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

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

MENDOZA

Congreso; CONGRESO SAIB 2022; 2022

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular

Hepatitis E virus (HEV) is an RNA virus responsible for the hepatitis E, a global emergingdisease. Camelids have heavy chain only antibodies from which the variable domain (VHH ornanobody) that specifically binds to antigens can be isolated. Different expression systems canbe used to produce VHH with high yield; VHH are easily modified and have superiorphysicochemical properties. In this work we obtained, selected, and modified nanobodies todetect the HEV capsid protein.Method: HEV-3 ORF2 protein was expressed and purified using NiNTA under nativeconditions. A llama was immunized following a protocol of five successive subcutaneousinjections with 150 g ORF2 at 2‐week intervals. Blood was taken 4 days after the lastimmunization; lymphocytes were isolated and RNA extracted. A nanobody library wasconstructed using a golden gate strategy. ORF2 specific nanobodies were selected after 3rounds of panning on transformed TG1 E. coli, using the phage display technology. Then, 96colonies 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 torecognize the ORF2 HEV-3 protein using an indirect ELISA. Positive clones were sequenced andnanobodies were selected according to the hypervariable complementary-determining region3 (CDR3) sequence. Scaling up of the selected nanobodies was done after transformation ofWK6 E. coli, the protein was extracted out of the periplasm and purified by affinitychromatography. Finally, the VHH were cloned and modified.Results: A nanobody library of 1.8x10 9 individual colonies was obtained and a 100% of thetested clones contained a VHH fragment. A phage library of 1x10 12 phages/ml was generatedafter infection of the TG1 E. coli with the M13K07 helper phage. After 3 rounds of panning, 96clones were randomly selected and successfully extracted from the periplasm to identifyspecific binders by ELISA. 86 individual positive colonies were identified, and 16 clones weresent for sequencing after a pre-selection by restriction enzyme analysis. Six different HEV-3ORF2 specific nanobodies were selected and modified with different tags (polystyrene bindingpeptide, biotin binding site, and proteins with enzymatic activity). These VHHs with andwithout tags were successfully expressed and purified by affinity chromatography.Conclusion: To our knowledge, this is the first report of the selection and production ofnanobodies specific for the ORF2 protein of HEV-3. We are currently working on the design ofa low-cost immunoassay, with different formats such as sandwich ELISA and competitive ELISA,to detect anti-HEV antibodies or the biomarker ORF2 in human or pig plasma/sera. It isimportant to mention that these nanoantibodies could also be used for research and passivetherapy to prevent HEV infection in pigs to avoid zoonotic transmission.