FR2600342A1 - METHOD FOR THE DETECTION OF DNA SEQUENCES OF RETROVIRUS HTLV III-LAV IN SEROPOSITIVE INDIVIDUALS AND ITS KIT - Google Patents

Abstract

Method, based on the general technique of molecular hybridation, for the detection in seropositive individuals of DNA sequences of the retrovirus HTLV III-LAV. The invention also relates to a kit for implementing such method. Application to the detection of AIDS in seropositive individuals .

Description

The present invention relates generally to
the one method of revealing the retroviral DNA sequences
HTLV III-LAV at the DNA level of T4 lymphocytes, by a particular use of the conventional molecular hybridization technique. Such a method is applicable to
the routine detection of the corresponding sequences in
HIV-positive individuals. The method according to the invention makes it possible to prove the direct presence (or absence) of the virus - in free form or its conformations - in the genome of T4 lymphocytes and corresponding individuals.

The invention also relates to a cabinet for the implementation of the method in question.

Recent publications have studied the retrovirus
HTLV III-LAV and showed that it was responsible for the AIDS disease. As an example of a relevant bibliographic reference in this field, mention may be made of the article by SHAW, GM, HAHN BH, ARYA SK, GROOPMAN JE, GALLO
RC and tONG-STAAL, F. (1984) Molecular characterization of human leukemia virus type III in the immune deficiency syndrome, Science, 226-1165-1171.

Although the HTLV III-LAV retrovirus is an RNA virus, it is already established (HIRSCH MS and KAPLAN JC (1985)
Prospects of therapeutics for human infections T-lymphotropic virus Type III. Annals of Internal Medicine, 103, 750755) at the time of integration into the T4 lymphocyte
it passes, thanks to its enzyme reverse transcriptase, in the state of simple DNA, then double-strand (it is not yet known very clearly whether it exists at this level in form
free or integrated into the genome of the host).

It has already been suggested to detect the presence of the virus at this time by the DNA-DNA molecular hybridization technique, much easier to implement than that between RNA molecules. However, the verification of
the presence of the viral sequences by this technique can not be practically achieved in a sufficiently safe manner. It has already been established that positive results have been determined only in a very small number of highly specific cases: 1 case out of 16 individuals with AIDS with regard to the detection of celiac cells.
peripheral blood mononuclear cells (SHAW GM, HAHN
BH, GROOPMAN JE, S. BRODER, U0NG-STAAL F. and GALLO RC

(1985) Molecular characterization of HTLV III DNA sequences
in fresh tissue (abstract, International Symposium on
AIDS Atlanta).

The idea underlying the present invention is to use the general technique of molecular hybridization in HIV-positive individuals, adapting it for
make it usable safely for practical purposes. It is known that when an individual has been in contact with the virus, without being
AIDS becomes HIV-positive and this state carries risks because the most recent studies show that there may be a slow and gradual evolution towards the actual disease of AIDS. For the purposes of prevention, it is therefore highly desirable to have a method for the diagnosis and screening of HTLV III-LAV virus in seropositive individuals. There are already tests that have become conventional, making it possible to establish whether an indiyidu is , or not, HIV-positive. But, to the knowledge of the applicant, there is no additional method for authenticating the presence of the virus itself in such seropositive individuals. Admittedly, one can think of using the usual techniques of culture, but these tests are too long and too expensive and, in addition, in the case of the virus
HTLV III-LAV, they are random.

 The object of the invention is to meet the above-mentioned practical needs by proposing a screening method in seropositive individuals, the results of which are obviously of extreme importance in deciding on the evolution of such individuals towards disease.

The invention therefore relates to a method for screening, in seropositive individuals, DNA sequences of the retrovirus HTLV III-LAV, in which method a general molecular hybridization technique comprising the following steps is applied:
a) the blood is collected from the individuals examined to isolate the white cells in known manner and to obtain lymphocyte DNA, which is used in unit quantity equal to at least 30
b) said amount of lymphocyte DNA is treated with a restriction enzyme which does not intersect within the sequence of the AON of the virus.

 c) passing the medium of step b) which contains the virus in free or integrated form, on a nitrocellulose filter able to retain up to 80 μg of DNA per cm 2 of surface.

 d) the product retained by the filter is brought into contact with a probe, which is a cloned and labeled probe of the HTLV III-LAV virus, said probe being a subcloned fragment of the DNA of the virus which does not contain the coding part for the envelope region of the virus, the labeling being capable of giving an activity as close as possible to the value of 5 × 10 9 cpm per 89 DNA of the probe, potentially an activity of 5 × 10 8 to 1 × 10 9 cpm / μl of DNA.

 e) the presence of characteristic bands of the virus is known in known manner.

 The molecular hybridization technique is known to those skilled in the art and it is therefore not necessary to describe it in detail. In the case of the HTLV III-LAV retrovirus, reference may be made to the previously mentioned article by SHAW et al (1985). The adaptation of this technique to the needs of the present invention will emerge from the description which follows and from the practical example given below to illustrate the invention.

In the first step of the method, the peripheral blood is removed from the individuals examined and the white cells are isolated in known manner. After digestion with an appropriate enzyme, such as XbaI, lymphocyte DNA is obtained. According to a feature of
the present invention uses the amounts of DNA
lymphocytes as high as possible compatible with the load of the filter used in step c). This unit quantity is at least equal to 30% of DNA. In practice, ranges of 30 to 50 μg can be used. Values below 30> 9 do not provide a sufficiently reliable response. Moreover, beyond 80
excessive amounts of blood must be taken; furthermore we come to the limit of use of the filter of
step c). Unit quantities of lymphocyte DNA between 30 and 50 μg can be easily obtained from about 10 million white blood cells of the examined individuals. As is customary in this art, these unit quantities are deposited in respective wells of gel, for example agarose gel.

In step b) of the method of the invention, the aforementioned amount of lymphocyte DNA is treated with a restriction enzyme selected such that it does not normally have a recognition site within the sequence of the virus DNA. This has the effect of concentrating the signal finally obtained into a small number of large autoradiographed bands, corresponding to the virus Vibre and its different conformations. As an enzyme that is suitable for the purposes of the process of the invention, mention should first be made of the Xbal enzyme, which is known to have no cleavage within the viral sequence, see HAHN BH, SHAW GM, ARYA SK, POPOVIC M., GALLO
RC, and WDNG-STAAL F., (1984) Molecular cloning and characterization of the HTLV I virus associated with AIDS. Nature, 312. 166-169. Other equivalent enzymes are however usable.

 In step c) of the process of the invention, a high-sensitivity nitrocellulose filter is used having, by cm 2 of surface, the highest possible charge. The characteristics of this filter are compatible with the unit quantities of lymphocyte DNA used in step a). The filter is capable of retaining at least 30 pv9 of DNA and preferably up to 80, 49 of DNA per cm 2 of surface area. In practice, the Millipore filters, or better still the Sartorius Schleicher & Schull BA85 filter can be used. In the latter case, preliminary experiments using the H9v3 culture DNA as a test showed that up to 0.1 μg of DNA from the cloned probe deposited was detectable by the technique described herein; this amounts to saying that about 104 molecules of DNA are thus detectable, ie in theory one infected cell out of 500.

In step d1, a probe is used which is a subcloned fragment of the virus DNA, which does not have the coding portion for the envelope region, variable in nature by different isolates. In practice, the best results have been obtained with a probe constituted by the Sst I-5 'fragment of o \ BH-10, which corresponds only to the 5' end and to the median part of the genome. Such probes are known to those skilled in the art. Op can refer in this respect to the European patent application published under No. 0173529. According to another very important feature of the method of the invention, the marking must be capable of providing a very high specific activity, as close as possible to the value of 5 × 10 9 cpm per DNA of the probe, preferably an activity of 5 × 10 8 to 1 × 10 9 cpm / μl of DNA. In practice we get such
activity with a labeling system using a large amount of small size primers and dCTP labeling using Klenow polymerase; or the marking by displacement of cut (said nick-translation) involving two radioelements (dCTP + dATP) at 800 Cu / mole (cold kit
N5500 and PB 10384 and PB10385 from Amersham - see example below).

 The revelation is carried out in a known manner by autoradiographic exposure. It has been found, in the practice of the invention, that an exposure time of a few days, for example 4 days, is sufficient for reading and, consequently, for screening.

The method according to the invention, which uses the molecular hybridization technique, can in particular comprise the following main steps: (1) extraction of long-stranded lymphocyte DNA by a phenol-chloroform mixture after proteinaceous digestion with proteinase K; (2) spectrophotometric assay of the obtained DNA, verification of its length on agarose minigel, and its digestibility by nuclease assay; (3) digestion with restriction enzyme XbaI, in excess of 5 units per 9 of DNA to be digested; (4) Stretching the 0.8% agarose gel restriction fragments in 10xSSC transfer buffer; (5) marking of the probe by 32PdCTP + dATP cleavage displacement at 800 mm for 250 Ci; (6) hybridization of the denatured probe with the 5xSSC buffer filter, 10xDenhardt, 0.1%
SDS, 50 μM EDTA, 50 μM Tris-HCl pH = 7.5 with 10 μg / ml of salmon sperm DNA sonicated and denatured for 48 hours at 6 ° C; (7) final wash in IxSSC buffer and 0.1% SDS; (8) drying of the filter and its inclusion in a cassette comprising two intensifying screens in the presence of sensitive films, the autoradiography being continued up to 1 month at -700C; (9) revelation of autoradiographed bands; (10) Reading of autoradiographed illuminated platform bands using HindIII digested phage DNA size scale.

 The Fiv. 1 of the appended drawing shows the radiographically obtained strips obtained in the case (1) of an HIV-positive individual> 0, (2) in that of an HIV-positive individual (O, and of the H9v3 culture, in the latter case, the accolade re presents the multiple integration sites.

Thus, the following Xbal fragments are observed in seropositive individuals:
a giant fragment, corresponding to the high molecular weight DNA, having no or little penetration into the gel,
a fragment at approximately 13 kob, corresponding to the virus in circular form and cut in a hap lotoni way,
a fragment at about 9 kb, corresponding to the virus in linear form. These last two bands correspond to the free form of the virus.

 By comparison, the H9v3 DNA under the same conditions gives a signal corresponding to the giant fragment, as well as another of large size whose weight limits, fuzzy, indicate the polyclonal integration of the virus.

The photograph of Figure I, which shows three gel migration channels, illustrates the signals observable on the autoradiogram, which materialize the results of the method of the invention. In the case of the lineage
H9v3 there is a weak signal in the region of the high molecular weight DNA that penetrates poorly into the wells of the gel; The essential of the signal is represented by a spot spread by weight (this spot is intense, because each cell of the culture comprises of the order of 50 to 100 viral particles); this spreading is due to the fact that the integration of the virus at the level of human DNA is polyclonal (there are multiple sites of insertion). In the case of channel 1, it concerns a positive seropositive individual for the signal. Three autoradiographed bands are present: one in the high molecular weight DNA, which does not enter the well of the gel; and two autoradiographed bands of approximate size 13 and 9 kb corresponding to the free form of the virus, probably in two different conformations (supercoiled and / or relaxed). Some individuals are strongly positive (this is the case
Fig. 1) with clearly marked bands, visible after 4 days of autoradiography. In the case of channel 2, it concerns an individual who is seropositive but negative for the signal (even after prolonged exposure).

 These results show that the reading of the results provided by the method of the invention can be made safely and reproducibly to detect the presence of HTLVIII-LAV virus in HIV-positive individuals.

 Out of a total of fourteen individuals examined, nine show the viral sequences. Of these nine positives, four of them show more intense bands and are therefore qualified as intense positive, the other five showing only weak bands, even after prolonged exposure; these differences in intensity are independent of the amount of DNA deposited in each well and undoubtedly reflect the number of viral copies present.

The fact that the three bands observed correspond to the sequences of the virus is indicated for the following reasons:
the signal obtained is after hybridization with a specific probe,
- the expected size bands are observed,
they are close to those of the control culture H9v3.

In the case of a control DNA taken at random from the population and digested with Xbal, the bands are not visible. They are not so in the case of a
Real AIDS under the same conditions; as already reported, the detection of the virus by this method is not practicable for patients (in this case, the amount of T4 cells is very low, and most of the virus is in the form of RNA).

 The observed bands are not the result of probe contamination by phage /. Digested with XbaI, this gives two 23.9 and 24.5 kb restriction fragments, much larger than those observed, as established in a control digestion of this type. In contrast, the presence of viral sequences in the lymphocyte DNA of seropositive individuals> O is proven by migrating DNA from these undigested individuals to the gel, and hybridizing the corresponding channels with the probe: in most In one case, a strong signal is obtained in the undigested DNA (integrated sequences), another, much weaker, in a molecular weight band ~ 10 kb (free form).

The invention also relates to a box or kit containing the ingredients required for the implementation of the method. The unit quantities of these ingredients will depend on the number of samples that are intended to be processed. The box according to the invention essentially comprises:
a HTLV III-LAV probe, for example the probe corresponding to subclone nBH-5, excised from phage A
a preparation of the digested phage A DNA
XbaI, used as a control,
XbaI enzyme in the enzyme conservation buffer,
the digestion buffer of the enzyme.

For marking, usable products are, for example, those put on the market by the Amersham Company.
France, Avenue du Canada 91944 Les Ulis France such as:
- cold kit N5500 (nick-translation buffer and enzymes)
p32 dCTP (PB 10385) and P32 dATP (PB 10384) at 800 Ci / mmole (250 ptCi each).

 A cold variant of labeling (for example, basic sulfonation labeling currently marketed by the company Organics, 12 Avenue Salvador Allende 94402 Vitry-sur-Seine, France) can also be used.

Alternatively, it is also possible to hybridize in situ using cold labeling or radioactive labeling, for example H3
The invention will be further illustrated by an example
complete illustrative of implementation.

EXAMPLE:
20 ml of whole blood are collected on 5% EDTA in physiological saline. Plasma is removed by vacuum aspiration after centrifugation for 5 minutes at 1500 rpm. Lysis of red blood cells is obtained by adding to the fresh pellet 50 ml of SLR buffer (10 mM Tris, 5 mM MgCl 2, 10 mM NaCl); a homogenization is obtained up to a lacquered appearance, then a centrifugation is carried out 5 to 10 minutes at 2000 rpm; the supernatant is then aspirated to the vacuum pump. Proteinase K solution is prepared in TE buffer (Tris-EDTA) at a final concentration of 0.2 mg / ml. The pellet obtained above is redissolved in 3 ml of the SLR buffer and redissolved in 20 ml of buffer SLB. (10 nM Tris, 10 mM EDTA, 50 mM NaCl, 0.2 SDS and 3 mg Proteinase K per tube to give a viscous block which is homogenized and then gently shaken (45 to 50 rpm in the bath). -marie orbital) overnight at 420C.

The phenol used for the extraction is saturated in
0.2 M Tris pH = 7.6. A first extraction is carried out in 314 phenol / 4 chloroform and 1/24 isoamyl alcohol; the upper aqueous phase is removed by pipette. A second extraction is then prepared in phenol-chloroform; the two phases then separate and the DNA is in the upper phase; the lower organic phase is removed by syringe. A third extraction is finally performed to remove the phenol with chloroform + isoamylic acid.

 In the residual phase the DNA is precipitated by addition of 300 F of 3M NaCl and by supplementing with 50 ml of absolute ethanol at -200C. Ethanol was pipetted off and the precipitated DNA was pipetted inverted; it is rinsed several times with absolute methanol.

 The DNA is resuspended in 2 to 4 ml of TE at 40C for several days. It is assayed for DO260, and the control of its size is carried out in 0.7% agarose minigel and staining with ethidium bromide in the TEA buffer.

 The indicated amount of DNA for each sample is digested with the XbaI restriction enzyme in excess of 5 units per $ Xg of DNA. The restriction fragments are then spread on a 0.8% horizontal agarose gel.

After migration, the gel is depurinated (0.25 M HCl), denatured (0.5 M NaOH in 1M NaCl) and neutralized (Tris 0.5
1.5M NaCl at pH = 7.0). The transfer buffer is 10 x
SSC, nitrocellulose filter transfer being performed overnight. The filter is finally dried and the DNA is fixed in the oven at 80 ° C. for 6 hours.

 The probe used is the probe consisting of the Sst I-5 'fragment of # -BH-10. It is marked by nicktranslation with dCTP + dATP at 8000 Cu / mole.

 The filter is prehybridized at 650C in a sealed plastic bag containing: 25ml of 5x SSC buffer (1 hour), then 5x SSC, 10xDenhardt (D), 0.1% SDS (1 hour), then 5xSSC , 10xD, 1rrM EDTA and 0.1% SDS supplemented with 100 g / ml of denatured and sonicated salmon sperm DNA (ss) (overnight).

 Hybridization with the denatured probe is carried out in 10 ml: 5xSSC, 10xD, 0.1% SDS, SrrM EDTA, 50mM Tris-HCl pH = 7.5 and 10 μg / ml ss DNA at 670 ° C in an orbital water bath at least 24 hours.

 After hybridization, the filters are washed in successive baths: (250 ml in a bowl at 660 ° C): 30 minutes in 5 × SSC, 10 × D, 0.148 SDS, 0.1% NaPPi, then 30 minutes in 3SSC, 10 × D, 1% SDS, 0. , 1% NaPPI, then 15 minutes in 2xSSC, 1xD, 0.1% SDS, 0.1% NaPPi and the time required (counter monitoring) in 1SSC buffer, IxD, 0.1% SDS, 0.1% NaPPi .

 The filters are then dried under the lamp, fixed on Wathman 3Mvl paper and coated with a plastic film, and then arranged for autoradiography at -700C for 1 to 30 days in a cassette with two intensifying screens.

The autoradiograms are revealed for 5 minutes, rinsed with water, fixed for 5 minutes, washed again and dried by drying. The size of the autoradiographed bands is calculated in relation to that of the size scale represented by Hind III fragments of phage 2 DNA.
The example detailed above is illustrative but not limiting of the process of the invention. Variations on the procedure can be made by those skilled in the art.

Claims (8)

 1. Method for the detection of HTLV retrovirus DNA sequences in seropositive individuals III-LAV, a method in which a general molecular hybridization technique is applied comprising the following steps:  a) the blood is collected from the individuals examined to isolate the white cells in known manner and obtain lymphocyte DNA, which is used in unit quantities of at least 30  b) said amount of lymphocyte DNA is treated with a restriction enzyme not intersecting within the sequence of the virus DNA.  c) the medium of step b), which contains the virus in free or integrated form, is passed through a nitrocellulose filter capable of retaining up to 80 DNA per cm 2 of surface.  d) the product retained by the filter is brought into contact with a probe, which is a cloned and labeled probe of the HTLV III-LAV virus, said probe being a subcloned fragment of the DNA of the virus which does not contain the coding part for the envelope region of the virus, the labeling being capable of giving an activity as close as possible to the value of 5 × 10 9 cpm per 69 of DNA of the probe, in particular an activity of 5 × 10 8 to 1 × 10 9 cpm /> 9 of DNA.  e) the presence of characteristic bands of the virus is known in known manner.  2. Method according to claim 1, characterized in that the restriction enzyme is Xba I or an equivalent enzyme.  3. Method according to one of claims I or 2, characterized in that the unit quantity of lymphocyte DNA is between 30 and 50 g approximately.  4. Method according to any one of claims I to 3, characterized in that a probe corresponding to subclone # BH-5, excised phage # is used.  5. Method according to any one of claims I s to 4, characterized in that the marking of the probe is a cold marking or a radioactive marking.  6. Method according to any one of claims I to 5, characterized in that it comprises the following main steps: (1) extraction of long-stranded lymphocyte DNA with a phenol-chloroform mixture after protein digestion with proteinase K ; (2) spectrophotometric assay of the obtained DNA, verification of its length on agarose minigel, and its digestibility by nuclease assay; (3) digestion with the restriction enzyme XbaI, in excess of 5 units per> g of DNA to be digested; (4) spreading the 0.8 agerose gel restriction fragments in 10xSSC transfer buffer; (5) marking this probe by 32PdCTP + dATP cleavage displacement at 800 cuprole for 250 ssCi; (6) hybridization of the denatured probe with the 5xSSC buffer filter, 10xDenhardt, 0.1% SDS, 50 mM EDTA, 50 mM Tris-HCl pH = 7.5 with 10 mglm of sonicated and denatured salmon sperm DNA for 48 hours at 670C; (7) final wash in IxSSC buffer and 0.1% SDS; (8) drying the filter and its inclusion in a cassette comprising two intensifying screens in the presence of sensitive films, the autoradiography being continued up to 1 month at -700C; (9) revelation of autoradiographed bands; (10) Reading the illuminated platform autoradiographed bands using HindIII-phage phage DNA size scale.  7. Box or kit for carrying out the method according to any one of claims I to 6, characterized in that it partially comprises:  an HTLV II1-LAV probe, for example the probe corresponding to the subclone / BH-5, excised from the phage  a preparation of DNA digested phage # XbaI, used as a witness,  XbaI enzyme in the enzyme conservation buffer,  the digestion buffer of the enzyme.  8. Box according to claim 7, characterized in that the marker is radioactive or cold.