Nanobodies: a versatile and low-cost tool for Hepatitis E virus research, diagnostics and therapeutics. *Equal contribution.

ARCE, LORENA PAOLA; PAVAN, MARIA FLORENCIA; BOK, MARINA; PARREÑO, VIVIANA; VIZOSO PINTO, MARIA GUADALUPE*; IBAÑEZ, LORENA ITATÍ*

Mendoza

Congreso; LVII Reunion Anual de la Sociedad Argentina de Invesigaciones Biológicas; 2022

SAIB

Hepatitis E virus (HEV) is an RNA virus responsible for the hepatitis E, a global emerging disease. Camelidshave heavy chain only antibodies from which the variable domain (VHH or nanobody) that specificallybinds to antigens can be isolated. Different expression systems can be used to produce VHH with highyield; VHH are easily modified and have superior physicochemical properties. In this work we obtained,selected, and modified nanobodies to detect the HEV capsid protein.Method: HEV-3 ORF2 protein was expressed and purified using NiNTA under native conditions. A llama wasimmunized following a protocol of five successive subcutaneous injections with 150 g ORF2 at 2‐weekintervals. Blood was taken 4 days after the last immunization; lymphocytes were isolated and RNAextracted. A nanobody library was constructed using a golden gate strategy. ORF2 specific nanobodieswere selected after 3 rounds of panning on transformed TG1 E. coli, using the phage display technology.Then, 96 colonies were randomly selected and the expression of HA-His-C-tagged nanobodies wasinduced with 1 mM IPTG. Recombinant VHH-HA-His proteins were tested for their capacity to recognize theORF2 HEV-3 protein using an indirect ELISA. Positive clones were sequenced and nanobodies wereselected according to the hypervariable complementary-determining region 3 (CDR3) sequence. Scalingup of the selected nanobodies was done after transformation of WK6 E. coli, the protein was extracted outof the periplasm and purified by affinity chromatography. Finally, the VHH were cloned and modified.Results: A nanobody library of 1.8x109 individual colonies was obtained and a 100% of the tested clonescontained a VHH fragment. A phage library of 1x1012 phages/ml was generated after infection of the TG1E. coli with the M13K07 helper phage. After 3 rounds of panning, 96 clones were randomly selected andsuccessfully extracted from the periplasm to identify specific binders by ELISA. 86 individual positivecolonies were identified, and 16 clones were sent for sequencing after a pre-selection by restrictionenzyme analysis. Six different HEV-3 ORF2 specific nanobodies were selected and modified with differenttags (polystyrene binding peptide, biotin binding site, and proteins with enzymatic activity). These VHHswith and without tags were successfully expressed and purified by affinity chromatography. Conclusion: To our knowledge, this is the first report of the selection and production of nanobodies specific for theORF2 protein of HEV-3. We are currently working on the design of a low-cost immunoassay, with differentformats such as sandwich ELISA and competitive ELISA, to detect anti-HEV antibodies or the biomarkerORF2 in human or pig plasma/sera. It is important to mention that these nanoantibodies could also beused for research and passive therapy to prevent HEV infection in pigs to avoid zoonotic transmission.