title: Generation of a nanobody targeting the paraflagellar rod protein of trypanosomes.
abstract: Trypanosomes are protozoan parasites that cause diseases in humans and livestock for which no vaccines are available. Disease eradication requires sensitive diagnostic tools and efficient treatment strategies. Immunodiagnostics based on antigen detection are preferable to antibody detection because the latter cannot differentiate between active infection and cure. Classical monoclonal antibodies are inaccessible to cryptic epitopes (based on their size-150 kDa), costly to produce and require cold chain maintenance, a condition that is difficult to achieve in trypanosomiasis endemic regions, which are mostly rural. Nanobodies are recombinant, heat-stable, small-sized (15 kDa), antigen-specific, single-domain, variable fragments derived from heavy chain-only antibodies in camelids. Because of numerous advantages over classical antibodies, we investigated the use of nanobodies for the targeting of trypanosome-specific antigens and diagnostic potential. An alpaca was immunized using lysates of Trypanosoma evansi. Using phage display and bio-panning techniques, a cross-reactive nanobody (Nb392) targeting all trypanosome species and isolates tested was selected. Imunoblotting, immunofluorescence microscopy, immunoprecipitation and mass spectrometry assays were combined to identify the target recognized. Nb392 targets paraflagellar rod protein (PFR1) of T. evansi, T. brucei, T. congolense and T. vivax. Two different RNAi mutants with defective PFR assembly (PFR2RNAi and KIF9BRNAi) were used to confirm its specificity. In conclusion, using a complex protein mixture for alpaca immunization, we generated a highly specific nanobody (Nb392) that targets a conserved trypanosome protein, i.e., PFR1 in the flagella of trypanosomes. Nb392 is an excellent marker for the PFR and can be useful in the diagnosis of trypanosomiasis. In addition, as demonstrated, Nb392 can be a useful research or PFR protein isolation tool.
authors.affiliation: Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium; Structural Biology Research Center, VIB (Flanders Institute for Biotechnology), Pleinlaan 2, B-1050 Brussels, Belgium.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Obishakin
authors.affiliation: Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium; Laboratory of Myeloid Cell Immunology, VIB, (Flanders Institute for Biotechnology) Pleinlaan 2, B-1050 Brussels, Belgium.
authors.forename: Benoit
authors.initials: B
authors.lastname: Stijlemans
authors.affiliation: Trypanosome Cell Biology Unit, Institut Pasteur & CNRS, URA 2581, 25 rue du Docteur Roux, 75015 Paris, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Santi-Rocca
authors.affiliation: Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Department of Biochemistry and Microbiology, Ghent University, K.L Ledeganckstraat 35, 9000, Ghent, Belgium.
authors.forename: Isabel
authors.initials: I
authors.lastname: Vandenberghe
authors.affiliation: Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Department of Biochemistry and Microbiology, Ghent University, K.L Ledeganckstraat 35, 9000, Ghent, Belgium.
authors.forename: Bart
authors.initials: B
authors.lastname: Devreese
authors.affiliation: Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium; Structural Biology Research Center, VIB (Flanders Institute for Biotechnology), Pleinlaan 2, B-1050 Brussels, Belgium.
authors.forename: Serge
authors.initials: S
authors.lastname: Muldermans
authors.affiliation: Trypanosome Cell Biology Unit, Institut Pasteur & CNRS, URA 2581, 25 rue du Docteur Roux, 75015 Paris, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Bastin
authors.affiliation: Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium; Structural Biology Research Center, VIB (Flanders Institute for Biotechnology), Pleinlaan 2, B-1050 Brussels, Belgium.
authors.forename: Stefan
authors.initials: S
authors.lastname: Magez
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000276:immunology*; D002161:Camelids, New World; D005407:Flagella / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000276:immunology*; D034622:RNA Interference; D034741:RNA, Small Interfering; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use; D016415:Sequence Alignment; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use*; D014344:Trypanocidal Agents / Q000276:immunology / Q000627:therapeutic use*; D014345:Trypanosoma / Q000276:immunology; D014352:Trypanosomiasis / Q000517:prevention & control* / Q000628:therapy
chemical_list: D000953:Antigens, Protozoan; D007143:Immunoglobulin Heavy Chains; D015800:Protozoan Proteins; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D014344:Trypanocidal Agents; C045519:paraflagellar rod proteins, Trypanosoma brucei