Glycosylation of Trypanosoma cruzi TcI antigen reveals recognition by chagasic sera

Chagas disease is considered the most important parasitic disease in Latin America. The protozoan agent, Trypanosoma cruzi, comprises six genetic lineages, TcI-TcVI. Genotyping to link lineage(s) to severity of cardiomyopathy and gastrointestinal pathology is impeded by the sequestration and replication of T. cruzi in host tissues. We describe serology specific for TcI, the predominant lineage north of the Amazon, based on expression of recombinant trypomastigote small surface antigen (gTSSA-I) in the eukaryote Leishmania tarentolae, to allow realistic glycosylation and structure of the antigen. Sera from TcI-endemic regions recognised gTSSA-I (74/146; 50.7%), with no cross reaction with common components of gTSSA-II/V/VI recombinant antigen. Antigenicity was abolished by chemical (periodate) oxidation of gTSSA-I glycosylation but retained after heat-denaturation of conformation. Conversely, non-specific recognition of gTSSA-I by non-endemic malaria sera was abolished by heat-denaturation. TcI-specific serology facilitates investigation between lineage and diverse clinical presentations. Glycosylation cannot be ignored in the search for immunogenic antigens

Expression of Trypanosoma brucei gambiense Antigens in Leishmania tarentolae. Potential for Use in Rapid Serodiagnostic Tests (RDTs)

The development of rapid serodiagnostic tests for sleeping sickness and other diseases caused by kinetoplastids relies on the affordable production of parasite-specific recombinant antigens. Here, we describe the production of recombinant antigens from Trypanosoma brucei gambiense (T.b. gambiense) in the related species Leishmania tarentolae (L. tarentolae), and compare their diagnostic sensitivity and specificity to native antigens currently used in diagnostic kits against a panel of human sera. A number of T.b. gambiense protein antigen candidates were chosen for recombinant expression in L. tarentolae based on current diagnostics in field use and recent findings on immunodiagnostic antigens found by proteomic profiling. In particular, the extracellular domains of invariant surface glycoprotein 65 (ISG65), variant surface glycoproteins VSG LiTat 1.3 and VSG LiTat 1.5 were fused with C-terminal histidine tags and expressed as soluble proteins in the medium of cultured, recombinant L. tarentolae. Using affinity chromatography, on average 10 mg/L of recombinant protein was purified from cultures and subsequently tested against a panel of sera from sleeping sickness patients from controls, i.e. persons without sleeping sickness living in HAT endemic countries. The evaluation on sera from 172 T.b. gambiense human African trypanosomiasis (HAT) patients and from 119 controls showed very high diagnostic potential of the two recombinant VSG and the rISG65 fragments with areas under the curve between 0.97 and 0.98 compared to 0.98 and 0.99 with native VSG LiTat 1.3 and VSG LiTat 1.5 (statistically not different). Evaluation on sera from 78 T.b. rhodesiense HAT patients and from 100 controls showed an acceptable diagnostic potential of rISG65 with an area under the curve of 0.83. These results indicate that a combination of these recombinant antigens has the potential to be used in next generation rapid serodiagnostic tests. In addition, the L. tarentolae expression system enables simple, cheap and efficient production of recombinant kinetoplatid proteins for use in diagnostic, vaccine and drug discovery research that does not rely on animal use to generate materials

Expression of Recombinant Receptors in Insect Cells

Membrane bound receptors are key molecules in intracellular communication systems but structure function studies are often hampered by low level of native expression of these proteins. An essential feature of any recombinant system used to over express receptors is the authenticity of the recombinant product. The Baculovirus/insect cell expression system BEVS has been widely used to express many mammalian proteins as it has been shown to carry out many eukaryotic post-translational modifications. This paper will describe its use to over express members of the single transmembrane domain, tyrosine kinase receptor family (PDGF-receptors), and members of the G-protein linked, seven transmembrane domain family (Dopamine receptors). In particular production and activity studies on full length and truncated forms of the PDGF-receptors and comparisons made with mammalian expressed constructs will be discussed. A full pharmacological profile of recombinant dopamine receptors will be presented and the significance of variations between sub-types will be discussed in the context of possible physiological significance

Towards an international system of professional recognition for public health nutritionists: a feasibility study within the European Union

Objective: to test the feasibility of a pan-European professional recognition system for public health nutrition.Design: a multistage consultation process was used to test the feasibility of a model system for public health nutritionist certification. A review of existing national-level systems for professional quality assurance was conducted via literature review and a web-based search, followed by direct inquiries among stakeholders. This information was used to construct a consultation document circulated to key stakeholders summarising the rationale of the proposed system and inviting feedback about the feasibility of the system. Two consultation workshops were also held. The qualitative data gathered through the consultation were collated and thematically analysed.Setting: EuropeSubjects: public health nutrition workforce stakeholders across twenty-nine countries in the European Union.Results: one hundred and forty-five contacts/experts representing twenty-nine countries were contacted with responses received from a total of twenty-eight countries. The system proposed involved a certification system of professional peer review of an applicant's professional practice portfolio, utilising systems supported by information technology for document management and distribution similar to peer-review journals. Through the consultation process it was clear that there was overall agreement with the model proposed although some points of caution and concern were raised, including the need for a robust quality assurance framework that ensures transparency and is open to scrutiny.Conclusions: the consultation process suggested that the added value of such a system goes beyond workforce development to enhancing recognition of the important role of public health nutrition as a professional discipline in the European public health workforc

Potential for Use in Rapid Serodiagnostic Tests (RDTs)

Abstract The development of rapid serodiagnostic tests for sleeping sickness and other diseases caused by kinetoplastids relies on the affordable production of parasite-specific recombinant antigens. Here, we describe the production of recombinant antigens from Trypanosoma brucei gambiense (T.b. gambiense) in the related species Leishmania tarentolae (L. tarentolae), and compare their diagnostic sensitivity and specificity to native antigens currently used in diagnostic kits against a panel of human sera. A number of T.b. gambiense protein antigen candidates were chosen for recombinant expression in L. tarentolae based on current diagnostics in field use and recent findings on immunodiagnostic antigens found by proteomic profiling. In particular, the extracellular domains of invariant surface glycoprotein 65 (ISG65), variant surface glycoproteins VSG LiTat 1.3 and VSG LiTat 1.5 were fused with C-terminal histidine tags and expressed as soluble proteins in the medium of cultured, recombinant L. tarentolae. Using affinity chromatography, on average 10 mg/L of recombinant protein was purified from cultures and subsequently tested against a panel of sera from sleeping sickness patients from controls, i.e. persons without sleeping sickness living in HAT endemic countries. The evaluation on sera from 172 T.b. gambiense human African trypanosomiasis (HAT) patients and from 119 controls showed very high diagnostic potential of the two recombinant VSG and the rISG65 fragments with areas under the curve between 0.97 and 0.98 compared to 0.98 and 0.99 with native VSG LiTat 1.3 and VSG LiTat 1.5 (statistically not different). Evaluation on sera from 78 T.b. rhodesiense HAT patients and from 100 controls showed an acceptable diagnostic potential of rISG65 with an area under the curve of 0.83. These results indicate that a combination of these recombinant antigens has the potential to be used in next generation rapid serodiagnostic tests. In addition, the L. tarentolae expression system enables simple, cheap and efficient production of recombinant kinetoplatid proteins for use in diagnostic, vaccine and drug discovery research that does not rely on animal use to generate materials. Funding: This work was funded via a Biotechnology and Biological Sciences Research Council (BBSRC, UK) Flexible Interchange Programme (FLIP) award (grant number BB/L026279/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Author Summary The development of rapid serodiagnostic tests for African sleeping sickness and other diseases caused by kinetoplastids relies in part on the affordable production of parasite-specific recombinant antigens. The majority of cases of sleeping sickness are caused by the parasite Trypanosoma brucei gambiense (T.b. gambiense) which is transmitted when bitten by an infected tsetse fly. Existing tests rely on the utilisation of extracts from the parasite or use antigens raised in animal models. In this study we have shown that using a cell culture system devised from a parasite similar to T.b. gambiense recombinant antigens can be produced that are as effective in rapid diagnostic tests as the native antigens purified from T.b. gambiense parasites grown in laboratory rodents. We compared the diagnostic sensitivity and specificity of the antigens we produced recombinantly to native antigens currently used in diagnostic kits against a panel of human sera. The evaluation on sera from 172 T.b. gambiense patients and from 119 controls without sleeping sickness showed very high diagnostic potential of two recombinant antigens where the response was not significantly different to that from the native antigens. These results indicate that a combination of these recombinant antigens has the potential to be used in next generation rapid serodiagnostic tests

Isolation and characterization of an insect cell line able to perform complex N-linked glycosylation on recombinant proteins

Site specific characterization of the N-glycan structures in human interferon gamma (IFN-gamma) derived from baculovirus-infected insect cells was performed using a combination of reverse-phase, high-performance liquid chromatography (rHPLC) and matrix assisted laser desorption time of flight (MALDI-TOF) mass spectrometry, IFN-gamma was produced in two cell lines, an Estigmena acrea-derived subclone (Ea4), and Spodoptera frugiperda cells (Sf9), Both IFN-gamma N-glycosylation sites (Asn(25) and Asn(97)) were characterized, Site-specific differences were observed in both the percentage of sites occupied by N-linked glycans and the types of structure associated with each site, The glycosylation capabilities and glycan processing of Sf9 were limited to the generation of chitobiose [GlcNAc(2)], truncated tri-mannose core [Man(3)GlcNAc(2)], or oligomannose structures, The glycosylation abilities of Ea4 cells were more extensive, producing IFN-gamma molecules incorporating oligosaccharides with GlcNAc and Gal residues on the outer arms (hybrid or complex type N-glycans), as well as oligomannose N-glycans, Incorporation of an alpha 1-6 linked fucose residue (<70% in Sf9 and <88% in Ea4) was confined to the Asn(25) glycosylation site. These findings demonstrate the more extensive N-glycosylation capabilities of the E(1) acrea-derived Ea4, compared to current insect cell lines used for the expression of recombinant proteins

Copper(I) bis(diphosphine) complexes as a basis for radiopharmaceuticals for positron emission tomography and targeted radiotherapy

Copper(I) bis(diphosphine) complexes provide an excellent basis for development of short/medium-lived PET (positron emission tomography) imaging and therapy agents containing copper radioisotopes, because of their extreme facility of synthesis and scope for derivatisation and bioconjugate formation