WO2022199526A1

WO2022199526A1 - Hepatitis e virus-like particles and uses thereof

Info

Publication number: WO2022199526A1
Application number: PCT/CN2022/081996
Authority: WO
Inventors: Siddharth Sridhar; Jianwen SITU; Jian-piao CAI; Kwok-Yung Yuen
Original assignee: The University Of Hong Kong
Priority date: 2021-03-26
Filing date: 2022-03-21
Publication date: 2022-09-29
Also published as: CN117083290A

Abstract

Disclosed are peptides, compositions, and assays for detecting Hepatitis E virus (HEV) species C (HEV-C) infections, distinguishing HEV-C infections from HEV-A infections, and protecting against HEV-C infections. The compositions typically include one or more peptides with an amino acid sequence identity similar to that of a portion of HEV-C genotype 1 (HEV-C1) open reading frame 2 (ORF-2) capsid protein. The HEV-C1 peptides are isolated and purified peptides that aggregate into immunogenic virus-like particles (VLPs). The HEV-C1 peptides have no substantial amino acid sequence identity with ORF2 peptides of HEV species A, and are used to distinguish human HEV-C infections from human HEV-A infections.

Description

HEPATITIS E VIRUS-LIKE PARTICLES AND USES THEREOF

This international patent application claims the benefit of U.S. Provisional Patent Application No.: 63/166,698 filed on March 26, 2021, the entire content of which is incorporated by reference for all purpose.

FIELD OF THE INVENTION

This invention is generally directed to compositions and assays detecting Hepatitis E virus (HEV) infections, distinguishing infections by different HEV species, and protecting against HEV.

BACKGROUND OF THE INVENTION

Hepatitis E virus (HEV) is a major cause of viral hepatitis globally. The clinical spectrum of acute hepatitis E includes asymptomatic infection, mild-to-moderate liver dysfunction, and fulminant hepatitis. Persistent hepatitis E can develop in immunocompromised persons, which can progress to liver cirrhosis if left untreated. HEV belongs to the family Hepeviridae, which includes two genera: Orthohepevirus (including variants that infect terrestrial vertebrates) and Piscihepevirus (cutthroat trout virus) . Hepatitis E in humans is mostly due to members of Orthohepevirus species A (HEV-A) . HEV-A includes eight genotypes that infect humans, pigs, wild boar, deer, rabbits, and camels. Four of the HEV-A eight genotypes commonly infect humans. HEV-A genotype 1 (HEV-A1) and genotype 2 are spread between humans via the fecal-oral route. HEV-A genotype 3 (HEV-A3) and genotype 4 (HEV-A4) circulate in swine and spread to humans via consumption of undercooked meat products. HEV-A3 circulates in Europe and the Americas while HEV-A4 circulates in China. HEV-A3 and HEV-A4 typically cause self-limiting hepatitis, but may progress to chronic infection in immunocompromised persons. A HEV-A1-based vaccine (Hecolin) is licensed in China following a clinical trial conducted in a HEV-A4 endemic area. The efficacy of this vaccine against other HEV-A genotypes is currently uncertain.

Human hepatitis E infections in industrialized countries are due to either HEV-A genotype 3 (Europe, Japan, and the Americas) or HEV-A genotype 4 (China) and in developing countries are due to HEV-

A genotypes

1 or 2. HEV-A

genotypes

3 and 4 are usually acquired by consumption of undercooked pork or game meat, but can also be transmitted through contaminated blood products or organs (Sridhar et al., Hepatology, January 2020, 1-13 (2020) ) .

Apart from HEV-A, the Orthohepevirus genus includes three other species: B (circulating in birds) , C (HEV-C; circulating in rodents and ferrets) and D (circulating in bats) . HEV-C, discovered in German rats in 2010, has since been detected in rats in Asia, Europe, and North America. Rats are susceptible to infection by HEV-C genotype 1 (HEV-C1) with other genotypes of HEV-C circulating in ferrets, shrews, voles, and so on. Hitherto, HEV-C1 was considered to have minimal zoonotic risk because of wide phylogenetic divergence from HEV-A and failure of experimental infection of pigs and nonhuman primates.

These HEV species are listed on ViralZone site maintained by the Swiss Institute of Bioinformatics. However, it is not uncommon in literature to refer to HEV-A and its genotypes as human Hepatitis E virus and to HEV-C as rat Hepatitis E virus.

HEV-C only shares 50%-60%nucleotide identity with HEV-A and has major differences in key epitopes of the putative receptor binding domain. However, a case study showed that HEV-C1 infected a liver transplant recipient even though the patient had pre-existing antibodies against HEV-A (Sridhar et al., Emerg Infect Dis; 24: 2241-2250 (2018) ) . That commonly used HEV-A nucleic acid amplification tests were unable to detect HEV-C1 infection because of significant sequence differences. Another study subsequently identified an immunocompetent adult with acute HEV-C1 infection, likely acquired in Africa (Andonov et al., J Infect Dis., ; 220: 951-955 (2019) ) . This raises the possibility that HEV-C1 is a globally prevalent zoonosis that is routinely missed by existing assays that are specific for HEV-A. An epidemiological study reports that HEV-C1 infection accounted for 8%of all genotyped hepatitis E cases in Hong Kong (Sridhar et al., Hepatology, January 2020, 1-13 (2020) ) .

The global prevalence of HEV-C1 infection is unknown due to a blind spot in HEV diagnostic testing. HEV-A based reverse-transcription polymerase chain reaction (RT-PCR) assays cannot detect HEV-C1 (Sridhar et al., Emerg Infect Dis; 24: 2241-2250 (2018) ) . The efficacy of hepatitis E enzymatic immunoassay (EIA) kits, which universally use HEV-A-derived target antigens, for diagnosing HEV-C1 is uncertain. A commercial EIA kit using the HEV-A1 pE2 antigen cross-reacted with some HEV-C1 patient sera; whether EIAs using other HEV-A peptides would be similarly cross-reactive is unknown (Andonov et al., J Infect Dis., 220: 951-955 (2019) ) .

There remains a need for compositions and assays that detect HEV-C infections, distinguish HEV-C infections from HEV-A infections, and protect against HEV-C infections.

SUMMARY OF THE INVENTION

It is the object of the present invention to provide compositions and assays that detect HEV-C infections, distinguish HEV-C infections from HEV-A infections, and protect against HEV-C infections.

It is another object of the present invention to provide methods of making the compositions and assays that detect HEV-C infections, distinguish HEV-C infections from HEV-A infections, and protect against HEV-C infections.

It is yet another object of the present invention to provide methods of using the compositions and assays that detect HEV-C infections, distinguish HEV-C infections from HEV-A infections, and protect against HEV-C infections.

Described are compositions and assays for detecting HEV-C infections and distinguishing HEV-C infections from HEV-A infections. Also described are compositions for protecting against HEV-C infections.

Typically, the compositions include a peptide, or a synthetic virus-like particle containing a plurality of peptides, having at least a 90%amino acid sequence identity with SEQ ID NO: 1, or an amino acid sequence as in SEQ ID NO: 1. The compositions may also include a peptide, or a synthetic virus-like particle containing a plurality of peptides, having at least a 90%amino acid sequence identity with SEQ ID NO: 3. The compositions may include an adjuvant. The compositions are typically for inducing an immune response against a portion of SEQ ID NO: 1. The compositions may also be bivalent compositions inducing an immune response against a portion of SEQ ID NO: 1 and against a portion of SEQ ID NO: 3. The composition typically induce an immune response against Hepatitis E virus (HEV) species A (HEV-A) , HEV-C genotype 1, or a combination thereof.

Also described are composition for detecting a HEV-C infection, or a combination of HEV-A and HEV-C infections. The compositions for detecting a HEV-C infection, or a combination of HEV-A and HEV-C infections typically include a plurality of peptides having at least a 90%amino acid sequence identity with SEQ ID NO: 1, at least 95 %amino acid sequence identity with SEQ ID NO: 1, or an amino acid sequence as in SEQ ID NO: 1. These compositions may also include a plurality of peptides having at least a 90%amino acid sequence identity with SEQ ID NO: 3. The plurality of peptides may be in a form of synthetic virus-like particles. The compositions may be included on assay platforms, or test vessels for an assay, for detecting a HEV-C infection, detecting a combination of HEV-A and HEV-C infections, or distinguishing between HEV-A and HEV-C infections.

Also described are assays, such as immunoassay or amplifications assays, for detecting a HEV-C infection in a sample, detecting a combination of HEV-A and HEV-C infections in a sample, or distinguishing between HEV-A and HEV-C infections in a sample. The immunoassays typically include contacting a sample or a test sample with a plurality of peptides having at least a 90%amino acid sequence identity with SEQ ID NO: 1, at least 95 %amino acid sequence identity with SEQ ID NO: 1, or an amino acid sequence as in SEQ ID NO: 1, and optionally, contacting the sample or the test sample with a plurality of peptides having at least a 90%amino acid sequence identity with SEQ ID NO: 3. The plurality of peptides may be in a form of synthetic virus-like particles. The contacting may be in on a platform or in a test vessel. The assays typically include a step of developing a signal from contacting.

The assays typically detect HEV-C infection when signal develops from the contacting of the sample or the test sample with the plurality of peptides having at least a 90%amino acid sequence identity with SEQ ID NO: 1, at least 95 %amino acid sequence identity with SEQ ID NO: 1, or an amino acid sequence as in SEQ ID NO: 1.

The assays typically detect HEV-A infection when signal develops from the contacting of the sample or the test sample with the plurality of peptides having at least a 90%amino acid sequence identity with SEQ ID NO: 3. The assays typically detect a HEV-C infection or a combination of HEV-A and HEV-C infections in the sample at a sensitivity of about or over 80%and a specificity of about or over 70%. The assays typically distinguish between HEV-A and HEV-C infections in the sample at a sensitivity of about or over 80%.

The sample is typically a sample obtained from a subject. Suitable subjects include a human, a non human primate, domestic animal, wild animal, farm animal, or a laboratory animal. The sample is a bodily fluid or mucus obtained from the subject and includes blood, serum, plasma, excrement, exudate, saliva, sputum, tear, sweat, urine, or a vaginal discharge. The sample may be diluted in a buffer to form a test sample. The sample may be diluted at a ratio between about 1: 5 and 1: 500 (v/v) of the sample to a buffer. The sample or the test sample may be treated to extract sample RNA for detection assays.

Also described are kits comprising a plurality of peptides having at least a 90%amino acid sequence identity with SEQ ID NO: 1, at least 95 %amino acid sequence identity with SEQ ID NO: 1, or having an amino acid sequence as in SEQ ID NO: 1.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A is a diagram showing a phylogenetic analysis of HEV-C1 ORF2 nucleotide sequences. The tree was constructed using neighbor-joining method with bootstrap values calculated from 1000 trees. Only bootstrap values > 700 are shown. HEV-C1 strains identified in human patients are highlighted in red while those derived from rats are in black. The SRN250811 strain is labelled in bold. Figure 1B is a diagram showing E2s amino acid sequence alignment of major HEV-A genotypes and the HEV-C1 LCK-3110 strain. Figure 1C shows residues bound by monoclonal antibodies (mAbs) , which are serially numbered in Figure 1B below the relevant alignment positions (digits) . Residues that are conserved between LCK-3110 and at least one of the HEV-A genotypes are boxed in as well as the non-conserved residues. MAbs binding to each residue are indicated in the key; those with overlapping or nested epitope specificity are represented together. MAbs annotated in bold were used in homology modeling and antigen EIAs. Ratio next to mAb labels indicates the number of conserved residues between HEV-C1 and HEV-A at these sites/number of positions involved in binding. GenBank accession numbers of sequences used in the alignment: L08816 (HEV-A1) , AB369687 (HEV-A3) , AJ272108 (HEV-A4) , and MG813927 (HEV-C1; LCK-3110) . Figure 1D is a diagram showing E2s amino acid sequence alignment of LCK-3110 and two divergent HEV-C1 strains infecting humans. Figure 1E is a diagram showing comparison of complex structures of mAbs 8C11 and 8G12 with E2s of HEV-A and three HEV-C1 strains representing the three strain groups infecting humans. 8C11 is shown on the bottom half of each of the top panels and E2s is shown on the top half of each of the top panels in these cartoon representations. 8G12 is shown on the bottom half of each of the bottom panels and E2s is shown on the top half of each of the bottom panels in these cartoon representations. Polar contacts between mAbs and E2s are depicted as dotted lines with interacting residues represented by sticks.

Figures 2A-2F are graphs showing optical density (OD) values of samples in panels A –D using three commercial IgG EIA (Figures 2A-2C) and IgM EIA (Figures 2D-2F) assays. Bars represent mean and standard error of mean (SEM) . Mean OD of each panel in each EIA is compared to mean OD of panel C using Student’s t-test with or without Welch’s correction as appropriate. P values are marked as ns: p value > 0.05, *: p value ≤ 0.05, **: p value ≤ 0.01, ***: p value ≤ 0.001, ****: p value ≤ 0.0001.

Figures 3A-3C are graphs showing Figure 3A –OD values of samples in panels B, C, and D using the Wantai antigen detection EIA. Dotted line represents assay cut-off. Dots in panel D represent healthy controls. Bars represent mean and standard error of mean. Mean ODs of panel B and panel D were compared to panel C by Student’s t-test. P values are marked as ns: p value >0.05, ****: p value ≤ 0.0001. Binding of mAbs #4 (Figure 3B) and 12F12 (Figure 3C) to HEV-A and HEV-C1 were assessed in an EIA format. ODs of HEV-negative control serum (sample 1) , HEV-A4 p239 and two HEV-A4 patient sera (samples 6-8) , HEV-C1 p241 and three HEV-C1 patient sera (samples 2-5) were measured. Each sample was tested in triplicate. Symbols represent mean and bars represent SD values of the triplicates. Mean OD of each sample was individually compared to OD of the negative control by Student’s t-test: ns: p value > 0.05, *: p value ≤ 0.05.

Figures 4A is a diagram showing the timeline of rat vaccination and infection challenge. On day 56, the rat livers were obtained for viral load testing. Figures 4B-4D are graphs showing Fecal viral load (Figure 4B) , plasma viral load (Figure 4C) , and day 28 liver tissue viral load (Figure 4D) of rats in each group. Bars represent mean and SEM. Undetectable viral load was represented as 3 log ₁₀ copies/mL (dotted line) , which was the limit of detection (LOD) of the HEV-C1 RT-PCR assay. For all time-points at which there was a significant difference between group means by one-way ANOVA, we compared the means of each group individually against the PBS group by the Tukey-Kramer method. P values are marked as ns: p value > 0.05, *: p value ≤ 0.05, **: p value ≤ 0.01, ***: p value ≤ 0.001, ****: p value ≤ 0.0001.

Figures 5A and 5B are graphs showing alanine aminotransferase (ALT, Figure 5A) and alkaline phosphatase (ALP, Figure 5B) of HEV-C1 challenged rats immunized with PBS, Hecolin, HEV-A4 p239 or HEV-C1 p241. Bars represent mean and standard error of mean.

Figure 6 is a diagram showing mixed vaccination scheme and HEV-C1 challenge experiment in rats with the timeline of rat vaccination and infection challenge. Four rats were given the Hecolin and HEV-C1 p241 vaccines two weeks apart and were then challenged with the SRN250811 strain. On day 56, the rat livers were obtained for viral load testing.

Figure 7 is a diagram showing assay evaluation of healthy organ donor sera who had previously tested negative in the Wantai HEV-IgG EIA kit. The mean OD values in both HEV-A and HEV-C IgG EIAs were broadly comparable.

Figure 8 is a diagram showing receiver operating characteristic (ROC) curves using RT-PCR results as the gold standard, which result in the generation of assay cut-offs for individual IgG EIAs.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

As used herein, the term “virus like particles” or “VLPs” refers to empty protein shells of viruses with the same or similar external form as the virus itself, but not containing any viral genome i.e. they are noninfectious particles. The VLPs may be peptide dimers, trimers, oligomers, or multimers of a peptide. The VLPs may be homodimers, homotrimers, homooligomers, or homomultimers of a peptide. The VLPs are typically synthetic particles formed from purified recombinant peptides.

As used herein, the term “peptides” includes proteins and fragments thereof. Peptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A) , Arginine (Arg, R) , Asparagine (Asn, N) , Aspartic Acid (Asp, D) , Cysteine (Cys, C) , Glutamine (Gln, Q) , Glutamic Acid (Glu, E) , Glycine (Gly, G) , Histidine (His, H) , Isoleucine (Ile, I) , Leucine (Leu, L) , Lysine (Lys, K) , Methionine (Met, M) , Phenylalanine (Phe, F) , Proline (Pro, P) , Serine (Ser, S) , Threonine (Thr, T) , Tryptophan (Trp, W) , Tyrosine (Tyr, Y) , and Valine (Val, V) .

As used herein, the term “variant” refers to a peptide or a polynucleotide that differs from a reference peptide or polynucleotide, but retains essential properties. A typical variant of a peptide differs in amino acid sequence from another, reference peptide. Generally, differences are limited so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions) . A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a peptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.

Modifications and changes can be made in the structure of the peptides and still obtain a molecule having similar characteristics as the peptide (e.g., a conservative amino acid substitution) . For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a peptide that defines that peptide’s biological functional activity, certain amino acid sequence substitutions can be made in a peptide sequence and nevertheless obtain a peptide with like properties.

As used herein, the term “identity, ” as known in the art, is a relationship between two or more peptide or two or more peptide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between sequences as determined by the match between strings of such sequences. “Identity” can also mean the degree of sequence relatedness of a peptide or a peptide compared to the full-length of a reference peptide. “Identity” and “similarity” can be readily calculated by known methods, including, but not limited to, those described in (Computational Molecular Biology, Lesk, A.M., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., Eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073 (1988) .

Various programs and alignment algorithms are described in: Smith &Waterman, Adv Appl Math 2, 482 (1981) ; Needleman &Wunsch, J Mol Biol 48, 443 (1970) ; Pearson &Lipman, Proc Natl Acad Sci USA 85, 2444 (1988) ; Higgins &Sharp, Gene 73, 237-244 (1988) ; Higgins &Sharp, CABIOS 5, 151-153 (1989) ; Corpet et al, Nuc Acids Res 16, 10881-10890 (1988) ; Huang et al, Computer App Biosci 8, 155-165 (1992) ; and Pearson et al, Meth Mol Bio 24, 307-331 (1994) . In addition, Altschul et al, J Mol Biol 215, 403-410 (1990) , presents a detailed consideration of sequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al, (1990) supra) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.

Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. The percent identity between two sequences can be determined using analysis software (i.e., Sequence Analysis Software Package of the Genetics Computer Group, Madison Wis. ) that incorporates the Needelman and Wunsch, (J. Mol. Biol., 48: 443-453, 1970) algorithm (e.g., NBLAST, and XBLAST) . The default parameters are used to determine the identity for the peptides.

By way of example, a peptide sequence may be identical to the reference sequence, that is be 100%identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the percent identity is less than 100%. Such alterations are selected from: at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino-or carboxy-terminal positions of the reference peptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of amino acid alterations for a given percent identity is determined by multiplying the total number of amino acids in the reference peptide by the numerical percent of the respective percent identity (divided by 100) and then subtracting that product from said total number of amino acids in the reference peptide.

The term “percent (%) sequence identity” is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical with the nucleotides or amino acids in a reference nucleic acid or peptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

As used herein, the term “substantial” generally refers to a comparative degree, such as at least about 85-90%, preferably about 95%or more for substantial similarity, and less than 15%, preferably less than 10%, or less than 5%for substantial difference. In the context of sequence identity, substantial sequence similarity, or substantial sequence identity, refers to at least about 85-90%, about 90%, preferably about 95%or more sequence identity.

As used herein, the term “recombinant polynucleotide” generally refers to a polynucleotide obtained through genetic engineering techniques.

As used herein, the term “recombinant peptide” generally refers to a peptide obtained from a recombinant polynucleotide. A recombinant nucleic acid or peptide is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. A recombinant peptide can also refer to a peptide that has been made using recombinant nucleic acids, including recombinant nucleic acids transferred to a host organism that is not the natural source of the peptide.

As used herein, the term “purified” and like terms relate to the molecule or compound in a form that is substantially free (at least 60%free, preferably 75%free, and most preferably 90%free) from other components normally associated with the molecule or compound in a native environment.

As used herein, the term “monomer” refers to a single peptide molecule.

As used herein, the terms “dimers” , “trimers” , “tetramers” , “oligomers” or “multimers” refer to two, three, four, or more monomers, respectively, forming a structure, such as an assembly of peptides, or a shell. The dimers, trimers, tetramers, or multimers may be homodimers, homotrimers, homotetramers, or homomultimers containing the same amino acid sequences for each of the monomers forming the dimers, trimers, tetramers, or multimers. The dimers, trimers, tetramers, or multimers may be heterodimers, heterotrimers, heterotetramers, or heteromultimers containing different amino acid sequences for each of the monomers forming the dimers, trimers, tetramers, or multimers.

As used herein, the term “detect” , “detecting” , “determine” or “determining” generally refers to obtaining information. Detecting or determining can utilize any of a variety of techniques available to those skilled in the art, including for example specific techniques explicitly referred to herein. Detecting or determining may involve manipulation of a physical sample, consideration and/or manipulation of data or information, for example utilizing a computer or other processing unit adapted to perform a relevant analysis, and/or receiving relevant information and/or materials from a source. Detecting or determining may also mean comparing an obtained value to a known value, such as a known test value, a known control value, or a threshold value. Detecting or determining may also mean forming a conclusion based on the difference between the obtained value and the known value.

As used herein, the term “sensitivity” refers to the ability of a test to correctly identify true positives, i.e., subjects infected with HEV-A or HEV-C. For example, sensitivity can be expressed as a percentage, the proportion of actual positives which are correctly identified as such (e.g., the percentage of test subjects having HEV-A or HEV-C infection correctly identified by the test as having the infection) . A test with high sensitivity has a low rate of false negatives, i.e., the cases of HEV-A or HEV-C infections not identified as such. Generally, the disclosed assays and methods have a sensitivity of about or over 70%, at least about 80%at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%.

As used herein, the term “specificity” refers to the ability of a test to correctly identify true negatives, i.e., the subjects that have no HEV-A or HEV-C infection. For example, specificity can be expressed as a percentage, the proportion of actual negatives which are correctly identified as such (e.g., the percentage of test subjects not having HEV-A or HEV-C infection correctly identified by the test as not having the infection) . A test with high specificity has a low rate of false positives, i.e., the cases of individuals not having HEV-A or HEV-C infection but suggested by the test as having the infection. Generally, the disclosed methods have a specificity of about or over 70%, at least about 80%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%.

As used herein, the term “accurate” refers to the ability of a test to provide a results with high sensitivity and high specificity, such as with sensitivity over about 70%and specificity over about 70%, with sensitivity over about 80%and specificity over about 80%, or with sensitivity over about 90%and specificity over about 90%.

As used herein, the term “sample” refers to body fluids, body smears, cell, tissue, organ or portion thereof that is isolated from a subject. A sample may be a single cell or a plurality of cells. A sample may be a specimen obtained by biopsy (e.g., surgical biopsy) . A sample may be cells from a subject that are or have been placed in or adapted to tissue culture. A sample may be one or more of cells, tissue, serum, plasma, urine, spittle, sputum, and stool. A sample may be one or more of a swab, fluid, blood, plasma, serum, urine, excrement, sputum, or exudate.

As used herein, the terms “subject, ” “individual” or “patient” refer to a human or a non-human mammal. A subject may be a non-human primate, domestic animal, wild animal, farm animal, or a laboratory animal. For example, the subject may be a dog, cat, goat, horse, pig, mouse, rabbit, rat, or the like. The subject may be a human. The subject may be healthy or suffering from or susceptible to a disease, disorder or condition. A patient refers to a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects.

A “control” sample or value refers to a sample that serves as a reference, usually a known reference, for comparison to a test sample. For example, a test sample can be taken from a test subject, and a control sample can be taken from a control subject, such as from a known normal (non-disease) individual. A control can also represent an average value gathered from a population of similar individuals, e.g., disease patients or healthy individuals with a similar medical background, same age, weight, etc. One of skill will recognize that controls can be designed for assessment of any number of parameters.

As used herein the terms “treatment” or “treating” refer to administering a composition to a subject or a system to treat one or more symptoms of a disease. The effect of the administration of the composition to the subject can be, but is not limited to, the cessation of a particular symptom of a condition, a reduction or prevention of the symptoms of a condition, a reduction in the severity of the condition, the complete ablation of the condition, a stabilization or delay of the development or progression of a particular event or characteristic, or minimization of the chances that a particular event or characteristic will occur.

As used herein the terms “effective amount” and “therapeutically effective amount, ” used interchangeably, as applied to the peptides, therapeutic agents, and pharmaceutical compositions described herein, refer to the quantity necessary to render the desired therapeutic result. For example, an effective amount is a level effective to treat, cure, or alleviate the symptoms of a disease for which the composition and/or therapeutic agent, or pharmaceutical composition, is/are being administered. Amounts effective for the particular therapeutic goal sought will depend upon a variety of factors including the disease being treated and its severity and/or stage of development/progression; the bioavailability and activity of the specific compound and/or antineoplastic, or pharmaceutical composition, used; the route or method of administration and introduction site on the subject.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. +/-10%; in other embodiments the values may range in value either above or below the stated value in a range of approx. +/-5%; in other embodiments the values may range in value either above or below the stated value in a range of approx. +/-2%; in other embodiments the values may range in value either above or below the stated value in a range of approx. +/-1%.

II. Peptides, Virus-Like Particles, and Compositions

Described are peptides, particles, compositions, kits, and assays that detect HEV-C infections in humans, protect against HEV-C infections, distinguishing between HEV-C and HEV-A infections and provide accurate and fast methods for diagnosing HEV-C infection.

The examples below show the failures of the existing compositions, kits, and methods to detect HEV-C infections or protect against HEV-C infections. There is no recognition with the existing diagnostic methods that detect HEV-A miss or misdiagnose a HEV-C infection in a subject.

The peptides, virus-like particles, and compositions include a peptide with substile amino acid similarity to a portion of HEV-C genotype 1 (HEV-C1) open reading frame 2 (ORF2) capsid protein, termed HEV-C1 p241 peptide. There is no substantial amino acid sequence identity between HEV-C1 p241 peptide and ORF2 capsid proteins of other HEV species, including as HEV-A. This makes HEV-C1 p241 and its substantially similar variants useful at detecting HEV-C infections in humans, protecting against HEV-C infections, providing accurate and fast methods for diagnosing HEV-C infections, and distinguishing between HEV-C and HEV-A infections.

A. Peptides

The peptides typically have at least a 90%amino acid sequence identity with SEQ ID NO: 1 or SEQ ID NO: 31, at least a 95%amino acid sequence identity with SEQ ID NO: 1 or SEQ ID NO: 31, or an amino acid sequence as in SEQ ID NO: 1 or SEQ ID NO: 31. Other peptides may have at least a 90%amino acid sequence identity with SEQ ID NO: 3 or SEQ ID NO: 29, at least a 95%amino acid sequence identity with SEQ ID NO: 3 or SEQ ID NO: 29, or an amino acid sequence as in SEQ ID NO: 3 or SEQ ID NO: 29.

The peptides typically include homologous peptides of hepatitis E virus genotype 4 and rat hepatitis E, called HEV-A4 p239 and HEV-C1 p241, respectively. These peptides share only 93%and 50-60%identity (i.e. %of amino acids similar) , respectively, with the original HEV-A1 p239. These peptides also form VLPs.

1. HEV-C1 p241 and its Variants

The HEV-C p241 (357I-597V, GenBank code: AYF53239.1) and its variants may form VLPs and compositions for detecting HEV-C infections in humans, providing accurate and fast methods for diagnosing HEV-C infection, distinguishing between HEV-C and HEV-A infections, and protecting against HEV-C infections.

The amino acid sequence for HEV-C1 p241 is as follows:

SEQ ID NO: 1 spans residues 357I-597V of the 644 amino acid long capsid protein of rat HEV, strain “LCK-3110” . The lowercase portion is shown as SEQ ID NO: 31 in Figure 1B. The peptide is encoded by the viruses’ ORF2. The variants of the SEQ ID NO: 1 or SEQ ID NO: 31 include peptides that have at least 90%amino acid sequence identity with SEQ ID NO: 1 or SEQ ID NO: 31. Examples of variant peptides include, but are not limited to, those containing SEQ ID NO: 32 or SEQ ID NO: 33 shown in Figure 1D. Other variants include OFR2 peptides of this region from HEV-C strains having GenBank Accession Nos: GU345042, GU345043, JN167537, AB847308, KM516906, AB847306, AB847309, AB847305, AB847307, JX120573, AB890001, MG020022, KU670940, and LC549186.

The nucleotide sequence for HEV-C1 p241 is as follows:

Variants of SEQ ID NO: 1 and SEQ ID NO: 2 include amino acid or nucleic acid sequences with substantial sequence identity, such as at least about 85-90%, about 90%, preferably about 95%or more sequence identity with SEQ ID NO: 1 or SEQ ID NO: 31 and SEQ ID NO: 2, respectively. Amino acid variability in the variants typically retains essential peptide properties. Generally, differences are limited so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions) . A variant of a peptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.

2. HEV-A4 p239

The HEV-A4 p239 (382I-620A, GenBank code: MW660888) has amino acid sequence as follows:

(SEQ ID NO: 3) . The lowercase portion is shown as SEQ ID NO: 29 in Figure 1B.

The HEV-A4 p239 has a substantial amino acid sequence identity with the p239 peptide of HEV-A genotype 1 (HEV-A1 p239) , and other capsid proteins of HEV species A.

The nucleotide sequence for HEV-A4 p239 is as follows:

The GenBank accession numbers of sequences at the corresponding regions in other HEV species A include: L08816 (HEV-A1) , AB369687 (HEV-A3) , and AJ272108 (HEV-A4) .

B. Virus-Like Particles

The peptides HEV-C1 p241, its variants, and HEV-A4 p239, are typically recombinant peptides purified from expression systems. They are typically isolated, purified, and folded to form virus-like particles (VLPs) .

The VLPs may include two, three, four, or more peptides assembled into one particle. Typically, the VLPs are homodimers, homotrimers, homooligomers, or homomultimers of the peptide. The VLPs may be homodimers, homotrimers, homooligomers, or homomultimers of HEV-C1 p241, where each monomer has a SEQ ID NO: 1 or its variant, such as monomer with a substantial amino acid sequence identity to SEQ ID NO: 1. Typically, the HEV-C1 VLPs include be homodimers, homotrimers, homooligomers, or homomultimers of HEV-C1 p241, or of monomers having at least 85%, preferably at least 90%, or 95%amino acid sequence identity with SEQ ID NO: 1.

The VLPs may be homodimers, homotrimers, homooligomers, or homomultimers of HEV-A4 p239, where each monomer has a SEQ ID NO: 3 or its variant, such as monomer with a substantial amino acid sequence identity to SEQ ID NO: 3. Typically, the HEV-A4 VLPs include be homodimers, homotrimers, homooligomers, or homomultimers of HEV-A4 p239, or of monomers having at least 85%, preferably at least 90%, or 95%amino acid sequence identity with SEQ ID NO: 3.

C. Compositions

The peptides or VLPs may be included in a composition. Typically, the peptides or the VLPs are included in a composition with a pharmaceutically acceptable excipient and/or an adjuvant.

The composition may include a plurality of peptides or VLPs where the peptides have at least a 90%or at least 95%amino acid sequence identity with SEQ ID NO: 1 or SEQ ID NO: 31. The composition may include a plurality of peptides or VLPs where the peptides have an amino acid sequence as in SEQ ID NO: 1 or SEQ ID NO: 31. The composition may include peptides or VLPs where the peptides have at least a 90%or at least 95%amino acid sequence identity with SEQ ID NO: 3 or SEQ ID NO: 29. The composition may include a plurality of peptides or VLPs where the peptides have an amino acid sequence as in SEQ ID NO: 3 or SEQ ID NO: 29.

The peptides or VLPs in a bivalent composition. The bivalent composition may include a mix of a plurality of peptides having at least a 90%or at least 95%amino acid sequence identity with SEQ ID NO: 1 or SEQ ID NO: 31 and a plurality of peptides having at least a 90%or at least 95%amino acid sequence identity with SEQ ID NO: 3 or SEQ ID NO: 29. The peptides of VLPs in the composition may be a mix of 1) a plurality of peptides having an amino acid sequence as in SEQ ID NO: 1 or SEQ ID NO: 31, and 2) a plurality of peptides having an amino acid sequence as in SEQ ID NO: 3 or SEQ ID NO: 29.

The compositions may contain effective amounts of peptides or the VLPs. Suitable effective amounts include between about 0.1 μg and about 10 000 μg, about 1 μg and about 10 000 μg, about 5 μg and about 10 000 μg, about 10 μg and about 9000 μg, about 10 μg and about 8000 μg, about 10 μg and about 7000 μg, about 10 μg0and about 6000 μg, about 10 μg and about 5000 μg, or between about 10 μg and about 1000 μg of the peptides or the VLPs.

The compositions may be provided in volumes containing between about 100 μl and about 5000 μl of the composition containing between about 0.1 μg and about 10 000 μg of the peptides or VLPs. The compositions may be provided in vials containing between about 200 μl and about 4000 μl, about 300 μl and about 3000 μl, about 300 μl and about 2000 μl, or between about 400 μl and about 1000 μl of the composition. These volumes of the composition may include between 0.1 μg and about 10 000 μg, about 1 μg and about 10 000 μg, about 5 μg and about 10 000 μg, about 10 μg and about 9000 μg, about 10 μg and about 8000 μg, about 10 μg and about 7000 μg, about 10 μg0and about 6000 μg, about 10 μg and about 5000 μg, or between about 10 μg and about 1000 μg of the peptides or the VLPs. In some aspects, the compositions are provided in volumes of about 200 μl, about 300 μl, about 400 μl, about 500 μl, about 600 μl, about 700 μl, about 800 μl, about 900 μl, or about 1000 μl and contain between about 10 μg and about 1000 μg, between about 10 μg and 800 μg, or between about 10 μg and 500 μg of the peptides or the VLPs in these volumes. Exemplary compositions may be provided in volumes of about 500 μl and may contain between about 10 μg and 1000 μg, or between about 10 μg and 100 μg of the peptides or VLPs.

The compositions may include a pharmaceutically acceptable excipient, carrier, and/or adjuvant.

1. Excipients

Excipients may be chosen based on the application of the composition, as would be understood by those of skill in the art. The excipients may include antioxidants, chelating agents, preservatives, suspending agents, and combinations thereof.

Suitable antioxidants include, but are not limited to, butylated hydroxytoluene, alpha tocopherol, ascorbic acid, fumaric acid, malic acid, butylated hydroxyanisole, propyl gallate, sodium ascorbate, sodium metabisulfite, ascorbyl palmitate, ascorbyl acetate, ascorbyl phosphate, Vitamin A, folic acid, flavons or flavonoids, histidine, glycine, tyrosine, tryptophan, carotenoids, carotenes, alpha-Carotene, beta-Carotene, uric acid, pharmaceutically acceptable salts thereof, derivatives thereof, and combinations thereof.

Suitable chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA) , and combinations thereof.

Suitable humectants include, but are not limited to, glycerin, butylene glycol, propylene glycol, sorbitol, triacetin, and combinations thereof.

Preservatives can be used to prevent the growth of fungi and other microorganisms. Suitable preservatives include, but are not limited to, benzoic acid, butylparaben, ethyl paraben, methyl paraben, propylparaben, sodium benzoate, sodium propionate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, thimerosal, and combinations thereof.

Excipients may include suspending agents such as sterile water, phosphate buffered saline, saline, or a non-aqueous solution such as glycerol.

Pharmaceutically acceptable excipients include compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio, in accordance with the guidelines of agencies such as the Food and Drug Administration.

2. Carriers

The skilled person can select the appropriate carrier based on the desired use. For example, in some embodiments the carrier can be polyethylene glycol, polypropylene glycol, a mixture or polymer of sugars (lactose, sucrose, dextrose, etc. ) , salts, poloxamers, hydroxypropylcellulose, polyvinyl alcohol, other water-soluble food grade excipients, or even other excipients.

The carrier may also include a water insoluble polymer. Examples of such polymers are ethylcellulose, acrylic resins, co-polymer of methacrylic acid and acrylic acid ethyl ester, polylactic acid, PLGA, polyurethane, polyethylene vinyl acetate copolymer, polystyrene-butadiene copolymer and silicone rubber, or mixtures thereof.

3. Adjuvants

Typically, the adjuvant is any substance stimulating an immune response against the administered peptides or VLPs. The adjuvants may be amorphous aluminum hydroxyphosphate sulfate (AAHS) , aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate (Alum) , Freund's complete adjuvant, Freund's incomplete adjuvant, Monophosphoryl lipid A (MPL) with aluminum salt, Oil in water emulsion composed of squalene, Quil A, MPL and QS-21, a natural compound extracted from the Chilean soapbark tree, combined in a liposomal formulation, or Cytosine phosphoguanine (CpG) -containing immunostimulatory oligonucleotides, a synthetic form of DNA that mimics bacterial and viral genetic material.

4. Exemplary compositions

In some aspects the compositions include the peptides or VLPs in a solid form containing, in addition to the peptides or the VLPs, one or more of the following: sodium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, aluminum hydroxide, and thiomersal.

In other aspects the compositions include the peptides or VLPs in a liquid form and containing, in addition to the peptides or the VLPs, one or more of the following: sodium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, aluminium hydroxide, thiomersal, and water.

III. Methods of Making

The HEV-A4 p239 and HEV-C1 peptides and VLPs are typically made using standard molecular biology techniques of any one or a combination of gene expression, cloning into expression vector, transforming bacterial cells with the expression vector, inducing expression, harvesting inclusion bodies from the bacterial cells, renaturation by step-wise dialysis in decreasing concentrations of urea, and purifying the peptides. This process induces folding into VLPs.

More specific methods include obtaining the gene encoding HEV-A4 p239 (382I-620A, GenBank code: MW660888) , the 239 amino acid peptide corresponding to HEV-A1 p239, and the gene encoding HEV-C1 p241 (357I-597V, GenBank code: AYF53239.1) . This may be achieved using amplification from a clinical isolate using primer pairs such as those of SEQ ID NO: 19 and SEQ ID NO: 20 to amplify the gene encoding HEV-A4 p239, or those of SEQ ID NO: 21 and SEQ ID NO: 22 to amplify the gene encoding HEV-C1 p241.

The amplified genes may then be cloned into expression cassettes of bacterial expression vectors, and downstream of a histidine tag. Expression vectors are well known in the art. Suitable sites in expression cassettes include the Nde I and Xol I sites of the bacterial expression vector pETH in-frame and downstream of a series of 6 histidine residues. Recombinant HEV-A4 p239 and HEV-C1 p241 peptides may then be overexpressed in E. coli. The produced protein may be harvested from inclusion bodies rather than supernatant due to different conformation and higher concentrations in the inclusion body fraction. Inclusion bodies may be harvested from E. coli sediment by liquid-solid separation followed by dissolution in urea.

Solubilized peptides may then be renatured by step-wise dialysis in decreasing concentrations of urea. Refolded proteins may be purified by gel filtration chromatography with methods well know in the art (BIO-RAD, Hercules, USA) . Concentration of protein may be determined by any suitable method, including by the BCA protein assay (Thermo Fisher, Waltham, USA) .

The purified HEV-C1 p241 and HEV-A4 p239 peptides may be allowed to refold into VLPs.

The purified HEV-C1 p241 and HEV-A4 p239 peptides and VLPs may be stored lyophilized, frozen, or in liquid form in suitable vials.

IV. Methods of Using

A. Protection against HEV-C

The peptides, VLPs, and compositions may be used to protect a subject from HEV-C infection. The peptides, VLPs, and compositions may be used to protect a subject from HEV-C and HEV-A infections.

Typically, the peptides or the VLPs are mixed with an adjuvant, and, optionally, with an excipient, and administered to a subject.

The compositions with peptides or the VLPs and the adjuvants may be administered at an effective amount of the peptides or the VLPs to elicit an immune response against the peptides or the VLPs. The effective amounts of peptides or the VLPs in the composition for inducing an immune response against the HEV-C include between about 0.1 μg/kg and about 1000 μg/kg, about 1 μg/kg and about 1000 μg/kg, about 5 μg/kg and about 1000 μg/kg, about 10 μg/kg and about 900 μg/kg, about 10 μg/kg and about 800 μg/kg, about 10 μg/kg and about 700 μg/kg, about 10 μg/kg and about 600 μg/kg, about 10 μg/kg and about 500 μg/kg, between about 50 μg/kg and about 500 μg/kg, or between about 0.1 μg/kg and about 10 μg/kg of the peptides or the VLPs.

The compositions providing protection against HEV-C may be provided in vials containing between about 100 μl and about 5000 μl of the composition containing between about 0.1 μg and about 10 000 μg of the peptides or VLPs. The compositions may be provided in vials containing between about 200 μl and about 4000 μl, about 300 μl and about 3000 μl, about 300 μl and about 2000 μl, or between about 400 μl and about 1000 μl of the composition. These volumes of the composition may include between 0.1 μg and about 10 000 μg, about 1 μg and about 10 000 μg, about 5 μg and about 10 000 μg, about 10 μg and about 9000 μg, about 10 μg and about 8000 μg, about 10 μg and about 7000 μg, about 10 μg0and about 6000 μg, about 10 μg and about 5000 μg, or between about 10 μg and about 1000 μg of the peptides or the VLPs. In some aspects, the compositions are provided in volumes of about 200 μl, about 300 μl, about 400 μl, about 500 μl, about 600 μl, about 700 μl, about 800 μl, about 900 μl, or about 1000 μl and contain between about 10 μg and about 1000 μg, between about 10 μg and 800 μg, or between about 10 μg and 500 μg of the peptides or the VLPs in these volumes. Exemplary compositions providing protection against HEV-C may be provided in volumes of about 500 μl and contain between about 10 μg and 1000 μg, or between about 10 μg and 100 μg of the peptides or VLPs.

The compositions may be administered, once, twice, three times, four times, or more times, as needed, to elicit an immune response against the peptides or the VLPs. Assays detecting immune response against the peptides or the VLPs include immunoassays for detecting IgG or IgM specific to the peptides or the VLPs.

The compositions containing dual VLPs system based on human hepatitis E virus genotype 4 and rat hepatitis E virus (the bivalent compositions) can be deployed in antibody tests for detection and differentiation of human and rat hepatitis E virus infection. Also, they trigger strong immune responses in animal models, and may be used in vaccines against rat hepatitis E infection or even bivalent vaccines against human and rat hepatitis E infection. The rat hepatitis E virus is a newly discovered infection in humans without commercially available antibody tests. Provided are:

1. Dual VLP based antibody tests that can detect and differentiate between human and rat hepatitis E infection. When these VLPs are deployed in commonly used antibody assay formats such as Western Blot and ELISA, they differentiate between the antibody profiles of human and rat hepatitis E infection.

2. VLPs can protect against hepatitis E challenge. The VLPs are immunogenic and administration of HEV-C 1 p241 can protect subjects against rat hepatitis E virus infection. These VLPs can be used as vaccines in humans.

B. Assays detecting HEV-C infections

The peptides, VLPs, and compositions may be used in a number of detection assays. Typical detection assays include immunoassays with a sample obtained from a subject.

The samples may also be used for HEV-C detection using amplification assays, such as polymerase chain reaction assays.

1. Immunoassays

Assays and conditions for the detection of immunocomplexes are known to those of skill in the art. Such assays include, for example, competition assays, direct reaction assays. sandwich-type assays, immunoblots, ELISA, EIA, competitive ELISA, and others. The assays may be quantitative or qualitative. There are a number of different conventional assays for detecting formation of an antibody-peptide complex. For example, the detecting step can include performing an ELISA assay, performing a lateral flow immunoassay, performing an agglutination assay, analyzing the sample in an analytical rotor, or analyzing the sample with an electrochemical, optical, or opto-electronic sensor. These different assays are well-known to those skilled in the art.

Detection may be either qualitative or quantitative and typically uses an antibody-detection label conjugate. The most commonly used detectable moieties in immunoassays are enzymes and fluorophores. In the case of an enzyme immunoassay (EIA or ELISA) , an enzyme such as horseradish perodixase, glucose oxidase, beta-galactosidase, alkaline phosphatase, and the like, is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. The substrates to be used with the specific enzymes are generally chosen for the production of a detectable color change, upon hydrolysis of the corresponding enzyme. In the case of immunofluorescence, the second antibody is chemically coupled to a fluorescent moiety without alteration of its binding capacity. After binding of the fluorescently labeled antibody to the immunocomplex and removal of any unbound material, the fluorescent signal generated by the fluorescent moiety is detected, and optionally quantified. Alternatively, the second antibody may be labeled with a radioisotope, a chemiluminescent moiety, or a bioluminescent moiety.

In some embodiments, the assay utilizes a solid phase or substrate to which the peptides or the VLPs are directly or indirectly attached. Accordingly in some embodiments, the peptides or the VLPs are attached to or immobilized on a substrate, such as a solid or semi-solid support. The attachment can be covalent or non-covalent, and can be facilitated by a moiety associated with the peptide that enables covalent or non-covalent binding, such as a moiety that has a high affinity to a component attached to the carrier, support or surface. The substrate may be a bead, such as a colloidal particle (e.g., a colloidal nanoparticle made from gold, silver, platinum, copper, metal composites, other soft metals, core-shell structure particles, or hollow gold nanospheres) or other type of particle (e.g., a magnetic bead or a particle or nanoparticle with silica, latex, polystyrene, polycarbonate, polyacrylate, or PVDF) . Such particles can include a label (e.g., a colorimetric, chemiluminescent, or fluorescent label) and can be useful for visualizing the location of the peptides or the VLPs during immunoassays. In some embodiments, the substrate is a dot blot or a flow path in a lateral flow immunoassay device. For example, the peptides or the VLPs can be attached or immobilized on a porous membrane, such as a PVDF membrane (e.g., an Immobilon ^TM membrane) , a nitrocellulose membrane, polyethylene membrane, nylon membrane, or a similar type of membrane.

In some embodiments, the substrate is a flow path in an analytical rotor. In some embodiments, the substrate is a tube or a well, such as a well in a plate (e.g., a microtiter plate) suitable for use in an ELISA assay. Such substrates can include glass, cellulose-based materials, thermoplastic polymers, such as polyethylene, polypropylene, or polyester, sintered structures composed of particulate materials (e.g., glass or various thermoplastic polymers) , or cast membrane film composed of nitrocellulose, nylon, polysulfone, or the like. A substrate can be sintered, fine particles of polyethylene, commonly known as porous polyethylene, for example, 0.2-15 micron porous polyethylene from Chromex Corporation (Albuquerque, N. Mex. ) . All of these substrate materials can be used in suitable shapes, such as films, sheets, or plates, or they may be coated onto or bonded or laminated to appropriate inert carriers, such as paper, glass, plastic films, or fabrics. Suitable methods for immobilizing peptides on solid phases include ionic, hydrophobic, covalent interactions and the like.

Immunoassay methods are well known in the art and include steps of contacting a test sample and a peptide or VLP on a substrate to initiate binding of the immunoglobulins in the test sample to the peptides or VLPs to form immunoglobulin-peptide complexes or immunoglobulin-VLP complexes. The immunoassays also include the step of detecting this binding by developing a signal from the binding. The methods may also include a comparison to a known control sample, control value, or a signal from a control sample. Positive detection of binding is typically indicative of a presence of anti-peptide or anti-VLP immunoglobulins in the test sample.

Generally, the immunoassay methods include (a) contacting the peptides or the VLPs with a test sample on a substrate or in a test vessel, (b) developing a signal, and (c) measuring the signal. The method may also include contacting a control sample with the peptides or the VLPs in the control vessel.

Generally, developing a signal includes a step of contacting an antibody-detection label conjugate with the complexes in the test vessels and/or control vessels.

The steps (a) and (b) may include additional steps of blocking, washing, and incubating, as needed per general immunoassay protocol.

2. Amplification assays

Another type of assay for detecting HEV-C or distinguishing between infections by HEV-C and by other HEV species include amplification assays. These include polymerase chain reactions (PCR) assays that are rapid, specific, and sensitive assay process for detection of HEV-C in biological samples by amplifying one or more nucleotide sequences. A process for RNA genomes may involve reverse transcriptase PCR (RT-PCR) or qRT-PCR methods. General principles of real time quantitative RT PCR are known in the art and are for instance described in Poitras et al., Reviews in Biology and Biotechnolog, 2: 1-11 (2002) and Gibson et al., Genome Research, 6: 995-1001 (1996) . Real-time PCR based on the

technology allows DNA or cDNA quantification over a large dynamic range (10 to 10 ⁷ copies) , and is therefore well adapted to the quantification of viral genomes. Moreover, the possibility of handling numerous samples and the absence of post-PCR handling makes it a safe and convenient approach for clinical diagnosis.

An oligonucleotide forward primer with a nucleotide sequence complementary to a unique sequence in a region of interest HEV-C nucleotide sequence is hybridized to its complementary sequence and extended. Similarly, a reverse oligonucleotide primer complementary to a second HEV-C nucleotide sequence in the same or an alternate region is hybridized and extended. This system allows for amplification of specific gene sequences and is suitable for simultaneous or sequential detection systems.

The polymerization assay detects the presence or absence of nucleic acid molecules of HEV-C in a biological sample. The process involves obtaining a biological sample contacting the sample with a compound or an agent capable of detecting a nucleic acid sequence of HEV-C or HEV-A, such that the presence of HEV-C or HEV-A is detected in the sample. Preferably, the assay is a quantitative real time polymerase chain reaction (qPCR) using the primers that are constructed based on a partial nucleotide sequence of cDNA corresponding to HEV-C or HEV-A genomic regions of interest. A forward primer and a reverse primer are typically used for these purposes. In some aspects, the assay includes primer and probe sequences. The polymerization process typically results in an amplicon.

The primer and probe sequences used in production of an amplicon are specifically tailored and designed to satisfy several different parameters depending on the primer or probe. In general, an amplicon is ideally less than 150 nucleotides, optionally from 75 to 150 nucleotides or any value or range therebetween.

The assay may be used for the simultaneous or sequential detection of the presence or absence of nucleic acid molecules of HEV-C and HEV-A, individually or in any combination, in a biological sample. The assay may be a real-time quantitative PCR assay (Holland et al., PNAS 88 (16) : 7276 (1991) ) . Other RT-PCR systems and protocols that can be used use Molecular Beacons probes, Scorpion probes, SYBRgreen, multiple reporters for multiplex PCR, combinations thereof, or other DNA detection systems.

The assays are performed on an instrument designed to perform such assays, for example those available from Applied Biosystems (Foster City, Calif. ) . The assays typically include subjecting a nucleic acid from the sample to RT-PCR or PCR reactions using specific primers, and detecting the amplified product.

The HEV virus nucleic acid sequences are typically converted to complementary DNA (cDNA) sequences and then amplified before being detected. The term “amplified” defines the process of making multiple copies of the nucleic acid in either RNA or cDNA form from a single or lower copy number of nucleic acid sequence molecule. The amplification of nucleic acid sequences is carried out in vitro by biochemical processes known to those of skill in the art. The amplification agent may be any compound or system that will function to accomplish the synthesis of primer extension products, including enzymes. Suitable enzymes for this purpose include, for example, E. coli DNA polymerase I, Taq polymerase, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, AmpliTaq Gold DNA Polymerase from Applied Biosystems, other available DNA polymerases, reverse transcriptase (preferably iScript RNase H+ reverse transcriptase) , ligase, and other enzymes, including heat-stable enzymes (i.e., those enzymes that perform primer extension after being subjected to temperatures sufficiently elevated to cause denaturation) .. Suitable enzymes will facilitate combination of the nucleotides in the proper manner to form the primer extension products that are complementary to each mutant nucleotide strand. Generally, the synthesis is initiated at the 3’-end of each primer and proceed in the 5’-direction along the template strand, until synthesis terminates, producing molecules of different lengths. There may be amplification agents, however, that initiate synthesis at the 5'-end and proceed in the other direction, using the same process as described above. In any event, the process of the invention is not to be limited to the embodiments of amplification described herein.

One process of in vitro amplification may include the polymerase chain reaction (PCR) such as those described in U.S. Pat. Nos. 4,683,202 and 4,683,195. The term “polymerase chain reaction” refers to a process for amplifying a DNA base sequence using a heat-stable DNA polymerase and two oligonucleotide primers, one complementary to the (+) -strand at one end of the sequence to be amplified and the other complementary to the (-) -strand at the other end. Because the newly synthesized DNA strands can subsequently serve as additional templates for the same primer sequences, successive rounds of primer annealing, strand elongation, and dissociation produce rapid and highly specific amplification of the desired sequence. Many polymerase chain processes are known to those of skill in the art and may be used in the process of the invention.

Primers used according to the process are complementary to each strand of nucleotide sequence to be amplified. The term “complementary” means that the primers must hybridize with their respective strands under conditions that allow the agent for polymerization to function. In other words, the primers that are complementary to the flanking sequences hybridize with the flanking sequences and permit amplification of the nucleotide sequence. Preferably, the 3’ terminus of the primer that is extended is perfectly base paired with the complementary flanking strand. In some embodiments, probes possess nucleotide sequences complementary to one or more of the strands.

Those of ordinary skill in the art will know of various amplification processes that can also be utilized to increase the copy number of a target HEV nucleic acid sequence. The nucleic acid sequences detected in the process of the invention are optionally further evaluated, detected, cloned, sequenced, and the like, either in solution or after binding to a solid support, by any process usually applied to the detection of a specific nucleic acid sequence such as another polymerase chain reaction, oligomer restriction (Saiki et al., BioTechnology 3: 1008 1012 (1985) ) , allele-specific oligonucleotide (ASO) probe analysis (Conner et al., PNAS 80: 278 (1983) ) , oligonucleotide ligation assays (OLAs) (Landegren et al., Science 241: 1077 (1988) ) , RNase Protection Assay and the like. Molecular techniques for DNA analysis have been reviewed (Landegren et al., Science 242: 229 237 (1988) ) . Following DNA amplification, the reaction product may be detected by the high level of the amplified signal from the probe. In another embodiment, amplification primers are fluorescently labeled and run through an electrophoresis system. Visualization of amplified products may be by laser detection followed by computer assisted graphic display.

i. Primer pairs for PCR

Primer pairs for detecting HEV-A may include nucleic acid sequences containing sequences as in SEQ ID NO: 19 and SEQ ID NO: 20.

Primer pairs for detecting HEV-C may include nucleic acid sequences containing sequences as in SEQ ID NO: 21 and SEQ ID NO: 22.

Specifically for detecting HEV-C, the primer pairs may include nucleic acid sequences containing sequences as in SEQ ID NO: 26 and SEQ ID NO: 27.

ii. Primer pairs for Nested PCR

An additional amplification step through polymerization may include a nested PCR of the PCR products.

For these nested PCR amplifications, the primer pairs may include nucleic acid sequences containing sequences as in SEQ ID NO: 23 and SEQ ID NO: 24.

C. Detecting HEV-C infections and Distinguishing between HEV-C and HEV-A infections

The described assays may be used to distinguish between HEV-C and HEV-A infections in a subject. The assays may have the steps of contacting a platform or test vials having a

(a) a plurality of peptides having at least a 90%amino acid sequence identity with SEQ ID NO:1 or SEQ ID NO: 31, at least 95 %amino acid sequence identity with SEQ ID NO: 1 or SEQ ID NO: 31, or an amino acid sequence as in SEQ ID NO: 1 or SEQ ID NO: 31, and optionally,

(b) a plurality of peptides having at least a 90%amino acid sequence identity with SEQ ID NO:3 or SEQ ID NO: 29,

with a sample or a test sample.

The plurality of peptides on the platform or in the test vials may be synthetic virus-like particles. Typically, a sample is a bodily fluid or mucus including blood, serum, plasma, excrement, exudate, saliva, sputum, tear, sweat, urine, or a vaginal discharge obtained from a subject. The sample may be diluted with a buffer forming a test sample. The sample may be diluted at a ratio between about 1: 5 and 1: 500 (v/v) of the sample to a buffer.

The method may include contacting a plurality of test samples with the plurality of peptides having at least a 90%amino acid sequence identity with SEQ ID NO: 1 or SEQ ID NO: 31 in a first set of platforms or test vessels. The method may also include contacting a plurality of test samples with the plurality of peptides having at least a 90%amino acid sequence identity with SEQ ID NO: 3 or SEQ ID NO: 29 in a second set of platforms or test vessels. The method then typically includes developing a signal from the contacting of the test sample with the plurality of peptides.

Typically, the method detects HEV-C infection when signal develops from the contacting of the test sample with the plurality of peptides having at least a 90%amino acid sequence identity with SEQ ID NO: 1 or SEQ ID NO: 31, at least 95 %amino acid sequence identity with SEQ ID NO: 1, or having an amino acid sequence as in SEQ ID NO: 1 or SEQ ID NO: 31. Typically, the method detects HEV-A infection when signal develops from the contacting of the test sample with the plurality of peptides having at least a 90%amino acid sequence identity with SEQ ID NO: 3 or SEQ ID NO: 29.

In some aspects, the method distinguishes between HEV-A and HEV-C infections in a subject. Typically, the method distinguishes between HEV-A and HEV-C infections when the method includes both steps (a) and (b) above, and the signal developed from the platforms or the test vials having (a) is significantly different from the signal developed from the platforms or the test vials having (b) .

In some aspects, the method detects both HEV-A and HEV-C infections in a subject. Typically, the method detects both HEV-A and HEV-C infections when the method includes both steps (a) and (b) above, and the signal developed from the platforms or the test vials having (a) is not significantly different from the signal developed from the platforms or the test vials having (b) .

Generally, the method detects a HEV-C infection or a combination of HEV-A and HEV-C infections in the sample at a sensitivity of about or over 80%and a specificity of about or over 70%. Generally, the method distinguishes between HEV-A and HEV-C infections in the sample at a sensitivity of about or over 80%.

D. Assay Accuracy

1. Early Detection

The assay compositions are highly specific to antibodies against HEV-C or HEV-A. Typically, the assay components have accuracy of greater than 70%specificity and greater than greater than 70%, greater than 80%, greater than 90%sensitivity for detecting HEV-C or HEV-A infection in the subject.

2. Rapid Detection

The assays described are typically performed within a time period between about 5 min and 5 hours, within about 15 min and 4 hours, within about 15 min and 3 hours, or within about 2 hours. The assays are rapid and provide fast and accurate results on presence of HEV-C or HEV-A infection in a sample.

Immunoassays in dipstick format typically are performed within less than one hour, such as within about 5-60 min.

Immunoassays in plate-form ELISA may be performed within 30 min to 3 hours.

Amplification assays using polymerization in thermocyclers may be performed within 30 min to 3 hours.

Generally, the assays are developed for fast and accurate detection of HEV-C or HEV-A in a sample, or for fast and accurate detection of HEV-C or HEV-A infection. The assays typically distinguish between HEV-C and HEV-A infections.

3. Accurate Detection

Typically the assay compositions and assays provide a results with high sensitivity and high specificity, such as with sensitivity over about 80%and specificity over about 80%, with sensitivity over about 85%and specificity over about 85%, or with sensitivity over about 90%and specificity over about 90%.

Sensitivity of a test is the ability to correctly identify true positives, i.e., subjects infected with HEV-C and/or HEV-A. For example, sensitivity can be expressed as a percentage, the proportion of actual positives which are correctly identified as such (e.g., the percentage of test subjects having HEV-C and/or HEV-A correctly identified by the test as having HEV-C and/or HEV-A) . A test with high sensitivity has a low rate of false negatives, i.e., the cases of HEV-C and/or HEV-A not identified as such. Generally, the disclosed assay compositions and assays have a sensitivity of about or above 70%, about 80%, about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

Specificity of a test is the ability to correctly identify true negatives, i.e., the individuals that have no HEV-C and/or HEV-A infection. For example, specificity can be expressed as a percentage, the proportion of actual negatives which are correctly identified as such (e.g., the percentage of test subjects not having HEV-C and/or HEV-A correctly identified by the test as not having HEV-C and/or HEV-A) . A test with high specificity has a low rate of false positives, i.e., the cases of individuals not having HEV-C and/or HEV-A but suggested by the test as having HEV-C and/or HEV-A. Generally, the disclosed assay compositions and assays have a specificity of about or over 70%, about 80%, about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

4. Detection and Diagnosis

The current or past exposure or infection with HEV-C and/or HEV-A can be detected and/or diagnosed using the disclosed compositions and methods. Typically the presence and/or elevated amount of HEV-C and/or HEV-A antibodies in subject’s biological sample as compared to a control is indicative of current or past exposure or an active infection with HEV-C and/or HEV-A. For example, the method for assisting in the detection or diagnosis of current or present exposure or infection with HEV-C and/or HEV-A in a subject can include determining the presence or level of antibodies against HEV-C1 p241 and/or HEV-A4 p239 protein or its fragments, or the presence or level of nucleic acids encoding these proteins, in a biological sample from the subject, wherein the presence of, or an elevated level of, the antibodies or nucleic acids in the biological sample relative to the level of antibodies in a control is indicative of current or past exposure or infection with HEV-C and/or HEV-A.

Any of the methods can be combined with a methods of treatment. In preferred embodiments, the method of treatment includes administering the subject an effective amount of an anti-viral therapy, analgesic therapy, fever reducers, or anti-hepatitis therapy.

IV. Kits

The peptides, VLPs, and compositions described above as well as other materials can be packaged together in any suitable combination as a kit useful for performing, or aiding in the performance of, the disclosed method. It is useful if the kit components in a given kit are designed and adapted for use together in the disclosed method. For example, disclosed are kits with one or more of assay platform, assay buffer, sample holder, reporter compound, and/or secondary detection compounds. The kits may include a sterile needle, ampule, tube, container, or other suitable vessels for holding assay components and/or performing the assay. The kits may include instructions use.

The kits for detecting the presence of HEV-C viral nucleic acids in a sample may include one or more primer pairs. The kit, for example, includes a labeled compound or agent capable of detecting a nucleic acid molecule in a test sample and, in certain embodiments, for determining the titer in the sample.

For oligonucleotide-based kits, the kit includes for example: (1) an oligonucleotide, e.g., a detectably labeled probe, which hybridizes to a nucleic acid sequence of the HEV virus and/or (2) a pair of primers (one forward and one reverse) useful for amplifying a nucleic acid molecule containing the HEV viral sequence. The kit can also include, e.g., a buffering agent, a preservative, or a protein stabilizing agent. The kit can also include components necessary for detecting the detectable agent (e.g., an enzyme or a substrate) . The kit can also contain a control sample or a series of control samples which is assayed and compared to the test sample contained. Each component of the kit is usually enclosed within an individual container and all of the various containers are usually enclosed within a single package along with instructions for use.

Examples

There is an existing assembly method for hepatitis E virus genotype 1 (HEV-A1) to make a peptide called p239 (so called because the protein comprises of 239 amino acids) . The p239 peptide forms VLPs and is the basis of the Hecolin vaccine. As shown below, the genetic segments that express a similar protein for hepatitis E virus genotype 4 (HEV-A4) do not protect against rat hepatitis E, caused by HEV-C. The examples show the expression of homologous peptides of hepatitis E virus genotype 4 and rat hepatitis E, called HEV-A4 p239 and HEV-C1 p241, respectively. These peptides share only 93%and 50-60%identity (i.e. %of amino acids similar) , respectively, with the original HEV-A1 p239. These peptides also form VLPs like the original HEV-A1 p239. The VLPs system based on human hepatitis E virus genotype 4 (HEV-A4 p239) and rat hepatitis E virus (HEV-C1 p241) may be deployed in antibody tests for detection and differentiation of human and rat hepatitis E virus infection.

The examples also show that the VLPs trigger strong immune responses in animal models, and can be used in vaccines against rat hepatitis E infection or bivalent vaccines against human and rat hepatitis E infection. Rat hepatitis E virus is a newly discovered infection in humans without commercially available antibody tests. The deployment of these VLPs as vaccines offers joint protection or monovalent protection against rat hepatitis E virus (HEV-C) .

The described VPLs provide the following advantages and uses:

1. Dual VLP based antibody test can detect and differentiate between human and rat hepatitis E infection. When these VLPs are deployed in commonly used antibody assay formats such as Western Blot, or ELISA, the VLPs differentiate between the antibody profiles of human and rat hepatitis E infection.

2. VLPs can protect against hepatitis E challenge. The VLPs are immunogenic in mice and administration of HEV-C1 p241 can protect rats against rat hepatitis E virus infection.

Example 1. Available kits and antibodies do not distinguish between HEV-A and HEV-C infections.

Materials and Methods

Multiple sequence alignment and phylogenetic analysis

HEV-C1 open reading frame 2 (ORF2) gene sequences from infected humans and commensal rats were retrieved from GenBank. ORF2 of three more HEV-C1 strains infecting patients in Hong Kong were sequenced using primers listed in (Table 1) . Multiple sequence alignments and phylogenetic analysis of ORF2 nucleotide sequences were performed using Clustal X version 2 (Larkin et al., Bioinformatics, 23: 2947-2948 (2007) ) . E2s peptide amino acid (aa) sequences of HEV-A and human HEV-C1 strains were compared. Degree of conservation at key amino acid residues targeted by monoclonal antibodies (mAbs) were analyzed using published toolboxes (Zhao et al., J Biol Chem., 290: 19910-19922 (2015) ; Gu et al., Cell Res., 25: 604-620 (2015) ) . Homology models of the E2s of human HEV-C1 strains were created as follows.

Homology modelling and docking

Available crystal structures of HEV-A E2s in complex with mAbs were downloaded from protein data bank (PDB ID: 3RKD and 4PLK) (Berman et al., Nucleic Acids Res., 28: 235-242 (2000) ) . Homology models of representative HEV-C1 strains causing human infection were modelled using iTASSER (Yang et al., Nat Methods., 12: 7-8 (2015) ) . Modelled HEV-C1 E2s peptides were docked against mAbs 8C11 and 8G12 with the Rosetta v3.5 protein-protein docking protocol (Das et al., Annu Rev Biochem., 77: 363-382 (2008) ) . Initial pose for docking sampling was generated by aligning HEV-C1 E2s to HEV-A E2s in the crystal structure. The high-resolution docking mode was used to search binding poses and 500 decoys were generated in each docking run. The pose with best interface score was used for interaction analysis. Polar interactions between E2s and mAbs was visualized with PyMOL.

Table 1. Primers used for sequencing the ORF2 genes of patients PC-2020, KW-2019, and PW-2020.

Patient samples

Archived sera or plasma from immunocompetent persons with RT-PCR-confirmed acute HEV-A infection (panel A) , immunocompromised patients with persistent HEV-A infection (panel B) , and patients with HEV-C1 infection (panel C) were retrieved. The earliest archived sample sent for evaluation of hepatitis E was selected for each patient. A fourth panel (panel D) included sera from organ donors who tested negative for HEV-A and HEV-C1 by RT-PCR and also tested negative in the Wantai HEV-IgG kit. Definitions of immunosuppressive conditions and persistent HEV infection are as follows.

Case definitions of persistent hepatitis E or immunosuppressive conditions

Patients were considered immunosuppressed if they a) had a hematological malignancy, b) were organ transplant recipients, c) were receiving disease-modifying anti-rheumatic drugs/marrow suppressive cancer chemotherapy, d) were taking steroids at doses above 0·5 mg/kg/day prednisolone-equivalent for at least one month, or e) were living with advanced HIV infection with CD4 T-lymphocyte counts less than 200 cells/mm ³. (Sridhar et al., Hepatology, 0: 1-13 (2020) ) . Patients were defined as having persistent hepatitis E if hepatitis E virus (HEV) species A (HEV-A) or C (HEV-C1) viremia persisted for more than three months as per Kamar et al., American journal of transplantation, 13: 1935-1936 (2013) . If sufficient archived samples were unavailable for viral load testing, duration of hepatitis was used to differentiate acute and persistent hepatitis E.

Collection of patient samples was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster.

EIA kits, monoclonal antibodies and polyclonal antisera

Samples were tested using HEV-IgM and IgG kits from Wantai (Beijing, China) , Beijing Bei’er Bioengineering Co. (Beijing, China) , and Novus Biologicals (Littleton, USA) . Samples were also tested using the Wantai HEV antigen detection kit. MAbs 12F12 and #4 were from Wen et al. (Wen et al., J Clin Microbiol., 53: 782-788 (2015) ) . WHO reference HEV antisera was procured from NIBSC (code 95/584, Potters Bar, UK) . Murine polyclonal antisera against HEV-A and HEV-C1 were prepared as previously described (Sridhar et al., Emerging infectious diseases, 24: 2241-2250 (2018) ) .

HEV-A4 p239 and HEV-C1 p241 peptide expression, immunoblot assays, and vaccines

Cloning and expression of HEV-A4 p239 and HEV-C1 p241, the HEV-A4 and HEV-C1 homologs of HEV-A1 p239 peptide used in the Hecolin vaccine (Xiamen Innovax Biotech, Xiamen, China) , were as follows. The gene encoding HEV-A4 p239 (382I-620A) , the 239 amino acid peptide corresponding to HEV-A1 p239, was amplified from a clinical isolate using primers 5’-CATATGATAGCATTGACCCTGTTTAATCT-3’ (SEQ ID NO: 19) and 5’-CTCGAGAGCAGAGTGGGGTGCTAAAACAC-3’ (SEQ ID NO: 20) . HEV-C1 p241 (357I- 597V, GenBank code: AYF53239.1) , the 241 amino acid peptide corresponding to HEV-A1 p239, was amplified using primers 5’-CATATGATTGTTCAGGTTTTGTTCAATAT-3’ (SEQ ID NO: 21) and 5’-CTCGAGAACGGGGTTGGGGCCGACAGCAC-3’ (SEQ ID NO: 22) .

Amplified genes were cloned into the Nde I and Xol I sites of the bacterial expression vector pETH in-frame and downstream of a series of 6 histidine residues. Recombinant HEV-A4 p239 and HEV-C1 p241 peptides were overexpressed in E. coli. Protein was harvested from inclusion bodies rather than supernatant due to different conformation and higher concentrations in the inclusion body fraction. Inclusion bodies were harvested from E. coli sediment by liquid-solid separation followed by dissolution in urea. Solubilized peptides were renatured by step-wise dialysis in decreasing concentrations of urea. Refolded proteins were purified by gel filtration chromatography (ENrich ^TM SEC 70 10 × 300 mm column, BIO-RAD, Hercules, USA) . Concentration of protein was determined by the BCA protein assay (Thermo Fisher, Waltham, USA) according to manufacturer instructions. HEV-C1 p241 and HEV-A4 p239 peptides were loaded and separated on 8–12%acrylamide gels with 0.1%sodium dodecyl sulphate (SDS) followed by staining with Coomassie Blue.

IgG immunoblots

Human and rat sera were tested in HEV-A4 p239 and HEV-C1 p241 IgG immunoblots as follows. Separated HEV-A4 p239 (22 μg) and HEV-C1 p241 peptides (22 μg) were transferred to a nitrocellulose membrane (Bio-Rad, Hercules, USA) . Blocking was done at 4 ℃ overnight using 10%skim milk in 1× phosphate-buffered saline (PBS) containing 0.1% (volume to volume, v/v) Tween 20. Immunoblot experiments were performed using the Mini-PROTEAN II Multiscreen Apparatus (BIO-RAD) , which allows lane-by-lane separation of the membrane and addition of different antibodies/sera to different lanes. The membranes were exposed to His-tag antibodies (Bio-Station Ltd, Hong Kong, China; diluted 1: 5000) , serially diluted WHO reference HEV antisera (from 0.02 U/mL to 0.00125 U/mL) , diluted human sera (1: 400) or rat sera (1: 5000) in blocking buffer at room temperature for 45 min for the immunoblot experiments. The blots were then washed in PBS containing 0.1%Tween 20. After exposure for one hour to horseradish peroxidase (HRP) -conjugated secondary antibodies and subsequent washes were performed as described for the primary antibodies. Membranes were visualized using the Luminescent image analyzer (GE Healthcare, Chicago, USA) .

Results

Phylogenetic analysis reveals three distinct HEV-C1 strain groups infecting humans

ORF2 encodes the HEV capsid protein, which includes most immunodominant epitopes. Of the 13 human HEV-C1 infection cases that had been reported worldwide, near-complete ORF2 nucleotide sequences of nine human-derived HEV-C1 strains were obtained and compared with 22 commensal rat-derived HEV-C1 for phylogenetic analysis. Eight of the human-derived HEV-C1 strain sequences were from Hong Kong patients while one (MK050105) was from a patient in Canada. Five Hong Kong strains were from patients diagnosed between August 2017 and July 2019 as described (Sridhar et al., Hepatology, 0: 1-13 (2020) ) , while three strains (PC-2020, KW-2019, and PW-2020) were from new patients diagnosed between September 2019 and May 2020. In the phylogenetic tree, strains from seven patients clustered together into a single strain group, which was named the ‘LCK-3110 strain group’ after the prototype LCK-3110 strain. The LCK-3110 strain was obtained from the first documented human HEV-C1 infection (Figure 1A) . Apart from LCK-3110-like strains, two other divergent HEV-C1 strains (MN450843 and MK050105) also infected humans; the ORF2 amino acid identity between these strains and the LCK-3110 strain was 95.8%&91.6%respectively.

E2s amino acid alignments show limited conservation of key residues targeted by anti-HEV mAbs.

The E2s peptide (amino acids (aa) 455-603 of HEV-A1 ORF2) corresponds to the protruding ‘P’ domain of the viral capsid, which contains most immunodominant epitopes targeted by neutralizing antibodies. The E2s regions of HEV-A1, HEV-A3, and HEV-A4 reference strains were aligned with the LCK-3110 HEV-C1 strain (Figure 1B) . The average inter-genotypic amino acid identity within HEV-A was 89.5%while the amino acid identity between HEV-A1 and LCK-3110 was only 48%. The 53 key residues involved in binding of 17 well-characterized anti-HEV-A mAbs were examined as per published toolkits (Zhao et al., J Biol Chem., 290: 19910-19922 (2015) ; Gu et al., Cell Res., 25: 604-620 (2015) ) . There was limited HEV-A inter-genotypic variation at these sites. However, only 23/53 (43.4%) of these residues were conserved between HEV-A genotypes and LCK-3110 (Figure 1B) . Amino acid identity between LCK-3110 and HEV-A at residues involved in mAb-E2s interactions was 50%or less for 13/17 (76.5%) mAbs. Residues involved in mAbs 8G12, 5H6, #4, and 12E11 binding showed more than 50%conservation, but none were completely conserved. Of the 30 non-conserved residues between HEV-A and HEV- C1, 19 (63.3%) were radical replacements, which resulted in a change in polarity and/or charge of the amino acid side chain at that residue (Table 2) .

Table 2. Effect of amino acid substitutions on side chain characteristics at key residues in E2s recognized by monoclonal antibodies

*residue position numbering follows order of key non-conserved residues in Figure 1B

Of the 11 conservative amino acid replacements, eight were concentrated in the linear epitope recognized by the mAb #4 (Figure 1B) . There was a high degree of E2s amino acid conservation between LCK-3110 and the other two divergent human-infecting HEV-C1 strains MN450843 and MK050105 (Figure 1D) confirming that the divergence from HEV-A E2s also held for these strains.

Protein-protein interaction models show lack of recognition of HEV-C1 by anti-HEV-A mAbs

MAbs 8C11 and 8G12 bound to various epitopes in HEV-A E2s (Figure 1E) . Binding interfaces were compact with multiple hydrogen-bonding contacts, which stabilized the binding. However, no comparable binding conformations were found between both mAbs and representative HEV-C1 strains (LCK-3110, MK050105, and MN450853) , indicating that the key residues for mAb recognition are mutated in HEV-C1 strains.

Commercial antibody EIAs are less sensitive for HEV-C1 serodiagnosis with considerable inter-assay variability

The impact of divergence of HEV-C1 antigenic sites on serodiagnosis was analyzed. The performance of six commercial HEV-IgG and IgM assays were compared using blood samples from 29 immunocompetent patients with HEV-A infection (panel A) , 10 immunocompromised patients with persistent HEV-A infection (panel B) , and 10 patients with HEV-C1 infection of whom five had an underlying immunosuppressive condition. Panel C included 10 of the 12 HEV-C1 infection cases reported worldwide at the time of this study. All blood samples had detectable HEV by RT-PCR. Demographic and clinical features of patients are summarized in Table 3. Underlying medical conditions of individual patients are listed in Table 4. HEV-PCR negative healthy organ donor sera (n = 10) constituted the negative controls (panel D) .

Table 3. Clinical and demographic characteristics of patients.

HEV-A: hepatitis E virus species A, HEV-A1: HEV genotype 1, HEV-A3 HEV genotype 3, HEV-A4: HEV genotype 4, HEV-C1: hepatitis E virus species C

Table 4. List of human blood samples included in this study.

*serum viral load was insufficient for genotyping, but HEV-A RT-PCR was positive

N/A: Not applicable; HEV-A1: HEV species A genotype 1; HEV-A3: HEV species A genotype 3;

HEV-A4: HEV species A genotype 4; HEV-C1: HEV species C genotype 1 (rat hepatitis E)

Table 5. Positive detection rate (sensitivity) of commercial HEV-IgG and IgM assays.

HEV-A: hepatitis E virus species A, HEV-C1: hepatitis E virus species C genotype 1

*inter-column p values calculated by Fisher’s exact test; intra-column p values (presented in text) calculated by McNemar’s test.

As shown in Table 5, with the exception of Wantai IgG, the positive detection rate (sensitivity) of all EIAs were significantly higher for HEV-A samples than HEV-C1 samples. The sensitivity of Wantai IgM tended to be higher than Bei’er IgM and Novus IgM assays for both HEV-A samples and HEV-C1 samples, although the differences did not reach statistical significance. However, Wantai IgG was significantly more sensitive than the other two IgG assays for both HEV-A samples (p = 0.013 for the comparison with Bei’er IgG and p = 0.041 for the comparison with Novus IgG) and HEV-C1 samples (p = 0.041 for both comparisons) . Mean OD values obtained in each IgG and IgM assay of the four panels were compared (Figures 2A-2F) . With the exception of the Wantai IgG assay, mean ODs of panel C HEV-C1 samples were not significantly different to panel D. Although only half of panel C patients were immunosuppressed, mean ODs of panel C was either significantly lower or did not differ from that of panel B (immunosuppressed HEV-A patients) . Again, with the exception of Wantai IgG, mean ODs of panel A (immunocompetent HEV-A patients) was consistently higher than panel C. Collectively, these results confirm that commercial anti-HEV antibody EIAs is not reliable for HEV-C1 diagnosis, irrespective of immune status of infected persons. The Wantai IgG assay was superior to comparator assays.

Parallel testing with recombinant peptide immunoblots can differentiate HEV-A and HEV-C1 serological profiles in rat and human sera

The pE2 capture antigen incorporates the entire E2s region maximizing cross-reactivity between HEV-A and HEV-C1. HEV-A1 p239 contains 26 additional amino acid residues (amino acids 368 –606) and is antigenically identical to pE2 (Zhang et al., Rev Med Virol., 22: 339-349 (2012) ) . The additional residues facilitate folding into a T=1 virus-like particle boosting immunogenicity and enabling it to be used as a vaccine. The HEV-A4 and HEV-C1 peptide homologs of HEV-A1 p239 were expressed, which were named HEV-A4 p239 and HEV-C1 p241 respectively. As has been described for HEV-A1 p239 (Li et al., Vaccine, 23: 2893-2901 (2005) ) , both these peptides formed 40-50 kDa dimers, which then resolved into about 30 kDa monomers upon boiling, indicating similar physicochemical characteristics to HEV-A1 p239. To assess serological cross-reactivity, serial dilutions of WHO reference HEV antisera were tested on HEV-A4 p239 and HEV-C1 p241 IgG immunoblots. Distinct bands were seen on the HEV-A4 p239 immunoblot at antisera concentrations as low as 0.005 U/mL, but bands on the HEV-C1 p241 immunoblot were weaker with a barely visible band at 0.02 U/mL.

Rats were immunized (n = 6 per group) with two intramuscular doses of HEV-A1 p239 (Hecolin) , HEV-A4 p239, HEV-C1 p241, or PBS spaced two weeks apart. Serological responses for all rats were simultaneously assessed in HEV-A4 p239 and HEV-C1 p241 IgG immunoblots at day 28 after the first vaccine dose. There was no cross-reactivity of rat sera in HEV-A and HEV-C1 IgG immunoblots. Hecolin and HEV-A4 p239-vaccinated rats showed minimal reactivity in the HEV-C1 immunoblot and vice versa. This shows that HEV species-specific immunoblots could detect and differentiate antibody responses triggered by HEV-A and HEV-C1 antigenic challenges.

Human serum panels were then retested using HEV-A4 p239 and HEV-C1 p241 IgG immunoblots. The positive detection rate of HEV-A4 p239 IgG immunoblot for HEV-A samples (panels A + B) was 29/39 (74.4%) and HEV-C1 p241 IgG immunoblot for panel C samples was 7/10 (70%) with lower sensitivity among immunocompromised persons. Most positive HEV-A samples (27/29; 93.1%) only reacted with the HEV-A4 p239 immunoblot and did not cross-react on the HEV-C1 immunoblot. Five panel C sera only reacted with the HEV-C1 p241 immunoblot while two cross-reacted in HEV-A4 and HEV-C immunoblots. Overall, for the 36 samples generating bands in either immunoblot, serological differentiation of HEV-A and HEV-C1 infection was possible in 32 samples (88.9%) . Infection by HEV-A or HEV-C1 elicits HEV species-specific serological responses in humans, which can be differentiated by parallel immunoblot testing.

Example 2. Lack of mAb cross-binding renders HEV antigen kits ineffective for HEV-C1 diagnosis

Materials and Methods

Antigen assays –12F12 and #4 monoclonal antibody antigen capture EIA

The ability of mAbs #4 and 12F12 to bind to HEV-A4 p239, HEV-C1 p241, HEV-A4 and HEV-C1 in clinical samples was assessed in an EIA format and compared to HEV-negative controls. The EIA design was as follows. Both monoclonal antibodies were diluted in coating buffer to a final concentration of 5 μg/ml and added to each well of 96-well plates (500 ng/well) . Plates were then incubated overnight at 4℃. Next, the plates were washed and incubated for 3h with 300 μl/well blocking buffer supplemented with 0.05%Tween 20 at 37℃. HEV-A4 p239 peptides, HEV-C1 p241 peptides, test, and control sera diluted 1: 4 in 1%BSA were distributed into the wells (100 μl/well) in triplicate and incubated for 1h at 37℃. Next, the sera/peptides were removed and the plates were washed six times with 0.3%Tween 20 + PBS (300 μl/well) . 100 μl of rat polyclonal sera against HEV-A or HEV-C1 as appropriate diluted 1: 2000 in 20%goat serum, was added to each well. After incubation at 37℃ for 1 h, the plates were washed six times and incubated with HRP-conjugated goat anti-rat IgG for 30 min at 37℃. The plates were washed six times and the reaction was detected by adding 1: 1 peroxidase solution/TMB substrate solution. Finally, after 10 min at 37℃, the reaction was stopped by adding 3M H ₂SO ₄ and the plates were read using a microplate reader at a wavelength of 450-620 nm.

Results

Given low conservation at key epitopes, the Wantai antigen kit, a sandwich EIA comprising mAbs 12F12 and #4, was examined for its ability to detect HEV-C1. Panels B and C were tested as they contained relatively higher viral loads exceeding 4 log ₁₀ copies/mL. All panel C HEV-C1 samples tested negative in the antigen assay with OD values similar to negative controls while all panel B samples tested positive (Figure 3A) .

The constituent mAbs of the Wantai antigen kit (12F12 and #4) were then examined if they could bind to selected panel B and panel C samples with high viral load. Panel B samples showed reactivity in both 12F12 and #4 EIAs while reactivity of panel C samples was not significantly different to negative controls (Figures 3B and 3C) . HEV-A4 p239 was bound by both mAbs, but not HEV-C1 p241. This again demonstrated that mAbs targeting HEV-A do not bind HEV-C1, significantly impacting efficacy of HEV antigen assays for HEV-C1 infection.

Example 3. Vaccination and infection challenge experiments with HEV-A4 p239 and HEV-C1 p241 peptides

Materials and Methods

Preparation of SRN250811 HEV-C1 strain

A rectal swab was obtained from a commensal rat (Rattus norvegicus) captured in the Southern District of Hong Kong and placed in virus transport medium (VTM) . The VTM was centrifuged and the supernatant was passed through a bacterial filter. Filtered supernatant was administered intravenously to an immunosuppressed 4-week old female SPF Sprague-Dawley rat. The immunosuppressive regimen was a combination of tacrolimus, prednisolone and mycophenolate mofetil given daily by oral gavage. The purpose of immunosuppression was to prolong the duration of HEV-C1 infection and increase virus shedding in fecal material, as HEV-C1 infection in rats is typically transient. Feces from the rat were collected in VTM. The VTM was centrifuged to pellet fecal material and the supernatant was passed through a bacterial filter. The fecal filtrate was then passaged once more in an immunocompromised SPF Sprague-Dawley rat to amplify the virus further. This passaged HEV-C1 strain was called SRN250811. Fecal suspension from this rat containing 10 ⁸ HEV-C1 genome equivalents/mL was used for the virus challenge in the vaccination experiments described in this study.

Vaccine preparation

For vaccine preparation, purified HEV-A4 p239 and HEV-C1 p241 peptides prepared as in Example 1 were adjuvanted with aluminum hydroxide and formulated at a concentration of 30 μg/0.5 mL to match the Hecolin vaccine.

Rat vaccination and infection challenge

Six-week old CD1 Sprague-Dawley rats (Rattus norvegicus; 3 males, 3 females (n = 6) per group) received two 10 μg intramuscular doses of HEV-A4 p239, HEV-C1 p241, HEV-A1 p239 (Hecolin) , or phosphate buffered saline (PBS mixed with adjuvant aluminum hydroxide) spaced two weeks apart. According to an a priori estimate that a) all PBS-vaccinated rats and 30%of vaccinated rats would develop infection and b) mean fecal viral load would be at least 2 log ₁₀ copies/mL higher in the PBS-vaccinated group, this sample size yields a power greater than 90%at an alpha level of 0.05. Serological responses in rats were monitored for four weeks after which they were intravenously administered 0.5 mL of a filtered fecal suspension containing 10 ⁶ HEV-C1 genome equivalents of SRN250811: a HEV-C1 strain derived from a captured commensal rat. Serial blood taking for HEV-C1 viral loads, serology and liver function tests was performed. Rats were sacrificed four weeks after infection. Liver tissue was obtained for HEV-C1 RT-PCR, H&E, and immunohistochemical staining (IHC) . Ethics approval for these experiments was obtained from the Committee on the Use of Live Animals in Teaching and Research of The University of Hong Kong. Protocols for these procedures were as follows.

Nucleic acid extraction, real-time RT-PCR, and nested RT-PCR assays

Total nucleic acid (TNA) extraction was performed using the EZ1 kit (Qiagen, Hilden, Germany) with 200 μL of plasma or stool in VTM eluted into 50 μL of TNA. Real-time RT-PCR for HEV-C1 detection was performed using primers and probes as described (Sridhar et al., Emerging infectious diseases, 24: 2241-2250 (2018) ) . Primers targeted the ORF1 gene of HEV-C1. Primer sequences are 5’-CTTGTTGAGCTYTTCTCCCCT-3’ (SEQ ID NO: 23, where “Y” is either C or T, as per IUPAC codes) (forward) and 5’-CTGTACCGGATGCGACCAA-3’ (SEQ ID NO: 24) (reverse) while the probe sequence is HEX-TGCAGCTTGTCTTTGARCCC –IABkFQ (SEQ ID NO: 25) . The amplicon size was 69 bp.

Real-time RT-PCR (qRT-PCR) assays were run using QuantiNova Probe RT-PCR Kit (Qiagen) in a LightCycler 480 Real-Time PCR System (Roche, Basel, Switzerland) . Each 20 μL-reaction mix contained 1X QuantiNova Probe RT-PCR Master Mix, 1X QN Probe RT-Mix, 0·8 μM forward and reverse primers, 0·2 μM probe and 5 μl template RNA. Reactions were incubated at 45℃ for 10 min and 95℃ for 5 min, followed by 50 cycles at 95℃ for 5 s and 55℃ for 30 s. Quantitation of HEV viral load in copies/mL or copies/g was performed using plasmid standards prepared using the pCRII-TOPO vector (Invitrogen, Carlsbad, CA, USA) cloned with the target insert. The limit of detection of the HEV-C1 RT-PCR assay was determined to be 3 log ₁₀ copies/mL.

For the nested HEV-C1 RT-PCR, the first reaction was performed using outer primers 5’-CAGCGGCTACCGCCTTTGCTAATGCTCAGGT-3’ (SEQ ID NO: 26) and 5’-GCGGCGGACGTACGCCTCCAGAAAATYATGAATA-3’ (SEQ ID NO: 27) for 40 cycles. This was followed by a second reaction using the amplicons from the first RT-PCR reaction as the template and inner primers 5’-CTTGTTGAGCTYTTCTCCCT-3’ (SEQ ID NO: 23) and 5’-CTGTACCGGATGCGACCAA-3’ (SEQ ID NO: 24) (same as the real-time RT-PCR primers listed above) . This reaction was run for another 40 cycles followed by PCR product detection by gel electrophoresis.

Rat liver histological analysis

Rat livers were fixed in 4%Formalin, embedded in paraffin, and then sectioned. The sections (4 mm thick) were deparaffinised in xylene and stained with hematoxylin and eosin (H&E) . For the immunohistochemical staining, tissues were deparaffinised, hydrated, and heated in a water bath for antigen retrieval and then treated with the addition of 3%hydrogen peroxide in PBS (pH 7.6) for 30 min and blocked with 1%BSA for 30 min. This was followed by incubation with streptavidin for 15 min and biotin solution for 15 min. The sections were then incubated at 4℃ overnight with primary antibody (polyclonal murine anti-HEV-C sera) . After washing with TBST, slides were incubated in secondary antibody conjugated with biotin for 30 min, followed by incubation with HRP streptavidin for 30 min. DAB substrate kit was used for color development. Sections were slightly counterstained with Mayer’s hematoxylin.

Statistical analysis

Throughout the examples, the charts were generated using GraphPad Prism Version 8.1 (GraphPad software, La Jolla, USA) . McNemar’s test and Fisher’s exact test were used to compare assay sensitivity. Student’s t-test with or without Welch’s correction were used for comparing mean ODs. One-way ANOVA was used to compare the mean viral loads of the four groups in the vaccination experiment.

Results

Vaccination with HEV-A antigens was not fully protective against HEV-C1 infection in rats

SRN250811, derived from a commensal rat, is a heterotypic HEV-C1 strain showing less than 92%amino acid identity with LCK-3110 (Figure 1A, Table 6) .

Table 6. Nucleotide and deduced amino acid sequence identities of HEV-C1 strain SRN250811 compared with human-infecting HEV-C1 isolates.

ORF1: open reading frame 1; ORF2: open reading frame 2; ORF3: open reading frame 3

Rats vaccinated with Hecolin (HEV-A1 p239) , HEV-A4 p239, HEV-C1 p241, or PBS were intravenously challenged with SRN250811 four weeks after the first dose of vaccine (Figure 4A, on day 56, rat livers were obtained for viral load testing and histology) . On day of challenge, vaccinated rats showed strong HEV antibody responses on species-specific immunoblots. All PBS-vaccinated rats had detectable virus in feces and serum; fecal viral loads peaked at day 7 and turned negative by day 28 (Figures 4B and 4C) . All six rats in the Hecolin group and 5/6 rats in the HEV-A4 p239 group had detectable virus in feces peaking at day 7 post-infection. None of the HEV-C1 p241-vaccinated rats had detectable virus in feces or plasma by either quantitative or nested RT-PCR assays, suggesting that the HEV-C1 p241 vaccine conferred sterilizing immunity. There was a trend towards lower viral loads in serum and feces of HEV-A4 p239 and Hecolin-vaccinated rats but this was not always statistically significant (Figures 4B and 4C) . At

day

28, 4/6 PBS-vaccinated rats still had detectable HEV-C1 RNA in liver tissue (Figure 4D) compared to none of the HEV-C1 p241-vaccinated rats. One of the HEV-A4 p239-vaccinated and none of the Hecolin vaccinated rats had detectable virus in liver tissue at day 28. Hecolin and HEV-A4 p239-vaccinated rats maintained strong HEV-A-specific antibody responses after infection with weak responses on the HEV-C1 p241 immunoblot. Liver function tests in all groups remained normal (Figures 5A and 5B) , which is typical of HEV-C1 infection in juvenile rats. Liver histology showed mild degrees of hepatitis in PBS and HEV-A vaccinated rats. HEV-C1 infected control rat showed acute hepatitis with swollen hepatocytes and apoptotic bodies. Sinusoidal mononuclear cell infiltrates and disarrayed plate architecture were observed. PBS-vaccinated rat infected with HEV-C1 showed disarrayed architecture of hepatocyte plates. Apoptotic cells with nuclear remnants were detected. Hecolin-vaccinated rat infected with HEV-C1 showed mild hepatitis with occasional apoptotic cells with nuclear remnants. HEV-A4 p239-vaccinated rat had mild hepatitis with occasional apoptotic cells with nuclear remnants. HEV-C1 p241-vaccinated rat showed normal hepatocytes and cord architecture except for a focus of mononuclear cell infiltration.

IHC staining of liver with anti-HEV-C1 polyclonal antisera showed positive signals in PBS-vaccinated rats infected with HEV-C1. Weaker signals were noted in HEV-A4 p239 and Hecolin-vaccinated rat livers and no signals were found in HEV-C1 p241-vaccinated rats infected with HEV-C1.

To further evaluate the effect of a mixed vaccine regimen, four rats were vaccinated with Hecolin at day 0 and HEV-C1 p241 at day 14, followed by SRN250811 challenge (Figure 6, n=4, on day 56, rat livers were obtained for viral load testing) . Only one (25%) rat had detectable HEV-C1 in feces at day 3 (4.75 log ₁₀ copies/mL) and day 7 (4.58 log ₁₀ copies/mL) , which was lower than the peak viral load observed in HEV-A vaccinated rats (Figure 4B) . All rat sera and day 28 post-infection liver tissue tested negative for HEV-C1 RNA. Rat sera showed stronger bands on the HEV-A4 immunoblot compared to the HEV-C1 immunoblot.

Taken together, these experiments demonstrate that prior HEV-A exposure is not protective against HEV-C1 infection in rats. This confirms the impact of HEV-C1 antigenic divergence on infection susceptibility.

The multimodal assessment of HEV-C1 antigenicity and the impact of divergent antigenicity on clinical diagnostics and vaccine prevention of HEV-C1 infection was assessed. Low homology at key epitopes between HEV-A and HEV-C1 E2s sequences were found with divergence often resulting in radical changes of side-chain characteristics at these residues. Due to the poor binding of HEV-C1 to anti-HEV mAbs, commonly used mAb-based HEV antigen EIAs cannot detect HEV-C1 infections. Specific mAbs against HEV-C1 have been raised but further work is necessary along this line to identify neutralizing epitopes in HEV-C1 E2s and also clarify the crystal structure of HEV-C1 E2s bound to mAbs. Pan-species HEV-antigen EIAs incorporating mAbs that bind to both HEV-A and HEV-C1 are required to ensure HEV-C1 infections are not missed.

The performance of antibody EIAs for HEV-C1 diagnosis shows high inter-assay variability. Using pE2 as a capture antigen, as in the Wantai IgG assay, enables detection of cross-reactive polyvalent antibody responses. HEV antibody assays currently in use worldwide require reevaluation for HEV-C1 using standards incorporating sera from HEV-C1 patients. The WHO reference HEV antisera is not representative of HEV-C1 infected patients. Taken together with the inability of HEV-A-based RT-PCR assays to detect HEV-C1, conventional hepatitis E diagnostics are inadequate for HEV-C1 infection. This may lead to systematic underestimation of HEV-C1 burden.

Rats and humans exposed to HEV-A or HEV-C1 antigens mount HEV species-specific humoral responses. Exploiting this it was demonstrated that parallel testing of sera using HEV-A4 p239 and HEV-C1 p241 immunoblots was able to determine whether patients were exposed to HEV-A or HEV-C1 with two caveats complicating interpretation. Firstly, immunocompromised patients may not mount sufficient antibody responses and secondly, anamnestic responses to HEV-A in HEV-C1-infected patients may result in erroneous assignment, especially in elderly patients who are more likely to have been exposed to HEV-A in the past. Differentiation assays will be useful for studying HEV-C1 population seroprevalence.

HEV-C1’s antigenic divergence raises the question of whether prior HEV-A immunity cross-protects against HEV-C1. The examples show that rats immunized with HEV-A antigens still develop infection after HEV-C1 challenge, although partial protection is apparent with lower viral loads and improved liver histology. HEV-C1 infection in a patient with baseline HEV-A seropositivity with a lack of cross-protection was previously identified, showing that this applies to humans (Sridhar et al., Emerging infectious diseases, 24: 2241-2250 (2018) ) . Interestingly, rats with prior exposure to HEV-A antigens developed an anamnestic response upon exposure to HEV-C1, showing that infection by a pathogen triggers an immune response against a related previously encountered antigen. Although this response did not worsen HEV-C1 infection outcomes in rats, it may contribute to ongoing HEV-C1 susceptibility and weak HEV-C1 p241 humoral responses in HEV-A vaccinated animals. A bivalent vaccine incorporating both HEV-C1 p241 and HEV-A p239 is required in areas like Hong Kong where HEV-C1 accounts for a significant portion of hepatitis E burden.

Although in the examples the HEV-C1 panel size was relatively small, panel C already included the majority of all reported HEV-C1 cases. As new cases emerge through surveillance, more samples will become available for immunoblots. Also, there is a lack of HEV-C1 cell culture model to test neutralization by anti-HEV-A mAbs to assist with in vitro assays. In silico experiments, mAb antigen EIAs using clinical samples, and rat infection models provided a surrogate assessment of cross-reactivity. A HEV-A infection model could not be developed because rats are not susceptible to HEV-A. A study has already been conducted in pigs showing that HEV-C1 vaccination is partially protective against HEV-A (Purcell et al., Emerg Infect Dis., 17: 2216-2222 (2011) ; Sanford et al., Vaccine, 30: 6249-6255 (2012) ) .

The antigenic diversity leads to frequent failures of HEV-A-based diagnostic assays in diagnosing HEV-C1 infections. Prior HEV-A infection or vaccination is not protective against HEV-C1. The examples show assays that can differentiate serological profiles of HEV-A and HEV-C1 infections and provide an immunogenic HEV-C1 peptide vaccine.

EXAMPLE 4. Development and evaluation of a parallel enzymatic immunoassay based on HEV-A4 p239 and HEV-C1 p241

The immunoblot system was adapted to an IgG EIA format to maximize convenience and sensitivity. HEV-A4 p239 &HEV-C1 p241 were coated in separate 96-well EIA plates at a concentration of 50 ng/well. For performing the test, sera or plasma samples were diluted 1: 200 with 1%casein and 100 μL of each diluted sample was tested in duplicate in HEV-A4 p239 and HEV-C1 p241 plate wells. Following a 30-minute incubation and washing step, 1: 8000 HRP-antibody complex was added to each well. Following another incubation and washing step, 100 μL chromogen solution was added to each well followed ten minutes later by addition of 3M H ₂SO ₄ stopping solution. Positive controls from patients with known HEV-A or HEV-C infection would be incorporated into each run. Pooled negative controls from healthy HEV IgG negative individuals would be used as negative controls. Shewhart charts would be maintained to ensure assay performance according to Westgard rule principles, especially when changing to a new batch of peptide.

For assay evaluation, the healthy organ donor sera who had previously tested negative in the Wantai HEV-IgG EIA kit was first tested (Figure 7) . The mean OD values in both HEV-A and HEV-C IgG EIAs were broadly comparable. In order to evaluate the assay and generate cut-offs, a panel of 195 human serum samples comprising 126 HEV negative organ donor sera (confirmed HEV-A and HEV-C RT-PCR and IgG negative) , 54 sera containing HEV-A RNA (confirmed by RT-PCR) , and 15 sera containing HEV-C RNA (confirmed by RT-PCR) were assembled. This panel of 15 HEV-C samples represents the largest repository of human HEV-C samples, comprising the majority of all known rat hepatitis E infections globally.

Each test sample was run in parallel on HEV-A4 p239 and HEV-C1 p241 EIAs as described above and OD values were generated. Assay cut-offs were then generated for individual IgG EIAs by receiver operating characteristic (ROC) curves using RT-PCR results as the gold standard (Figure 8) .

Using these cut-offs, the performance of HEV-A4 p239 and HEV-C1 p241 IgG EIAs against RT-PCR assays are presented in Table 7 and Table 8. The performance characteristics of the HEV-A p239 and HEV-C p241 IgG EIAs for detecting HEV-A and HEV-C infection is presented in Table 9.

Table 7. The performance of HEV-A4 p239 IgG EIA against RT-PCR assays

Table 8. The performance of HEV-C1 p241 IgG EIA against RT-PCR assays

Table 9. The performance characteristics of the HEV-A p239 and HEV-C p241 IgG EIAs for detecting HEV-A and HEV-C infection

After establishing the performance of the individual EIAs, a simple algorithm for result interpretation was proposed (Table 10) .

Table 10. A simple algorithm for result interpretation

For the final category (positive in both EIAs) , a cut-off for the ratio of normalized ODs for the samples in the HEV-A4 p239 and HEV-C1 p241 IgG EIAs was evaluated. With a C/Acut-off ratio of 1.971, a sensitivity of 100%and a specificity of 88.9%were found.

Using the cut-offs for the individual assays and the cut-off of C/Afor samples positive in both assays according to the algorithm presented in Table 10, the performance of the entire algorithm against RT-PCR was evaluated again (Table 11) .

Table 11. The performance of the entire algorithm against RT-PCR

The Cohen’s kappa value of the EIA against RT-PCR was 0.883 indicating excellent inter-rater agreement.

The overall performance characteristics of the entire algorithm for discriminating between different categories of HEV is presented in Table 12 below:

Table 12. The overall performance characteristics of the entire algorithm for discriminating between different categories of HEV

Claims

A synthetic virus-like particle comprising a plurality of peptides each having at least a 90%amino acid sequence identity with SEQ ID NO: 31.
The synthetic virus-like particle of claim 1 comprising a plurality of proteins each having at least a 95%amino acid sequence identity with SEQ ID NO: 31.
The synthetic virus-like particle of claim 1 or 2 comprising a plurality of proteins having an amino acid sequence as in SEQ ID NO: 1 or as in SEQ ID NO: 31.
A composition comprising the synthetic virus-like particle of any one of claims 1-3.
The composition of claim 4 comprising a synthetic virus-like particle comprising a plurality of peptides having at least a 90%amino acid sequence identity with SEQ ID NO: 3 or SEQ ID NO: 29.
The composition of claim 4 or 5 comprising an adjuvant.
The composition of any one of claims 4-6, comprising an adjuvant selected from the group consisting of amorphous aluminum hydroxyphosphate sulfate (AAHS) , aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate (Alum) , Freund's complete adjuvant, Freund's incomplete adjuvant, Monophosphoryl lipid A (MPL) with aluminum salt, Oil in water emulsion composed of squalene, Quil A, MPL and QS-21, and Cytosine phosphoguanine (CpG) -containing immunostimulatory oligonucleotides.
The composition of any one of claims 4-7 comprising one or more compounds selected from the group consisting of sodium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, aluminium hydroxide, and thiomersal.
The composition of any one of claims 4-8 for inducing an immune response against a portion of SEQ ID NO: 31 and against a portion of SEQ ID NO: 29.
The composition of any one of claims 4-9 for inducing an immune response against Hepatitis E virus (HEV) species A (HEV-A) genotype 4, HEV-C genotype 1, or a combination thereof.
A composition for detecting a HEV-C infection, or a combination of HEV-Aand HEV-C infections, the composition comprising

a plurality of peptides each having at least a 90%amino acid sequence identity with SEQ ID NO: 31, at least 95 %amino acid sequence identity with SEQ ID NO: 31, or an amino acid sequence as in SEQ ID NO: 31, and optionally,

a plurality of peptides each having at least a 90%amino acid sequence identity with SEQ ID NO: 29.
The composition of claim 11, wherein the plurality of peptides are synthetic virus-like particles.
A kit comprising a plurality of peptides each having at least a 90%amino acid sequence identity with SEQ ID NO: 31, at least 95 %amino acid sequence identity with SEQ ID NO: 31, or an amino acid sequence as in SEQ ID NO: 31, and optionally,

a plurality of peptides having at least a 90%amino acid sequence identity with SEQ ID NO: 29.
The kit of claim 13, wherein the plurality of peptides are synthetic virus-like particles.
An assay platform comprising

a plurality of peptides each having at least a 90%amino acid sequence identity with SEQ ID NO: 31, at least 95 %amino acid sequence identity with SEQ ID NO: 31, or an amino acid sequence as in SEQ ID NO: 31, and optionally,

a plurality of peptides each having at least a 90%amino acid sequence identity with SEQ ID NO: 29.
The assay platform of claim 15, wherein the plurality of peptides are synthetic virus-like particles.
An immunoassay comprising a step of contacting a test sample with

a plurality of peptides each having at least a 90%amino acid sequence identity with SEQ ID NO: 31, at least 95 %amino acid sequence identity with SEQ ID NO: 31, or an amino acid sequence as in SEQ ID NO: 31, and optionally, with

a plurality of peptides each having at least a 90%amino acid sequence identity with SEQ ID NO: 29.
The immunoassay of claim 17, wherein the plurality of peptides are synthetic virus-like particles.
A method of detecting a HEV-C infection, or a combination of HEV-Aand HEV-C infections in a sample, the method comprising contacting a sample or a test sample with

a plurality of peptides each having at least a 90%amino acid sequence identity with SEQ ID NO: 31, at least 95%amino acid sequence identity with SEQ ID NO: 31, or an amino acid sequence as in SEQ ID NO: 31, and optionally, with

a plurality of peptides each having at least a 90%amino acid sequence identity with SEQ ID NO: 29.
The method of claim 19, wherein the plurality of peptides are synthetic virus-like particles.
The method of claim 19 or 20, wherein contacting is in a test vessel.
The method of any one of claims 19-21, wherein contacting is contacting a plurality of test samples with the plurality of peptides having at least a 90%amino acid sequence identity with SEQ ID NO: 31 in a first set of test vessels.
The method of any one of claims 19-22, wherein contacting is contacting a plurality of test samples with the plurality of peptides having at least a 90%amino acid sequence identity with SEQ ID NO: 29 in a second set of test vessels.
The method of any one of claims 19-23, further comprising developing a signal from the contacting of the test sample with the plurality of peptides.
The method of any one of claims 19-24, wherein the method detects HEV-C infection when signal develops from the contacting of the test sample with the plurality of peptides having at least a 90%amino acid sequence identity with SEQ ID NO: 31, at least 95 %amino acid sequence identity with SEQ ID NO: 31, or an amino acid sequence as in SEQ ID NO: 31.
The method of any one of claims 19-25, wherein the method detects HEV-Ainfection when signal develops from the contacting of the test sample with the plurality of peptides having at least a 90%amino acid sequence identity with SEQ ID NO: 29.
The method of any one of claims 19-26, wherein the method detects a HEV-C infection or a combination of HEV-Aand HEV-C infections in the sample at a sensitivity of about or over 80%and a specificity of about or over 70%.
The method of any one of claims 19-27, wherein the method distinguishes between HEV-A and HEV-C infections in the sample at a sensitivity of about or over 80%.
A method of detecting Hepatitis E virus species C (HEV-C) in a sample, the method comprising testing RNA from the sample in a polymerase chain reaction with primers specific to HEV-C open reading frame 1 or open reading frame 2.
The method of claim 29, wherein the primers specific for HEV-C comprise primers as in SEQ ID NO: 26 and SEQ ID NO: 27.
The method of claim 29 or 30, wherein the primers specific for HEV-C comprise primers as in SEQ ID NO: 23 and SEQ ID NO: 24.
The method of any one of claims 29-31, wherein the RNA from the sample is turned to complementary DNA prior to the polymerase chain reaction.
The method of any one of claims 29-32, wherein the method detects a HEV-C infection in the sample at a sensitivity of about or over 90%and a specificity of about or over 90%.
The method of any one of claims 19-33, wherein the sample is sample obtained from a subject, wherein the subject is a human, a non human primate, domestic animal, wild animal, farm animal, or a laboratory animal.
The method of any one of claims 19-34, wherein the sample is a bodily fluid or mucus selected from the group consisting of blood, serum, plasma, excrement, exudate, saliva, sputum, tear, sweat, urine, or a vaginal discharge.
The method of any one of claims 19-35, wherein the test sample is a diluted sample of a bodily fluid or mucus selected from the group consisting of blood, serum, plasma, excrement, exudate, saliva, sputum, tear, sweat, urine, and a vaginal discharge.
The method of any one of claims 19-36, wherein the test sample is the sample diluted at a ratio between about 1: 5 and 1: 500 (v/v) of the sample to a buffer.