A multispecies competitive nanobody-based ELISA for the detection of antibodies against hepatitis E virus

Abstract

The hepatitis E virus (HEV) is an emergent zoonotic virus causing viral hepatitis worldwide. Clinically, hepatitis E is not easily distinguished from other types of acute viral hepatitis. There is a need for HEV diagnostic assays to detect and prevent interspecies transmission among susceptible populations. Nanobodies (Nbs) are expressed recombinantly in different systems, produced with high yields, and have superior physicochemical properties compared with conventional antibodies (Ab). Several Nbs against ORF2, the capsid protein and main antigen, were selected and produced in E. coli. Nb39 and Nb74 specifically recognized HEV ORF2 (genotypes 3 and 4). A competitive ELISA (cELISA) was developed and validated using a reference panel of human (n = 86) and swine sera (n = 116) tested in comparison with a commercial kit. The optimal cutoff values determined by ROC analysis were 69.16% (human) and 58.76% (swine); the sensitivity and specificity were high: 97.4% (95% CI 86.5-99.5%) and 95.8% (95% CI 86.0-98.8%) for human vs. 100% (95% CI 93.5-100%) and 98.3% (95% CI 91.0-99.7%) for swine. Further, the cELISA detected total anti-HEV antibodies in wild boar, deer, and mice. To our knowledge, this is the first report of production of Nbs against HEV-3 ORF2 for diagnostic purposes.

Figure 1

Llama immunization and screening of Nbs against the recombinant HEV-3 ORF2 protein. (A) SDS-PAGE and Coomassie blue staining showing the expression and purification of the recombinant HEV-3 ORF2 protein. (B) Schematic representation of the llama immunization protocol depicting boosting, antigen dose and blood extraction time (p.i. post immunization). (C) Antibody titer against HEV-3 ORF2 in llama sera after immunization, a maximum response is reached at PID 32. (D) Agarose gel showing the ~ 700 bp PCR fragments corresponding to VHHs of variable size amplified from randomly picked individual colonies. (E) Identification of positive clones that bind specifically to the HEV-3 ORF2 protein by ELISA, 95 periplasmic extracts from positive colonies and from a control colony were tested. (F) Sequence logo plot using 12 Nbs sequences (WebLog3). High variability can be observed on the AA composition of the CDR3 domains.

Figure 2

Characterization of Nbs against HEV-3 ORF2 protein. (A,B) An ELISA was performed to select the most suitable Nbs able to bind to the ORF2 protein at 2 different concentrations. A 96-well microtiter plate was coated with 0.62 (A) and 1.25 μg/ml (B) of ORF2 protein, respectively. (C) Further characterization of the 2 best Nbs to develop the immunoassay. A 96-well microtiter plate was coated with 0.62 μg/ml and incubated with different concentrations of Nb39 and Nb74. (D) Binding capacity of Nb39 and Nb74 to different form, native (nORF2) vs. denaturized (dORF2), of ORF2 proteins from 4 HEV genotypes.

Figure 3

Parameter optimization of the cELISA. (A) iELISA. Selection of concentration of Ag and Nb. The figure shows the selected three points (red triangle) for Ag and Nb that can be observed on the right table. (B,C) cELISA. The optimal conditions of Ag, Nb, and serum dilution were selected. Human sera (B). Swine sera (C). The figure shows a lighter color in the positive serum using the following conditions: 50 ng/ml of Ag, undiluted serum, and 60 ng/ml of Nb39.

Figure 4

Determination of cutoff, sensitivity, and specificity for the cELISA. (A) Receiver operating characteristics (ROC) analysis shows sensitivity vs specificity for discrimination between positive and negative human serum samples that were screened for in house and commercial tests. AUC = 0.976. (B). The optimal values of the sensitivity and specificity curves were used to determine the cELISA cutoff = 69.16%, for human samples, full blue line. (C) ROC curve for swine serum samples. AUC = 1.000 (D) The cELISA cutoff for swine serum samples was 58.76%, dashed sky-blue line.

Figure 5

Detection of specific total anti-HEV antibodies with the novel cELISA in serum samples of different species (human, swine, wild boar, deer, dog, and mice).