Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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Immune-relevant thrombocytes of common carp undergo parasite-induced nitric oxide-mediated apoptosis
Fink, I.R. ; Ribeiro, C.M.S. ; Forlenza, M. ; Taverne-Thiele, J.J. ; Rombout, J.H.W.M. ; Savelkoul, H.F.J. ; Wiegertjes, G. - \ 2015
Developmental and Comparative Immunology 50 (2015)2. - ISSN 0145-305X - p. 146 - 154.
lymphocyte maturation factor - cyprinus-carpio - expression analysis - human platelets - functional-characterization - monoclonal-antibodies - cell-differentiation - teleost fish - genes - l.
Common carp thrombocytes account for 30–40% of peripheral blood leukocytes and are abundant in the healthy animals' spleen, the thrombopoietic organ. We show that, ex vivo, thrombocytes from healthy carp express a large number of immune-relevant genes, among which several cytokines and Toll-like receptors, clearly pointing at immune functions of carp thrombocytes. Few studies have described the role of fish thrombocytes during infection. Carp are natural host to two different but related protozoan parasites, Trypanoplasma borreli and Trypanosoma carassii, which reside in the blood and tissue fluids. We used the two parasites to undertake controlled studies on the role of fish thrombocytes during these infections. In vivo, but only during infection with T. borreli, thrombocytes were massively depleted from the blood and spleen leading to severe thrombocytopenia. Ex vivo, addition of nitric oxide induced a clear and rapid apoptosis of thrombocytes from healthy carp, supporting a role for nitric oxide-mediated control of immune-relevant thrombocytes during infection with T. borreli. The potential advantage for parasites to selectively deplete the host of thrombocytes via nitric oxide-induced apoptosis is discussed.
A sensitive epitope-blocking ELISA for the detection of Chikungunya virus-specific antibodies in patients
Goh, L.Y.H. ; Kam, Y.W. ; Metz, S.W.H. ; Hobson-Peters, J. ; Prow, N.A. ; McCarthy, S. ; Smith, D.W. ; Pijlman, G.P. ; Ng, L.F.P. ; Hall, R.A. - \ 2015
Journal of Virological Methods 222 (2015). - ISSN 0166-0934 - p. 55 - 61.
west-nile-virus - linked-immunosorbent-assay - valley encephalitis-virus - monoclonal-antibodies - diagnostic-accuracy - universal detection - reunion island - insect cells - ns1 protein - pcr assay
Chikungunya fever (CHIKF) has re-emerged as an arboviral disease that mimics clinical symptoms of other diseases such as dengue, malaria, as well as other alphavirus-related illnesses leading to problems with definitive diagnosis of the infection. Herein we describe the development and evaluation of a sensitive epitope-blocking ELISA (EB-ELISA) capable of specifically detecting anti-chikungunya virus (CHIKV) antibodies in clinical samples. The assay uses a monoclonal antibody (mAb) that binds an epitope on the E2 protein of CHIKV and does not exhibit cross-reactivity to other related alphaviruses. We also demonstrated the use of recombinant CHIK virus-like particles (VLPs) as a safe alternative antigen to infectious virions in the assay. Based on testing of 60 serum samples from patients in the acute or convalescent phase of CHIKV infection, the EB-ELISA provided us with 100% sensitivity, and exhibited 98.5% specificity when Ross River virus (RRV)- or Barmah Forest virus (BFV)-immune serum samples were included. This assay meets the public health demands of a rapid, robust, sensitive and specific, yet simple assay for specifically diagnosing CHIK-infections in humans.
Molecular and functional characterization of the scavenger receptor CD36 in zebrafish and common carp
Fink, I.R. ; Benard, E.L. ; Hermsen, G.J. ; Meijer, A.H. ; Forlenza, M. ; Wiegertjes, G. - \ 2015
Molecular Immunology 63 (2015)2. - ISSN 0161-5890 - p. 381 - 393.
toll-like receptors - salmon salmo-salar - cyprinus-carpio - density-lipoprotein - neutrophilic granulocytes - monoclonal-antibodies - accessory molecules - innate immunity - gene family - in-vivo
CD36 is a scavenger receptor which has been studied closely in mammals where it is expressed by many different cell types and plays a role in highly diverse processes, both homeostatic and pathologic. It is among other things important in the innate immune system, in angiogenesis, and in clearance of apoptotic cells, and it is also involved in lipid metabolism and atherosclerosis. Recently, in the cephalochordate amphioxus a primitive CD36 family member was described, which was present before the divergence of CD36 from other scavenger receptor B family members, SCARB1 and SCARB2. Not much is known on the Cd36 molecule in teleost fish. We therefore studied Cd36 in both zebrafish and common carp, two closely related cyprinid fish species. Whereas a single cd36 gene is present in zebrafish, carp has two cd36 genes, and all show conserved synteny compared to mammalian CD36. The gene expression of carp cd36 is high in brain, ovary and testis but absent in immune organs. Although in mammals CD36 expression in erythrocytes, monocytes and macrophages is high, gene expression studies in leukocyte subtypes of adult carp and zebrafish larvae, including thrombocytes and macrophages provided no indication for any substantial expression of cd36 in immune cell types. Surprisingly, analysis of the cd36 promoter region does show the presence of several binding sites for transcription factors known to regulate immune responses. Overexpression of carp cd36 locates the receptor on the cell surface of mammalian cell lines consistent with the predicted topology of cyprinid Cd36 with a large extracellular domain, two transmembrane domains, and short cytoplasmic tails at both ends. Gene expression of cd36 is down-regulated during infection of zebrafish with Mycobacterium marinum, whereas knockdown of cd36 in zebrafish larvae led to higher bacterial burden upon such infection. We discuss the putative role for Cd36 in immune responses of fish in the context of other members of the scavenger receptor class B family.
Detection, identification and differentiation of Pectobacterium and Dickeya species causing potato blackleg and tuber soft rot: a review
Czajkowski, R.L. ; Pérombelon, M.C.M. ; Jafra, S. ; Lojkowska, E. ; Potrykus, M. ; Wolf, J.M. van der; Sledz, W. - \ 2015
Annals of Applied Biology 166 (2015)1. - ISSN 0003-4746 - p. 18 - 38.
carotovora subsp atroseptica - fragment-length-polymorphism - plant-pathogenic bacteria - erwinia-chrysanthemi strains - polymerase-chain-reaction - 16s ribosomal-rna - monoclonal-antibodies - genetic diversity - ssp-carotovorum - peel extracts
The soft rot Enterobacteriaceae (SRE) Pectobacterium and Dickeya species (formerly classified as pectinolytic Erwinia spp.) cause important diseases on potato and other arable and horticultural crops. They may affect the growing potato plant causing blackleg and are responsible for tuber soft rot in storage thereby reducing yield and quality. Efficient and cost-effective detection and identification methods are essential to investigate the ecology and pathogenesis of the SRE as well as in seed certification programmes. The aim of this review was to collect all existing information on methods available for SRE detection. The review reports on the sampling and preparation of plant material for testing and on over thirty methods to detect, identify and differentiate the soft rot and blackleg causing bacteria to species and subspecies level. These include methods based on biochemical characters, serology, molecular techniques which rely on DNA sequence amplification as well as several less-investigated ones.
Salmonid alphavirus glycoprotein E2 requires low temperature and E1 for virion formation and induction of protective immunity
Hikke, M.C. ; Braaen, S. ; Villoing, S. ; Hodneland, K. ; Geertsema, C. ; Verhagen, L. ; Frost, P. ; Vlak, J.M. ; Rimstad, E. ; Pijlman, G.P. - \ 2014
Vaccine 32 (2014)47. - ISSN 0264-410X - p. 6206 - 6212.
pancreas disease virus - atlantic salmon - monoclonal-antibodies - insect cells - salar l - replicon - line
Salmonid alphavirus (SAV; also known as Salmon pancreas disease virus; family Togaviridae) causes pancreas disease and sleeping disease in Atlantic salmon and rainbow trout, respectively, and poses a major burden to the aquaculture industry. SAV infection in vivo is temperature-restricted and progeny virus is only produced at low temperatures (10–15 °C). Using engineered SAV replicons we show that viral RNA replication is not temperature-restricted suggesting that the viral structural proteins determine low-temperature dependency. The processing/trafficking of SAV glycoproteins E1 and E2 as a function of temperature was investigated via baculovirus vectors in Sf9 insect cells and by transfection of CHSE-214 fish cells with DNA constructs expressing E1 and E2. We identified SAV E2 as the temperature determinant by demonstrating that membrane trafficking and surface expression of E2 occurs only at low temperature and only in the presence of E1. Finally, a vaccination-challenge model in Atlantic salmon demonstrates the biological significance of our findings and shows that SAV replicon DNA vaccines encoding E2 elicit protective immunity only when E1 is co-expressed. This is the first study that identifies E2 as the critical determinant of SAV low-temperature dependent virion formation and defines the prerequisites for induction of a potent immune response in Atlantic salmon by DNA vaccination.
Monomeric IgA can be produced in planta as efficient as IgG, yet receives different N-glycans
Westerhof, L.B. ; Wilbers, R.H.P. ; Raaij, D.R. van; Nguyen, D. ; Goverse, A. ; Henquet, M.G.L. ; Hokke, C.H. ; Bosch, D. ; Bakker, J. ; Schots, A. - \ 2014
Plant Biotechnology Journal 12 (2014)9. - ISSN 1467-7644 - p. 1333 - 1342.
monoclonal-antibodies - subcellular-localization - recombinant antibodies - hybrid immunoglobulin - terminal propeptide - insect cells - heavy-chain - glycosylation - protein - infliximab
The unique features of IgA, such as the ability to recruit neutrophils and suppress the inflammatory responses mediated by IgG and IgE, make it a promising antibody isotype for several therapeutic applications. However, in contrast to IgG, reports on plant production of IgA are scarce. We produced IgA1¿ and IgG1¿ versions of three therapeutic antibodies directed against pro-inflammatory cytokines in Nicotiana benthamiana: Infliximab and Adalimumab, directed against TNF-a, and Ustekinumab, directed against the interleukin-12p40 subunit. We evaluated antibody yield, quality and N-glycosylation. All six antibodies had comparable levels of expression between 3.5 and 9% of total soluble protein content and were shown to have neutralizing activity in a cell-based assay. However, IgA1¿-based Adalimumab and Ustekinumab were poorly secreted compared to their IgG counterparts. Infliximab was poorly secreted regardless of isotype backbone. This corresponded with the observation that both IgA1¿- and IgG1¿-based Infliximab were enriched in oligomannose-type N-glycan structures. For IgG1¿-based Ustekinumab and Adalimumab, the major N-glycan type was the typical plant complex N-glycan, biantennary with terminal N-acetylglucosamine, ß1,2-xylose and core a1,3-fucose. In contrast, the major N-glycan on the IgA-based antibodies was xylosylated, but lacked core a1,3-fucose and one terminal N-acetylglucosamine. This type of N-glycan occurs usually in marginal percentages in plants and was never shown to be the main fraction of a plant-produced recombinant protein. Our data demonstrate that the antibody isotype may have a profound influence on the type of N-glycan an antibody receives.
Characterization and expression analysis of an interferon-y2 induced chemokine receptor CXCR3 in common carp (Cyprinus carpio L.)
Chadzinska, M.K. ; Golbach, L.A. ; Pijanowski, L. ; Scheer, M.H. ; Verburg-van Kemenade, B.M.L. - \ 2014
Developmental and Comparative Immunology 47 (2014)1. - ISSN 0145-305X - p. 68 - 76.
central-nervous-system - rheumatoid-arthritis - t-cells - monoclonal-antibodies - molecular evolution - endothelial-cells - phagocytic-cells - teleost fish - b-cells - inflammation
Chemokine and chemokine receptor signalling pairs play a crucial role in regulation of cell migration, morphogenesis, and cell activation. Expressed in mammals on activated T and NK cells, chemokine receptor CXCR3 binds interferon-¿ inducible chemokines CXCL9–11 and CCL21. Here we sequenced the carp CXCR3 chemokine receptor and showed its relationship to CXCR3a receptors found in other teleosts. We found high expression of the CXCR3 gene in most of the organs and tissues of the immune system and in immune-related tissues such as gills and gut, corroborating a predominantly immune-related function. The very high expression in gill and gut moreover indicates a role for CXCR3 in cell recruitment during infection. High in vivo expression of CXCR3 at later stages of inflammation, as well as its in vitro sensitivity to IFN-¿2 stimulation indicate that in carp, CXCR3 is involved in macrophage-mediated responses. Moreover, as expression of the CXCR3 and CXCb genes coincides in the focus of inflammation and as both the CXCb chemokines and the CXCR3 receptor are significantly up-regulated upon IFN-¿ stimulation it is hypothesized that CXCb chemokines may be putative ligands for CXCR3.
Immune escape mutants of highly pathogenic avian influenza H5N1 selected using polyclonal sera: Identification of key amino acids in the HA protein
Sitaras, I. ; Kalthof, D. ; Beer, M. ; Peeters, B.P.H. ; Jong, M.C.M. de - \ 2014
PLoS ONE 9 (2014)2. - ISSN 1932-6203 - 13 p.
virus hemagglutinin - antigenic drift - a viruses - monoclonal-antibodies - evolution - poultry - egypt - vaccination - molecule - neutralization
Evolution of Avian Influenza (AI) viruses – especially of the Highly Pathogenic Avian Influenza (HPAI) H5N1 subtype – is a major issue for the poultry industry. HPAI H5N1 epidemics are associated with huge economic losses and are sometimes connected to human morbidity and mortality. Vaccination (either as a preventive measure or as a means to control outbreaks) is an approach that splits the scientific community, due to the risk of it being a potential driving force in HPAI evolution through the selection of mutants able to escape vaccination-induced immunity. It is therefore essential to study how mutations are selected due to immune pressure. To this effect, we performed an in vitro selection of mutants from HPAI A/turkey/Turkey/1/05 (H5N1), using immune pressure from homologous polyclonal sera. After 42 rounds of selection, we identified 5 amino acid substitutions in the Haemagglutinin (HA) protein, most of which were located in areas of antigenic importance and suspected to be prone to selection pressure. We report that most of the mutations took place early in the selection process. Finally, our antigenic cartography studies showed that the antigenic distance between the selected isolates and their parent strain increased with passage number.
Identification and functional characterization of nonmammalian Toll-like receptor 20
Pietretti, D. ; Scheer, M.H. ; Fink, I.R. ; Taverne, N. ; Savelkoul, H.F.J. ; Spaink, H.P. ; Forlenza, M. ; Wiegertjes, G. - \ 2014
Immunogenetics 66 (2014)2. - ISSN 0093-7711 - p. 123 - 141.
carp cyprinus-carpio - leucine-rich repeats - common carp - ictalurus-punctatus - monoclonal-antibodies - pathogen recognition - accessory molecules - expression analysis - sequence-analysis - channel catfish
Like other vertebrate Toll-like receptors (TLRs), the TLRs of teleost fish can be subdivided into six major families, each of which recognize a general class of molecular patterns. However, there also are a number of Tlrs with unknown function, the presence of which seems unique to the bony fish, among which is Tlr20. We identified full-length complementary DNA (cDNA) sequences for tlr20 of zebrafish and common carp, two closely related fish species. Zebrafish have six copies of tlr20, whereas carp express only a single copy. Both zebrafish Tlr20 (at least Tlr20a–d) and carp Tlr20 have 26 leucine-rich repeats (LRRs). Three-dimensional modeling indicates a best fit to the crystal structure of TLR8. Phylogenetic analyses place Tlr20 in the TLR11 family closest to Tlr11 and Tlr12, which sense ligands from protozoan parasites in the mouse. Conservation of genes on zebrafish chromosome 9, which carries tlr20, with genes on mouse chromosome 14, which carries tlr11, indicates Tlr11 could be a possible ortholog of Tlr20. Confocal microscopy suggests a subcellular localization of Tlr20 at the endoplasmatic reticulum. Although in vitro reporter assays could not identify a ligand unique to Tlr20, in vivo infection experiments indicate a role for Tlr20 in the immune response of carp to protozoan parasites (Trypanoplasma borreli). Carp tlr20 is mainly expressed in peripheral blood leukocytes (PBL) with B lymphocytes, in particular, expressing relatively high levels of Tlr20. In vitro stimulation of PBL with T. borreli induces an upregulation of tlr20, supportive of a role for Tlr20 in the immune response to protozoan parasites.
Preliminary mapping of non-conserved epitopes on envelope glycoprotein E2 of bovine viral diarrhea virus type 1 and 2
Jelsma, H. ; Loeffen, W.L.A. ; Beuningen, A.R. van; Rijn, P.A. van - \ 2013
Veterinary Microbiology 166 (2013)1-2. - ISSN 0378-1135 - p. 195 - 199.
swine-fever virus - random peptide library - hog-cholera virus - b-cell epitope - monoclonal-antibodies - strain brescia - vaccines - protein - identification - neutralization
Bovine viral diarrhea virus (BVDV) belongs together with Classical swine fever virus (CSFV) and Border disease virus (BDV) to the genus Pestivirus in the Flaviviridae family. BVDV has been subdivided into two different species, BVDV1 and BVDV2 based on phylogenetic analysis. Subsequent characterization of both strains revealed major antigenic differences. Because the envelope glycoprotein E2 is the most immunodominant protein for all pestiviruses, the present study focused on epitope mapping by constructing chimeric BVDV type 1 and 2 E2 genes in expression plasmids. These plasmids with chimeric E2-genes were transfected in SK6 cells and transient expression was studied by immunostaining with a panel of MAbs specific for E2 of BVDV1 or BVDV2, resulting in the localization of type-specific antigenic domains at similar regions. These results indicate that E2 glycoproteins of both BVDV types exhibit a comparable antigenic structure, but with type specific epitopes. In addition, the antigenic resemblance with envelope glycoprotein E2 of Classical swine fever virus is discussed.
Efficacy of chimeric Pestivirus vaccine candidates against Classical Swine Fever: protection and DIVA characteristics
Eble, P.L. ; Geurts, Y. ; Quak, J. ; Moonen-Leusen, H.W.M. ; Blome, S. ; Hofmann, M.A. ; Koenen, F. ; Beer, M. ; Loeffen, W.L.A. - \ 2013
Veterinary Microbiology 162 (2013)2-4. - ISSN 0378-1135 - p. 437 - 446.
subunit marker vaccine - hog-cholera virus - monoclonal-antibodies - pigs - transmission - differentiation - strains - infection - virulent - induce
Currently no live DIVA (Differentiating Infected from Vaccinated Animals) vaccines against classical swine fever (CSF) are available. The aim of this study was to investigate whether chimeric pestivirus vaccine candidates (CP7_E2alf, Flc11 and Flc9) are able to protect pigs against clinical signs, and to reduce virus shedding and virus transmission, after a challenge with CSF virus (CSFV), 7 or 14 days after a single intramuscular vaccination. In these vaccine candidates, either the E2 or the Erns encoding genome region of a bovine viral diarrhoea virus strain were combined with a cDNA copy of CSFV or vice versa. Furthermore, currently available serological DIVA tests were evaluated. The vaccine candidates were compared to the C-strain. All vaccine candidates protected against clinical signs. No transmission to contact pigs was detected in the groups vaccinated with C-strain, CP7_E2alf and Flc11. Limited transmission occurred in the groups vaccinated with Flc9. All vaccine candidates would be suitable to stop on-going transmission of CSFV. For Flc11, no reliable differentiation was possible with the current Erns-based DIVA test. For CP7_E2alf, the distribution of the inhibition percentages was such that up to 5% false positive results may be obtained in a large vaccinated population. For Flc9 vaccinated pigs, the E2 ELISA performed very well, with an expected 0.04% false positive results in a large vaccinated population. Both CP7_E2alf and Flc9 are promising candidates to be used as live attenuated marker vaccines against CSF, with protection the best feature of CP7_E2alf, and the DIVA principle the best feature of Flc9.
In vitro neutralization of viral hemorrhagic septicemia virus by plasma from immunized zebrafish
Chinchilla, B. ; Gomez-Casado, E. ; Encinas, P. ; Falco Gracia, J.A. ; Estepa, A. ; Coll, J. - \ 2013
Zebrafish 10 (2013)1. - ISSN 1545-8547 - p. 43 - 51.
hematopoietic necrosis virus - rainbow-trout - egtved virus - danio-rerio - spring viremia - monoclonal-antibodies - carp virus - vhs virus - g-protein - infection
We studied humoral long-term adaptive viral neutralization responses in zebrafish (Danio rerio), an increasingly useful vertebrate model for viral diseases actually limited by the absence of standardized anti-zebrafish immunoglobulin M (IgM) antibodies. We established an alternative method, similar to those used in other fish, to achieve a first estimation of zebrafish anti-viral antibody-like responses. We used the viral hemorrhagic septicemia virus (VHSV) model because, although protection after this non-natural infection was demonstrated in cold-acclimatized zebrafish, little is known about their induced anti-VHSV antibody-like responses. Therefore, we first optimized a micro-neutralization method based on immunostaining VHSV-infected fish cell monolayers to detect zebrafish neutralizing activity in plasma samples in one day. We then used the method to measure the specific anti-VHSV neutralization in plasma obtained from individual zebrafish under various VHSV challenges or immunization protocols. The neutralizing activity was inhibited by protein A-sepharose and rabbit anti-zebrafish IgM antibodies, suggesting the implication of IgM zebrafish antibodies in such responses. To our knowledge, this is the first report to demonstrate detectable and significant VHSV neutralization titers in zebrafish surviving VHSV infections. This micro-method might be useful, not only for the follow-up of infection/vaccine development in the zebrafish/VHSV model in particular, but also for similar work involving other in vitro neutralizable zebrafish pathogens. This technique might also further the development of alternative ELISA assay methods to measure specific immunoglobulins in zebrafish.
Model-based rational methodology for protein purification process synthesis
Nfor, B. ; Ahamed, T. ; Dedem, G. ; Verhaert, P. ; Wielen, L. ; Eppink, M.H.M. ; Sandt, E. van de; Ottens, M. - \ 2013
Chemical Engineering Science 89 (2013). - ISSN 0009-2509 - p. 185 - 195.
pore-size distributions - in-process synthesis - chromatographic-separations - monoclonal-antibodies - ion-exchange - hydrophobic interaction - integrated bioprocess - liquid-chromatography - expert-systems - design
A model-basedrationalproteinpurificationprocesssynthesismethodologythataddressesthe challengeofselectingthemostoptimalprocessschemefromseveralpossiblealternativesispresented in thisstudy.Themainrationalebehindthemethodologyistokeepthenumberofpurificationunitsin the finalprocesstotheminimumthroughasystematiccycleofflowsheetsynthesis,optimization, evaluationandtherationaleliminationoftheleastfeasibleprocessoptionsateachpurificationstep, takingintoaccountthespecificneedsofthepurificationstep.Processevaluationisbasedontechno- economicperformanceobtainedbymodel-basedoptimizationoftheintegratedprocessesusing validatedcolumnmodels.Themethodologywasillustratedbysynthesizingaprocessforthe purificationofmonoclonalantibodyfromcrudehybridomacellculturesupernatantusingfournon- affinitychromatographicmethods(AEX,CEX,SECandHIC).Theresultsshowedthatfouroutofthirteen evaluatedprocessoptionssatisfiedallpre-definedproductspecifications,withoverallyields Z90%. In order ofincreasingproductcost,thesewereCEX–AEXoAEX–CEXoAEX–HICoHIC–AEX,takinginto accounttheneedforinter-stageconditioning.Themainadvantagesofthemethodologyincludesavings in experimentation,computationaltimeandeffort;unbiasedevaluationofpurificationschemesby using theiridealoperatingconditions;andtheconsiderationofthespecificneedsofeachpurification step duringprocessevaluation.Finally,themethodologyisgenericforcell-derivedproteins,irrespec- tive ofthehostorganismorapplication.
Truncated VP28 as oral vaccine candidate against WSSV infection in shrimp: an uptake and processing study in the midgut of Penaeus monodon
Kulkarni, V. ; Rombout, J.H.W.M. ; Singh, I.S.B. ; Sudheer, N.S. ; Vlak, J.M. ; Caipang, C.M.A. ; Brinchmann, M. ; Kiron, V. - \ 2013
Fish and Shellfish Immunology 34 (2013)1. - ISSN 1050-4648 - p. 159 - 166.
spot syndrome virus - prophenoloxidase activating system - cyprinus-carpio l - monoclonal-antibodies - hemocytes - arthropods - exocytosis - protection - immersion - decapoda
Several oral vaccination studies have been undertaken to evoke a better protection against white spot syndrome virus (WSSV), a major shrimp pathogen. Formalin-inactivated virus and WSSV envelope protein VP28 were suggested as candidate vaccine components, but their uptake mechanism upon oral delivery was not elucidated. In this study the fate of these components and of live WSSV, orally intubated to black tiger shrimp (Penaeus monodon) was investigated by immunohistochemistry, employing antibodies specific for VP28 and haemocytes. The midgut has been identified as the most prominent site of WSSV uptake and processing. The truncated recombinant VP28 (rec-VP28), formalin-inactivated virus (IVP) and live WSSV follow an identical uptake route suggested as receptor-mediated endocytosis that starts with adherence of luminal antigens at the apical layers of gut epithelium. Processing of internalized antigens is performed in endo-lysosomal compartments leading to formation of supra-nuclear vacuoles. However, the majority of WSSV-antigens escape these compartments and are transported to the inter-cellular space via transcytosis. Accumulation of the transcytosed antigens in the connective tissue initiates aggregation and degranulation of haemocytes. Finally the antigens exiting the midgut seem to reach the haemolymph. The nearly identical uptake pattern of the different WSSV-antigens suggests that receptors on the apical membrane of shrimp enterocytes recognize rec-VP28 efficiently. Hence the truncated VP28 can be considered suitable for oral vaccination, when the digestion in the foregut can be bypassed.
Model-based high-throughout process development for chromatographic whey proteins separation
Nfor, B. ; Ripic, J. ; Padt, A. van der; Jacobs, M. ; Ottens, M. - \ 2012
Biotechnology Journal 7 (2012)10. - ISSN 1860-6768 - p. 1221 - 1232.
ion-exchange chromatography - mixed matrix membrane - raw whole milk - monoclonal-antibodies - beta-lactoglobulin - alpha-lactalbumin - optimization - fractionation - purification - design
In this study, an integrated approach involving the combined use of high-throughput screening (HTS) and column modeling during process development was applied to an industrial case involving the evaluation of four anion-exchange chromatography (AEX) resins and four hydrophobic interaction chromatography (HIC) resins for the separation of whey proteins having close pIs. From the HTS data, one resin of each type was selected (Capto Q and Octyl Sepharose 4 FF). Next, batch uptake experiments were performed to determine the adsorption isotherms of the major whey proteins on the selected resins, followed by isotherm parameters regression. Using the obtained isotherm parameters, the candidate chromatographic operations were modeled and experimentally validated. Finally, these were model-optimized and evaluated based on their optimized performances. In this example, Capto Q performed much better than Octyl Sepharose 4 FF in terms of column capacity, loading, throughput and productivity; hence, Capto Q was selected as the resin of choice for whey proteins separation. This operation was further scaled up from the lab scale (1 mL) to a preparative scale (35 L), with reproducible column elution profile. By this approach, a much wider space of operating variables could be investigated, thereby increasing the chances of finding the ideal operating conditions while keeping experimentation to the minimum.
All major prion types recognised by a multiplex immunofluorometric assay for disease screening and confirmation in sheep
Tang, Y. ; gielbert, A. ; Jacobs, J.G. ; Baron, T. ; Andreoletti, O. ; Langeveld, J.P.M. ; Sauer, M.J. - \ 2012
Journal of Immunological Methods 380 (2012)1-2. - ISSN 0022-1759 - p. 30 - 39.
bovine spongiform encephalopathy - atypical scrapie - natural scrapie - monoclonal-antibodies - molecular analysis - protein - bse - ch1641 - strains - isolate
Prion diseases or transmissible spongiform encephalopathies (TSEs) in small ruminants are presented in many forms: classical scrapie, Nor98/atypical scrapie, CH1641 scrapie and bovine spongiform encephalopathy (BSE). We previously described a multiplex immunofluorometric assay (mIFMA), based on a bead array flow cytometry technology, which provided, in a single assay, discrimination between BSE (in cattle and sheep) and classical scrapie (Tang et al., 2010). In this study, we extended the mlFMA to differentiate classical scrapie, atypical scrapie, BSE (experimentally infected sheep and naturally infected cattle) and CH1641 (both experimental and natural CH1641-like infections in sheep). Three capture antibodies were used, two distinct PrP N-terminus specific antibodies 12B2 and 9A2, and a PrP core specific antibody 94B4. All three antibodies were shown to bind classical scrapie PrPres strongly, whereas in Nor98/atypical scrapie PrPres only 12B2 and 9A2 binding was observed. PrPres binding of 12B2 was low for both BSE and CH1641, as expected. Furthermore, analysis of serially diluted samples indicated that the assay provided a similar level of sensitivity for atypical scrapie as that found using a well established commercial test. Unexpectedly, 9A2 binding to CH1641 PrPres was reduced by 2.1 fold both for experimental CH1641 and CH1641-like scrapie when compared with BSE, suggesting that major cleavage of the N-terminus occurs further towards the C-terminus in CH1641 than in BSE. The ratios of 12B2/94B4 and 9A2/94B4 were similar between experimental CH1641 and CH1641-like cases, although two CH1641-like subjects displayed slightly elevated ratios of both 12B2/94B4 and 9A2/94B4. To verify this finding for PrPres, mass spectrometry based quantification was used to determine the absolute abundance of the peptides associated with all three antibody binding regions. There was a 2.2 fold reduction of peptides containing the 9A2 epitope for experimental CH1641 PrPres in comparison to BSE PrPres. Observation of reduced PrPres may serve as a new marker for CH1641. This mIFMA may thus provide the basis for simplified TSE diagnosis with capability for simultaneous screening and differential diagnosis
Effect of thiomersal on dissociation of intact (146S) foot-and-mouth disease virions into 12S particles as assessed by novel ELISAs specific for either 146S or 12S particles
Harmsen, M.M. ; Fijten, H.P.D. ; Westra, D.F. ; Coco-Martin, J.M. - \ 2011
Vaccine 29 (2011)15. - ISSN 0264-410X - p. 2682 - 2690.
domain antibody fragments - whole virus-particles - monoclonal-antibodies - thermal-stability - passive-immunization - international bank - protein subunit - sandwich elisa - field strains - vaccine
Intact (146S) foot-and-mouth disease virions (FMDVs) can dissociate into specific (12S) viral capsid degradation products. Using two single-domain antibody fragments that bind specifically to either 146S or 12S particles we developed two ELISAs for the quantification of these particles in FMDV antigen preparations used for vaccine manufacturing. Only O serotype strains are detected in the 146S specific ELISA whereas strains of most serotypes are detected in the 12S specific ELISA. However, the 146S concentration of A and Asia 1 serotype strains could be measured indirectly using the 12S specific ELISA by prior conversion of 146S into 12S particles by heat treatment. This allowed us to demonstrate that addition of the preservative thiomersal to FMDV antigens stimulates the dissociation into 12S particles of O, A and Asia 1 serotype strains upon prolonged storage at 4 °C. FMDV dissociation is known to result in a strongly reduced immunogenicity, which was experimentally confirmed here. Therefore, we recommend to omit thiomersal from FMD vaccines to increase its shelf life
Four independent molecular prion protein parameters for discriminating new cases of C, L, and H BSE in cattle.
Langeveld, J.P.M. ; Erkens, J.H.F. ; Rammel, I. ; Jacobs, J.G. ; Davidse, A. ; Zijderveld, F.G. van; Bossers, A. ; Schildorfer, H. - \ 2011
Journal of Clinical Microbiology 49 (2011)8. - ISSN 0095-1137 - p. 3026 - 3028.
monoclonal-antibodies - great-britain - atypical bse - identification - similarities - diseases - prpsc - rich
In anticipation of the emergence of more variants of bovine spongiform encephalopathy (BSE), a semiquantitative display of the following four independent molecular diagnostic prion parameters was designed: N terminus, proteinase K (PK) resistance, glycoprofile, and mixed population. One H BSE case, three L BSE cases, six C BSE cases, and one unusual classical BSE (C BSE) case are reported.
Proteinase K resistant material in ARR/VRQ sheep brain affected with classical scrapie is composed mainly of VRQ prion protein
Jacobs, J.G. ; Bossers, A. ; Rezaei, H. ; Keulen, L. Van; McCutcheon, S. ; Sklaviadis, T. ; Lantier, I. ; Berthon, P. ; Lantier, F. ; Zijderveld, F.G. van; Langeveld, J.P.M. - \ 2011
Journal of Virology 85 (2011)23. - ISSN 0022-538X - p. 12537 - 12546.
bovine spongiform encephalopathy - creutzfeldt-jakob-disease - susceptibility-linked polymorphisms - straussler-scheinker disease - in-vitro conversion - allelic variants - amino-acid - monoclonal-antibodies - natural scrapie - prp
Classical scrapie is a prion disease in sheep and goats. In sheep, susceptibility to disease is genetically influenced by single amino acid substitutions. Genetic breeding programs aimed at enrichment of arginine-171 (171R) prion protein (PrP), the so called ARR allele, in the sheep population have demonstrated to be effective in reducing the occurrence of classical scrapie in the field. Understanding the molecular basis for this reduced prevalence would serve the assessment of ARR-adaptation. The prion formation mechanism and conversion of PrP from the normal form (PrPC) to scrapie associated form (PrPSc) could play a key-role in this process. Therefore, we investigated whether the ARR allele substantially contributes to scrapie prion formation in naturally infected heterozygous 171Q/R animals. Two methods were applied to brain tissue of 171Q/R heterozygous sheep with natural scrapie to determine the relative amount of the 171R PrP fraction in PrPres, the proteinase K-resistant PrPSc core. An antibody test differentiating between 171Q and 171R PrP fragments showed that PrPres mostly was composed of the 171Q allelotype. Furthermore using a novel tool for prion research, endoproteinase Lys-C digested PrPres yielded substantial amounts of non-glycosylated and mono-glycosylated 114-188 PrP fragment. Following two-dimensional gel electrophoresis only marginal amounts (
A novel soluble immune-type receptor (SITR) in teleost fish: carp SITR is involved in the nitric oxide-mediated response to a protozoan parasite
Ribeiro, C.M.S. ; Raes, G. ; Ghassabeh, G.H. ; Schijns, V.E.J.C. ; Pontes, M.J.S.L. ; Savelkoul, H.F.J. ; Wiegertjes, G.F. - \ 2011
PLoS ONE 6 (2011)1. - ISSN 1932-6203 - 20 p.
immunoglobulin-like transcripts - ig-like domains - cyprinus-carpio - trypanoplasma-borreli - gene-cluster - monoclonal-antibodies - natural cytotoxicity - evolution - molecules - recognition
Background- The innate immune system relies upon a wide range of germ-line encoded receptors including a large number of immunoglobulin superfamily (IgSF) receptors. Different Ig-like immune receptor families have been reported in mammals, birds, amphibians and fish. Most innate immune receptors of the IgSF are type I transmembrane proteins containing one or more extracellular Ig-like domains and their regulation of effector functions is mediated intracellularly by distinct stimulatory or inhibitory pathways. Methodology/Principal Findings - Carp SITR was found in a substracted cDNA repertoire from carp macrophages, enriched for genes up-regulated in response to the protozoan parasite Trypanoplasma borreli. Carp SITR is a type I protein with two extracellular Ig domains in a unique organisation of a N-proximal V/C2 (or I-) type and a C-proximal V-type Ig domain, devoid of a transmembrane domain or any intracytoplasmic signalling motif. The carp SITR C-proximal V-type Ig domain, in particular, has a close sequence similarity and conserved structural characteristics to the mammalian CD300 molecules. By generating an anti-SITR antibody we could show that SITR protein expression was restricted to cells of the myeloid lineage. Carp SITR is abundantly expressed in macrophages and is secreted upon in vitro stimulation with the protozoan parasite T. borreli. Secretion of SITR protein during in vivo T. borreli infection suggests a role for this IgSF receptor in the host response to this protozoan parasite. Overexpression of carp SITR in mouse macrophages and knock-down of SITR protein expression in carp macrophages, using morpholino antisense technology, provided evidence for the involvement of carp SITR in the parasite-induced NO production. Conclusion/Significance - We report the structural and functional characterization of a novel soluble immune-type receptor (SITR) in a teleost fish and propose a role for carp SITR in the NO-mediated response to a protozoan parasite.
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