title: Llama-derived single domain antibodies to build multivalent, superpotent and broadened neutralizing anti-viral molecules.
abstract: For efficient prevention of viral infections and cross protection, simultaneous targeting of multiple viral epitopes is a powerful strategy. Llama heavy chain antibody fragments (VHH) against the trimeric envelope proteins of Respiratory Syncytial Virus (Fusion protein), Rabies virus (Glycoprotein) and H5N1 Influenza (Hemagglutinin 5) were selected from llama derived immune libraries by phage display. Neutralizing VHH recognizing different epitopes in the receptor binding sites on the spikes with affinities in the low nanomolar range were identified for all the three viruses by viral neutralization assays. By fusion of VHH with variable linker lengths, multimeric constructs were made that improved neutralization potencies up to 4,000-fold for RSV, 1,500-fold for Rabies virus and 75-fold for Influenza H5N1. The potencies of the VHH constructs were similar or better than best performing monoclonal antibodies. The cross protection capacity against different viral strains was also improved for all three viruses, both by multivalent (two or three identical VHH) and biparatopic (two different VHH) constructs. By combining a VHH neutralizing RSV subtype A, but not subtype B with a poorly neutralizing VHH with high affinity for subtype B, a biparatopic construct was made with low nanomolar neutralizing potency against both subtypes. Trivalent anti-H5N1 VHH neutralized both Influenza H5N1 clade1 and 2 in a pseudotype assay and was very potent in neutralizing the NIBRG-14 Influenza H5N1 strain with IC(50) of 9 picomolar. Bivalent and biparatopic constructs against Rabies virus cross neutralized both 10 different Genotype 1 strains and Genotype 5.The results show that multimerization of VHH fragments targeting multiple epitopes on a viral trimeric spike protein is a powerful tool for anti-viral therapy to achieve "best-in-class" and broader neutralization capacity.
authors.affiliation: Department of Biology, Cellular Architecture and Dynamics, University of Utrecht, Utrecht, The Netherlands.
authors.forename: Anna
authors.initials: A
authors.lastname: Hultberg
authors.forename: Nigel J
authors.initials: NJ
authors.lastname: Temperton
authors.forename: Valérie
authors.initials: V
authors.lastname: Rosseels
authors.forename: Mireille
authors.initials: M
authors.lastname: Koenders
authors.forename: Maria
authors.initials: M
authors.lastname: Gonzalez-Pajuelo
authors.forename: Bert
authors.initials: B
authors.lastname: Schepens
authors.forename: Lorena Itatí
authors.initials: LI
authors.lastname: Ibañez
authors.forename: Peter
authors.initials: P
authors.lastname: Vanlandschoot
authors.forename: Joris
authors.initials: J
authors.lastname: Schillemans
authors.forename: Michael
authors.initials: M
authors.lastname: Saunders
authors.forename: Robin A
authors.initials: RA
authors.lastname: Weiss
authors.forename: Xavier
authors.initials: X
authors.lastname: Saelens
authors.forename: José A
authors.initials: JA
authors.lastname: Melero
authors.forename: C Theo
authors.initials: CT
authors.lastname: Verrips
authors.forename: Steven
authors.initials: S
authors.lastname: Van Gucht
authors.forename: Hans J
authors.initials: HJ
authors.lastname: de Haard
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000096:biosynthesis* / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D000998:Antiviral Agents / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D002161:Camelids, New World; D003429:Cross Reactions; D004306:Dose-Response Relationship, Immunologic; D000939:Epitopes / Q000276:immunology; D005838:Genotype; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis* / Q000276:immunology* / Q000302:isolation & purification; D053124:Influenza A Virus, H5N1 Subtype / Q000276:immunology; D018114:Lyssavirus / Q000235:genetics / Q000276:immunology; D012136:Respiratory Syncytial Viruses / Q000276:immunology; D014764:Viral Proteins / Q000276:immunology; D014780:Viruses / Q000276:immunology*
chemical_list: D057134:Antibodies, Neutralizing; D000998:Antiviral Agents; D000939:Epitopes; D007143:Immunoglobulin Heavy Chains; D014764:Viral Proteins