Patent Grant
7479386
Hepatitis is the most important of the transmissible diseases. The mode of transmission is most often transfusion, organ transplants and haemodialysis, but hepatitis can also be transmitted by ingestion of contaminated food or water and by contact between individuals.
Viral hepatitis are induced by various viral agents which are distinguishable from each other by their genomes and their modes of replication. Viral hepatitis cause damage to the liver with varying degrees of severity. More than a billion people worldwide suffer from viral hepatitis. Serious risks exist in the chronic forms of hepatitis which can progress to cirrhosis or hepatocarcinoma. Viral hepatitis can be diagnosed by the detection of well-defined symptoms, such as jaundice, high levels of transaminases (aspartate transaminase or AST, alanine transaminase or ALT, lactate dehydrogenase or LDH), and hepatic lesions. Despite the knowledge of various hepatitis A, B, C, D, E, G and TTV viruses, 5% of all hepatitis and 40% of fulminant hepatitis remain unexplained, hence the hypothesis of the existence of unknown hepatitis viruses. These hepatitis of unknown aetiology are both post-transfusional and sporadic, chronic or fulminant. They are commonly called hepatitis X.
Hepatitis G (GBV-A, GBV-B, GBV-C) and TTV viruses recently identified do not appear to be pathogenic in humans and cannot therefore explain the cases of hepatitis of unknown aetiology or hepatitis X.
From one case of serious hepatitis of unknown aetiology, in a patient in whom a treatment with interferon made it possible to normalize transaminases, the present inventors were able to clone, by means of the “RDA” (Representational Difference Analysis) subtractive hybridization technique, the nucleic acids differentially expressed between the serum at the transaminase peak and the serum after normalization of the transaminases. 643 clones representing DNA sequences specific to serum at the transaminase peak were screened. In order to exclude the possibility of homologies with the human genome or any other known sequence, various routes were explored:
At the end of these various steps, 2 DNA clones were selected of which the sequences were unknown in data banks. By virtue of PCRs performed within the cloned sequences, tested on human genomic DNA obtained from blood donors, in the end a single clone containing an insert of 1400 base pairs was selected for its lack of homology with the human genomic DNA and with any sequences present in the data banks. The sequence of this clone was called XH. This sequence is rich in GC (62%) and has four open reading frames. This isolated sequence was then characterized by four parallel studies:
The results show that:
The FIGURE is a graph of results of ELISA test using SEQ ID NO:6 for the capture of antibodies specific for the HXHV virus potentially present in patients having chronic non-A, non-E hepatitis.
Accordingly, the present invention relates to the isolated HXHV virus and provides nucleic acids and polypeptides which are capable of being derived from HXHV. Furthermore, the invention provides the means for diagnosing an HXHV virus infection in patients or animals suspected of having a viral hepatitis and the pharmaceutical or vaccine compositions for preventing or treating the disease associated with the infection.
Thus, the present invention relates to
The above homology covers the functional equivalents of the sequence SEQ ID NO: 1, that is to say the DNA sequences in which at least one codon can be replaced by another codon while encoding an identical amino acid. This is referred to as degeneracy of the genetic code. Thus, the codes for argininine, for serine and for leucine exhibit a degeneracy of the order 6 (that is to say that there are six different codons for each of them), whereas the codes for the other amino acids, such as glutamic acid, glutamine, tyrosine, histidine and a few others exhibit a degeneracy of the order 2. Of all the amino acids, only tryptophan and methionine have a degeneracy of the order 1. It is therefore clear that for the expression of a polypeptide whose sequence is for example represented in SEQ ID NO: 6, it is possible to use variants and functional nucleic acid sequences in which the codon compositions are different from the nucleic acid sequence represented in SEQ ID NO: 1.
The homology defined above also relates to the mutants of the HXHV virus, and in particular those derived from natural variability. Indeed, it is well known to specialists that the viruses have relatively high levels of spontaneous or induced mutations.
The invention also relates to
“Polypeptide” denotes a peptide, in the isolated state, having a succession of a variable number of amino acids, such as an oligopeptide, a protein, a fusion protein, a fusion peptide, a synthetic peptide. A polypeptide may be obtained by various techniques well known to a person skilled in the art, and in particular by chemical synthesis or by genetic recombination techniques. The polypeptides according to the invention may be obtained by conventional methods of synthesis, for example with an automated peptide synthesizer, or by genetic engineering techniques comprising the insertion of a DNA sequence encoding the said polypeptide into an expression vector such as a plasmid or a virus, and the transformation of cells with this expression vector and culturing of these cells.
The expression peptide sequence equivalent to a reference peptide sequence is understood to mean an amino acid sequence modified by insertion and/or deletion and/or substitution and/or extension and/or shortening and/or chemical modification of one or more amino acids, as long as these modifications substantially preserve or even develop the immunoreactive properties of the said reference peptide sequence.
Thus, the expression functionally equivalent sequences which preserve the immunoreactive properties of SEQ ID NO: 5 is understood to mean in particular the sequences in which one or more amino acids are substituted by one or more other amino acids; the sequences in which one or more amino acids of the L series are replaced by an amino acid of the D series, and vice versa; the sequences into which a modification of the amino acid side chains is introduced, such as an acetylation of the amine functional groups, a carboxylation of the thiol functional groups, an esterification of the carboxylic functional groups; a modification of the peptide bonds such as for example of the carba, retro, inverso, retro-inverso, reduced and methyleneoxy bonds.
For example, one or more amino acids in the sequences of the polypeptides of the invention may be substituted by one or more other amino acids of similar polarity which act as functional equivalents. Substitutions for an amino acid in polypeptide sequences of interest may be determined from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids comprise alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids comprise glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids comprise arginine, lysine and histidine. The negatively charged (acidic) amino acids comprise aspartic acid and glutamic acid. Other substitutions for an amino acid in polypeptide sequences of interest may be determined from the information contained in the article by Kramer A. et al. (Molecular Immunology, Vol. 32, No. 7, pp. 459-465 (1995)). These authors have set up banks in which, in order to reduce the problem of combinatory explosion of the number of molecules, they used groups of amino acids consisting of amino acids having similar physicochemical properties and it is the amino acids grouped together in each of these six groups, listed below, which are considered mainly as equivalents in the present invention.
The equivalence for a peptide sequence with respect to a reference peptide sequence may be defined by its identity or its homology, expressed as a percentage, with the said reference sequence. This percentage is determined, for a sequence of a given number of contiguous amino acids, by alignment of the two sequences, moving one with respect to the other, and comparing the amino acids in the two sequences. The percentage of identity is determined from the number of amino acids which are identical to the amino acids of the reference sequence, in the same position. The percentage homology is determined from the number of amino acids which are equivalent to amino acids of the reference sequence, in the same position.
The invention also relates to an expression cassette which is functional in a cell derived from a prokaryotic or eukaryotic organism allowing the expression of a nucleic acid sequence or of a DNA fragment or of a DNA molecule as described above, placed under the control of elements necessary for its expression. The expression cassette is characterized in that it is functional in a cell derived from a prokaryotic organism, in particular E. coli, or from a eukaryotic or lower eukaryotic organism, in particular COS and CHO cells and Saccharomyces cerevisiae and Pichia pastoris cells.
The invention also relates to a vector comprising the said expression cassette; a cell derived from a prokaryotic organism, such as E. coli or a eukaryotic organism, preferably a eukaryotic or lower eukaryotic organism, and advantageously a COS or CHO cell or a cell derived from Saccharomyces cerevisiae or Pichia pastoris comprising an expression cassette or a vector as defined above; and the polypeptide produced by the expression cassette, the vector or the cell.
The subject of the invention is a method for preparing a polypeptide or a polypeptide fragment as defined above which consists in cultivating a host cell satisfying the preceding definitions, in an appropriate culture medium, the said host cell being transformed with an expression vector which contains a DNA nucleic acid sequence as defined above or a DNA nucleic fragment as defined above or a DNA molecule as defined above, and in purifying the said polypeptide produced to a required degree of purity.
The subject of the invention is also an immunogenic polypeptide, characterized in that it has a peptide sequence or in that it consists of a polypeptide as defined above, in particular the polypeptide represented in SEQ ID NO: 6. Such an immunogenic polypeptide is used for the production of monoclonal or polyclonal antibodies or of fragments of the said antibodies and the invention encompasses the monoclonal or polyclonal antibodies or fragments thereof, being obtained by immunizing a mammalian animal (rabbit, rat, mouse) with such an immunogenic peptide.
The production of monoclonal or polyclonal antibodies is well known to a person skilled in the art. There may be mentioned by way of reference Köhler G. and Milstein C. (1975): Continuous culture of fused cells secreting antibody of predefined specificity, Nature 256:495-497 and Galfre G. et al. (1977) Nature, 266: 522-550 for the production of monoclonal antibodies and Roda A., Bolelli G. F.: Production of high-titer antibody to bile acids, Journal of Steroid Biochemistry, Vol. 13, pp 449-454 (1980) for the production of polyclonal antibodies. Antibodies may also be produced by immunizing mice, rats or rabbits with the HXHV viral particles. For the production of polyclonal and monoclonal antibodies, the immunogen may be coupled to serum albumin (peptide SA) or to Keyhole Lympet haemocyanin (peptide KLH) as support for the immunization. In the context of the present invention, the immunogen identified in SEQ ID NO: 6 is coupled to BSA (bovine serum albumin). The animals are subjected to several injections of immunogen, the antibodies are collected from the serum, purified (brought into contact with a “normal” liver powder) and are then screened for their specificity using the usual techniques, such as ELISA or Western blot tests. For the production of monoclonal antibodies, the animals are subjected to an injection of immunogen using Freund's complete adjuvant. The sera and the hybridoma culture supernatants obtained from the immunized animals are analysed for their specificity and their selectivity using conventional techniques, such as for example ELISA or Western blot tests. The hybridomas producing the most specific and the most sensitive antibodies are selected. Monoclonal antibodies may also be produced in vitro by cell culture of the hybridomas produced or by recovering ascitic fluid, after intraperitoneal injection of the hybridomas into mice. Regardless of the mode of production, as a supernatant or as an ascitic fluid, the antibodies are then purified. The methods of purification used are mainly filtration on an ion-exchange gel and exclusion chromatography or affinity chromatography (protein A or G). A sufficient number of antibodies are screened in functional tests for identifying the most efficient antibodies. The in vitro production of antibodies, of antibody fragments or of antibody derivatives, such as chimeric antibodies produced by genetic engineering, is well known to persons skilled in the art. It is advantageous to use humanized antibodies. The “humanized” forms of nonhuman antibodies, for example murine antibodies, are chimeric antibodies which comprise a minimal sequence derived from a nonhuman immunoglobulin. For the majority, the humanized antibodies are human immunoglobulins (receptor antibody) in which the residues of a hypervariable region of the receptor are replaced by residues of a hypervariable region of a nonhuman donor species (donor antibody), such as mice, rats, rabbits or nonhuman primates, having the desired specificity, affinity and capacity. In some cases, the residues (FR) of the Fv region of the human immunoglobulin are replaced by corresponding nonhuman residues. Furthermore, the humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are performed in order to improve the performance of the antibody. In general, the humanized antibody will comprise at least, and preferably, two variable domains, in which all or nearly all the hypervariable loops correspond to a nonhuman immunoglobulin and all or nearly all the FR regions will be those of a human immunoglobulin. The humanized antibodies may optionally also comprise at least part of a constant (Fc) region of an immunoglobulin, such as a human immunoglobulin (Jones et al., Nature 321: 522-525 (1986); Reichmann et al., Nature 332: 323-329 (1988); and Presta et al., Curr. Op. Struct. Biol. 2: 593-596 (1992).
More particularly, the expression antibody fragment is understood to mean the F(ab)2, Fab, Fab′, sFv fragments (Blazar et al., 1997, Journal of Immunology 159: 5821-5833 and Bird et al., 1988, Science 242: 423-426) of a native antobidy and the expression derived is understood to mean, inter alia, a chimeric derivative of a native antibody (see for example Arakawa et al., 1996, J. Biochem 120: 657-662 and Chaudray et al., 1989, Nature 339: 394-397). These antibody fragments and antibody derivatives preserve the capacity to selectively bind to the target antigen.
The monoclonal or polyclonal antibody thus obtained or its fragment is incorporated into a diagnostic composition which is used in a method for detecting at least one polypeptide or one polypeptide fragment as defined above in a biological sample, according to which the biological sample is brought into contact with the composition under predetermined conditions which allow the formation of antibody/antigen complexes and the formation of the said complexes is detected.
The subject of the invention is also a diagnostic composition which comprises a polypeptide or a polypeptide fragment as defined above and a method for detecting antibodies directed against the HXHV virus or at least against a polypeptide or a polypeptide fragment of the invention, according to which a biological sample suspected of being or of having possibly been infected with the HXHV virus is brought into contact with the diagnositic composition under predetermined conditions which allow the formation of antibody/antigen complexes and the formation of the said complexes is detected. The method described in the experimental part of the appended figure clearly shows that the polypeptide of the invention or the polypeptide fragment SEQ ID NO: 6 makes it possible to detect antibodies specific for the HXHV virus in patients having a non-A, non-E hepatitis.
It is known that during an infection by a viral agent, the host develops antibodies directed against this viral agent (humoral response). It is shown in the experimental part that the immunization of rabbits with an immunogen of the invention allows the producction of antibodies by the rabbits. It has also been shown that patients having a non-A, non-E hepatitis developed antibodies directed against the HXHV virus. The subject of the present invnetion is therefore also the biological material for the preparation of a pharmaceutical composition intended for the treatment of human beings or of animals infected with at least the HXHV virus and immunogenic preparations which may be used to produce therapeutic vaccines against an HXHV virus infection and prophylactic vaccines for preventing a potential HXHV virus infection, the said immunogenic preparations comprising:
(i) either at least one natural, recombinant or synthetic polypeptide or polypeptide fragment of the invention; or
(ii) the HXHV virus, for example in an attenuated or mutated form.
The subject of the present invention is also the use of at least one monoclonal or polyclonal antibody or of at least one fragment of the said antibodies of the invention, specific for at least one polypeptide or a polypeptide fragment as defined above in (i) for the preparation of a pharmaceutical composition which, when administered to a patient infected with the HXHV virus, has the capacity to reduce or even inhibit the proliferation and/or replication of the virus. These antibodies or fragments thereof are called neutralizing antibodies.
Consequently, the invention relates to an immunogenic or vaccine composition, characterized in that it comprises a polypeptide or a polypeptide fragment of the invention or the HXHV virus, optionally combined with an appropriate vehicle and/or adjuvant and/or diluent and/or with a pharmaceutically acceptable excipient; and to a pharmaceutical composition comprising a biological material as defined above, optionally combined with an appropriate vehicle and/or adjuvant and/or diluent and/or with a pharmaceutically acceptable excipient.
The expression biological sample is understood to mean for example blood, serum, plasma, tissue samples, such as liver biopsy extracts.
The vaccines prepared are injectable, that is to say in liquid solution or in suspension. Optionally, the preparation may also be emulsified. The antigenic molecule may be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Examples of favourable excipients are water, a saline solution, dextrose, glycerol, ethanol or equivalents and combinations thereof. If desired, the vaccine may contain minor quantities of auxiliary substances such as wetting or emulsifying agents, pH-buffering agents or adjuvants such as aluminium hydroxide, the muramyl dipeptide or variations thereof. In the case of peptides, their coupling to a larger molecule (KLH, tetanus toxin) sometimes increases immunogenicity. The vaccines are conventionally administered by injection, for example intramuscularly. Additional favourable formulations with other modes of administration include suppositories and sometimes oral formulations.
The expression “pharmaceutically acceptable vehicle” is understood to mean carriers and vehicles administrable to human beings or to an animal, as described for example in Remington's Pharmaceutical Sciences 16th ed., Mack Publishing Co. The pharmaceutically acceptable vehicle is preferably isotonic, hypotonic or has low hypertonicity and a relatively low ionic strength. The definitions of pharmaceutically acceptable excipients and adjuvants are also given in Remington's Pharmaceutical Sciences cited above.
The subject of the invention is also
The production of polynucleotides, probes or primers forms part of the general knowledge of persons skilled in the art. There may be mentioned in particular the use of restriction enzymes, and chemical synthesis on an automated synthesizer. The probes and primers capable of hybridizing, under determined stringency conditions, with a DNA or RNA nucleotide sequence or with a nucleotide fragment as defined above form part of this definition. It is within the capability of persons skilled in the art to define the appropriate stringency conditions. Characteristic stringency conditions are those which correspond to a combination of the temperature and of the salt concentration chosen approximately between 12 and 20° C. under the Tm (“melting temperature”) of the hybrid to be studied. Reference may thus be made to the book by George H. Keller et Mark M. Manak, DNA PROBES, second edition, Stockton Press, 1993, 49 West 24th St., New York, N.Y. 10010 USA. The stringency conditions for differentiation of even a single point mutation have been known since at least the period 1979; there may be mentioned by way of examples Wallace R. B et al., DNA. Nucleic. Acids. Res. 6, 3543-3557 (1979), Wallace R. B et al., Science, 209, 1396-1400 (1980), Itakura K. and Riggs A. D., Science, 209, 1401-1405 (1980), Suggs S. V. et al., PNAS, 78, 6613-6617 (1981), Wallace R. B et al. DNA. Nucleic. Acids. Res., 9, 3647-3656 (1981), Wallace R. B et al. DNA. Nucleic. Acids. Res., 9, 879-894 (1981) et Conner B. J. et al, PNAS, 80, 278-282 (1983). Moreover, techniques are known for the production of anti-nucleic acid antibodies. There may be mentioned, by way of examples, Philippe Cros et al., Nucleic Acides Researc, 1994, Vol. 22, No. 15, 2951-2957; Anderson, W. F. et al. (1988) Bioessays, 8 (2), 69-74; Lee, J. S. et al. (1984) FEBS Lett., 168, 303-306; Malfoy, B. et al. (1982) Biochemistry, 21(22), 5463-5467; Stollar, B. D. et al., J. J. (eds) Methods in Enzymology, Academic Press, pp 70-85; Traincard, F. et al. (1989) J. Immunol. Meth., 123, 83-91 and Traincard, F. et al. (1989) Mol. Cell. Probes, 3, 27-38).
The invention also relates to:
The pharmaceutical compositions defined above are DNA vaccine compositions which are particularly advantageous, in particular compared with “conventional” vaccine compositions based on a recombinant protein. Indeed, the use for vaccine purposes of recombinant proteins is a cumbersome and expensive system, in particular because it requires very important steps of purification of recombinant antigens. Furthermore, one of the difficulties encountered is of obtaining a sufficiently long persistence of the vaccine to maintain good immune memory. By contrast, the DNA vaccination method, whose advantages are inherent in the intrinsic properties of the DNA, is simple and not very expensive and is simply carried out by intramuscular or intradermal injection. Furthermore, it should be noted that:
The figure labeled “FIGURE, ELISA 1/50” show the results of an ELISA assay to detect anti-HXHV antibodies in 8 control samples and 16 patient samples.
The material used for the RDA consists of sera archived in a serum bank. The RDA was applied to a case of serious hepatitis of unknown aetiology, successfully treated with IFN. The normalization of the transaminases in this patient after antiviral treatment and especially the relapse after stopping the treatment, with another response during reintroduction of the treatment are elements which militate in favour of a viral aetiology of the disease. A serum sample before treatment and a serum sample after treatment were used for the RDA, the sample before treatment potentially containing the HXHV virus and the sample after treatment few or no viruses.
I-1.1 Preliminary Search for Known Hepatitis Viruses
Screening by Ultrasensitive PCRs:
In order to eliminate the presence of known hepatitis viruses, which could be present in very low quantities or in the form of mutants, very sensitive PCR tests for the hepatitis viruses responsible for chronic hepatitis (B, C, G and TTV) were developed (Chemin et al, J of Hepatol., 2001) which were highly sensitive (detection of 10 viral genomes).
The successive sera from the patient treated with interferon and intended for the subtractive hybridization method remained negative by these tests for all known hepatitis viruses and for the SEN virus (Minoru Shibata et al. The Journal of Infectious Diseases 2001; 184:400-404).
I-1.2 Choice of the Samples for the RDA Procedure
Tester samples or positive controls at the transaminase peak: total nucleic acids extracted from the serum collected at the transaminase peak.
Driver samples or negative controls after treatment: total nucleic acids extracted from the serum collected at the lowest transaminase level.
The extractions of the nucleic acids are carried out with the QIAGEN QIAamp® Viral RNA Mini Kit. The differential technique is carried out using the CLONTECH PCR—Select™ cDNA Subtraction Kit from CLONTECH according to the manufacturer's instructions.
I-2.1 Principle
The method developed by Lysitsyn et al. in 1993 is based on the following principle and the Clontech kit (Clontech PCR-select™ cDNA substraction kit) was used:
Reduction of the Complexity of the Genomic DNA.
The 1st step of the RDA consists in reducing the complexity of the genome. For that, it is necessary to use a restriction enzyme which has a fairly frequent cleavage site, making it possible to reduce the complexity of the genome. The result of this step is the production of a set of sequences of lesser complexity although representative of the initial sequences, hence the term “representational”. The preliminary reduction of the complexity of the genome to be studied improves the efficiency of the enrichment steps by facilitating the complete hybridization of the samples.
Enrichment
Adaptors are joined (by means of a ligase) to each end of the DNA fragments of the tester sample containing the viral genome. The driver sample has no adaptors.
During the subtractive hybridization, the tester is exposed to an excess of driver. The driver D contains numerous DNA fragments in common with the tester T and therefore plays the role of competitive inhibitors by limiting self-hybridization of the DNA fragments of the tester common to the two populations.
The whole is subjected to a process of denaturation/renaturation during which three types of association will be produced according to the complementarity of the sequences.
The driver being in a large excess, the fragments common to both pools form mainly D/D hybrids (without adaptors) and also T/D hybrids (with an adaptor at the end of one of the two strands). Only the T/T hybrids possess at each end the adaptors which will be supplemented during the extension step preceding the PCR. By repeating the procedure several times after having selected the T/T duplexes, it is theoretically possible to purify the sequences present only in the tester. However, the efficiency of the subtrative hybridization technique is partly limited because of the great complexity of the human genome and of the abundance of genomic DNA sequences in any serum sample. The repetition of subtractive step results in a final enrichment factor of 102 to 103, whereas a factor of 106 is sought.
It is therefore necessary to combine this method with a second so-called kinetics enrichment method. This is carried out by means of a PCR step which uses primers complementary to the adaptors added during the preceding step. The T/T duplexes are therefore exponentially amplified by PCR and the T/D duplexes undergo a linear amplification in which the derived fragments are single-stranded. The T/D hybrids are, for their part, not amplified. It is possible to destroy the single-stranded DNAs with a nuclease which spares the double-stranded DNAs. This method of selective enrichment of the double-stranded testers can be repeated with different adaptors in order to enrich the target DNA with respect to the other sequences of the tester after amplification. In general, three cycles of this reaction make it possible to obtain the enrichment factor of 106 for the viral sequence to be sought. Subsequently, the products obtained are subcloned into a plasmid for sequencing and analysis.
I-2.2 Protocol
The RDA is carried out on the tester and driver samples using the DNAs from the patient previously mentioned.
Digestion of the Samples with the Restriction Enzyme RsaI
The procedure is carried out separately for the DNA of the tester and the DNA of the driver.
In order not to miss any lane, an RDA experiment was carried out in parallel with the RNAs on the same samples. However, no clones, whose sequence does not exhibit homology with known sequences, was able to be obtained from the viral RNA.
Preparation of the Following Mixture:
The mixture 10× RsaI buffer (5 μl), RsaI (10 U/μl) (1.5 μl), 43.5 μl nucleic acids (2 μg) is prepared. The mixture is incubated for 1 h 30 min at 37° C. and the reaction is stopped with 2.5 μl of 20× EDTA/glycogen. An extraction is carried out with 50 μl of phenol:chloroform:isoamyl alcohol (25:24:1). A centrifugation is carried out for 10 min at 14 000 rpm and the top aqueous phase is collected. Another extraction is carried out with 50 μl of chloroform:isoamyl alcohol (24:1), followed by a centrifugation for 10 min at 14 000 rpm and collection at the top aqueous phase. The aqueous phase is precipitated with 25 μl of 4M NH4OAc and 187.5 μl of 95% ethanol, followed by centrifugation for 20 min at 14 000 rpm and removal of the supernatant. The pellet is washed with 200 μl of 80% ethanol. Another centrifugation is carried out for 5 min at 14 000 rpm and the supernatant is removed. The pellet is air-dried for 5 to 10 min and then dissolved in 5.5 μl of H2O.
Ligation of the Tester to the Adaptors (which Contain the Sequences of the PCR Primers Necessary for the Final Phase of the Procedure):
1 μl of the tester is diluted in 5 μl of water. The ligation mixture is prepared as follows: H2O (15 μl), 5× ligation buffer (BIOLABS) (10 μl), T4 DNA ligase (400 U/μl) BIOLABS (5 μl).
The mixture 1.1 was prepared as follows: tester 2 μl, adaptor 1 (10 μM) 2 μl, Master mix 6 μl.
The mixture 1.2 was prepared as follows: tester 2 μl, adaptor 2R (10 μM) 2 μl, Master mix 6 μl.
Preparation of the nonsubtracted control: 2 μl of the mixture 1.1 were added to 2 μl of the mixture 1.2. The mixture thus prepared is incubated overnight at 16° C. and the reaction is stopped by adding 1 μl of 20× EDTA/glycogen. The ligase is inactivated at 72° C. for 5 min and the mixture is cooled in ice.
Subtractive Hybridization: Tester—Driver.
The procedure is carried out separately for the DNA of the tester and the DNA of the driver.
First Hybridization:
Two mixtures H1 and H2 were prepared separately, consisting of the DNA of the tester ligated to the adaptor 1 (mixture H1) or the adaptor 2R (mixture H2). Mixture H1: driver 1.5 μl, “tester 1-1” 1.5 μl, 4× hybridization buffer 1 μl. Mixture H2: driver 1.5 μl, “tester 1-2” 1.5 μl, 4× hybridization buffer 1 μl. Oil was added to each of these mixtures and each mixture is incubated for 1 min 30 sec at 98° C. then for 6 h at 68° C. before immediately proceeding to the second hybridization.
Second Hybridization:
The following mixture is prepared: driver 1 μl, 4× hybridization buffer, 1 μl, H2O 2 μl.
1 μl of the mixture is denatured at 98° C. for 1 min 30 sec.
15 μl of the mixture H2 are collected at the oil/sample interface. The air is aspirated, the driver is collected and the whole is added to the mixture H1. The whole is incubated at 68° C. overnight.
Dilution of the Subtracted DNAs:
To the hybridized tubes are added 200 μl of dilution buffer. The mixture, after aspirations and dischargings, is incubated at 68° C. for 7 min and the reaction is stopped in ice.
PCR on the RDA Products
The procedure is carried out separately on:
First Round of PCR:
The following mixture is prepared: H2O 19.5 μl, 10× PCR buffer 2.5 μl, dNTPs (10 mM of each) 0.5 μl, PCR primer 1 (10 μM) 1 μl, 50× advantage cDNA polymerase mix 0.5 μl, RDA samples 1 μl.
The programme used is the following: 75° C. 5 min, 94° C. 30 sec
Then 25 cycles: 94° C. 10 sec, 66° C. 30 sec, 72° C. 1 min 30 sec
Second Round of PCR:
Use of 1 μl of first PCR product: the same mixture is used.
20 cycles are performed: 94° C. 10 sec, 68° C 30 sec, 72° C. 1 min 30 sec.
The pairs of primers of the Clontech PCR-select™ cDNA subtraction kit are used, they are located in the adaptors “linked” to the tester DNA at the beginning of the RDA procedure.
Analysis of 8 μl of sample on 1% agarose gel in 1× TBE with an ethidium bromide staining.
The RDA products are then ligated into the plasmid pTOPO using topoisomerase I, and then cloned into E. coli using the Invitrogen TOPO TA Cloning® Kit.
The RDA technique makes it possible to obtain, from tester and driver samples, PCR products which reflect what is potentially different between the two samples. This constitutes the material subtracted.
After cloning of the RDA products, 643 clones were screened according to the steps described below.
In order to remove a maximum of clones of human origin, the replicas of the “DNA” RDA clones are hybridized with probes corresponding to human genomic DNA digested with EcoRI/PstI and radiolabelled with 32P.
The results at the end of this screening are presented below.
The fifty clones selected at the end of the first step are sequenced. Their respective sequences are compared to the known sequences in the data banks Swissprot, Embl, Genbank, Draft of the human genome. A number which are unknown appear. The results of the distribution of the sequences are given below.
Additional screening steps were carried out on the 4 clones selected in the preceding step according to the process described below.
At the end of these various steps, two “DNA” clones, whose sequences are unknown in the data banks, were selected.
The following step is the validation of the relevance of these data both to the samples obtained from patients suffering from hepatitis of undetermined aetiology and the controls consisting, on the one hand, of blood donors and, on the other hand, of patients suffering from pathologies of the liver of nonviral origin. For that, primers were synthesized within the DNA of the two clones and PCRs were performed using these primers on the genomic DNA extracted from the serum of blood donors in order to eliminate the possibility of the DNA of the clones corresponding to a DNA sequence which is frequent in the human genome.
One clone containing an insert of 1400 bp (XH) obtained from the RDA on the DNA was selected for its lack of homologies with the human genomic DNA and with any sequence present in the databases.
This sequence, rich in GC (62%) has several open reading frames. It is identified in SEQ ID NO: 1. The open reading frames are identified in SEQ ID NO: 2, 3, 4 and 5.
Four parallel approaches were used in order to characterize and specify the nature of the XH sequence isolated:
III-1. Bioclinical and Epidemiological Studies
It is possible to use the following combination of primers:
PCR cycles: 75° C. 5 min, 94° C. 2 min, 94° C. 10 s×28, 66° C. 30 s×28, 72° C. 1 min 30×28 and 72° C. 7 min.
Second round of PCR:
3 μl of samples from the 1st round were diluted with 27 μl of water.
The following mixture was prepared: H2O 17.5 μl, 10× PCR buffer 2.5 μl, dNTPs (10 μM of each) 0.5 μl, nested PCR sense primer (10 μM) 1 μl, nested PCR anti-sense primer 2R (10 μM) 1 μl, 50× advantage cDNA polymerase mix 0.5 μl, RDA samples 1/10 2 μl.
The following pairs of primers were used:
PCR cycles: 94° C. 2 min, 94° C. 10 s×10-15, 68° C. 30 s×10-15, 72° C. 1 min 30×10-15 and 72° C. 7 min.
First of all, 90 blood donors having normal transaminases were screened by PCR. All are negative for the XH sequence.
In order to determine the nature of the DNA isolated (single-stranded or double-stranded), the XH sequence was captured with alternately two oligonucleotides specific for either of the two DNA strands of the XH sequence attached to magnetic particles (Gene Trapper cDNA positive selection system, Gibco BRL (trade name)). The oligonucleotides used are the following:
In a second instance, the products released after capture with the specific oligonucleotides are analysed by:
This approach was validated on the XH sequence cloned into the TOPO vector. In this form, the sequence derived from the RDA is double-stranded since it is derived from a PCR. The capture procedure carried out on the cloned sequence finally makes it possible to capture the two DNA strands present.
The nature of the XH sequence which circulates in the patients was examined using several sera of “hepatitis X” patients, who were positive by PCR for the XH sequence. The sera are ultracentrifuged or not before the capture procedure.
All of these results demonstrate that only one of the oligonucleotides (the sense oligonucleotide, S6M13) is capable of retaining the XH sequence which circulates in the serum of the patients. Consequently, this sequence is at least partially single-stranded. This hypothesis on the single-stranded nature of the XH DNA is supported by trials of preliminary treatments of the XH DNA with S1 nuclease, an enzyme which is specific for the single-stranded forms of DNA. Preliminary treatment of extracts of sera positive for XH DNA abolishes the PCR signal and therefore confirms the single-stranded nature of the sequence.
For the immunization of the rabbits, a cysteine (C) was added to the COOH-terminal end of this polypeptide for coupling to BSA. The immunogen RRAAELHRRDQYRLC-BSA was injected into rabbits in an amount of 100 μg of immunogen per rabbit and per injection. The immunization protocol used is the following:
D0: 1st taking of 10 ml of blood, immunization: 0.1 mg of immunogen (1 mg/ml)+0.4 ml of physiological saline+0.5 ml CFA 1 ml ID (0.1 ml×10)
D28: 0.1 mg of immunogen (1 mg/ml)+0.4 ml of physiological saline+0.5 ml IFA ID-1 ml ID (0.1 ml×10)
D56: 0.1 mg of immunogen (1 mg/ml)+0.4 ml of physiological saline+0.5 ml IFA-1 ml SC (0.25 ml×4)
D63: 1st taking of 3 ml of blood from the ear without anticoagulant
D84: 0.1 mg of immunogen (1 mg/ml)+0.4 ml of physiological saline+0.5 ml AFI 1 ml IM (0.25 ml×4)
D91: 2nd taking of 9 ml of blood from the ear without anticoagulant
D112: 0.1 mg of immunogen (1 mg/ml)+0.4 ml of physiological saline+0.5 ml IFA-1 ml ID (0.1 ml×10)
D119: 3rd taking of 9 ml of blood from the ear without anticoagulant.
The production of specific antibodies is a very effective tool for screening for the expression of the XH sequence on biopsies from non-A, non-E patients in parallel with other categories of patients or controls.
Sucrose gradients were prepared in order to isolate possible viral particles associated with the presence of the XH sequence. For that, the serum of a patient of the non-A, non-E hepatitis cohort was used. Two consecutive fractions of this gradient appeared positive by PCR for the XH sequence. These fractions correspond to densities of the order of 1.2 to 1.5 g/cm3. The electron microscopy study made it possible to observe particles whose size and regularity (110 nm diameter) are compatible with that of a viral agent. The frequency with which these particles are observed in electron microscopy suggests a concentration of the order of 5×105 particles/ml of serum. The ELISA test developed allowed us to search for the presence of “anti-XH” antibodies in blood donors and in various categories of patients.
The search for anti-XH antibodies in blood donors and in various categories of patients was carried out by the ELISA test. The results are presented in the table below.
The difference in prevalence of the anti-XH antibodies between the 408 blood donors having normal transaminases and the 389 blood donors having high transaminases is significant. This difference, observed between these two groups tested according to a double blind design, suggests a causal relationship between the presence of the anti-XH antibodies and the hepatic disease. A fraction of the blood donors having high transaminases (20%) is found to be positive by PCR for the XH sequence. This sequence was never found in the blood donors having normal transaminases.
A higher percentage of positivity in anti-XH antibodies is found for the HIV-positive patients (35%), compared to the 16% in the patients who are chronic carriers of HBV and to the 3% in the chronic carriers of HCV. Detailed study of the risk factors to which the patients were exposed demonstrates that the XH sequence is transmissible by the parenteral and sexual route.
Only the patients having a chronic or fulminant non-A, non-E hepatitis positive in ELISA are positive by PCR in 50% of the cases, whereas no positive for DNA was detected in the HCV or HBV group of patients or among the haemophiliacs.
A study was also carried out on patients suffering from acute hepatitis of unknown aetiology progressing to chronicity or cure. Of 13 cases studied, 11 proved positive for anti-XH antibodies during the acute phase of the disease. Among the cases which were able to be monitored, all remained positive for antibodies during this time.
A real-time quantitative PCR method using the Roche® Light cycler was developed in order to amplify the XH sequence. It was used, inter alia, to amplify the XH sequence in the ORF 4 in the 11 patients who were found to be positive for anti-XH antibodies.
The four specific primers described below were synthesized and purified by HPLC.
The PCR is carried out using a Qiagen® kit “QuantiTect SYBR Green PCR Kit” (trade name).
The composition of the reaction mixture is the following
Once the 20 μl have been depositied, the capillary tubes are sealed, briefly centrifuged and placed in the rotor of the Light Cycler.
PCR Protocol
Activation:
Amplification/quantification:
Analysis of the Melting Curve:
Cooling:
Characterization of the Threshold for Detection of the Sequence of Interest
The theoretical detection threshold for the XH sequence inserted into the plasmid is between 1 and 2 copies per capillary tube. The Tm of the amplicon S1/AS2 (312 bp) most frequently used is 88° C.
The application of this technique shows, inter alia, that four out of five of the patients who progressed to a chronic hepatitis are positive by PCR for the XH sequence. Conversely, five out of six of the patients who were cured became negative by PCR for the XH sequence.
| Number | Date | Country | Kind |
|---|---|---|---|
| 01 17048 | Dec 2001 | FR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/FR02/04578 | 12/27/2002 | WO | 00 | 10/12/2004 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO03/055994 | 7/10/2003 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 6551795 | Rubenfield et al. | Apr 2003 | B1 |
| Number | Date | Country | |
|---|---|---|---|
| 20050037336 A1 | Feb 2005 | US |
