JPH04501210A - Cell lines that can be infected with retroviruses and contain markers that are activated upon infection - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] 3. Indicator cells are usually transformed with the complete HIV sequence of these cells. and the ability to fuse with each other to form a syncytium when expressed. It is derived from a certain cell and therefore has low spontaneous fusion ability and is an infectious natural retrovirus. recombinant vector or DNA modified with a complete envla sequence It is characterized by showing great fusion ability when transfected with A. The cell line according to claim 1 or 2, which is a cell.

4. For example, from HTA strains such as HT4 LacZ4 (CNCM l-899) The cell line according to any one of claims 1 to 3, characterized in that the cell line is a cell line that has been grown from the United States.

5. The marker imparts a specific color to the indicator cells or invades into these indicator cells. When exposed to light, it causes an in-situ color change, or more generally a change in the optical radiation absorption spectrum. from claim 1, characterized in that it encodes an enzyme that confers the ability to act on a substrate. 4. The cell line according to any one of 4.

6. Cell line according to claim 5, characterized in that the 68 marker is the sequence LacZ.

7. A device according to claim 6, characterized in that the marker has the sequence n1sLacZ. Cell strain.

8. The recombinant gene sequence has a second protein immediately upstream of the marker in the direction of transcription. According to any one of claims 1 to 7, comprising a motor. cell line.

9. Using the indicator cell according to any one of claims 1 to 8, (e.g., to determine whether they are infected with the virus FIIV) human serum collection containing lymphocytes T4) or previously contacted with these cells. A method for detecting retroviral infection in a medium such as a culture supernatant comprising: - If a retrovirus is present in the sample, the indicator cell will be infected with that retrovirus. The sample can be processed according to the invention under conditions (particularly at appropriate incubation temperatures and times) such that contact with an indicator cell of - Clarify the amplified expression of markers and, in some cases, the formation of syncytia. detecting whether the cell is infected by A method characterized by:

10. A kit for carrying out the method according to claim 9, comprising: - an indicator cell as described above; −Means for determining the expression of the marker, especially if the marker is an enzyme, - contacting the indicator cells with the biological sample and the reaction mixture; Conditions such that indicator cells that come into contact with a test sample become infected (in the presence of a virus) Optionally also includes buffers and reagents necessary for incubation with - Possibly a medium for preserving or culturing the aforementioned healthy and modified cells. A kit comprising:

Specification Cell lines that can be infected with retroviruses and contain markers that are activated upon infection The present invention is directed to biological samples, particularly humans suspected of harboring retroviruses. Infectious retrow in samples taken from patients or carriers or infected animals Means that can be used to detect the presence of viruses in vitro (activated cell 0 present invention related to (cellules activatrices> and method) One of the preferred uses for infectious or defective AIDS virus (HIV or 5IV ) is detected.

Another application of the present invention is to reduce the effects of foreign substances, particularly in vitro test retroviruses. may suppress or at least modulate infectivity in vivo or in vitro It is also used to investigate the effects of active ingredients in medicines.

The development of ultra-sensitive systems to directly detect viruses, especially the HIV virus, is still underway. There is an urgent need in the fields of science and medicine.

French patent application No. 8714384 claims that the host (human or animal) is infected with a retrovirus It is possible to detect infection in vitro even at an extremely early stage of the infection process. In particular, human and higher mammalian retroviruses such as HTL The gene activity of V, BLV, etc. is usually caused by endogenous Transactivation (trans- activation, or trans-stabilization For example, retroviruses In the case of HIV, the gene activity of the virus is determined by the gene HIM-tat (r gene q depends on trans-activation (or trans-stabilization) by do. At least one of the targets of the protein encoded by the gene tat (sequence t ar) is usually found in the R region included in the LTR” region of this retrovirus gene. Consists of an array. Transactivation of retroviruses under the control of the gene tat (or trans-stabilized) specifically encodes proteins g&g, env and pol One of the important conditions for efficient replication of retroviruses at the gene transcription level. It is one.

The method for detecting retrovirus infection described in the above-mentioned European patent application For example, if a biological sample that may be infected with HIV is treated with a defective combination such as Transformed cells containing the modified gene or provirus within their entire genetic material (hereinafter referred to as (referred to as cells). said defective recombinant gene or prowi rus encodes the retroviral transactivator, especially HIV-tat. Although essential parts of the retroviral transactivator sequence have been deleted, contains a recombinant sequence resulting from the fusion of a heterologous marker with a sequence that can be activated by I'm reading. This recombinant sequence is transmitted to the retrovirus during infection with a wild retrovirus. virus in the corresponding region of the retroviral genome (particularly tat in the case of old V). said by exogenous (“trans”) introduction of a coded transactivator. It is under the control of a promoter that causes the marker to be expressed in the indicator cell.

A preferred form of the recombinant gene derived from HIV as described in said European patent application is: - DNA corresponding to essentially the entire retroviral genome, including the promoter is activated by the tat retroviral transactivator placed under the control of This gene (especially the R region of retrovirus IIV) is included in the tat sequence. essential parts, and in some cases proteins gag, env, pol, etc. DNA that does not contain the deleted part of the coding sequence, - protein gag, pol and under the control of the promoter normally associated with the env encoding sequence. If the case is a marker placed under the control of LTR5°, the defective recombinant gene or is a marker encoding an enzyme produced in the indicator cell transformed by the virus. The enzyme imparts a specific color to the indicator cells, or an in situ change in color or, more generally, a change in the optical radiation absorption spectrum. This type of enzyme is preferably one that gives the ability to act on a substrate that causes Examples include β-galactosidase, β-glucuronidase or luciferase. It will be done.

The advantage of marker-containing indicator cells as mentioned above is that marker expression is clearly When these indicator cells are infected with the test retrovirus, Cells containing the defective recombinant gene or provirus can be observed individually. It is in.

It is advantageous to use markers localized within the cell chamber that allow for easy identification of indicator cells. Therefore, in the prior art, Ce1l, 39, 499-509 (1984) The gene referred to as rnlsLae ZJ by Kalderon, D., et al. Suggestions have been made that it is preferable to use arrays. This sequence is gene La c Localization signal region of antigen T (nfs) of simian virus SV40 fused to Z The whole cell is under the control of, for example, the HIV LTR5'. corresponding fusion The protein nfs-β-Gal has enzymatic activity and does not accumulate in the morphotype, cells that contain this kind of gene sequence, and more specifically on the membrane of the cell nucleus of said cells. maintain a consistent state. Therefore, this arrangement improves marking sensitivity. Ru.

This type of defective recombinant virus (pHIV-1n1sLaeZ, hereinafter referred to as 1 recombinant virus) Indicator cell lines transformed with It is shown. If this indicator cell line is infected with a wild virus, the gene La Expression of cZ can be detected histochemically at the cellular level. infected instructions Each cell of the LacZ cell line is clearly marked with histochemical markings in situ. The cell βgal” that is clarified corresponds to the cell βgal”.

The activator of the gene n1sLaeZ introduces the sequence tat in a different form in trans. for example by transforming said indicator cells with a plasmid containing said sequence. Therefore, it can also be induced within these indicator cells. C. Bennerot et al. is Ie C,R.

^cad, sci Paris, T, 307. S&rie111. p, 311- 316.1988, cells of the transformable strain were transformed with pHIV-1n1sLacZ. defective recombinant retrovirus of type (hereinafter also referred to as recombinant) and sequence ta A plasmid containing T (M, The E, M, B, 0J by Emer+san et al. own-al, 6, 1987. p, described in p. 3755-3760 A similar effect was observed when co-transformed with LET or pSET). ing.

C. Bennerot et al. also tested cells of the same type of cell line with two other recombinants. retroviruses, i.e. (B) HIV-1n1sLaeZta Engineering and (C) HIV-I 5VnlsL It has been reported that similar observations were made in experiments involving co-transformation with aeZ tat. Ru.

Recombinant B has a non-deleted portion of the early retrovirus downstream of the LacZ sequence. It differs from a recombinant in this respect. R, N, ^.

Tumor Viruses, Cold Spring Harbor Lab oratory, ColdSpring Harbor, N.Y., 1985 According to the paper by R. Leiss et al. described in A gene containing a nucleotide sequence extending between nucleotide 5289 and nucleotide 9194. This sequence of nom HIV-1 encodes tat, rev, nef, env It contains a gene that targets the protein REV and a sequence targeted by the protein REV.

Recombinant C has a complementary promo downstream of the sequence n1sLacZ and adjacent to the sequence LaeZ. recombinant B, except that it contains a promoter (hereinafter referred to as an internal promoter). It's the same. That is, in this recombinant, the sequence LacZ is located in the internal promoter. It is also under control. In this structure, the internal promoter is of SV40. Corresponds to the promoter of the early region (rI!gion pr&coce).

Attached are the structures of the two retroviruses mentioned later and the retrovirus mentioned first. This is briefly shown in Figure 1 of the drawings.

In FIG. 1, the sequence n1sLacZ is indicated by a gray bar. These arrays are It has sequences derived from the proviral copy of virus HTV-1 at the 3' and 3' positions.

These recombinant retroviruses contain the complete viral LTR (white bar in Figure 1). The sequence between the U5 and 5ph1 sites of these three recombinants is the same as that of the gag gene. For the 0 recombinant containing the beginning, there is a link between the BamHI site downstream of LacZ and u3. In the zero recombinants (B) and (C) whose sequences only contain information about the gene nef, Ee The sequence between the oR1 site (downstream of LacZ) and 03 is the gene tat, rev, Parts encoding nef and env and target sequences used for regulation by REV The initial region of SV40, including the columns, is indicated (in (C)) by a short black bar. 0 sites 5phI and EcoRI were destroyed during formation. Details of these structures Please refer to the paper by Bonnerot et al.

Using the three recombinant retroviruses and plasmids psET and hippLET, Bonnerot et al.'s observations when transfecting the cell system used. Based on the findings, the transactivator encoded by the tat region of pSET and LET When introduced in trans, the stimulation of β-galactosidase was shown by the results of the previous patent. only seen in indicator cells transformed by recombinant retrovirus 8. but also observed in indicator cells transformed by recombinant retrovirus B. 1. Indicator cells transformed with recombinant retrovirus B. was already observed to be expressed to a certain extent before transduction, but this expression Of course, the stimulation induced by the internal tat sequence of this recombinant retrovirus (internal This is the result of local stimulation). On the other hand, there is a complement immediately upstream of n1sLaeZ. In the case of cells transformed with recombinant C containing a foot internal promoter, The clearly increased expression observed under the action of internal stimuli is due to the transtransduction. It does not seem to be increased by external stimuli.

The present invention is based on the following discovery. - Contains receptors for retroviruses and has the following gene sequence, i.e. A sequence derived from a retrovirus or a homologous retrovirus; The internal tat region encoding the virus transactivator and the tat-encoded Can be activated by trans-activators or trans-introduced activators Cells transformed with a gene sequence containing the tar region at the same time are gene rearrangement that confers it-characteristics (rl!arrange+5ent g!n For example, the above-mentioned rB, type structure The converted cells do not express the gene LaeZ after the rearrangement, and are "C" type structures. In the case of cells transformed with β-galactosidase expression decreases compared to when it is not used, and background noise is reduced. It has come to the point where it can be seen as the same as Z. Infection of cells in the presence of infectious retroviruses When transactivation occurs, the marker is activated in the case of transformation with rB, type constructs. expressed, and the expression rate of the marker increases in the case of transformation with the “C” type construct. Ru. Marker expression rates observed before contact with an infectious agent and markers observed after contact - The difference in car expression rate is extremely large, more than 10 times, and often 50 to 100 times higher. twice as much.

- contains within the entire genetic material the same type of nuclear sequence as that of the recombinant (C) and therefore In addition to the sequence tat and the following regulatory markers, namely LTR5', a separate internal protein Even in the case of indicator cells that simultaneously contain regulatory markers that are also under the control of motors. , marker-containing indicator cells become native retroviruses or homologous retroviruses. When infected, the expression reserve (r6serve d’express) of the marker ion).

- due to the effect of internal stimuli, possibly maintained by complementary internal promoters The natural expression of the marker that occurs may be due to the presence of the corresponding retrovirus in the biological sample. In vitro diagnosis of whether infection by homologous retrovirus exists or not Natural expression of markers and infectious retrovirus under experimental conditions performed at early stages of infection. It is possible to control the expression so that it can be clearly distinguished from the expression induced by the presence of the virus. Ru.

This control is achieved in the present invention by using a recombinant vector of this type with a corresponding natural retrovirus. The markings are then clearly observed in other m cells. This can be carried out by selecting cells that do not appear as indicator cells. This selects When these indicator cells are infected with natural or homologous retroviruses, This is because it indicates the “marking reserve” revealed by amplified marking. Ru.

Therefore, the present invention more particularly includes receptors for retroviruses and the following: recombinant gene sequences, i.e., identical or homologous retroviruses. The internal tat region is derived from a virus and encodes a retroviral transactivator. and the transactivator encoded by the tat region under the control of the first promoter. natural receptors that are activated by sexualization factors (internal stimulation) or that infect the indicator cells. transactivation introduced by retroviruses or foreign retroviruses A recombinant gene sequence containing a tar region that can be activated (externally stimulated) by an activation factor. , preferably in the form of an integral part of the genome, in the total genetic material of the indicator cell line. . This indicator cell is such that the recombinant gene sequence has a marker such as is the control of a second promoter, an internal promoter different from the first promoter. such that the marker is activated as a result of activation of the region tar. Also includes a marker inserted between the regions tar and tat under the condition, and The activation of the tar area by the action of internal stimulation is caused by the activation of the tar region by the action of internal stimulation. Cells that cannot be activated by both stimuli or It is characterized by being a cell that has been In other words, the detection of retrovirus infection is , whether or not the transactivator tit has been trans-introduced into the indicator cells of the present invention. assessed by the differential intensity of marker expression in these indicator cells, depending on the be done. Therefore, in the following explanatory text, we will discuss the -King (marquage diff!-rentiel) J is sometimes mentioned. ing. In one preferred embodiment, the internal tat) transactivator The resulting internal stimulus causes genetic rearrangement in the indicator cell, giving the cell the tat-characteristic. As a result of genetic rearrangement, it becomes zero.

The above-mentioned "gene sequence derived from homologous retrovirus" means that the retrovirus is The same retro virus as the "infectious retrovirus" that is specific to the CD4 receptor of indicator cells. It means a sequence that may not be a virus. However, these retroviruses must be functionally equivalent; in particular, the region tar must be functionally equivalent to the infectious retro It must be able to be activated in trans by the tat region of the virus. No - for example, if the infectious retrovirus in question was Htvz or even older v-1. However, the retrovirus from which the recombinant gene sequence is derived is derived from SIV. There are cases where you do. Also in the regions tat and tar contained in the same recombinant sequence. The same can be said. Although these regions can be derived from two different retroviruses, , must be functionally homogeneous.

Indicator cells of the invention may be formed by the following steps: i) Cells containing retrovirus receptors are injected with the same retrovirus or homologous receptors. An internal virus derived from a retrovirus and encoding a retroviral transactivator t which can be activated by a tat region and an activator encoded by the tat region. Transfection with a recombinant gene sequence containing both the ar region and a marker sequence. action. The marker generates a marker gene by activation of the tar region. or so that this marker is under the control of an internal promoter. is located between regions tLt and tar and is therefore expressed without transactivation. Ru. The gene sequence may also include the retroviral gene env.

ii) Cells endowed with tat-characteristics by gene rearrangement among transformed cells Select. The taj characteristic is that expression of the marker gene leads to transactivation of the tar region. In cases of dependence, the phenomenon appears as a phenomenon in which the marker gene is not expressed at all. child On the other hand, if the expression of the marker gene is controlled by an internal promoter, Due to the phenomenon that the expression rate is lower than that of cells that have not undergone rearrangement, the tadji characteristic is shown. If the gene sequence also includes the gene env, this rearrangement env-characteristics are given. This env-characteristic has low suitability for natural fusion. and when cells are infected with a retrovirus or infected with a vector containing the complete env sequence. When dyed, it appears as a phenomenon that shows great fusion aptitude. This env-characteristic Select cells with .

1ii) For each cell selected in (ii), transactivation of the labeled gene to investigate whether this leads to differential intensities of expression and, in some cases, syncytial forms. Check to see if it is connected to success.

As will be apparent to those skilled in the art, it is clear that the transformed cells exhibit "differential markings" under the conditions described above. In order to select cells that give a by means of a suitable vector containing the recombinant gene sequence that has to be integrated into the plasma. When performing transformation in these cell lines, the cells used are Parameters can be changed depending on the type.

Indicator cells of the invention are typically transformed by full env expression of HIV and Recombination of cells that exhibit fusion fitness and form syncytia when this sequence is expressed These instructions are preferably formed by transformation with the genetic sequences. When infected with a virus or modified by a complete env mother or It is characterized by showing great fusion suitability when transformed with recombinant DN8. .

A selection protocol that can be used to obtain indicator cell lines of this type is summarized below. child The protocol will be followed later in the transformation culture of HT4LaeZ type cells. Cell selection will be explained in more detail.

(1) Plasmid transfer by the method of ManiaLis et al. (1982) ection.

(2) Passage when cells reach confluence R (1-10xlO'/c cell 2). 2 Dissociation occurred in 1/3 of the week.

(3) “Cell sorter (cell 5-orter)” CNoan et al., 1988 ) Selection of cells LacZ"" using a cell sorter (or any other method) .

(4) After isolation of clones and division of these clones, perform the procedure for each cloned cell. The following tests: (a) Unfused state and nuclear coloration (visual) when cells were kept confluent for 3 days. inspection).

(b) transactivation and fusion of colored cells after transfection or infection; This is investigated in particular by co-cultivation with cells infected with retroviruses.

(5) Results in colored and fused cells when tested under conditions 4(b) Cell clones are maintained as indicator cells that can be used in the present invention. On the contrary, 4(b) Conditions that did not cause fusion and/or did not induce transactivation must be discarded.

As in the case of the recombinant retrovirus of European Patent Application No. 87.14384, this The recombinant nucleic acid sequence of the invention has the effect of a transactivator encoded by the sequence tat. All elements that act in ``cis'' to activate transcription of the sequence tar are shown below. Including, and in some cases additionally, acting on the ``cis'' of wild viruses and usually retroviruses. Viral packaging (empaquetage) and transactivation factor t The first promoter controlling the a sequence tar that can be activated by at A promoter that can be recognized by a competent cellular host polymerase. array t ar can be placed under the direct control of the U3 region of the LTR'° of retroviruses. Ru. Alternatively, one or both of the LTR regions lack a promoter. If transactivation region R is used, the sequence tar and a marker that places the marker under the control of another promoter that replaces region U3. encodes a colorable enzyme or a sequence that acts on a colorable substrate, as described above. Preferably consisting of a column 0 normally disclosed as preferred in said European patent application. The characteristics described are supersedes of those described for defective recombinant retroviruses. In other words, features other than those directly associated with the region tat (deletion) are also preferred in the present invention. For example, nuclear localization peptides of genes LacZ and SV40 can be used as new features. One of these is the fusion with the code sequence (nlsLacZ).

An internal promoter immediately upstream of the marker sequence (if a marker N sequence is present) The promoter or second promoter is any promoter that can be recognized by the polymerase of the cellular host. It may be a promoter of any kind.

Recombinant strain HT4LacZ-1 was acquired on July 27, 1989 with No. l-899. Ia Co11ection Nationale des Cultures de I’1stitut Pasteur de Paris (CNCM) Deposited in.

The present invention uses the indicator cells to identify samples containing cells suspected of infection (e.g. If you want to check whether or not you are infected with the virus HIV, use human cells containing T4 lymphocytes. (serum collection) or in a medium such as culture supernatant that has been previously contacted with these cells. It also relates to methods for detecting retrovirus infections.

This method is characterized in that it includes the following steps: under conditions (particularly suitable contacting the sample with indicator cells of the invention (at a suitable incubation temperature and time); - Marker By revealing amplified expression, it is detected whether the cells are infected or not.

Using such a method, retroviral infection can be detected in the initial cell sample being tested. The presence of The degree of retrovirus infection in a sample can be determined at the time of testing.

Amplification of transactivation can be observed in the same indicator cells in the presence of uninfected samples. The amplification can be 10 times or more compared to the amplification that can be regarded as background noise, and is preferable. 50~1o.

It's double.

This test is used to detect infection in patients with disease induced by wild retroviruses. It can also be used to check progress. In that case, blood drawn from the patient or other infected body fluids or tissue in the previously described manner at intervals. and inspect.

The inventor has provided instructions #1II12! of the present invention! , especially the aptitude for natural fusion is small. Furthermore, cells that exhibit great fusion aptitude when infected with natural retroviruses are syncytial. retroviral strains, such as H, 1, and V, that have the ability to induce tumor formation in the patient's serum. It has been proven that this method can be used for detection. According to the results of the detection method of the present invention The ability of an HIV strain to induce syncytium formation depends on the degree of disease, the percentage of CD4+ cells, and the ability of an HIV strain to induce syncytium formation. and is an indicator of the presence of detectable antigen p24. In addition, syncytium formation induction A competent strain may form part of an isolat with a high rate of replication. found.

Therefore, the method of the present invention first detects the presence of a retrovirus and then detects its syncytia. Suitable for investigating the ability to induce umbilical formation.

The method of the invention also provides, particularly when carried out in the presence of said contacting operation and a pharmaceutically active ingredient, The active pharmaceutical ingredient counteracts the interaction between the retrovirus in question and infectious cells. It can also be used to find out whether or not it is effective. This kind of inspection If this is done, it is possible to select the active pharmaceutical ingredient that can have the greatest activity, and also to It is possible to determine which regimen is likely to be most effective in treating the patient undergoing treatment. child In particular, the effective dose of These indicator cells are removed when the indicator cells are contacted with the supernatant of the medium in which the sample was maintained. determined based on the dose that does not induce amplification of the cell marker.

The invention relates more generally to the in vitro study of the influence of factors that can interfere with viral infection. ro test, in which case the method is used to test the indicator cells of the invention for the presence of the factor. By contacting with a predetermined amount of foreign retrovirus and activating the marker, detectable factors for preventing retroviral infection of said cells, especially for the use of pharmaceutically active ingredients; It consists of measuring a quantity.

The invention therefore also provides supplies or kits for carrying out the method. this The kit is - an indicator cell as described above; -7- A means of clarifying the expression of the marker, especially if the marker is an enzyme, - contacting the indicator cells with the biological sample and the reaction mixture; Conditions such that indicator cells that come into contact with a test sample become infected (in the presence of a virus) Optionally also includes buffers and reagents necessary for incubation with - including media for preserving or culturing the healthy and modified cells mentioned above, if necessary; nothing.

The indicator cells used in these kits are infected with the corresponding recombinant retrovirus. Advantageously, the cell line consists of an "infinitely amplifiable" cell line that can be used. For example, the virus Hiv In the case of kits used for the detection of infectious, modified and healthy cells, Both can belong to m-foam-like types such as tlT4, CEN, I(tlT).

In the Examples below, the possibilities of the present invention will be demonstrated for the production of indicator cells derived from the cell line HT4. Let's take an example.

As is clear from the above explanation, the present invention exhibits infectivity against certain cell cultures. The basic f! Any type of letrow with symptoms The present invention is particularly applicable to the detection of HTLV I, tl virus under the conditions described above. TLV II,) IIV I (or LAV or HTLV III), HIV II (,51LAY II), Leto known as HTLV IV Production of indicator cells that can detect the infectivity (or adjust the infectivity) of the virus Applies to construction. The same applies to many retroviruses that infect animals. Wear. A specific example is the bovine leukemia virus (Bovine Leukem virus). ia Viruses) and Feline Leukemia Virus (FeLV).

Another feature of the invention is that the defective recombinant retrovirus of the invention derived from l'1IV-1 As disclosed in the description of the example during the production of an indicator cell line containing the gene sequence of Good morning.

The following description will be made with reference to the accompanying drawings. In these drawings, -Figure 1 shows the structures of the three recombinant retroviruses (^), (B) and (C) mentioned above. FIG. 2 is an explanatory diagram briefly showing the essential elements of

- The second section is β-galactin, which was measured by marker activation under various conditions described below. It is a graph showing the magnitude of sidase activity.

-Figure 3 shows the response time of the indicator cells of the present invention infected with the virus under the conditions described below. This is a curve graph.

- 4th (2I is the virus dilution obtained from the various retroviruses listed in this figure) Figure 2 is a curve graph showing the titer of indicator cells when used together.

- Figure 5 shows instructions of the invention in which the protein CD4 was infected in the presence of increasing amounts. 1 is a curve graph showing changes in marker activation in cells.

- Figure 6 is under the same conditions as Figure 5, but induced by different amounts of ΔZT. Correlation between the number of phosphonates and the proportion of antigen p24 detected in the corresponding supernatant FIG.

- Figure 8 shows the concentration of anti-J'Xp24 in the supernatant of a co-culture of highly replicating PBL. It is a graph showing the degree of linkage shown by the indicator cell of the present invention. -9 means that it has the ability to induce lynchium formation, and -9 has no ability to induce lynchium formation. It means that.

,' HeLa T4 HIV infected with n1sLacZ multivirus HIV Human adherent cell lines were selected because they have the ability to form lynchium. . Formation of lynchatium facilitates detection of infected cells when present in suspension This is because it does. 1 (T4 is a mouse-derived retrovirus recombinant vector T This is a cell clone expressing the molecule CD4 derived from C. 4. Therefore, this type of cell can be infected with HIV (in Chesebro, B. and Ehrlly, (1) 988), J. of Virol, 62°3779-3788), for transformation. The vector used is the vector described above, namely pHIVsnlsLacZtat. (Bonnerot et al., 1988).

Cells HeLa T4 are cultured in medium RPMI 1640 containing 10% fetal bovine serum. do. The plasmid was prepared using C,R, except that the precipitate was added directly to the culture medium. , ^cad, sci, Paris, 299, 271-274 (1988) Rubenstein, J.R., Colas, J.F., & Jacob, F. .

Primary cells transfected by phosphocalcium co-precipitation using the same method as in RN was analyzed by the Sachin method and dot plot hybridization. Transformed cells were analyzed using standard methods of detection.

Detection of β-galactosidase expression and cell sorting were performed as follows: For detection of X-gal, cells were incubated with 150+aM NaCl+15- pi47.3 1% formaldehyde and 0.2% glutarium in sodium phosphate (PBS). Quick fixation (2-5 min) with rudehyde, then placed twice in PBS, 4-chloro-5-bromo-3-indolyl-β-galactosylate at 1 mH/a+1 (X-gal), 5mM potassium ferrocyanide, and 2mM MgC1z. The cells were incubated at 30° C. for 10-60 minutes in the reaction medium. X-4al was dissolved in dimethyl sulfoxide at a rate of 40 ng/ml. X-gal's Removal stopped the enzymatic reaction. Store colored cells in PBS or water It is possible. FACS method was used for cell sorting, and fluorescein di-β-loga Detection of cells labeled by lactopyranoside (FD) staining Proc. Nat,^cad,sci,Us^85°2603-2607 (1988). No1an, G.P., Fiering, S., N1colas.

It was performed according to the method of J.F. and Herzenberg, L.^. protein The ONPC test of the quality extract was carried out by the method of Rubenstein et al. mentioned above.

In the first two studies, performed in total and in the absence of infectious virus, β-galactin Several colonies showing expression of tosidase were isolated. You can also individually select multiple blue The presence of phosphocium containing dark nuclei was also confirmed. To isolate stably transformed cells, For this purpose, these cells were cultured for 2 weeks after transfection to ensure that the markers were expressed. The resulting cell clones were tested for the strength of β-galactosidase activity in living cells. It was isolated by a screening method based on its appearance. More specifically, evaluation of the fluorescence profile β-galactosidase activity was evaluated using a sorting device that allows.

HT4-LacZ-1 is a clone isolated as a result of this selection.

This clone has a gene sequence derived from the sequence encoding the ring protein env. This sequence, which contains but does not undergo spontaneous fusion, is said to express the viral receptor. As shown by the ability of this gene sequence to It is present on the plasma membrane of cells of this clone at high levels. This clone is described below. As such, transactivation is possible. Minutes of sequence LacZ by Southern plot Analysis reveals that this sequence characteristic must be due to rearrangements within the region envTAT. It was confirmed that there were no. The sequence LTRSVnI5LaeZ is clearly complete. It turns out that there is something. Therefore, the selected clones are provirus tat-/en The biological properties of v- and the biological properties of the genetically engineered regulatory region 3. Combined with motsu.

HT4-LacZ-1 is a small β-galactosidase in exponentially growing cultures. However, this activity decreases markedly when the culture becomes confluent (back ground noise). This background noise causes the clone to The derived subclones were cultured in the absence of selective pressure for β-galactosidase. If you keep doing this, it will stabilize.

Introduction of exogenous virus material of HT4LaeZ to the tone of Tampa TAT Detection of transactivation of the integrated gene HIV LaeZ by HT4Mac Transfection of indicator cells with appropriate plasmids in Z-1 It was analyzed by pSVtat examines cells colored by X-gal Enzyme activity quantification using 0NPC as substrate inducing amplification of LacZ expression confirmed by measurement.

FIG. 2 is a graph showing the results observed during the measurement described above. This graph is H β generated under the action of transactivation by the protein TAT within T4LacZ - Shows the degree of galactosidase expression. On the ordinate, protein extraction The β-galactosidase activity measured by hydrolysis of the substrate 0NPC by has been done.

The height of the vertical bar indicates the intensity of enzyme activity measured in indicator cells in culture according to the conditions below. is a direct function of: <a) the absence of any substance that induces the expression of the gene LacZ; Measured at the start of culture without

(b) After 4 weeks of culture under the same conditions as above.

(c) After 11 weeks of culturing under the same conditions as above.

(d) In the presence of pRev.

(f) In the presence of pTat.

(g) In the presence of pRev and pTat.

In the absence of pRev, expression of the gene LacZ becomes background noise. (Fig. 2g), and no increase in syncytium formation is observed.

On the other hand, in addition to proteins TAT and REV, gp120, that is, [IIV-1] In the presence of plasmid p97 expressing the envelope gene, infection a Indicated by the intense coloration of the T cells and the production of syncytia, which may contain up to 100 nuclei. A high degree of cell fusion is observed. The same results were obtained in the presence of pLet. It will be done. This plasmid is present at levels below those detectable by immunoprecipitation. TAT, REV and I (IV envelope genes are expressed. This result indicates that fusion This demonstrates the sensitivity of HT4LacZ to

[11V-L HIM-2SIVmac 5IVa cells Sup T-1 pre-prepared Virus old v-1 (isolate BRU), which was cultured and recovered from these cells, was cells BT4LacZ were infected. Coloring with X-Hat at various time points after infection provoked.

Syncytia with blue nuclei were first observed after 40-50 hours. β-gala The number of syncytia colored by tosidase reached its maximum value after 60-80 hours. . This number then decreases.

The sensitivity of indicator cells is determined by:) Infection with IIV is carried out in the presence of dextran to DE8. and can increase. The amount of polyga-dion is lhg/+ 1, the number of syncytia colored with β-galactosidase increases several times.

Therefore, all other experiments described below were performed in the presence of DEAE dextran. It was performed at a cell density of 1. Isolates of other viruses, especially nrv-Z (RO[l), Similar experiments were conducted using SIV, , , SIV, , and SIV, ,c.

The results are shown in Figure 4, which shows the amount of infectious virus using the obtained results. It is expressed as the number of colored syncytia as a function. As is clear from this figure, the finger The degree of coloration of the displayed cells was greater when using tllV- than when using other viruses. Therefore, the indicator cell containing the recombinant sequence derived from fllV-1 in its genome. T4LacZ is used to detect the presence of infectious virus of the same species in a biological sample in the above sense. It is clear that it can also be used very effectively for detection. There are various differences Various trans-activations that can be induced by proteins derived from test viruses This is explained by the strength of oxidation.

FIGS. 5 and 6 illustrate the above-mentioned principle. That is, the indicator cell of the present invention We are testing the effectiveness of active ingredients that are presumed to have neutralizing activity against retroviruses. This shows that it can be used to For example, FIG. Various effects on transactivation ability induced by existing retroviruses Showing the effect of molar concentration of protein CD4, Section 6 shows the influence of ^ZT at various molar concentrations. The above-mentioned influence is shown. The action of these infection-interfering substances is to prevent pigmented syncytia. Indicated by a decrease in the number and relative intensity of the coloration [ It has been demonstrated that the method of the present invention can detect syncytium formation induced by HIV. To demonstrate, fresh PBLs freshly isolated from patients infected with IIIV were pH^2ug/ml and IL-25μ8 contained in the supernatant of activated normal PBL. PBLs were then co-cultured for 4 days with normal PBLs activated with /-1. was contacted with cells tlT4LacZ-1 (CNCM l-899).

[lIV infection in these patients was determined by anti-HIV-1 antibody detection by ELIS^ and Confirmed by Western plot. Zidovuci for these patients No inine treatment was given. The clinical stage of the disease in these patients is C, D, C, layer separation method (M, M, 11.R.

1986.35:334-339). 2 out of 40 patients 0 people rated it as stage II, 3 people as stage III, and 17 people as stage was valued.

Syncytia formed after contacting cells HT4LacZ-1 with IIIV isolate The detection method of the present invention cannot detect a single syncytium. I can do it. Therefore, this test requires that both infected and 3xlO' (co-cultured cells tested) The fused cells can be identified by the number of cells (number of cells). The specificity of this reaction is From serologically negative (Saron&gative) subjects for HIV-1. This was confirmed by the absence of syncytium formation by co-cultured PBLs. 4 days When the test of the present invention was performed after co-culturing for a period of time, out of 47 test isolates, Fourteen isolates with the ability to induce cystium formation were detected.

In these experiments, syncytia containing three or more colored nuclei are considered positive. . The results show that the detection compound per 10' co-culture supernatant added to HT4LacZ-1 Expressed in cytium numbers.

The detection method of the present invention is used to detect free virus in the supernatant of PBL co-cultured for 4 days. It was also used. The average number of syncytia induced by the supernatant is the same as that of the corresponding simultaneous It was lower than the average number of syncytia induced by cultured PBL.

The co-cultivation step is essential for the production of transactivated syncytia. We also investigated whether this is the case. To this end, P. BL was added directly to cell HT4LacZ-1 cultures. 3 out of 19 PBL samples , induced the formation of pigmented syncytia after only 4 days. induce syncytium Of the 16 samples that did not, 6 were co-treated with activated normal PBL for 4 days. When cultured and tested, it induced the formation of syncytia.

In conclusion, depending on the patient, there is no need to perform a co-culture step with normal PBL. This means that the presence of cells infected with HIV can be detected.

b) to 24 Transactivated syncytia induced by PBL co-cultured for 4 days. The number of antigens detected in the supernatants of these co-cultures was determined by the method of the invention. compared with the concentration of p24. The concentration of antigen p24 was determined by the method of Goudsmit J, et al. (Lancet 1986 ii: 177-180). This concentration “threshold” The value is 30 pg/l. The average number of syncytia observed was was correlated with the amount of antigen p24 produced during nutrition (, = 0.77, Spear man test, p = 0.01). These results are shown in FIG. Testing of the present invention Applying the method, two samples were found to be positive on the fourth day; The presence of antigen p24 in the samples was unknown until day 7. These results The method of the present invention has a high degree of anger and the production of anti-[p24] cannot be detected. This means that it can be used to detect the presence of viruses.

C) Shinshi Ram Ta... Isolates found to have the ability to induce syncytium formation by the method of the present invention were tested for the presence of antigen p24 on the 4th and 7th day of co-culture. this If antigen p24 is present at this stage, the replication rate will be high. 10 clinical trials tested All materials were found to have high replication rates. These results indicate that the syncytial form This means that isolates that have the ability to induce growth have a high replication rate.

Additionally, some isolates without the ability to induce syncytium formation also have high replication rates. It turns out that there is something. Isolates with high replication rates are generally stage IV patients. isolates with low replication rates were isolated from stage TI and III patients. It was isolated. Table 1 below shows the replication rate of isolate 1 (IV) and the clinical stage of the patients. It shows the relationship with the floors.

Table 1 = Numbers represent number of patients in each group, S, 1. :Ability to induce syncytium formation Powerful isolate; N, S, 1. : Isolate without the ability to induce syncytium formation.

Then, on days 4, 7 and 111, in the co-culture supernatant of the monolayer with high replication rate. The concentration of antigen p24 was measured.

The concentration of antigen 1)24 was higher in the supernatant of the isolate capable of inducing syncytium formation than in the other. was higher than that of the supernatant of the isolate. These results are shown in Figure 8 (-Rho2 replication rate Isolate with high replication rate and ability to induce syncytium formation -+-: High replication rate and isolates without the ability to induce syncytium formation).

d) Shinshi Ram Ta = and 11 to 11 Isolates with the ability to induce syncytia formation are most often found in stage IV patients, circulating cells. Patients with CD4+ cells less than 200 cells/pl and antigen 24 detected in blood Detected in patients with These results indicate that the ability to induce syncytium formation is This means that it is one of the viral characteristics associated with severe disease. Therefore, A.I. DS can exist even in the absence of isolates with syncytium-inducing ability.

The inventors have further demonstrated that the method of the present invention provides a clinical trial in stage II and III patients. We also demonstrated the presence of phytocytosis-induced isolates. These patients have antigen ρ24 in their blood. I don't have one.

The percentage of circulating cells CD4+ was also low. Therefore, if the method of the present invention is used, AIDS It is possible to detect patients infected with isolates with syncytium-inducing potential before the onset of symptoms. can.

rtq, L Fig, 2 Fig, 3 Fiq, 4 *HIV + Column 3 *Column 5 dots F Engineering GURE 7 F Engineering GURE 8 international investigation complaint □□- to T/FR 匍lα 19 m stillness-1 luster-,, PCT/FR90100619 international investigation report FR9000619 SA　39981

Claims (10)

[Claims] 1. Contains receptors for retroviruses and the following recombinant gene sequences: sequence, i.e. derived from the same or homologous retrovirus and retrovirus The internal tat region encoding the viral transactivator and the first promoter is under the control and activated by the transactivator encoded by the tat region. (internal stimulation) or a natural retrovirus or external virus that infects the indicator cells. Activated by trans-activating factors introduced in trans by retroviruses ( A recombinant gene sequence containing a tar region that can be stimulated (externally stimulated) is preferably combined with the genome. An indicator cell line containing in an integral form the entire genetic material, said recombinant genetic material The sequence acts as a marker, i.e. the result of activation of the region tar. and the marker is inserted between the regions tar and tar under conditions such that the marker is activated. These indicator cells also contain the entered sequences and these indicator cells are activated by the action of internal stimuli. Activation of the region does not extend to activation produced by both internal and external stimuli. An indicator cell line characterized by being a cell that is or has been made to be such a cell. . 2. There is no activation of area tar due to the action of internal stimulation, so The effect of both external stimulation and external stimulation is equal to the effect of external stimulation alone. The cell line according to claim 1. 3. Indicator cells are usually used to transform these cells with the complete env sequence of HIV. When the env sequences are expressed, they fuse together to form a syncytium. is derived from competent cells and therefore has low spontaneous fusion potential and infectious natural recombination. or a recombinant vector modified with the complete env sequence or It has the characteristic of showing great fusion ability when transfected with DNA. The cell line according to claim 1 or 2, characterized in that the cell line is a cell that does 4. For example, in HT4 strains such as HT4 LacZ-1 (CNCM 1-899) 4. The cell line according to claim 1, wherein the cell line is derived from 5. The marker imparts a specific color to the indicator cells or invades into these indicator cells. When exposed to light, it causes an in-situ color change, or more generally a change in the optical radiation absorption spectrum. from claim 1, characterized in that it encodes an enzyme that confers the ability to act on a substrate. 4. The cell line according to any one of 4. 6. Cell line according to claim 5, characterized in that the marker is the sequence LacZ. 7. A device according to claim 6, characterized in that the marker is the sequence nlsLacZ. Cell strain. 8. The recombinant gene sequence has a second protein immediately upstream of the marker in the direction of transcription. According to any one of claims 1 to 7, comprising a motor. cell line. 9. The indicator cells according to any one of claims 1 to 8 are used to treat suspected cases of infection. (e.g., when testing whether or not they are infected with the virus HIV) human serum collection containing lymphocytes T4) or previously contacted with these cells. A method for detecting retroviral infection in a medium such as a culture supernatant comprising: If a retrovirus is present in a sample, the indicator cells will be infected with that retrovirus. The sample can be processed according to the invention under conditions (particularly at appropriate incubation temperatures and times) such that contact with an indicator cell of 1. Clarify the amplified expression of the marker and, in some cases, the formation of syncytia. detecting whether the cell is infected by determining whether the cell is infected or not. A method characterized by: 10. A kit for carrying out the method according to claim 9, comprising: 1. An instruction cell as described above, 1. A means of clarifying the expression of the marker, especially if the marker is an enzyme, that enzyme a substrate specific for the reaction; The mixture is applied in such a way that indicator cells that come into contact with the test sample become infected (in the presence of virus). Optionally also includes buffers and reagents necessary to incubate under the conditions; 1. Media for preserving or culturing the above-mentioned healthy cells and modified cells, as the case may be. A kit characterized in that it also includes: