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ABSTRACT: Due to the advantageous properties of synthetic molecules compared to biological ones biological molecules in diagnostic tests are replaced increasingly by synthetic ones, usually synthetic peptides or related molecules. The replacement of biological antigens by synthetic peptides is most advanced at present, as well as the use of site-specific antibodies induced with synthetic peptides. Moreover recent results indicate that synthetic molecules may also replace antibodies. Ultimately this will lead to diagnostic assays built of synthetic molecules only.
Current Protein and Peptide Science 09/2003; 4(4):253-60. · 2.89 Impact Factor
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ABSTRACT: Porcine parvovirus (PPV) virus-like particles (VLPs) constitute a potential vaccine for prevention of parvovirus-induced reproductive failure in gilts. Here we report the development of a large scale (25 l) production process for PPV-VLPs with baculovirus-infected insect cells. A low multiplicity of infection (MOI) strategy was efficiently applied avoiding the use of an extra baculovirus expansion step. The optimal harvest time was defined at 120 h post-infection at the MOI used, with the cell concentration at infection being 1.5x10(6) cells/ml. An efficient purification scheme using centrifugation, precipitation and ultrafiltration/diafiltration as stepwise unit operations was developed. The global yield of the downstream process was 68%. Baculovirus inactivation with Triton X-100 was successfully integrated into the purification scheme without an increase in the number of process stages. Immunogenicity of the PPV-VLPs tested in guinea pigs was similar to highly purified reference material produced from cells cultured in the presence of serum-containing medium. These results indicate the feasibility of industrial scale production of PPV-VLPs in the baculovirus system, safety of the product, and the potency of the product for vaccine application.
Applied Microbiology and Biotechnology 07/2002; 59(1):45-50. · 3.42 Impact Factor
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In: Immunological recognition of peptides in medicine and biology/ eds. N. Zegers, W. Boersma, E. Claassen. - Boca Raton : CRC Press, 1995. - pp. 15-31. 03/1995;
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01/1995: pages 15-31;
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Journal peptide research 50, 1997, 357-364.
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Vaccine 13, 1995, 1033-1037.
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In: Vaccines 95 / eds. R.M. Chanock [et al.]. - Cold Spring Harbor, N.Y. Cold Spring Harbor Laboratory Press, 1995. - pp. 399-401.
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Vaccine 13, 1995, 885-886.
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Journal of virology 69, 1995, 7274-7277.
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Molecular diversity 1, 1996, 87-96.
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Veterinary quarterly 20 Supp. 3, 1998, S92-S95.
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Veterinary quarterly 19, 1997, 122-126.
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In: Vaccines: new generation immunological adjvant /eds. G. Gregoriadis [et al.]. - New York: Plenum, 1995. - pp. 127-133.
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A.F.G. Antonis,
C.J.M. Bruschke,
P Rueda,
L Maranga,
J. Casal,
C Vela,
L.A.T. Hilgers,
P.B.G.M. Belt,
K. Weerdmeester,
M.J. Carrondo, J.P.M. Langeveld
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ABSTRACT: A novel vaccine against porcine parvovirus (PPV), composed of recombinant virus-like particles (PPV-VLPs) produced with the baculovirus expression vector system (BEVS) at industrial scale, was tested for its immunogenicity and protective potency. A formulation of submicrogram amounts of PPV-VLPs in a water-in-mineral oil adjuvant evoked high serum antibody titres in both guinea pigs, used as reference model, and target species, pigs. A single immunisation with 0.7 ¿g of this antigen yielded complete foetal protection against PPV infection after challenge with a virulent strain of this virus. Furthermore, also in the presence of mild adjuvants the protective action of these PPV-VLPs is excellent. This recombinant subunit vaccine overcomes some of the drawbacks of classical PPV vaccines
Vaccine 24 (2006) 26.
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ABSTRACT: Complement activation products C1q and C3d, serum amyloid P component (SAP) and activated glial cells accumulate in amyloid deposits of conformationally changed prion protein (PrPSc) in Creutzfeldt¿Jakob disease, Gerstmann¿Sträussler¿Scheinker disease and scrapie-infected mouse brain. Biological properties, including the potential to activate microglia, relate to prion (PrP) peptide fibrillogenic abilities. We investigated if SAP and C1q influence the fibrillogenic properties of human and mouse PrP peptide and concomitantly their stimulatory effects on human microglia in vitro. PrP-peptide induced microglial IL-6 and TNF-¿ release significantly increased in the presence of SAP and C1q. Also, SAP and C1q enhanced PrP-peptide fibril formation as revealed by electron microscopy and thioflavin S-based quantitative assays. This suggests that SAP and C1q contribute to fibrillar state-dependent cellular effects of PrP. Combined, ultrastructural and thioflavin assays, together with microglial cytokine release measurements, provide a test system to screen potential, fibrillarity impeding therapeutics for prion disease
Neurobiology of Disease 19 (2005) 1-2.
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ABSTRACT: Transmissible spongiform encephalopathies are neurodegenerative diseases and are considered to be caused by malformed prion proteins accumulated into fibrillar structures that can then aggregate to form larger deposits or amyloid plaques. The identification of fibril-interfering compounds is of therapeutic and prophylactic interest. A robust and easy-to-perform, high-throughput, in vitro fluorescence assay was developed for the detection of such compounds. The assay was based on staining with the fluorescent probe thioflavin S in polystyrene microtiter plates to determine the amyloid state of synthetic peptides, representing a putative transmembrane domain of human and mouse prion protein. In determining optimal test conditions, it was found that drying peptides from phosphate buffer prior to staining resulted in good reproducibility with an interassay variation coefficient of 8%. Effects of thioflavin S concentration and staining time were established. At optimal thioflavin S concentration of 0.2 mg/ml, the fluorescence signals of thioflavin S with five different prion protein-based fibrillogenic peptides, as well as peptide Aß(1¿42), were found to show a peptide-dependent linear correlation within a peptide concentration range of 10¿400 ¿M. The ability of the assay to identify compounds that interfere with fibril formation and/or dissociate preformed fibrils was demonstrated for tetracyclic compounds by preceding coincubation with human prion protein peptide huPrP106¿126
Analytical Biochemistry 333 (2004) 2.
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ABSTRACT: Prion diseases are characterized by accumulation of protease resistant isoforms of prion protein (termed PrPSC), glial activation and neurodegeneration. The time course of PrP deposition, appearance of activated microglia, and of neuronal apoptosis in experimentally-induced prion disease suggests that microglial activation precedes the process of neuronal loss. Activated microglia and inflammatory mediators, including cytokines and prostaglandin E2 (PGE2) co-localize with PrP deposits. In vitro, mouse microglia secrete neurotoxic agents and interleukins (IL)-1 and IL-6, when exposed to synthetic peptides representing the neurotoxic fragment of PrP. In this study, adult human microglia were found to secrete IL-6 and TNF- upon exposure to synthetic fibrillar PrP105-132, the putative transmembrane domain of PrP. Little cytokine release occurred following exposure of microglia to C-terminally amidated, nonfibrillar PrP105-132, suggesting that the degree of fibrillarity of PrP peptides affects their biological properties. Non-steroidal anti-inflammatory drugs (NSAIDs) are thought to exert beneficial effects in neurodegenerative disorders through suppressive effects on microglial activation and on cyclooxygenase (COX) activity. Since microglial COX-2 expression and PGE2 synthesis are increased in human and experimental prion diseases, we investigated the effects of the NSAIDs indomethacin and BF389, an experimental COX-2 selective inhibitor, on the PrP105-132-induced microglial IL-6 and TNF- synthesis in vitro. No inhibitory effects of the NSAIDs were observed. Furthermore, PrP105-132 did not stimulate microglial PGE2 synthesis. We conclude that, unlike IL-1-induced IL-6 synthesis in astrocytes, the PrP-induced IL-6 synthesis in human adult microglia is not PGE2 mediated.
Brain research 925, 195-203. - ISSN 0006-8993.
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ABSTRACT: The influence of the nature of the bond between a peptide and a (lipidic) carrier molecule on the immunogenicity of that construct was investigated. As types of bonds a thioester-, a disulfide-, an amide- and a thioether bond were investigated. As carrier molecules a peptide, an N-palmitoylated peptide or a C16-hydrocarbon chain were used. The biostability of the bond between peptide and carrier molecule is thioether > amide > disulfide thioester. However, the immunogenic potency of the constructs used was found to be thioester > disulfide > amide > thioether. In conclusion, a construct with a bond between peptide and (lipidic) carrier molecule that is more susceptible to biological degradation is more immunogenic when used in a peptide-based vaccine than a bond that is less susceptible to biological degradation.
Journal of peptide research 58, 237-245.
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ABSTRACT: Previous studies have demonstrated a role for microglia in the neuronal loss that occurs in the transmissible spongiform encephalopathies or prion diseases. In the present studies, the processes that lead to the death of neurones treated with synthetic peptides derived from the prion protein (PrP) were fully activated within 1 h, although neuronal cell death was not seen until 24 h later. Similarly, neurones exposed to PrP peptides for only 1 h activated microglia and a temporal relationship between the production of interleukin-6, an indicator of microglial activation, and microglial killing of PrP-treated neurones was also demonstrated. Activation of microglia and microglia-mediated killing of PrP-treated neurones or scrapie-infected neuroblastoma cells were maximal only when microglia were in direct contact with neurones.
NeuroReport 13 (2002) 13.
