-compatible antigen delivery system. Both cyclized and linear versions of the peptide antigen elicited antibodies which specifically bound to parasite-expressed AMA-1 in Western blotting with parasite lysates as well as in immunofluorescence assays with blood stage parasites. All 11 peptidomimetic-specific monoclonal antibodies generated were cross-reactive with parasite-expressed AMA-1. Antigen binding assays with a library of overlapping cyclic peptides covering the target sequence revealed differences in the fine specificity of these monoclonal antibodies and provided evidence that at least some of them recognized discontinuous epitopes. The two immunodominant epitopes comprised the conserved linear sequences K(459)RIKLN(464) and D(467)DEGNKKII(475). A key feature of the synthetic vaccine formulation proposed here is the display of the peptide antigen in a native-like state on the surface of the Virosome.
Jan Wilschut - One of the best experts on this subject based on the ideXlab platform.
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EFFICIENT GENE TRANSFER MEDIATED BY INFLUENZA VirosomeS WITH ENCAPSULATED PLASMID DNA
2015Co-Authors: Peter Cullis, Jan Wilschut, A HuckriedeAbstract:CHAPTER 3 Reconstituted influenza virus membrane envelopes (Virosomes) have been used previously to deliver plasmid DNA (pDNA) bound to their external surface to a variety of target cells. While high transfection efficiencies can be obtained with these complexes, the Virosome-associated DNA is readily accessible for nucleases and is prone to rapid degradation. Here we present a new method for the production of DNA-Virosomes resulting in complete protection of the DNA from nucleases. This method relies on the use of the short-chain phospholipid dicaproylphosphatidylcholine (DCPC) for solubilization of the viral membrane. The solubilized viral membrane components are mixed with pDNA and cationic lipid. Reconstitution of the viral envelopes and simultaneous encapsulation of pDNA is achieved by removal of the DCPC from the mixture through dialysis. Analysis by linear sucrose density gradient centrifugation revealed that protein, phospholipid and pDNA physically associated to particles which appeared as vesicles with spike proteins inserted in their membranes when analyzed by electron microscopy. The DNA-Virosomes retained the membrane fusion properties of the native influenza virus. The Virosome-associated pDNA was completely protected from degradation by added nucleases, providing evidence for the DNA being highly condensed and encapsulated in the lumen of the Virosomes. DNA-Virosomes, containing reporter gene constructs, transfected a variety of cell lines, with efficiencies approaching 90%. Transfection was completely dependent on the fusogenic properties of the viral spike protein haemagglutinin. Thus, DNA-Virosomes prepared by the new procedure are highly efficient vehicles for DNA delivery offering the advantage of complete DNA protection which is especially important for future in vivo applications
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In vitro analysis of RSV and RSV-MPLA Virosomes.
2013Co-Authors: Tobias Kamphuis, Jan Wilschut, Toon Stegmann, Tjarko Meijerhof, Julia Lederhofer, Aalzen De HaanAbstract:(A,B) RSV Virosomes and RSV-MPLA Virosomes were spun on an equilibrium density sucrose gradient. Subsequently, density, protein concentration, and phosphate concentrations of each fraction was determined. (C,D) Fractions from A and B were analyzed for their TLR4-signaling ability using Hek-Blue TLR4 cells. To assess non-TLR specific activation of cells, control cells (Null2 cells) were incubated with the same Virosome fractions. As a control for activation both Hek blue TLR4 and Hek blue null2 cells were stimulated with 100 ng/ml TNF-α. Bars represent TLR activation relative to that of the TNF-α control (E) Upregulation of DCs costimulatory molecules CD40, CD86, CD80. Unfractionated Virosome preparations were used to stimulate ex vivo cultured mouse DCs overnight. Cells were stained for expression of costimulatory molecules using specific monoclonal antibodies and analyzed by FACS. Bars represent the percentage of positive cells. The data shown are a representative of three individual experiments.
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In vitro analysis of RSV Virosomes and RSV Virosomes adjuvanted with TLR2 and/or NOD2ligands.
2013Co-Authors: Muhammad Shafique, Jan Wilschut, Tjarko Meijerhof, Aalzen De HaanAbstract:RSV Virosomes and RSV Virosomes adjuvanted with TLR2 and/or NOD2 ligands were spun on an equilibrium density sucrose gradient. Subsequently, density, phospholipids and protein concentrations of each fraction was determined. Panel A shows a representative profile of a Virosome purification gradient. Fractions (1,5,10; representing bottom, virosomal and top gradient fractions, respectively) were analyzed to determine their capacity to activate NF-κB in mouse macrophages (RAW-Blue cells; panel B) and human embryonic kidney cells (HEK-BlueTLR2 & HEK-Blue NOD2 cells; panel C). The level of NF-κB induced in RAW-blue cells was expressed as values relative to levels of NF-κB induced by CpG ODN, the positive control. To assess non-specific NF-κB activation by TLR2 and NOD2 ligand-carrying Virosomes in HEK cells, control cells (HEK-Blue Null1 & HEK-Blue Null2 cells, respectively) were incubated with the same fractions and these values were subtracted from values obtained with HEK-BlueTLR2 & HEK-Blue NOD2 cells, respectively. As a control, HEK-Blue TLR2 and HEK-Blue NOD2 cells were stimulated with 100 ng/ml TNF-α. Bars represent the NF-κB activation relative to TNF-α control.
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lipopeptide adjuvanted respiratory syncytial virus Virosomes a safe and immunogenic non replicating vaccine formulation
Vaccine, 2010Co-Authors: Toon Stegmann, Tobias Kamphuis, Tjarko Meijerhof, Ellen Goud, Aalzen De Haan, Jan WilschutAbstract:Respiratory syncytial virus (RSV) causes severe respiratory disease in children and the elderly. There is no registered RSV vaccine. Early experimental non-replicating vaccines have been found to exacerbate RSV symptoms upon infection causing enhanced respiratory disease. Here we show that immunization of mice with reconstituted Virosomes produced from RSV envelopes and containing the lipopeptide adjuvant (P3CSK4), induces high-titer virus-neutralizing antibodies, and the secretion of IFN-γ through both MHC-I and MHC-II presentation of antigen, with a balanced Th1/Th2 profile. Immunization with RSV Virosomes provides sterilizing immunity to virus challenge in mice and cotton rats, while not producing symptoms of enhanced disease. Therefore, these Virosomes represent a promising candidate inactivated RSV vaccine formulation.
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gene transfer mediated by fusion protein hemagglutinin reconstituted in cationic lipid vesicles
Gene Therapy, 1999Co-Authors: Pieter Schoen, Arcadio Chonn, Pieter R Cullis, Jan Wilschut, Peter ScherrerAbstract:Hemagglutinin, the membrane fusion protein of influenza erythrocyte ghosts. Efficient cell transfection of BHK-21 virus, is known to mediate a low-pH-dependent fusion cells was observed with Virosomes containing 30 mol% reaction between the viral envelope and the limiting mem- DODAC and plasmid encoding for b-galactosidase (pCMV brane of the endosomal cell compartment following cellular b-gal) associated to their surface. The transfection activity uptake of the virus particles by receptor-mediated endo- observed was dependent on the functional activity of hemcytosis. Here we exploited this activity of hemagglutinin to agglutinin. Contrary to DNA/cationic lipid complexes the achieve efficient gene delivery to cultured cells. Hemagglu- transfection was not dependent on the cationic lipid to DNA tinin was reconstituted in the presence of the monocationic charge ratio. Importantly, transfection of BHK-21 cells with lipid dioleoyldimethylammonium chloride (DODAC) to per- pCMV b-gal by DODAC-containing Virosomes did not show mit plasmid binding to the Virosome surface. Virosomes any significant signs of cytotoxicity that is commonly with 30 mol% DODAC exhibited a distinct binding capacity observed with DNA/cationic lipid complexes. Together with for plasmid without causing aggregation. The Virosome the high levels of expression of the transgene this highfusion activity was not affected by the cationic lipid DODAC lights the potential of DODAC-containing Virosomes as a as demonstrated by low-pH-dependent lipid mixing with novel approach in nonviral gene transfer.
Wilschut J - One of the best experts on this subject based on the ideXlab platform.
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Incorporation of LpxL1, a detoxified lipopolysaccharide adjuvant, in influenza H5N1 Virosomes increases vaccine immunogenicity
2009Co-Authors: De Vries J. J. C., Wilschut J, Bungener L, Ter Veer W, Van Alphen L., Van Der Ley R., Huckriede AAbstract:The increasing number of human influenza H5N1 infections accentuates the need for the development of H5N1 vaccine candidates to prevent a potential influenza pandemic. The use of adjuvants in such vaccines can contribute significantly to antigen dose-sparing. In this study, we evaluated the capacity of the non toxic Neisseria meningitidis lipopolysaccharide analog LpxL1 to function as an adjuvant for an influenza H5N1 virosomal vaccine. Inactivated influenza H5N1 virus (NIBRG-14) was used to construct Virosomes (reconstituted virus envelopes) with LpxL1 incorporated in the virosomal membrane thus combining the influenza hemagglutinin (HA) antigen and the adjuvant in the same particle. Mice were immunized in a one- or two-dose immunization regimen with H5N1 Virosomes with or without incorporated LpxL1 After a single immunization, H5N1 Virosomes with incorporated LpxL1 induced significantly enhanced H5N1-specific total IgG titers as compared to non-adjuvanted Virosomes but hemagglutination inhibition (HI) titers remained low. In the two-dose immunization regimen, LpxL1-modified H5N1 Virosomes induced HI titers above 40 which were significantly higher than those obtained with non-adjuvanted Virosomes. Incorporation of LpxL1 had little effect on Virosome-induced IgG1 levels, but significantly increased IgC2a levels in both the one- and two-close immunization regimen. Compared to non-adjuvanted Virosomes, LpxL1-modified Virosomes induced similar numbers of IFN gamma-producing T cells but decreased numbers of IL-4-producing T cells irrespective of the number of immunizations. We conclude that LpxL1 incorporated in H5N1 influenza Virosomes has the capacity to function as a potent adjuvant particularly Stimulating Th1-type immune reactions. (C) 2008 Elsevier Ltd. All rights reserved
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Use of a dialyzable short-chain phospholipid for efficient solubilization and reconstitution of influenza virus envelopes
'Elsevier BV', 2006Co-Authors: De Jonge J, Stegmann T., Wilschut J, Ter Veer W, Schoen P, Huckriede AAbstract:Virosomes are reconstituted viral envelopes that can serve as vaccines and as vehicles for Cellular delivery of various macromolecules. To further advance the use of Virosomes, we developed a novel dialysis procedure for the reconstitution of influenza virus membranes that is easily applicable to industrial production and compatible with encapsulation of a variety of compounds. This procedure relies on the use of 1,2-dicaproyl-sn-glycero-3-phosphocholiiie (DCPC) as a solubilizing agent. DCPC is a short-chain lecithin with detergent-like properties and with a critical micelle concentration of 14 mM. DCPC effectively dissolved the influenza virus membranes after which the nucleocapsids could be removed by ultracentrifugation. The solubilized membrane components were reconstituted either by removal of DCPC by dialysis or by a procedure involving initial dilution of the solubilized membrane components followed by dialysis. Both protocols resulted in removal of 99.9% of DCPC and simultaneous formation of Virosomes. Analysis of the Virosome preparations by equilibrium sucrose density gradient centrifugation revealed co-migration of phospholipid and protein for Virosomes produced by either method. Moreover, both Virosome preparations showed morphological and fusogenic characteristics similar to native influenza virus. Size, homogeneity and spike density of the Virosomes varied with the two different reconstitution procedures employed. The recovery of viral membrane proteins and phospholipids in the Virosomes was found to be higher for the dilution/dialysis procedure than for the simple dialysis protocol. This novel procedure for the production of Virosomes is straightforward and robust and allows further exploitation of Virosomes as vaccines or as drug delivery vehicles not only in academia, but also in industrial settings. (c) 2006 Elsevier B.V All rights reserved
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Virosome-mediated delivery of protein antigens in vivo: efficient induction of class I MHC-restricted cytotoxic T lymphocyte activity
2005Co-Authors: Bungener L, Wilschut J, Huckriede A, De Mare A, De Vries-idema J, Daemen TAbstract:Induction of CTL responses against protein antigens is an important aim in vaccine development. In this paper we present fusion-active Virosomes as a vaccine delivery system capable of efficient induction of CTL responses in vivo. Virosomes are reconstituted viral membranes, which do not contain the genetic material of the virus they are derived from. Foreign macromolecules, including protein antigens, can be encapsulated in Virosomes during the reconstitution process. Functionally reconstituted Virosomes retain the cell binding and fusion characteristics of the native virus. Thus, upon uptake by cells through receptor-mediated endocytosis, Virosomes will deliver their content to the cell cytosol. In a previous study, we demonstrated that protein antigens delivered in this manner to dendritic cells are efficiently processed for both MHC class I and class 11 presentation. Here, we studied in vivo induction of cellular immune responses against Virosome-encapsulated ovalbumin (OVA) in mice. As little as 0.75 mug OVA delivered by fusion-active Virosomes was sufficient to induce a powerful class I MHC-restricted CTL response. All immunization routes that were used (i.m., i.p. and s.c.) resulted in efficient induction of CTL activity. The CTLs induced were cytotoxic in a standard Cr-51-release assay and produced IFN-gamma in response to OVA peptide. Thus, Virosomes represent an ideal antigen delivery system for induction of cellular immunity against encapsulated protein antigens. (C) 2004 Elsevier Ltd. All rights reserved
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Virosomes for antigen and DNA delivery
'Elsevier BV', 2005Co-Authors: Daemen T, Bungener L, Huckriede A, De Jonge J, De Mare A, Wilschut JAbstract:Specific targeting and delivery as well as the display of antigens on the surface of professional antigen-presenting cells (APCs) are key issues in the design and development of new-generation vaccines aimed at the induction of both humoral and cell-mediated immunity. Prophylactic vaccination against infectious diseases in general aims at the induction of humoral immune responses to prevent infection. This humoral immune response is mediated by antibody-producing B cells. On the other hand, therapeutic immunisation against virally infected cells and tumour cells requires the induction of cytotoxic T lymphocytes (CTLs) that can specifically recognise and lyse infected cells or transformed tumour cells. The induction of Major Histocompatibility Complex (MHC) class I restricted CTL activity is optimally achieved by synthesis of antigens within APCs, for example, after immunisation with live attenuated virus. However, immunisation with live vaccines bears the risk of causing disease. Therefore, alternative vaccine delivery systems, which enable introduction of nonreplicating antigen into the MHC class I presentation pathway, are sought. Furthermore, for the induction of effective humoral and cellular responses, MHC class II restricted activation of T helper cells (Th cells) is required. Among other delivery systems, as described in this theme issue of Advanced Drug Delivery Reviews, Virosomes seem ideally suited for delivery of antigens into both MHC pathways. In this review, we will focus on the use of Virosomes as carrier vehicles for the intracellular delivery of protein antigens and DNA, and the induction of a cellular immune response against encapsulated protein antigens and proteins expressed by Virosome-associated plasmids. (C) 2004 Elsevier B.V. All rights reserved
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The Virosome concept in influenza vaccines
MEDIMOND PUBLISHING CO, 2004Co-Authors: Wilschut J, Stegmann T., Bungener L, Huckriede A, Daemen T, Palache AAbstract:Reconstituted influenza virus envelopes (Virosomes) represent efficient influenza vaccines inducing high antibody titers upon intramuscular adminsitration. Virosomes are reconstituted viral envelopes which retain the cell entry and membrane fusion characteristics of native influenza virus. Here, we show that Virosomes, by virtue of their membrane fusion activity, have the capacity to deliver a Virosome-encapsulated protein antigen (ovalbumin, OVA) to the cytosol of cultured murine bone-marrow-derived dendritic cells (DCs) and thus to the MHC class I presentation pathway. Accordingly, immunization of mice with OVA-containing Virosomes resulted in efficient priming of an OVA-specific class I MHC-restricted cytotoxic T lymphocyte (CTL) response. These results indicate that Virosomes have the capacity to prime a broad immune response, inducing not only humoral but also cellular immunity
Shinji L. Okitsu - One of the best experts on this subject based on the ideXlab platform.
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A Randomized Placebo-Controlled Phase Ia Malaria Vaccine Trial of Two Virosome-Formulated Synthetic Peptides in Healthy Adult Volunteers
2013Co-Authors: Blaise Genton, Gerd Pluschke, Lukas Degen, Andreas R. Kammer, Nicole Westerfeld, Shinji L. Okitsu, Ro Schroller, Markus M. Mueller, Marcel Tanner, Rinaldo ZurbriggenAbstract:Background and Objectives. Influenza Virosomes represent an innovative human-compatible antigen delivery system that has already proven its suitability for subunit vaccine design. The aim of the study was to proof the concept that Virosomes can also be used to elicit high titers of antibodies against synthetic peptides. The specific objective was to demonstrate the safety and immunogenicity of two Virosome-formulated P. falciparum protein derived synthetic peptide antigens given in two different doses alone or in combination. Methodology/Principal Findings. The design was a single blind, randomized, placebo controlled, dose-escalating study involving 46 healthy Caucasian volunteers aged 18–45 years. Five groups of 8 subjects received virosomal formulations containing 10 mg or50mg of AMA 49-CPE, an apical membrane antigen-1 (AMA-1) derived synthetic phospatidylethanolamine (PE)-peptide conjugate or 10 ug or 50 ug of UK39, a circumsporozoite protein (CSP) derived synthetic PE-peptide conjugate or 50 ug of both antigens each. A control group of 6 subjects received unmodified Virosomes. Virosomal formulations of the antigens (designated PEV301 and PEV302 for the AMA-1 and the CSP virosomal vaccine, respectively) or unmodified Virosomes were injected i. m. on days 0, 60 and 180. In terms of safety, no serious or severe adverse events (AEs) related to the vaccine were observed. 11/46 study participants reported 16 vaccine related local AEs. Of these 16 events, all being pain, 4 occurred after the 1 st, 7 after the 2 nd and 5 after the 3 rd vaccination. 6 systemic AEs probably related to the study vaccine were reported after the 1 st injection, 10 after the 2 nd and 6 after the 3 rd. Generally, no difference in the distribution of the systemic AEs between either the doses applied (10 respectively 50 mg) or th
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a virosomal malaria peptide vaccine elicits a long lasting sporozoite inhibitory antibody response in a phase 1a clinical trial
PLOS ONE, 2007Co-Authors: Shinji L. Okitsu, Blaise Genton, Andreas R. Kammer, Nicole Westerfeld, Markus S Mueller, John A Robinson, Olivier Silvie, Marija Curcic, Robert W Sauerwein, Dominique MazierAbstract:Objectives. Peptides delivered on the surface of influenza Virosomes have been shown to induce solid humoral immune responses in experimental animals. High titers of peptide-specific antibodies were also induced in a phase 1a clinical trial in volunteers immunized with virosomal formulations of two peptides derived from the circumsporozoite protein (CSP) and the apical membrane antigen 1 (AMA-1) of Plasmodium falciparum. The main objective of this study was to perform a detailed immunological and functional analysis of the CSP-specific antibodies elicited in this phase 1a trial. Methodology/Principal Findings. 46 healthy malaria-nao ¨ve adults were immunized with virosomal formulations of two peptide-phosphatidylethanolamine conjugates, one derived from the NANP repeat region of P. falciparum CSP (designated UK-39) the other from P. falciparum AMA-1 (designated AMA49-C1). The two antigens were delivered in two different concentrations, alone and in combination. One group was immunized with empty Virosomes as control. In this report we show a detailed analysis of the antibody response against UK-39. Three vaccinations with a 10 mg dose of UK-39 induced high titers of sporozoite-binding antibodies in all volunteers. This IgG response was affinity maturated and long-lived. Co-administration of UK-39 and AMA49C1 loaded Virosomes did not interfere with the immunogenicity of UK-39. Purified total IgG from UK-39 immunized volunteers inhibited sporozoite migration and invasion of hepatocytes in vitro. Sporozoite inhibition closely correlated with titers measured in immunogenicity assays. Conclusions. Virosomal delivery of a short, conformationally constrained peptide derived from P. falciparum CSP induced a long-lived parasite-inhibitory antibody response in humans. Combination with a second virosomally-formulated peptide derived from P. falciparum AMA-1 did not interfere with the immunogenicity of either peptide, demonstrating the potential of influenza Virosomes as a versatile, human-compatible antigen delivery platform for the development of multivalent subunit vaccines. Trial Registration. ClinicalTrials.gov NCT00400101
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A randomized placebo-controlled phase Ia malaria vaccine trial of two Virosome-formulated synthetic peptides in healthy adult volunteers.
Public Library of Science (PLoS), 2007Co-Authors: Blaise Genton, Gerd Pluschke, Lukas Degen, Andreas R. Kammer, Nicole Westerfeld, Shinji L. Okitsu, Markus M. Mueller, Sandro Schroller, Penelope Vounatsou, Marcel TannerAbstract:Influenza Virosomes represent an innovative human-compatible antigen delivery system that has already proven its suitability for subunit vaccine design. The aim of the study was to proof the concept that Virosomes can also be used to elicit high titers of antibodies against synthetic peptides. The specific objective was to demonstrate the safety and immunogenicity of two Virosome-formulated P. falciparum protein derived synthetic peptide antigens given in two different doses alone or in combination.The design was a single blind, randomized, placebo controlled, dose-escalating study involving 46 healthy Caucasian volunteers aged 18-45 years. Five groups of 8 subjects received virosomal formulations containing 10 microg or 50 microg of AMA 49-CPE, an apical membrane antigen-1 (AMA-1) derived synthetic phospatidylethanolamine (PE)-peptide conjugate or 10 ug or 50 ug of UK39, a circumsporozoite protein (CSP) derived synthetic PE-peptide conjugate or 50 ug of both antigens each. A control group of 6 subjects received unmodified Virosomes. Virosomal formulations of the antigens (designated PEV301 and PEV302 for the AMA-1 and the CSP virosomal vaccine, respectively) or unmodified Virosomes were injected i. m. on days 0, 60 and 180. In terms of safety, no serious or severe adverse events (AEs) related to the vaccine were observed. 11/46 study participants reported 16 vaccine related local AEs. Of these 16 events, all being pain, 4 occurred after the 1(st), 7 after the 2(nd) and 5 after the 3(rd) vaccination. 6 systemic AEs probably related to the study vaccine were reported after the 1(st) injection, 10 after the 2(nd) and 6 after the 3(rd). Generally, no difference in the distribution of the systemic AEs between either the doses applied (10 respectively 50 microg) or the synthetic antigen vaccines (PEV301 and PEV302) used for immunization was found. In terms of immunogenicity, both PEV301 and PEV302 elicited already after two injections a synthetic peptide-specific antibody response in all volunteers immunized with the appropriate dose. In the case of PEV301 the 50 microg antigen dose was associated with a higher mean antibody titer and seroconversion rate than the 10 microg dose. In contrast, for PEV302 mean titer and seroconversion rate were higher with the lower dose. Combined delivery of PEV301 and PEV302 did not interfere with the development of an antibody response to either of the two antigens. No relevant antibody responses against the two malaria antigens were observed in the control group receiving unmodified Virosomes.The present study demonstrates that three immunizations with the virosomal malaria vaccine components PEV301 or/and PEV302 (containing 10 microg or 50 microg of antigen) are safe and well tolerated. At appropriate antigen doses seroconversion rates of 100% were achieved. Two injections may be sufficient for eliciting an appropriate immune response, at least in individuals with pre-existing anti-malarial immunity. These results justify further development of a final multi-stage virosomal vaccine formulation incorporating additional malaria antigens.ClinicalTrials.gov NCT00400101
