CN117083290A - Hepatitis E virus-like particles and uses thereof - Google Patents

Abstract

Peptides, compositions, and assays for detecting Hepatitis E Virus (HEV) C (HEV-C) infection, distinguishing HEV-C infection from HEV-A infection, and protecting against HEV-C infection are disclosed. The composition generally comprises one or more peptides having amino acid sequence identity with the amino acid sequence of a portion of the HEV-C genotype 1 (HEV-C1) open reading frame 2 (ORF-2) capsid protein. The HEV-C1 peptide is an isolated and purified peptide that aggregates into immunogenic virus-like particles (VLPs). The HEV-C1 peptide has no substantial amino acid sequence identity with the ORF2 peptide of HEV A species and is used to distinguish between human HEV-C infection and human HEV-A infection.

Description

Hepatitis E virus-like particles and uses thereof

The international patent application claims the benefit of U.S. provisional patent application No. 63/166,698, filed on 26, 3, 2021, the entire contents of which are incorporated by reference for all purposes.

Technical Field

The present invention is generally directed to compositions and assays for detecting Hepatitis E Virus (HEV) infection, differentiating between different HEV species of infection, and protecting against HEV.

Background

Hepatitis E Virus (HEV) is the leading cause of viral hepatitis worldwide. Clinical manifestations of acute hepatitis E include asymptomatic infection, mild to moderate liver dysfunction, and fulminant hepatitis. Persistent hepatitis E may develop in immunocompromised individuals and may progress to cirrhosis if left untreated. HEV belongs to the hepatitis virus family (famly Hepeviridae) which contains two genera: orthohepatics (including variants that infect terrestrial vertebrates) and piscihepatics (Piscihepvirus) (oncorhynchus mykiss virus). Human hepatitis E is mainly due to the genus A. HEV-A contains eight genotypes, which can infect humans, pigs, boars, deer, rabbits, and camels. Four of the eight genotypes of HEV-A are commonly found to infect humans. HEV-A genotype 1 (HEV-A1) and genotype 2 spread between humans viSup>A the faecal route. HEV-A genotype 3 (HEV-A3) and genotype 4 (HEV-A4) are transmitted in pigs and spread to humans by eating uncooked meat products. HEV-A3 is transmitted in Europe and America, while HEV-A4 is transmitted in China. HEV-A3 and HEV-A4 typically cause self-limiting hepatitis, but may progress to chronic infection in immunocompromised individuals. HEV-A1 based vaccines (Hecolin) were licensed in China after clinical trials in the HEV-A4 epidemic area. The efficacy of this vaccine against other HEV-A genotypes is currently uncertain.

The hepatitis E infection of human in industrialized countries is caused by HEV-A genotype 3 (Europe, japan and AmericSup>A) or HEV-A genotype 4 (ChinSup>A), while in developing countries it is caused by HEV-A genotype 1 or 2. HEV-A genotypes 3 and 4 are typically obtained by eating uncooked pork or game meat, but may also be transmitted through contaminated blood products or organs (Sridhar et al, hepatology, january 2020,1-13 (2020)).

In addition to HEV-A, the genus orthopoxvirus includes three other species: b (spread in birds), C (HEV-C; spread in rodents and ferrets) and D (spread in bats). HEV-C was found in German rats in 2010 and was later detected in Asia, europe and North America rats. Rats are susceptible to HEV-C genotype 1 (HEV-C1), while other HEV-C genotypes are transmitted in ferrets, shrew murines, field rats, and the like. To date, HEV-C1 is considered to have minimal risk of human and animal co-morbidity due to the wide phylogenetic difference with HEV-A and the failure to experimentally infect pigs and non-human primates.

These HEV species are listed on the ViralZone website maintained by swiss bioinformatics institute. However, it is not uncommon in the literature to refer to HEV-A and its genotype as human hepatitis E virus and HEV-C as rat hepatitis E virus.

HEV-C shares only 50% to 60% nucleotide identity with HEV-A and has significant differences in the key epitopes of the putative receptor binding domain. However, case studies have shown that HEV-C1 infects liver transplant recipients even if the patient has an existing antibody against HEV-A (Sridhar et al, emerg effect Dis;24:2241-2250 (2018)). Due to the significant sequence differences, the common HEV-A nucleic acid amplification test is unable to detect HEV-C1 infection. Another study subsequently identified immunocompromised adults with acute HEV-C1 infection, likely in Africa (Andonov et al, J select Dis.; 220:951-955 (2019)). This opens the possibility that HEV-C1 is Sup>A worldwide epidemic of human and animal co-occurrence, and that existing HEV-A specific assays are often missed. An epidemiological study reports that HEV-C1 infection accounts for 8% of all genotypes of hong Kong hepatitis E cases (Sridhar et al, hepatology, january 2020,1-13 (2020)).

The global prevalence of HEV-C1 infection is not clear due to the blind spot in HEV diagnostic testing. HEV-A based reverse transcription polymerase chain reaction (RT-PCR) assay was unable to detect HEV-C1 (Sridhar et al, emerg information Dis;24:2241-2250 (2018)). The efficacy of hepatitis E Enzyme Immunoassay (EIA) kits for diagnosing HEV-C1, which commonly use HEV-A derived target antigens, has not been determined. Commercial EIA kits using HEV-A1 pE2 antigen cross-react with some HEV-C1 patient sera; it is unclear whether EIA using other HEV-A peptides are similarly cross-reactive (Andonov et al, J select Dis.,220:951-955 (2019)).

There remains Sup>A need for compositions and assays for detecting HEV-C infection, distinguishing HEV-C infection from HEV-A infection, and protecting against HEV-C infection.

Disclosure of Invention

It is an object of the present invention to provide compositions and assays for detecting HEV-C infection, distinguishing HEV-C infection from HEV-A infection, and protecting against HEV-C infection.

It is another object of the present invention to provide methods for preparing compositions and assays for detecting HEV-C infection, distinguishing HEV-C infection from HEV-A infection, and protecting against HEV-C infection.

It is yet another object of the present invention to provide Sup>A method of using the composition and assay to detect HEV-C infection, distinguish HEV-C infection from HEV-A infection, and protect against HEV-C.

Described are compositions and assays for detecting HEV-C infection and distinguishing HEV-C infection from HEV-A combination infection. Compositions for protection against HEV-C are also described.

Typically, the composition comprises a peptide or synthetic virus-like particle comprising a plurality of peptides having at least 90% amino acid sequence identity to SEQ ID NO. 1 or having the amino acid sequence as set forth in SEQ ID NO. 1. The composition may further comprise a peptide or a synthetic virus-like particle comprising a plurality of peptides having at least 90% amino acid sequence identity to SEQ ID No. 3. The composition may comprise an adjuvant. The composition is typically used to induce an immune response against a portion of SEQ ID NO. 1. The composition may also be a bivalent composition inducing an immune response against a portion of SEQ ID NO. 1 and against a portion of SEQ ID NO. 3. The composition generally induces an immune response against Hepatitis E Virus (HEV) type A (HEV-A), HEV-C genotype 1, or Sup>A combination thereof.

Compositions for detecting HEV-C infection or Sup>A combination HEV-A and HEV-C infection are also described. Compositions for detecting HEV-C infection or Sup>A combination of HEV-A and HEV-C infection typically comprise Sup>A plurality of peptides having at least 90% amino acid sequence identity to SEQ ID NO. 1, at least 95% amino acid sequence identity to SEQ ID NO. 1, or an amino acid sequence as in SEQ ID NO. 1. These compositions may also comprise a plurality of peptides having at least 90% amino acid sequence identity to SEQ ID NO. 3. The plurality of peptides may be in the form of synthetic virus-like particles. The composition may be contained on an assay platform or test container for assay for detecting HEV-C infection, detecting HEV-A and HEV-C combined infection, or distinguishing HEV-A from HEV-C infection.

Assays, such as immunoassays or amplification assays, are also described for detecting HEV-C infection in Sup>A sample, detecting HEV-A and HEV-C combined infection in Sup>A sample, or distinguishing between HEV-A and HEV-C infection in Sup>A sample. The immunoassay typically comprises contacting the sample or test sample with a plurality of peptides having at least 90% amino acid sequence identity to SEQ ID No. 1, at least 95% amino acid sequence identity to SEQ ID No. 1, or at least 95% amino acid sequence identity to SEQ ID No. 1, and optionally contacting the sample or test sample with a plurality of peptides having at least 90% amino acid sequence identity to SEQ ID No. 3. The plurality of peptides may be in the form of synthetic virus-like particles. The contacting may be performed on a platform or in a test vessel. The assay typically includes the step of forming a signal from the contact.

Typically, the assay detects HEV-C infection when a signal is formed from the contact of a sample or test sample with a plurality of peptides having at least 90% amino acid sequence identity to SEQ ID NO. 1, a plurality of peptides having at least 95% amino acid sequence identity to SEQ ID NO. 1, or a plurality of peptides having an amino acid sequence as in SEQ ID NO. 1.

Typically, the assay detects HEV-A infection when Sup>A signal is formed from the contact of the sample or test sample with Sup>A plurality of peptides having at least 90% amino acid sequence identity to SEQ ID NO: 3. Typically, the assay detects HEV-C infection or Sup>A combination of HEV-A and HEV-C infection in Sup>A sample with Sup>A sensitivity of about or more than 80% and Sup>A specificity of about or more than 70%. Typically, the assay distinguishes between HEV-A infection and HEV-C infection in the sample with Sup>A sensitivity of about or more than 80%.

The sample is typically a sample obtained from a subject. Suitable subjects include humans, non-human primates, domestic animals, wild animals, farm animals, or laboratory animals. The sample is a body fluid or mucus obtained from a subject and includes blood, serum, plasma, fecal matter, exudates, saliva, sputum, tears, sweat, urine, or vaginal secretions. The sample may be diluted in a buffer to form a test sample. The sample may be diluted at a sample to buffer ratio of between about 1:5 to 1:500 (v/v). The sample or test sample may be processed to extract sample RNA for detection assays.

Also described are kits comprising a plurality of peptides having at least 90% amino acid sequence identity to SEQ ID No. 1, a plurality of peptides having at least 95% amino acid sequence identity to SEQ ID No. 1, or a plurality of peptides having the amino acid sequence as in SEQ ID No. 1.

Drawings

FIG. 1A is a diagram showing phylogenetic analysis of HEV-C1 ORF2 nucleotide sequences. The tree was constructed using the adjacency method with bootstrap values calculated from 1000 trees. Only the bootstrap values >700 are shown. HEV-C1 strains identified in human patients are highlighted in red, while HEV-C1 strains derived from rats are highlighted in black. The SRN250811 strain is marked in bold. FIG. 1B is Sup>A diagram showing alignment of the major HEV-A genotypes and the E2s amino acid sequences of the HEV-C1 LCK-3110 strain. Figure 1C shows monoclonal antibody (mAb) binding residues numbered consecutively below the relevant alignment position (number) in figure 1B. Conserved residues and non-conserved residues between LCK-3110 and at least one HEV-A genotype are boxed. Monoclonal antibodies that bind to each residue are indicated in the legend; those with overlapping or nested epitope specificities are displayed together. MAbs in bold are used for homology modeling and antigen EIA. The ratio beside the mAb tag indicates the number of conserved residues between HEV-C1 and HEV-Sup>A at these sites/the 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). FIG. 1D is a diagram showing an alignment of the E2s amino acid sequences of LCK-3110 and two different HEV-C1 strains of infected humans. FIG. 1E is Sup>A graph showing Sup>A comparison of the complex structures of mAbs 8C11 and 8G12 with HEV-A and E2 representing three HEV-C1 strains of three strain groups that infect humans. In these cartoon representations, 8C11 is displayed in the lower half of each top picture and E2 is displayed in the upper half of each top picture. In these cartoon representations, 8G12 is displayed in the lower half of each lower picture and E2 is displayed in the upper half of each lower picture. Polar contacts between mAb and E2 are depicted as dashed lines, and residues of interactions are represented by bars.

FIGS. 2A-2F are graphs showing optical density values of samples in groups A-D measured using three commercial IgG EIAs (FIGS. 2A-2C) and IgM EIAs (FIGS. 2D-2F). Bars represent mean and Standard Error of Mean (SEM). The mean OD for each group in each EIA was compared to the mean OD for group C using Student's t-test, with or without Welch's correction, as appropriate. The P value is marked ns: p value >0.05,: p value +.0.05,: p value +.0.01,: p value +.0.001,: the p value is less than or equal to 0.0001.

Fig. 3A-3C are diagrams showing the following: FIG. 3A-OD values of samples in groups B, C and D of EIA were detected using the Wantai antigen. The dashed line represents the measurement cut-off value. The points in group D represent healthy controls. Bars represent the mean and standard error of the mean. The average OD values of group B and group D were compared to group C by t-test. The P value is marked ns: p value >0.05,/x: the p value is less than or equal to 0.0001. Binding of mAb #4 (FIG. 3B) and 12F12 (FIG. 3C) to HEV-A and HEV-C1 was evaluated in EIA format. OD's of HEV negative control serum (sample 1), HEV-A4p239, and two HEV-A4 patient sera (samples 6-8), HEV-C1p241, and three HEV-C1 patient sera (samples 2-5) were measured. Each sample was tested in triplicate. Symbols represent mean values and bars represent SD values for the triplicate. The average OD of each sample was compared by t-test alone to the OD of the negative control: ns: p value >0.05,: the p value is less than or equal to 0.05.

Fig. 4A is a graph showing the time line of vaccination and infection challenge for rats. On day 56, rat livers were obtained for viral load testing. Fig. 4B-4D are graphs showing fecal viral load (fig. 4B), plasma viral load (fig. 4C), and day 28 liver tissue viral load (fig. 4D) of rats in each group. Bars represent mean and SEM. Undetectable viral load was expressed as 3 logs ₁₀ copy/mL (dashed line), which is the limit of detection (LOD) of the HEV-C1 RT-PCR assay. For all time points where there was a significant difference between the group averages by one-way anova, we compared the average of each group separately to the PBS group by Tukey-Kramer method. The P value is marked ns: p value>0.05,*: p value +.0.05,: p value +.0.01,: p value +.0.001,: the p value is less than or equal to 0.0001.

FIGS. 5A and 5B are graphs showing alanine aminotransferase (ALT, FIG. 5A) and alkaline phosphatase (ALP, FIG. 5B) in rats challenged with HEV-C1 immunized with PBS, hecolin, HEV-A4 p239 or HEV-C1p 241. Bars represent the mean and standard error of the mean.

FIG. 6 is a graph showing the time lines of the mixed vaccination protocol and HEV-C1 challenge experiment in rats with vaccination and infection challenge in rats. Four rats were given Hecolin and HEV-C1p241 vaccine two weeks apart and then challenged with SRN250811 strain. On day 56, rat livers were obtained for viral load testing.

FIG. 7 is a graph showing the assay evaluation of healthy organ donor serum previously tested negative in the Wantai HEV-IgG EIA kit. The average OD values in both HEV-A and HEV-C IgG EIA were substantially comparable.

FIG. 8 is a graph showing a subject operating characteristic (ROC) curve using RT-PCR results as gold standards, which results in the generation of an assay cut-off for each IgG EIA.

Disclosure of Invention

I. Definition of the definition

As used herein, the term "virus-like particle" or "VLP" refers to empty protein shells of viruses that have the same or similar external form as the virus itself, but do not contain any viral genome, i.e., they are non-infectious particles. VLPs may be peptide dimers, trimers, oligomers or polymers of peptides. VLPs may be homodimers, homotrimers, homooligomers, or homomultimers of peptides. VLPs are typically synthetic particles formed from purified recombinant peptides.

As used herein, the term "peptide" includes proteins and fragments thereof. Peptides are disclosed herein as amino acid residue sequences. These sequences are written left to right in the direction from the amino-terminus to the carboxy-terminus. According to standard nomenclature, the amino acid residue sequence is named by a three letter or one letter code as shown below: alanine (Ala, a), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (gin, 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 polynucleotide that differs from a reference peptide or polynucleotide but retains essential characteristics. A variant of a typical peptide differs in amino acid sequence from another reference peptide. In general, the differences are limited such that the sequences of the reference peptide and variants are very similar overall and identical in many regions. The variant and reference peptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). The substituted or inserted amino acid residues may or may not be those encoded by the genetic code. The variant of the peptide may be naturally occurring, such as an allelic variant, or it may be a variant that is not considered to be naturally occurring.

Modifications and changes can be made in the structure of a peptide and still obtain a molecule (e.g., conservative amino acid substitutions) with similar properties to the peptide. For example, certain amino acids may be substituted for other amino acids in the sequence without significant loss of activity. Because it is the interactive capacity and nature of the peptide that determines the biological functional activity of the peptide, certain amino acid sequence substitutions may be made in the peptide sequence but still obtain peptides with similar properties.

As used herein, the term "identity", as known in the art, is the relationship between two or more peptides 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" may also mean the degree of sequence relatedness of a peptide or the degree of sequence relatedness of a peptide as 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 below: computational Molecular Biology, lesk, a.m. plaited, oxford University Press, new York,1988; biocomputing: informatics and Genome Projects, smith, d.w. editions, academic Press, new York,1993; computer Analysis of Sequence Data, part I, griffin, a.m., and Griffin, h.g., editions, humana Press, new Jersey,1994; sequence Analysis in Molecular Biology von Heinje, g., academic Press,1987; and Sequence Analysis Primer, grisskov, M.and Devereux, J.ed., M Stockton Press, new York,1991; and Carilo, H., and Lipman, D., SIAM J Applied Math.,48:1073 (1988).

Various procedures and alignment algorithms are described in the following documents: 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, 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), present detailed considerations for sequence alignment methods and homology calculations.

NCBI's search tool based on the local alignment algorithm (BLAST) (Altschul et al, (1990) supra) are available from a number of sources including the national center for biological information (NCBI, national Library of Medicine, building38A, room 8N805, bethesda, md.20894) and on the Internet for use in connection with sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. More information can be found at the NCBI website. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. If two compared sequences share homology, the designated output file will present these regions of homology as aligned sequences. If the two compared sequences do not share homology, the designated output file will not present the aligned sequences.

The preferred method of determining identity is designed to give the greatest match between the sequences tested. Methods of determining identity and similarity are encoded in publicly available computer programs. The percent identity between two sequences may be determined using analysis software (e.g., sequence analysis software package of a genetic computer group, madison wis.) in which needlelman and Wunsch (j. Mol. Biol.,48:443-453,1970) algorithms (e.g., NBLAST and XBLAST) are combined. Default parameters are used to determine peptide identity.

For example, a peptide sequence may be identical to a reference sequence, i.e., 100% identical, or it may contain up to an integer number of amino acid changes compared to the reference sequence such that the percent identity is less than 100%. Such changes are selected from: at least one amino acid deletion, substitution (including conservative substitutions and non-conservative substitutions) or insertion, and wherein the change may occur at the amino-or carboxy-terminal positions of the reference peptide sequence or at any position between these terminal positions, interspersed either alone between amino acids within the reference sequence or in one or more contiguous groups within the reference sequence. The number of amino acid changes for a given percentage of identity is determined by multiplying the total number of amino acids in the reference peptide by the percentage value of the corresponding percentage of identity (divided by 100) and then subtracting this product from the 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 to nucleotides or amino acids in a reference nucleic acid or peptide sequence after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percent sequence identity. Alignment to determine percent sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN-2, or Megalign (DNASTAR) software. The appropriate parameters for measuring the alignment, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared, can be determined by known methods.

As used herein, the term "substantially" generally refers to a degree of comparison, 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 variability. In the context of sequence identity, substantial sequence similarity, or substantial sequence identity, refers to a sequence identity of at least about 85-90%, about 90%, preferably about 95% or more.

As used herein, the term "recombinant polynucleotide" generally refers to a polynucleotide obtained by 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 a nucleic acid or peptide having a non-naturally occurring sequence or having a sequence produced by artificial combination of two or more otherwise isolated sequence fragments. Such artificial combination is often achieved by chemical synthesis or more commonly by manual manipulation of isolated nucleic acid fragments, for example by genetic engineering techniques. Recombinant peptide may also refer to peptides prepared using recombinant nucleic acids, including recombinant nucleic acids transferred to a host organism of natural origin other than the peptide.

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

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

As used herein, the terms "dimer", "trimer", "tetramer", "oligomer" or "multimer" refer to two, three, four or more monomers, respectively, that form a structure such as an assembly or a shell of a peptide. For each dimer, trimer, tetramer or multimer-forming monomer, the dimer, trisomy, tetramer or multimer may be a homodimer, homotrimer, homotetramer or homomultimer containing the same amino acid sequence. For each dimer, trimer, tetramer or multimer-forming monomer, the dimer, trimer, tetramer or multimer may be a heterodimer, heterotrimer, heterotetramer or heteromultimer containing different amino acid sequences.

As used herein, the term "detect" or "determining" generally refers to obtaining information. The detection or determination may utilize any of a variety of techniques available to those skilled in the art, including, for example, the specific techniques explicitly referred to herein. The detection or determination may involve manipulation of the physical sample, consideration of data or information, and/or manipulation, e.g., using a computer or other processing unit adapted to perform a correlation analysis, and/or receiving the correlation information and/or material from a source. Detection or determination may also mean comparing the obtained value with 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 Sup>A test to correctly identify Sup>A true positive (i.e., sup>A subject is infected with HEV-Sup>A or HEV-C). For example, sensitivity can be expressed as Sup>A percentage, i.e., the proportion of actual positives that are correctly identified (e.g., test subjects with HEV-Sup>A or HEV-C infection are correctly identified by testing as having Sup>A percentage of infection). Assays with high sensitivity have low false negative rates (i.e., cases where HEV-A or HEV-C infection is not identified). Typically, the disclosed assays and methods have a sensitivity of about or more than 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 Sup>A test to correctly identify true negatives (i.e., sup>A subject does not have an HEV-Sup>A or HEV-C infection). For example, specificity may be expressed as Sup>A percentage, i.e., the proportion of actual negatives that are correctly identified (e.g., test subjects without HEV-Sup>A or HEV-C infection are correctly identified by testing as Sup>A percentage without infection). Tests with high specificity have Sup>A low false positive rate (i.e., cases without HEV-Sup>A or HEV-C infection but the test suggests individuals with infection). Typically, the disclosed methods have a specificity of about or more than 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 to test results that provide high sensitivity and high specificity, such as having a sensitivity of greater than about 70% and a specificity of greater than about 70%, having a sensitivity of greater than about 80% and a specificity of greater than about 80%, or having a sensitivity of greater than about 90% and a specificity of greater than about 90%.

As used herein, the term "sample" refers to a bodily fluid, body smear, cell, tissue, organ, or portion thereof isolated from a subject. The sample may be a single cell or a plurality of cells. The sample may be a specimen obtained by biopsy (e.g., surgical biopsy). The sample may be cells from the subject that are or have been placed in tissue culture or are suitable for tissue culture. The sample may be one or more of cells, tissue, serum, plasma, urine, saliva, sputum, and stool. The sample may be one or more of a swab, liquid, blood, plasma, serum, urine, faeces, sputum or exudates.

As used herein, the term "subject," "individual," or "patient" refers to a human or non-human mammal. The subject may be a non-human primate, livestock, wild animal, farm animal or 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 suffering from a disease or disorder. The term "patient" includes both human and veterinary subjects.

A "control" sample or value refers to a sample that is used as a reference (typically a known reference) for comparison with a test sample. For example, a test sample may be taken from a test subject and a control sample may be taken from a control subject, such as from a known normal (non-disease) individual. The control may also represent an average value taken from a population of similar individuals, such as a disease patient or healthy individual with similar medical background, the same age, weight, etc. The skilled artisan will recognize that controls may be designed to evaluate the number of any parameters.

As used herein, the term "treating" refers to administering a composition to a subject or system to treat one or more symptoms of a disease. The effect of administering the composition to a subject may be, but is not limited to, stopping a particular symptom of the condition, alleviating or preventing a symptom of the condition, alleviating the severity of the condition, completely ablating the condition, stabilizing or delaying the development or progression of a particular event or feature, or minimizing the chance that a particular event or feature will occur.

As used herein, the terms "effective amount" and "therapeutically effective amount" are used interchangeably, as applied to the peptides, therapeutic agents, and pharmaceutical compositions described herein, which are the amounts necessary to direct the desired therapeutic result. For example, an effective amount is a level that is effective to treat, cure, or alleviate the symptoms of a disease being treated with the composition and/or therapeutic agent or pharmaceutical composition. The amount effective for the particular therapeutic target sought will depend on a variety of factors, including the disease being treated and its severity and/or the stage of development/progression; bioavailability and activity of the particular compound and/or antineoplastic agent or pharmaceutical composition used; a route or method of administration and an 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.

The term "about" is used to describe a value within about + -10% of the specified value or below; in other embodiments, the value may vary within a value within ±5% above or below the specified value; in other embodiments, the value may vary within a value within ±2% above or below the specified value; in other embodiments, the value may vary within ±1% above or below a specified value.

II, peptides, virus-like particles and compositions

Peptides, particles, compositions, kits and assays are described that detect HEV-C infection, protect against HEV-C, distinguish HEV-C infection from HEV-A infection, and provide accurate and rapid methods for diagnosing HEV-C infection in humans.

The following examples illustrate that existing compositions, kits, and methods fail to detect HEV-C infection or protect against HEV-C. There is no recognition that existing diagnostic methods for detecting HEV-A may miss or misdiagnose HEV-C infection in Sup>A subject.

Peptides, virus-like particles and compositions comprise peptides having subtle amino acid similarity to a portion of the HEV-C genotype 1 (HEV-C1) open reading frame 2 (ORF 2) capsid protein, which is referred to as HEV-C1p241 peptide. There is no substantial amino acid sequence identity between the HEV-C1p241 peptide and the ORF2 capsid proteins of other HEV species (including, e.g., HEV-A). This makes HEV-C1p241 and its substantially similar variants useful in detecting HEV-C infection in humans, protecting against HEV-C, providing an accurate and rapid method of diagnosing HEV-C infection, and distinguishing between HEV-C infection and HEV-A infection.

A. Peptides

The peptide will typically have at least 90% amino acid sequence identity to SEQ ID NO. 1 or SEQ ID NO. 31, at least 95% amino acid sequence identity to 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. The other peptide may have at least 90% amino acid sequence identity to SEQ ID NO. 3 or SEQ ID NO. 29, at least 95% amino acid sequence identity to SEQ ID NO. 3 or SEQ ID NO. 29, or an amino acid sequence as shown in SEQ ID NO. 3 or SEQ ID NO. 29.

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

HEV-C1 p241 and variants thereof

HEV-C p241 (357I-597V, genBank code: AYF 53239.1) and variants thereof can form VLPs and compositions for detecting HEV-C infection in humans, providing accurate and rapid diagnosis of HEV-C infection, distinguishing HEV-C infection from HEV-A infection, and protecting against HEV-C.

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

IVQVLFNIADTLLGGLPTDLVSNAGGQLFYGRPQVSENGEPSVKLYTSVEAAQLDHGVTIPHDIDLGVSAITLQDFDNQHLQDRPTPspaparpitnwrsgdvvwvtlpsaeyaqsqsamgshpaywseeatiinvatgqraavssikwdqvtlngkalhkethsglvyyqlplmgkinfwqqgttkagytynynttdsdslwvwwdggskaylyistyttmlgagpvnitglgavgPNPV(SEQ ID NO:1)。

SEQ ID NO. 1 spans residues 357I-597V of the 644 amino acid long capsid protein of the rat HEV "LCK-3110" strain. The lower case portion is shown as SEQ ID NO. 31 in FIG. 1B. The peptide is encoded by ORF2 of the virus. Variants of SEQ ID NO. 1 or SEQ ID NO. 31 include peptides having at least 90% amino acid sequence identity to SEQ ID NO. 1 or SEQ ID NO. 31. Examples of variant peptides include, but are not limited to, variant peptides comprising SEQ ID NO. 32 or SEQ ID NO. 33 as shown in FIG. 1D. Other variants include those from having GenBank accession numbers: OFR2 peptide of this region of HEV-C strain of GU345042, GU345043, JN167537, AB847308, KM516906, AB847306, AB847309, AB847305, AB847307, JX120573, AB890001, MG020022, KU670940 and LC 549186: .

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

ATTGTTCAGGTTTTGTTCAATATTGCGGATACCCTGTTAGGCGGCCTGCCAACGGACCTTGTTTCTAACGCTGGGGGCCAACTGTTCTATGGCCGACCGCAGGTGTCTGAGAATGGTGAACCATCGGTTAAGCTTTATACATCTGTGGAAGCCGCCCAGCTGGACCATGGGGTTACCATTCCCCATGATATTGACTTGGGTGTGTCCGCCATCACACTACAGGACTTTGATAACCAGCATTTGCAGGACCGCCCTACGCCCTCACCAGCGCCAGCTCGCCCGATCACGAACTGGCGCTCTGGTGATGTGGTGTGGGTCACATTACCATCGGCCGAATATGCGCAGTCTCAGAGCGCAATGGGTTCCCACCCGGCCTACTGGTCCGAGGAGGCGACTATAATCAACGTCGCTACGGGCCAACGGGCCGCCGTGTCTAGCATAAAGTGGGATCAGGTCACTCTTAACGGCAAGGCCCTGCATAAGGAGACTCATTCAGGCTTGGTTTATTACCAGCTGCCATTGATGGGGAAGATCAATTTTTGGCAGCAGGGTACCACCAAAGCCGGTTATACTTATAATTACAACACTACTGATTCAGACAGTTTGTGGGTGTGGTGGGATGGGGGCTCTAAGGCTTATCTCTATATATCTACTTATACTACTATGCTAGGTGCTGGACCTGTTAACATCACGGGGCTGGGTGCTGTCGGCCCCAACCCCGTTG。(SEQ ID NO:2)。

variants of SEQ ID NO. 1 and SEQ ID NO. 2 include amino acid or nucleic acid sequences having substantial sequence identity, such as having at least about 85-90%, about 90%, preferably about 95% or more sequence identity to SEQ ID NO. 1 or SEQ ID NO. 31 and SEQ ID NO. 2, respectively. Amino acid variability in variants generally retains important peptide properties. In general, the differences are limited such that the sequences of the reference peptide and variants are very similar overall and identical in many regions. The amino acid sequences of the variant and reference peptides may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). The variant of the peptide may be naturally occurring, such as an allelic variant, or it may be a variant that is not considered to be naturally occurring.

Modifications and changes can be made in the structure of a peptide and still obtain a molecule (e.g., conservative amino acid substitutions) with similar properties to the peptide. For example, certain amino acids may be substituted for other amino acids in the sequence without significant loss of activity. Indeed, because the interactive capacity and nature of a peptide determines the biological functional activity of the peptide, certain amino acid sequence substitutions may be made in the peptide sequence, but still obtain a peptide with similar properties.

2.HEV-A4 p239

HEV-A4 p239 (382I-620A, genBank code: MW 660888) has the following amino acid sequence:

IALTLFLFANLADTLLGGLPTELSSAGGQLFYSRPVVSANGEPTVKLYTQENAQDKGAIPHDIDLGESRVVIQDYQHEQDRPTPspaprpsfsvrandwlsltaaaey dqttygsntmyvsttvtvvanvaqgsktswtldgrpttigplttiqysktffvlplrgklgfwe, agttkagypypynydttashdaglagngyttttttttgntsgstsgstsgshvgstsgshvgshvgslasvgsvvalavglavglavglavvala (SEQ ID NO: (SEQ ID NO: 3). The lower case portion is shown as SEQ ID NO. 29 in FIG. 1B.

HEV-A4 p239 shares substantial amino acid sequence identity with the p239 peptide of HEV-A genotype 1 (HEV-A1 p 239) and with other capsid proteins of HEV A species.

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

ATAGCATTGACCCTGTTTAATCTTGCTGATACGCTTCTCGGCGGGCTCCCGACAGAATTAATTTCGTCGGCTGGTGGCCAGCTGTTTTACTCTCGCCCCGTCGTCTCAGCCAATGGCGAGCCGACTGTGAAACTCTACACTTCAGTCGAGAATGCCCAGCAGGATAAGGGTATAGCTATCCCACATGATATTGATCTTGGTGAGTCCCGAGTAGTTATTCAGGATTATGACAACCAGCATGAGCAAGATCGCCCTACTCCATCTCCTGCTCCCTCTCGCCCTTTTTCTGTTCTTCGTGCTAATGATGTGCTTTGGCTTTCACTTACAGCTGCTGAATACGATCAGACTACCTATGGCTCTTCTACTAATCCTATGTATGTCTCTGACACCGTAACATTTGTTAATGTGGCCACTGGCGCCCAGGGGGTGGCACGCTCTCTGGACTGGTCCAAGGTCACCCTTGATGGGCGCCCACTTACCACTATTCAGCAGTACTCTAAGACTTTCTTTGTCCTACCCCTCCGTGGTAAACTTTCTTTTTGGGAGGCTGGTACAACTAAAGCTGGCTACCCATATAATTATGATACTACTGCCAGTGACCAGATTTTGATTGAGAATGCGGCAGGTCATCGTGTCTGTATTTCTACTTACACTACTAACTTAGGTTCTGGGCCTGTCTCTATTTCTGCTGTTGGTGTTTTAGCACCCCACTCTGCT

(SEQ ID NO:4)。

GenBank accession numbers for sequences at corresponding regions in other HEV a species include: l08816 (HEV-A1), AB369687 (HEV-A3) and AJ272108 (HEV-A4).

B. Virus-like particles

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

VLPs may comprise two, three, four or more peptides assembled into one particle. Typically, VLPs are homodimers, homotrimers, homooligomers or homomultimers of the peptide. The VLP may be a homodimer, homotrimer, homooligomer or homomultimer of HEV-C1 p241, wherein each monomer has SEQ ID NO. 1 or a variant thereof, such as a monomer having substantial amino acid sequence identity with SEQ ID NO. 1. Typically, HEV-C1 VLPs comprise HEV-C1 p241 or homo-, co-trimers, homo-oligomers or homo-multimers of monomers having at least 85%, preferably at least 90% or 95% amino acid sequence identity with SEQ ID NO. 1.

The VLP may be a homodimer, homotrimer, homooligomer or homomultimer of HEV-A4 p239, wherein each monomer has SEQ ID NO. 3 or a variant thereof, such as a monomer having substantial amino acid sequence identity with SEQ ID NO. 3. Typically, HEV-A4 VLPs comprise HEV-A4 p239 or homo-, homo-or homo-multimers of monomers having at least 85%, preferably at least 90% or 95% amino acid sequence identity with SEQ ID NO. 3.

C. Composition and method for producing the same

The peptide or VLP may be comprised in a composition. Typically, the peptide or VLP is included in the composition together with pharmaceutically acceptable excipients and/or adjuvants.

The composition may comprise a plurality of peptides or VLPs, wherein the peptides have at least 90% or at least 95% amino acid sequence identity to SEQ ID NO. 1 or SEQ ID NO. 31. The composition may comprise a plurality of peptides or VLPs, wherein the peptides have the amino acid sequence as in SEQ ID NO. 1 or SEQ ID NO. 31. The composition may comprise a peptide or VLP, wherein the peptide has at least 90% or at least 95% amino acid sequence identity with SEQ ID NO. 3 or SEQ ID NO. 29. The composition may comprise a plurality of peptides or VLPs, wherein the peptides have the amino acid sequence as in SEQ ID NO. 3 or SEQ ID NO. 29.

A peptide or VLP in a bivalent composition. The divalent composition may comprise a mixture of a plurality of peptides having at least 90% or at least 95% amino acid sequence identity to SEQ ID NO. 1 or SEQ ID NO. 31 and a plurality of peptides having at least 90% or at least 95% amino acid sequence identity to SEQ ID NO. 1 or SEQ ID NO. 31. The peptides of the VLPs in the composition may be: 1) A plurality of peptides having the amino acid sequence as set forth in SEQ ID NO. 1 or SEQ ID NO. 31, and 2) a mixture of a plurality of peptides having the amino acid sequence as set forth in SEQ ID NO. 1 or SEQ ID NO. 29.

The composition may contain an effective amount of peptide or VLP. Suitable effective amounts comprise peptides or VLPs from about 0.1 μg to about 10000 μg, from about 1 μg to about 10000 μg, from about 5 μg to about 10000 μg, from about 10 μg to about 9000 μg, from about 10 μg to about 8000 μg, from about 10 μg to about 7000 μg, from about 10 μg to about 6000 μg, from about 10 μg to about 5000 μg, or from about 10 μg to about 1000 μg.

The composition may be provided in a volume containing from about 100. Mu.l to about 5000. Mu.l of the composition containing from about 0.1. Mu.g to about 10000. Mu.g of the peptide or VLP. The composition may be provided in a vial containing about 200 μl to about 4000 μl, about 300 μl to about 3000 μl, about 300 μl to about 2000 μl, or about 400 μl to about 1000 μl of the composition. These volumes of the composition may comprise about 0.1 μg to about 10000 μg, about 1 μg to about 10000 μg, about 5 μg to about 10000 μg, about 10 μg to about 9000 μg, about 10 μg to about 8000 μg, about 10 μg to about 7000 μg, about 10 μg to about 6000 μg, about 10 μg to about 5000 μg, or about 10 μg to about 1000 μg of peptide or VLP. In some aspects, the composition is provided in a volume 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 the composition contains about 10 μg to about 1000 μg, about 10 μg to 800 μg, or about 10 μg to 500 μg of peptide or VLP. Exemplary compositions may be provided in a volume of about 500 μl and may contain about 10 μg to 1000 μg, or about 10 μg to 100 μg of peptide or VLP.

The composition may comprise pharmaceutically acceptable excipients, carriers and/or adjuvants.

1. Excipient

As will be appreciated by those skilled in the art, the excipients may be selected based on the application of the composition. Excipients may include antioxidants, chelating agents, preservatives, suspending agents, and combinations thereof.

Suitable antioxidants include, but are not limited to, butylhydroxytoluene, alpha tocopherol, ascorbic acid, fumaric acid, malic acid, butyl hydroxyanisole, propyl gallate, sodium ascorbate, sodium metabisulfite, ascorbyl palmitate, ascorbyl acetate, ascorbyl phosphate, vitamin a, folic acid, flavone or flavonoid, 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, ethylenediamine tetraacetic 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, butyl parahydroxybenzoate, ethyl parahydroxybenzoate, methyl parahydroxybenzoate, propyl parahydroxybenzoate, sodium benzoate, sodium propionate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenethyl alcohol, thimerosal, and combinations thereof.

Excipients may include suspending agents such as sterile water, phosphate buffered saline, saline or non-aqueous solutions 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 contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio, commensurate with a reasonable guideline for such an organization such as the food and drug administration.

2. Carrier body

The skilled artisan can select an appropriate carrier based on the intended use. For example, in some embodiments, the carrier may be a polymer or mixture of polyethylene glycol, polypropylene glycol, sugar (lactose, sucrose, dextrose, etc.), salts, poloxamers, hydroxypropyl cellulose, 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 resin, copolymers of methacrylic acid and ethyl acrylate, polylactic acid, PLGA, polyurethane, polyethylene vinyl acetate copolymer, polystyrene-butadiene copolymer and silicone rubber, or mixtures thereof.

3. Adjuvant

In general, an adjuvant is any substance that stimulates an immune response against the peptide or VLP administered. The adjuvant may be amorphous aluminum hydroxy phosphate sulfate (AAHS), aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), freund's complete adjuvant, freund's incomplete adjuvant, lipid a Monophosphate (MPL) with aluminum salts, oil-in-water emulsions composed of squalene, quil a, MPL and QS-21 (natural compounds extracted from tree of chile soap (Chilean soapbark tree), incorporated in liposomal formulations), or immunostimulatory oligonucleotides containing cytosine-phosphate guanosine (CpG) (a form of DNA synthesis mimicking bacterial and viral genetic material).

4. Exemplary compositions

In some aspects, the composition comprises a peptide or VLP in solid form, and in addition to the peptide or VLP, one or more of the following: sodium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, aluminum hydroxide and thimerosal.

In other aspects, the composition comprises a peptide or VLP in liquid form and, in addition to the peptide or VLP, contains one or more of the following: sodium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, aluminum hydroxide, thimerosal, and water.

III preparation method

HEV-A4 p239 and HEV-C1 peptides and VLPs are typically prepared using standard molecular biology techniques of any one or a combination of the following: gene expression, cloning into expression vectors, transformation of bacterial cells with expression vectors, induction of expression, harvesting of inclusion bodies from bacterial cells, renaturation by progressive dialysis with decreasing urea concentration, and purification of peptides. This process induces folding into VLPs.

More specific methods include obtaining a gene encoding HEV-A4 p239 (382I-620A, genBank code: MW 660888), a 239 amino acid peptide corresponding to HEV-A1 p239, and a gene encoding HEV-C1 p241 (357I-597V, genBank code: AYF 53239.1). This can be accomplished using amplification from a clinical isolate using a primer pair, such as the primer pair of SEQ ID NO. 19 and SEQ ID NO. 20 to amplify the gene encoding HEV-A4 p239 or the primer pair of SEQ ID NO. 21 and SEQ ID NO. 22 to amplify the gene encoding HEV-C1 p 241.

The amplified gene can then be cloned into the expression cassette of a bacterial expression vector and located downstream of the histidine tag. Expression vectors are well known in the art. Suitable sites in the expression cassette include Nde I and Xol I sites within the frame of the bacterial expression vector pETH and are located downstream of a series of 6 histidine residues. The recombinant HEV-A4 p239 and HEV-C1 p241 peptides can then be overexpressed in E.coli. Due to the different conformations and higher concentrations of the inclusion body fraction, the produced protein can be harvested from inclusion bodies instead of supernatant. By liquid-solid separation, inclusion bodies can be harvested from E.coli pellet and then solubilized in urea.

The dissolved peptide can then be renatured by progressive dialysis with gradually decreasing urea concentration. Refolded proteins can be purified by gel filtration chromatography using methods well known in the art (BIO-RAD, hercules, USA). Protein concentration may be determined by any suitable method, including by BCA protein assay (thermo fisher, waltham, USA).

Purified HEV-C1 p241 and HEV-A4 p239 peptides can allow refolding into VLPs.

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

IV method of use

A. Protection for HEV-C

The peptides, VLPs and compositions are useful for protecting a subject from HEV-C infection. The peptides, VLPs and compositions are useful for protecting subjects from HEV-C infection and HEV-A infection.

Typically, the peptide or VLP is mixed with an adjuvant, and optionally an excipient, and administered to a subject.

In general, an adjuvant is any substance that stimulates an immune response against the peptide or VLP administered. The adjuvant may be amorphous aluminum hydroxy phosphate sulfate (AAHS), aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), freund's complete adjuvant, freund's incomplete adjuvant, lipid a Monophosphate (MPL) with aluminum salts, oil-in-water emulsions consisting of squalene, quil a, MPL and QS-21 (natural compounds extracted from the tree of chile's soap, incorporated in liposomal formulations), or immunostimulatory oligonucleotides containing cytosine-guanine phosphate (CpG) (a form of DNA synthesis mimicking bacterial and viral genetic material).

The composition with the peptide or VLP and the adjuvant may be administered in an effective amount of the peptide or VLP to elicit an immune response against the peptide or VLP. An effective amount of peptide or VLP in the composition for inducing an immune response against HEV-C comprises peptide or VLP of about 0.1 μg/kg to about 1000 μg/kg, about 1 μg/kg to about 1000 μg/kg, about 5 μg/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 about 600 μg/kg, about 10 μg/kg to about 500 μg/kg, about 50 μg/kg to about 500 μg/kg, or about 0.1 μg/kg to about 10 μg/kg.

The composition providing protection against HEV-C may be provided in a vial containing about 100. Mu.l to about 5000. Mu.l of the composition containing about 0.1. Mu.g to about 10000. Mu.g of peptide or VLP. The composition may be provided in a vial containing about 200 μl to about 4000 μl, about 300 μl to about 3000 μl, about 300 μl to about 2000 μl, or about 400 μl to about 1000 μl of the composition. These volumes of the composition may comprise about 0.1 μg to about 10000 μg, about 1 μg to about 10000 μg, about 5 μg to about 10000 μg, about 10 μg to about 9000 μg, about 10 μg to about 8000 μg, about 10 μg to about 7000 μg, about 10 μg to about 6000 μg, about 10 μg to about 5000 μg, or about 10 μg to about 1000 μg of peptide or VLP. In some aspects, the composition is provided in a volume 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 the composition contains about 10 μg to about 1000 μg, about 10 μg to 800 μg, or about 10 μg to 500 μg of peptide or VLP. Exemplary compositions that provide protection against HEV-C can be provided in a volume of about 500. Mu.l and contain about 10. Mu.g to 1000. Mu.g, or about 10. Mu.g to 100. Mu.g, of peptide or VLP.

The composition may be administered once, twice, three times, four times or more as desired to elicit an immune response against the peptide or VLP. Assays for detecting an immune response against a peptide or VLP include immunoassays for detecting IgG or IgM specific for a peptide or VLP.

Compositions containing dual VLP systems based on human hepatitis E virus genotype 4 and rat hepatitis E virus (bivalent compositions) can be utilized in assays for detecting and distinguishing between human and rat hepatitis E virus infection antibodies. Furthermore, in animal models they trigger strong immune responses and may be used in vaccines against hepatitis E infection in rats or even bivalent vaccines against hepatitis E infection in humans and rats. The rat hepatitis E virus is a newly discovered human infection and no commercially available antibody test is available. Providing:

1. a dual VLP-based antibody test that can detect and distinguish human hepatitis e infection from rat hepatitis e infection. When these VLPs are utilized in commonly used antibody assay formats such as western blot and ELISA, they can distinguish between antibody profiles of human and rat hepatitis e infections.

Vlps may be protected against hepatitis e attack. VLPs are immunogenic and administration of HEV-C1 p241 can protect a subject from a hepatitis e virus infection in rats. These VLPs are useful as vaccines in humans.

B. Assay for detecting HEV-C infection

Peptides, VLPs and compositions are useful in a variety of detection assays. Typical detection assays include immunoassays with samples obtained from subjects.

The sample may also be used for HEV-C detection using an amplification assay (such as a polymerase chain reaction assay, etc.).

1. Immunoassay method

The assays and conditions for detecting immune complexes are known to those skilled in the art. Such assays include, for example, competition assays, direct reaction assays, sandwich assays, immunoblots, ELISA, EIA, competitive ELISA, and the like. The assay may be quantitative or qualitative. There are many different conventional assays for detecting antibody-peptide complex formation. For example, the detection step may comprise 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 photoelectric sensor. These different assays are well known to those skilled in the art.

Detection may be qualitative or quantitative, and typically uses antibody-detection label conjugates. The most commonly used detectable moieties in immunoassays are enzymes and fluorophores. In the case of an enzyme immunoassay (EIA or ELISA), enzymes such as horseradish peroxidase, glucose oxidase, β -galactosidase, alkaline phosphatase, etc., are typically conjugated to a secondary antibody by glutaraldehyde or periodate. The substrate to be used with a particular enzyme is typically selected to produce a detectable color change upon hydrolysis of the corresponding enzyme. In the case of immunofluorescence, the secondary antibody is chemically coupled to the fluorophore without altering its binding capacity. After the fluorescently labeled antibody binds to the immunocomplexes and any unbound material is removed, 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 group, or a bioluminescent group.

In some embodiments, the assay utilizes a solid phase or substrate to which the peptide or VLP is directly or indirectly attached. Thus, in some embodiments, the peptide or VLP is attached to or immobilized on a substrate, such as a solid or semi-solid support. Attachment may be covalent or non-covalent and may be facilitated by groups associated with peptides capable of covalent or non-covalent binding, such as groups having high affinity for components attached to a carrier, support or surface. The substrate may be a bead such as a colloidal particle (e.g., a colloidal nanoparticle made of gold, silver, platinum, copper, a metal composite, other soft metals, core-shell structured particles, or hollow gold nanospheres) or other types of particles (e.g., magnetic beads or particles or nanoparticles with silica, latex, polystyrene, polycarbonate, polyacrylate, or PVDF). Such particles may include labels (e.g., colorimetric, chemiluminescent, or fluorescent) and may be used to visualize the location of the peptide or VLP during an immunoassay. In some embodiments, the substrate is a flow path in a dot blot or lateral flow immunoassay device. For example, the peptide or VLP may be attached or immobilized on a porous membrane, such as a PVDF membrane (e.g., immobilon ^TM Membranes), nitrocellulose membranes, polyethylene membranes, nylon membranes, or similar types of membranes.

In some embodiments, the substrate is a flow path in an analytical rotor. In some embodiments, the substrate is a tube or well, such as a well in a plate (e.g., a microtiter plate) suitable for use in an ELISA assay. Such substrates may 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 film membranes (cast membrane film) composed of nitrocellulose, nylon, polysulfone, or the like. The substrate may be sintered polyethylene fines, commonly referred to as porous polyethylene, such as 0.2-15 micron porous polyethylene from Chromex Corporation (Albuquerque, n.mex.). All of these substrate materials may be used in suitable shapes such as films, sheets or plates, or they may be coated or adhered or laminated to a suitable inert carrier such as paper, glass, plastic film or fabric. Suitable methods for immobilizing peptides on a solid phase include ionic, hydrophobic, covalent interactions, and the like.

Immunoassay methods are well known in the art and comprise the steps of: the test sample is contacted with the peptide or VLP on the substrate to initiate binding of the immunoglobulin in the test sample to the peptide or VLP, thereby forming an immunoglobulin-peptide complex or an immunoglobulin-VLP complex. The immunoassay further comprises the step of detecting the binding by forming a signal from the binding. The method may further comprise comparing to a known control sample, a control value, or a signal from a control sample. Positive detection of binding is typically indicative of the presence of an anti-peptide or anti-VLP immunoglobulin in the test sample.

Typically, the immunoassay method comprises: (a) contacting the peptide or VLP with a test sample on a substrate or in a test container, (b) forming a signal, and (c) measuring the signal. The method may further comprise contacting the control sample with the peptide or VLP in the control container.

Typically, forming the signal comprises the step of contacting the antibody-detection label conjugate with the complex in the test container and/or the control container.

Steps (a) and (b) may comprise additional steps of blocking, washing and incubation, as required for each general immunoassay protocol.

2. Amplification assay

Another type of assay for detecting HEV-C or distinguishing HEV-C infection from other HEV species includesAnd (5) amplification measurement. These include Polymerase Chain Reaction (PCR) assays, which are rapid, specific and sensitive assay procedures for detecting HEV-C in a biological sample by amplifying one or more nucleotide sequences. The process of RNA genome may involve reverse transcriptase PCR (RT-PCR) or qRT-PCR methods. The general principles of real-time quantitative RT PCR are known in the art and are described, for example, in Poitras et al, reviews in Biology and Biotechnolog,2:1-11 (2002) and Gibson et al, genome Research,6:995-1001 (1996). Taq-based Real-time PCR of technology allows for a large dynamic range (10 to 10 ⁷ Copy) and thus is well suited for quantification of viral genomes. In addition, a large number of samples can be processed and no post-PCR processing makes it a safe and convenient clinical diagnostic method.

An oligonucleotide forward primer having a nucleotide sequence complementary to a unique sequence in a region of the HEV-C nucleotide sequence of interest hybridizes to and extends the complementary sequence. Similarly, a reverse oligonucleotide primer complementary to a second HEV-C nucleotide sequence in the same or a replacement region hybridizes to and extends. The 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 HEV-C nucleic acid molecules in a biological sample. The process involves obtaining Sup>A biological sample, contacting the sample with Sup>A compound or agent capable of detecting Sup>A nucleic acid sequence of HEV-C or HEV-A, thereby detecting the presence of HEV-C or HEV-A in the sample. Preferably, the assay is Sup>A quantitative real-time polymerase chain reaction (qPCR) using primers constructed based on Sup>A portion of the nucleotide sequence of the cdnSup>A corresponding to the HEV-C or HEV-Sup>A genomic region of interest. Forward and reverse primers are commonly used for these purposes. In some aspects, the assay comprises primer and probe sequences. The polymerization process typically results in amplicons.

The primer and probe sequences used to generate the amplicon are specifically tailored and designed to meet several different parameters depending on the primer or probe. Generally, the amplicon is desirably less than 150 nucleotides, optionally from 75 to 150 nucleotides or any value or range therebetween.

The assay can be used to detect the presence or absence (alone or in any combination) of HEV-C and HEV-A nucleic acid molecules in Sup>A biological sample, either simultaneously or sequentially. 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 may be used use molecular beacon probes, scorpion probes, SYBRgreen, multiple reporter genes for multiplex PCR, combinations thereof, or other DNA detection systems.

The assays are performed on instruments designed to perform such assays, such as those available from Applied Biosystems (FosterCity, CA). Assays typically involve performing an RT-PCR or PCR reaction on nucleic acids from a sample using specific primers and detecting amplification products.

HEV virus nucleic acid sequences are typically converted to complementary DNA (cDNA) sequences and then amplified prior to detection. The term "amplification" defines the process of preparing multiple copies of a nucleic acid in the form of RNA or cDNA from a single copy number or lower copy number nucleic acid sequence molecule. Amplification of the nucleic acid sequence is carried out in vitro by biochemical processes known to the person skilled in the art. The amplification agent may be any compound or system, including enzymes, capable of completing the synthesis of the primer extension product. Suitable enzymes for this purpose include, for example, E.coli DNA polymerase I, taq polymerase, the Klenow fragment of E.coli DNA polymerase I, T4 DNA polymerase, the AmpliTaq Gold DNA polymerase of Applied Biosystems, other available DNA polymerases, reverse transcriptases (preferably, iScript Rnase H+ reverse transcriptases), ligases, and other enzymes including thermostable enzymes (those that undergo primer extension after being subjected to a temperature sufficient to cause denaturation). Suitable enzymes will facilitate the nucleotide combinations in an appropriate manner to form primer extension products that are complementary to each mutant nucleotide strand. Typically, synthesis starts from the 3 'end of each primer and proceeds in the 5' direction along the template strand until synthesis terminates, which results in molecules of different lengths. However, there may be an amplification agent which starts synthesis at the 5' end and proceeds in the other direction using the same procedure as described above. In any event, the process of the present invention will not be limited to the amplification embodiments described herein.

An in vitro amplification process may include a 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 of amplifying a base sequence of DNA using a thermostable 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 strand can then serve as an additional template for the same primer sequence, successive rounds of primer annealing, strand extension and dissociation produce rapid and highly specific amplification of the desired sequence. Many polymerase chain methods are known to those skilled in the art and can be used in the process of the present invention.

The primers used according to this procedure are complementary to each strand of the nucleotide sequence to be amplified. The term "complementary" means that the primer must hybridize to its respective strand under conditions that allow the polymerization agent to function. In other words, the primer complementary to the flanking sequence hybridizes to the flanking sequence and allows for amplification of the nucleotide sequence. Preferably, the 3' end of the extended primer perfectly base pairs with the complementary flanking strand. In some embodiments, the probe has a nucleotide sequence complementary to one or more strands.

Those of ordinary skill in the art will recognize a variety of amplification methods that may also be used to increase the copy number of the target HEV nucleic acid sequence. Optionally, the nucleic acid sequences detected in the process of the invention are optionally further evaluated, detected, cloned, sequenced, etc., in solution or after binding to a solid support by any process commonly applied to detect specific nucleic acids, 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 Assay (OLA) (Landegren et al, science 241:1077 (1988)), RNase protection assay, etc. Molecular techniques for DNA analysis have been reviewed (Landegren et al Science 242:229 237 (1988)). After DNA amplification, the reaction product can be detected by a high level of amplified signal from the probe. In another embodiment, the amplification primers are fluorescently labeled and run by an electrophoresis system. Visualization of the amplified product can be performed by: laser detection and then computer aided graphical display.

i. Primer pair for PCR

Primer pairs for detection of HEV-A may include nucleic acid sequences comprising the sequences as set forth in SEQ ID NO. 19 and SEQ ID NO. 20.

Primer pairs for detecting HEV-C may include nucleic acid sequences comprising the sequences as set forth in SEQ ID NO. 21 and SEQ ID NO. 22.

Specifically for detection of HEV-C, the primer pair may include a nucleic acid sequence comprising the sequences as set forth in SEQ ID NO. 26 and SEQ ID NO. 27.

Primer pairs for nested PCR

Additional amplification steps by polymerization may include nested PCR of the PCR products.

For these nested PCR amplifications, the primer pair may include a nucleic acid sequence comprising the sequences as set forth in SEQ ID NO. 23 and SEQ ID NO. 24.

C. Detecting HEV-C infection and distinguishing between HEV-C infection and HEV-A infection

The described assays can be used to distinguish between HEV-C infection and HEV-A infection in Sup>A subject. The assay may have a platform or test vial that will contain:

(a) A plurality of peptides having at least 90% amino acid sequence identity to SEQ ID NO. 1 or SEQ ID NO. 31, a plurality of peptides having at least 95% amino acid sequence identity to SEQ ID NO. 1 or SEQ ID NO. 31, or a plurality of peptides having the amino acid sequence as in SEQ ID NO. 1 or SEQ ID NO. 31, and optionally,

(b) A plurality of peptides having at least 90% amino acid sequence identity to SEQ ID NO. 3 or SEQ ID NO. 29,

a step of contacting with the sample or the test sample.

The plurality of peptides on the platform or in the test vial may be synthetic virus-like particles. Typically, the sample is a body fluid or mucus, including blood, serum, plasma, fecal matter, exudates, saliva, sputum, tears, sweat, urine, or vaginal secretions obtained from the subject. The sample may be diluted with a buffer, which forms a test sample. The sample may be diluted at a ratio of sample to buffer of between about 1:5 to 1:500 (v/v).

The method may comprise contacting a plurality of test samples with a plurality of peptides having at least 90% amino acid sequence identity to SEQ ID NO. 1 or SEQ ID NO. 31 in a first set of platforms or test receptacles. The method may further comprise contacting the plurality of test samples with a plurality of peptides having at least 90% amino acid sequence identity to SEQ ID NO. 3 or SEQ ID NO. 29 in a second set of platforms or test receptacles. The method then generally includes forming a signal from the contact of the test sample with the plurality of peptides.

Typically, the method detects HEV-C infection when a signal is formed from contact of a test sample with a plurality of peptides having at least 90% amino acid sequence identity to SEQ ID NO. 1 or SEQ ID NO. 31, a plurality of peptides having at least 95% amino acid sequence identity to SEQ ID NO. 1, or a plurality of peptides having the amino acid sequence as in SEQ ID NO. 1 or SEQ ID NO. 31. Typically, this method detects HEV-A infection when Sup>A signal is formed from Sup>A test sample and Sup>A plurality of peptides having at least 90% amino acid sequence identity to SEQ ID NO. 3 or SEQ ID NO. 29.

In some aspects, the method distinguishes between HEV-A infection and HEV-C infection in Sup>A subject. Generally, when the method includes both steps (Sup>A) and (b) above, and the signal formed from the platform or test vial having (Sup>A) is significantly different from the signal formed from the platform or test vial having (b), the method distinguishes HEV-Sup>A infection from HEV-C infection.

In some aspects, the method detects both HEV-A infection and HEV-C infection in Sup>A subject. In general, the method detects both HEV-Sup>A and HEV-C infections when the method includes both steps (Sup>A) and (b) above, and the signal formed from the platform or test vial with (Sup>A) is not significantly different from the signal formed from the platform or test vial with (b).

Typically, the method detects HEV-C infection or Sup>A combination of HEV-A and HEV-C infection in Sup>A sample with Sup>A sensitivity of about or more than 80% and Sup>A specificity of about or more than 70%. Typically, the method distinguishes between HEV-A infection and HEV-C infection in the sample with Sup>A sensitivity of about or more than 80%.

D. Measurement accuracy

1. Early detection

The assay composition is highly specific for antibodies directed against HEV-C or HEV-A. Typically, the assay component has an accuracy of greater than 70% and Sup>A sensitivity of greater than 70%, greater than 80%, greater than 90% for detecting HEV-C infection or HEV-Sup>A infection in Sup>A subject.

2. Rapid detection

The determination is typically performed within a period of time between about 5 minutes and 5 hours, within about 15 minutes and 4 hours, within about 15 minutes and 3 hours, or within about 2 hours. The assay is rapid and can provide rapid and accurate results for the presence of HEV-C infection or HEV-A infection in the sample.

Immunoassays in the form of dipsticks are typically performed in less than one hour, such as, for example, within about 5-60 minutes.

Immunoassays for plate-format ELISA can be performed within 30min to 3 hours.

Amplification assays using polymerization in a thermocycler can be performed within 30 minutes to 3 hours.

Typically, these assays are developed for the rapid and accurate detection of HEV-C or HEV-A in Sup>A sample, or for the rapid and accurate detection of HEV-C or HEV-A infection. These tests can generally distinguish between HEV-C and HEV-A infections.

3. Accurate detection

Generally, assay compositions and assays provide highly sensitive and highly specific results, such as a sensitivity of greater than about 80% and a specificity of about 80%, a sensitivity of greater than about 85% and a specificity of greater than about 85%, or a sensitivity of greater than about 90% and a specificity of greater than about 90%.

The sensitivity of the test is the ability to correctly identify true positives (i.e., subjects with HEV-C and/or HEV-A infection). For example, sensitivity may be expressed as Sup>A percentage, i.e., the proportion of actual positives that are correctly identified (e.g., test subjects with HEV-C infection and/or HEV-Sup>A infection are correctly identified by testing as having Sup>A percentage of HEV-C infection and/or HEV-Sup>A infection). Detection with high sensitivity has Sup>A low false negative rate (i.e., no HEV-C and/or HEV-A cases are identified). Typically, 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%.

The specificity of the test refers to the ability to positively identify true negatives (i.e., individuals do not have HEV-C and/or HEV-A infection). For example, specificity may be expressed as Sup>A percentage, i.e., the percentage of actual negatives that are correctly identified (e.g., test subjects without HEV-C and/or HEV-A are correctly identified as being without HEV-C and/or HEV-A by testing). Tests with high specificity have Sup>A low false positive rate (i.e., an individual does not have HEV-C and/or HEV-A but the test suggests cases with HEV-C and/or HEV-A). Typically, the disclosed assay compositions and assays have a specificity of about or more than 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 disclosed compositions and methods can be used to detect and/or diagnose current or past HEV-C and/or HEV-A exposures or infections. Typically, an elevated presence and/or amount of HEV-C and/or HEV-A antibodies in Sup>A biological sample of Sup>A subject as compared to Sup>A control is indicative of current or past exposure or active infection with HEV-C and/or HEV-A. For example, sup>A method for aiding in detecting or diagnosing Sup>A current or present HEV-C and/or HEV-Sup>A exposure or infection in Sup>A subject may comprise determining the presence or level of proteins or fragments thereof directed against HEV-C1 p241 and/or HEV-Sup>A 4 p239, or the presence or level of nucleic acids encoding these proteins, in Sup>A biological sample from the subject, wherein the presence or level increase of antibodies or nucleic acids in the biological sample relative to the level of antibodies in Sup>A control is indicative of current or past contact or infection with HEV-C and/or HEV-Sup>A.

Any method may be combined with the therapeutic method. In preferred embodiments, the method of treatment comprises administering to the subject an effective amount of an antiviral treatment, analgesic treatment, antipyretic, or anti-hepatitis treatment.

V. kit

The peptides, VLPs and compositions, as well as other materials, may be packaged together in any suitable combination as a kit for or to aid in the practice of the disclosed methods. This is useful if the kit components in a given kit are designed and adapted for use together in the disclosed methods. For example, disclosed are kits having one or more of an assay platform, an assay buffer, a sample holder, a reporter compound, and/or a second detector compound. The kit may comprise a sterile needle, ampoule, tube, container or other suitable vessel for holding the assay components and/or performing the assay. The kit may contain instructions for use.

A kit for detecting the presence of HEV-C viral nucleic acid in a sample may comprise one or more primer pairs. For example, the kit comprises 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 comprises, for example: (1) Oligonucleotides, such as detectably labeled probes, that hybridize to nucleic acid sequences of HEV viruses, and/or (2) a pair of primers (one forward and one reverse) that are used to amplify nucleic acids containing HEV virus sequences. The kit may also comprise, for example, buffers, preservatives or protein stabilizers. The kit may also contain components (e.g., enzymes or substrates) necessary for detection of the detectable agent. The kit may also contain a control sample or series of control samples, which are assayed and compared to the test samples contained. Each component of the kit is typically packaged in a separate container, and all of the various containers are typically packaged in a single package with instructions for use.

Examples

There is an existing assembly method for preparing a peptide called p239 (this is called because the protein consists of 239 amino acids) of hepatitis E virus genotype 1 (HEV-A1). The p239 peptide forms VLPs and is the basis of the Hecolin vaccine. As shown below, a gene fragment of a similar protein expressing hepatitis E virus genotype 4 (HEV-A4) was not protected against hepatitis E in rats caused by HEV-C. The examples show the expression of homologous peptides of hepatitis E virus genotype 4 and rat hepatitis E (HEV-A4 p239 and HEV-C1 p241, respectively). These peptides share only 93% and 50-60% identity (i.e., similar amino acid%) with the original HEV-A1 p239, respectively. These peptides also formed VLPs like the original HEV-A1 p 239. VLP systems based on human hepatitis E virus genotype 4 (HEV-A4 p 239) and rat hepatitis E virus (HEV-C1 p 241) can be used for antibody testing for detecting and distinguishing human hepatitis E virus infection from rat hepatitis E virus infection.

The examples also show that VLPs trigger a strong immune response in animal models and can be used in vaccines against hepatitis e infection in rats or bivalent vaccines against hepatitis e infection in humans and rats. The rat hepatitis E virus is a newly discovered infection of humans and no commercially available antibody test is available. The use of these VLPs as vaccines provides combined protection or monovalent protection against the rat hepatitis e virus (HEV-C).

The described VPL provides the following advantages and uses:

1. the dual VLP-based antibody test can detect and distinguish human hepatitis e infection from rat hepatitis e infection. When these VLPs are utilized in a commonly used antibody assay format, such as western blot or ELISA, the VLPs distinguish antibody profiles of human hepatitis e infection from rat hepatitis e infection.

Vlps may be protected against hepatitis e attack. VLPs are immunogenic in mice and administration of HEV-C1 p241 protects rats from hepatitis e virus infection.

Example 1. Available kits and antibodies fail to distinguish HEV-A infection from HEV-C infection.

Materials and methods

Multiple sequence alignment and phylogenetic analysis

HEV-C1 open reading frame 2 (ORF 2) gene sequences from infected humans and symbiotic rats were retrieved from GenBank. ORF2 of the other 3 HEV-C1 strains of hong Kong's infected patients was sequenced using the primers listed in Table 1. Multiple sequence alignment and phylogenetic analysis of ORF2 nucleotide sequences was performed using ClustalX version 2 (Larkin et al, bioinformation, 23:2947-2948 (2007)). The E2s peptide amino acid (aSup>A) sequences of HEV-A and human HEV-C1 strains were compared. The degree of conservation at key amino acid residues targeted by monoclonal antibodies (mAbs) was analyzed using published kits (Zhao et al, J Biol chem.,290:19910-19922 (2015); gu et al, cell Res.,25:604-620 (2015)). A homology model of E2 of the human HEV-C1 strain was created as follows.

Homology modeling and docking

The available crystal structures of HEV-AE2 complexed with mAb were downloaded from a protein database (PDB ID:3RKD and 4 PLK) (Berman et al, nucleic Acids Res.,28:235-242 (2000)). Homology models of representative HEV-C1 strains that lead to human infection were modeled using iTASSER (Yang et al, nat methods, 12:7-8 (2015)). Modeled HEV-C1E 2s peptides were docked against mAbs 8C11 and 8G12 using the Rosettav3.5 protein-protein docking protocol (Das et al, annu Rev biochem.,77:363-382 (2008)). The starting pose of the butt-sampling is generated by comparing HEV-C1E 2 with HEV-AE2 in the crystal structure. The high resolution docking mode is used to search for the joint gesture and 500 baits are generated in each docking run. The pose with the best interface score is used for interaction analysis. Polar interactions between E2 and mAb were visualized with PyMOL.

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

Patient sample

The archived serum or plasmSup>A from immunocompromised patients with RT-PCR confirmed acute HEV-Sup>A infection (group Sup>A), immunocompromised patients with persistent HEV-Sup>A infection (group B) and patients with HEV-C1 infection (group C) was retrieved. The earliest archive samples for the assessment of hepatitis E were selected for each patient. The fourth group (group D) contained serum 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. Immunosuppressed conditions and persistent HEV infection are defined as follows.

Case definition of persistent hepatitis E or immunosuppression conditions

If the patient: a) suffering from hematological malignancy, b) being an organ transplant recipient, c) being subjected to disease-modifying antirheumatic/myelosuppressive cancer chemotherapy, d) being taking a steroid in an amount higher than 0.5 mg/kg/day prednisolone equivalent for at least one month, or e) concomitantly having less than 200 cells/mm ³ Advanced HIV infection of the CD4T lymphocyte count of these patients is considered immunosuppressive. (Sridhar et al, hepatology,0:1-13 (2020)). If the Hepatitis E Virus (HEV) A (HEV-A) or C (HEV-C1) viremiSup>A persists for more than three months, these patients are defined as suffering from persistent hepatitis E according to Kamar et al, americanjournal of transplantation,13:1935-1936 (2013). If sufficient archived samples are not available for viral load testing, the duration of hepatitis is used to distinguish between acute and persistent hepatitis E.

Patient sample collection has been approved by the university of hong Kong ethical review Committee/the western Hospital administration of hong Kong.

EIA kit, monoclonal antibody and polyclonal antiserum

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

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

HEV-A4 p239 and HEV-C1 p241, HEV-A4 and HEV-C1 homologs of HEV-A1p239 peptides used in the Hecolin vaccine (Xiamen Innovax Biotech, xiamen, china) were cloned and expressed as follows. The gene encoding HEV-A4 p239 (382I-620A) (corresponding to 239 amino acid peptides of HEV-A1p 239) was amplified from the 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: AYF 53239.1), a 241 amino acid peptide corresponding to HEV-A1p239, was amplified using primers 5'-CATATGATTGTTCAGGTTTTGTTCAATAT-3' (SEQ ID NO: 21) and 5'-CTCGAGAACGGGGTTGGGGCCGACAGCAC-3' (SEQ ID NO: 22).

The amplified gene was cloned into the bacterial expression vector pETH in frame downstream of the Nde I and Xol I sites and a series of 6 histidine residues. Recombinant HEV-A4 p239 and HEV-C1 p241 peptides were overexpressed in E.coli. Due to the different conformations and higher concentrations of the inclusion body fraction, proteins were harvested from inclusion bodies instead of supernatant. The inclusion bodies were harvested from E.coli pellet by liquid-solid separation and then dissolved in urea. The dissolved peptide is then renatured by progressive dialysis with progressively lower urea concentrations. By gel filtration chromatography (ENrich ^TM. SEC 70 10X 300mm column, BIO-RAD, hercules, USA) to purify refolded proteins. The concentration of protein was determined by BCA protein assay (Thermo Fisher, waltham, USA) according to the manufacturer's instructions. HEV-C1 p241 and HEV-A4 p239 peptides were loaded and separated on 8-12% acrylamide gel with 0.1% Sodium Dodecyl Sulfate (SDS), followed by staining with Coomassie blue.

IgG immunoblotting

Human and rat sera were tested in HEV-A4 p239 and HEV-C1 p241 IgG immunoblots as follows. The isolated HEV-A4 p239 (22 μg) and HEV-C1 p241 peptides (22 μg) were transferred to nitrocellulose membranes (Bio-Rad, hercules, USA). Blocking was performed overnight at 4℃using 10% skim milk in 1 Xphosphate buffered saline (PBS) containing 0.1% (v/v) Tween 20. Immunoblotting experiments were performed using Mini-PROTEAN II Multiscreen Apparatus (BIO-RAD) which allowed separation of the membrane lane by lane and addition of different antibodies/serum to different lanes. For immunoblotting experiments, the membranes were exposed to His-tagged antibodies (Bio-Station Ltd, hong Kong, china; dilution 1:5000), serial dilutions of WHO-referenced HEV antisera (from 0.02U/mL to 0.00125U/mL), diluted human serum (1:400) or rat serum (1:5000) for 45min in blocking buffer at room temperature. The blots were then washed in PBS containing 0.1% tween 20. After exposure to horseradish peroxidase (HRP) -conjugated secondary antibodies for one hour, subsequent washes were performed as described for primary antibodies. The films were visualized using a luminescence image analyzer (GE Healthcare, chicago, USA).

Results

Phylogenetic analysis revealed three different HEV-C1 strain groups that infest humans

ORF2 encodes the HEV capsid protein, which contains most of the immunodominant epitopes. In 13 cases of human HEV-C1 infection reported worldwide, nearly all ORF2 nucleotide sequences of 9 human-derived HEV-C1 strains were obtained and compared with 22 symbiotic rat-derived HEV-C1 for phylogenetic analysis. Eight human derived HEV-C1 strain sequences were from hong Kong patients, while one (MK 050105) was from Canadian patients. As mentioned, five hong Kong strains are from patients diagnosed between 8 months 2017 and 7 months 2019 (Sridhar et al, hepatology,0:1-13 (2020)), while three strains (PC-2020, KW-2019 and PW-2020) are from new patients diagnosed between 9 months 2019 and 5 months 2020. In the phylogenetic tree, strains from seven patients were clustered together to form a single strain set, designated "LCK-3110 strain set" as prototype LCK-3110 strain. The LCK-3110 strain was obtained from the first recorded human HEV-C1 infection (FIG. 1A). In addition to the LCK-3110 strain, two other HEV-C1 strains (MN 450843 and MK 050105) also infested humans; the amino acid identity of ORF2 between these strains and the LCK-3110 strain was 95.8% and 91.6%, respectively.

E2s amino acid alignment shows limited conservation of key residues targeted by anti-HEVmAb.

The E2s peptide (amino acids (aa) 455-603 of HEV-A1ORF 2) corresponds to the protruding "P" domain of the viral capsid, which contains the majority of immunodominant epitopes targeted by neutralizing antibodies. The E2s regions of HEV-A1, HEV-A3 and HEV-A4 reference strains were aligned with LCK-3110HEV-C1 strain (FIG. 1B). The average inter-genotype amino acid identity within HEV-A was 89.5%, while the amino acid identity between HEV-A1 and LCK-3110 was only 48%. 53 key residues involved in binding 17 well-characterized anti-HEV-AmAb were examined according to published kits (Zhao et al, J Biol chem.,290:19910-19922 (2015); gu et al, cell Res.,25:604-620 (2015)). HEV-A genotypes at these sites have limited variation. However, only 23/53 (43.4%) of these residues are conserved between HEV-A genotype and LCK-3110 (FIG. 1B). For 13/17 (76.5%) mAb, the amino acid identity between LCK-3110 and HEV-A at residues involved in mAb-E2 interactions was 50% or less. Residues involved in mAb 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 free radical substitutions, which resulted in Sup>A change in the polarity and/or charge of the amino acid side chain at that residue (Table 2).

TABLE 2 influence of amino acid substitutions on side chain characteristics at key residues in E2 recognized by monoclonal antibodies

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* The numbering of residue positions follows the order of key non-conserved residues in FIG. 1B

Of the 11 conservative amino acid substitutions, 8 focused on the linear epitope recognized by mAb #4 (fig. 1B). LCK-3110 and two other different human infectious HEV-C1 strains MN450843 and MK050105 have a high degree of E2 amino acid conservation (FIG. 1D), which confirms that these strains also hold differences from HEV-AE 2.

The protein-protein interaction model shows that anti-HEV-A mAb lacks recognition of HEV-C1

MAbs 8C11 and 8G12 bind to various epitopes in HEV-AE2 (FIG. 1E). The bonding interface is compacted with multiple hydrogen bond contacts, thereby stabilizing the bond. However, no similar binding conformation was found between these two mAbs and the representative HEV-C1 strain (LCK-3110, MK050105 and MN 450853), indicating that the key residues recognized by the mAbs were mutated in the HEV-C1 strain.

Commercial antibody EIA is less sensitive to HEV-C1 serodiagnosis and has considerable inter-assay variability

The effect of the difference in HEV-C1 antigenic sites on serum diagnosis was analyzed. Six commercial HEV-IgG and IgM assays were compared for performance using blood samples from 29 patients with immune function from HEV-Sup>A infection (group Sup>A), 10 immunocompromised patients with persistent HEV-Sup>A infection (group B), and 10 patients with HEV-C1 infection in which 5 people had Sup>A potential immunosuppressive condition (group C). Group C contained 10 out of 12 HEV-C1 infection cases reported globally as the study was conducted. All blood samples were HEV detectable by RT-PCR. Demographic and clinical characteristics of the patients are summarized in table 3. The potential medical conditions for an individual patient are listed in table 4. HEV-PCR negative healthy organ donor serum (n=10) constitutes the negative control (group D).

Table 3. Clinical and demographic characteristics of patients.

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HEV-A: hepatitis E Virus A species, HEV-A1: HEV genotype 1, HEV-A3HEV genotype 3, HEV-A4: HEV genotype 4, HEV-C1: hepatitis E virus seed C

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

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* Serum viral load was insufficient for genotyping, but HEV-ART-PCR was positive N/a: inapplicable; HEV-A1: HEVA genotype 1; HEV-A3: HEVA genotype 3; HEV-A4: HEV a genotype 4; HEV-C1: HEV C genotype 1 (rat hepatitis E)

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

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

* The inter-column p-value is calculated by Fisher's exact test; the intra-column p-value is calculated (presented in text form) by McNemar's test.

As shown in Table 5, the EIA positive detection rate (sensitivity) was significantly higher for all HEV-A samples than for HEV-C1 samples, except for the Wantai IgG. For both HEV-A and HEV-C1 samples, the sensitivity of the Wantai IgM detection tended to be higher than that of the Bei' er IgM and Novus IgM assays, although the differences did not reach statistical significance. However, for HEV-Sup>A samples (p=0.013 compared to Bei' ergg and p=0.041 compared to novusig) and HEV-C1 samples (p=0.041 compared to both), wantai IgG is significantly more sensitive than the other two IgG assays. The average OD values obtained in each IgG and IgM assay for the four panels were compared (fig. 2A-2F). The average OD of group C HEV-C1 samples was not significantly different from group D except for the Wantai IgG assay. Although only half of group C patients were immunosuppressed, the average OD of group C was significantly lower than that of group D or no difference from group B (immunosuppressed HEV-Sup>A patients). Also, the average OD of group Sup>A (HEV-Sup>A patients with normal immune function) was consistently higher than group C, except for Wantai IgG. Overall, these results demonstrate that the commercial anti-HEV antibody EIA is not reliable for HEV-C1 diagnosis, regardless of the immune status of the infected person. The Wantai IgG assay is superior to the comparator assay.

Parallel testing using recombinant peptide immunoblots can distinguish between HEV-A and HEV-C1 serological profiles in rat and human serum

pE2 capture antigen incorporates the entire E2s region, which maximizes cross-reactivity between HEV-Sup>A and HEV-C1. HEV-A1p239 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)). Additional residues facilitate folding into t=1 virus-like particles, which enhances immunogenicity and enables their use as vaccines. HEV-A4 and HEV-C1 peptide homologs, designated HEV-A4p239 and HEV-C1 p241, respectively, were expressed in HEV-A1p 239. As described for HEV-A1p239 (Li et al, vaccine,23:2893-2901 (2005)), both peptides formed 40-50kDa dimers, which then decomposed into about 30kDa monomers upon boiling, indicating similar physicochemical characteristics as HEV-A1p 239. To assess serological cross-reactivity serial dilutions of WHO referenced HEV antisera were tested on HEV-A4p239 and HEV-C1 p241 IgG immunoblots. At antiserum concentrations as low as 0.005U/mL, a distinct band was seen on the HEV-A4p239 immunoblot, but a weaker band was seen on the HEV-C1 p241 immunoblot, with almost no visible band at 0.02U/mL.

Rats were immunized with two intramuscular doses of HEV-A1 p239 (Hecolin), HEV-A4 p239, HEV-C1p241 or PBS (n=6 per group) two weeks apart. On day 28 after the first vaccine dose, all rats were simultaneously assessed for serological response in HEV-A4 p239 and HEV-C1p241 IgG immunoblots. Rat serum did not cross-react in HEV-A and HEV-C1 IgG immunoblots. Rats vaccinated with Hecolin and HEV-A4 p239 showed minimal reactivity in HEV-C1 immunoblots and vice versa. This suggests that HEV species-specific immunoblots can detect and distinguish antibody responses triggered by HEV-A and HEV-C1 antigen attacks.

Human serogroups were then retested using HEV-A4 p239 and HEV-C1p241 IgG immunoblots. The positive detection rate of HEV-A4 p239 IgG immunoblots of HEV-A samples (group A+B) was 29/39 (74.4%), and that of HEV-C1p241 IgG immunoblots of group C samples was 7/10 (70%), while having lower sensitivity among immunocompromised persons. Most positive HEV-A samples (27/29; 93.1%) reacted only with HEV-A4 p239 immunoblots and did not cross-react on HEV-C1 immunoblots. Five group C sera reacted only with HEV-C1p241 immunoblots, while two reacted cross-wise in HEV-A4 and HEV-C immunoblots. In general, for 36 samples producing 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 will elicit Sup>A serological response in humans that is specific for HEV species, which can be distinguished by parallel immunoblotting tests.

Example 2 lack of mAb cross-binding results in HEV antigen kits that are not effective for HEV-C1 diagnosis

Materials and methods

Antigen assay-12F 12 and #4 monoclonal antibody antigen capture EIA

mAb #4 and 12F12 in clinical samples were evaluated in EIA format for their ability to bind HEV-A4 p239, HEV-C1 p241, HEV-A4 and HEV-C1 and compared to HEV negative controls. The EIA design is as follows. Both monoclonal antibodies were diluted to a final concentration of 5 μg/ml in coating buffer and added to each well of a 96-well plate (500 ng/well). Plates were then incubated overnight at 4 ℃. Next, the plates were washed and incubated at 37 ℃ with 300 μl/well of blocking buffer supplemented with 0.05% tween 20 for 3h. HEV-A4 p239 peptide, HEV-C1 p241 peptide, test serum and control serum were 1:4 diluted in 1% BSA, aliquoted into wells (100 μl/well), and incubated at 37℃for 1h. Next, serum/peptide was removed and the plates were washed six times with 0.3% tween 20+pbs (300 μl/well). Mu.l of rat polyclonal serum 1:2000 against HEV-A or HEV-C1 was diluted appropriately in 20% goat serum and added to each well. After incubation at 37 ℃ for 1h, plates were washed six times and incubated with HRP conjugated goat anti-rat IgG for 30min at 37 ℃. Plates were washed six times and the reaction was detected by adding 1:1 peroxidase solution/TMB substrate solution. Finally, at 37℃1 After 0min, by adding 3M H ₂ SO ₄ To terminate the reaction and read the plate at a wavelength of 450-620nm using a microplate reader.

Results

Given the low conservation at the key epitopes, the ability to detect HEV-C1 for the Wantai antigen kit (a sandwich EIA comprising mabs 12F12 and # 4). Test group B and group C because they contain more than 4 logs ₁₀ Copy/ml of relatively high viral load. All group C HEV-C1 samples tested negative in the antigen assay, with OD values similar to the negative control, while all group B samples tested positive (fig. 3A).

It was then examined whether the constituent mabs of the Wantai antigen kit (12F 12 and # 4) could bind to the selected group B and group C samples with high viral load. Group B samples showed reactivity in both 12F12 and #4EIA, while group C samples had no significant difference in reactivity from the negative control (fig. 3B and 3C). HEV-A4 p239 binds to both mAbs, but not HEV-C1 p 241. Again, this demonstrates that mabs targeting HEV-Sup>A do not bind to HEV-C1, which significantly affects the 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 SRN250811HEV-C1 Strain

Rectal swabs were obtained from symbiotic rats (brown rats) captured in the south hong Kong and placed in a Viral Transport Medium (VTM). VTM was centrifuged and the supernatant passed through a bacterial filter. The filtered supernatant was administered intravenously to immunosuppressive 4 week old female SPF Sprague-Dawley rats. The immunosuppression regimen is a combination of tacrolimus, prednisolone and mycophenolate mofetil administered daily by oral gavage. The purpose of immunosuppression is to extend the duration of HEV-C1 infection and increase virus removal in fecal material, as HEV-C1 infection in rats is typically transient. Feces from rats were collected in VTM. VTM was centrifuged to pellet fecal material and the supernatant passed through a bacterial filter. The fecal filtrate was then passaged again in immunocompromised SPF Sprague-Dawley rats for further amplificationAnd (3) viruses. This passaged HEV-C1 strain was designated SRN250811. From this rat, it contains 10 ⁸ HEV-C1 genome equivalent/mL fecal suspension was used for viral challenge in the vaccination experiments described in this study.

Vaccine preparation

For vaccine preparation, purified HEV-A4 p239 and HEV-C1p241 peptides as prepared in example 1 were adjuvanted with aluminum hydroxide and formulated at a concentration of 30 μg/0.5mL to match the Hecolin vaccine.

Vaccination and infection challenge of rats

Six week old CD1 Sprague-Dawley rats (brown rats; 3 males, 3 females (n=6) per group) received 10 μg intramuscular doses of HEV-A4 p239, HEV-C1 p241, HEV-A1p239 (Hecolin), or phosphate buffered saline (PBS mixed with adjuvant aluminum hydroxide) two weeks apart. From the a priori estimates: a) All PBS vaccinated rats and 30% vaccinated rats will develop infections, b) the average fecal viral load in the PBS vaccinated group will be at least higher than 2log ₁₀ Copy number/mL, this sample amount yields an index of greater than 90% at an alpha level of 0.05. The serological response of rats was monitored for 4 weeks and then given intravenous administration of 0.5mL of the composition containing 10 ⁶ HEV-C1 genome equivalent SRN250811 (HEV-C1 strain derived from captured synbiotic rats). Serial blood tests, serological and liver function tests were performed for HEV-C1 viral load. Rats were sacrificed four weeks after infection. Liver tissue obtained for HEV-C1RT-PCR, H&E and immunohistochemical staining (IHC). Ethical approval of these experiments was obtained from the university of hong Kong using living animals for teaching and research committee. The schemes for these procedures are 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), and 200. Mu.L of plasma or feces was eluted into 50. Mu.L of TNA in VTM. Real-time RT-PCR for HEV-C1 detection was performed using the primers and probes described (Sridhar et al Emerging infectious diseases,24:2241-2250 (2018)). The primer is directed to the ORF1 gene of HEV-C1. The primer sequences are 5'-CTTGTTGAGCTYTTCTCCCCT-3' (SEQ ID NO:23, wherein "Y" is C or T, encoded according to IUPAC) (forward) and 5'-CTGTACCGGATGCGACCAA-3' (SEQ ID NO: 24) (reverse), and the probe sequence is HEX-TGCAGCTTGTCTTTGARCCC-IABkFQ (SEQ ID NO: 25). The amplicon size was 69bp.

Real-time RT-PCR (qRT-PCR) assays were run in a LightCycler 480 real-time PCR system (Roche, basel, switzerland) using a QuantiNova Probe RT-PCR kit (Qiagen). Each 20. Mu.L reaction Mix contained 1X QuantiNova ProbeRT-PCR Master Mix, 1 XQN Probe RT-Mix, 0.8. Mu.M forward and reverse primers, 0.2. Mu.M Probe and 5. Mu.L template RNA. The reaction was incubated at 45℃for 10min and 95℃for 5min, followed by 50 cycles of 5s at 95℃and 30s at 55 ℃. Quantification of HEV viral load (in copies/mL or copies/g) was performed using plasmid standards prepared with pcr ii-TOPO vectors (Invitrogen, carlsbad, CA, USA) cloned with target inserts. The detection limit of HEV-C1 RT-PCR assay was determined to be 3log ₁₀ copy/mL.

For nested HEV-C1RT-PCR, the first reaction was performed using the outer primers 5' -CAGCGGCTACCGCCTTTGCTAATGCTCAGGT-3SEQ ID NO: 26) and 5' -GCGGCGGACGTACGCCTCCAGAAAATYATGAATA-3' (SEQ ID NO: 27) for 40 cycles. A second reaction was then performed using the amplicon from the first RT-PCR reaction as template and the inner primers 5'-CTTGTTGAGCTYTTCTCCCT-3' (SEQ ID NO: 23) and 5'-CTGTACCGGATGCGACCAA-3' (SEQ ID NO: 24) (identical to the real-time RT-PCR primers listed above). This reaction run was continued for another 40 cycles, followed by detection of the PCR product by gel electrophoresis.

Rat liver histological analysis

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

Statistical analysis

In the entire example, the graph was generated using GraphPad Prism version 8.1 (GraphPad software, la Jolla, USA). The McNemar's test and Fisher's precision test were used to compare assay sensitivity. Student's t-test with or without Welch's correction was used to compare the average OD. One-way anova was used to compare the average viral load of four groups in vaccination experiments.

Results

Vaccination with HEV-A antigen did not completely prevent HEV-C1 infection in rats

SRN250811, which is derived from symbiotic rats, is a heterotypic HEV-C1 strain that exhibits less than 92% amino acid identity with LCK-3110 (FIG. 1A, table 6).

Table 6. Nucleotide and deduced amino acid sequence identity of HEV-C1 strain SRN250811 compared to human infectious HEV-C1 isolate.

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

Rats vaccinated with Hecolin (HEV-A1 p 239), HEV-A4p239, HEV-C1p241 or PBS were challenged intravenously with SRN250811 4 weeks after the first dose of vaccine (fig. 4A, on day 56, rat livers were obtained for viral load testing and histology). On the day of challenge, vaccinated rats showed strong HEV antibody responses on the species-specific immunoblots. All PBS vaccinated rats had detectable virus in feces and serum; fecal viral load peaked at day 7 and turned negative before day 28 (fig. 4B and 4C). All 6 rats of the Hecolin group and 5/6 rats of the HEV-A4p239 group had detectable virus in the feces, peaking at day 7 post infection. Rats not vaccinated with HEV-C1p241 had detectable virus in feces or plasma as determined by quantitative or nested RT-PCR, indicating that HEV-C1p241 vaccine confers an eliminant immunity. The serum and fecal viral load tended to decrease in HEV-A4p239 and Hecolin vaccinated rats, but this was not always statistically significant (fig. 4B and 4C). On day 28, 4/6 PBS vaccinated rats still had detectable HEV-C1RNA in liver tissue compared to no HEV-C1p241 vaccinated animals which still had detectable HEV-C1RNA in liver tissue (FIG. 4D). On day 28, one of the HEV-A4p239 vaccinated rats had detectable virus and none of the Hecolin vaccinated rats had detectable virus in liver tissue. Rats vaccinated with Hecolin and HEV-A4p239 maintained Sup>A strong HEV-Sup>A specific antibody response after infection, accompanied by Sup>A weak response on the HEV-C1p241 immunoblot. Liver function tests were kept normal for all groups (fig. 5A and 5B), which is typical of HEV-C1 infection in young rats. Liver histology showed mild hepatitis in PBS and HEV-Sup>A vaccinated rats. HEV-C1 infected control rats showed acute hepatitis with swollen hepatocytes and apoptotic bodies. Sinusoidal mononuclear cell infiltration and chaotic plate structure were observed. PBS vaccinated HEV-C1 infected rats showed chaotic structure of the hepatocyte plaques. Apoptotic cells with nuclear remnants were detected. HECOLIN vaccine vaccinated rats infected with HEV-C1 showed mild hepatitis with occasional apoptotic cells with nuclear residues. Rats vaccinated with HEV-A4p239 had mild hepatitis with occasional apoptotic cells with nuclear residues. In addition to mononuclear cell infiltration foci, HEV-C1p241 vaccine vaccinated rats showed normal hepatocytes and cord structures.

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

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

Taken together, these experiments demonstrate that prior HEV-A exposure does not prevent HEV-C1 infection in rats. This demonstrates the effect of HEV-C1 antigen variation on susceptibility to infection.

The effect of multimodal assessment of HEV-C1 antigenicity and different antigenicity on clinical diagnosis and vaccine prophylaxis of HEV-C1 infection was evaluated. The low homology at the key epitopes between HEV-A and HEV-C1E2 sequences was found to be different, which generally results in fundamental changes in the side chain characteristics at these residues. Because HEV-C1 binds poorly to anti-HEV mAb, the common mAb-based HEV antigen EIA is unable to detect HEV-C1 infection. Specific mAbs for HEV-C1 have been generated, but further work along this line is required to identify neutralizing epitopes in HEV-C1E2, and also elucidate the crystal structure of HEV-C1E2 that binds to mAb. Sup>A generic HEV antigen eiSup>A that combines mabs that bind to both HEV-Sup>A and HEV-C1 is needed to ensure that HEV-C1 infection is not missed.

The performance of the antibody EIA for HEV-C1 diagnosis shows high inter-assay variability. The use of pE2 as capture antigen, as in the Wantai IgG assay, enables detection of cross-reactive multivalent antibody reactions. The currently globally used HEV antibody assay requires re-assessment of HEV-C1 using a standard that incorporates serum from HEV-C1 patients. HEV antisera referenced by WHO could not represent HEV-C1 infected patients. Taking into account the inability of HEV-A based RT-PCR assays to detect HEV-C1, traditional hepatitis E diagnosis is inadequate for HEV-C1 infection. This may result in a systematic underestimation of HEV-C1 loading.

Rats and humans exposed to HEV-A or HEV-C1 antigen will develop HEV species specific humoral responses. With this, it was demonstrated that parallel testing of serum using HEV-A4 p239 and HEV-C1 p241 immunoblots can determine whether Sup>A patient is exposed to HEV-A or HEV-C1, but there are two descriptions that complicate interpretation. First, immunocompromised patients may not develop Sup>A sufficient antibody response, and second, recall responses to HEV-Sup>A from HEV-C1 infected patients may lead to misdistribution, especially in elderly patients, who are more likely to be exposed to HEV-Sup>A in the past. The differential assay will help to study the seropositive rate of HEV-C1 population.

The antigen differentiation of HEV-C1 causes problems: whether the prior HEV-A immunity could be cross-protected against HEV-C1. These examples show that rats immunized with HEV-A antigen still develop infection after HEV-C1 challenge, despite the apparent partial protection, with low viral load and improved liver histology. HEV-C1 infection has been previously identified in patients with baseline HEV-A seropositivity and lack of cross-protection, indicating that this also applies to humans (Sridhar et al Emerging infectious diseases,24:2241-2250 (2018)). Interestingly, previously exposed rats with HEV-A antigen developed Sup>A recall response after HEV-C1 exposure, suggesting that pathogen infection triggered an immune response against the relevant previously encountered antigen. Although this response does not worsen HEV-C1 infection outcome in rats, it may lead to sustained HEV-C1 susceptibility and weaker HEV-C1 p241 humoral responses in HEV-Sup>A vaccinated animals. Regions like hong Kong require bivalent vaccines combining both HEV-C1 p241 and HEV-Ap 239, where HEV-C1 accounts for a significant portion of the hepatitis E load.

Although in the example, the HEV-C1 group size is relatively small, group C already contains the majority of all reported HEV-C1 cases. More samples will become available for immunoblotting when new cases are detected. In addition, HEV-C1 cell culture models were lacking to test for neutralization of anti-HEV-AmAb to aid in vitro assays. Computer simulation experiments, mAb antigen EIA using clinical samples, and rat infection models provided alternative assessments of cross-reactivity. Because rats are insensitive to HEV-A, HEV-A infection models cannot be developed. One study that has been conducted on pigs shows that vaccination with HEV-C1 Vaccine is partially protective against HEV-A (Purcell et al, emerg information Dis.,17:2216-2222 (2011); sanford et al, vaccine,30:6249-6255 (2012)).

Antigen diversity results in frequent failure of HEV-A based diagnostic assays in diagnosing HEV-C1 infection. Previous HEV-A infections or vaccinations were not protected against HEV-C1. These examples show that serological profiles of HEV-A and HEV-C1 infections can be distinguished and that immunogenic HEV-C1 peptide vaccines are provided.

Example 4 development and evaluation of parallel enzyme immunoassays based on HEV-A4 p239 and HEV-C1 p241

The immunoblotting system was adapted to the IgG EIA format to maximize convenience and sensitivity. HEV-A4p239 and HEV-C1 p241 were coated in separate 96-well EIA plates at a concentration of 50 ng/well. To perform this test, serum or plasma samples were diluted 1:200 with 1% casein and 100 μl of each diluted sample was tested in duplicate in HEV-A4p239 and HEV-C1 p241 wells. After 30 minutes incubation and washing steps, 1:8000 HRP-antibody complex was added to each well. After another incubation and washing step, 100 μl of the developer solution was added to each well, ten minutes later, 3M H was added ₂ SO ₄ The solution was terminated. Positive controls from patients with known HEV-Sup>A or HEV-C infections will be incorporated into each run. Pooled negative controls from healthy HEV IgG negative individuals will be used as negative controls. Shewhart's chart will be maintained to ensure assay performance according to the Westgard rule principles, especially when changing new batches of peptides.

For assay evaluation, healthy organ donor serum previously tested negative in the Wantai HEV-IgG EIA kit was first tested (fig. 7). The average OD values in both HEV-A and HEV-C IgG EIA were substantially comparable. To evaluate the assay and generate the cut-off value, sup>A panel of 195 human serum samples was assembled comprising 126 HEV negative organ donor serSup>A (confirmed HEV-Sup>A and HEV-C RT-PCR and IgG negative), 54 serSup>A containing HEV-arnSup>A (confirmed by RT-PCR) and 15 serSup>A containing HEV-C rnSup>A (confirmed by RT-PCR). The group of 15 HEV-C samples represents the largest pool of largest human HEV-C samples that contains the majority of all known rat hepatitis E infections worldwide.

As described above, each test specimen was run in parallel on HEV-A4 p239 and HEV-C1 p241 EIA, and OD values were generated. The RT-PCR results were then used as gold standard to generate the assay cut-off for each IgG EIA by a subject operating characteristic (ROC) curve (FIG. 8).

The performance of HEV-A4 p239 and HEV-C1 p241IgG EIA for RT-PCR assays using these cut-offs is shown in tables 7 and 8. Performance characteristics of HEV-Ap239 and HEV-C p241IgG eiSup>A for detecting HEV-Sup>A infection and HEV-C infection are shown in table 9.

TABLE 7 Performance of HEV-A4 p239 IgG EIA for RT-PCR assay

TABLE 8 Performance of HEV-C1 p241IgG EIA against RT-PCR assay

TABLE 9 Performance characteristics of HEV-Ap239 and HEV-C p241IgG EIA for detecting HEV-A and HEV-C infections

After establishing the performance of each EIA, a simple algorithm for interpretation of the results was presented (table 10).

TABLE 10 algorithm for simple result interpretation

For the final class (positive for both EIAs), cut-off values for normalized OD ratios of samples in HEV-A4 p239 and HEV-C1 p241IgG EIAs were evaluated. It was found to have a C/A cutoff ratio of 1.971, a sensitivity of 100% and a specificity of 88.9%.

The performance of the overall algorithm for RT-PCR was again assessed using the cut-off values for each assay and the C/a cut-off value for sample positivity in both assays according to the algorithm shown in table 10 (table 11).

TABLE 11 Performance of the entire algorithm against RT-PCR

EIA Cohen's kappa number for RT-PCR was 0.883, indicating very good inter-evaluator consistency.

The overall performance characteristics of the overall algorithm for distinguishing between different classes of HEVs are shown in table 12 below:

TABLE 12 integral performance characterization of the entire algorithm for differentiating different classes of HEVs

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Sequence listing

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<223> primer

<400> 6

acgtagactc ctcttgtggc 20

<210> 7

<211> 19

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 7

cgatgtctag caagcccaa 19

<210> 8

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> primer

<220>

<221> misc_feature

<222> (14)..(14)

<223> n is a, c, g or t

<400> 8

cgactgaggc ytcnaattat gc 22

<210> 9

<211> 19

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 9

tgcacrtcct gcatraacc 19

<210> 10

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 10

attcagtgcc acaggaggag t 21

<210> 11

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 11

tggagtatag gaacctgaca c 21

<210> 12

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 12

agagcgccag ttcgtgatcg g 21

<210> 13

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 13

tgtcaggttc atgcaggacg t 21

<210> 14

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 14

gataagcctt agagccccca t 21

<210> 15

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 15

gaagatagcc cggccaaagc a 21

<210> 16

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 16

tggggaagat caatttttgg c 21

<210> 17

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 17

tgggggctct aaggcttatc t 21

<210> 18

<211> 39

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 18

gaccacgcgt atcgatgtcg actttttttt ttttttttv 39

<210> 19

<211> 29

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 19

catatgatag cattgaccct gtttaatct 29

<210> 20

<211> 29

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 20

ctcgagagca gagtggggtg ctaaaacac 29

<210> 21

<211> 29

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 21

catatgattg ttcaggtttt gttcaatat 29

<210> 22

<211> 29

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 22

ctcgagaacg gggttggggc cgacagcac 29

<210> 23

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 23

cttgttgagc tyttctcccc t 21

<210> 24

<211> 19

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 24

ctgtaccgga tgcgaccaa 19

<210> 25

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Probe sequence

<400> 25

tgcagcttgt ctttgarccc 20

<210> 26

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 26

cagcggctac cgcctttgct aatgctcagg t 31

<210> 27

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 27

gcggcggacg tacgcctcca gaaaatyatg aata 34

<210> 28

<211> 149

<212> PRT

<213> artificial sequence

<220>

<223> amino acid sequence of HEV-A genotype 3

<400> 28

Ser Pro Ala Pro Ser Arg Pro Phe Ser Val Leu Arg Ala Asn Asp Val

1 5 10 15

Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr Asp Gln Thr Thr Tyr Gly

20 25 30

Ser Ser Thr Asn Pro Met Tyr Val Ser Asp Thr Val Thr Phe Val Asn

35 40 45

Val Ala Thr Gly Ala Gln Ala Val Ala Arg Ser Leu Asp Trp Ser Lys

50 55 60

Val Thr Leu Asp Gly Arg Pro Leu Thr Thr Ile Gln Gln Tyr Ser Lys

65 70 75 80

Thr Phe Tyr Val Leu Pro Leu Arg Gly Lys Leu Ser Phe Trp Glu Ala

85 90 95

Gly Thr Thr Lys Ala Gly Tyr Pro Tyr Asn Tyr Asn Thr Thr Ala Ser

100 105 110

Asp Gln Ile Leu Ile Glu Asn Ala Ala Gly His Arg Val Ala Ile Ser

115 120 125

Thr Tyr Thr Thr Ser Leu Gly Ala Gly Pro Val Ser Val Ser Ala Val

130 135 140

Gly Val Leu Ala Pro

145

<210> 29

<211> 149

<212> PRT

<213> artificial sequence

<220>

<223> amino acid sequence of HEV-A genotype 4

<400> 29

Ser Pro Ala Pro Ser Arg Pro Phe Ser Val Leu Arg Ala Asn Asp Val

1 5 10 15

Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr Asp Gln Thr Thr Tyr Gly

20 25 30

Ser Ser Thr Asn Pro Met Tyr Val Ser Asp Thr Val Thr Phe Val Asn

35 40 45

Val Ala Thr Gly Ala Gln Gly Val Ser Arg Ser Leu Asp Trp Ser Lys

50 55 60

Val Thr Leu Asp Gly Arg Pro Leu Thr Thr Ile Gln Gln Tyr Ser Lys

65 70 75 80

Thr Phe Tyr Val Leu Pro Leu Arg Gly Lys Leu Ser Phe Trp Glu Ala

85 90 95

Gly Thr Thr Lys Ala Gly Tyr Pro Tyr Asn Tyr Asn Thr Thr Ala Ser

100 105 110

Asp Gln Ile Leu Ile Glu Asn Ala Ala Gly His Arg Val Cys Ile Ser

115 120 125

Thr Tyr Thr Thr Asn Leu Gly Ser Gly Pro Val Ser Val Ser Ala Val

130 135 140

Gly Val Leu Ala Pro

145

<210> 30

<211> 149

<212> PRT

<213> artificial sequence

<220>

<223> amino acid sequence of HEV-A genotype 1

<400> 30

Ser Pro Ala Pro Ser Arg Pro Phe Ser Val Leu Arg Ala Asn Asp Val

1 5 10 15

Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr Asp Gln Ser Thr Tyr Gly

20 25 30

Ser Ser Thr Gly Pro Val Tyr Val Ser Asp Ser Val Thr Leu Val Asn

35 40 45

Val Ala Thr Gly Ala Gln Ala Val Ala Arg Ser Leu Asp Trp Thr Lys

50 55 60

Val Thr Leu Asp Gly Arg Pro Leu Ser Thr Thr Gln Gln Tyr Ser Lys

65 70 75 80

Thr Phe Phe Val Leu Pro Leu Arg Gly Lys Leu Ser Phe Trp Glu Ala

85 90 95

Gly Thr Thr Lys Ala Gly Tyr Pro Tyr Asn Tyr Asn Thr Thr Ala Ser

100 105 110

Asp Gln Leu Leu Val Glu Asn Ala Ala Gly His Arg Val Ala Ile Ser

115 120 125

Thr Tyr Thr Thr Ser Leu Gly Ala Gly Pro Val Ser Ile Ser Ala Val

130 135 140

Ala Val Leu Ala Pro

145

<210> 31

<211> 151

<212> PRT

<213> artificial sequence

<220>

<223> amino acid sequence of HEV-C LCK-3110

<400> 31

Ser Pro Ala Pro Ala Arg Pro Ile Thr Asn Trp Arg Ser Gly Asp Val

1 5 10 15

Val Trp Val Thr Leu Pro Ser Ala Glu Tyr Ala Gln Ser Gln Ser Ala

20 25 30

Met Gly Ser His Pro Ala Tyr Trp Ser Glu Glu Ala Thr Ile Ile Asn

35 40 45

Val Ala Thr Gly Gln Arg Ala Ala Val Ser Ser Ile Lys Trp Asp Gln

50 55 60

Val Thr Leu Asn Gly Lys Ala Leu His Lys Glu Thr His Ser Gly Leu

65 70 75 80

Val Tyr Tyr Gln Leu Pro Leu Met Gly Lys Ile Asn Phe Trp Gln Gln

85 90 95

Gly Thr Thr Lys Ala Gly Tyr Thr Tyr Asn Tyr Asn Thr Thr Asp Ser

100 105 110

Asp Ser Leu Trp Val Trp Trp Asp Gly Gly Ser Lys Ala Tyr Leu Tyr

115 120 125

Ile Ser Thr Tyr Thr Thr Met Leu Gly Ala Gly Pro Val Asn Ile Thr

130 135 140

Gly Leu Gly Ala Val Gly Pro

145 150

<210> 32

<211> 151

<212> PRT

<213> artificial sequence

<220>

<223> amino acid sequence of HEV-C MN450853

<400> 32

Ser Pro Ala Pro Ala Arg Pro Ile Thr Asn Trp Arg Ser Gly Asp Val

1 5 10 15

Val Trp Val Thr Leu Pro Ser Ala Glu Tyr Ala Gln Ser Gln Ser Ala

20 25 30

Met Gly Ser His Pro Ala Tyr Trp Ser Glu Glu Ala Ser Ile Ile Asn

35 40 45

Val Ala Thr Gly Gln Arg Ala Ala Val Ser Gly Ile Lys Trp Asp Gln

50 55 60

Val Ile Leu Asn Gly Arg Ala Leu His Lys Glu Thr His Ser Gly Leu

65 70 75 80

Val Tyr Tyr Gln Leu Pro Leu Met Gly Lys Ile Ser Phe Trp Gln Gln

85 90 95

Gly Thr Thr Lys Ala Gly Tyr Thr Tyr Asn Tyr Asp Thr Thr Asp Ser

100 105 110

Asp Ser Leu Trp Val Trp Trp Asp Gly Gly Ser Lys Ala Tyr Leu Tyr

115 120 125

Val Ser Thr Tyr Thr Ala Met Leu Gly Ala Gly Pro Val Asn Ile Thr

130 135 140

Gly Val Gly Ala Val Gly Pro

145 150

<210> 33

<211> 151

<212> PRT

<213> artificial sequence

<220>

<223> amino acid sequence of HEV-C MK050105

<400> 33

Ser Pro Ala Pro Ala Arg Pro Ile Thr Asn Trp Arg Ser Gly Asp Val

1 5 10 15

Val Trp Val Thr Leu Pro Ser Ala Glu Tyr Ala Gln Ser Gln Ser Ala

20 25 30

Met Gly Ser His Pro Ala Tyr Trp Ser Glu Glu Ala Thr Ile Ile Asn

35 40 45

Val Ala Thr Gly Gln Arg Ala Ser Val Ser Ser Ile Lys Trp Asp Gln

50 55 60

Val Thr Leu Asn Gly Lys Ala Leu His Lys Glu Thr His Ser Gly Leu

65 70 75 80

Val Tyr Tyr Gln Leu Pro Leu Met Gly Lys Ile Ser Phe Trp Gln Gln

85 90 95

Gly Thr Thr Lys Ala Gly Tyr Thr Tyr Asn Tyr Asn Thr Thr Asp Ser

100 105 110

Asp Ser Leu Trp Val Trp Trp Asp Gly Ala Ser Lys Ala Tyr Leu Tyr

115 120 125

Leu Ser Thr Tyr Thr Thr Met Leu Gly Ala Gly Pro Val Asn Ile Thr

130 135 140

Gly Leu Gly Ala Ile Gly Pro

145 150

Claims (37)

1. A synthetic virus-like particle comprising a plurality of peptides each having at least 90% amino acid sequence identity to SEQ ID No. 31.

2. The synthetic virus-like particle of claim 1, comprising a plurality of proteins each having at least 95% amino acid sequence identity to SEQ ID No. 31.

3. The synthetic virus-like particle of claim 1 or 2, comprising a plurality of proteins having the amino acid sequence as set forth in SEQ ID No. 1 or as set forth in SEQ ID No. 31.

4. A composition comprising the synthetic virus-like particle of any one of claims 1-3.

5. The composition of claim 4, comprising a synthetic virus-like particle comprising a plurality of peptides having at least 90% amino acid sequence identity to SEQ ID No. 3 or SEQ ID No. 29.

6. The composition of claim 4 or 5, comprising an adjuvant.

7. The composition of any one of claims 4-6, comprising an adjuvant selected from the group consisting of: amorphous aluminum hydroxy phosphate sulfate (AAHS), aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), freund's complete adjuvant, freund's incomplete adjuvant, oil-in-water emulsions of lipid a (MPL) with aluminum salts, consisting of squalene, quil a, MPL and QS-21, and immunostimulatory oligonucleotides containing cytosine-guanine phosphate (CpG).

8. 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, aluminum hydroxide and thimerosal.

9. 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.

10. The composition of any one of claims 4-9 for inducing an immune response against Hepatitis E Virus (HEV) type Sup>A (HEV-Sup>A) genotype 4, HEV-C genotype 1, or Sup>A combination thereof.

11. Sup>A composition for detecting HEV-C infection or Sup>A combined HEV-Sup>A and HEV-C infection, the composition comprising

A plurality of peptides each having at least 90% amino acid sequence identity to SEQ ID NO. 31, a plurality of peptides having at least 95% amino acid sequence identity to SEQ ID NO. 31 or a plurality of peptides having the amino acid sequence as in SEQ ID NO. 31, and optionally,

a plurality of peptides each having at least 90% amino acid sequence identity to SEQ ID NO. 29.

12. The composition of claim 11, wherein the plurality of peptides are synthetic virus-like particles.

13. A kit comprising a plurality of peptides each having at least 90% amino acid sequence identity to SEQ ID NO. 31, a plurality of peptides having at least 95% amino acid sequence identity to SEQ ID NO. 31, or a plurality of peptides having the amino acid sequence as in SEQ ID NO. 31, and optionally,

a plurality of peptides having at least 90% amino acid sequence identity to SEQ ID No. 29.

14. The kit of claim 13, wherein the plurality of peptides are synthetic virus-like particles.

15. An assay platform comprising

A plurality of peptides each having at least 90% amino acid sequence identity to SEQ ID NO. 31, a plurality of peptides having at least 95% amino acid sequence identity to SEQ ID NO. 31, or a plurality of peptides having the amino acid sequence as in SEQ ID NO. 31, and optionally,

a plurality of peptides each having at least 90% amino acid sequence identity to SEQ ID NO. 29.

16. The assay platform of claim 15, wherein the plurality of peptides are synthetic virus-like particles.

17. An immunoassay comprising the step of contacting a test sample with:

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

A plurality of peptides each having at least 90% amino acid sequence identity to SEQ ID NO. 29.

18. The immunoassay of claim 17, wherein the plurality of peptides are synthetic virus-like particles.

19. Sup>A method of detecting HEV-C infection or Sup>A combination of HEV-Sup>A and HEV-C infection in Sup>A sample, the method comprising contacting the sample or test sample with:

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

A plurality of peptides each having at least 90% amino acid sequence identity to SEQ ID NO. 29.

20. The method of claim 19, wherein the plurality of peptides are synthetic virus-like particles.

21. The method of claim 19 or 20, wherein the contacting is in a test vessel.

22. The method of any one of claims 19-21, wherein contacting is contacting a plurality of test samples with a plurality of peptides having at least 90% amino acid sequence identity to SEQ ID No. 31 in a first set of test receptacles.

23. The method of any one of claims 19-22, wherein contacting is contacting the plurality of test samples with a plurality of peptides having at least 90% amino acid sequence identity to SEQ ID No. 29 in a second set of test receptacles.

24. The method of any one of claims 19-23, further comprising forming a signal from contact of the test sample with the plurality of peptides.

25. The method of any one of claims 19-24, wherein the method detects HEV-C infection when a signal is formed from contact of the test sample with a plurality of peptides having at least 90% amino acid sequence identity to SEQ ID No. 31, a plurality of peptides having at least 95% amino acid sequence identity to SEQ ID No. 31, or a plurality of peptides having an amino acid sequence as in SEQ ID No. 31.

26. The method of any one of claims 19-25, wherein the method detects HEV-Sup>A infection when Sup>A signal is formed from contact of the test sample with Sup>A plurality of peptides having at least 90% amino acid sequence identity to SEQ ID No. 29.

27. The method of any one of claims 19-26, wherein the method detects HEV-C infection or Sup>A combined HEV-Sup>A and HEV-C infection in the sample with Sup>A sensitivity of about or more than 80% and Sup>A specificity of about or more than 70%.

28. The method of any one of claims 19-27, wherein the method distinguishes between HEV-Sup>A infection and HEV-C infection in the sample with Sup>A sensitivity of about or more than 80%.

29. A method of detecting hepatitis C virus type C (HEV-C) in a sample, the method comprising testing RNA from the sample in a polymerase chain reaction using primers specific for HEV-C open reading frame 1 or open reading frame 2.

30. 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.

31. The method of claim 29 or 30, wherein the primers specific for HEV-C comprise the primers as in SEQ ID No. 23 and SEQ ID No. 24.

32. The method of any one of claims 29-31, wherein RNA from the sample is converted to complementary DNA prior to the polymerase chain reaction.

33. The method of any one of claims 29-32, wherein the method detects HEV-C infection in the sample with a sensitivity of about or more than 90% and a specificity of about or more than 90%.

34. The method of any one of claims 19-33, wherein the sample is a sample obtained from a subject, wherein the subject is a human, a non-human primate, a livestock animal, a wild animal, a farm animal, or a laboratory animal.

35. 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, faeces, exudates, saliva, sputum, tears, sweat, urine or vaginal secretions.

36. The method of any one of claims 19-35, wherein the test sample is a diluted sample of a body fluid or mucus selected from the group consisting of: blood, serum, plasma, fecal matter, exudates, saliva, sputum, tears, sweat, urine and vaginal secretions.

37. The method of any one of claims 19-36, wherein the test sample is a sample diluted with a ratio of the sample to buffer of between about 1:5 and 1:500 (v/v).