Formatting single-domain antibodies for the detection and inhibition of influenza virus infection

PAVAN MF; SPERAT W; GOMEZ JM; MATTION N; IBAÑEZ LI

Mar del Plata

Congreso; Reunión Conjunta SAIC SAF SAI; 2018

Influenza is an enveloped RNA virus that causes acute respiratory infections in both humans and animals and is responsible for epidemics all over the world. Even though, vaccines are the most effective way to prevent influenza virus infection, antivirals are also useful tools to control the disease. The emergence of multidrug-resistant viruses has prompted the development of novel antiviral drugs. Single domain antibodies (sdAbs) are small molecules with high stability, solubility and affinity, that can be produced at low cost. For this reason, in this work we propose developing sdAbs capable of binding influenza?s hemagglutinin protein (HA), and therefore have the potential to act as antiviral molecules.In order to develop anti-HA sdAbs, llamas were immunized with recombinant HA, and subsequently a phage library containing genes encoding sdAbs was generated. Molecules obtained after panning the library were modified to obtain bivalent derivates, which bind with higher affinity to the HA protein. sdAbs were expressed in Escherichia coli strain BL21 and extracted from the periplasm. Antibodies were subsequently purified by immobilized metal chelate chromatography (IMAC) and protein expression and purification were checked by Western Blot and SDS-PAGE. Binding capacity of mono- and bivalent sdAbs was assessed by ELISA, in plates coated with viral particles of different influenza serotypes. The tested sdAbs displayed a wide heterotypic recognition pattern, since they were capable of recognizing H1, H3, H5, H7 and H9 influenza subtypes.We have also generated chromobodies by fusing mCherry and GFP coding sequences to those of the selected sdAbs, which were expressed in HEK-293 cells at high levels as assessed by confocal microscopy.The anti-HA modified sdAbs produced in this work are capable of binding strongly to different types of influenza viral strains and therefore will be used to study their ability to inhibit viral infection in vitro and in vivo.