MUTUATED HEV POLYPEPTIDES AND THEIR USE FOR THE ASSAY OF ANTI-HEV ANTIBODIES

专利摘要:
The present invention provides polypeptides derived from the hepatitis E virus p-ORF2 protein comprising at least the amino acid sequence 394-660, numbered with respect to a p-ORF2 protein of 660 amino acids, wherein three cysteines at positions 627, 630 and 638 have been mutated or, for a p-ORF2 protein of different length, at least the amino acid sequence corresponding to amino acids 394-660 of the p-ORF2 protein of 660 amino acids, wherein the three cysteines at the three positions corresponding to positions 627, 630 and 638 of the p-ORF2 protein of 660 amino acids were mutated. It also relates to methods for determining the presence of the humoral response or the level of antibodies directed against the p-ORF2 protein using these polypeptides, as well as their use in the context of a virus infection context. hepatitis E.
公开号:FR3044312A1
申请号:FR1561596
申请日:2015-11-30
公开日:2017-06-02
发明作者:Yasemin Ataman-Onal；Soizic Daniel；Nadege Goutagny；Francoise Luciani
申请人:Biomerieux SA；
IPC主号:

专利说明:

The present invention relates to the field of infections with the hepatitis E virus (HEV). In particular, the invention relates to the detection of hepatitis due to the hepatitis E virus.
Hepatitis is an inflammatory lesion of the liver whose causes can be multiple: infectious, medicated, autoimmune, etc. Acute hepatic disorders of viral origin are frequent, often asymptomatic. They are due either to a direct cytopathic action of the virus, or, most often, to the immune reaction directed against the infected liver cells. Symptoms, when present, include febrile jaundice, itching, discolouration of the stool, browning of the urine, and a greater or smaller increase in transaminases, indicating cytolysis and hepatic dysfunction.
Many viruses are capable of causing liver damage, such as Epstein-Barr virus (EBV) or Cytomegalovirus (CMV), but only six viruses are known to cause the so-called "hepatitis viral ". These viruses are hepatitis viruses A, B, C, Delta, E and G, which are viruses belonging to very different families.
The hepatitis G virus is poorly described.
Hepatitis A virus, or HAV or HAV, belongs to the family Picornaviridae and is the only representative of the genus Hepatovirus. This is a naked RNA virus. The reservoir of virus is the infected subject, sick or not. The modes of transmission are determined by the exceptional resistance 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 dirty shells and crudités.
Hepatitis B virus, or HBV or HBV, belongs to the family of hepadnaviridae. It is a circular, double-stranded DNA virus about 3/4 of its circumference. This virus carries the risk of fulminant hepatitis, chronic active hepatitis, cirrhosis and hepatocarcinoma. The main vector of the virus is blood, but it can be transmitted sexually. Globally, an estimated 350 million people are chronically infected with this virus and cause more than one million deaths annually.
Hepatitis C virus, or HCV or HCV, or NANBH for "Non-A, Non-B Hepatitis", a virus with a positive-RNA genome, has an organization close to that of flaviviruses with 9500 nucleotides (9.5 kb), 5 'and 3' non-coding ends, and starting from the 5 'end of the capsid (C), envelope (E1 and E2) and nonstructural protein (NSI to NS5) genes. HCV is a strictly human virus. The main route of infection is through the venous route, for example through the use of unsterilized needles, with blood transfusion contamination still present in developing countries where donor screening is not available. The most worrying element of hepatitis C is that, beyond a generally asymptomatic primary infection (90% of cases), the evolution occurs in 70 to 80% of cases towards chronicity, with 20 % of chronically infected persons at risk for cirrhosis and primary liver cancer after an average of 20 years of incubation for cirrhosis and 30 years of cancer.
The hepatitis delta virus, or HDV or HDV, is a very small RNA virus that can not replicate without HBV, which lends it HBs surface antigen. DELTA virus infection occurs only at the same time as an HBV infection with a worsening prognosis: increased risk of fulminant hepatitis and progression to chronic active hepatitis.
Hepatitis E virus, or HEV or HE-NANBH for "Enterically Transmitted Non-A, Non-B Hepatitis", is a small, non-enveloped, naked virus whose genome is a single-stranded RNA of positive polarity. Originally classified in the family of Caliciviridae of which he is close, the knowledge of his entire genome leads today to classify him apart, as the only member of the genus Hepevirus, from the Hepeviridæ family (Emerson, SU, & Purcell, RH , 2007). The inter-human transmission of this virus is mainly through the fecal-oral route (contaminated water, food). Infections are endemic in parts of Asia, Africa and Central and South America. The hepatitis E virus 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 genuine sporadic cases of acute hepatitis in industrialized countries in patients who have never been in an endemic area. It is now clearly demonstrated that hepatitis E is a zoonosis and that many domesticated and wild animal species are infected with HEV, constituting the reservoir of viruses. Hepatitis E, like hepatitis A, does not usually go into chronicity except for certain groups of patients such as those who have received a solid organ transplant. However, it has a poorly explained particularity: although generally self-limiting, it has been observed that in India, mortality can reach 20% in pregnant women, as gestational age increases, which could make HEV infection the most serious hepatitis of all viral hepatitis during pregnancy. It is therefore essential to have powerful and reliable tools for detecting HEV infection.
The genome of the hepatitis E virus is approximately 7.5 kb in length and has 3 partially overlapping frames (ORF1, ORF2 and ORF3) framed at the 5 'end of a non-coding sequence of 27 to 32 nucleotides and at the 3 'end of a sequence of 65 to 74 bases followed by a polyadenylated end of variable length according to the viruses. ORF1 encodes a polyprotein of about 186 kDa, termed p-ORF1 protein, subsequently cleaved into nonstructural proteins including a methyltransferase, demonstrating that the virus is capped at its 5 'end, and RNA dependent RNA polymerase. LORF2 codes for the glycosylated capsid protein, called p-ORF2 protein, having from 659 to 674 amino acids according to the variants described to date, the majority of the p-ORF2 proteins of the variants having 660 amino acids. This p-ORF2 protein has several immunogenic sites, including a conformational immunodominant epitope between amino acids 394 and 457, numbered relative to the 660 amino acid protein, and a target epitope of neutralizing antibodies, also conformational, located between amino acids 452. and 617, with the same numbering (Meng J, et al., 2001). It also contains another immunodominant epitope, called epitope 406.3-2, which corresponds to amino acids 613-654 of an ORF2 variant of 660 amino acids (WO93 / 14116). The phosphoprotein with a molecular weight of 13 kDa, encoded by 1ORF3, called p-ORF3 protein, is highly variable according to the viruses. This protein whose role remains to be clarified, would be involved in the regulatory functions of viral replication or in the assembly of the nucleocapsid.
The current diagnosis is based on the detection of the virus by gene amplification from stool and serum samples, or even bile or liver biopsy, or on the detection of the anti-HEV serum antibody response. 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 to 103 cDNA molecules / reaction, according to the techniques, the viral excretion in the stool can reach 106 cDNA molecules. Characterization of the genotype can be performed in a second step. These techniques are essentially useful for detecting viremia in the blood early in relation to the infection, before onset of symptoms and antibodies. However, these techniques aimed at detecting viral nucleic acids have the drawbacks that the period of viremia is short (1 to 2 weeks in the blood, 3 to 4 weeks in the stool) and that they require expensive equipment that can not be used at the same time. closer to the patient.
The serological diagnosis of HEV infection is based on the detection of specific anti-HEV IgM and / or IgG antibodies whose main target is p-ORF2. Several kits are marketed. MP Diagnostic ™ offers the ASSURE® HEV IgM kit which is an immuno-chromatographic test device for the rapid detection of IgM antibodies against the p-ORF2 protein of the hepatitis E virus. the kit implements a recombinant polypeptide, polypeptide 394-660, numbered with respect to the 1-660 sequence of p-ORF2, otherwise called p-ORF2.1 polypeptide, corresponding to the last 267 amino acids of the protein. Mouse antibodies against human IgM are immobilized on the immunochromatography membrane, allowing the capture of the different human IgM present in the sample. The presence of IgM specifically directed against HEV is revealed using, as a detection partner, the recombinant polypeptide 394-660 complexed with a gold-labeled anti-HEV monoclonal antibody. Reasons for using recombinant polypeptide 394-660 rather than whole protein are disclosed in WO95 / 08632. According to the teachings of this patent application, the immunological reactivity of the complete p-ORF2 protein expressed in E. coli is not optimal, since part of the molecule can reduce or even inhibit the immunoreactivity of another part of the molecule. . To overcome this inhibitory effect, the patent application WO95 / 08632 proposed using p-ORF2 proteins deleted or truncated. Among the various constructs tested, the recombinant polypeptide 394-660, deleted from the first 393 amino acids, had the best immunoreactivity.
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 in vitro self-assembly and antigenically close to the HEV viral particle, are described in Riddell MA, et al. The disadvantage of using the 394-660 polypeptide is that it contains in its C-terminal part a domain which at least partially inhibits self-assembly of the polypeptide into oligomers and VLPs. This can interfere with the good presentation of conformational epitopes.
To overcome these drawbacks, Wantai Corporation has modified the polypeptide 394-660 by removing amino acids 607-660 (numbered relative to a 1-660 sequence of p-ORF2) that interfere with oligomerization and auto-ability. -assembly. The polypeptide thus obtained was called the pE2 polypeptide, as described in patent application WO01 / 22916. The advantage of this pE2 polypeptide, of sequence 394-606, is that it dimerizes naturally and that the immunoreactivity of the dimeric pE2 is much greater than that of the monomeric pE2 by promoting the good presentation of the conformational epitopes. The disadvantage is that such a truncated polypeptide does not comprise an important epitope, the 406.3-2 epitope corresponding to amino acids 613-654 of an ORF2 variant of 660 amino acids, as indicated in patent application WO93 / 14116. . Such a deletion can then lead to a decrease in the sensitivity of a diagnostic test using such a truncated polypeptide.
The Applicant has unexpectedly discovered that it is possible to overcome the disadvantages of the polypeptides of the prior art by performing, in the peptide 394-660 of HEV ORF2, numbered with respect to a p-ORF2 protein of 660 amino, 3 mutations at positions 627, 630 and 638, by improving its antigenicity and immunoreactivity. Thus, the mutated peptide, which can be called p-ORF2-MUT, has all the important epitopes, naturally dimerizes non-covalently, is capable of oligomerization without any aggregation, and has greater immunoreactivity than the recombinant polypeptide. 394-660 not mutated.
Also, the invention relates to a polypeptide derived from the p-ORF2 protein of the hepatitis E virus comprising (i) at least the amino acid sequence 394-660, numbered relative to a p-ORF2 protein of 660 acids amino, wherein the three cysteines at positions 627, 630 and 638 have been mutated, or (ii) for a p-ORF2 protein of different length, at least the amino acid sequence corresponding to amino acids 394-660 of the protein p-ORF2 of 660 amino acids, wherein the three cysteines at the three positions corresponding to positions 627, 630 and 638 of the p-ORF2 protein of 660 amino acids were mutated.
Another subject of the invention relates to isolated nucleic acids comprising a nucleotide sequence coding for the polypeptides of the invention or a sequence complementary to said coding sequence, as well as the expression vectors comprising these sequences.
Yet another object relates to host cells comprising these same nucleic sequences, directly inserted or via expression vectors.
It also relates to the use of the polypeptides of the invention for determining the presence of an antibody response directed against the p-ORF-2 protein of the hepatitis E virus or for determining the level of these antibodies. .
Thus, another subject of the invention relates to a method for immunoassay determination of the presence of an antibody response directed against the p-ORF-2 protein of the hepatitis E virus in a biological sample from a subject, capable of containing the antibodies of said response, which comprises the following steps: - bringing said biological sample into contact with a polypeptide of the invention, - detecting a signal emitted by the binding between said polypeptide and said antibodies, if they are present using a marker capable of emitting a detectable signal, - comparing the signal thus obtained with a previously determined reference signal S with two control populations, one having developed said antibodies and the other having not developed said antibody, a signal below said reference signal S meaning that the sample does not contain said antibodies, and a signal greater than said reference signal S meaning that the sample contains said antibodies.
Another subject also relates to a method for immunoassay determination of the level of antibodies directed against the hepatitis E virus p-ORF-2 protein in a biological sample derived from a subject, capable of containing said antibodies, which comprises the following steps: - contacting said biological sample with a polypeptide of the invention, - detecting a signal emitted by the binding between said polypeptide and said antibodies, if present, using a marker capable of emitting a signal detectable, - transform the detected signal into an antibody level.
Yet another object relates to the use of these methods for the aid in vitro diagnosis, for the in vitro diagnosis of a hepatitis E virus infection in a subject likely to be infected, for the therapeutic monitoring of a subject infected with the hepatitis E virus, to make epidemiological studies of the seroprevalence of anti-HEV antibodies in a given population or geographic area or to determine whether a subject needs to be vaccinated or revaccinated against the hepatitis E virus
Finally, a last object concerns the kits for the immunoassay determination of the presence of the humoral response or the level of antibodies directed against the p-ORF2 protein of the hepatitis E virus in a subject likely to have produced these antibodies. comprising a polypeptide of the invention. The invention will be better understood on reading the nonlimiting description which follows and the appended FIGS. 1 to 6, in which: FIG. 1 gives an alignment of amino acid sequences of different p-ORF2 proteins of the main virus variants HEV obtained from the Uniprot database, the first column corresponding to the UNIPROT reference, the second column corresponding to the name of the HEV strain and the last column corresponding to the alignment of the sequences. The sequence alignment was realized by the Clustal Omega program accessible on the UNIPROT website. The different parts of the alignment are distributed from Figures IA to IL. The sequence of the Q81871 variant, of 660 amino acids, is underlined as a reference. The arrows in FIG. 1G indicate the first amino acid of the minimal sequence of the polypeptides of the invention and the rectangle in FIG. 1K shows the 12 amino acid sequence of the Q81871 variant in which the 3 cysteines to be mutated are located. The last line under each sequence alignment shows the amino acid identity or not between each variant, "*" indicating a fully conserved position, with identical amino acids in all variants, ":" indicating a well-conserved position, with amino acids having strongly similar properties and a score> 0.5 in the matrix Gonnet PAM 250, ". Indicating a fairly conserved position, with amino acids having weakly similar properties and a score = <0.5 in the matrix Gonnet PAM 250. The other positions are marked by "°". FIG. 2 shows the representations of the X-ray diffraction of natural amino acid side chain maps, calculated at a resolution of 1.5 Angstrom, printed from the site (reference dated November 13, 2015) : http://people.mbi.ucla.edu/sawava/m230d/Modelbuilding/modelbuilding.html. 3 is a photograph of an SDS-PAGE analysis gel (4-12%) stained in Coomassie blue to visualize a polypeptide of the invention, ORF2-MUT, and an unmutated polypeptide, ORF2- REF which corresponds to the polypeptide p-ORF2.1 (amino acids 394-660) disclosed in the application WO95 / 08632. Prior to gel analysis, purified and dialyzed ORF2-REF and ORF2-MUT polypeptides were either reduced by addition of dithiothreitol (DTT), denaturation by heating (10 min at 75 ° C), or both treatments. either, no treatment, as shown on the chart above the gel. The M line corresponds to the Ruler molecular weight marker (Pierce), the apparent molecular weights of the bands are indicated on the left in kilo Daltons (kDa). FIG. 4 represents the size exclusion chromatograms obtained by following the UV absorbance at 280 nm for the polypeptide of the prior art ORF2-REF (FIG. 4A) and the polypeptide of the invention ORF2-MUT (FIG. 4B) . To allow a good visualization of the different peaks of FIG. 4A, the two chromatograms are not presented at the same scale for the ordinate axis. FIG. 5 represents the graph giving the results obtained by the AsFIFFF-MALS ("asymmetric flow-field-flow fractionation-multi angle light scattering") technique for the polypeptide of the invention ORF2-MUT. The UV absorbance at 280 nm (solid line), the multi-angle light scattering signal (MALS, hatched line) and the molar mass estimate (thick solid line) are represented superimposed on the y-axis. analysis time (min) FIG. 6 is a box-and-mustache representation of the distributions of the RFV signals obtained with an immunoassay (VIDAS® automaton, bioMérieux) using as capture antigen the polypeptide of the prior art ORF2-REF or the polypeptide of the invention ORF2 -MUT, on samples not containing anti-ORF2 antibody (Neg) and positive HEV samples, containing anti-ORF2 antibodies (Pos). Boxplots were plotted according to Tukey's method: the upper and lower limits of the box correspond to the 25th and 75th percentile distributions, respectively. The value traced to half of the box is the median. The high mustache is the 75th percentile +1.5 χ between the interquartile range and the low mustache at the 25th percentile - 1.5 χ the interquartile range. Values beyond and under whiskers are represented as individual dots because they are extreme, infrequent values.
The Applicant has therefore shown, against all expectations, that it is possible, with a view to the management of subjects concerned by a hepatitis E infection, to use polypeptides derived from the p-ORF2 protein of the hepatitis E comprising at least the amino acid sequence 394-660, numbered with respect to a p-ORF2 protein of 660 amino acids, while avoiding the disadvantages of the prior art when amino acids 607-660 are included in the polypeptides, namely, that they possess all important epitopes, naturally dimerize non-covalently, are capable of oligomerizing without any aggregation. Moreover, the polypeptides of the invention are produced homogeneously, unlike the polypeptides of the prior art. Indeed, during their production, the final product reproducibly exhibits more than 75% of non-covalent dimers, the remainder being composed of dodecamers, whereas the proportion of non-covalent dimers, covalent dimers and aggregates of the polypeptides of the art the former varies from one production to another. In addition, the polypeptides of the invention exhibit greater immunoreactivity than the unmutated recombinant polypeptide 394-660. Finally, the polypeptides of the invention make it possible, when used in an immunoassay, to increase the diagnostic specificity of the test, without modifying its diagnostic sensitivity, which is fundamental for a test for detecting the hepatitis virus. E.
As indicated previously and as well illustrated in FIG. 1, the p-ORF2 protein of the HEV virus has different lengths, from 659 to 674 amino acids (see FIG. 11 giving the last amino acids of the p-ORF2 protein). The majority of the proteins having 660 amino acids, it is a protein of 660 amino acids which is often taken as reference, as in the present application. However, the other variants, although having a different amino acid sequence, are well within the scope of the invention.
Thus, to find all the polypeptides of the invention, being defined as comprising: when the p-ORF2 protein has 660 amino acids, at least the amino acid sequence 394-660, numbered with respect to a p-ORF2 protein of 660 amino acids, wherein 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 protein p-ORF2 of 660 amino acids, wherein the three cysteines located at the three positions corresponding to the positions 627, 630 and 638 of the p-ORF2 protein of 660 amino acids are mutated, it is sufficient for a person skilled in the art to perform a alignment with a protein of 660 amino acids. Thus, for example, with reference to FIG. 1, the variant Q81871 (sequence underlined in FIG. 1) is taken as a reference variant of 660 amino acids and that, for example, the variants Q2PYP3 and B6VC89 are considered, respectively of 668 and 674 amino acids, the polypeptides of the invention comprise at least: the amino acid sequence 394-660 whose cysteines at positions 627, 630 and 638 are mutated (variant Q81871), or - the sequence of amino acids 405-668 whose cysteines at positions 638, 641 and 649 are mutated (variant Q2PYP3) or - the amino acid sequence 408-674 whose cysteines at positions 641, 644 and 652 are mutated (variant B6VC89).
As shown in Figure 1K, the 3 cysteines to be mutated are in a 12 amino acid sequence defined as follows: CPECRX1LGX2QGC (SEQ ID NO: 25), wherein X1 is P, T, S or A and X2 is L or F.
The mutations at the level of the three cysteines above are made by substitution of said cysteines by any amino acid different from cysteine well known to those 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 preferable to choose the amino acid substitution according to the following two criteria: 1) the "size or volume" of the amino acid side chain based on the representations of electron density maps obtained by diffraction with X-rays, as for example shown in Figure 2 giving such a representation, calculated at a resolution of 1.5 Angstrom, and from the site (printing dated November 13, 2013): http: //people.mbi.ucla. edu / sawaya / m230d / Modelbuilding / modelbuilding.html.
In fact, from these maps, one chooses the amino acids whose electronic density is the most similar to that of cysteine (for example Serine, Valine, and Threonine) or amino acids "smaller" than cysteine (by example Glycine, Alanine). Amino acids that are too "fat" (for example Lysine, Histidine, Phenylalanine, Tyrosine, Arginine and Tryptophan) are preferably discarded. 2) Possible reactivities. The substituted amino acid is not desired to react readily with other surrounding amino acids. The charged amino acids are preferably separated such as basic amino acids (already excluded with the first criterion) and acidic amino acids.
According to one embodiment, the mutations in the polypeptides of the invention are implemented by replacing the three cysteines with any amino acid except for proline, amino acids whose side chains are loaded such as lysine, arginine , histidine, aspartic acid, glutamic acid and amino acids whose side chains include a benzene aromatic ring, such as tyrosine, phenylalanine, tryptophan.
Preferably, the mutations in the polypeptides of the invention are implemented by replacing the three cysteines with an amino acid selected from alanine, glycine, threonine, valine and serine.
The 3 cysteines may be substituted with the same amino acid or with different amino acids, preferably according to the above criteria.
According to another embodiment, the mutations used consist in substituting the 3 cysteines with the same amino acid and preferably with serine.
The polypeptides of the invention comprise at least the amino acid sequence 394-660, numbered with respect to a p-ORF2 protein of 660 amino acids, and, for a p-ORF2 protein of different length, they comprise at least the amino acid sequence corresponding to amino acids 394-660 of the p-ORF2 protein of 660 amino acids, said sequences being mutated as previously indicated.
By polypeptide derived from the p-ORF2 protein of the hepatitis E virus is meant a continuous sequence of amino acids at positions 394-660 or equivalent positions, derived from the p-ORF2 protein of the hepatitis E virus. can also speak indifferently of protein derived from the p-ORF2 protein.
By the expression "comprises at least the sequence", it is meant that the polypeptide has said continuous sequence of amino acids derived from the p-ORF2 protein, or it possesses this sequence of amino acids to which may be added (s): (i) one or more amino acids belonging to the p-ORF2 protein, located before said sequence, and / or (ii) one or more amino acids not belonging to the p-ORF2 protein, such as a polyhistidine tail, a polysin tail, or a fusion protein, for example GST (Glutathione S Transferase), MBP (Maltose Binding Protein), CBP (Calmodulin Binding Peptide), CBD (Chitin Binding Domain), Protein A, Thioredoxin and / or (iii) labeling, for example (a) by coupling to a marker 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 further, or (b) by phosphorylation.
Thus, according to one embodiment, the polypeptides of the invention comprise one or more of the following characteristics: they consist of amino acid sequence polypeptides 394-660, numbered with respect to a 660-acid p-ORF2 protein amino, wherein the three cysteines at positions 627, 630 and 638 have been mutated or, for a p-ORF2 protein of different length, amino acid sequence corresponding to amino acids 394-660 of the p-ORF2 protein of 660 amino acids, wherein the three cysteines at the three positions corresponding to positions 627, 630 and 638 of the p-ORF2 protein of 660 amino acids were mutated; they comprise one or more amino acids not belonging to the p-ORF2 protein; they are marked, for example as illustrated above.
The polypeptides of the invention may be produced by techniques well known to those skilled in the art. For example, the polypeptides of the invention can be obtained by genetic engineering using steps, conventionally known to those skilled in the art, consisting in: - disposing of the DNA coding for the polypeptides of the invention, - inserting this DNA by cloning into an expression vector such as a plasmid, a cosmid, a phage λ or a viral vector (baculovirus (Autographa califomica Nuclear Polyhedrosis Virus), vaccinia virus, Semliki's forest virus, adenovirus, lentivirus , ...), which vector also comprises an origin of replication (for plasmids or cosmids) or replication system allowing its amplification in the host cell and one or more promoters allowing the transcription of messenger RNAs which will be translated into proteins, introducing the vector for expression into a host cell, such as a prokaryotic cell (e.g., bacteria such as Escherichia coli, Bacillus subtilis) by transformation or infection, or ekaryotic cell (for example yeasts (Saccharomyces cerevisiae, Pichia pastoris), insect cells (Sf9, Sf21, High5 cells), mammalian cells (CHO, 293, Per.C6, BHK-21, Vero, etc.) by transient or permanent transfection, or viral infection, - culture and possibly multiplication of the host cell containing the expression vector, possibly with amplification of the vector in the host cell, - if necessary, induction of transcription and protein synthesis for the production of the recombinant polypeptides of the invention, and - purification for extracting said polypeptides, for example by means of a polyHistidine tail. The polypeptides are then called recombinant.
Also, the subject of the invention is also: isolated nucleic acids comprising nucleotide sequences coding for the polypeptides of the invention as defined above or sequences complementary to said coding sequences; expression vectors comprising a sequence of nucleotides; nucleic acid as defined above. host cells, prokaryotic or eukaryotic, comprising a nucleotide sequence coding for 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 comprise other components such as polypeptide markers or fusion proteins, as described above, the nucleic acid sequence encoding these components can also be inserted in the same reading frame. in the vector to allow melt production. The addition of non-protein markers to the polypeptides of the invention can be implemented by techniques known to those skilled in the art using -NH-OC- bonds formed from -NH2 and -COOR groups (R being for example an activated ester group) of the markers and polypeptides of the invention. Thus, for example, when the label is biotin, those skilled in the art may use commercial reagents, such as the EZ-Link® NHS-Biotin reagents (ThermoScientific No. 20217, 21336 and 21343), which comprise a COO-ester activated to react with the -NH2 group of the 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 p-ORF-2 protein of the hepatitis E virus.
The determination of the presence of an antibody response directed against the hepatitis E virus p-ORF-2 protein in a biological sample derived from a subject, capable 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 emitted by the binding between said polypeptide and said antibodies, if present, using a marker capable of emitting a detectable signal, - comparing the signal thus obtained with a reference signal S determined beforehand with two populations of controls, one having developed said antibodies and the other having not developed said antibodies, - a signal lower than said reference signal S meaning that the sample does not contain said antibodies, and - a signal greater than said signal reference S signifying that the sample contains said antibodies.
The subjects likely to be infected by the HEV virus, in which the determination of the presence of the antibody response or of the antibody level is implemented, can be any subject and in particular: o the subjects having symptoms of acute hepatitis, such as yellowing of the skin and eyes (jaundice or jaundice), dark urine, discolored stools, extreme tiredness, nausea, vomiting, fever, abdominal pain or "flu-like" syndrome. These symptoms may be accompanied by elevated liver enzymes (ALAT / ASAT) or not. These subjects have already been tested positive for HAV, HBV or HCV viruses or not; o asymptomatic subjects with elevated liver enzymes (ALAT / ASAT). These subjects have already been tested positive for HAV, HBV or HCV viruses or not; o individuals belonging to a population at risk of either chronicization or severe fulminant disease, such as: • immunocompromised for any cause, including transplanted subjects, subjects receiving immunomodulatory or immunosuppressive therapy (s) such as chemotherapy, anti-TNF alpha treatment or a corticosteroid treatment, subjects with HIV co-infection, the elderly (immunosenescence), • pregnant women, • subjects with chronic hepatopathy.
The subjects may be mammals such as men, domestic animals (dogs, cats, horses, etc.) and livestock (sheep, cattle, goats), preferably men. As biological samples of the subjects likely to contain the anti-p-ORF2 antibodies of the hepatitis E virus, mention may be made of biological fluids such as whole blood or its derivatives, for example serum or plasma, urine, saliva and effusions, as well as stool. Blood or its derivatives and stool are preferred. These samples can be used as such in the process of the invention or have undergone pretreatment by methods known to those skilled in the art.
By determining the antibody response directed against the hepatitis E virus p-ORF-2 protein in the biological sample derived from a subject, is meant the determination of the presence or absence of antibodies produced by the subject in a subject. the case of an infection with the HEV virus, these antibodies being directed against the p-ORF2 protein.
This determination is implemented by immunoassay which is a test widely known to those skilled in the art. In short, it consists in determining an analyte, in this case the anti-p-ORF2 antibodies of the antibody response (also called humoral), by implementing at least one binding partner to the analyte.
Of course, the prefix "immuno" in the term "immunoassay", for example, is not to be considered in the present application as strictly indicating that the binding partner is necessarily a partner of immunological origin, such as an antibody or an antibody fragment. Indeed, as is well known to those skilled in the art, this term is more widely used to designate also tests and methods in which the binding partner is not a partner of origin / immunological nature but consists, for example, an analyte receptor that one wishes to detect and / or quantify. The essential condition is that the binding partner concerned is capable of binding to the desired analyte, in this case of the antibody nature, preferably in a specific manner. Thus, it is known to speak of the ELISA test for tests that use non-immunological binding partners in the strict sense of the term, called "ligand binding assay", which could be translated into French by "test using ligand binding ", while the term" immuno "is included in the verbatim title corresponding to the acronym ELISA. For the sake of clarity and uniformity, the term "immuno" is used in this application to refer to any biological assay using at least one binding partner adapted to bind to the analyte of interest and detect and / or quantify the latter. preferably, specifically, even when said binding partner is not of a strictly immunological nature or origin.
By binding partner to the anti-p-ORF2 antibodies is meant any molecule capable of binding to these antibodies. By way of example of such binding partners, mention may be made of antigens such as native or recombinant p-ORF2 protein, fragments of this protein, and in particular the polypeptides as described above, antibodies such as -Ig, for example total anti-Ig for a given species, or anti-IgG or anti-IgM depending on whether IgG or IgM is being sought (using an anti-IgG or anti-IgM species for the detection of IgG or IgM in this species), antibody analogues (molecules capable of mimicking antibodies) such as nanofitins, aptamers or "DARPins", or any other molecule that is known to interact with antibodies. The essential condition for the implementation of the method for determining the presence of the antibody response according to the invention is the use, as a binding partner, of at least the polypeptides of the invention as described above.
The antibody binding partners are, for example, either polyclonal antibodies or monoclonal antibodies, the production of which is widely known to those skilled in the art. By way of example of antibody fragments, mention may be made of the Fab, Fab ', F (ab') 2 fragments as well as the scFvs (single chain variable fragment) and dsFv (double-stranded variable fragment). These functional fragments can in particular be obtained by genetic engineering.
Nanofitin antibody analogues are small proteins that, like antibodies, are able to bind to a biological target, allowing it to be detected, captured, or simply targeted within an organism.
The aptamer antibody analogs are oligonucleotides, generally RNA or DNA, identified in libraries containing up to 1015 different sequences, by an in vitro selection combinatorial method called SELEX for "Systematic Evolution of Ligands by Exponential Enrichment" (Ellington AD and Szostak JW., 1990). Most aptamers are composed of RNA, because of the ability of RNA to adopt varied and complex structures, which allows to create on its surface cavities of various geometries, to fix various ligands. These are biochemical tools of interest that can be used in biotechnological, diagnostic or therapeutic applications. Their selectivity and ligand binding properties are comparable to that of antibodies.
The "DARPins" antibody analogues for Designed Ankyrin Repeat ProteINS (Boersma YL and Plutkthun A, 2011) are another class of proteins that mimic antibodies and can bind with high affinity and selectivity to target proteins. They derive from the family of ankyrin proteins that are adapter proteins that make it possible to bind integral membrane proteins to the spectrin / actin network that constitutes the "spine" of the cellular plasma membrane. The structure of the ankyrins is based on the repetition of a pattern of about 33 amino acids and so are the DARPins. Each pattern has a secondary structure of helix-helix-helix type. DARPins contain at least three, preferably four to five repeating units and are obtained by screening combinatorial libraries. The immunoassay of determining the antibody response is a qualitative, semi-quantitative or quantitative assay widely known to those skilled in the art preferably employing two antibody binding partners. One of the two partners may be coupled to a label to form a conjugate or a tracer. The other link partner can be captured on a solid support. This is called capture partner for the latter and detection partner for the first.
The formats using two binding partners are sandwich formats well known to those skilled in the art, namely: a format commonly called double antigen sandwich, using in capture and detection two antigens of identical or different nature, capable of being recognized by the desired antibody, it being understood that at least one of the antigens is a polypeptide of the invention A format commonly called immunocapture, using in capture an antibody, an antibody fragment or an antibody analogue, as described previously and in detecting a polypeptide of the invention and a format commonly referred to as an indirect sandwich, which captures a polypeptide of the invention and detects an antibody, an antibody fragment or an antibody analogue.
Preferably, the capture partner is a polypeptide of the invention and the detection partner is an anti-human IgG or IgM antibody (indirect sandwich format).
The measured signal emitted during the immunoassay is then proportional to the amount of antibody in the biological sample.
Marker means, in particular, any molecule containing a group reactive with a group 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-limiting list of these direct detection markers consists of: enzymes that produce a detectable signal, for example by colorimetry, fluorescence, luminescence, such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose-6- phosphate dehydrogenase, • chromophores such as fluorescent compounds, luminescent compounds, dyes,
ΛΛ ΛΓ 1AP • radioactive molecules such as P, S or I, • fluorescent molecules such as Alexa or phycocyanins, and • electrochemiluminescent salts such as organometallic derivatives based on acridinium or ruthenium.
Indirect detection systems can also be used, such as, for example, ligands capable of reacting with an anti-ligand. The ligand then corresponds to the marker to form, with the binding partner, the conjugate.
The ligand / anti-ligand pairs are well known to those skilled in the art, which is the case, for example, of the following pairs: biotin / streptavidin, hapten / antibody, antigen / antibody, peptide / antibody, sugar / lectin, polynucleotide / complementary polynucleotide. The anti-ligand can then be detectable directly by the direct detection markers described above or be itself detectable by another pair ligand / anti-ligand, and so on.
These indirect detection systems can lead, under certain conditions, to amplification of the signal. This signal amplification technique is well known to those skilled in the art, and reference may be made to prior patent applications FR 2781802 or WO 95/08000 of the Applicant.
These different markers 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 allowing the visualization of the marking or the emission of a detectable signal by any type of suitable measuring device, such as for example a spectrophotometer, a spectrofluorimeter, a densitometer , a luminometer or a high definition camera. The immunoassay may also include other steps known to those skilled in the art, such as washing steps and incubation steps. The immunoassay may be a one-step or two-step assay, as is widely known to those skilled in the art. In a few words, a one-step immunoassay comprises bringing the sample to be tested simultaneously with the two binding partners, including the polypeptides of the invention as defined above, whereas a two-step immunoassay comprises the placing the sample to be tested on the one hand with the first binding partner, then the analyte-first binding partner complex thus formed is brought into contact with 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 method according to the invention is a signal obtained beforehand with two populations of controls, one having developed an antibody response directed against the p-ORF2 protein following an infection with the HEV virus and the another who has not developed such an antibody response. Such a determination is widely known to those skilled in the art. It consists in particular in implementing an immunoassay identical to that used in the method of the invention, in biological samples of these two populations (identical in nature to the samples which will be used in the process for determining the presence the antibody response in the tested subjects), and to determine the value of the test (signal) to discriminate between these two populations.
The detected signal, compared to the reference signal, used to know whether the sample contains the desired antibodies or not, can correspond to the signal as such emitted by the marker, or it can be transformed into an index which is signal ratio detected / reference signal. According to a simple example, for which no gray area exists, if the reference index is set to "1", an index for the tested sample greater than "1" means that the sample contains said antibodies and an index less than "1" means that the sample does not contain the said antibodies.
Of course, all of the above definitions regarding polypeptides apply to the method of determining the presence of an antibody response directed against the hepatitis E virus p-ORF-2 protein described above.
The polypeptides of the invention may also be useful for determining the level of antibodies directed against the hepatitis E virus p-ORF-2 protein in a biological sample from a subject, which may contain said antibodies. This determination may be carried out by immunoassay and comprises or consists of the following steps: - bringing said biological sample into contact with a polypeptide as defined above, - detecting a signal emitted by the binding between said polypeptide and said antibodies, they are present, using a marker capable of emitting a detectable signal, transforming the detected signal into an antibody level.
Of course, again, all the definitions previously given about the polypeptides, as well as those related to the method for determining the presence of the antibody response apply to the method of determining the level of antibodies. The only difference is the given result, which is not a "yes" / "no" result following the comparison of the signal detected with a reference signal, but a result of the type concentration, or title, or quantity following the last step of transforming the detected signal into an antibody level.
This step of transforming the detected signal into an antibody level is widely known to those skilled in the art. It consists of using a pre-established mathematical model from a standard range. This standard range will be obtained previously in a known manner. In a few words, obtaining a standard range consists in measuring the signal generated by increasing or known quantities or concentrations of the target antibody, in plotting the curve giving the signal as a function of the antibody level and in finding a mathematical model that represents this relationship as faithfully as possible. The mathematical model will be used to determine the amounts, titers or concentrations of unknown anti-p-ORF2 antibodies contained in the biological sample to be tested.
The antibodies sought in the biological sample of the subjects are of various types: IgM, IgG, IgA, IgE, the IgG and IgM type antibodies being preferred. Antibodies of the same type can be investigated, for example IgG alone or IgM alone, or antibodies of different types can be tested in combination, for example IgG and IgM simultaneously or all types of anti-ORF2 immunoglobulins. at the same time (total Ig).
Whatever the nature of the antibodies sought, and preferably when they are IgG or IgM, the methods for determining the presence of the antibody response or the level of antibodies as described above are particularly useful for the management of subjects related to an infection with the hepatitis E virus.
Infection with the hepatitis E virus means both an infection present, that is, the subject in whom the immunoassay is being tested is past, that is to say that the subject in whom the immunoassay test is done has no more symptoms, but has been in contact first with either the virus or with a vaccine against the virus.
Also, another subject of the invention relates to the use of a method as defined above for the aid in vitro diagnosis, for the in vitro diagnosis of a hepatitis E virus infection in a subject susceptible to to be infected, for the therapeutic follow-up of a subject infected with the hepatitis E virus or to make epidemiological studies of the seroprevalence of anti-HEV antibodies in a given population or geographic area.
All these uses are well known to those skilled in the art, the only condition being that they are implemented with the methods described above and therefore the polypeptides described above.
When the desired antibodies are IgG, the methods as defined above are also particularly useful for determining whether a subject needs to be vaccinated or revaccinated against the hepatitis E virus, which is another object of the invention. .
Indeed, to determine whether the subject needs to be vaccinated or revaccinated against HEV or not, the following steps can be implemented: 1. Determine the level of anti-HEV IgG antibodies in a biological sample, especially in a sample of blood or blood derivative, according to a method as defined above, in a healthy subject or preferably in patients at risk, such as those described above 2. compare the response obtained to a threshold, such a threshold being determined beforehand according to the requirements in force, 3. the response obtained being below the threshold meaning that it is appropriate to vaccinate or revaccinate the subject 4. the response obtained being greater than the threshold meaning that it is not necessary to vaccinate or to revaccinate the subject.
Of course, the characteristics described previously in the context of the methods for determining the presence of the antibody response or the level of antibodies apply to the uses made of these methods, for example the polypeptides and their various lengths and mutations, the biological samples and the subjects concerned.
To implement the methods of the invention, used in particular according to the uses described above, the polypeptides of the invention may be contained in kits.
Another object of the invention is therefore to provide kits for the immunoassay determination of the presence of the antibody response or the level of antibodies directed against the hepatitis E virus p-ORF-2 protein in a susceptible subject. to have produced these antibodies, comprising a polypeptide as defined above. Here again, the characteristics described above in the context of the polypeptides and methods of the invention apply to the kits of the invention.
According to a particular embodiment, the kits also comprise or contain at least one positive control. This positive control comprises a compound capable of binding to the binding partners implemented during the use of the kit, the compound being present at a predetermined rate. By way of nonlimiting examples of such compounds, mention may be made of natural anti-ORF2 immunoglobulins (in this case, the positive control may be a seropositive biological sample ORF2), non-natural anti-ORF2 immunoglobulins, for example humanized, the anti-ORF2 monoclonal antibodies, for example mouse.
The kits may also contain all the compounds necessary for demonstrating the reaction between the binding partner (s) and the target antibodies, such as washing buffers or reagents allowing the visualization of a labeling or the emission of a detectable signal. The invention will be better understood with the aid of the following examples which are given by way of illustration and not limitation.
EXAMPLES
Example 1 Construction, expression and purification of mutated and non-mutated fragments 394-660 of the capsular protein ORF2 of the hepatitis E virus
The ORF2 sequence expressed is that of the Human / China / HeBei / 1987 isolate of the hepatitis E virus which is of genotype 1 (Uniprot Accession No. Q81871 - See also Figure 1 - SEQ ID No. 11). For the reference construct (ORF2-REF), the sequence corresponding to amino acids 394-660 of ORF2 (SEQ ID No. 26) was fused on the N-terminal side with a polyhistidine tag (8-his). For the construction according to the invention (ORF2-MUT), 3 non-conservative mutations (cysteine to serine) were carried out in the fragment 394-660 of ORF2 at the 3 cysteines at positions 627, 630 and 638 (SEQ ID No. 27). Like ORF2-REF, ORF2-MUT has an 8-his tag on the N-terminal side. SEQ ID NO: 26:
QLFYSRP WSANGEPTV KLYTSVENAQ QDKGIAIPHD IDLGESRWI
QDYDNQHEQD RPTPSPAPSR PFSVLRANDV LWLSLTAAEY DQSTYGSSTG
PVYVSDSVTL VNVATGAQAV ARSLDWTKVT LDGRPLSTTQ QYSKTFFVLP
LRGKLSFWEA GTTKAGYPYN YNTTASDQLL VENAAGHRVA ISTYTTSLGA
GPVSISAVAV LAPHSALALL EDTMDYPARA HTFDDFCPEC RPLGLQGCAF
QSTVAELQRL KMKVGKTREL SEQ ID NO: 27:
QLFYSRP WSANGEPTV KLYTSVENAQ QDKGIAIPHD IDLGESRWI
QDYDNQHEQD RPTPSPAPSR PFSVLRANDV LWLSLTAAEY DQSTYGSSTG
PVYVSDSVTL VNVATGAQAV ARSLDWTKVT LDGRPLSTTQ QYSKTFFVLP
LRGKLSFWEA GTTKAGYPYN YNTTASDQLL VENAAGHRVA ISTYTTSLGA
GPVSISAVAV LAPHSALALL EDTMDYPARA HTFDDFSPES RPLGLQGSAF
QSTVAELQRL KMKVGKTREL
The DNA fragments corresponding to the ORF2-REF and ORF2-MUT constructs were obtained in the form of synthetic genes from GeneArt® (Life Technologies). They were cloned between the Nco I (5 ') and Bam FU (3') sites in the pET3d vector (Novagen, EMD Millipore) under the control of the IPTG-inducible T7 promoter (isopropyl beta-D-1-thiogalactopyranoside). The plasmids obtained were verified by sequencing at the inserts to ensure that they did not contain errors.
Expression plasmids are introduced into E. coli BL21 DE3 bacteria (Stratagene, Agilent Technologies) by heat shock transformation. After isolation of the colonies on an LB-agar plate containing ampicillin, a colony corresponding to ORF2-REF and one corresponding to ORF2-MUT are removed and inoculated in 200 ml of culture medium. 2x YT, 0.5% glucose. in the presence of ampicillin 100 μg / mL, overnight at 37 ° C, with stirring of 250 rpm. A volume of 16 ml of each preculture is used to inoculate 400 ml of medium 2x YT-glucose 0.5% -ampicillin 100 μg / ml. These cultures are incubated at 37 ° C. with stirring at 250 rpm. When the optical density (OD) measured at 600 nm reaches about 1 unit OD, the induction of protein expression is done by adding 1 mM IPTG. Crop growth is monitored by measuring optical density at regular intervals. After about 3 hours of induction, when the cultures arrive in the stationary phase, the cultures are stopped and the bacteria are collected by centrifugation (5000 g, 20 min, + 2/8 ° C.). The bacterial pellets are weighed and then frozen at -80 ° C until purification.
For purification, the pellets (2 to 2.2 g) are taken up in 30 ml of lysis buffer (20 mM Tris HCl, 100 mM NaCl, 5% glycerol, 5U Benzonase Nuclease / mL (Novagen), MgCl 2 O, 48 g / L, protease inhibitors supplemented EDTA free (Roche, Ref 045-66462) 1 pellet / 50 mL, pH 7.4). The bacteria are disrupted by using a Cell Disruption System (Constant Systems Ltd, Northants, UK) at 1600 bar while maintaining system refrigeration at + 2/8 ° C. The disintegrator is rinsed with an additional 30 ml of lysis buffer to recover the entire lysate. The lysates are then centrifuged at 10,000 g, 40 min, + 2/8 ° C and the pellets are recovered.
In order to solubilize the inclusion bodies, each pellet is taken up in 30 ml of a 20 mM Tris-HCl buffer, 100 mM NaCl, 5% glycerol, 5 M urea, pH 7.4 and stirred for 1 h 30 at + 18/25 ° C. . The supernatants are recovered by centrifugation at 10,000 g, 20 min, at room temperature, and then successively filtered through nitrocellulose filters of 1.2 μιτι and 0.8 μηι.
The ORF2-REF and ORF2-MUT proteins are purified by one-step chelate metal affinity chromatography, using their poly-histidine tags. The purification is carried out on an automated system of the type AKTA (GE Healthcare Lifesciences). The supernatant obtained after centrifugation is loaded onto a column of Ni-NTA resin (Roche, Ref 058-93682001) equilibrated in 20 mM Tris HCl buffer, 100 mM NaCl, 5% glycerol, 5M urea, pH 7.4 (buffer of equilibration, identical to the solubilization buffer). The elution buffer is equilibration buffer containing 300 mM imidazole and whose pH was readjusted to 7.4. A wash cycle is performed with the equilibration buffer containing 40 mM imidazole. Then the protein is eluted with a 100% plateau of elution buffer, ie 300 mM imidazole. The purification fractions are analyzed on SDS-PAGE gel stained with Coomassie blue. This analysis makes it possible to check the course of the purification process and the selection of the fractions containing the protein of interest.
The selected fractions are pooled and dialyzed in 40 mM Tris HCl buffer, 250 mM NaCl, 10% mannitol, 0.4 M arginine, 2M urea, pH 7.4. Two successive dialyses are performed at + 18/25 ° C against a buffer volume 100 times greater than that of the sample. The dialyzed proteins are assayed as total protein by measuring the optical density at 280 nm and then stored at -80 ° C.
Example 2 Characterization by SDS-PAGE Analysis of the ORF2-REF and ORF2-MUT Proteins
A first characterization of purified proteins ORF2-REF and ORF2-MUT was performed by SDS-PAGE analysis on a NuPAGE® Bis-Tris 4-12% gel in NuPAGE® MES SDS buffer (Life Technologies). Before loading on the gel (10 μl / well), the proteins were diluted in NuPAGE® LDS Sample Buffer 4X buffer (Life Technologies) (3/1, volume / volume) and underwent various treatments. The reduction is done by adding 50 mM final dithiothreitol (DTT). Heating is 10 minutes at 75 ° C. The tested combinations are as follows: HEATED and REDUCED (with DTT) HEATED and UNLIMITED (without DTT) UNHEATED and REDUCED (with DTT) NOT HEATED and UNLIMITED (without DTT)
A photograph of the SDS-PAGE gel stained with Coomassie blue to visualize total proteins is shown in Figure 3. Reduced and heated (bands under + and + columns in the table), ORF2-REF and ORF2-MUT proteins have the same molecular weight which is slightly greater than 30 kDa. This analysis condition makes it possible to visualize the monomeric form of the two proteins.
In a non-reduced and heated condition (strips under the columns + for heating and - for reduced in the table), the ORF2-REF has 4 bands, a majority of apparent molecular weight less than 70 kDa. This band corresponds to a dimeric form of the ORF2-REF protein: the two monomers are connected by at least one covalent bond (disulfide bridge) which is not destroyed by heat denaturation and which requires the addition of a reducer. Under the same analysis conditions, 1ORF2-MUT has a single band, so it is monomeric.
In unheated conditions, with or without the presence of a reductant (under-column band - for heating and respectively + or - for reduced in the table), 1ORF2-REF presents a complex migration profile with many bands, highlighting the diversity of interactions taking place between the monomers. The heterogeneity oligomeric forms in the presence in 1ORF2-REF is well demonstrated in the line analyzed non-denaturing conditions, that is to say unheated and not reduced. The presence of at least 5 high molecular weight bands is observed in addition to the bands corresponding to the covalent and non-covalent dimer. On the contrary 1ORF2-MUT unheated, reduced or not (band under the columns - for heating and respectively + or - for reduced in the table), presents a very simple migration profile, with a largely majority band which corresponds to the dimer non-covalent. There are also traces of monomer and a band that migrates at about 80 kDa which is most likely the non-covalent tetrameric form.
Thus, the ORF2-MUT protein is much more homogeneous than the ORF2-REF protein and is essentially in the form of a non-covalent dimer. LORF2-REF, highly heterogeneous, contains both covalent dimers (predominant form), non-covalent dimers and various forms of high molecular weight.
Example 3 Characterization of the OKF2-REF and ORF2-MUT Proteins by Fluorescent Marking of Free Cysteines
To refine the above 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 in two: the first half is directly alkylated free thiols accessible cysteines; the second half undergoes alkylation after reduction and heating, a treatment that makes all cysteines accessible. The alkylation is carried out using 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, it is necessary to extemporaneously prepare a stock solution of BODIPY® FL iodoacetamide at 1 or 10 mM and to dilute the proteins to 100 μΜ. In the dark, BODIPY® FL iodoacetamide is added dropwise to the protein solution to be labeled (10 to 20 moles of BODIPY® FL iodoacetamide per 1 mole of protein) and incubated 30 to 60 min. 'darkness. The protein thus labeled is migrated on an 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 the fluorescence intensity at the protein band is measured. This fluorescence is specific and proportional to the number of labeled cysteines. The analysis is carried out in relative quantity, taking as a reference the fluorescence intensity of the monomer ORF2-REF obtained after heating and reduction. In this molecule, there are 3 cysteines and in theory under these conditions all the cysteines are labeled (100% fluorescence). The ORF2-MUT protein is not labeled with BODIPY® FL iodoacetamide. About 1% fluorescence is detected for the 0RF2-MUT protein, this is the non-specific background. Regarding the ORF2-REF protein, there is no fluorescence detected in the non-reduced unheated sample. This indicates that no cysteine is accessible by the alkylating agent, which is consistent with the profile observed in SDS-PAGE (Figure 3). For the heated ORF2-REF sample, the monomer band corresponds to a fluorescence intensity of 5%, which indicates that 5% of ORF2-REF cysteines are not engaged in disulfide bridges but buried in the core. of the protein and therefore not accessible when the sample is not heated.
This analysis makes it possible to confirm that the ORF2-REF protein is predominantly non-monomeric. Forming both covalent dimers and non-covalent dimers, the ORF2-REF protein is much more heterogeneous than the ORF2-MUT protein.
EXAMPLE 4 Characterization of the ORF2-REF and ORF2-MUT Proteins by Size Exclusion Chromatography (SEC)
Steric exclusion chromatography makes it possible to separate the molecules according to their size. Each exclusion chromatography resin is characterized by a specific fractionation domain, expressed in molecular weight, within which the separation of the molecules is possible. Molecules whose size is below the lower limit of the fractionation domain or greater than its upper limit are not fractionated efficiently. Molecules whose size exceeds the exclusion limit, also expressed in molecular weight, are not fractionated and are eluted together in the dead volume of the column.
Size exclusion chromatography analyzes were performed on a Waters Alliance high performance liquid chromatography (HPLC) chain with a Superdex 200 10/300 GL column (GE Healthcare) in PB S buffer ("phosphate buffered saline"). The effective fractionation domain of Superdex 200 resin is 10 to 600 kDa and its exclusion limit is 1300 kDa. For each ORF2 protein, 100 μL of sample (approximately 175 μg) was injected at 0.5 mL / min. The detection is done by measuring the absorbance at 280 nm. The chromatograms obtained for each protein are shown in Figure 4. The ORF2-REF chromatogram (Figure 4A) shows 3 populations, a majority population representing 86.9% of the observed forms, and two additional populations, corresponding to 8.5% and 4.2% of the forms observed, eluting a little before and a little after the majority peak, respectively. On the other hand, on the dORF2-MUT chromatogram (FIG. 4B), the presence of a single peak is observed, representing 99.9% of the observed forms.
For each of the chromatograms, integration of the absorbance signal at 280 nm at the peaks makes it possible to determine the total amount of protein that was fractionated during the analysis. For the ORF2-REF protein, the sum of the areas under each of the 3 peaks is 6800 mU * sec. For the ORF2-MUT protein, make under the single peak is 16100 mU * sec. The amount of fractionated ORF2-REF in the assay represents only 42% of the amount of fractionated ORF2-MUT (area ratio), whereas initially the same amount of each protein was injected. It can be deduced that a large fraction of ORF2-REF has not entered the resin and is therefore in the form of a precipitate retained at the level of the prefilter of the column. Aggregation precipitation is favored because unlike SD S-PAGE electrophoresis, no SEC chromatographic reagent contains SD S or any other detergent that could contribute to protein solubilization.
In conclusion, the steric exclusion chromatography analysis made it possible to confirm by an independent technique that the ORF2-MUT protein (1 form observed) is much more homogeneous than the ORF2-REF protein (3 forms observed). Under the conditions of the analysis, a large part of the ORF2-REF protein is in the form of a precipitate and can not therefore be studied. Moreover, it can not be excluded that a phenomenon of similar precipitation or self-assembly also occurs for the ORF2-MUT protein and that at least a part of it could not be analysis. In order to complete the SEC analysis and to be more certain to demonstrate that the ORF2-MUT protein does not contain aggregates, it is necessary to use an alternative biophysical characterization technique, allowing analyzes to be carried out on a very large scale. wide range of molecular sizes.
EXAMPLE 5 Characterization of the ORF2-REF and ORF2-MUT Proteins by the AsFIFFF-MALS ("asymmetric flow-field flow fractionation-multi-anele lieht scattering" 1 technique
To study the state of aggregation of ORF2-REF and ORF2-MUT proteins in native conditions, we implemented a technique that allows the separation of a wide range of molecules ranging from 5 kDa to 10 pm. This is flow / strength coupling with asymmetric flow ("asymmetry of field flow fractionation", AsFIFFF or AF4) coupled with multi-angle light scattering (MALS) detection. The macromolecules are separated according to their diffusion coefficient, under the effect of cross flows, without any stationary phase and in native conditions. The absence of stationary phase is a considerable advantage because it can interact with one or more of the molecular species that one seeks to separate and thus bias the analysis. The AsFIFFF-MALS analysis was carried out by the Biological and Technological Qualities team of Plant Raw Materials at the INP of Toulouse (Purpan School of Engineering, Toulouse). The experimental conditions of the analysis as performed are as follows: HPLC Ultimate 3000 Dionex
AsFIFFF Eclipse 2 Wyatt Fleleos II MALS Wyatt (633nm)
Eluent PBS 1x + 500 mM NaCl
Sample volume injected 30 μL and 60 μL
AsFIFFF Small Cell
RC membrane 5 kDa
Spacer 350 pm W
Linear flow 1 mL / min
Cross flow 3 to 0.1 Injection rate 0.2 mL / min
UV detectors 280 nm_lA
Treatment parameters MALS data: Model Zimm dn / dc: 0.185 mL / g UV extinction: 1246 mL / (g cm)
The profile of the fractogram obtained for the ORF2-REF protein is not presented because the analysis is difficult to interpret. Indeed a first peak overwrites the entire MALS signal of the analysis, making the molecular weight estimates unreliable and unreliable. However one can conclude to the presence of very large aggregates whose size is estimated at 105 - 106 kDa.
The fractogram profiles obtained for the UV (full-line) and MALS (hatched line) signals of the ORF2-MUT protein are given in Figure 5. A peak is observed in UV (fine line) with a shoulder that elutes from 9 to 15 minutes. The bimodal character of this peak, suggested in UV, appears very clearly in MALS (hatched line). In the latter, there is a first population of molar mass estimated at about 70 kDa (75% of the sample, elution between 9.2 and 11.7 min) and a second population of molar mass. estimated at about 356 kDa (25% of the sample, eluting between 11.7 and 15.0 min). For a monomer of the ORF2-MUT protein, the theoretical molar mass calculated from its sequence is 31 kDa. This theoretical calculation was experimentally confirmed during the SDS-PAGE analysis presented in Example 2. Thus, the observed molar mass of about 70 kDa corresponds to a dimer and that of 356 kDa corresponds to a dodecamer (12-mer ) of ORF2-MUT. Finally, and unlike ORF2-REF, ORF2-MUT does not contain large detectable aggregates that elute in steric mode at the beginning of the fractogram.
In conclusion, the AsFIFFF-MALS analysis, a sophisticated method allowing a characterization of the molecular species in native conditions, without any interactions with a stationary phase, made it possible to assemble that the ORF2-MUT protein i) is a mixture of 75% non-covalent dimers and 25% non-covalent dodecamers, ii) contains no aggregates in the native state, and iii) is much more homogeneous than FORF2-REF. The heterogeneity of the molecular species in the ORF2-REF protein is so important that even a technique as sophisticated and resolutive as the AsFIFFF-MALS does not allow to characterize reliably the distribution of the different forms.
EXAMPLE 6 Comparison of Immunological Reactivities of the ORF2-REF and ORF2-MUT Antigens and the Diagnostic Performance of Immunoassays Using these Antigens for the Detection of Anti-ORF2 IgMs The Antigenicity of the ORF2-REF and ORF2-MUT Proteins Was Compared by Immunoassay using the VIDAS® immunoassay automaton (bioMérieux). The disposable cone serves both as a solid phase for the reaction and as a pipetting system. The cartridge consists of 10 wells (X0 to X9) covered with sealed and labeled aluminum foil. The first well (X0) has a pre-cut portion to facilitate the introduction of the sample. The last well (X9) is an optical cuvette in which the fluorescence of the substrate is measured. The different reagents necessary for the analysis are contained in the intermediate wells (XI to X8). All the test steps are performed automatically by the instrument. They consist of a succession of suction / discharge cycles of the reaction medium. a) Sensitization and passivation of cones (coating)
The cones were sensitized with 300 μL of ORF2-REF or ORF2-MUT solution at 2 μg / mL in 77 mM carbonate buffer, pH 9.2. After approximately 20 hours of incubation at +18 / 25 ° C. with the sensitization solution, the cones are emptied. Then 300 μl of a 200 mM Tris solution containing 5 g / l bovine albumin is added. Passivation continues at + 18/25 ° C overnight. The cones are emptied, dried and stored at + 4 ° C until use, protected from moisture. b) Immunoassay Procedure The VIDAS® Automate mixes 600 μL sample diluent containing 20 mM Tris pH 7.4, 300 mM NaCl and 5 g / L serum albumin with 38.3 μL of the sample. of serum or plasma to be tested. As soon as the VIDAS® cone is in contact with the sample, the first step of the immunological reaction begins. This step allows the specific binding of the anti-ORF2 IgM, present or not the serum or plasma sample, to the ORF2 protein adsorbed on the cone. After 4 minutes of incubation at 37 ° C., unbound components are removed by washing with a 200 mM Tris buffer pH 9, 300 mM NaCl, 0.275% Triton X-100. In the second step, the cone is incubated with a solution of conjugate containing about 60 ng / ml of a mouse anti-human IgM mouse (bioMérieux), coupled to alkaline phosphatase, in a 10 mM phosphate buffer, containing 300 μg / ml. mM NaCl and 5 g / L bovine serum albumin. Well X5 contains 400 μL of this solution as the cone draws / squeezes for 5 minutes, still at 37 ° C. The second step results in the formation of a complex between the anti-ORF2 IgM present in the sample and the anti-IgM conjugate coupled to the alkaline phosphatase. This step is followed by 2 successive washes in order to eliminate the non-fixed compounds.
During the final revelation step, the 4-methylumbelliferyl phosphate substrate is sucked and then forced back into the cone; the alkaline phosphatase of the conjugate catalyzes the hydrolysis reaction of this substrate to 4-methylombelliferone whose emitted fluorescence is measured at 450 nm. The value of the fluorescence signal (RFV = relative fluorescence value) is proportional to the concentration of anti-ORF2 IgM present in the sample.
The immunoassay procedure for the detection of anti-ORF2 IgM was performed in 18 HEV positive IgM samples and 21 HEV negative IgM samples. These samples, serums or plasmas, were mainly obtained via the French Blood Establishments (EFS) and were previously characterized by various commercial tests: Wantai HEV-IgM ELISA (Ref.WE.-7196), recomWell HEV IgM (Ref. 5005, Mikrogen Diagnostik) or EIAgen HEV IgM kit (Ref 071050 Adaltis). The positive IgM HEV status of the samples was thus defined if the sample was positive at least in one of the tests mentioned above. The so-called negative HEV samples are negative in all the commercial techniques used.
Immunoreactivity. At an equal amount, the ORF2-MUT protein has an antigenic reactivity which is much greater than that of the ORF2-REF protein. This superiority is very statistically significant (PO.OOO1, one-way matched Wilcoxon test) and is illustrated in FIG. 6 which represents the distributions of the RFV signals obtained by the IgM immunoassays using either the ORF2-REF antigen (hereinafter referred to as the ORF2-IgM test). REF), the ORF2-MUT antigen (hereinafter referred to as the ORF2-MUT IgM test), on positive HEV samples (Table 1) and negative HEV samples (Table 2). For all positive samples, the RFV signals obtained with the 0RF2-MUT antigen are higher than those obtained with the 0RF2-REF antigen. For samples 155797, 154183, 154053 and 154050, the gain in RFV, very consistent, reaches about 1000 RFV. In addition, the RFV signals obtained on the negative HEV samples by the IgM ORF2-REF and ORF2-MUT tests are comparable and remain very low (FIG. 6).
Diagnostic sensitivity. On the panel of positive samples analyzed presented in Table 1, the IgM ORF2-REF test, according to the prior art, has two false negatives (samples 155118 and 136997), which corresponds to a sensitivity of 88.9%. only while the ORF2-MUT IgM test has no false-negatives resulting in 100% increased sensitivity.
In addition, the panel analyzed was pre-tested by the Wantai test in order to identify samples for which the latter was negative, but which were confirmed positive by two other IgM kits. The purpose of this selection was to highlight the advantages of the polypeptides 394-660 of the invention. The Wantai IgM kit is the only IgM commercial kit comprising only an ORF2 antigen, called pE2, which is directly comparable to an IgM immunoassay using ORF2-REF or ORF2-MUT. However, unlike the 394-660 polypeptides, the pE2 antigen sequence does not include the C-terminal epitope (aa 613-654). On the panel of positive samples tested, the Wantai test has 6 false negatives, a sensitivity of only 66.6%. Among these samples, 4/6 are detected positive by the IgM ORF2-REF test, illustrating the diagnostic interest of the C-terminal epitope and especially 6/6 are detected by the IgM ORF2-MUT test, again illustrating the superiority of this polypeptide, as well as its contribution to the improved sensitivity of an immunoassay.
Table 1. Investigation of anti-ORF2 IgM in the sera of patients with definite acute hepatitis E infection. Sensitivity study on confirmed positive samples.
Neg = Negative and Pos = Positive
Diagnostic specificity. On the panel of negative samples analyzed (Table 2), the IgM ORF2-REF test has two false positives (samples 129534 and 137163), which corresponds to a specificity of only 88.9% whereas the IgM test ORF2- MUT has no false positives resulting in 100% increased specificity.
It should be noted that the improved sensitivity of the ORF2-MUT IgM test is not at the expense of its specificity.
Table 2. Investigation of anti-ORF2 IgM in the sera of patients without hepatitis E infection with certainty. Specificity study on confirmed negative samples.
In conclusion, the ORF2-MUT antigen has better immunoreactivity than the ORF2-REF antigen, which results in superior diagnostic performance in both sensitivity and specificity. This better immunoreactivity of the ORF2-MUT protein could be explained by a better presentation of the immunodominant conformational epitopes because of its more homogeneous and oligomeric structure, as shown in Example 2 (more non-covalent dimers) and in the Example 5 (formation of dodecamers), which would allow it to present globally an antigenic structure much closer to that of the viral particle.
Example 7 Reproducibility of Hepatitis E Virus IgM Detection Tests Using O RM-RE F or ORF2-MUT
The same positive sample was assayed in duplicate, in two different series, 3 days in a row, by the IgM ORF2-REF test and the IgM ORF2-MUT test according to the procedure described in Example 6. The results are presented in FIG. Table 3. The coefficient of variation (CV) is the ratio of the standard deviation to the mean and allows the comparison of distributions of values whose scales of measurement are not comparable. The lower the value of the coefficient of variation, the smaller the dispersion around the mean, so the more reproducible the measurement. The coefficient of variation is 5.4% for the ORF2-REF IgM test and 2.1% for the ORF2-MUT IgM test. Both immunoassays are well reproducible, the IgM ORF2-MUT test seems better.
Table 3. Reproducibility of the anti-ORF2 IgM detection assays using the ORF2-REF or ORF2-MUT protein.
In order to be able to determine if the observed difference between the 2 CVs is statistically significant, the uncertainty of each of them is estimated. By accepting a risk a = 0.05, (95% confidence interval CI) and assuming that the risk is distributed symmetrically and bilaterally (ie as much risk that the CV is overestimated as it is underestimated), we deduce the upper limit CV by applying the following formula:
Khi2 (0.025, dof) is the value of the Khi2 distribution for a risk of 0.025 (half of a = 0.05) and a given degree of freedom (dof). For the series presented, the number of repetitions is n = 12 and ddl = n-1, ie 11. The value of the Khi2 law (0.025, 11) is 21.92. The upper limit of the 95% CI of the CV is given by the formula. The lower limit of the 95% CI is deduced by subtracting the difference between the upper limit and the observed CV from the observed CV. This leads to the following estimates:. , CV Limit CV Limit CV observes,. . ,,. lower upper ORF2-REF 5.4% 7.7% 3.2% ORF2-MUT 2.1% 3.0% 1.3%
According to these calculations, the CV of the ORF2-REF IgM test can be between 3.2% and 7.7% and that of the ORF2-MUT IgM test between 1.3% and 3.0%. The two intervals do not overlap, so the two observed CVs of 5.4% and 2.1% are therefore significantly different.
As a result, the ORF2-MUT IgM test is more reproducible than the ORF2-REF IgM test.
Bibliographical References - Boersma YL, Plückthun A, 2011, Curr. Opin. Biotechnol, 22: 849-857 - Ellington AD and Szostak JW., 1990, Nature. 346: 818-822 - Emerson, S. U., &amp; Purcell, R.H., 2007, Hepatitis E Virus. In D. M. Knipe, P.M. Howley, D.E.GrifFin, R.A. Lamb, M.A. Martin &amp; B. a. S. Roizman, S.E. (Eds.), Fields Virology (5th ed., Pp. 3047-3058). Philadelphia, USA: Lippincott Williams &amp; Wilkins - Fields and Noble, 1990, Int J Pept Protein Res., 35: 161-214 - Meng J, et al., 2001, Virology, 288: 203-211 - Merrifield 1963, J Am Chem Soc. 85: 2149-2154 - Riddell M.A., et al., 2000, Journal of Virology, 74 (17): 8011-8017

权利要求:
Claims (17)
[1" id="c-fr-0001]
A polypeptide derived from the hepatitis E virus p-ORF2 protein comprising at least the amino acid sequence 394-660, numbered with respect to a p-ORF2 protein of 660 amino acids, wherein the three cysteines positions 627, 630 and 638 have been mutated or, for a p-ORF2 protein of different length, at least the amino acid sequence corresponding to amino acids 394-660 of the p-ORF2 protein of 660 amino acids, wherein three cysteines at the three positions corresponding to positions 627, 630 and 638 of the p-ORF2 protein of 660 amino acids were mutated.
[2" id="c-fr-0002]
2. Polypeptide according to claim 1, characterized in that the mutations are implemented by replacing the three cysteines with any amino acid except proline, amino acids whose side chains are charged and amino acids whose side chains have a benzene aromatic ring.
[3" id="c-fr-0003]
3. Polypeptide according to claim 1 or 2, characterized in that the mutations are implemented by replacing the three cysteines with an amino acid selected from alanine, glycine, threonine, valine and serine.
[4" id="c-fr-0004]
4. Polypeptide according to any one of claims 1 to 3, characterized in that the mutations consist in substituting the cysteines with the same amino acid.
[5" id="c-fr-0005]
5. Polypeptide according to claim 4, characterized in that the mutations are implemented by replacing the three cysteines with serine.
[6" id="c-fr-0006]
Polypeptide according to any one of the preceding claims, characterized in that it consists of the amino acid sequence polypeptide 394-660, numbered with respect to a p-ORF2 protein of 660 amino acids, in which the three cysteines at positions 627, 630 and 638 were mutated or, for a p-ORF2 protein of different length, amino acid sequence corresponding to amino acids 394-660 of the p-ORF2 protein of 660 amino acids, in which the three cysteines at the three positions corresponding to positions 627, 630 and 638 of the p-ORF2 protein of 660 amino acids were mutated.
[7" id="c-fr-0007]
7. 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" id="c-fr-0008]
An isolated nucleic acid comprising a nucleotide sequence encoding the polypeptide as defined in any one of claims 1 to 7 or a sequence complementary to said coding sequence.
[9" id="c-fr-0009]
An expression vector comprising a nucleic acid sequence as defined in claim 8.
[10" id="c-fr-0010]
10. Host cell comprising a nucleotide sequence coding for the polypeptide as defined in one of claims 1 to 7 or a sequence complementary to said coding sequence or an expression vector as defined in claim 9.
[11" id="c-fr-0011]
11. A method for immunoassay determination of the presence of an antibody response directed against the p-ORF-2 protein of the hepatitis E virus in a biological sample from a subject, which may contain the antibodies of 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 emitted by the binding between said polypeptide and said antibodies, if present using a marker capable of emitting a detectable signal, - comparing the signal thus obtained with a previously determined reference signal S with two control populations, one having developed said antibodies and the other having not developed said antibody, - a signal below said reference signal S meaning that the sample does not contain said antibodies, and - a higher signal said reference signal S signifying that the sample contains said antibodies.
[12" id="c-fr-0012]
12. A method for immunoassay determination of the level of antibodies directed against the p-ORF-2 protein of the hepatitis E virus in a biological sample derived from a subject, capable of containing said antibodies, 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 emitted by the binding between said polypeptide and said antibodies, if present, using a marker capable to emit a detectable signal, - transform the detected signal into an antibody level.
[13" id="c-fr-0013]
13. Method according to one of claims 11 or 12, characterized in that the desired antibodies are IgM or IgG.
[14" id="c-fr-0014]
14. Use of a method as defined in any one of claims 11 to 13 for in vitro diagnostic assistance, for the in vitro diagnosis of a hepatitis E virus infection in a subject susceptible to to be infected, for the therapeutic follow-up of a subject infected with the hepatitis E virus or to make epidemiological studies of the seroprevalence of anti-HEV antibodies in a given population or geographic area.
[15" id="c-fr-0015]
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 virus, wherein the desired antibodies are IgG.
[16" id="c-fr-0016]
16. A kit for the immunoassay determination of the presence of the antibody response or the level of antibodies to the hepatitis E virus p-ORF-2 protein in a subject who may have produced these antibodies, including polypeptide as defined in any one of claims 1 to 7.
[17" id="c-fr-0017]
The kit of claim 16, further comprising at least one positive control sample which is a sample containing a given level of antibodies to the hepatitis E virus p-ORF-2 protein.

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同族专利:

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公开号 | 公开日

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FR3044312B1|2017-12-08|

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BR112018009851A2|2018-11-13|

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EP3383887B1|2022-03-16|

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KR20180083941A|2018-07-23|

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WO2017093649A1|2017-06-08|

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US20180328929A1|2018-11-15|

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CN108473540A|2018-08-31|

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EP3383887A1|2018-10-10|

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US10408841B2|2019-09-10|

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CN109765373A|2019-01-29|2019-05-17|中国医学科学院输血研究所|A kind of detection method and kit of viral hepatitis type E IgA antibody|

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CN109765372A|2019-01-29|2019-05-17|中国医学科学院输血研究所|A kind of detection method and kit of viral hepatitis type E IgM antibody|

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CN109765374A|2019-01-29|2019-05-17|中国医学科学院输血研究所|A kind of detection method and kit of viral hepatitis type E IgG antibody|

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CN110927374A|2019-12-02|2020-03-27|昆明理工大学|Colloidal gold test strip for detecting hepatitis E virus IgG antibody and preparation method thereof|

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FR1561596A|FR3044312B1|2015-11-30|2015-11-30|MUTUATED HEV POLYPEPTIDES AND THEIR USE FOR THE ASSAY OF ANTI-HEV ANTIBODIES|FR1561596A| FR3044312B1|2015-11-30|2015-11-30|MUTUATED HEV POLYPEPTIDES AND THEIR USE FOR THE ASSAY OF ANTI-HEV ANTIBODIES|

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US15/774,366| US10408841B2|2015-11-30|2016-11-29|Mutated HEV polypeptides and the use thereof for assaying anti-HEV antibodies|

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EP16815613.1A| EP3383887B1|2015-11-30|2016-11-29|Mutated hev polypeptides and the use thereof for assaying anti-hev antibodies|

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KR1020187018464A| KR20180083941A|2015-11-30|2016-11-29|Mutant HEV polypeptides and their uses for assaying anti-HEV antibodies|

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PCT/FR2016/053127| WO2017093649A1|2015-11-30|2016-11-29|Mutated hev polypeptides and the use thereof for assaying anti-hev antibodies|

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BR112018009851A| BR112018009851A2|2015-11-30|2016-11-29|hev mutated polypeptides and their use for anti-hev antibody count|

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CN201680079160.7A| CN108473540A|2015-11-30|2016-11-29|The HEV polypeptides of mutation and its purposes for measuring Anti-HEV antibody|