title: Novel camelid antibody fragments targeting recombinant nucleoprotein of Araucaria hantavirus: a prototype for an early diagnosis of Hantavirus Pulmonary Syndrome.
abstract: In addition to conventional antibodies, camelids produce immunoglobulins G composed exclusively of heavy chains in which the antigen binding site is formed only by single domains called VHH. Their particular characteristics make VHHs interesting tools for drug-delivery, passive immunotherapy and high-throughput diagnosis. Hantaviruses are rodent-borne viruses of the Bunyaviridae family. Two clinical forms of the infection are known. Hemorrhagic Fever with Renal Syndrome (HFRS) is present in the Old World, while Hantavirus Pulmonary Syndrome (HPS) is found on the American continent. There is no specific treatment for HPS and its diagnosis is carried out by molecular or serological techniques, using mainly monoclonal antibodies or hantavirus nucleoprotein (N) to detect IgM and IgG in patient serum. This study proposes the use of camelid VHHs to develop alternative methods for diagnosing and confirming HPS. Phage display technology was employed to obtain VHHs. After immunizing one Lama glama against the recombinant N protein (prNΔ₈₅) of a Brazilian hantavirus strain, VHH regions were isolated to construct an immune library. VHHs were displayed fused to the M13KO7 phage coat protein III and the selection steps were performed on immobilized prNΔ₈₅. After selection, eighty clones recognized specifically the N protein. These were sequenced, grouped based mainly on the CDRs, and five clones were analyzed by western blot (WB), surface plasmon resonance (SPR) device, and ELISA. Besides the ability to recognize prNΔ85 by WB, all selected clones showed affinity constants in the nanomolar range. Additionaly, the clone KC329705 is able to detect prNΔ₈₅ in solution, as well as the native viral antigen. Findings support the hypothesis that selected VHHs could be a powerful tool in the development of rapid and accurate HPS diagnostic assays, which are essential to provide supportive care to patients and reduce the high mortality rate associated with hantavirus infections.
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, RO, Brazil.
authors.forename: Soraya S
authors.initials: SS
authors.lastname: Pereira
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, RO, Brazil.
authors.forename: Leandro S
authors.initials: LS
authors.lastname: Moreira-Dill
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, RO, Brazil.
authors.forename: Michelle S S
authors.initials: MS
authors.lastname: Morais
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, RO, Brazil.
authors.forename: Nidiane D R
authors.initials: ND
authors.lastname: Prado
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, RO, Brazil.
authors.forename: Marcos L
authors.initials: ML
authors.lastname: Barros
authors.affiliation: Instituto Carlos Chagas, Fiocruz Paraná, Curitiba, PA, Brazil.
authors.forename: Andrea C
authors.initials: AC
authors.lastname: Koishi
authors.affiliation: Instituto Carlos Chagas, Fiocruz Paraná, Curitiba, PA, Brazil.
authors.forename: Giovanny A C A
authors.initials: GA
authors.lastname: Mazarrotto
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, RO, Brazil.
authors.forename: Giselle M
authors.initials: GM
authors.lastname: Gonçalves
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, RO, Brazil; Departamento de Medicina, Universidade Federal de Rondônia, UNIR, Porto Velho, RO, Brazil.
authors.forename: Juliana P
authors.initials: JP
authors.lastname: Zuliani
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, RO, Brazil; Departamento de Medicina, Universidade Federal de Rondônia, UNIR, Porto Velho, RO, Brazil.
authors.forename: Leonardo A
authors.initials: LA
authors.lastname: Calderon
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, RO, Brazil.
authors.forename: Andreimar M
authors.initials: AM
authors.lastname: Soares
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, RO, Brazil.
authors.forename: Luiz H
authors.initials: LH
authors.lastname: Pereira da Silva
authors.affiliation: Instituto Carlos Chagas, Fiocruz Paraná, Curitiba, PA, Brazil.
authors.forename: Claudia N
authors.initials: CN
authors.lastname: Duarte dos Santos
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, RO, Brazil; Centro de Pesquisa em Medicina Tropical, CEPEM, Porto Velho, RO, Brazil.
authors.forename: Carla F C
authors.initials: CF
authors.lastname: Fernandes
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, RO, Brazil; Departamento de Medicina, Universidade Federal de Rondônia, UNIR, Porto Velho, RO, Brazil.
authors.forename: Rodrigo G
authors.initials: RG
authors.lastname: Stabeli
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000096:biosynthesis; D002162:Camelus / Q000276:immunology*; D042241:Early Diagnosis; D006476:Orthohantavirus / Q000276:immunology*; D018804:Hantavirus Pulmonary Syndrome / Q000175:diagnosis* / Q000276:immunology; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000276:immunology*; D008297:Male; D008969:Molecular Sequence Data; D009698:Nucleoproteins / Q000276:immunology*; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance
chemical_list: D000914:Antibodies, Viral; D007128:Immunoglobulin Fragments; D009698:Nucleoproteins