Novel nanobodies-based diagnostic tools for the detection of Hepatitis E Virus for zoonotic reservoirs.

ARCE LORENA PAOLA; PAVAN MARIA FLORENCIA; BRANCHER JULIA MATIAS; BOK MARINA; PARREÑO VIVIANA; GUTIERREZ SILVINA ELENA; ESTEIN SILVIA MARCELA; TAMMONE AGOSTINA; CONDORI WALTER EZEQUIEL; UHART MARCELA MARIA; VISOZO-PINTO MARIA GUADALUPE; IBAÑEZ LORENA ITATI

Londres

Simposio; Second International Hepatitis E Symposium; 2023

Background and aims: HEV (genotypes 3 and 4) is an emergent and zoonotic virus causing hepatitis worldwide. Acute HEV infection can be diagnosed by detecting the capsid antigen in plasma and stool or by detecting specific antibodies against HEV. Our aim was to obtain nanobodies (VHH) to develop novel diagnostic tools for HEV infection in clinical and veterinary samples.Methods: A llama was immunized subcutaneously with HEV-3 ORF2. Lymphocytes were isolated from blood and RNA extracted. A nanobody library was constructed using a golden gate strategy. ORF2 specific nanobodies were selected by panning using phage display. An indirect ELISA was used to test the VHH capacity to recognize ORF2 and were selected according to the hypervariable CDR3 sequence. VHH were scaled up, extracted out of the periplasm and purified by affinity chromatography.We developed a multispecies competitive ELISA (cELISA) to screen total anti-HEV antibodies testing different concentrations of HEV-3 ORF2 and VHH, serum dilutions and conjugated antibody (anti-HisHRP). We validated the cELISA (cutoff, sensitivity, specificity, area under curve (AUC), k-index, reproducibility, and limit detection) and challenged it with human and animal sera.The sandwich ELISA (sELISA) to detect antigen (glycosylated ORF2) and viral particles was developed using VHH cloned and modified with plastic (VHHPSW) and biotin (VHHbio) binding tags. ORF2 was expressed in HEK 293 cells and purified with affinity chromatography to obtain the glycosylated protein. For sELISA, we tried concentrations of VHH and VHHPSW, different blocking agents, concentration of gORF2, and dilutions of VHHbio and streptavidin HRP antibodies.Results: A phage library of 1x10¹² phages/ml was generated. After panning, 96 clones were randomly selected and extracted from the periplasm to identify specific binders by ELISA. 16 positive colonies were selected by restriction analysis and sequenced. Six nanobodies were selected, modified, expressed and purified by affinity chromatography. The VHH39 and VHH74 with the best affinity were used.The cELISA was established for humans and animals samples as follows: 0.05 μg/ml ORF2, 5% skimmed milk, undiluted serum, 60 ng/ml of VHH39, and 1/10000 conjugated antibody. Validation data of cELISA is shown in Table1. The cELISA allowed it to detect positive 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 a commercial 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 conjugated streptavidin. The detection limit was 100 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 for diagnostic purposes. We developed two low-cost immunoassays, one for detecting antibodies, the other for detecting antigens which can be used in human and animal plasma/sera. These nanobodies could also serve for research and passive therapy.Table 1. Validation of a novel cELISAValidationHumanSwineCut off 69.16%58.76% Sensitivity97.4 % (IC95%; 86.5-99.5%)100 % (IC95%: 93.5-100%)Specificity95.8 % (IC95%; 86.0-98.8%)98.3 % (IC95%: 91.0-99.7%)Kappa index0.93 (IC95%; 0.85-1.00) 0.95 (IC95%; 0.89-1.00) AUC0.976 (0.938 – 1)1 (1-1)