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

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

Mendoza

Congreso; LVIII Congreso Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular; 2022

SAIB

HepatitisE virus (HEV) is an RNA virus responsible for the hepatitis E, a globalemerging disease. Camelids have heavy chain only antibodies from which thevariable domain (VHH or nanobody) that specifically binds to antigens can beisolated. Different expression systems can be used to produce VHH with high yield;VHH are easily modified and have superior physicochemical properties. In thiswork we obtained, selected, and modified nanobodies to detect the HEV capsidprotein. Method: HEV-3 ORF2 protein was expressed and purified using NiNTAunder native conditions. A llama was immunized following a protocol of fivesuccessive subcutaneous injections with 150 g ORF2 at 2-weekintervals. Blood was taken 4 days after the last immunization; lymphocytes wereisolated and RNA extracted. A nanobody library was constructed using a goldengate strategy. ORF2 specific Nanobodies were selected after 3 rounds of panningon transformed TG1 E. coli, using the phage display technology. Then, 96colonies were randomly selected and the expression of HA-His-C-taggednanobodies was induced with 1 mM IPTG. Recombinant VHH-HA-His proteins weretested for their capacity to recognize the ORF2 HEV-3 protein using an indirectELISA. Positive clones were sequenced and nanobodies were selected according tothe hypervariable complementary-determining region 3 (CDR3) sequence. Scaling upof the selected nanobodies was done after transformation of WK6 E. coli, theprotein was extracted out of the periplasm and purified by affinitychromatography. Finally, the VHH were cloned and modified. Results: A nanobodylibrary of 1.8x109 individual colonies was obtained and a 100% of the testedclones contained a VHH fragment. A phage library of 1x1012 phages/ml wasgenerated after infection of the TG1 E. coli with the M13K07 helper phage.After 3 rounds of panning, 96 clones were randomly selected and successfullyextracted from the periplasm to identify specific binders by ELISA. 86individual positive colonies were identified, and 16 clones were sent forsequencing after a pre-selection by restriction enzyme analysis. Six differentHEV-3 ORF2 specific nanobodies were selected and modified with different tags(polystyrene binding peptide, biotin binding site, and proteins with enzymaticactivity). These VHHs with and without tags were successfully expressed andpurified by affinity chromatography. Conclusion: To our knowledge, this is thefirst report of the selection and production of nanobodies specific for the ORF2protein of HEV-3. We are currently working on the design of a low-costimmunoassay, with different formats such as sandwich ELISA and competitiveELISA, to detect anti-HEV antibodies or the biomarker ORF2 in human or pigplasma/sera. It is important to mention that these nanoantibodies could also beused for research and passive therapy to prevent HEV infection in pigs to avoidzoonotic transmission.