FR2656627A1 - PAPILLOMAVIRUS PROBE (HVPV63), ESPECIALLY FOR THE IN VITRO DIAGNOSIS OF PAPILLOMAVIRUS INFECTIONS, WHICH MAY BE ACCOMPANIED BY GENITAL NEOPLASIA, AND GENETICALLY AND IMMUNOLOGICALLY LINKED PRODUCTS TO THIS PAPILLOMAVIRUS. - Google Patents

Abstract

A probe of papillomaviruses derived from DNA-HPV63, which was registered with the CNCM on December 21, 1989 under registration number I-918. This probe can be used for the in vitro detection of genital neoplasias or cervical cancers using hybridization with the DNAs contained in a human biological sample.

Description

PAPILLOMAVIRUS PROBE (HPV63), PARTICULARLY FOR THE
IN VITRO DIAGNOSIS OF PAPILLOMAVIRUS INFECTIONS,
TO BE ACCOMPANIED BY GENITAL NEOPLASIA, AND
PRODUCTS GENETICALLY AND IMMUNOUXICALLY BOUND TO THIS PAPILLOMAVIRUS.

 The invention relates to a papillomavirus DNA (HPV63) or variants of this papillomavirus, and more particularly to probes derived from this papillomavirus.

It also relates to products genetically and immunologically linked to this papillomavirus, as well as to methods using these various products for the in vitro diagnosis of papillomavirus infections and, for some of them, for vaccination against these same papillomaviruses or papillomavirus variants.

 The expression “product genetically or immunologically linked to a papillomavirus” means the various products derived from its initial DNA, whether they are corresponding RNAs or recombinant DNAs containing all or part of this initial DNA, expression products of these DNAs, where appropriate recombinant, in competent cell hosts and antibodies directed against these products. They are therefore polypeptides resulting from the transcription and translation of all or part of the different open reading phases of the initial DNA. They are also antibodies induced in vivo by said polypeptides.

 The expression "papillomavirus" covers a large number of viruses which have in common being held responsible for several forms of viral infections spreading between skin warts or relatively benign mucous membranes and hyperplasias liable to degenerate into intra- neoplasias. epithelial and in skin or mucosal cancers. Among papillomavirus infections, mention will also be made more particularly of skin warts (in particular, vulgar and plantar warts), epidermodysplasia verruciformis, flat or intermediate cutaneous or mucous warts, intraepithelial neoplasias and skin cancers, cancers of the wart shaped epidermodysplasia, intraepithelial neoplasia and cancers of the cervix and external genitalia, genital warts and papillomas.

 A number of types of papillomavirus have already been described. By way of example, mention will be made of those described in the main French patent n "84.18369 / 2.578.267 and its certificates of addition n" 85.07073 / 2.581.655, patent n "86.01425 / 2.593.828 and patent n" 88.08324 filed in June 1988, all relating to new types and subtypes of papillomavirus which have been isolated from warts or disseminated macular lesions, some of which are more likely than others to give rise to the early development of genital neoplasias, in particular cervical cancer in patients who are affected by it.

 In general, it has been noted in previous patents that papillomaviruses, although very different from one another, have sizes of the order of 7000-8000 base pairs. In addition, their genomes may exhibit certain degrees of homology, evaluated in hybridization tests carried out under conditions known as "non-stringent" or "not strict" or, on the contrary, under conditions of "stringent hybridization" or " strict ".

Hybridization tests under non-strict or non-stringent conditions involve the mutual contacting of DNAs originating from two virus isolates under the following conditions described by HEILMAN CA et al, 1980, J. Virol., 36, 395- 407, and CROISSANT et al, 1982,
CR Acad. Sc. Paris, 294, 581-586 (heteroduplex molecules).

It will be recalled that these non-strict or non-stringent conditions imply carrying out hybridization tests under the following conditions, in particular of medium and temperature
Non-strict conditions (Tm-40 C):
Hybridization is carried out at 42 "C in a solution containing: 50 mM sodium phosphate buffer pH = 6.5 5xSSC (lxSSC = 0.15 M NaCl, 0.015 M Na Citrate); 20% formamide; 200 ug / ml yeast transfer RNA; 0.02% Denhart solution.

Hybridization tests under strict or stringent conditions involve the mutual contacting of DNAs originating from two virus isolates under the conditions described by KREMSDORF, D. ET AL. ((1982),
J. Virol. 43: 436-447 and 1983, J. Virol. 48: 340-351) and
Davis RW et al., 1971, Methods Enzymol., 21, 413-418 (heteroduplex molecules).

It will be recalled that these strict or stringent conditions imply carrying out hybridization tests under the following conditions, in particular of medium and temperature,
Strict conditions (Tm-20eC)
Hybridization is carried out at 42 "C in a solution containing: 50 mM sodium phosphate buffer pH = 6.5 5xSSC (lxxSSC = 0.15 M NaCl, 0.015 M Na Citrate); 50% formamide; 200 ug / ml of yeast transfer RNA 0.02% Denhart solution.

 It has been said in previous patents that papillomaviruses which exhibit percentages of cross-hybridization of less than 50% under strict or stringent conditions belong to different types. Viruses for which we observe, under these strict or stringent conditions, percentages (or degrees) of hybridization greater than 50% are considered to belong to the same type. They can form different subtypes within this same type. As a reminder, it will be recalled that two HPVs belong to different types if their genomes exhibit less than 50% of cross hybridization under strict hybridization conditions, that is to say after hybridization in saturated liquid medium and treatment of S1 nuclease hybrids. Two HPVs represent subtypes of the same type if their genomes have incomplete homology, but greater than 50%. Differences in only a small number of cleavage sites by restriction enzymes define variants.

 Earlier patents have also described the use of DNAs derived from these papillomaviruses, in particular genetic recombinants comprising all or part of the genomes (called DNA-HPVs) of these papillomaviruses, as hybridization probes.

The use of mixtures or "cocktails" of DNA-HPVs, for the preparation of mixtures of hybridization probes, has also been described. These mixtures of probes are then capable of being used, sometimes more effectively than isolated probes, for the in vitro diagnosis of various categories of infections, or even of the levels of risk which accompany the discovery in a patient of certain papillomaviruses. These categories of infections include those that are likely to be accompanied by genital neoplasia and, in particular, uterine cancer: see for example the cocktails indicated below (if we disregard DNA-HPV which is the object of the present invention).

 For the record, mention may be made, among the probes capable of being used in the above-mentioned in vitro diagnostic tests, those which can be formed from probes HPV54, HPV55 (a and b), described in patent No. 88.08324 and whose restriction maps have been reproduced in FIGS. 1 and 2 of the attached drawings.

 The invention relates to a newly isolated papillomavirus, the genomic DNA which can be obtained from this new papillomavirus, fragments of this genomic DNA, as well as the new hybridization probes which can be formed from this DNA-HPV. or these DNA-HPV fragments.

 The DNA of this new HPV (hereinafter called HPV63), which has been isolated several times, from lesions having the clinical characteristics of genital intraepithelial neoplasias of the cervix, is characterized by the map of restriction shown in Figure 3 attached.

 The physical map gives the position of sites of cleavage by various restriction endonucleases.

The origin of each card is constituted by a single cut-off site, in this case a BamH1 site. The distances of other sites from the origin are expressed as a percentage of genome length.

 The discovery of this new papillomavirus and the production of DNAs which can be derived therefrom therefore provides the possibility of carrying out more refined diagnoses, in particular of infections causing genital lesions attributable to papillomaviruses or liable to develop under the effect. of these, and better predict the degree of risk that these infections will turn into more dreaded diseases.

 The invention also relates to all the variants of these papillomaviruses, belonging respectively to the same subtypes, therefore capable of hybridizing with DNA-HPV63 under stringent conditions.

 The invention also relates to "cocktails" of probes already described in the previous patents, but nevertheless supplemented with a probe derived from the papillomavirus according to the present application and, consequently, even more sophisticated diagnostic kits or "kits".

 Hybridization probes constructed from the genome of HPV63 are particularly useful, for diagnosis and / or screening in vitro, under the conditions which have been described in previous patents and which will be recalled below, of the types of papillomavirus which constitute a risk, in particular of genital neoplasias and cancers of the cervix.

 These hybridization probes are advantageously incorporated into "cocktails" for diagnosing conditions of the same type. Reference is also made to the prior patents in which the restriction maps of the constituents other than HPV54, 55a and 55b of these diagnostic mixtures appear. As such, the prior patents should be considered as part of the present description, with regard to the identification of the other DNA-HPVs used in the composition of these "cocktails".

 The invention also relates to fragments of each of the DNA-HPV63 or to DNA fragments, in particular of equivalent lengths, capable of hybridizing with the preceding one, in particular under strict conditions. Likewise, it relates to the recombinant DNAs containing all or part of the DNA-HPV mentioned above, and more particularly to the recombinant DNAs containing, respectively, fragments corresponding to the genes E1, E2, E4, E6-E7, L1, L2 and the region intergenic, NC, or fragments containing sequences corresponding to the intergenic regions of each of said DNA-HPV. It also relates to the probes which can be formed from these DNA-HPV or from any fragment contained in this DNA-HPV , since it is capable of hybridizing with the previous one under strict conditions and under non-strict conditions with DNA-HPV corresponding to the previously described types HPV16, HPV18 and HPV33 and the in vitro diagnostic methods involving said probes or the mixtures containing them.

Molecular cloning and characterization of a new type of HPV associated with genital neoplasias (HPV63)
The new type of HPV has been demonstrated in DNA extracted from mild dysplasia (grade I intraepithelial neoplasia), by hybridization under non-strict conditions (Tm-40%), using a mixture of specific probes for HPV16, 18 and 33. The study of the sensitivity of this DNA to various restriction endonucleases has shown that the enzyme BamHI once cuts the viral sequences, generating molecules of about 8
Kb (size of a linearized genome of HPV). After digestion of the DNA preparation with BamHI, the 8 Kb DNA molecules were inserted into the plasmid pUC18, at the unique BamHI site, and cloned into Escherichia coli K12 (strain C600). The recombinant plasmids were detected by hybridization of replicates of the infected bacterial cultures, with a mixture of the probes corresponding to types 16, 18 and 33, under non-strict conditions. Several recombinant plasmids, containing all of the viral sequences, have been isolated. The cleavage of the recombinant plamides by the insertion enzyme BamHI generates an 8 Kb fragment which hybridizes with probes 16, 18 and 33 under non-strict conditions. Cleavage of recombinant plasmids and DNA from the original lesion by mixing enzymes
PstI plus BamHI generates the same three fragments whose sum of molecular weights is equal to the size of a papillomavirus genome. A restriction map of viral DNA was established from the study of the sensitivity of this DNA to 19 enzymes, which made it possible to locate 8 cleavage sites (Figure 1). The map thus established is different from that of the HPV genomes described to date. The degree of sequence homology between the DNA of the new HPV and the DNA of the other HPVs was analyzed by transfer-hybridization experiments carried out under very strict conditions (Tm-100C). No cross-hybridization was detected under these conditions with the DNA of the HPVs already described. The new HPV thus characterized therefore constitutes a new type of HPV, called
HPV63.

 The analysis, using an electron microscope, of heteroduplex molecules formed, under different conditions, between the DNA of HPV63 and the DNA of HPV16 made it possible to align the physical maps of the two genomes and to define the theoretical position of the different genes carried by HPV63 DNA.

Putative localization of the main genes and of the HPV63 regulatory region on the map of this genome:
End coordinates
5 '3'
E6-E7 .......... 71 81
El 19 5.8
E2 ........ r 4.7 19
L2 ................. 22.5 41.2 L1 .. 40 60.7
Regulatory region ......... 60.7 71
In general, the invention therefore also relates to any recombinant DNA containing the above-mentioned DNA-HPV or fragments of this DNA-HPV, in particular hybridization probes formed from these recombinant DNAs and specially adapted for the detection of infection by the papillomavirus according to the invention or of a variant or subtype of this papillomavirus. These probes can either be labeled themselves or be modified at the level of certain nucleotides, in particular with a view to their coupling, direct or indirect, with a separate marker.

 It goes without saying that in these probes the parts foreign to the nucleotide sequence corresponding to the papillomavirus DNA and normally originating from a cloning vector, are such that they are not likely to hybridize under stringent conditions with the others. nucleic acids possibly contained in the test sample for its possible DNA content of the corresponding papillomavirus or one of its variants.

 The method according to the invention for the in vitro diagnosis on a biological sample to be tested, normally coming from a human patient, of an infection by a papillomavirus which can cause or has led to genital neoplasia and / or cervical cancer. uterus, is therefore characterized by bringing a probe as defined above into contact with the nucleic acids of this sample, if necessary previously made accessible to the probe, preferably under stringent hybridization conditions, and by the detection of the hybrid formed between the viral DNA sought, possibly present in the sample and said probe.

 The use of radioactive probes prepared from the DNA of the purified HPV63 made it possible to determine the pathogenic power of this virus. The search for HPV63 DNA in 94 DNA preparations extracted from genital lesions containing HPV genomes different from those identified to date, revealed the presence of HPV63 DNA in six cervical intraepithelial neoplasias uterus (6.4%). HPV63 is therefore a type of genital tropism HPV with oncogenic potential. It is very desirable to incorporate it into any mixture of HPV DNA intended for the preparation of molecular probes, for the diagnosis or screening of the types of HPV constituting a risk for the development of genital neoplasias and, in particular , cervical cancer.

In a variant of the invention, the probe is therefore associated with probes derived from other papillomaviruses, in particular from those designated below - HPV16, 18, 33, 39, 54 or alternatively still, with the
HPV6, 11, 42, 54, for the in vitro diagnosis of neo
genital plasias and cervical cancers,
genital warts and papillomas, - HPV6, 11, 42, 55, especially when the
grouped diagnoses also relate to genital warts
and papillomas.

 Each of the probes according to the invention or the mixtures containing the above-mentioned probe can in particular be used as follows, it being understood of course that the diagnostic tests described cannot be considered as limiting the conditions of use of the probes or mixtures of probes according to the invention.

 In the example considered, this involves, for example, identifying an HPV in a biopsy, in cells obtained by scraping lesions, or in biopsy sections fixed by the Carnoy mixture (ethanol, chloroform, acetic acid 6 : 3: 1) and included in the paraffin. The examination requires the prior extraction of DNA from the samples according to methods the principle of which is known and involves the analysis of this DNA by molecular hybridization experiments, carried out under strict or less strict conditions, to using radioactive probes (labeled with 32p or 35S) prepared from the HPV according to the invention or from mixtures of DNAs or HPVs containing it.

 Several hybridization methods can be used. One can, for example, implement the hybridization method on spot. This method comprises, after denaturation of the DNA, the deposition of an aliquot of DNA on membranes (nitrocellulose or "Genescreenplus"), the hybridization of each membrane, under the usual conditions, with a mixture of probes and the detection of radioactive hybrids, by exposure of the membranes to contact with an x-ray film, or a replica hybridization method can also be used.

This method comprises the electrophoretic separation in agarose gel of the DNA fragments generated after treatment of the DNA with restriction enzymes, the transfer of the fragments, after alkaline denaturation, on membranes (nitrocellulose, "Genescreenplus") and their hybridization, under usual conditions, with the appropriate mixture of probes. The formation of radioactive hybrids is detected after exposure of the membranes in contact with an X-ray film.

 The radioactive probes are constituted either by HPV DNAs labeled by the nick-translation method, or by RNAs prepared by transcription of viral DNAs inserted into a vector, for example of the SP6 type. The use of radioactive probes has the advantage of great sensitivity, but this does not exclude the use of non-radioactive probes, for example biotinylated probes which are capable of being recognized by antibodies or labeled themselves, either themselves recognized by antibodies carrying an enzymatic, fluorescent marker, etc.

 The invention also relates to competent cell cultures transformed with recombinant DNAs of the above-mentioned type, in particular those in which the nucleotide sequence corresponding to the papillomavirus DNA is placed under the control of transcription and regulatory elements for this. nucleotide sequence in said cell culture.

 Consequently, it also relates to the expression products of these recombinant DNAs in the corresponding competent cell hosts and the corresponding antibodies capable of being produced against these expression products.

As such, the invention relates to the polypeptides resulting in particular from the respective expressions of the genes
E1, E2, E4, E6, E7, L1, L2 of DNA-HPV63.

 The process according to the invention for the production of these polypeptides consequently comprises the transformation of competent cell cultures with the recombinant DNAs containing the corresponding nucleotide sequences originating from HPV63, so that the nucleotide sequence corresponding to one of said proteins can be expressed in this cell host, the recovery of these polypeptides from the products synthesized by the competent cell host and the purification (for example by contacting the expression products previously extracted from the cell cultures or from the medium in which these have been developed, with antibodies previously formed against such polypeptides).

 The expression products of the L2 sequences of each of the papillomavirus genomes according to the invention are however of very particular interest, in that they can themselves be used for the in vivo production of antibodies capable of recognizing the products of expression of the L2 gene in biological samples affected by a papillomavirus of the HPV63 type or a variant thereof, and this more particularly when the preparations of the genus in question have been previously fixed.

 The invention also relates to hybrid polypeptides containing the above-mentioned polypeptides and respectively derived from HPV63, for example the L2 protein fused to other polypeptide sequences, insofar as these do not essentially modify the immunogenic properties of the protein. L2. The presence of these other polypeptide fragments may in particular result from the mode of production used for these hybrid polypeptides, for example by genetic engineering.

For example, these hybrid polypeptides contain a sequence derived from B-galactosidase. Such products can in particular be obtained by transformation of E.

coli with appropriate vectors (phages or plasmids) modified by all or part of the lactose operon and comprising, in addition, inserted downstream of the lacton operon promoter (or any other suitable promoter, for example phage A) , the nucleotide sequence derived from an L2 gene derived from the papillomavirus concerned according to the invention. Advantageously use is made of plasmids or phages of this type comprising at least part of the ss-galactosidase gene of the lacton operon.

 The polypeptides according to the invention can also, when they have been purified, be used in techniques for purifying the antibodies which correspond to them, in particular from sera from animals which had been immunized with these polypeptides.

In particular, these polypeptides can be attached to affinity columns. The purification operations of the antibodies then consist in passing the serum containing them in contact with affinity columns carrying the above-mentioned polypeptides. The antibodies selectively attached to these columns can then be recovered by dissociation of the antigen-antibody complexes, using an appropriate buffer, having an adequate ionic strength, for example an aqueous solution of a salt such as ammonium acetate. . Acidified solutions can also be used.

The invention also relates to a method for producing antibodies against said polypeptides, in particular against gene expression products.
E6, E7 or, preferably L2 of HPV63, this method comprising the immunization of a suitable living host with said polypeptides and the recovery of the antibodies formed from a serum of the immunized host, in particular by contacting these sera with the polypeptides corresponding to the purified state and by the recovery of these antibodies from the antigen-antibody complexes formed.

 Finally, the invention relates to the compositions involving the antibodies according to the invention (or groups containing these antibodies in association with antibodies from other papillomaviruses), in particular the compositions containing the antibodies derived from the compositions or "cocktails" of DNA-HPVs listed above and to which it is again referred to in the following (or groups of corresponding antibodies).

 In particular, the invention relates to the above-mentioned antibodies or mixtures of antibodies, previously purified, in association with an appropriate pharmaceutical vehicle.

This composition is then capable of being used for the treatment of the given condition, as soon as it has been diagnosed clinically, after an in vitro diagnostic test on a histological or cytological sample originating from of the patient. This composition (in particular in the form of a serum) can be administered, preferably parenterally. This serum is then capable of causing a regression of the infections induced by papillomaviruses of the HPV63 type.

 These antibodies can more particularly be used in tests for the diagnosis of an infection with respect to one of the papillomaviruses according to the invention or of variants of these papillomaviruses, insofar as histological sections coming from infected persons may also contain expression products of some of the structural genes, in particular L2, of papillomaviruses of the same type.

 The invention therefore also relates to a method for in vitro diagnosis, in particular of general neoplasias and of cervical cancers, comprising the contacting of histopathological sections originating from lesions induced in the persons concerned under conditions allowing production of an antigen-antibody complex and the detection of this antigen-antibody complex. Advantageously, the detection is done on preparations fixed beforehand under dissociating conditions, for example with the medium or mixture of CARNOY already mentioned above (also described in the work of L. LISON, entitled "Histochemistry and cytochemistry of animals") .

 The anti-L2 antibodies which may be attached can be recognized by other antibodies formed against the former, these other antibodies carrying appropriate markers, preferably non-radioactive. These markers are for example of an enzymatic or fluorescent nature.

 The antibodies thus selected, as well as the above-defined hybridization probes which correspond to them, can therefore be used to diagnose in vitro the types of corresponding conditions.

 Finally, the invention relates to the corresponding vaccinating compositions containing one or preferably several other L2 proteins, in combination with a pharmaceutically acceptable vehicle suitable for the chosen mode of administration, in particular parenterally. These compositions can be used to protect people subjected to high risks of infection by HPV63 or by papillolaviruses of the corresponding type.

The strain containing DNA-HPV63 was deposited at the
CNCN December 21, 1989 under the deposit number I-918.

The strain deposited consists of a culture of E. coli K12 strain NM522 harboring a recombinant plasmid BamHI itself containing the DNA of HPV63.

 I1 is reminded that the HPV54, 55A and 55B were deposited at the CNCM (in the forms also indicated below) on May 6, 1988, under the following numbers - PSP 64 HPV54: I-756 - PGM 4 HPV55A: I-757 - PGM 4 HPV55B: I-758.

Claims (14)

 1 - papillomavirus DNA characterized in that it essentially consists of the DNA characterized by the restriction map of FIG. G or a fragment thereof, or alternatively in a corresponding DNA or DNA fragment hybridizing with the precedents under strict conditions.  2 - papillomavirus DNA according to claim 1, characterized in that it corresponds to that contained in the deposit made with the CNCM on December 21, 1989 under the number I-918 or to a fragment thereof, or even in a corresponding DNA or DNA fragment hybridizing with the preceding ones under strict conditions.  3 - DNA according to claim 1 or claim 2, characterized in that it consists of a fragment hybridizing under strict conditions with HPV63 and in non-strict conditions with HPV16, HPV18 or HPV33.  4 - DNA according to claim 3, characterized in that said fragment corresponds to one of the following genes of HPV63: E1, E2, E4, E6-E7, L1, L2 or to all or part of the NC intergenic region.  5 - Hybridization probe constituted by a DNA derived from the DNA according to any one of claims 1 to 4.  6 - Polypeptide corresponding to the expression product of one of the structural genes of one of the Papillomavirus DNAs according to any one of claims 1 to 4.  7 - Polypeptide according to claim 6, characterized in that it consists more particularly in an expression product of the L2 sequence of the genome of the HPV63 papillomavirus.  8 - Antibodies directed against the polypeptide according to claim 6 or claim 7.  9 - Method for in vitro detection on a biological sample to be tested, of an infection with a HPV63 type papillomavirus, characterized by bringing a corresponding probe, as defined in claim 5, into contact with the nucleic acids of this sample, if necessary previously made accessible to the probe, preferably under stringent hybridization conditions, and by the detection of the hybrid formed between the viral DNA sought, possibly present in the sample, and said probe.  10 - Process according to claim 9, characterized in that the in vitro diagnosis is oriented towards the detection of genital neoplasias, and cervical cancers.  11 - Application of the antibodies according to claim 8 to the in vitro diagnosis of an infection by a papillomavirus of a type capable of inducing genital neoplasias and cervical cancers, characterized by bringing a antibody, as defined in claim 8, with a biological sample from the person subjected to the in vitro diagnosis, and by the detection of the antigen-antibody complex formed.  12 - Mixture of papillomavirus DNAs comprising a DNA according to any one of claims 1 to 4 or a probe according to claim 5, associated with several corresponding DNAs of distinct papillomaviruses, in particular of the following - HPVs 16, 18, 33, 39, 54 - HPVs 6, 11, 42, 54 - HPVs 6, 11, 42, 55, - HPVs 6, 11, 42, 54 and 55.  13 - Mixture of polypeptides corresponding to the expression products of structural genes of the HPV DNAs of claim 12.  14 - Mixture of antibodies directed against the polypeptides according to claim 13.