WO1987007912A1

WO1987007912A1 - Method for detecting in seropositive individuals dna sequences of the retrovirus htlv iii-lav and kit for implementing such method

Info

Publication number: WO1987007912A1
Application number: PCT/FR1987/000228
Authority: WO
Inventors: Pierre Reveilleau
Original assignee: Laboratoires Eurobio
Priority date: 1986-06-19
Filing date: 1987-06-18
Publication date: 1987-12-30
Also published as: FR2600342A1; JPH01500008A; FR2600342B1; EP0270642A1

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

Method for screening, in HIV-positive individuals, DNA sequences of the HTLV III-LAV retrovirus and kit for its implementation.

The present invention relates generally to a process for revealing the DNA sequences of the HTLV III-LAV retrovirus at the level of the DNA of the T4 .lymphocytes, by a particular use of the conventional technique of molecular hybridization. Such a method can be applied to the on-line 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 conforma ions - in the genome of T4 lymphocytes and of the corresponding individuals. The invention also relates to a box allowing the implementation of the method, in ques t ion.

Recent publications have studied the HTLV III-LAV retrovirus and have shown that it is responsible for AIDS disease. As an example of relevant bibliographic reference in this area, we can cite the article by SHAW, GM, HAHN BH, A YA., SK GROOPMAN JE, GALLO RC and VONG-STAAL, F. (1984) Molecular charac eri zat ion of human ce 11 leukemia virus type III in the acquired immune deficiency syndrome, Se ience, 226 -1165-1171.

Although the HTLV III-LAV r trovirus is an RNA virus, it has already been established (HIRSCH M. S. And KAPLAN JC (1985) Prospects of therapy for infec¬ tions wi th human T- 1 ymphot rop ic virus Type III Annal s of Internai Med ic i ne, 103, 750-755) that at the moment of its integration in the T4 lymphocyte it passes, thanks to its reverse transcriptase enzyme, to the state of single DNA, then double -strand (we do not know

* f * however still not very precisely if it exists at this level in free form or integrated in 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, which is much easier to use than that between RNA molecules. However, the verification of the presence of viral sequences by this technique cannot be carried out in practice in a sufficiently safe manner. It has already been established that positive results have only been determined in a very small number highly specific cases: 1 case in 16 individuals suffering from AIDS with regard to the detection of peripheral blood mononuclear cells (SHAW GM, HAHN BH, GROOPMAN JE, BRODER S., VvONG-STAAL F. and GALLO RC ( 1985) Molecular character iza ion of HTLV III DNA sequences in french tissue (abstract, International Symposium on AIDS Atlanta).

The idea which is the basis of the present invention is to use the general technique of molecular hybridization in seropositive individuals, by adapting it. to make it safe to use for practical purposes. We know, in fact, that when an individual has been in contact with the virus, but without having AIDS, he becomes HIV positive and this state involves risks because the most recent studies show that there may be a slow and progressive evolution towards the actual AIDS disease. For prevention purposes, it is therefore highly desirable to have a diagnostic and screening process for the HTLV III-LAV virus in individuals. due to HIV positive. We already have tests that have become conventional, making it possible to establish whether or not an individual is HIV positive. However, as far as the applicant is aware, there is no additional method for authenticating the presence of the virus itself in such seropositive individuals. Admittedly, one can think of using usual culture techniques, but these tests are too long and too expensive and, moreover, in the case of the HTLV III-LAV virus, they are random.

The object of the invention is to respond to the aforementioned practical needs, by proposing a screening method in HIV-positive individuals, the results of which are obviously of extreme importance in deciding on the development of such individuals. towards disease.

The invention therefore relates to a method for screening, in HIV-positive individuals, DNA sequences of the HTLV III-LAV r trovirus, method in which a general molecular hybridization technique is applied comprising the following steps: a) the blood of the individuals examined is drawn to isolate the white cells in a known manner and to obtain DNA 1 ymphocyta i re, which is used in a unit quantity equal to at least 30 A / g. b) treating said quantity of lymphocyte DNA with a restriction enzyme which does not cut within the DNA sequence of the virus, c) passing the medium from step b) which contains the virus in free or integrated form, on a ni trocel 1 ulose filter capable of retaining up to 80 g of DNA per cm of surface. d) the product retained by the filter is brought into contact with a probe, which is a cloned probe, and marked with the HTLV III-LAV virus, said probe being a subcloned fragment of the DNA of the virus which does not carry the coding part for the envelope region of the virus, the labeling being capable of giving an activity as close as possible of the value of 5.10 cpm per ,. g of probe DNA, in particular an activity of 5.10 ⁸ to 2.10 ⁹ cpm // * / g of DNA. e) the presence of characteristic bands of the virus is revealed in a 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 article by SHAW et al (1985) mentioned above. 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 process, the peripheral blood is taken from the individuals examined and the white cells are isolated in a known manner. After digestion with an appropriate enzyme, such as Xba I, the lymphocyte DNA is obtained. According to a feature of the present invention, the highest possible quantities of lymphocytic DNA are used which are compatible with the charge of the filter used in step c). This unit quantity is at least equal to 30 μg of DNA. In practice, ranges from 30 to 50 g can be used. Values less than ^~ θ À / g do not provide a sufficiently reliable response. By the way, beyond 80 / g, it is necessary to draw excessively large quantities of blood; in addition we arrive at the limit of use of the filter of step c). Unit quantities of lymphocyte DNA included between 30 and 50 ^ / g can be easily obtained from about 10 million ions of white cells from the blood of the individuals examined. As is usual in this technique, these unit quantities are deposited in respective wells of gel, for example of agarose gel.

In step b) of the process of the invention, the abovementioned quantity of DNA 1 ymphocyta i re is treated with a restriction enzyme chosen in such a way that it does not normally have a recognition site inside the DNA sequence of the virus This has the effect of concentrating the signal finally obtained into a small number of large autoradiographed bands, corresponding to the free virus and to its various conforma ions. As an enzyme suitable for the needs of the process of the invention, mention must first be made of the enzyme XbA I, which is known to not cut within the viral sequence: see HAHN BH, SHAW GM , ARYA SK, POPOVIC M. GALLO RC, and VDNG-STAAL F., (1984) Molecular cloning and charac er i zat ion of the HTLV I virus associated with AIDS. Nature, 312: 166-169. Other equivalent enzymes are, however, ut i 1 i sab les. In step c) of the process of the invention, a ni t rocel lulose filter of great sensitivity is used, presenting, per cm of surface, the highest possible load. The characteristics of this filter are compatible with the unitary quantities of DNA 1 ymphocyta i re used in step a). The filter is capable of retaining at least 30κ ^ g of DNA and preferably up to 80 / ^ g of DNA per cm ² of surface. In practice, Millipore filters, or better still the Sartorius Schleicher & Schull BA85 filter can be used. In this last In this case, preliminary experiments using DNA from the H9v3 culture as a test have shown that up to 0.1 μg of DNA from the cloned probe deposited was detectable by the technique described here, this amounts to saying that approximately 10 DNA molecules are well dé¬ nizable or in an infected cell theory of ^'500.

In step d), a probe is used which is a subclone fragment of the DNA of the virus, which does not include the coding part for the enveloped region, variable, by nature, in the different isolates. In practice, good results have been obtained with a probe constituted by the fragment Sst 1-5 'of BH-10, which corresponds only to the 5' end and to the middle part of the genome. Such probes are known to those skilled in the art. In this regard, reference may be made to the European patent application published under the number 0173529. According to another very important characteristic of the inventive method, the marking must be capable of providing a very high specific activity, as close as possible as the value of 5.10 cpm per / g of probe DNA, preferably an activity of

5.10 ⁸ to 2.10 ⁹ cpm / Lj g of DNA. In practice, such an activity is obtained with a labeling system using a large quantity of primers of small sizes and labeling with dCTP using Kleno polymerase; or marking by cut displacement (nick-translat ion) involving two radioelements (dCTP + dATP) at 8CO Cu / mole (cold kit N5500 and marking PB 10384 and PB10385 from Amersham - see example below);

The best results have been obtained by quantifying the probe by the method of oligonucleotides synthesis ides, used as a primer, by making intervene a single radioelement (dCTP) with labeling at ^{• 52} P. In routine and using the corresponding kit from Amersham available on the market, we obtained a specific activity of the order of 1 - 2 x 10 cpm / J g. It has also been found that, under these conditions, the process consumes only very small quantities of probe (of the order of 100 ng at each marking) and does not require very high purity of the probe compared to the contaminants. protein. Therefore, the method is easier and less expensive to implement.

The revelation is carried out in a known manner by exposure to autorad iograph i que. It has been found, during the implementation of the invention, that a period of exposure of a few days, for example 4 days,. was sufficient for reading and therefore screening. The best results were obtained with the hypersensitive car radio films (Amersham). The method according to the invention, which uses the technique of molecular hybridization, can in particular comprise the following main steps: (1) extraction of long stranded 1ymphocytic DNA by a phenol -choroform mixture after digestion proteinase by proteinase K; (2) spectrophotometric assay of the DNA obtained, verification of its length on agarose minigel, and of its digestibility by nuclease test; (3) digestion with the restriction enzyme Xbal, in excess of 5 units per A g of DNA to be digested; (4) spreading of the restriction fragments on agarose gel at 0.8% in lOxSSC transfer buffer; (5) marking of the probe by cut-off displacement with PdCTP + dATP at 8CO cu / mmol for 250 Ci or with ⁵² PdCTP alone; (6) hybridization of the denatured probe with the filter in buffer 5xSSC, lOXDenhardt, 0.1% SDS, 50 rrM EDTA; 50 rrM Tris-HCl pH = 7.5 with 10 mg / ml of DNA from sperm of sonicated and denatured salmon for 48 hours at 67 ° C; (7) final washing in IxSSC buffer and 0.1% SDS; (8) drying of the filter and its inclusion in a casette comprising two reinforcing screens in the presence of sensitive films, the autoradiograph being carried out at -70 ° C; (9) revelation of the autoradiographed tapes; (lb) reading of the autoradiographed bands on an illuminating platform using the DNA III phage DNA size scale digested with Hind III.

FIG. 1 of the appended drawing represents the radiographed bands obtained in the case (1) of an HIV-positive individual> O, (2) in that of an HIV-positive individual / O, and of the H9v3 culture; in the latter case, the brace represents multiple integration sites.

Thus, the following Xbal fragments are observed in HIV-positive individuals:

a giant fragment, corresponding to DNA of high molecular weight which has not, or only slightly, penetrated into the gel,

-a fragment at around 13 kb, corresponding to the virus in circular form and cut haplotonically,

-a fragment at approximately 9kb, corresponding to the virus in linear form. These last two bands correspond to the free form of the virus. By comparison, the DNA of H9v3 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 modalities of polyclonal integration of the virus. The photograph of FIG. 1, which represents three channels of migration of the gel illustrates the signals observable on the autoradiogram, which materialize the results of the process of the invention. In the case of the H9v3 line, there is a weak signal in the region of high molecular weight DNA which does not penetrate well into the gel wells; the main part of the signal is represented by a spot spread by weight (this spot is intense, since each cell of the culture comprises of the order of 50 to 100 viral particles); this spreading comes from the fact that the integration of the virus at the level of human DNA takes place in a polyclonal manner (there are multiple sites of insertion). In the case of channel l, this concerns an individual who is positive for the signal. Three autoradiographed bands are present: one in high molecular weight DNA, which does not enter the gel well; and two autoradiographed bands of approximate size 13 and 9kb corresponding to the free form of the virus, probably in two different conformations (rolled up and / or released) Some individuals are strongly positive (this is the case represented in figure l) with clear bands particularly marked, visible after 4 days of autoradiography. In the case of channel 2, this concerns an individual who is HIV positive, but negative for the signal (even after prolonged exposure).

These results show that the reading of the results obtained by the process of the invention can be done in a safe and reproducible manner to detect the presence of the HTLVIII-LAV virus in seropositive individuals. 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 positives, 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. no doubt reflect the number of viral copies present.

In an advantageous variant of the process of the invention, the samples are simultaneously deposited on the gel, some being undigested and the others digested with the enzyme such as Xbal. Typical results obtained are shown in FIGS. 2 and 3.

It can be seen in FIG. 2, which is similar to FIG. 1, but includes in duplicate, the undigested samples and the digested samples, that the signal obtained corresponds respectively to giant fragments, to a fragment integrated into 13 kb and a free fragment at 9 kb. In such an embodiment, there is first of all an indication of the presence of the virus in the undifferentiated sample, which is verified and confirmed after digestion.

FIG. 3 illustrates the influence of the duration of exposure for the characteristic bands at 13 and 9 kb, respectively at 10 days and 1 month.

Using the mode of implementation with double sample (undigested and digested), the method of the invention has been applied to 25 seropositive subjects. Each test was repeated twice. The results are collated in the following table. In this table: -the indication "^ / Tg sample" means the ratio between T ^ lymphocytes and lymphocytes

Tg, the measurement being made at the time of the sample and constituting a presumption that the subject is seropos i t i f.

the indication "HBS / HBC antigens" signifies the presence or absence of the antigens corresponding to the viral hepatitis.

Other ^'légences the table are common. The process of the invention makes it possible, as is apparent from the results of the table, to state that 16 individuals, or 64% of the total tested, are positive.

The fact that the three bands observed correspond well 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 H9v3 culture.

In the case of a control DNA taken at random from the population and digested with Xba I, the bands are not visible. Nor are they in the case of true 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 T ^ cells is very low, and most of the virus is in the form of RNA) .

The bands observed are not the result of a contamination of the probe by the phage ^ Digested by Xbal, this 1 u ic i gives two restriction fragments at 23.9 and 24.5 kb, clearly larger than those observed, as established in a control digestion of this type. On the other hand, the presence of free viral sequences in the lymphocytic DNA of HIV-positive individuals is proven by causing the DNA of these undigested individuals to migrate onto the gel, and hybridizing the corresponding channels with the probe: in most cases, a signal is obtained, naked, intense, in the undigested DNA (integrated sequences), another, much weaker, in a band of molecular weight di 10 kb (free form).

The subject of the invention is also a box or kit, containing the ingredients necessary for carrying out the process. The unit quantities of these ingredients will depend on the number of samples which it is intended to process. The box according to the invention essentially comprises:

an HTLV III-LAV probe, for example the probe corresponding to the π BH-5 subclone, excised from phage A,

a preparation of the phage n DNA digested with Xbal, used as a control,

-the enzyme XbaI in the enzyme preservation buffer, -the enzyme digestion buffer.

For marking, the products that can be used are, for example, those put on the market by the company Amersham France, Avenue du Canada 91944 Les Ulis France such as: - cold kit N55CO (nick-1rans 1at ion buffer and enzymes)

- \ p ³² dCTP (PB 10385) and <P ³² dATP (PB 10384) at 800 Ci / mmole (250 / t / Ci of each).

A cold variant of marking (for example marking by basic sulfonation currently marketed by the company Organics, 12 Avenue du Président Salvador Allende 94402 Vi try-sur-Se ine, France) can also be used. practice in situ hybridizations using cold labeling or radioactive labeling, for example H.

The invention will be further illustrated by a complete illustrative example of implementation. EXEM ^P LE: 20 ml of whole blood are taken from EDTA at..5% in physiological water. The plasma is removed by vacuum suction after centr i fugat ion for 5 minutes at 1500 rpm. The lysis of the red blood cells is obtained by adding to the fresh pellet 50 ml of SLR buffer (Tris ÎO rrM, MgCl ₂ , 5rτM, NaCl 10 rrM); homogenization is obtained until a lacquered appearance, then a centr i fugat ion is carried out 5 to 10 minutes at 2000 rpm; the supernatant is then aspirated with the vacuum tube. The proteinase K solution is prepared in the TE buffer (Tris-EDTA) at a final concentration of 0.2 mg / ml. The pellet obtained précé¬ ously is redissolved in 3 ml of buffer and SLR r ^'edissous again in 20 ml of PES buffer (Tris IO RRM RRM 10 EDTA, 50mM NaCl RRM, 0.2% SDS) and proteinase K at .3 mg per tube; a viscous block is thus obtained which is homogenized and then stirred gently (45 to 50 rpm in an orbital water bath) overnight at 42 ° C.

The phenol used for the extraction is saturated with 0.2 M Tris at pH = 7.6. A first extraction is carried out in 3/4 of phenol / 1/4 of chloroform and 1/24 of isoamyl alcohol; the upper aqueous phase is removed with a pipette. A second extraction is then prepared in pheno 1 -ch loroform; the two phases then separate and the DNA is in the upper phase; the lower organic phase is eliminated with a syringe. A third extraction is finally carried out to remove the phenol with chloroform + isoamylic acid. In the residual phase, the DNA is precipitated by adding 300 ^μl of 3M NaCl and supplementing with 50 ml of absolute ethanol at -20 ° C. The ethanol is removed with a pipette and the precipitated DNA recovered with an inverted pipette; rinsed several times with absolute ethanol. the DNA is resuspended in 2 to 4 ml of TE at 4 ° C for several days. It is dosed at DOogg _^"and ^e * control of the size is done by agarose minigel O, 7% and staining with ethidium bromide in the TEA buffer.

The indicated quantity of DNA for each sample is digested by the restriction enzyme Xbal in excess of 5 units per fg of DNA. The restriction fragments are then spread on a 0.8% horizontal agarose gel.

After migration the gel was depurinated (0.25 M HC1) ^', denatured (0.5 M NaOH in lM NaCl) and neutralized (0.5M Tris in 1.5 M NaCl at pH = 7.0). The transfer buffer is 10 × SSC, the transfer to the ni troce11ulose filter being carried out 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 constituted by the Sst 1-5 'fragment of

-BH-10. It is marked by nick-translat ion with dCTP + dATP at 8000 Cu / mole, or with dCTP alone.

The filter is prehybridized at 65 ° C in an orbital water bath in a sealed plastic bag containing: 25 ml of buffer 5x SSC (1 hour), then 5x SSC, 10 x Denhardt (D), 0.1% SDS (1 hour ), then 5xSSC, 10xD, lmM EDTA and O, 1% SDS added with

g / ml denatured and sonicated salmon sperm DNA (ss) (one nu it).

Hybridization with the denatured probe is carried out in 10 ml: 5xSSC, 10xD, 0, 1% SDS, 5nrM EDTA, 50rrM Tris-HCl pH = 7.5 and 10 mg / ml of ss DNA at 67 ° C in an orbital water bath for at least 24 hours.

After hybridization, the filters are washed in successive baths: (250 ml in a box at 66 ° C): 30 minutes in 5xSSC, lOxD, 0.1% SDS, 0.1% NaPPi, then 30 minutes in 3SSC, lOxD, 1% SDS, O, 1% NaPPI, then 15 minutes in 2xSSC, lxD, O, 1% SDS, O, 1% NaPPi and the time required (metering) in ISSC buffer, lxD, 0.1% SDS, 0.1% NaPPi. The filters are then dried under the lamp, fixed on Wathman 3Mv paper | and coated with a plastic film, then placed for autoradiography at -7O ⁰ C for 1 to 30 days in a cassette comprising two reinforcing screens.

The autoradiograms are revealed for 5 minutes, rinsed with water, fixed for 5 minutes, washed and dried in the dryer. The size of the autoradiographed bands is calculated relative to that of the size scale represented by the Hind I I I fragments of phage A DNA.

The example detailed above is illustrative but in no way limitative of the process of the invention. Variants on the operating mode can be made by the person skilled in the art, as has been illustrated previously with the mode of implementation with or without digestion (double sample).

The invention thus provides a sensitive method capable of detecting in vivo and under the conditions of practical reality, the retrovirus HTLV III-LAV, which is known to be present in minute quantities in seropositive individuals. Such a result is very advantageous and all the more surprising since the specialized literature teaches that such a detection technique cannot be practically used.

Furthermore, work on the probe was carried out under optimal conditions and only in vitro on culture cells, which is completely different from in vivo application.

Claims

o

CLAIMS 1. Method for screening, in HIV-positive individuals, DNA sequences of the HTLV III-LAV retrovirus, process in which a general molecular hybridization technique is applied comprising the following steps: a) the blood of individuals examined to isolate known manner the white cells and obtain DNA 1ymphocytai re, which is used in equal unit amount to at least 30 _/ ^ 'g, b) treating said amount of DNA lymphocyta i re by a restriction enzyme which does not cut within the DNA sequence of the virus, c) the medium of step b) which contains the virus in free or integrated form is passed through a filter of ni troce11 u lose capable of retaining up to 80 / C / g of DNA per cm of surface, d) the product retained by the filter is brought into contact with a probe, which is a probe cloned and labeled with the virus HTLV III-LAV, said probe being a subcloned fragment of the DNA of the virus which i does not include the coding part for the enveloped region of the virus, the labeling being capable of giving an activity as close as possible to the value g of 5.10 'cpm by> cg of DNA of the probe, in particular an activity of 5.10 ⁸ to 2.10 ⁹ cpm ^ g of DNA. e) the presence of characteristic bands of the virus is revealed in a known manner.

2. Process according to claim 1, characterized in that the restriction enzyme is XbaI or an equivalent enzyme.

3. Method according to one of claims l or 2, characterized in that the unit quantity of DNA 1 lymphocyte i is between 30 and 50 θ g envi¬ ron.

4. Method according to any one of the claims 1 to 3, characterized in that a probe corresponding to the subclone BH-5 is used, excised from phage utilise

5. Process according to any one of claims I. 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 1 to 5, characterized in that it comprises the main following steps: (1) extraction of the DNA lymphocyte i re long strand by a phenol-chloroform mixture after protein digestion by protei¬ nase K; (2) spectrophotometric assay of the DNA obtained, verification of its length on agarose minigel, and of its digest ib i 1 i ed by nucleic acid test; (3) digestion with the restriction enzyme Xba I, in excess of 5 units per> g of DNA to be digested;

(4) spreading the restriction fragments on 0.8% agarose gel in lOxSSC transfer buffer;

(5) marking of the probe by cut-off displacement at ³² PdCTP + dATP at 800 cu / mole for 250 y (Ci or at

PdCTP alone; (6) hybridization of the denatured probe with the 5xSSC buffer filter, lOxDenhardt, 0.1% SDS, 50 rrM EDTA, 50 rrM Tris-HCl pH = 7.5 with 10 mg / ml of sonicated salmon sperm DNA and denatured for 48 hours at 67 ° C; (7) final washing in lxSSC and 0.1% SDS tam¬ pon; (8) drying of the filter and its inclusion in a cassette comprising two reinforcing screens in the presence of sensitive films, the autoradiograph being carried out at -70 ° C; (9) revelation of the autoradiograph bands; (10) reading of the autoradiographed strips on an illuminating platform, using the phage DNA digested by Hi nd III as a size scale.

7. Box or kit for implementing the method according to any one of claims 1 to 6, characterized in that it partially comprises:

an HTLV III-LAV probe, for example the probe corresponding to the Λ BH-5 subclone, excised from the phage

-a preparation of phage DNA - digested with Xbal, used as a control,

-the enzyme XbaI in the enzyme conservation buffer,

-the enzyme digestion buffer.

8. Box according to claim 7, characterized in that the marker is radioactive or cold.