OA10208A - Recombinant gene including recombinant retrovirus defective and activatable by a transactivator - Google Patents

Description

1 010208

Recombinant gene defective and activatable by a transactivator. The invention relates to means (products andprocesses) applicable to the in vitro detection of the presence of an infectious retrovirus in a biological sample, especially in a sample from a patient or suspected carrier of the retrovirus. A preferred application of the invention is directed to the detection of an AIDS virus (HIV) or nucleic acid sequences of ceretroviruses in such biological samples.

In another variant of application, the purpose of the invention is to enable the study of the influence of external agents, in particular active principles of medicinal products, which can inhibit, at least modulate, the infectious character in vivo of the retrovirus studied

The development of a direct and ultrasensitive detection system, particularly HIV viruses, comes urgently. Sero-positivity tests involving the detection of antibodies against the HIV retrovirus, the potential carriers, may prove to be insufficient, in view of the observation that the desired antibodies have often appeared in the subject in question. -infected by the HIV virus after an incubation period of 12 to 18 months. Other tests currently used, such as the detection and assay of the reverse transcriptase activity of the virus possibly present in the samples studied, do not always have the degree of sensitivity required to conclude usefully with the infection or not the subject from which the sampling took place. . The object of the invention is to overcome at least a large part of the disadvantages of existing tests, in particular to provide extremely sensitive means and methods for detecting a retroviral infection and itself at a very early stage of infection. .

In particular, a first object of the invention is to allow the detection, i of the existence or not of a retroviral infection in a host (human or animal) susceptible to said retroviral infection, and this even at a very early stage. early evolution of this infection.

Another object of the invention is the study of the influence of external agents on the capacity of infection of cell lines by this retrovirus originating from this host (or related thereto), for example from active drugs to inhibit the in vivo infectiousness of this retrovirus. But these external agents may also consist of other factors, on the contrary able to accelerate the retroviral infection. Still further objects of the invention are to enable the detection of the ability of a specific retrovirus to infect a given cell line, or the identification of those of cellular clones which, at the heart of a particular culture. These viruses are irreversible by this retro-virus or the in vitro study of the poten-tial capacity of various agents, among which potential drug-active principles to affect the retroviral infection of susceptible cells exposed to the retrovirus studied. The invention exploits the ability of recombinant retroviruses (or recombinant genes derived from these retroviruses) containing an exogenous gene whose expression is sought to be incorporated into the genome of individual cells infected by the corresponding retrovirus and to allow Expression of the exogenous gene, if appropriate under appropriate activation conditions, in the infected individual cells to which the retroviral recombinant has been incorporated, wherein the exogenous gene is transmissible from cell-to-cell generation. The invention takes advantage of the fact that the gene activity of several retroviruses, in particular human and mammalian retroviruses (HTLV, BLV), generally depends on a trans-activation (or trans-stabilization) by a viral protein. encoded by a gene part of the retroviral gene. For example, the HIV gene activity of a trans-activation (or trans-HIV-tat gene (also known as "Q gene"), at least one of the lapar targets being constituted by a The endogenous protein coding equivalents in the region R, itself normally the LTR region of the retroviral genome. The trans-activation (or trans-stabilization) of the retrovirus under the control of the tat gene is an essential condition for an erosion. effective retrovirus, particularly at the protein level transcription of genes encoding gag, env and pol. The lack of transactivation is not a total barrier to viral replication capacity. Nevertheless, it can be estimated that the efficiency of reéplication under the effect of this trans-activation is 500 to 1000 times greater than that of a defective retrovirus at the tat region. of detecting the invention of an in-based on the idea of ensuring in already a gene sequence or a defective virus at the level of the region tat (or the analogous region with respect to another retrovirus ), the aforementioned trans-activation or trans-stabilization by an external input (in "trans") of a transactivator coded by the tat region of the retroviral genome of the retrovirus, if any

The method of retroviral cells containing 010208 present in study. Also for the result of the ransactivator, the sample or biological sample to achieve for this purpose, the invention provides means for carrying out this process and the revelation of the trans-activation thus in-duite in the aforesaid cells, as soon as when they have been in contact with the sample to be studied and in-fected by the retrovirus possibly present in this sample. The invention more particularly relates to a recombinant gene that can be incorporated into infectious cell lines paa: the retrovirus whose presence is sought after, this recombinant gene being itself activatable by a retroviral transactivator of the desired virus, and more particularly characterized in that it comprises a recombinant sequencefusion of an activatable sequence by said sequence being derived from the genome of the homologous retrovirus and fused to a heterologous marker, said recombinant sequence being itself placed under the control of a promoter present in the gene recombinant and allowing expression of the marker in a susceptible cell host, infectable by the corresponding wild-type retrovirus; in that this recombinant gene lacks the essential of the coding sequence for the aforementioned retroviral transactivator. The invention therefore also relates to the cells to which the aforesaid activatable recombinant gene has been incorporated, whether by transfection or infection of these cells with a vector containing the aforesaid recom-binant gene which can be activated under conditions allowing the expression of heterologous marker within this cell or cells.

Advantageously in a preferred embodiment of these cells, the recombinant gene is incorporated into their own genetic inheritance.

In a preferred form of the recombinant gene according to the invention, the recombinant gene contains a DNA substantially corresponding to the entire genome of a retrovirus comprising a gene activatable by a retroviral transactivator placed under the control of a promotion. However, this DNA is, on the one hand, devoid of the frequency coding for the retroviral transactivator and, on the other hand, provided with a heterologous marker placed under the control of the promoter, in this case the LTR. , normally associated with the sequences coding for the proteins Ssa and Sqv.

The sequences encoding gag proteins. env pol can be deleted at least in part. In other words, and more particularly in the case where the retro-virus concerned is an HIV virus, the corresponding recombinant gene contains the R region of an HIV retrovirus and is devoid of the tat region of an HIV retrovirus. More particularly, the invention relates to a defective recombinant retrovirus-derived or native retrovirus containing the cis-acting elements for activating transcription of viral protein-encoding sequences by transactivator-encoding another region of the retroviral genome. As well as the parts acting in "cis" of the wild-type virus and providing normal packaging of the retrovirus, its reverse transcription into a DNA molecule and its integration into the genome of the host cell, as well as the polyadenylation site of this RNA, this recombinant retrovirus being more particularly characterized in that it comprises: a deletion of at least a part if not all of the aforesaid coding region for the aforesaid transactivator of the wild or native virus, the released portion being sufficiently large that either the endogenous production, in the host cell, of a polypeptide having the property It has been necessary to activate the aforementioned activating elements and a sequence coding for a marker of host cells infected with the defective recombinant retrovirus thus constituted, either in the state incorporated in the retroviral region normally comprising the gag.moi and env genes. replacing all or part of this region.

Contacting a host cell thus infected with a wild or native virus preparation then induces the expression of the activating elements, thanks to the external input of the transactivator. When the tag encodes an enzyme acting on a seedable substrate, the labeled cells can be identified by their selective staining.

The promoter under whose control the activatable sequence is placed by the transactivator is a promoter capable of being recognized by the pelymerases of the infected cell host. The gene sequence may be substantially limited to the marker coding sequence under control; direct from the U35 1 region of the retrovirus LTR. In another variant, either or both of the LTR regions are devoid of promoter (s), the gene pool encoding the label then being under the control of a distinct promoter replacing the U3 region. so that the transcription is always done from the region Rtransactivatrice.

For example, a defective recombinant retrovirus conforming to the invention, derived from HIV, comprises successively; The LTR region R of the corresponding wild-type retrovirus, preceded by the LTR region J3 or a distinct substitu 010208 promoter substituted for the above, this region R being, where appropriate, followed by the region of packaging, the RNA initiation site corresponding to viral pro-proteins normally encoded by the gag, polet env genes, the coding sequence for the retroviral incorporated marker having the gag, col and env genes, or substituted for any or part of this retroviral region, a deletion or a mutation in the tat region, and the LTR region, which coincides with the polyadenylation site of the RNA.

The recombinant gene sequences or recombinant defective retro-vectors according to the invention can be manufactured by conventional techniques in genetic degeneration, in particular by in vitro recombination of the characteristic constitutive elements entering into the recombinant gene sequence or the defective retrovirus re-combining such as they have been defined above. In this regard, the invention also relates to the recombinant vectors containing the DNA of the defective-defined recombinant virus, incorporates any vector allowing the amplifi cation of the assembly in a competent cellular host, for example a bacterial plasmid. The invention also relates to cells or cell lines that are infected with the wild-type or native retrovirus corresponding to the retrovirus, some parts of which intervene in the sequence of the aforementioned defective retroviruses and gene sequences, these cells being themselves modified by incorporation into their own genetic patrimony. said gene sequences or recombinant proviruses corresponding to said defective retroviruses.

These indicator cells can be produced by any conventional method, in particular by infection of cells susceptible to infection by a recombinant defective retrovirus produced in vitro or by transformation of the same cells by a vector containing the recombinant gene sequence or the entire genome. of recombinant defective retro-virus.

The following observations may be made to the various components of the recombinant genetic sequence or defective recombinant retrovirus according to the invention.

The marker itself is advantageously constituted by an enzyme produced in the cells transformed by the defective recombinant gene or virus, in order to confer on it a particular coloration or the capacity to act on a substrate penetrating the transformed or disinfected cells, in particular to undergo a modification of coloring or more generally its absorption spectrum lightradiations, under the action of this enzyme A.titre examples of enzymes coded by the coding sequence, contained in the gene sequence implemented in the invention, it mention will be made of β-galactosidase, β-glucuronidase or luciferase. It goes without saying that any other coding sequence may be used as soon as it codes for a protein capable of playing. The role of a marker makes it possible to easily identify the transformed cells. The use of such types of markers in the frame of the invention is particularly advantageous, since it allows an individualized observation of cells to which the above-defined recombinant defective viruses or proviruses have been incorporated, in particular on the occasion of the revelation of the expression of the marker, for example under the conditions described below.

It is advantageous, in order to obtain a sensitive marking, to use the marker which will be located in a cell compartment which is easily identifiable with the cells studied, the whole forming the above-defined gene sequence, itself placed under the control of the C 1 LTR region of the retrovirus concerned. An avan-tagging gene sequence comprises the sequence "nls-Lac Z" described by Kalderon, D. et al (1984), Cell JjL, 499-509, and containing a signaling region of the antigen T (nls) of the monkey virus. SV40 fused to the Lac Z gene, this Nils-LacZ sequence being, for example, under the control of HIV LTr5. The corresponding fusion protein p-Gal is enzymatically active and remains associated with the nucleus of the cells to which such a gene sequence is incorporated, more particularly with the membranes of the cell nuclei of said cells, rather than accumulating in the nucleoplasm. This gives a maximum sensitivity. When the indicator line is infected with the wild-type virus, expression of the LacZ gene can be easily detected histochemically at the cell level. Each cell of the infected indicator line LacZ then corresponds to a pgal + cell which can be detected by histochemical labeling in situ.

Advantageously, the promoter under whose control the marker coding sequence is placed is a weak or weak promoter. In particular, a weakened pro-motor derived from the sequence of the LTR virus HIV is constituted by this LTR itself, partially dé-lété. In particular, this promoter can be deleted from part of the region 03, without suppressing all the promoter activity of the region 03.

Indeed, as indicated above, the deletion of the tat region does not form a complete obstacle to virus replication, in which case the marker protein may sometimes be slightly expressed in a cell line infected with the defective retrovirus. The weakening of the promoter is intended to make the expression of the marker even less in the absence of contact between these cell lines and an external medium containing the wild-type retrovirus. This is only an unnecessary enhancement, since even with a non-deleted promoter the degree of expression of the marker is higher when these cells are brought into contact with a wild-type retrovirus supply. only in the case where it is protected that confusion between the degrees of marking observable in the two cases is practically impossible.

The method according to the invention for the detection of a retroviral infection in a sample containing optionally infected cells (such as a human serum sample containing T4 cells, in order to assess whether it is infected with an HIV virus) or the medium, such as a culture supernatant, with which these cells had previously been in contact, is characterized by the steps of: contacting this sample with the cells containing the gene sequence according to the invention, in particular a defective provirus capable of itself being transactivated by the external contribution of retroviruses possibly contained in the sample, and this under conditions (in particular of temperature and incubation time appropriate) allowing the infection of said cells with leretrovirus possibly present in the sample, and detecting the possible infection of said cells by revealing the expression of the marker resulting from the retransactivation of the gene sequence or of the de-fective provirus present in these latter cells, using a suitable reporter.

Such a method permits the very sensitive detection, within a very short time, of the presence of a retroviral infection in the initial cell sample that was to be tested, and even the costimation of the degree of retroviral infection in the patient. the sample tested, at the time the test was performed.

It will be appreciated that this test also makes it possible to study the evolution of the infection for a patient suffering from wild retrovirus-induced disease, if it is repeated over time, on samples of sera, any other fluid or infected biological tissue from of cepatient.

This method can also be used for the study of the possible efficacy of a drug active ingredient vis-à-vis the interaction between the retrovirusconsiderere and sensitive cells, especially when lasusdite put -in-indicated contact is operated in presence of this active ingredient. It is appreciated that the test as modified allows both to select the drug active ingredient that could be the most active, to determine the doses that could be effective to treat the patient who provided the sample, these doséseffaces resulting in particular from those who, in the setting The above-mentioned test does not allow the infection of cells containing the defective retrovirus or provirus in contact with specimens from the patient to be treated or the supernatant of the medium in which this cell sample had been maintained.

More generally, the invention allows the invitro study of the influence of factors likely to interfere with the viral infection, the above-defined method thencontaining the cells containing the gene sequence or the recombinant provirus according to the invention. invention, in the presence of this factor, with determined amounts of external retrovirus and the detection of the doses of factors, in particular drug active principle, which precede the retroviral infection, detectable by the activa-tion of the marker , said cells. The invention therefore also relates to necessary devices or kits for carrying out the method that must be described, these kits comprising: modified sensitive cells containing the defective retrovirus or defective recombinant provirus, revelation means the expression of the marker, especially when the marker is an enzyme, a specific substrate of the enzyme, optionally the buffers and reagents necessary for the realization of the contacting, with the biological sample, of the cells containing the gene sequence or the recombinant retrovirus defective to be studied and incuba-tion of the reaction mixture under conditions allowing the possible infection of these cells in contact with the sample studied, - where appropriate conservation media or cultured said healthy and modified cells.

Advantageously, the indicator cells used in these kits are constituted by "immortalized" cell lines that are infectable by the corresponding retrovirus, for example, in the case of a kit intended for the detection of an HIV virus, the Modified sensitive cells and healthy cells would both belong to cell lines of the type CEM, HUT, etc.

In the examples given below, the possibilities of the invention are illustrated in connection with the production of a defective recombinant virus derived from an HIV-1 virus.

It is therefore quite clear that the invention is directed to the detection of any type of retrovirus, possibly infectious with respect to specific cell cultures and having the essential structural characteristics discussed above. In particular infectious retrovirus, it applies to the manufacture of defective recombinant retro-viruses allowing, under the conditions that have been indicated, the detection of the power (or the nodulation of infectivity) known under the names HTLV I , HTLV II, HIV I (or LAV or HTLV III), HIV II (or LAV II), HTLV IV. It is still the same for many retro-viruses infectious with regard to the animal. By way of example, mention may be made of leukemic retroviruses of cattle (Bovine Leukemia Viruses) and felines (FeLV).

Additional features of the invention will become apparent in the description of embodiments, applied to the production of recombinant defective recombinant viruses according to the invention derived from HIV-1. Other objects of the invention and the means for achieving them will still result from the description of some of the embodiments presented, either by way of preference or as an illustration of the possibilities offered by the invention for the invention. study of all aspects of the various possible interactions between certain wild-type retroviruses and indicative cells that are detectable by these retroviruses and the modulation of these interactions.

In the following, reference will be made to the figures in which: FIGS. 1a and 1b schematically show the characteristics of recombinant gene sequences in accordance with the invention; FIGS. 2a, 2b and 2c are diagrammatic representations of examples of defective recombinant retroviruses according to the invention.

Figures 1a and 1b are illustrative of two recombinant genes according to the invention, both lacking the region âcfc. The recombinant gene of FIG. 1b differs from that of FIG. 1a by a partial deletion of the LTR. These genes, incorporated into the legenoma of cells susceptible to HIV infection, are functional when the cells in question are placed in contact with a source of wild-type HIV virus; EXAMPLES test for detection of a wild-type retrovirus of the HIV type by trans-activation of a defective copy integrated into an infected cell The recombinant DNA whose expression must be amplified by the presence of the product of the external tat gene possesses the following elements: a promoter (pro in FIG. 2a) allowing the transcription of a sequence that may be ex-awarded, placed under its control; 2 'the R region of HIV-1 to the Hind III site (nucleotide # 83 according to WRIGHT et al., Science 234. p.988 or nucleotide # 77 in CSH, RNA tumor virus, volume 2, p.1 104, 1985) as demonstrated in the article by WRIGHT et al., Science 234, p. 988. This fragment indeed contains the elements which confer in cis the trans-activation by the tat gene products; A marker gene such as, for example, the 3.3 kb fragment of the lacZ nls gene as described in the article by KALDERON and SMITH; 4 'a polyadenylation signal of RNA.

This structure is shown schematically in Figure 2a.

Another particular example is given in FIG. 2b in which the recombinant sequence comprises the complete LTR of an HIV-1 retrovirus thus comprising the promoter (U3), the cis (R) region required for the trans-activation, fused by genetic recombination with a nlsLacZ fragment extracted using restriction enzymes of the L7RH vector (3gal described in the article by KALDERON and SMITH before being placed by in vitro ligation under the control of the R region of the LTR and linked, also by ligation, to the 010208 polyadenylation signal of the HBV virus,

Another particular example is given in FIG. 2c where the infectious provirus DNA described in the article of M. MARTIN et al. (Journal of Virol., Vol 59, pp. 284-291) was fragmented by the restriction enzymes Sph1 (nucleotide # 989 of the sequence published in RNAt.umor viruses, Cold Spring Harbor 1985) and BamHI (nucleotide). 832, same reference) as shown in Figure 2c and where the lacZ nls gene (SalI BamHI fragment of plasmid pMMuLVnls LacZ) was put in place of gag. pol and approx.

These fragments are cut, isolated and lined as indicated in MANIATIS.

The plasmids described in FIGS. 1a, 1b or 2a, 2b, 2c can be introduced, preferably by co-transfection, using the usual methods described for example in GRAHAM and VANDER Eb 19873, Virclogy 12 "456 and NICOLAS and BERG , CHS, Cell proliferation, 12 "469, in cells infected with wild-type HIV heterotirovirus, such as LeuKOK4 cell lines referred to in the article by MALCOLM MARTIN or CEM (CNCM 1-416 and CNCM 1-417) or SupTI.

Regardless of the recombinant gene introduced, it will only result in minimal expression of the LacZ gene in the cells into which it has been introduced. This indicator cell has one or more LacZ constructs. These cell cells (forming a plurality of indicator copies can then be used in a biological sample of an indicatricetrans cell) of the LacZ gene and, by detection assay in a wild-type virus. The infection will lead to activation (hence its expression).

This activation can be demonstrated by the histochemical test based for example on the use of X-gal according to the technique described by SANES et al. Embo J. 1986) which allows in situ detection of p-galactosidase activity. Each event of infection with a wild virus will correspond to a detectable βgal + cell more than 48 hours later.

Viruses present in the humors or cells or even in human embryos can be detected very rapidly, and at a very early stage of the retroviral infection. i

Claims (11)

17 "10208 resulting from the trans-activator, placed under recombinant recombinant 1. Recombinant gene that can be incorporated into cell lines that are infectable by the retrovirus whose presence is sought, this recombinant gene being itself reactive by a retroviral transactivator of the retrovirusesearched, characterized in that it comprises a recombinant sequencefusion of an activatable sequence by this sequence being derived from the genome of the homologous retrovirus and fused to a heterologous marker, said recombinant sequence being itself a control of a promoter present in the leet and allowing the expression of the marker in a susceptible cellular host, infectable by the corresponding wild-type retrovirus ; in that this recombinant gene lacks the essential of the coding sequence for the aforementioned retroviral transactivator. in that the marker itself is constituted by an enzyme which, when produced in the cells transformed by the defective recombinant gene or virus, a particular coloration or capacity, a substrate penetrating the transformed or infected cells and undergoing a change in coloring; or more generally of its spectrum of absorption of the luminous radiations, under the action of this enzyme. give him to act on 2. Recombinant gene according to claim 1, characterized in that it contains a DNA corresponding essentially to the entire genome of a retroviruscomportant a gene activatable by a troviral trans-activator placed under the control of a promoter, this DNA is, however, d on the one hand, lacking the coding sequence for the retroviral transactivator and, on the other hand, provided with a heterologous marker placed under the control of the promoter, in this case the LTR, normally associated with the sequences coding for gag.Do1 and env proteins. 3. Recombinant gene according to claim 1 or larevendication 2, characterized in that it is derived fromHIV, 4. Recombinant gene according to any one of claims 1 to 3, characterized in that it is consti-killed by a defective recombinant retrovirus comprising. a deletion of at least a part, if not all, of the aforesaid coding region for the wild-type or native virus susidiotransactivator, the partial elongation being sufficiently large that the endogenous production, in the host cell, of a polypeptide having the property of activating the aforementioned activating elements and a sequence coding for a host cell marker infected with the defective recombinant retrovirus thus constituted, either in the state incorporated in the retroviral region normally comprising the gag, pol and env genes. replacing all or part of this region. Defective recombinant retrovirus according to any one of claims 1 to 4, characterized in that it comprises: the LTR region of the corresponding wild-type retrovirus preceded by the U3 region of the LTR or a distinct promoter substituted for the preceding one, this region R being, where appropriate, followed by the packaging region; the RNA initiation site corresponding to the viral pro-proteins normally coded by the gag poland £ QV genes; the coding sequence for the marker incorporated into the retroviral region comprising the gag, pol and env genes, or substituted for all or part of this retroviral region, such as cells, had a deletion or mutation. in the region tat. and here LTR region, which coincides with the polyadenylation site of RNA. 6. Recombinant gene according to any one of claims 1 to 5, characterized in that the marker is 0-galactosidase. 7. Cells or cell lines that are infectable by wild-type or native retrovirus corresponding to the retrovirus, some parts of which intervene in the sequence above said defective retroviruses and geric sequences according to any one of Claims 1 to 6, these cells being themselves modified by incorporation into their own genetic inheritance of said recombinant defective gene sequences or proviruses corresponding to said defective retroviruses. 8. Method for the detection of atravirus infection in a sample containing potentially infected cells (such as a human serum sample containing T4 cells, in order to assess whether it is infected with an HIV virus) or the mid-supernatant culture, with which these previously been in contact, characterized by the steps that constitute: the contacting of this sample with the cellscontenant the recombinant sequence according to any one of claims 1 to 6, including a defective provirususceptible to be himself transactivated by the external retrovirus carrier optionally contained in the sample, and under conditions (including temperature and incubation time appropriate) for the infection of said cells by the retrovirus optionally present in the sample, and - · the detection of the eventual infection of said cells by revealing the expression of the resulting marker transactivation of the gene sequence or the defective provirus present in these latter cells. 9. The method according to claim 8, characterized in that the marker consists of a LacZ gene and the expression of the LacZ gene is detected by a specific substrate for β-galactosidase. 10. The method of claim 9, characterized in that the aforesaid contact is made in the presence of a principle whose ability to interfere with the infectivity of the retrovirus considered 11. Kit or kit for implementing the method according to claim K), this kit comprising: - modified sensitive cells containing the retrovirus or defective recombinant provirus above-defined means for revealing the expression of the marker, notarunent a substrate specific to the enzyme, optionally the buffers and reagents necessary for the realization of contacting, with the biological sample, the cells containing the defective recombinant gene sequence or retrovirus to be studied and the incubation of the reaction mixture under conditions allowing the possible infection of these cells in contact with the sample studied, - if appropriate conservation media or cultured said healthy and modified cells.