Novel nanobodies-based diagnostic tools for the detection of Hepatitis E Virus in humans and zoonotic reservoirs

LORENA PAOLA ARCE; MARÍA FLORENCIA PAVAN; JULIA MATÍAS BRANCHER; MARINA BOK; VIVIANA PARREÑO; SILVINA ELENA GUTIERREZ; SILVIA MARCELA ESTEIN; AGOSTINA TAMMONE SANTOS; WALTER EZEQUIEL CONDORÍ; MARCELA MARÍA UHART; MARÍA GUADALUPE VIZOSO-PINTO; LORENA ITATÍ IBAÑEZ

Londres

Simposio; 2nd international hepatitis e symposium; 2023

International Hepatitis E Symposium

Background and aims: HEV (genotypes 3 and 4) is an emergent and zoonotic viruscausing hepatitis worldwide. Acute HEV infection can be diagnosed by detecting thecapsid antigen in plasma and stool or by detecting specific antibodies against HEV. Ouraim was to obtain nanobodies (VHH) to develop novel diagnostic tools for the detectionof HEV infection in clinical and veterinary samples.Methods: A llama was immunized subcutaneously with HEV-3 ORF2. Lymphocyteswere isolated from blood and RNA extracted. A nanobody library was constructed usinga golden gate strategy. ORF2 specific nanobodies were selected by panning usingphage display. An indirect ELISA was used to test the VHH capacity to recognize ORF2and were selected according to the hypervariable CDR3 sequence. VHH were scaledup, extracted out of the periplasm, and purified by affinity chromatography.We developed a multispecies competitive ELISA (cELISA) to screen total anti-HEVantibodies testing different concentrations of HEV-3 ORF2 and VHH, serum dilutions andconjugated antibody (anti-HisHRP). We validated the cELISA (cutoff, sensitivity,specificity, area under curve (AUC), k-index, reproducibility, and limit detection) andchallenged it with human and animal sera.The sandwich ELISA (sELISA) to detect antigen -glycosylated ORF2-(gORF2) and viralparticles was developed using VHH cloned and modified with plastic (VHHPSW) andbiotin (VHHbio) binding tags. ORF2 was expressed in HEK 293 cells and purified withaffinity chromatography to obtain the glycosylated protein. For sELISA, we triedconcentrations of VHH and VHHPSW, different blocking agents, concentration ofgORF2, and dilutions of VHHbio and HRP-conjugated streptavidin.Results: A phage library of 1x1012 phages/ml was generated. After panning, 96 cloneswere randomly selected and extracted from the periplasm to identify specific binders byELISA. 16 positive colonies were selected by restriction analysis and sequenced. Sixnanobodies were selected, modified, expressed, and purified by affinity chromatography.The VHH39 and VHH74 with the best affinity were used.The cELISA was established for human and animal samples as follows: 0.05 μg/mlORF2, 5% skimmed milk, undiluted serum, 60 ng/ml of VHH39, and 1/10000 conjugatedantibody. Validation data of cELISA is shown in Table1. The cELISA allowed it to detectpositive samples in panels of sera from humans and animals: 5/64 human, 58/116 swine,8/22 wild board, 1/20 deer, and 3/5 mice. These results highly correlated with those of acommercial ELISA kit.The conditions of the sELISA for Ag detection are: 1 μg/ml VHHPSW-74, 1% gelatin,1/200 VHHbio-39, and 1/10000 HRP-conjugated streptavidin. The detection limit was100 ng/ml of gORF2. ORF2 was detected in human samples of patients with liver disease(3/64).Conclusion: This is the first report of production of nanobodies against HEV-3 ORF2 fordiagnostic purposes. We developed two low-cost immunoassays, one for detectingantibodies, the other for detecting antigens which can be used in human and animalplasma/sera. These nanobodies could also be used? for research and passive therapy.