Patent Grant
8507196
The invention relates to cloned DNA sequences indistinguishable from genomic RNA and DNA of lymphadenopathy-associated virus (LAV), a process for their preparation and their uses. It relates more particularly to stable probes including a DNA sequence which can be used for the detection of the LAV virus or related viruses or DNA proviruses in any medium, particularly biological samples containing, any of them. The invention also relates to polypeptides, whether glycosylated or not, encoded by said DNA sequences.
Lymphadenopathy-associated virus (LAV) is a human retrovirus first isolated from the lymph node of a homosexual patient with lymphadenopathy syndrome, frequently a prodrome or a benign form of acquired immune deficiency syndrome (AIDS). Subsequently, other LAV isolates have been recovered from patients with AIDS or pre-AIDS. All available data are consistent with the virus being the causative agent of AIDS.
A method for cloning such DNA sequences has already been disclosed in British Patent Application Nr. 84 23659 filed on Sep. 19, 1984. Reference is hereafter made to that application as concerns subject matter in common with the further improvements to the invention disclosed herein.
The text of British Patent Application Number 84 23659, filed on Sep. 19, 1984, is as follows:
The invention relates to cloned DNA sequences hybridizable to genomic RNA and DNA of lymphadenopathy-associated virus (LAV), a process for their preparation and their uses. It relates more particularly to stable probes including a DNA sequence which can be used for the detection of the LAV virus or related viruses or DNA pro-viruses in any medium, particularly biological, samples containing of any them.
Lymphadenopathy-associated virus (LAV) is a human retrovirus first isolated from the lymph node of a homosexual patient with lymphadenopathy syndrome, frequently a prodrome or a benign form of acquired immune deficiency syndrome (AIDS) (cf. 1). Subsequently other LAV isolates have been recovered from patients with AIDS or pre-AIDS (Cf. 2-5). All available data are consistent with the virus being the causative agent of AIDS (cf. 11).
The virus is propagated on activated T lymphocytes and has a tropism for the T-cell subset OKT4 (cf. 2-6), in which it induces a cytopathic effect. However, it has been adapted for growth in some Epstein-Barr virus transformed B-cell lines (cf. 7), as well as in the established T-lymphoblastic cell line, CEM.
LAV-like viruses have more recently been independently isolated from patients with AIDS and pre-AIDS. These viruses called HTLV-III (Human T-cell Leukemia/Lymphoma virus type III (cf. 12-15) and ARV (AIDS-associated retrovirus) seem to have many characteristics similar to those of LAV and it is thus probable that they represent independent isolates of the LAV prototype.
Detection methods so far available are based on the recognition of core proteins. Such a method is disclosed in European application titled “Antigènes, moyens et méthode pour le diagnostic de lymphadénopathie et du syndrome d'immunodépression acquise” filed on Sep. 14, 1984, under the priority of British application Serial Nr. 83 24800 filed on Sep. 15, 1983. As a matter of fact, a high prevalence of anti-p25 antibodies has been found in the sera of AIDS and pre-AIDS patients and to a lower but significant extent in the high-risk groups for AIDS (cf. 8-10). However the same sera were found not to recognize the virus as a whole, in a non-disintegrated state.
The present invention aims at providing new means which should not only also be useful for the detection of LAV or related viruses (hereafter more generally referred to as “LAV viruses”), but also have more versatility, particularly in detecting specific parts of the genomic DNA of said viruses whose expression products are not always detectable by immunological methods.
The DNAs according to the invention consist of DNAs which contain DNA fragments, hybridizable with the genomic RNA of LAV. Particularly said DNAs consist of said cDNAs or cDNA fragments or of recombinant DNAs containing said cDNAs or cDNA fragments.
Preferred cloned cDNA fragments respectively contain the following restriction sites in the respective orders which follow (from the 3′ end to the 5′ end):
1) HindIII, SacI, BglII (LAV75)
2) HindIII, SacI, BglII, BglII, KpnI (LAV82)
3) HindIII, SacI, BglII, BglII, KpnI, XhoI, BamHI, HindIII, BglII (LAV13).
The LAV75, LAV82, and LAV13 designations correspond to the designations of the recombinant plasmids designated as pLAV 75, pLAV 82 and pLAV 13, respectively, in which they were first cloned. In other words, LAV 75, LAV 82, and LAV 13 respectively present as inserts in said recombinant plasmids. For convenience, the designations LAV 75, LAV 82 and LAV 13 will be further used throughout this specification to designate the cDNA fragments, whether the latter are in isolated form or in a plasmid forms, whereby the other DNA parts of said last mentioned recombinants are identical to or different from the corresponding parts of pLAV 75, pLAV 82, and pLAV 13, respectively.
Preferred cDNAs also (like LAV 75, LAV 82, and LAV 13) contain a region corresponding to the R and U 3 regions of the LTR (Long Terminal Repeat) as well as the 3′ end of the coding region of the retroviral DNA. Particularly, if it is assumed that the retroviral structure of LAV is in general agreement with the retroviral genomic structures to date.
LAV 13, which has a size of about 2.5 Kbp has been found of particular advantage. It is highly specific of LAV or LAV related viruses and does also recognizes more of the LAV retroviral genomes than do LAV75 or LAV82. Particularly, LAV 13 enabled the identification of the RU 5 junction of the retroviral genomes within the LTR and, subsequently, the sizes of the LAV genomes, which average from about 9.1 to about 9.2 kb.
LAV 13 is free of restriction sites for the following enzymes Eco RI, Nru I, Pvu I, Sal I, Sma I, Sph I, Stu I, and Xba I.
LAV 13 further appears to contain at least part of the DNA sequences corresponding to those which, in retroviral genomes, code for the envelope protein.
The invention further relates to any of the fragments contained in the cDNA, which seems to correspond to part of the whole of the LAV retroviral genome, which is characterized by a series of restriction sites in the order hereafter (from the 5′ end to the 3′ end).
The coordinates of the successive sites of the whole LAV genome (restriction map) are indicated hereafter too, with respect to the Hind III site (selected as of coordinate 1), which is located in the R region. The coordinates are estimated to within ±200 bp. Some coordinates are better established than others.
The abovesaid DNA according to the invention optionally contains an additional Hind III approximately at the 5 550 coordinate.
The invention further relates to other preferred DNA fragments corresponding substantially to those which in relation to the abovesaid restriction map extend respectively:
More particularly, the invention relates to any fragment corresponding to the above ones, having substantially the same sites at substantially same distances from one another, all of these fragments having in common the capability of hybridizing with the LAV retroviral genomes. It is of course understood that fragments which would include some deletions or mutation which would not substantially alter their capability of also hybridizing with the LAV retroviral genomes are to be considered as forming obvious equivalents of the DNA fragments more specifically referred to hereabove.
Additional features of the invention will appear in the course of the disclosure of additional features of preferred DNAs of the invention, the preparation conditions and the properties of which will be illustrated hereafter in a non limitative manner. Reference will also be had to the drawings in which:
1.1 Virus Purification
Virions were purified from FR8, an immortalized, permanent LAV producing B-Lymphocyte line (cf. 7) (deposited at the “Collection Nationale de Cultures de Micro-organismes” of the INSTITUT PASTEUR of Paris, under Nr. 1-303 on May 9, 1984). The purification protocol was described (cf. 1). The main steps were:
1.2 First-Strand cDNA Synthesis
The virus associated detergent activated endogenous reaction is a technique bringing into play the reverse transcriptase of the virus, after purification thereof and lysis of its envelope.
For each reaction, purified virus corresponding to 250-300 ml of FR8 supernatant was used. Final reaction volume was 1 ml. Incubation was at 37° C. for 45 nm. Protein concentration was about 250 microg/ml. Buffer was: NaCl 25 mM; Tris HCl pH 7.8 50 mM, dithiothreitol 10 mM, MgCl2 6 mM, each of dATP, dGTP, dTTP at 0.1 mM, Triton X-100 0.02%; oligo dT primer 50 microg/ml. The cDNA was labelled 15 nm with alpha 32P-dCTP 400 Ci/rnmole to 0.6 microM plus cold dCTP to 4 microM. Afterwards, cold dCTP was added to 25 microM to ensure optimal elongation of the first strand.
The reaction was stopped 30 nm after the dCTP chase by adding EDTA to 20 mM, SDS to 0.5%, digesting one hour with proteinase K at 100 microg/ml and phenol-chloroform extraction.
cDNA was then purified on G-50 Sephadex (Pharmacia) and ethanol precipitated.
1.3 2nd Strand Synthesis and Cloning
Purified cDNA-RNA hybrids were treated with DNA polymerase I and RNase H, according to GUBLER and HOFFMAN (cf. 17). Double-stranded cDNA was dC-tailed with terminal transferase and annealed to dG-tailed Pst-digested pBR 327 (cf. 34) a derivative of pBR 322.
A cDNA library was obtained by transfection of E. coli C 600 recBc strain.
2. Detection of LAV-Specific Clones
2.1 Screening of the Library
recombinant clones were grown on nitrocellulose filters and in situ colony hybridization (cf. 35) was performed with another batch of cDNA made in endogenous virus-associated reaction as described (cf. 1.2) and labelled with 32P. About 10% of the clones could be detected.
A major family was obtained by small-scale amplification of these clones and cross-hybridization of their inserts. Among these clones a major family of hybridizing recombinants was identified. Three of these cDNA clones, named pLAV 13, 75 and 82, carrying inserts of 2.5, 0.6, and 0.8 kb respectively were further characterized (
All three inserts have a common restriction pattern at one end, indicating a common priming site. The 50 bp long common Hind III-Pst I fragment was sequenced (
The LAV 13 specificity was shown by different assays.
The specificity of pLAV 13 was determined in a series of filter hybridization experiments using nick-translated pLAV 13 as a probe. Firstly, the probe hybridized to purified LAV genomic RNA by dot and Northern blotting (data not shown). pLAV 13 also hybridizes to the genomic RNA of virus concentrated from culture supernatant directly immobilized on filters (dot blot technique). LAV RNA from different sources: normal T-cells, FR8 and other B-cell LAV producing lines, CEM cells and, although less strongly, LAV from the bone marrow culture from a haemophiliac with AIDS (cf. 3) were detected in a similar manner. Uninfected cultures proved negative. This rapid dot blot technique can be adapted with minor modifications to the detection of LAV in serum or other body fluids.
Secondly, the probe detected DNA in the Southern blots of LAV-infected T-lymphocytes and in the LAV-producing CEM cell line. No hybridization was detected in the DNA of uninfected lymphocytes nor in the DNA from normal liver (data not shown) under the same hybridization conditions.
A third characteristic resulted from the possibility of using LAV 13 to identify the whole retroviral genome of the LAV viruses as disclosed hereafter. Particularly characteristic 1.45 kb Hind III fragment which co-migrates with an internal viral fragment in Hind III cleaved pLAV 13 was detected. Bands at 2.3 and 6.7 kb were also detected. As the probe was only 2.5 kb long and as no junction fragments could be detected, it is probable that these extra-bands represent internal fragments arising from a Hind III polymorphism of the LAV genome.
Together, these data show that pLAV 13 DNA is exogenous to the human genome and detects both RNA and integrated DNA forms derived from LAV infected cells. Thus, pLAV 13 is LAV specific. Being oligo-dt primed, pLAV 13 must contain the R and U3 regions of the LTR as well as the 3′ end of the coding region, assuming a conventional retroviral genome structure.
Having found a HindIII site within the R region of the LTR, it was decided to clone the LAV genome by making a partial Hind III digest of proviral DNA from LAV infected cells. It was found that: (a) partial digestion increased the chance of isolating complete clones and (b) Hind III fragments were easily cloned in lambda replacement vectors. The DNA isolated from T-cells of a healthy donor after in vitro infection with LAV was partially digested with Hind III and fractionated. A 9+ 1.5 kb DNA containing fraction was precipitated and ligated into the Hind III arms of lambda-L47.1 (cf. 18).
The cloning of LAV genomic DNA was carried out more particularly as follows:
cDNAs was prepared from LAV infected T cells as described above, then partially digested with Hind III and fractionated on a 5-40% sucrose gradient in 10 mM Tris.Cl pH 8. 10 mM EDTA, 1 M NaCl (SW41 rotor, 16 hours at 40 000 rpm). A single fraction (9±0.5 kb) was precipitated with 20 microg/ml Dextran T40 as carrier and taken up in TE-buffer (10 mM Tris.Cl pH 8, 1 mM EDTA). Lambda-L47.1 Hind III arms were prepared by first ligating the cos sites followed by Hind III digestion and fractionation through a 5-40% sucrose gradient. Fractions containing only the lambda-Hind III arms were pooled, precipitated and taken up in TE-buffer. Ligation of arms to DNA was made at approximately 200 microg DNA/ml using a 3:1 molar excess of arms and 300 units of T4 DNA ligase (Biolabs). In vitro packaging lysates were made according to (38). After in vitro packaging the phage lysate was plated out on NM538 on a C600 recBC strain. Approximately two million plaques were screened by in situ hybridization (cf. 39) using nitrocellulose filters. Hybridization was performed at 68° C. in 1×Denhardt solution, 0.5% SDS, 2×SSC, 2 mM EDTA. Probe: 32P nick-translated LAV insert of pLAV 13 at >108 cpm/microg: Filters were washed 2×30 minutes in 0-1 SSC, 0.1% SDS at 68° C., and exposed to Kodak XAR-5 film for 29-40 hours. Seven positive clones were identified and plaque purified on a C 600 rec BC strain. Liquid cultures were grown and the recombinant phages banded in CsCl. Plage DNA was extracted and digested under the appropriate conditions.
Seven independent clones were so derived from approximatively two million phage plaques after screening in situ with a nick-translated pLAV 13 insert as a probe. Restriction maps of lambda-J19 as well as of a Hind III polymorph lambda-J81 are shown in
The restriction maps of
The restriction map of the LAV 13 cDNA clone is also shown in
There may be other Bam HI sites in the 5′ 0.52 kb Hind III fragment of lambda-J19. They generate fragments that are too small to be detected.
More particularly lambda-319 shows four Hind III bands of 6.7, 1.45, 0.6, and 0.52 kb the first two of which correspond to bands in the genomic blot of Hind III restricted DNA. The smallest bands of 0.6 and 0.52 kb were not seen in the genomic blot but the fact that they appear in all the independently derived clones analyzed indicates that they represent internal and not junction fragments, assuming a random integration of LAV proviral DNA. Indeed, the 0.5 kb band hybridizes with pLAV 13 DNA (
It appears that lambda-J81 is a restriction site polymorph of lambda-J19. Lambda-J81 shows five Hind III bands of 4.3, 2.3, 1.45, 0.6, and 0.52 kb. The 2.3 kb band is readily detected in the genomic blot by a pLAV 13 probe, but not the 4.3 kb fragment. That lambda-J81 is a Hind III polymorph and not a recombinant virus is shown by the fact that nick-translated lambda-J19 DNA hybridizes to all five Hind III bands of lambda-J81 under stringent hybridization and washing conditions. Also other restrictions sites in lambda-J81 are identical to those of lambda-J19.
HTLV-I and HTLV-II constitute a pair of C-type transforming retroviruses with a tropism for the T-cell subset, OKT4 (cf. 20). An isolate of HTLV-I has been totally sequenced (cf. 21) and partial sequencing of an HTLV-II has been reported (cf. 22-24). Both genomes (one LTR) were approximately 8.3 kb in length, have a pX region and show extensive sequence homology. They hybridize between themselves under reasonably stringent conditions (40% formamide, 5×SSC) and even at 60% formamide the pX regions hybridize (cf. 26). Thus a conserved pX region is a hallmark of this class of virus.
We have compared cloned LAV DNA and cloned HTLV-II DNA (pMO (cf. 27)) by blot-hybridization and found no cross-hybridization under low stringency conditions of hybridization and washing. For example, Hind III digested lambda-J19, lambda-J27 and lambda-J81 were electrophoresed, blotted and hybridized overnight with 32P nick-translated pMO (HTLV-II) DNA (having a specific activity greater than 0.5×108 cpm/microg) in 20% formamide, 5×SSC, 1×Denhardt's solution, 10% Dextran sulphate, at 37° C. Filters were washed at 37° C. (tm-50) tm-50 using a 53.1% GC content derived from the HTLV-I sequence (21). The washings were repeated at 50° C. and 65° C. in 1×SSX, 0.1% SDS. Even when hybridized in 20% formamide, 8×SSC (tm-50) and washed at 37° C. in 2×SSC (tm-50) no hybridization was detected after two days exposure at −70° C. using an intensifying screen.
Thus, there is no molecular evidence of a relationship between LAV and the HTLV viruses. In addition, the LAV genome is approximately 9 kb long in contrast to 8.3 kb for the HTLV viruses. Despite their comparable genome sizes LAV and Visna (cf. 29) cloned viral genomes do not cross-hybridize, nor does LAV with a number of human endogenous viral genomes (cf. 30) under non stringent conditions (hybridization—20% formamide, 8 SSC, 37° C.; washing—2 SSC, 0.1% SDS, 37° C.
The invention also relates more specifically to cloned probes which can be made starting from any DNA fragment according to the invention, thus to recombinant DNAs containing such fragments, particularly any plasmids amplifiable in procaryotic or eucaryotic cells and carrying said fragments. As mentioned earlier a preferred DNA fragment is LAV 13.
Using the cloned provirus DNA as a molecular hybridization probe—either by marking with radionucleotides or with fluorescent reagents—LAV virion RNA may be detected directly in the blood, body fluids and blood products (e.g. of the antihemophylic factors such as Factor VIII concentrates) and vaccines, i.e. hepatitis B vaccine. It has already been shown that whole virus can be detected in culture supernatants of LAV producing cells. A suitable method for achieving that detection comprises immobilizing virus onto said a support e.g. nitrocellulose filters, etc., disrupting the virion and hybridizing with labelled (radiolabelled or “cold” fluorescent- or enzyme-labelled) probes. Such an approach has already been developed for Hepatitis B virus in peripheral blood (according to SCOTTO J. et al. Hepatology (1983), 3, 379-384).
Probes according to the invention can also be used for rapid screening of genomic DNA derived from the tissue of patients with LAV related symptoms, to see if the proviral DNA or RNA is present in host tissue and other tissues.
A method which can be used for such screening comprise the following steps: extraction of DNA from tissue, restriction enzyme cleavage of said DNA, electrophoresis of the fragments and Southern blotting of genomic DNA from tissues, subsequent hybridization with labelled cloned LAV provival DNA. Hybridization in situ can also be used.
Lymphatic fluids and tissues and other non-lymphatic tissues of humans, primates and other mammalian species can also be screened to see if other evolutionary related retrovirus exist. The methods referred to hereabove can be used, although hybridization and washings would be done under non stringent conditions.
The DNA according to the invention can be used also for achieving the expression of LAV viral antigens for diagnostic purposes as well as far the production of a vaccine against LAV. Of particular advantage in that respect are the DNA fragments coding core (gag region) and for envelope proteins, particularly the DNA fragment extending from Kpn I (6 100) to BglII(9 150).
The methods which can be used are multifold:
All of the above (a-d) can be used in diagnostics as sources of immunogens or antigens free of viral particles, produced using non-permissive systems, and thus of little or no biohazard risk.
The invention further relates to the hosts (procaryotic or eucaryotic cells) which are transformed by the above mentioned recombinants and which are capable of expressing said DNA fragments.
Finally, it also relates to vaccine compositions whose active principle is to be constituted by any of the expressed antigens, i.e. whole antigens, fusion polypeptides, or oligopeptides.
The invention finally refers to the purified genomic mRNA, which can either be extracted as such from the LAV viruses or resynthesized back from the cDNA, particularly to a purified mRNA having a size approximating 9.1 to 9.2 kb, hybridizable to any of the DNA fragments defined hereabove or to parts of said purified mRNA. The invention also relates to parts of said RNA. The nucleotidic structures of this purified RNA or of the parts thereof can indeed be deduced from the nucleotidic sequences of the related cDNAs.
It will finally be mentioned that lambda-J19 and lambda-J81 have been deposited at the Collection Nationale des Cultures de Micro-organismes (C.N.C.M.) of the INSTITUT PASTEUR of Pasteur (France) under Nr. 1-338 and 1-339, respectively, on Sep. 11, 1984.
The invention finally refers to the genomic DNA, the DNA sequence of which can be determined and used to predict the amino acid sequences of the viral protein (antigens) and to the RNA probes which can be derived from the cDNA.
There follows the bibliography to which references have been made throughout this specification by bracketed numbers.
All the publications referred to in this bibliography are incorporated herein by reference.
1. A cloned DNA which contains a DNA which is hybridizable with the genomic RNA of the LAV, viruses or a fragment of said hybridizable DNA.
2. The DNA of claim 1 which is a recombinant of said hybridizable DNA or DNA fragment hybridizable with the genomic RNA of the LAV virus.
3. The DNA of claim 1 or 2 wherein said hybridizable DNA or DNA fragment is a cDNA.
4. The DNA of claims 1 to 3 which contains the following restriction sites in the—following order (from the 3′ end to the 5′ end): Hind III, Sac I, Bgl II (LAV 75).
5. The DNA of claim 4 which contains the following restriction sites in the following order: Hind III, Sac I, Bgl II, Bgl II, Kpn I (LAV 82).
6. The DNA of claim 4 which contains the following restriction sites in the following order: Hind III, Sac I, Bgl II, Bgl II, Kpn I, XHo I, Bam HI, Hind III, 891 II (LAV 13)
7. The DNA of claim 6 which has a size of about 2.5 kb.
8. The DNA of any of claims 1 to 7 which contains a region corresponding to the R and U3 regions of the LTR as well as to the 3′ end of the coding region of the retroviral DNA.
9. The DNA of claim 1 which has a size from about 9.1 to 9.2 kb.
10. The DNA of claim 9 which contains the following series of restriction sites:
11. The DNA of claim 10 which contains an additional Hind III approximately at the 5 550 coordinate.
12. A DNA fragment according to claim 1 which comprises a sequence extending from approximately Kpn I (6100) to approximately Bam HI (8150) of the sequence defined in claim 11.
13. A DNA fragment according to claim 1 which comprises a sequence extending from approximately Kpn I (3500) to approximately Bgl II (6500) of the sequence defined in claim 11
14. A DNA fragment according to claim 1 which comprises a sequence extending from approximately Pst (800) to approximately Kpn 1 (3500) of the sequence defined in claim 11.
15. A DNA fragment of claim 1 which codes for the envelope proteins.
16. A DNA fragment of claim 1 which codes for the retroviral polymerase.
17. A DNA fragment which codes for the core proteins.
18. A probe for the in vitro detection of LAV which consists of a DNA according to any of claims 1 to 17.
19. An expression vector, particularly a plasmid, for the transformation of procaryotic or eucaryotic cells which contains an insert consisting of a DNA fragment hybridizable with the retroviral genome of LAV viruses as defined in any of claims 1 to 17.
20. The vector of claim 18 which contains the DNA fragment of claim
21. A microorganism, eukaryotic or prokaryotic cell which is transformed by a vector according to claim 19 or 20 and which expresses the polypeptide encoded by the corresponding DNA fragment.
22. The purified RNAs of LAV viruses which have sizes from 9.1 to 9.2
The present invention aims at providing additional new means which should not only also be useful for the detection of LAV or related viruses (hereafter more generally referred to as “LAV viruses”), but also have more versatility, particularly in detecting specific parts of the genomic DNA of said viruses whose expression products are not always directly detectable by immunological methods.
The present invention further aims at providing polypeptides containing sequences in common with polypeptides encoded by the LAV genomic RNA. It relates even more particularly to polypeptides comprising antigenic determinants included in the proteins encoded and expressed by the LAV genome occurring in nature. An additional object of the invention is to further provide means for the detection of proteins related to LAV virus, particularly for the diagnosis of AIDS or pre-AIDS or, to the contrary, for the detection of antibodies against the LAV virus or proteins related therewith, particularly in patients afflicted with AIDS or pre-AIDS or more generally in asymtomatic carriers and in blood-related products. Finally the invention also aims at providing immunogenic polypeptides, and more particularly protective polypeptides for use in the preparation of vaccine compositions against AIDS or related syndromes.
The present invention relates to additional DNA fragments, hybridizable with the genomic RNA of LAV as they will be disclosed hereafter, as well as with additional cDNA variants corresponding to the whole genomes of LAV viruses. It further relates to DNA recombinants containing said DNAs or cDNA fragments.
The invention relates more particularly to a cDNA variant corresponding to the whole of LAV retroviral genomes, which is characterized by a series of restriction sites in the order hereafter (from the 5′ end to the 3′ end).
The coordinates of the successive sites of the whole LAV genome (restriction map) are indicated hereafter too, with respect to the Hind III site (selected as of coordinate 1) which is located in the R region. The coordinates are estimated with an accuracy of ±200 bp:
Another DNA variant according to this invention optionally contains an additional Hind III approximately at the 5 550 coordinate.
Reference is further made to
An even more detailed nucleotide sequence of a preferred DNA according to the invention is shown in
The invention further relates to other preferred DNA fragments which will be referred to hereafter.
Additional features of the invention will appear in the course of the non-limitative disclosure of additional features of preferred DNAs of the invention, as well as of preferred polypeptides according to the invention. Reference will further be had to the drawings in which:
The sequencing and determination of sites of particular interest was carried out on a phage recombinant corresponding to λJ19 disclosed in the above said British Patent application Nr. 84 23659. A method for preparing it is disclosed in that application.
The whole recombinant phage DNA of clone λJ19 (disclosed in the earlier application) was sonicated according to the protocol of DEININGER (1983), Analytical Biochem. 129, 216, the DNA was repaired by a Klenow reaction for 12 hours at 16° C. The DNA was electrophoresed through 0.8% agarose gel and DNA in the size range of 300-600 bp was cut out and electroeluted and precipitated. Resuspended DNA (in 10 mM Tris, pH 8:0.1 mM EDTA) was ligated into M13 mp8 RF DNA (cut by the restriction enzyme SmaI and subsequently alkaline phosphated), using T4 DNA- and RNA-ligases (Maniatis T et al (1982)-Molecular cloning—Cold Spring Harbor Laboratory). An E. coli strain designated as TG1 was used for further study. This strain has the following genotype Δlac pro, supE, thi.F'traD36, proAB, lacIq, ZΔM15, r−.
This E. coli TGI strain has the peculiarity of enabling recombinants to be recognized easily. The blue colour of the cells transfected with plasmids which did not recombine with a fragment of LAV DNA is not modified. To the contrary, cells transfected by a recombinant plasmid containing a LAV DNA fragment yield white colonies. The technique which was used is disclosed in Gene (1983), 26, 101.
This strain was transformed with the ligation mix using the Hanahan method (Hanahan D (1983) J. Mol. Biol. 166, 557). Cells were plated out on tryptone-agarose plate with IPTG and X-gal in soft agarose. White plaques were either picked and screened or screened directly in situ using nitrocellulose filters. Their DNAs were hybridized with nick-translated DNA inserts of pUC18 Hind III subclones of λJ19. This permitted the isolation of the plasmids or subclones of λ which are identified in the table hereafter. In relation to this table it should also be noted that the designation of each plasmid is followed by the deposition number of a cell culture of E. coli TGI containing the corresponding plasmid at the “Collection Nationale des Cultures de Micro-organismes” (C.N.C.M.) of the Pasteur Institute in Paris, France. A non-transformed TGI cell line was also deposited at the C.N.C.M. under Nr. 1-364. All these deposits took place on Nov. 15, 1984. The sizes of the corresponding inserts derived from the LAV genome have also been indicated.
Positively hybridizing M13 phage plates were grown up for 5 hours and the single-stranded DNAs were extracted.
M13 mp8 subclones of J19 DNAs were sequenced according to the dideoxy method and technology devised by Sanger et al. (Sanger et al (1977), Proc. Natl. Acad. Sci. USA, 74, 5463, and M13 cloning and sequencing handbook, AMERSHAM (1983), the 17-mer oligonucleotide primer α-35SdATP (400 Ci/mmol, AMERSHAM), and 0.5×-5× buffer gradient gels (Biggen M. D. et al. (1983, Proc. Natl., Acad. Sci. USA, 50, 3963) were used. Gels were read and put into the computer under the programs of Staden (Staden R. (1982), Nucl. Acids Res. 10, 4731). All the appropriate references and methods can be found in the AMERSHAM M13 cloning and sequencing handbook.
The complete sequence of J19 was deduced from the experiments as further disclosed hereafter.
Above each of the lines of the successive nucleotidic sequences there are provided three lines of single letters corresponding to the aminoacid sequence deduced from the DNA sequence (using the genetic code) for each at the three reading phases, whereby said single letters have the following meanings.
A: alanine
R: arginine
K: lysine
H: histidine
C: cysteine
M: mthionine
W: tryptophan
F: phenylalanine
V: tyrosine
L leucine
V: valine
I: isoleucine
G: glycine
I: threonine
S: serine
E: glutamic acid
D Aspartic acid
N: asparagine
Q: glutamine
P: proline
The asterisk signs “*” correspond to stop codons (i.e. TAA, TAG and TGA).
Starting above the first line of the DNA nucleotidic sequence of
The same relative presentation of the three theoritical reading phases is then used all over the successives lines of the LAV nucleotidic sequence.
1) The “gag gene” (or ORF-gag)
The “gag gene” codes for core proteins. Particularly it appears that a genomic fragment (ORF-gag) thought to code for the core antigens including the p25, p18, and p13 proteins is located between nucleotidic position 236 (starting with 5′ CTA GCG GAG 3′) and nucleotidic position 1759 (ending by CTCG TCA CAA 3′). The structure of the peptides or proteins encoded by parts of said ORF is deemed to be that corresponding to phase 2.
The methionine aminoacid “M” coded by the ATG at position 260-262 is the probable initiation methionine of the gag protein precursor. The end of ORF-gag and accordingly of gag protein appears to be located at position 1759.
The beginning of p25 protein, thought to start by a P-I-V-Q-N-I-Q-G-Q-M-V-H . . . aminoacid sequence is thought to be coded for by the nucleotidic sequence CCTATA . . . , starting at position 656.
Hydrophilic peptides in the gag open reading frame are identified hereafter. They are defined starting from aminoacid 1=Met (M) coded by the ATG starting from 260-2 in the LAV DNA sequence.
Those hydrophilic peptides are
The invention also relates to any combination of these peptides.
2) The pol gene” (or ORF-pol)
These genes are thought to code for the virus polymerase or reverse transcriptase.
The envelope gene (or ORF-env)
The DNA sequence thought to code for envelope proteins is thought to extend from nucleotidic position 5670 (starting with 5′AAA GAG GAG A . . . 0.3′) up to nucleotidic position 8132 (ending by . . . A ACT AAA GAA 3′). Polypeptidic structures of sequences of the envelope protein correspond to those read according to the “phase 3” reading phase.
The start of env transcription is thought to be at the level of the ATG codon at positions 5691-5693.
Additional features of the envelope protein coded by the env genes appear on
It is to be mentioned that because of format difficulties.
Thus, for instance,
Particularly the sequence shown on the first line on the top of
The locations of neutralizing epitopes are further apparent in
The invention thus concerns with more particularity peptide sequences included in the env-proteins and excisable therefrom (or having the same aminoacid structure), having sizes not exceeding 200 aminoacids.
Preferred peptides of this invention (referred to hereafter as a, b, c, d, e, f) are deemed to correspond to those encoded by the nucleotide sequences which extend respectively between the following positions:
Other hydrophilic peptides in the any open reading frame are identified hereafter. they are defined starting from aminoacid 1=lysine (K) coded by the AAA at position 5670-2 in the LAV DNA sequence.
These hydrophilic peptides are
The invention also relates to any combination of these peptides.
4) The other ORFs
The invention further concerns DNA sequences which provide open reading frames defined as ORF-Q, ORF-R and as “1”, “2”, “3”, “4”, “5”, the relative position of which appears more particularly in
These ORFs have the following locations:
The LTR (long terminal repeats) can be defined as lying between position 8560 and position 160 (end extending over position 9097/1). As a matter of fact the end of the genome is at 9097 and, because of the LTR structure of the retrovirus, links up with the beginning of the sequence:
The invention concerns more particularly all the DNA fragments which have been more specifically referred to hereabove and which correspond to open reading frames. It will be understood that the man skilled in the art will be able to obtain them all, for instance, by cleaving an entire DNA corresponding to the complete genome of a LAV species, such as by cleavage by a partial or complete digestion thereof with a suitable restriction enzyme and by the subsequent recovery of the relevant fragments. The different DNAs disclosed in the earlier mentioned British Application can be resorted to also as a source of suitable fragments. The techniques disclosed hereabove for the isolation of the fragments which were then included in the plasmids referred to hereabove and which were then used for the DNA sequencing can be used.
Of course other methods can be used. Some of them have been exemplified in the earlier British Application, reference is for instance made to the following methods.
The invention also relates more specifically to cloned probes which can be made starting from any DNA fragment according to this invention, thus to recombinant DNAs containing such fragments, particularly any plasmids amplifiable in procaryotic or eucaryotic cells and carrying said fragments.
Using the cloned DNA fragments as a molecular hybridization probe—either by marking with radionucleotides or with fluorescent reagents—LAV virion RNA may be detected directly in the blood, body fluids and blood products (e.g. of the antihemophylic factors such as Factor VIII concentrates) and vaccines, i.e., hepatitis B vaccine. It has already been shown that whole virus can be detected in culture supernatants of LAV producing cells. A suitable method for achieving that detection comprises immobilizing virus onto said a support e.g. nitrocellulose filters, etc. disrupting the virion and hybridizing with labelled (radiolabelled or “cold” fluorescent- or enzyme-labelled) probes. Such an approach has already been developed for Hepatitis B virus in peripheral blood (according to SCOTTO J. et al. Hepatology (1983), 3, 379-384).
Probes according to the invention can also be used for rapid screening of genomic DNA derived from the tissue of patients with LAV related symptoms, to see if the proviral DNA or RNA is present in host tissue and other tissues.
A method which can be used for such screening comprise the following steps: extraction of DNA from tissue, restriction enzyme cleavage of said DNA, electrophoresis of the fragments and Southern blotting of genomic DNA from tissues, subsequent hybridization with labelled cloned LAV proviral DNA. Hybridization in situ can also be used.
Lymphatic fluids and tissues and other non-lymphatic tissues of humans, primates, and other mammalian species can also be screened to see if other evolutionarily related retrovirus exist. The methods referred to hereabove can be used, although hybridization and washings would be done under non stringent conditions.
The DNA according to the invention can be used also for achieving the expression of LAV viral antigens for diagnostic purposes.
The invention also relates to the polypeptides themselves which can be expressed by the different DNAs of the inventions, particularly by the ORFs or fragments thereof, in appropriate hosts, particularly procaryotic or eucaryotic hosts, after transformation thereof with a suitable vector previously modified by the corresponding DNAs.
These polypeptides can be used as diagnostic tools, particularly for the detection of antibodies in biological media, particularly in sera or tissues of persons afflicted with pre-AIDS or AIDS, or simply carrying antibodies in the absence of any apparent disorders. Conversely, the different peptides according to this invention can be used themselves for the production of antibodies, preferably monoclonal antibodies specific of the different peptides, respectively. For the production of hybridomas secreting said monoclonal antibodies conventional production and screening methods are used. These monoclonal antibodies, which themselves are part of the invention, then provide very useful tools for the identification and even determination of relative proportions of the different polypeptides or proteins in biological samples, particularly human samples containing LAV or related viruses.
Thus, all of the above peptides can be used in diagnostics as sources of immunogens or antigens free of viral particles, produced using non-permissive systems, and thus of little or no biohazard risk.
The invention further relates to the hosts (procaryotic or eucaryotic cells) which are transformed by the above mentioned recombinants and which are capable of expressing said DNA fragments.
Finally, it also relates to vaccine compositions whose active principle is to be constituted by any of the expressed antigens, i.e., whole antigens, fusion polypeptides, or oligopeptides in association with a suitable pharmaceutical or physiologically acceptable carrier.
Preferably, the active principles to be considered in that field consist of the peptides containing less than 250 amino acid units, preferably less than 150, as deducible for the complete genome of LAV, and even more preferably those peptides which contain one or more groups selected from N-X-S and N-X-T as defined above. Preferred peptides for use in the production of vaccinating principles are peptides (a) to (f) as defined above. By way of example having no limitative character, there may be mentioned that suitable dosages of the vaccine compositions are those which enable administration to the host, particularly human host ranging from 10 to 500 micrograms per kg, for instance 50 to 100 micrograms per kg.
For the purpose of clarity,
Needless to say,
Finally, the invention also concerns vectors for the transformation of eucaryotic cells of human origin, particularly lymphocytes, the polymerases of which are capable of recognizing the LTRs of LAV. Particularly said vectors are characterized by the presence of a LAV LTR therein, said LTR being then active as a promoter enabling the efficient transcription and translation in a suitable host of the above defined, of a DNA insert coding for a determined protein placed under its controls.
Needless to say, the invention extends to all variants of genomes and corresponding DNA fragments (ORFs) having substantially equivalent properties, all of said genomes belonging to retroviruses, which can be considered as equivalents of LAV.
| Number | Date | Country | Kind |
|---|---|---|---|
| 8324800 | Sep 1983 | GB | national |
| 84/7005 | Sep 1984 | ZA | national |
| 8423659 | Sep 1984 | GB | national |
This is a continuation of application Ser. No. 07/158,652, filed Feb. 22, 1988 (pending), which is a division of application Ser. No. 06/771,248, filed Aug. 30, 1985 (now abandoned). This application is also a continuation-in-part of application Ser. No. 07/999,410, filed Dec. 31, 1992 (pending), which is a continuation of application Ser. No. 07/499,210 filed Mar. 19, 1990 (pending), which is a continuation of application Ser. No. 06/771,230, filed Aug. 30, 1985 (now abandoned), which is a continuation-in-part of application Ser. No. 06/706,562, filed Feb. 28, 1985 (now abandoned), which is a continuation-in-part of application Ser. No. 06/558,109, filed Dec. 5, 1983 (now abandoned).
| Number | Name | Date | Kind |
|---|---|---|---|
| 4520113 | Gallo et al. | May 1985 | A |
| 4629783 | Cosand | Dec 1986 | A |
| 4677054 | White et al. | Jun 1987 | A |
| 4708818 | Montagnier et al. | Nov 1987 | A |
| 4725669 | Essex et al. | Feb 1988 | A |
| 4808536 | Chang et al. | Feb 1989 | A |
| 5156949 | Luciw et al. | Oct 1992 | A |
| 5665536 | Goldstein et al. | Sep 1997 | A |
| 6001977 | Chang et al. | Dec 1999 | A |
| 6531276 | Luciw et al. | Mar 2003 | B1 |
| 6627395 | Montagnier et al. | Sep 2003 | B1 |
| Number | Date | Country |
|---|---|---|
| 0 113 078 | Jul 1984 | EP |
| 0 115 459 | Aug 1984 | EP |
| 0 199 301 | Oct 1986 | EP |
| 0 258 404 | Mar 1988 | EP |
| WO 8404327 | Nov 1984 | WO |
| WO 8705399 | Sep 1987 | WO |
| WO 9104051 | Apr 1991 | WO |
| Entry |
|---|
| L. Ratner et al. (1985) Nature 313: 636-637, [Exhibit 2]. |
| S. Wain-Hobson et al. (1985) Cell 40:9-17. [Exhibit 3]. |
| L. Ratner et al. (1985) Nature 313: 277-284. [Exhibit 4]. |
| R. Sanchez-Pescador et al. (1985) Science 227: 484-492, [Exhibit 5]. |
| M. Guyader et al. (1987) Nature 326: 662-669. [Exhibit 7]. |
| F. Clavel et al. (1986) Science 233:343-346, [Exhibit 8]. |
| M.A. Muesing et al. (1985) Nature 313:450-458. [Exhibit 9]. |
| M. Alizon et al. (1984) Nature 312: 757-760. [Exhibit 10]. |
| B.H. Hahn et al. (1984) Nature 312: 166-169. |
| Richterich, Peter, “Estimation of errors in ‘Raw’ DNA sequences: a validation study”, Genome Research (1998) 8:251-259. |
| Hogrefe, http://www.trilinkbiotech.com/tech/pdf/A%20Short%20History%20of%20Oligonucleotide%20Synthesis.pdf, accessed 2004. |
| Sch üpbach et al., Science, 224, 503-505 (1984). |
| Kalyanaraman et al., Science, 225, 321-323 (1984). |
| Robey et al., Science, 228, 593-595 (1985). |
| Allan et al., Science, 228, 1091-1094 (1985). |
| Crowl et al., Chemical Abstracts, 103, p. 190, No. 154983e (1985). |
| Montagnier et al., Chemical Abstracts, 103, p. 263, No. 34641v (1985). |
| Weiss et al., Nature, 316, 69-72 (1985). |
| Barin et al., Science, 228, 1094-1096 (1985). |
| Wain-Hobson et al., Cell, 40, 9-17 (1985). |
| Muesing et al., Nature, 313, 450-458 (1985). |
| Schneider et al., Chemical Abstracts, 103, p. 430, No. 52370k (1985). |
| Sarngadharan et al., Chemical Abstracts, 103, p. 551, No. 121329t (1985). |
| Alizon et al., Nature, 312, 757-760 (1984). |
| Chang et al., Nature, 315, 151-154 (1985). |
| Chang et al., Biotechnology, 3, 905-909 (1985). |
| Ratner et al., Nature, 313, 277-284 (1985). |
| Dreesman et al., Nature, 295, 158-160 (1982). |
| Kitchen et al., Nature, 312, 367-369 (1984). |
| Essex et al., Science, 220, 859-862 (1983). |
| Seiki et al., Proc. Natl. Acad. Sci., USA, 80, 3618-3622 (1983). |
| Biotechnol. Newswatch, vol. 5, p. 3, abstract No. 03-10-003226 (1985). |
| Paper No. 300 in Interference No. 102,822; APJs Metz, Pate, and Martin; Mar. 9, 2001; pp. 1-6. |
| Paper No. 271 in Interference No. 102,822; APJs Metz, Pate, and Martin; Apr. 21, 1999; pp. 1-3. |
| Paper No. 282 in Interference No. 102,822; APJs Metz, Pate, and Martin; Jul. 30, 1999; pp. 1-2. |
| Paper No. 289 in Interference No. 102,822; APJs Metz, Pate, and Martin; Oct. 19, 1999; pp. 1-3. |
| Claims 30, 31, 57-61, 109-115, and 132 in Interference No. 102,822. |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 06771248 | Aug 1985 | US |
| Child | 07158652 | US | |
| Parent | 08051226 | US | |
| Child | 07158652 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 07158652 | Feb 1988 | US |
| Child | 08051226 | US | |
| Parent | 07499210 | Mar 1990 | US |
| Child | 07999410 | US | |
| Parent | 06771230 | Aug 1985 | US |
| Child | 07499210 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 07999410 | Dec 1992 | US |
| Child | 08051226 | US | |
| Parent | 06706562 | Feb 1985 | US |
| Child | 06771230 | US | |
| Parent | 06558109 | Dec 1983 | US |
| Child | 06706562 | US |
