The invention relates to the field of infections with hepatitis E (HEV). In particular, the invention relates to the detection of hepatitis due to hepatitis E.
Hepatitis is an inflammatory liver injury, the causes can be many: infectious, drug, autoimmune, etc. Acute liver damage caused by viruses are common, often asymptomatic. They are due either to direct cytopathic action of the virus or, more often, the immune response against the infected liver cells. Symptoms, when they exist, associate febrile jaundice, itching, discoloration of feces, urine and browning of a more or less significant increase in transaminases, reflecting cytolysis and liver dysfunction.

Many viruses can cause liver damage, such as the Epstein-Barr (EBV) and cytomegalovirus (CMV), but only six viruses are recognized as responsible for what is commonly called the "hepatitis viral ". These viruses are hepatitis viruses A, B, C, Delta, E and G, which are viruses belonging to many different families.

The hepatitis G is little described.

The hepatitis A, or HAV or HAV, belongs to the family

Picornaviridae and is the only representative of the genus Hepato virus. This is a naked RNA viruses. The reservoir of the virus is infected individual, sick or not. Modes of transmission are determined by the exceptional strength of the virus and its high concentration in the stool. The main mode of transmission is mainly fecal-oral. A particular risk is related to the consumption of contaminated shellfish and vegetables.

The Hepatitis B or HBV or HBV belongs to the family of hepadnaviridas. This is a circular DNA virus, double-stranded about 3/4 of its circumference. This virus exposed to the risk of fulminant hepatitis, chronic active hepatitis, cirrhosis and hepatocellular carcinoma. The main vector of the virus is blood, but it can be transmitted sexually. Globally, an estimated 350 million people chronically infected with this virus and is responsible for over one million deaths annually.

The hepatitis C, or HCV or HCV or NANBH for "Non-A, Non-B Hepatitis" genome RNA virus of positive polarity, has an organization similar to that of flaviviruses with 9500 nucleotides (9.5 kb), the 5 'and 3' non-coding, and starting from the 5 'end of the capsid gene (C), envelope (El and E2) and nonstructural protein (NS5 NSI). The HCV virus is strictly human. The mode of transmission is mainly by intravenous, for example by using unsterilized needles, blood transfusion contamination is still present in developing countries where there is no donor screening. The most disturbing element of hepatitis C is that beyond a generally asymptomatic primary infection (90% of cases), the

The hepatitis Delta or HDV or HDV is a very small RNA virus unable to replicate without HBV who lends his HBs surface antigen. DELTA virus infection occurs at the same time that HBV infection in which the prognosis is worse: increased risk of fulminant hepatitis and transition to chronic active hepatitis.

The hepatitis E, or HEV or HEV or ET-NANBH for "enterically Transmitted Non-A, Non-B Hepatitis" is a small naked non-enveloped viruses whose genome is a single strand of positive polarity RNA. Initially classified in the family Caliciviridae he is near, the knowledge of its entire genome leads today to classify it separately, as one member of the genus Hepevirus, the family of Hepeviridœ (Emerson, SU, & Purcell, RH, 2007). The inter-human transmission of this virus is mainly through fecal-oral route (contaminated water, food). Infections are endemic in parts of Asia, Africa and Central and South America. The hepatitis E is identified as the main agent of acute hepatitis epidemics in countries with low levels of hygiene. More recently, it has been clearly defined as responsible for real sporadic cases of acute hepatitis in industrialized countries in patients who have never stayed in endemic areas. It is now clear that hepatitis E is a zoonotic disease and many domesticated and wild animals are infected with HEV, constituting the virus reservoir. Hepatitis E, like hepatitis A, generally do not go to chronicity, except for certain groups of patients such as those who have received a solid organ transplant. However, a poorly explained characteristic: although usually self-limiting, it was observed that in India, the mortality could reach 20% in pregnant women, as gestational age increases, This could make the infection HEV hepatitis worst of all viral hepatitis during pregnancy. It is therefore essential to have the infection detection tools for efficient and reliable HEV.

The genome of hepatitis E has an approximate length of 7.5 kb and present three reading frames (ORF1, ORF2 and ORF3) partly overlapping boxes in the 5 'end of non-coding sequence from 27 to 32 nucleotide and the 3 'end of a sequence of 65-74 bases followed by a polyadenylated end of variable length depending on the virus. The ORFl encodes a polyprotein of 186 kDa, called p-ORFl protein subsequently cleaved into nonstructural proteins including a methyl transferase, showing that the virus is capped at its 5 'end, and AR-dependent RNA polymerase. LORF2 encodes the capsid protein of glycosylated protein called p-ORF2, having 659-674 amino acids according to the variants described to date, the majority of p-ORF2 protein variants having 660 amino acids. This p-ORF2 protein is more immunogenic sites immuno dominant epitope conformational between amino acids 394 and 457, numbered relative to the protein 660 amino acids, and a target epitope neutralizing antibodies, also conformational, located between amino acids 452 and 617, with the same numbering (Meng J, et al, 2001). It also comprises another immunodominant epitope, called 406.3-2 epitope, which corresponds to amino acids 613-654 of ORF2 variant 660 amino acids (W093 / 14116). Phosphoprotein with a molecular weight of 13 kDa, encoded by 1ORF3, called p-ORF3 protein is very This p-ORF2 protein is more immunogenic sites immuno dominant epitope conformational between amino acids 394 and 457, numbered relative to the protein 660 amino acids, and a target epitope neutralizing antibodies, also conformational, located between amino acids 452 and 617, with the same numbering (Meng J, et al, 2001). It also comprises another immunodominant epitope, called 406.3-2 epitope, which corresponds to amino acids 613-654 of ORF2 variant 660 amino acids (W093 / 14116). Phosphoprotein of a molecular weight of 13 kDa, encoded by 1ORF3, called p-ORF3 protein is very This p-ORF2 protein is more immunogenic sites immuno dominant epitope conformational between amino acids 394 and 457, numbered relative to the protein 660 amino acids, and a target epitope neutralizing antibodies, also conformational, located between amino acids 452 and 617, with the same numbering (Meng J, et al, 2001). It also comprises another immunodominant epitope, called 406.3-2 epitope, which corresponds to amino acids 613-654 of ORF2 variant 660 amino acids (W093 / 14116). Phosphoprotein with a molecular weight of 13 kDa, encoded by 1ORF3, called p-ORF3 protein is very with the same numbering (Meng J, et al, 2001). It also comprises another immunodominant epitope, called 406.3-2 epitope, which corresponds to amino acids 613-654 of ORF2 variant 660 amino acids (W093 / 14116). Phosphoprotein with a molecular weight of 13 kDa, encoded by 1ORF3, called p-ORF3 protein is very with the same numbering (Meng J, et al, 2001). It also includes immunodominant epitope another, called 406.3-2 epitope, which corresponds to amino acids 613-654 of a variant ORF2 660 amino acids (W093 / 14116). Phosphoprotein with a molecular weight of 13 kDa, encoded by 1ORF3, called p-ORF3 protein is very

variable depending on the virus. This protein, whose role remains unclear, would be involved in the control functions of viral replication or assembly of the nucleocapsid.

The current diagnosis is based either on the detection of gene amplification by virus from stool samples and serum or bile or liver biopsy, either on the detection of the response anti-HEV serum antibodies.

Gene amplification is carried out by RT-PCR, nested PCR, or real-time PCR using several pairs of primers according to the genotypes from the most conserved regions of the genome. With a detection threshold of 10 10 to 3 cDNA molecules / reaction, according to the techniques, viral shedding in feces can reach 10 6cDNA molecules. The genotype characterization can be performed in a second time. These techniques are primarily useful for the detection of viremia in the blood early with respect to the infection before symptoms appear and antibodies. However, these techniques for the detection of viral nucleic acids have disadvantages that viremia period is short (1 to 2 weeks in the blood, 3 to 4 weeks in the stool) and require expensive equipment and not used to closer to the patient.

The serological diagnosis of HEV infection rests on the detection of specific IgM anti-HEV antibodies and / or IgG which is the main target p-ORF2. Several kits are marketed. Thus, the company MP Diagnostics ™ kit offers Assure® HEV IgM is an immuno-chromatographic test device for the rapid detection of IgM antibodies against p-ORF2 protein of hepatitis E. To do this, the kit implements a recombinant polypeptide, the polypeptide 394-660, numbered relative to the sequence 1-660 p-ORF2, otherwise known as p-ORF2.1 polypeptide, corresponding to the last 267 amino acids of the protein. Mouse antibodies directed against human IgM antibodies are immobilized on the membrane for immunochromatography, thereby capture the different human IgM in the sample. The presence of IgM specifically directed against HEV is revealed using, as a detection partner, the recombinant polypeptide 394-660 complexed to an anti-HEV monoclonal antibody labeled with gold. The reasons for

the use of the recombinant polypeptide 394-660 rather than the whole protein are disclosed in WO95 / 08632 application. According to the teachings of this patent application, the logic immuno reactivity of the complete protein p-ORF2 expressed in E. coli is not optimal, a part of the molecule that can reduce or inhibit another part of the immunoreactivity molecule. To overcome this inhibitory effect, the patent application WO95 / 08632 proposed the use of the deleted or truncated p-ORF2 proteins. Of the various constructions tested, the recombinant polypeptide 394-660, deleted the first 393 amino acids, presented the best immunoreactivity.

The detailed characterization of the antigenic structure of the 394-660 polypeptide and its comparison with that of the "virus-like particles" or VLPs formed by self-assembly in vitro and close antigenically to the HEV virus particle, are described in Riddell MA, et al, 2000.

The disadvantage of using the 394-660 polypeptide is that it contains in its C-terminal domain which inhibits, at least partially, self-assembly of the polypeptide oligomers and VLPs. This can interfere with the proper presentation of conformational epitopes.

To overcome these drawbacks, the Wantai Company changed the 394-660 polypeptide by deleting amino acids 607-660 (numbered relative to a sequence of 1-660 p-ORF2) which interfere with the oligomerization and the capacity for self -assembly. The resulting polypeptide was called pE2 polypeptide, as described in the patent application WO01 / 22916. The advantage of this pE2 polypeptide sequence of 394-606, is that dimerizes naturally and immunoreactivity dimeric pE2 is much higher than that of monomeric pE2 promoting good presentation of conformational epitopes. The disadvantage is that such a truncated polypeptide does not include an important epitope, the epitope 406.3-2 corresponding to amino acids 613-654 of ORF2 variant 660 amino acids, as described in the patent application W093 / 14116. Such deletion can then lead to a decrease in sensitivity of a diagnostic test implementing such a truncated polypeptide.

We have found against all expectations it was possible to overcome the drawbacks of the polypeptides of the prior art by carrying out, in the peptide 394-660 of HEV ORF2, numbered with reference to a p-ORF2 protein of 660 amino acids, 3 mutations at positions 627, 630 and 638, and by improving its antigenicity and immunoreactivity. Thus, the mutated peptide may be called p-ORF2-MUT, has all the important epitopes naturally dimerizes noncovalently, is able to oligomerize without aggregation and has immunoreactivity than that of the recombinant polypeptide 394-660 unmutated.

Also, the invention relates to a polypeptide derived from p-ORF2 protein of hepatitis E comprising (i) at least the amino acid sequence 394-660, numbered relative to a p-ORF2 protein acid 660 amino, wherein the three cysteines at positions 627, 630 and 638 have been mutated, or (ii) for a p-ORF2 protein of different lengths, at least the amino acid sequence corresponding to amino acids 394-660 of the protein p-ORF2 of 660 amino acids, in which the three cysteines located in the three positions corresponding to positions 627, 630 and 638 of the p-ORF2 protein of 660 amino acids have been mutated.

Another object of the invention relates to isolated nucleic acids comprising a nucleotide sequence encoding the polypeptides of the invention or a sequence complementary to said coding sequence, as well as expression vectors comprising these sequences.

Yet another object relates to host cells comprising these nucleic acid sequences, inserted directly or via expression vectors.

It further relates to the use of polypeptides of the invention for determining the presence of antibodies directed response against p-ORF-2 protein of the virus of hepatitis E or for determining the levels of these antibodies .

Thus, another object of the invention relates to a method for determining by immunoassay the presence of an antibody directed response against p-ORF-2 protein of the virus of hepatitis E in a biological sample from a subject, suspected of containing the antibodies of said response, which comprises the steps of:

- contacting said biological sample with a polypeptide of the invention,

- detecting a signal transmitted by the binding between said polypeptide and said antibody, if present, using a label capable of emitting a detectable signal,

- comparing the thus obtained signal with a signal of predetermined reference S with two populations of controls, one having developed said antibody and the other having not developed said antibodies,

- a less than said reference signal S meaning that the sample does not contain said antibodies, and

- a higher than said reference signal S indicating that the sample contains said antibody.

Another object relates to a method of determination by immunoassay of antibodies directed against the p-ORF-2 protein of the virus of hepatitis E in a biological sample from a subject, suspected of containing said antibodies, which comprises the following steps:

- contacting said biological sample with a polypeptide of the invention,

- detecting a signal transmitted by the binding between said polypeptide and said antibody, if present, using a label capable of emitting a detectable signal,

- convert the signal detected in antibody levels.

Yet another object is the use of these processes to aid in vitro diagnostics for in vitro diagnosis of infection with hepatitis E in a subject susceptible to infection, for therapeutic monitoring of a subject infected with hepatitis E, for epidemiological studies of the prevalence of anti-HEV antibodies in a population or in a given geographical territory or to determine if a subject needs to be vaccinated or revaccinated against the hepatitis E.

Finally, a last subject relates to kits for the determination by immunoassay of the presence of the humoral response or antibodies directed against the protein p-ORF2 from hepatitis E in a subject likely to produce these antibodies comprising a polypeptide of the invention.

The invention will be better understood upon reading the nonlimiting description which follows and the appended Figures 1 to 6, wherein:

- Figure 1 shows an alignment of amino acid sequences of different p-ORF2 proteins of the major variant HEV virus obtained from the Uniprot database, the first column corresponding to the reference UniProt, the second column is the name of HEV strain and the last column corresponding to the alignment of the sequences. Sequence alignment was performed by the Clustal Omega program available on the UniProt website. The last line in each sequence alignment showing the amino acid identity or not between each variant, "*" indicating a fully retained position, with the amino acids identical in all variants, ":" indicating a well conserved position, with amino acids with highly similar properties and a score> 0 5 in the Gonnet matrix PAM 250 ". "Indicating a fairly conserved position with amino acids having similar properties and a low score = <0.5 in the Gonnet matrix PAM 250. Other positions are marked by" ° ". The different parts of the alignment are distributed 1A to 1R. Figures 1A IL provide alignment of whole proteins of different variants. The 394-660 sequence of the variant Q81871, 660 amino acids (SEQ ID NO: ll), is highlighted as reference. The arrows in Figure 1G indicate the first amino acid of the minimal sequence of the polypeptides of the invention and the rectangle in Figure 1K shows the sequence of 12 amino acids of the variant Q81871 wherein the three cysteines are found to be mutated. The Figures 1M to 1R give the alignment of the minimal sequences of the polypeptides of the invention various variants extracted 1A IL from arrows. The 394-660 sequence of the reference polypeptide variant Q81871 (SEQ ID NO: 26) is underlined.

- Figure 2 shows representations of the electron density maps of the side chains of naturally occurring amino acids, obtained by X-ray diffraction, calculated to a resolution of 1.5 Angstrom, printed site (reference, dated November 13, 2015) :

http://people.mbi. ucla.edu/sawaya/m230ci/Modelbuiklin g/modelbuilding.htinL

- Figure 3 is a photograph of a SDS-PAGE gel analysis (4-12%) stained with Coomassie blue to visualize polypeptide of the invention, ORF2-MUT, and a non-mutated polypeptide ORF2- REF corresponding to p-ORF2.1 polypeptide (amino acids 394-660) disclosed in WO95 / 08632 application. Before gel analysis, the REF-ORF2 ORF2 polypeptides and MUT-purified and dialyzed underwent a reduction by adding dithiothreitol (DTT), an heating by denaturation (10 min at 75 ° C), or both treatments both or no treatment, as shown in the table above the gel. Line M corresponds to molecular weight marker Page Ruler (Pierce), the apparent molecular weights bands are indicated to the left in kilo Daltons (kDa).

- Figure 4 shows chromatograms steric exclusion obtained by following the UV absorbance at 280 nm for the polypeptide of the prior art ORF2-REF (Figure 4A) and the polypeptide of the invention ORF2-MUT (Figure 4B) . For proper viewing of the various peaks in Figure 4A the two chromatograms are not presented on the same scale for the y-axis.

- Figure 5 shows the graph of the results obtained by the AsFlFFF-MALS technique ( "asymmetry fiow field fiow fractionation multi-angle light scattering") for the polypeptide of the invention ORF2-MUT. The UV absorbance at 280 nm (thin solid line), the multi-angle light scattering signal (MALS, hatched line) and the estimated molecular weight (thick full line) are shown superimposed manner on the ordinate as a function analysis time (min).

- Figure 6 is a box plot representation of the distribution of RFV signals obtained with an immunoassay (VIDAS PLC, bioMérieux) using as capture antigen polypeptide of the prior art ORF2-REF or the polypeptide of the invention ORF2 -MUT ,, on samples containing no anti-ORF2 antibody (Neg) and positive HEV samples containing anti-ORF2 antibody (Pos). The box plots were plotted according to Tukey's method: the upper and lower limits of the box represent the 25 th and 75 th percentile distributions, respectively. The value plotted in the middle of the box is the median. High mustache matches the 75 th percentile + 1.5 xl 'and percentile - 1.5 x interquartile talented. Les valeurs plates and en-deca des

mustaches are represented as individual points because it is of extreme values, infrequent.

The Applicant has thus shown, against all expectations, it was possible for the management of the subjects concerned by infection with hepatitis E, using polypeptides derived from the p-ORF2 virus protein hepatitis E comprising at least the sequence of amino acids 394-660, numbered relative to a p-ORF2 protein of 660 amino acids, while avoiding the drawbacks of the prior art when the 607-660 amino acids are included in polypeptides, ie they possess all the important epitopes naturally non-covalently dimerize, are capable of oligomerization without any aggregation. In addition, the polypeptides of the invention are produced seamlessly, unlike the polypeptides of the prior art. Indeed, during production, the final product has a reproducible manner over 75% of non-covalent dimer, the remainder being composed of dodecamers, while the proportion of non-covalent dimer, covalent dimers and aggregates of polypeptides of the prior art varies from one production to the 'other. In addition, polypeptides of the invention exhibit immunoreactivity higher than the non-mutated recombinant polypeptide 394-660. Finally, the polypeptides of the invention allow, when used in an immunoassay, increase the diagnostic specificity of the test, without changing the diagnostic sensitivity, which is fundamental for a detection test of hepatitis virus E. while the proportion of non-covalent dimer, covalent dimers and aggregates prior art polypeptides vary from one production to another. In addition, polypeptides of the invention exhibit immunoreactivity higher than the non-mutated recombinant polypeptide 394-660. Finally, the polypeptides of the invention allow, when used in an immunoassay, increase the diagnostic specificity of the test, without changing the diagnostic sensitivity, which is fundamental for a detection test of hepatitis virus E. while the proportion of non-covalent dimer, covalent dimers and aggregates prior art polypeptides vary from one production to another. In addition, polypeptides of the invention exhibit immunoreactivity higher than the non-mutated recombinant polypeptide 394-660. Finally, the polypeptides of the invention allow, when used in an immunoassay, increase the diagnostic specificity of the test, without changing the diagnostic sensitivity, which is fundamental for a detection test of hepatitis virus E. invention exhibit immunoreactivity higher than the non-mutated recombinant polypeptide 394-660. Finally, the polypeptides of the invention allow, when used in an immunoassay, increase the diagnostic specificity of the test, without changing the diagnostic sensitivity, which is fundamental for a detection test of hepatitis virus E. invention exhibit immunoreactivity higher than the non-mutated recombinant polypeptide 394-660. Finally, the polypeptides of the invention allow, when used in an immunoassay, increase the diagnostic specificity of the test, without changing the diagnostic sensitivity, which is fundamental for a detection test of hepatitis virus E.

As indicated above and as clearly illustrated in Figure 1, the p-ORF2 protein of HEV has different lengths of 659-674 amino acids (see Figure IL giving the last amino acids of p-ORF2 protein). The majority of proteins with 660 amino acids, is a protein of 660 amino acids which is often taken as a reference. In the present application the reference sequence of 660 amino acids is that of the variant Q81871 (SEQ ID NO: ll). However other variants of proteins, although having a different amino acid sequence, for example 674 amino acids (SEQ ID NO: 7), 672 amino acids (SEQ ID NO: 8), 671 acid amino (SEQ ID NO: 9), 668 amino (SEQ ID NO: 10) or 659 amino acids (SEQ ID No. 24),

So to find all the polypeptides of the invention are defined as including:

- when the p-ORF2 protein has 660 amino acids, at least the sequence of amino acids 394-660, numbered relative to a p-ORF2 protein of 660 amino acids, in which the three cysteines at positions 627, 630 and 638 are mutated and,

- when the p-ORF2 protein is of different length, at least the amino acid sequence corresponding to amino acids 394-660 of the p-ORF2 protein of 660 amino acids, in which the three cysteines located in the three positions corresponding to positions 627, 630 and 638 of the p-ORF2 protein of 660 amino acids are mutated,

it is sufficient for the skilled person to perform an alignment with respect to a protein of 660 amino acids. For example, if one refers to Figure 1, one takes as reference protein of 660 amino acids belonging to one variant Q81871 (wherein the 394-660 sequence is underlined in Figure 1) and that consider for example the protein variants, 672, 671, 659, 668 and 674 amino acids, the polypeptides of the invention comprise at least:

- the sequence of amino acids 394-660 (SEQ ID NO: 26, and SEQ ID NO: 38 to 49) of which the cysteines at positions 627, 630 and 638 are mutated (outcome of variants Q81871, P29326, Q6J8F7, Q04611, Q68965, Q9YLQ9, P33426, Q9YLR2, Q0QC51, Q69411, A0A024D9U6, A0A024D9R2, Q8V729), or

- the sequence of amino acids 408-674 (SEQ ID No. 28 to 34) of which the cysteines at positions 641, 644 and 652 are mutated (outcome of Q8JJN2 variants, Q80IR5, Q806D7, Q6BD83, Q6BD78, B6VC89, Q6PMR3).

- the sequence of amino acids 406-672 (SEQ ID NO: 35), the cysteines at positions 639, 642 and 650 are mutated (from variant Q9IVZ8), or

- the sequence of amino acids 405-671 (SEQ ID NO: 36), the cysteines at positions 638, 641 and 649 are mutated (from variant Q8JJM1), or

- the sequence of amino acids 405-668 (SEQ ID NO: 37), the cysteines at positions 638, 641 and 649 are mutated (from variant Q2PYP3) or

- the sequence of amino acids 393-659 (SEQ ID NO: 50), the cysteines at positions 626, 629 and 637 are mutated (from variant Q03500).

Thus the different fragments 394-660, 408-674, 406-672, 405-671, 405-668 and 393-659 of the variants depicted in Figure 1M 1R and extracts Figures 1G IL from the arrow Figure 1G correspond to the following sequences:

Fragments 394-660

Q81871 P29326 Q6J8F7 Q04611 Q68985 Q9YLQ9

SEQIDN°26 SEQIDN°38 SEQIDN°39 SEQ ID SEQIDN°41 SEQIDN°42

N°40

P33426 Q9YLR2 Q0QC51 Q69411 A0A024D9U6 A0A024D9R2

SEQIDN°43 SEQIDN°44 SEQIDN°45 SEQ ID SEQIDN°47 SEQIDN°48

N°46

Q8V729

SEQ ID N°49

Fragments 408-674

Q8JJN2 Q80IR5 Q806D7 Q6BD83 Q6BD78 B6VC89

SEQ ID N°28 SEQ ID N°29 SEQ ID N°30 SEQ ID N°31 SEQ ID N°32 SEQ ID N°33

Q6PMR3

SEQ ID N°34

Fragment 406-672

Q9IVZ8

SEQ ID N°35

Fragment 405-671

Q8JJM1

SEQ ID N°36

Fragment 405-668

Q2PYP3

SEQ ID N°37

Fragment 393-659

Q03500

SEQ ID N°50

Figure 1 also shows that if we take the amino acid sequence of the variant Q81871 394- 660 as a reference sequence (SEQ ID NO: 26) for all the proteins of 660 amino acids, the amino acid sequences 394-660 other variants (SEQ ID NO: 38 to 49) have between 90.64% and 99.25% identity with the sequence SEQ ID No. 26, while if one takes the total sequence of the protein of 660 acid the same variant (SEQ ID NO: 11) sequences of 660 amino acid other variants (SEQ ID No. 12 to 23) present between 90.45% and 99.09% identity with the sequence SEQ ID NO: 11.

Likewise if one takes the amino acid sequence 408-674 of the variant Q8JJN2 as a reference sequence (SEQ ID NO: 28) for all the proteins of 674 amino acids, the amino acid sequences 408-674 of the other variants (SEQ ID No. 29 to 34) have between 98.50% and 98.88% identity with the sequence SEQ ID N ° 28, while if one takes the total sequence of the protein of 674 acids of this same variant (SEQ ID NO: l), the sequences of 674 amino acid other variants (SEQ ID NOS 2-7) have between 98.22% and 98.37% identity with the sequence SEQ ID No. l.

More generally, if we take the 394-660 amino acid sequence of the variant

Q81871 as a reference sequence (SEQ ID NO: 26), corresponding amino acid sequences of other variants (SEQ ID NOs: 28 to 50) have between 90.64%> and 99.25%) identity with the sequence SEQ ID N ° 26, so at least 90%> identity, while if one takes the total sequence of the protein of 660 acids of this same variant (SEQ ID NO: ll), total protein sequences of other variant (SEQ ID NO: to 10 and 12 to 24) present between 89.97%> 99.09% and> identity with the sequence SEQ ID NO: 1, so at least 89% identity.

The percent identity between two sequences is calculated from the alignment of multiple sequences. The program Clustal Omega available in a customizable version on the EMBL-EMI website (http://www.ebi.ac.uk/Tools/msa/clustalo/) generates along with the multiple alignment, a score of 'alignment. This score is related to the degree of similarity between two sequences compared and that for all sequences, as shown in Figure 1.

As shown in Figure 1K, the three cysteines to be mutated are located in a sequence of 12 amino acids defined as follows: CPECRXiLGX 2 CHQ (SEQ ID NO: 25), wherein Xi is P, T, S or A and X 2 is L or F.

Mutations in the three cysteines above are made by substitution of said cysteine ​​by any amino acid other than the well-known cysteine ​​skilled in the art, such as for example the proteinogenic amino acids Histidine, Isoleucine, Leucine, Lysine, Methionine , Phenylalanine, Threonine, Tryptophan, Valine, Alanine, Arginine, aspartic acid, Asparagine, glutamic acid, Glutamine, Glycine, Proline, Serine and Tyrosine.

However, it is best to choose the substitute amino acid according to the following two criteria:

1) the "size or volume" of the side chain of amino acids by relying on the representations of the electron density maps obtained by X-ray diffraction, such as shown in Figure 2 giving such a representation, calculated at a resolution of 1.5 Angstroms, and after the site (print November 13, 2013)

http://people.mbi.ucla.edu/sawaya/rn230d/Modelbuilding/modelbuilding.html.

Indeed, from these cards, the amino acids are chosen which electron density is most similar to cysteine ​​(eg Serine, Valine, and threonine) or more "small" amino acids cysteine ​​(by example Glycine, Alanine). amino acids is preferably deviates too "big" (eg, lysine, histidine, phenylalanine, tyrosine, arginine and tryptophan)

2) The possible reactivity. We do not want that the substituted amino acid reacts

easily with other surrounding amino acids. Is discarded preferably charged amino acids such as basic amino acids (already excluded with the 1 st test) and amino acids.

According to one embodiment, the mutations in the polypeptides of the invention are implemented by replacing the three cysteines by any amino acid except proline, amino acids whose side chains are loaded such as lysine, arginine , histidine, aspartic acid, glutamic acid and amino acids whose side chains contain a benzene aromatic ring, such as tyrosine, phenylalanine, tryptophan.

Preferably, mutations in the polypeptides of the invention are implemented by replacing the three cysteines by an amino acid selected from alanine, glycine, threonine, valine and serine.

3 cysteines may be substituted by the same amino acid or with different amino acids, preferably according to the above criteria.

According to another embodiment, mutations implemented include substituting 3 cysteines by the same amino acid, preferably by serine.

The polypeptides of the invention comprise at least the sequence of amino acids 394-660, numbered relative to a p-ORF2 protein of 660 amino acids, and for a p-ORF2 protein of different length, they comprise at least amino acid sequence corresponding to amino acids 394-660 of ORF2 protein p 660 amino acids, said sequences being mutated as described above.

By polypeptide derived from p-ORF2 protein of hepatitis E is meant a continuous sequence of amino acids 394-660 or equivalent positions positions, from the p-ORF2 protein of hepatitis E. We can speak as either of polypeptide derived from the p-ORF2 protein, polypeptide p-ORF2 protein, p-ORF2 polypeptide, polypeptide mutated p-ORF2 protein derived from p-ORF2 protein, protein from p-ORF2 protein or mutated p-ORF2 protein.

The expression "includes at least the sequence" means that the

polypeptide has said continuous chain of amino acids from the p-ORF2 protein, or it has this sequence of amino acids which can be added (s):

(I) one or more amino acids of the p-ORF2 protein, located prior to said sequence, and / or

(Ii) one or more amino acids not belonging to the p-ORF2 protein, such as a polyhistidine tail, a tail polysine, or a fusion protein, such as GST (Glutathione S-transferase), MBP (Maltose Binding Protein), CBP (Calmodulin-binding peptide), CBD (Chitin Binding Domain), Protein A, thioredoxin, and / or

(Iii) a marking, for example (a) by coupling to a reporter molecule known to those skilled in the art such as biotin, an enzyme, a fluorescent label, a radioactive molecule or any other label as defined below, or (b) by phosphorylation.

Thus, in one embodiment, the polypeptides of the invention comprise one or more of the following:

- they consist of the amino acid sequence of polypeptides 394-660, numbered relative to a p-ORF2 protein of 660 amino acids, in which the three cysteines at positions 627, 630 and 638 have been mutated or, for a protein p-ORF2 of different length of amino acid sequence corresponding to amino acids 394-660 of the p-ORF2 protein of 660 amino acids, in which the three cysteines located in the three positions corresponding to positions 627, 630 and 638 of the p-ORF2 protein of 660 amino acids have been mutated;

- they comprise one or more amino acids not belonging to the p-ORF2 protein;

- they are labeled, for example as illustrated above.

The polypeptides of the invention can be produced by techniques well known in the art. For example, the polypeptides of the invention can be obtained by genetic engineering using the steps, conventionally known to those skilled in the art, of:

- have the DNA encoding the polypeptides of the invention,

- insert this DNA by cloning into an expression vector such as a plasmid, cosmid, phage λ, or a viral vector (baculovirus (Autographa californica Nuclear Polyhedrosis Virus) virus, vaccinia, Semliki Forest virus, adenovirus, lentivirus, ...), which vector also includes an origin of replication (for plasmids or cosmids) or replication system allowing its amplification in the host cell and one or more promoters for the transcription of messenger RNA to be translated into protein,

- introducing the vector for expression in a host cell, such as a prokaryotic cell (e.g., bacteria such as Escherichia coli, Bacillus subtilis) by transformation or infection or a eukaryotic cell (e.g. yeast cells (Saccharomyces cerevisiae, Pichia pastoris) cells insect (Sf9 cell, Sf21, High5), mammalian cells (CHO, 293, Per.C6, BHK-21, Vero, ...) by transient or permanent transfection, or viral infection,

- culture and optionally propagating the host cell containing the expression vector, optionally with amplification of the vector in the host cell,

- where necessary, inducing transcription and protein synthesis for the production of recombinant polypeptides of the invention, and

- purifying said polypeptides to extract, for example by a polyhistidine tail. The polypeptides are said to recombinants.

Also, the invention also relates to:

- the isolated nucleic acids comprising nucleotide sequences encoding the polypeptides of the invention as defined above or sequences complementary to said coding sequences,

- the expression vectors comprising a nucleic acid sequence as defined above.

- the host cell, prokaryotic or eukaryotic, comprising a nucleotide sequence encoding the polypeptides of the invention as defined above or a sequence complementary to said coding sequence or an expression vector as defined above.

When the polypeptides of the invention include other components such as marker polypeptide nature or fusion proteins as described above, the nucleic acid sequence encoding these components may also be inserted in the same reading frame in the vector to allow molten production.

The addition of nonproteinaceous labels to polypeptides of the invention can be implemented by techniques known to those skilled in the art using -NH-OC- bonds formed from -NH 2 and -COOR (R being for example an activated ester group) markers and polypeptides of the invention. Thus, for example, where the label is biotin, those skilled in the art may use commercial reagents, such as reagents, EZ-Link ® NHS-Biotin (ThermoScientific No. 20217, 21336 and 21343), which include a group - COO-activated ester to be reacted with the -NH 2 polypeptides of the invention according to the supplier's recommendations.

As indicated above, the polypeptides of the invention are particularly useful for determining the presence of an antibody response directed against the protein p-ORF-2 of hepatitis E.

Determining the presence of antibody response directed against the protein p-ORF-2 of hepatitis E in a biological sample from a subject, suspected of containing the antibodies of said response, can be implemented by immunoassay and comprises or consists of the following steps:

- contacting said biological sample with a polypeptide as defined above,

- detecting a signal transmitted by the binding between said polypeptide and said antibody, if present, using a label capable of emitting a detectable signal,

- comparing the thus obtained signal with a signal of predetermined reference S with two populations of controls, one having developed said antibody and the other having not developed said antibodies,

- a less than said reference signal S meaning that the sample does not contain said antibodies, and

- a higher than said reference signal S indicating that the sample contains said antibody.

Topics likely to be infected with HEV, in which the determination of the presence of the antibody response and antibody levels is implemented, can be any subject in particular:

o subjects with symptoms of acute hepatitis, such as yellowing of the skin and eyes (jaundice), dark urine, pale stools, extreme fatigue, nausea, vomiting, fever, abdominal pain syndrome or "flu-like ". These symptoms may be accompanied by elevated liver enzymes (ALT / AST) or not. These subjects have been tested positive for HAV, HBV or HCV or not;

o asymptomatic subjects with elevated liver enzymes (ALT / AST). These subjects have been tested positive for HAV, HBV or HCV or not;

o individuals of this population at risk either chronicity or severe fulminant form such as:

• immuno depressed for any reason, including transplanted subjects, subjects receiving one or immunomodulatory or immunosuppressive therapies such as chemotherapy, an anti-TNF alpha therapy, or steroid therapy, subjects co-infected with HIV, seniors (immunosenescence)

• pregnant women,

• subjects with prior chronic liver disease.

The subjects can be mammals such as humans, domestic animals (dogs, cats, horses, etc.) and livestock (sheep, cattle, goats), preferably men.

For biological samples of subjects may contain anti-p-ORF2 antibody hepatitis E include biological fluids such as whole blood or derivatives thereof, for example serum or plasma, urine, saliva and effusions and feces. Preferred is blood or its derivatives, as well as feces. These samples can be used as is in the process of the invention or be pretreated by known methods of the art.

By determining the antibody response directed against the protein p-ORF-2 of Hepatitis E in the biological sample from a subject refers to the determination of the presence or absence of antibodies produced by the subject in the case of an infection with HEV, these antibodies are directed against the p-ORF2 protein.

This determination is implemented by immunoassay which is a widely known test in the art. In short, it consists of determining an analyte, in this case the anti-p-ORF2 antibodies antibody response (also called humoral), by implementing at least one binding partner for the analyte.

Of course, the prefix "immune" in the term "immunoassay" for example, is not to be considered in this application as strictly indicating that the link partner is necessarily an immune-partner, such as an antibody or an antibody fragment. Indeed, as is well known in the art, this term is widely used to also refer to tests and processes in which the binding partner is not an original partner / immunological nature, but is, for example, a receptor for the analyte that is desired to detect and / or quantify. The essential condition is that the binding partner in question is capable of binding to the analyte, in the present case such antibodies, preferably specifically. Thus, it is known to speak of the ELISA assay for testing with non immuno logical link partners strictly speaking, known more widely in English "ligand binding assay" that could be translated in French by "trial using the ligand binding, "while the term" immunoassay "is included in the title verbatim corresponding to the acronym ELISA. For the sake of clarity and consistency, the term "immunoassay" is used herein to refer to any biological analysis using at least one binding partner adapted to bind to the analyte and detecting and / or quantifying the latter preferably specifically, even when said binding partner is not liable or immunological origin in the strict sense. it is known to speak of the ELISA assay for testing with non immuno logical link partners strictly speaking, known more widely in English "ligand binding assay" that could be translated in French by "trial using the ligand binding, "while the term" immunoassay "is included in the title verbatim corresponding to the acronym ELISA. For the sake of clarity and consistency, the term "immunoassay" is used herein to refer to any biological analysis using at least one binding partner adapted to bind to the analyte and detecting and / or quantifying the latter preferably specifically, even when said binding partner is not liable or immunological origin in the strict sense. it is known to speak of the ELISA assay for testing with non immuno logical link partners strictly speaking, known more widely in English "ligand binding assay" that could be translated in French by "trial using the ligand binding, "while the term" immunoassay "is included in the title verbatim corresponding to the acronym ELISA. For the sake of clarity and consistency, the term "immunoassay" is used herein to refer to any biological analysis using at least one binding partner adapted to bind to the analyte and detecting and / or quantifying the latter preferably specifically, even when said binding partner is not liable or immunological origin in the strict sense. ELISA for tests using non immuno logical strictly speaking binding partners, more widely known in English "ligand binding assay" that could be translated in French by "test using the ligand binding" while the term "immunoassay" is included in the title verbatim corresponding to the acronym ELISA. For the sake of clarity and consistency, the term "immunoassay" is used herein to refer to any biological analysis using at least one binding partner adapted to bind to the analyte and detecting and / or quantifying the latter preferably specifically, even when said binding partner is not liable or immunological origin in the strict sense. ELISA for tests using non immuno logical strictly speaking binding partners, more widely known in English "ligand binding assay" that could be translated in French by "test using the ligand binding" while the term "immunoassay" is included in the title verbatim corresponding to the acronym ELISA. For the sake of clarity and consistency, the term "immunoassay" is used herein to refer to any biological analysis using at least one binding partner adapted to bind to the analyte and detecting and / or quantifying the latter preferably specifically, even when said binding partner is not liable or immunological origin in the strict sense. French language could be translated as "test using the ligand binding," while the term "immunoassay" is included in the title verbatim corresponding to the acronym ELISA. For the sake of clarity and consistency, the term "immunoassay" is used herein to refer to any biological analysis using at least one binding partner adapted to bind to the analyte and detecting and / or quantifying the latter preferably specifically, even when said binding partner is not liable or immunological origin in the strict sense. French language could be translated as "test using the ligand binding," while the term "immunoassay" is included in the title verbatim corresponding to the acronym ELISA. For the sake of clarity and consistency, the term "immunoassay" is used herein to refer to any biological analysis using at least one binding partner adapted to bind to the analyte and detecting and / or quantifying the latter preferably specifically, even when said binding partner is not liable or immunological origin in the strict sense.

By binding partner anti-p-ORF2 antibody is any molecule that can bind to these antibodies. Examples of such binding partners include antigens such as native or recombinant p-ORF2 protein, fragments of this protein, and in particular the polypeptides as described above, antibodies such as anti -ig, for example total anti-Ig for a given species, or anti-IgG or anti-IgM as that for IgG or IgM (using an anti-IgG or anti-IgM species for the detection of IgG or IgM in this species), antibody analogs (molecules capable of mimicking the antibody) such as nanofïtines, aptamers or the "DARPins," or any other molecule which is known to interact with the antibody.

antibody binding partners are for example be polyclonal or monoclonal antibodies, whose production is widely known in the art.

Examples of antibody fragments include Fab, Fab ', F (ab') 2 and scFv (single chain variable fragment), dsFv (Double-stranded fragment variable). These functional fragments can in particular be obtained by genetic engineering.

Analogs nanofïtines antibodies are small proteins, such as antibodies, are capable of binding to a biological target and to detect, to capture or just the target within an organization.

Analogs antibody aptamers are oligonucleotides, usually AR or DNA identified in libraries containing up to 10 15 different sequences, by a combinatorial method for selecting in vitro called SELEX "Systematic Evolution of Ligands by Exponential Enrichment" (Ellington AD and Szostak JW., 1990). Most Aptamers are RNA compounds, due to the ability to

RA to adopt varied and complex structures, which allows to create on its surface cavities of various geometries, for setting various ligands. It is of interest biochemical tools that can be used in biotechnology applications, diagnosis or treatment. Their selectivity and their ligand binding properties comparable to that of antibodies.

The antibody analogues "DARPins" for Ankyrin Designed Repeat Proteins (YL Boersma and Plutckthun A, 2011) are another class of proteins to mimic the antibodies and is able to fix with high affinity and selectivity of target proteins. They derive from the family of proteins that are ankyrins adapter proteins for fixing the integral membrane protein spectrin network / actin which is "the backbone" of the cell plasma membrane. Ankyrins structure is based on the repetition of a motif of approximately 33 amino acids and it is the same of DARPins. Each pattern has a secondary structure helix-turn-helix ( "helix-turn-helix"). The DARPins contain at least three,

The immunoassay of determining the antibody response is a qualitative test, semi-quantitative or quantitative widely known to those skilled in the art keeping implemented preferably two antibody binding partners. One of the two partners can be coupled to a label to form a conjugate or a plotter. The other binding partner can be captured on a solid support. This is known as capture partner for the latter and detection partner for the first.

The formats using two connection partners are well-known sandwich formats of the art, namely:

a common format called Double antigen sandwich, using capture and detection both antigens, of identical or different nature, capable of being recognized by the desired antibody, provided that at least one antigen is a polypeptide of the invention

A format commonly referred to immunocapture using a capture antibody, an antibody fragment or an analog of antibodies, as described

above, and detecting a polypeptide of the invention and

A commonly known as indirect sandwich format using capture a polypeptide of the invention and detecting antibody, an antibody or the like antibody fragment.

Preferably, the capture partner is a polypeptide of the invention and the detection partner is an anti-IgG or human IgM antibody (indirect sandwich format).

The measured signal at Pimmunoessai is then proportional to the amount of antibody from the biological sample.

By marker is intended, in particular, any molecule containing a reactive group with a moiety of the binding partner, directly without chemical modification, or after chemical modification to include such a group, which molecule is capable of directly or indirectly generating a detectable signal. A non-exhaustive list of these direct detection of labels consists of:

• enzymes which produce a detectable signal, for example by colorimetry, fluorescence, luminescence, such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, the enzyme glucose-6-phosphate,

• chromophores such as fluorescent, luminescent dyes,

32 35 125

• radioactive molecules such as P, S or I,

• fluorescent molecules such as Alexa or phycocyanins and

• electrochemiluminescent salts such as derivatives organo -métalliques based acridinium or ruthenium.

Indirect detection systems may also be used, such as ligands capable of reacting with an antiligand. The ligand corresponds to the marker to form, with the binding partner conjugate.

Couples ligand / anti- ligand are well known to those skilled in the art, which is the case eg of the following pairs: biotin / streptavidin, hapten / antibody, antigen / antibody, peptide / antibody, sugar / lectin, polynucleotide / complementary to the polynucleotide.

The anti-ligand may then be directly detectable by the markers

direct detection described above or be itself be detectable by another ligand / anti-ligand, and so on.

These indirect detection systems can, under certain conditions, to a signal amplification. This signal amplification technique is well known in the art, and reference may be made to prior patent applications FR 2781802 or WO 95/08000 of the Applicant.

These different labels can be coupled to the polypeptides of the invention as indicated above.

Depending on the type of labeling used, those skilled in the art will add reagents for visualizing the labeling or the emission of a detectable signal by any type of suitable measuring instrument, such as a spectrophotometer, a spectrofluorimeter, a densitometer , luminometer or a high definition camera.

The immunoassay may also include other steps known in the art, such as washing steps and incubation steps.

The immunoassay can be a test in one step or two steps, as is widely known to those skilled in the art. Briefly, an immunoassay in a step comprises the presence of the sample to be tested simultaneously with the two binding partners, including the polypeptides of the invention as defined above, while a two-step immunoassay comprises placed in the presence of the test sample on the one hand with the first binding partner and the analyte-first binding partner complex thus formed is brought into the second binding partner, one of the two binding partners being a polypeptide of the invention as defined above.

The reference signal S used in the process according to the invention is a previously obtained signal with two populations of controls, one having developed an antibody response directed against the p-ORF2 protein following infection with HEV and the other has not developed such antibody response. Such a determination is well known to the skilled person. It is particularly to implement the same immunoassay to that implemented in the process of

the invention in biological samples of both populations (identical in nature to the samples that will be implemented in the method of determining the presence of the antibody response in subjects tested), and determining the test value (signal ) to discriminate between these two populations.

The detected signal compared to the reference signal, used to determine whether the sample contains the tested for or antibodies, may correspond to the signal as such from the label, or it may be converted into an index that is ratio signal detected / reference signal. In one simple example, for which no gray area exists, if the reference index is set to "1", an index for the test sample than "1" means that the sample contains said antibody and an index less than "1" means that the sample does not contain said antibodies.

Of course, all the definitions given above with respect to polypeptides apply to the method for determining the presence of antibodies directed response against p-ORF-2 protein of hepatitis E described above.

Polypeptides of the invention may also be useful for determining the level of antibodies directed against the protein p-ORF-2 of hepatitis E in a biological sample from a subject, suspected of containing said antibodies. This determination can be implemented by immunoassay and comprises or consists of the steps:

- contacting said biological sample with a polypeptide as defined above,

- detecting a signal transmitted by the binding between said polypeptide and said antibody, if present, using a label capable of emitting a detectable signal,

- convert the signal detected in antibody levels.

Of course, again, all the definitions given above with respect to polypeptides, as well as those related to the presence of the antibody response determination method apply to the antibody levels of the determining method. The only difference is the given result, which is not a result of "yes" / "no" after comparing the detected signal with a reference signal, but a result of type concentration, or title or quantity following the last step of transforming the signal detected in antibody levels.

This signal processing step detected in antibody level is widely known to those skilled in the art. It consists of using a predetermined mathematical model from a standard range. This standard range is previously obtained in a known manner. Briefly, obtaining a calibration range is to measure the signal generated by the quantities or increasing concentrations and the known target antibody, to trace the curve giving the signal as a function of antibody levels and find mathematical model that represents the most accurate way possible this relationship. The mathematical model is used to determine the amounts, securities or unknown concentrations of anti-p-ORF2 antibodies contained in the biological sample to be tested.

The desired antibodies in the biological sample subjects are diverse: IgM, IgG, IgA, IgE, IgG and IgM antibodies are preferred. of similar antibodies can be sought, eg only IgG or IgM only, or can search for different kinds of antibodies in combination, for example, IgG and IgM simultaneously or all types of anti-immunoglobulin ORF2 at the same time (total Ig).

Whatever the nature of the desired antibodies, and preferably when they are IgGs or IgMs, the methods for determining the presence of antibody response or antibody level as described above are particularly useful for the treatment topics related to infection with hepatitis E.

By infection with hepatitis E is defined as an infection present, that is to say that the subject in which they made the immunoassay test is doing the infection, an infection past, that is to say that the subject in which they made the immunoassay test has no symptoms, but was previously in contact with either the virus or with a vaccine against the virus.

Also, another object of the invention relates to the use of a method as defined above for diagnosis using in vitro, for the in vitro diagnosis of infection with hepatitis E in a susceptible subject to be infected, for therapeutic monitoring of a subject infected with hepatitis E or for epidemiological studies of the prevalence of anti-HEV antibodies in a population or in a given geographical territory.

All of these uses are well known to the skilled person, the only condition being that they are implemented with the methods described above and therefore the above described polypeptides.

When the desired antibodies are IgG, the processes as defined above are also particularly useful for determining if a subject needs to be vaccinated or revaccinated against the hepatitis E, which is another object of the invention .

In order to determine whether the subject needs to be vaccinated or revaccinated against HEV or not, the following steps can be implemented:

1. determine the rate of anti-HEV IgG antibodies in a biological sample, particularly a sample of blood or blood derived, according to a method as defined above, in a healthy subject or, preferably, in patients at risk, such as previously described

2. compare the response to a threshold, such a threshold is determined beforehand according to the requirements in force,

3. the response is below the threshold meaning that it be vaccinated or revaccinated topic

4. the response obtained is greater than the threshold meaning that it is not necessary to vaccinate or revaccination the subject.

Of course, the characteristics described above in connection with methods for determining the presence of the antibody response or antibody rates apply to the uses made of these processes, such as polypeptides and various lengths and mutations, biological samples and topics concerned.

To implement the methods of the invention, used in particular by the uses described above, the polypeptides of the invention may be contained in kits.

Also, another object of the invention relates to kits for the determination by immunoassay of the presence of the antibody response or antibody levels directed against the protein p-ORF-2 of hepatitis E in a susceptible subject to have produced these antibodies comprising a polypeptide as defined above.

Again, the features described above in the context of polypeptides and methods of the invention apply to the kits of the invention.

According to a particular embodiment, the kits comprise also contain at least one positive control. The positive control comprises a compound capable of binding to binding partners implemented in use of the kit, the compound is present at a predetermined rate.

As non-limiting examples of such compounds include anti-ORF2 native immunoglobulins (in this case, the positive control can be an HIV-positive biological sample ORF2), non-natural anti-ORF2 immunoglobulins, e.g. humanized, the anti-ORF2 monoclonal antibodies, e.g., mouse.

The kits may also contain all the chemicals necessary for the identification of the reaction between the binding partners and target antibodies, such as washing buffers or reagents for visualizing a marking or issue a detectable signal.

L'invention is elucidated with following examples which are given for illustrative and not restrictive.

EXAMPLES

Example 1: Construction, expression and purification of mutated fragments 394-660 and non-mutated capsid protein OR 1 2 of Hepatitis E

The ORF2 sequence expressed is that of the isolate Human / China / HeBei / 1987 of Hepatitis E which is genotype 1 (accession No. Q81871 Uniprot - See also 1 - SEQ ID NO: ll). For the reference structure (REF-ORF2), the sequence corresponding to amino acids 394-660 of ORF2 (SEQ ID N ° 26) was fused N-terminal side with a polyhistidine tag (8-his). In the construction according to

the invention (ORF2-MUT), three non-conservative mutations (cysteine ​​to serine) were performed in the 394-660 fragment of ORF2 in the 3 cysteines at positions 627, 630 and 638 (SEQ ID NO: 27). As ORF2-REF, MUT-ORF2 comprises a tag 8-his N-terminal side.

SEQ ID N°26 :

QLFYSRP VVSANGEPTV KLYTSVENAQ QDKGIAIPHD IDLGESRVVI QDYDNQHEQD RPTPSPAPSR PFSVLRANDV LWLSLTAAEY DQSTYGSSTG PVYVSDSVTL VNVATGAQAV ARSLDWTKVT LDGRPLSTTQ QYSKTFFVLP LRGKLSFWEA GTTKAGYPYN YNTTASDQLL VENAAGHRVA I STYTTSLGA GPVSISAVAV LAPHSALALL EDTMDYPARA HTFDDFCPEC RPLGLQGCAF QSTVAELQRL KMKVGKTREL

SEQ ID N°27 :

QLFYSRP VVSANGEPTV KLYTSVENAQ QDKGIAIPHD IDLGESRVVI QDYDNQHEQD RPTPSPAPSR PFSVLRANDV LWLSLTAAEY DQSTYGSSTG PVYVSDSVTL VNVATGAQAV ARSLDWTKVT LDGRPLSTTQ QYSKTFFVLP LRGKLSFWEA GTTKAGYPYN YNTTASDQLL VENAAGHRVA I STYTTSLGA GPVSISAVAV LAPHSALALL EDTMDYPARA HTFDDFSPES RPLGLQGSAF QSTVAELQRL KMKVGKTREL

The DNA fragments corresponding to ORF2-REF buildings and ORF2-MUT was obtained in the form of synthetic genes with the GeneArt® company (Life Technologies). They were cloned between the Nco I site (5 ') and Bam HI (3') into the pET3d vector (Novagen, EMD Millipore) under control of the T7 inducible promoter with IPTG (isopropyl-beta-Dl-thiogalactopyranoside). The plasmids were verified by sequencing in the inserts to ensure they do not contain errors.

The expression plasmids are introduced into E. coli BL21 DE3 bacteria (Stratagene, Agilent Technologies) by heat shock transformation. After isolation of the colonies on petri dish LB-agar containing ampicillin, a colony corresponding to ORF2-REF and one corresponding to ORF2-MUT are picked and inoculated into 200 mL of 2x YT culture medium, glucose 0.5%, by presence

ampicillin 100 ug / mL, 1 night at 37 ° C with shaking at 250 rpm. A volume of 16 ml of each preculture is used to inoculate 400 mL medium 2x YT-glucose 0.5% -ampicilline 100 mcg / mL. These cultures were incubated at 37 ° C with shaking 250 rpm. When the optical density (OD) measured at 600 nm reaches about 1 OD unit, induction of protein expression is effected by addition of 1 mM IPTG. Crop growth is monitored by measuring optical density at regular intervals. After about 3 hours of induction, when the cultures reach stationary phase, the cultures are stopped and the bacteria were collected by centrifugation (5000 g, 20 min, + 2/8 ° C). Bacterial pellets are weighed and frozen at -80 ° C until purification.

For purification, the pellets (2 to 2.2 g) are taken up in 30 mL of lysis buffer (Tris HC1 20 mM, 100 mM NaCl, 5% glycerol, Benzonase Nuclease 5U / mL (Novagen), MgC12 0, 48 g / L, complete EDTA free protease inhibitors (Roche, Ref 045-66462) 1 tablet / 50 mL, pH 7.4). Bacteria are lysed by disintegration using a Cell Disruption System (Constant Systems Ltd., Northants, UK) at 1600 bar maintaining a refrigeration system at + 2/8 ° C. The disintegrator is rinsed with additional 30 ml lysis buffer to recover all of the lysate. The lysates are then centrifuged at 10 000 g, 40 min, + 2/8 ° C and the pellets were recovered.

To solubilize the inclusion bodies, each pellet is taken up in 30 ml of buffer Tris HC1 20 mM, 100 mM NaCl, 5% glycerol, 5 M urea, pH 7.4 and stirred + h 30 at 18/25 ° C . Supernatants are recovered by centrifugation at 10 000 g, 20 min, room temperature and then filtered successively on nitrocellulose filters in 1.2 μιη and 0.8 μιη.

Purification of the ORF2 protein and ORF2-REF-MUT is effected by metal chelate affinity chromatography step, thanks to their poly-histidine tags. Purification is performed on an automated system type Akta (GE Healthcare Lifesciences). The supernatant obtained after centrifugation is loaded onto a resin Ni-NTA column (Roche, Ref 058-93682001) equilibrated in buffer Tris HC1 20 mM, 100 mM NaCl, 5% glycerol, 5 M urea, pH 7.4 (buffer equilibration, the same as the solubilizing buffer). The elution buffer is of the equilibration buffer containing 300 mM imidazole and whose H was readjusted to 7.4. A washing cycle is performed with the equilibration buffer containing 40 mM imidazole. Then the protein is eluted with a plateau with 100% elution buffer, 300 mM imidazole. Purification fractions were analyzed on SDS-PAGE gel stained with Coomassie blue. This analysis allows to check the progress of the purification process and selection of fractions containing the protein of interest.

Selected fractions are pooled and dialyzed in Tris HC1 40 mM, NaCl 250 mM, mannitol 10%, arginine 0.4 M, 2M urea, pH 7.4. Two successive dialyses are performed at + 18/25 ° C against a buffer volume 100 times larger than that of the sample. The dialyzed proteins are assayed in total protein by measuring the optical density at 280 nm, and stored at -80 ° C.

Example 2 Characterization by SDS-PAGE analysis of the ORF2 protein and ORF2-REF-MUT

A first characterization of the purified proteins and ORF2 ORF2-REF-MUT was performed by SDS-PAGE analysis on a gel NuPAGE® Bis-Tris 4-12% NuPAGE ® in MES SDS buffer (Life Technologies). Before loading on the gel (10 μΕ / well), the proteins were diluted in the buffer NuPAGE ® LDS Sample Buffer 4X (Life Technologies) (3/1, v / v) and underwent various treatments. The reduction is done by adding 50 mM final dithiothreitol (DTT). Heating was for 10 min at 75 ° C. The combinations tested are:

HEATED and reduced (with DTT)

HEATED NOT REDUCED (without DTT)

Unheated and reduced (with DTT)

NO HEATED NOT REDUCED (without DTT)

A photograph of the stained SDS-PAGE gel with Coomassie blue to visualize the total protein is shown in Figure 3. Reduced and heated (lanes under columns + and + in the table), the ORF2 protein and ORF2-REF-MUT have the same molecular weight which is slightly greater than 30 kDa. This analysis condition can view the monomeric form of the two proteins.

Under non-reduced and heated (lanes under columns + for heating and - lowered in the table), PORF2-REF has four bands for which a majority of apparent molecular weight less than 70 kDa. This band corresponds to a dimeric form of the ORF2 protein-REF: the two monomers are linked by at least one covalent bond (disulfide bridge) which is not destroyed by heat denaturation and requires the addition of a reducer. Under the same conditions of analysis, PORF2-MUT has a single band, so it is monomeric.

Under non-heated, with or without the presence of reducing agent (lane under columns - for heating and + or respectively - for reduced in the table), 1ORF2-REF has a complex migration profile with many bands, emphasizing the diversity of interactions occuring between the monomers. Heterogeneity in oligomeric forms in the presence 1ORF2-REF is well demonstrated in the analyzed line non-denaturing conditions, that is to say non-heated and non-reduced. It is observed the presence of at least 5 bands of high molecular weight in addition to the bands corresponding to dimeric covalent and non-covalent. Conversely 1ORF2-MUT unheated reduced or not (lane under columns - and heated to respectively + or - to reduce in the table), has a very simple migration profile, with a largely predominant band corresponding to the dimeric non-covalent. Note also traces of monomer and a band migrating at about 80 kDa which is probably non-covalent tetrameric form.

Thus, the ORF2-MUT protein is more homogeneous than the ORF2 protein-REF and lies substantially in the form of a non-covalent dimer. LORF2-REF, very heterogeneous, containing both covalent dimers (major form), non-covalent dimers and various forms of high molecular weight.

Example 3: Characterization of the ORF2 protein and ORF2-REF-MUT by fluorescent labeling of the free cysteines

In order to refine the previous results, we wanted to determine, for each protein preparation, the proportion of free cysteines and cysteines involved in disulfide bridges. The protein sample is divided into two: the first half undergoes direct alkylation of free thiols accessible cysteines; the second half undergoes alkylation after reduction and heating, a treatment which makes accessible all cysteines.

Alkylation is done through the fluorescent reagent BODIPY® FL iodoacetamide (Life Technologies, Ref. D-6003) which has spectral characteristics very similar to fluorescein. The marking is done according to the manufacturer's instructions. Very briefly, prepare extemporaneously a stock solution of iodoacetamide BODIPY® FL 1 or 10 mM and dilute proteins to 100 μΜ. Protect from light, BODIPY® FL was added dropwise iodoacetamide in protein solution at mark (10 to 20 moles of BODIPY® FL iodoacetamide for 1 mole of protein) and incubated 30-60 min at 'darkness. The thus labeled protein is migrated on a SDS-PAGE gel to separate it from the excess fluorophore. The gel is then visualized on a fluorescence imaging system (ChemiDocTM XRS +, Bio-Rad) and fluorescence intensity at the protein band is measured. This fluorescence is specific and proportional to the number of labeled cysteine.

The analysis is performed in relative amount taking as reference the fluorescence intensity of the ORF2-REF monomer obtained after heating and reduction. In this molecule there are three cysteines in theory and in these conditions all cysteines are marked (100% fluorescence). The MUT-ORF2 protein is not marked by the BODIPY® FL iodoacetamide. About 1% of fluorescence is detected to the MUT-ORF2 protein, it is the non-specific background noise. Concerning REF-ORF2 protein, there is no fluorescence detected in the unheated non-reduced sample. This indicates that no cysteine ​​is accessible by the alkylating agent, which is consistent with the pattern observed in SDS-PAGE (Figure 3). For the sample heated ORF2-REF,

This analysis confirmed that the well-REF ORF2 protein is predominantly non-monomeric. Forming both covalent dimers and

noncovalent dimers, the ORF2-REF protein is much more heterogeneous than the MUT-ORF2 protein.

Example 4: Characterization of the ORF2 protein and ORF2-REF-MUT by steric exclusion chromatography (SEC - "size exclusion chromatography")

The size exclusion chromatography is used to separate molecules according to size. Each exclusion chromatography resin is characterized by a domain specific fractionation, expressed in molecular weight, within which the separation of molecules is possible. Molecules whose size is smaller than the lower limit of the fractionation range or greater than the upper limit are not divided effectively. Molecules whose size exceeds the exclusion limit, as expressed in molecular weight, are not split and are eluted together in the void volume of the column.

CLAIMS
1. The polypeptide of p-ORF2 protein of hepatitis E comprising at least the sequence of amino acids 394-660, numbered relative to a p-ORF2 protein of 660 amino acids, in which the three cysteines in positions 627, 630 and 638 have been mutated or, for a p-ORF2 protein of different lengths, at least the amino acid sequence corresponding to amino acids 394-660 of the p-ORF2 protein of 660 amino acids, wherein the three three cysteines located at positions corresponding to positions 627, 630 and 638 of the p-ORF2 protein of 660 amino acids have been mutated.

2. The polypeptide of claim 1, characterized in that the mutations are implemented by replacing the three cysteines by any amino acid except proline, amino acids whose side chains are loaded and amino acids whose side chains comprise a benzene aromatic ring.

3. The polypeptide of claim 1 or 2, characterized in that the mutations are implemented by replacing the three cysteines by an amino acid selected from alanine, glycine, threonine, valine and serine.

4. A polypeptide according to any one of claims 1 to 3, characterized in that the mutations consist of substituting the cysteines by the same amino acid.

5. A polypeptide according to claim 4, characterized in that the mutations are implemented by replacing the three cysteines by serine.

6. A polypeptide according to any one of the preceding claims, characterized in that it consists of the polypeptide of amino acid 394-660, numbered relative to a p-ORF2 protein of 660 amino acids, wherein the three cysteines at positions 627, 630 and 638 have been mutated or, for a p-ORF2 protein of length

different sequence of amino acids corresponding to amino acids 394-660 of the p-ORF2 protein of 660 amino acids, in which the three cysteines located in the three positions corresponding to positions 627, 630 and 638 of the p-ORF2 protein 660 amino acids have been mutated.

7. A polypeptide according to any one of the preceding claims, characterized in that it also comprises one or more amino acids not belonging to the p-ORF2 protein or in that it is labeled.

8. An isolated nucleic acid comprising a nucleotide sequence encoding the polypeptide as defined in any of claims 1 to 7 or a sequence complementary to said coding sequence.

9. An expression vector comprising a nucleic acid sequence as defined in claim 8.

10. A host cell comprising a nucleotide sequence encoding the polypeptide as defined in any of claims 1 to 7 or a sequence complementary to said coding sequence or an expression vector as defined in claim 9.

11. A method of determining by immunoassay the presence of an antibody directed response against p-ORF-2 protein of the virus of hepatitis E in a biological sample from a subject, suspected of containing antibodies to said response, which comprises the following steps:

- contacting said biological sample with a polypeptide as defined in any one of claims 1 to 7,

- detecting a signal transmitted by the binding between said polypeptide and said antibody, if present, using a label capable of emitting a detectable signal,

- comparing the signal thus obtained with a predetermined reference signal S with two populations of controls, one having developed said antibody and the other

having developed not said antibodies,

- a less than said reference signal S meaning that the sample does not contain said antibodies, and

- a higher than said reference signal S indicating that the sample contains said antibody.

12. A method of determining by immunoassay the level of antibodies directed against the protein p-ORF-2 of hepatitis E in a biological sample from a subject, suspected of containing said antibodies, which comprises the steps of:

- contacting said biological sample with a polypeptide as defined in any one of claims 1 to 7,

- detecting a signal transmitted by the binding between said polypeptide and said antibody, if present, using a label capable of emitting a detectable signal,

- convert the signal detected in antibody levels.

13. A method according to one of claims 11 or 12, characterized in that the desired antibodies are IgM or IgG.

14. Use of a process as defined in any one of claims 11 to 13 for in vitro diagnostic aid for the in vitro diagnosis of infection with hepatitis E in a subject likely to being infected, for therapeutic monitoring of a subject infected with hepatitis E or for epidemiological studies of the prevalence of anti-HEV antibodies in a population or in a given geographical territory.

15. Use of a method as defined in claim 11 or 12 for determining whether a subject needs to be vaccinated or revaccinated against the hepatitis E, wherein the desired antibodies are IgG.

16. Kit for the determination by immunoassay of the presence of the response

antibody or antibody levels directed against the p-ORF-2 protein of the virus of hepatitis E in a subject likely to have produced these antibodies comprising a polypeptide as defined in any one of claims 1 to 7 .

17. The kit of claim 16, further comprising at least one positive control sample is a sample containing a given level of antibodies directed against the protein p-ORF-2 of hepatitis E.