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 GUTIÉRREZ; SILVIA MARCELA ESTEIN; AGOSTINA TAMMONE SANTOS; WALTER EZEQUIEL CONDORÍ; MARCELA MARÍA UHART; LORENA ITATÍ IBAÑEZ; MARIA GUADALUPE VIZOSO PINTO

Londres

Simposio; Second International Hepatitis E Symposium Londres; 2023

Background and aims: Hepatitis E virusgenotypes 3 and 4 (HEV-3 and HEV-4) are emergent and zoonotic viruses causing hepatitis worldwide. Acute HEVinfection can be diagnosed by detecting the capsid antigen in plasma and stoolor by detecting specific antibodies against HEV. Ouraim was to obtain nanobodies (Nbs) to develop novel diagnostic tools forthe detection of HEV infection in clinical and veterinary samples.Methods: A llama was immunized subcutaneously with HEV-3ORF2. Lymphocytes were isolated from blood and RNA extracted. A nanobodylibrary was constructed using a golden gate strategy. ORF2 specific Nbs wereselected by panning using phage display. An indirect ELISA was used to test theNbs capacity to recognize ORF2 and were selected according to the hypervariableCDR3 sequence. Nbs were scaled up, extracted out of the periplasm, and purifiedby affinity chromatography.Wedeveloped a multispecies competitive ELISA (cELISA) to screen total anti-HEVantibodies testing different concentrations of HEV-3 ORF2 and Nbs, serumdilutions 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 animalsera.Thesandwich ELISA (sELISA) to detect the antigen -glycosylated ORF2 (gORF2)- andviral particles was developed using Nbs cloned and modified with plastic (Nbs-PSW)and biotin (Nbs-bio) binding tags. ORF2 was expressedin HEK 293 cells and purified with affinity chromatography to obtain the glycosylated protein.For sELISA, we tried concentrations of Nbs andNbs-PSW, different blockingagents, concentration of gORF2, and dilutionsof Nbs-bio and HRP-conjugatedstreptavidin.Results: A phage library of 1x10¹² phages/ml wasgenerated. After panning, 96 clones were randomly selected and extracted fromthe periplasm to identify specific binders by ELISA. 16 positive colonies wereselected by restriction analysis and sequenced. Six Nbs were selected,modified, expressed, and purified by affinity chromatography. The Nb39 and Nb74with the best affinity were used.The cELISA was established for human and animal samples as follows: 0.05μg/ml ORF2, 5% skimmed milk, undiluted serum, 60 ng/ml of Nb39, and 1/10000conjugated antibody. Validation data of cELISA is shown in Table1. The cELISAallowed it to detect positive samples in panels of sera from humans andanimals: 5/64 human, 58/116 swine, 8/22 wild board, 1/20 deer, and 3/5 mice. Theseresults highly correlated with those of a commercial ELISA kit.Theconditions of the sELISA for Ag detection are: 1 μg/ml Nbs-PSW-74,1% gelatin, 1/200 Nbs-bio-39, and 1/10000 HRP-conjugated streptavidin. Thedetection limit was 100 ng/ml of gORF2. ORF2 was detected in human samples ofpatients with liver disease (3/64).Conclusion: This is the first report of production of Nbsagainst HEV-3 ORF2 for diagnostic purposes. We developed two low-costimmunoassays, one for detecting antibodies, the other for detecting antigenswhich can be used in human and animal plasma/sera. These Nbs could also be usedfor research and passive therapy.