Isolation of Panels of Llama Single-Domain Antibody Fragments Binding All Nine Neuraminidase Subtypes of Influenza A Virus

• Main Authors: Guus Koch, Herman van der Burg, Willem Bartelink, Juliette C. Blokker, Sylvia B. Pritz-Verschuren, Michiel M. Harmsen
• Format: Article
• Language: English
• Published: MDPI AG 2013-04-01
• Series: Antibodies
• Subjects: recombinant neuraminidase; nanobody; single-domain antibody; phage display; enzymatic activity; antigenic site
• Online Access: http://www.mdpi.com/2073-4468/2/2/168

Avian influenza A virus comprises sixteen hemagglutinin (HA) and nine neuraminidase (NA) subtypes (N1–N9). To isolate llama single-domain antibody fragments (VHHs) against all N subtypes, four llamas were immunized with mixtures of influenza viruses. Selections using influenza virus yielded predominantly VHHs binding to the highly immunogenic HA and nucleoprotein. However, selection using enzymatically active recombinant NA (rNA) protein enabled us to isolate NA binding VHHs. Some isolated VHHs cross-reacted to other N subtypes. These were subsequently used for the capture of N subtypes that could not be produced as recombinant protein (rN6) or were enzymatically inactive (rN1, rN5) in phage display selection, yielding novel VHHs. In total we isolated 188 NA binding VHHs, 64 of which were expressed in yeast. Most VHHs specifically recognize a single N subtype, but some VHHs cross-react with other N-subtypes. At least one VHH bound to all N subtypes, except N4, identifying a conserved antigenic site. Thus, this work (1) describes methods for isolating NA binding VHHs, (2) illustrates the suitability of llama immunization with multiple antigens for retrieving many binders against different antigens and (3) describes 64 novel NA binding VHHs, including a broadly reactive VHH, which can be used in various assays for influenza virus subtyping, detection or serology.