FR2599756A1 - Streptococcal mitogens specific for T4 lymphocytes and their application to stimulating the production of the LAV virus - Google Patents

Abstract

The invention relates to the application of streptococcal mitogens to stimulating the capacity of T4 lymphocytes to produce the LAV virus. It also relates more particularly to two possible fields of application of this technique. The first application relates to the production of the LAV virus in sufficient amounts, especially for the production of the active ingredients required for the preparation of streptococcal mitogens for the in vitro diagnosis of the absence or, on the contrary, of the presence of a risk of developing AIDS in carriers of anti-LAV antibodies.

Description

"STREPTOCOCCAL MITOGENES SPECIFIC TO LYMPHOCYTES
T4, THEIR APPLICATION TO THE STIMULATION OF PRODUCTION
OF VIRUS LAV "
The invention relates to the application of streptococcal mitogens to the stimulation of T4 lymphocytes infected or infectable with the LAV virus considered to be one of the etiological agents of lymphadenopathy syndromes (ALS) and of acquired immunodeficiency syndrome or AIDS, or to the stimulation of permanent cell lines derived from these T4 lymphocytes. In other words, the invention relates more particularly to the application of these streptococcal mitogens to the stimulation of the possible production of LAV virus by these T4 lymphocytes or these cell lines, as soon as they are infected with this virus.

It has already been known for a few years that liquids supernatant from the culture of group
A had mitogenic properties on human, rabbit, guinea pig and mouse lymphocytes, as well as on thymocytes and splenocytes of these animals. (J. Lab. Clin. Immunol. 2 155-163 (1979)).

Purification of the supernatant liquids from culture media of Streptococcus pyogenes (especially strain of
Streptococcus pyogenes S 84 Griffith referenced under number 9994 of the catalog of the NCTC (National Collection of Type Cultures, Central Public Pealth Laboratory,
Colindale Avenue London, NW9 sHT3) made it possible to isolate three fractions called Y,, corresponding to proteins characterized by their molecular weights of the order of 72,000, 30,000 and 17,000 respectively and their isoelectric points, which are respectively 4 , 2 - 4.8 and 10.3 measured under the conditions described in the article by
CAVAILLON et al, Immunology Letters, 5: 317-322 (1982).

Furthermore, these mitogenic fractions do not interact with the F C positions of the IgGs, also under the conditions described in this last article. These three fractions have mitogenic properties on human T lymphocytes at very low doses (nJ0.1 to 2 pg), and are known to be endowed with certain immunological properties which make them capable of inducing interferon (Immunol.

Letters 5: 323-326 (1982)) or to allow immunomodulation and polyclonal activation of the immune response in vitro (Cell. Immunol, 76: 200-206 (1983)). The most effective mitogenic fraction corresponds to fraction X (PN-30 K Dal) which has been identified as the erythrogenic (or pyrogenic) toxin responsible for scarlet fever toxin which will be referred to hereinafter as scarlet fever toxin for the convenience of language.

The invention stems first from the discovery that these streptococcal mitogens stimulate preferential
The division of T4 lymphocytes, which are known to be the main target of the LAV virus.

It is certainly known that other substances endowed with mitogenic properties with regard to lymphocytes
T, among which are essentially the lectins of plant origin, such as concanavalin A, phytohemagglutin (PHA), could exert an action of stimulating the production in vitro of virus LAV by T4 lymphocytes, (also known under the designation
"Leu-3 + cell">. These are more particularly
lymphocytes that have a surface glycoprotein
(called "T4 antigen" or even "T4 protein") of
62 k Dal, specifically recognized by mono antibodies
clonal, for example those marketed by the firm
ORTHO Pharmaceutical Corporation, under the designation
OKT4.This T4 protein is the main target of
LAV virus (SINOUSSI BAR et al; Science 225: 59-63 (1984) and
KLATZMAN-et al, Nature (London) 317: 395-403 (1984). The application
of PHA for the stimulation of T4 lymphocytes in vitro used
for the production of LAV virus has been described by these authors.

 The production of the LAV virus is appreciated by the appearance of reverse transcriptase which is then secreted into the lymphocyte culture medium. The authors of the present invention have realized that not only the streptococcal mitogens preferentially stimulate the transformation of T4 lymphocytes, but also that, to this selective stimulation action, a significant amplification was added.

of the production of LAV virus, when these lymphocytes
T4 were the site of viral activity.

 While the streptococcal fractions have been shown to exert an activity stimulating the mitogenicity of T4 lymphocytes of the same order of magnitude as that of PHA, it has been found that. the yield of LAV virus produced was of the order of three to six times higher than that resulting from stimulation induced by PHA. In addition, the onset of viral expression, detected through reverse transcriptase activity in the culture medium, manifests itself much more rapidly than in prior systems involving human lymphocytes. an appearance of reverse transcriptase activity is noted only after six days, under the conditions described in the article by BAR SINOUSSI et al already mentioned, the reverse transcriptase activity is observed from the third day when the mitogenic preparation used is based on a streptococcal fraction, more particularly "scalatinous toxin".

 The invention therefore relates to a method of stimulating lymphocytes or cell lines, carrying T4 receptors, infected or liable to be infected with the LAV virus, by culturing these lymphocytes or cell lines under conditions allowing their development, and this in the presence of mitogenic fractions of streptococcal origin, and by the detection of the reverse transcriptase activity observed and, if necessary, the recovery of the LAV virus produced in the culture medium.

 In one of these first modes of application, the invention applies to the production of LAV virus with increased yields. It applies to the production of LAV virus, both on cultures of fresh non-immortalized lymphocytes, cultures which must then be periodically renewed, and in immortalized or permanent cell lines carrying the above-mentioned T4 receptors, for example CEM type lines. , HT, HUT, MOLT, etc.

Other types of infectious cell lines will also be mentioned, such as those made up of B lymphocytes transformed by the virus.
EPSTEIN-BARR, for example the lines described in European patent application No. 165.120
It will be clear to those skilled in the art that the culture conditions can in each case be in accordance with those described in the prior literature, except that within the framework of the present invention, the mitogenic agent used consists of a fraction streptococcal.

In general, the invention is applicable to any virus having a tropism for T4 lymphocytes, which normally tend to destroy them and to which can be attributed the development in humans of ALS or of
AIDS. Consequently, the expression LAV does not only extend to the culture of those viruses which have been designated by this abbreviation by the research teams of the Institut Pasteur, under the direction of Professor MONTAGNIER, but also to production viruses designated by the abbreviations HTLV III, as isolated by RC GALLO et al; Science, 224, 500 (1984) or by
MG SARNGADHARAN et al; Science, 224, 506 (1984), or to viruses isolated by M. JAY LEVY et al; Science, 225, 840-842 (1984), the latter type of virus having been designated by the abbreviation ARV. Reference may also be made to European patent application No. 138.667.

filed on 14.09.1984 with regard to the general definition of the viruses concerned. It should be clear that in what follows the expression LAV relates to all these types of virus.

With regard to the streptococcal fractions capable of being used, it is possible to use mitogenic fractions extractable from the supernatants Qn ts of streptococci. In this regard, mention will be made, in addition to the mitogenic fractions of streptococci of the Streptococcus pyogenes group A type, referred to in the article by CAVAILLON et al; Immunology letters, 5 (1982) 317-322, other strains of group A streptococci, for example those cited under the abbreviations NY 5 Doquer by Y. ABE et al, Infection and Immunity; 29: 814-818 (1980)> C 203S and
C 203U, Richards 814, 594 by JE Alouf (1980), and more generally all the strains of streptococci of the groups
A, C and G. These mitogenic fractions can be used at different levels of separation. It is possible to use either raw products, semi-purified or purified products. Mention will be made, as crude products, of the extracellular products released into the culture media, in which the strains of streptococci used have previously been grown. Preferably, more purified mitogenic products will be used, more particularly scarlet toxin.

 It is also possible to use mixtures of different mitogens, for example the fractions, mentioned above, etc.

 Good production conditions are obtained when the concentration of a streptococcal mitogen varies from 0.1 to 2 μg / 2.10 cells, per ml of culture medium. In addition, it is advantageous to operate in the presence of non-T helper cells, it being understood that this expression denotes monocytes from human peripheral blood or from other tissues and organs.

 Preferably, the infection of the lymphocytes is also made in the presence, not only of at least one of the above-mentioned streptococcal mitogens, but also of 5 to 7% of non-T helper cells per 2 .106 T4 lymphocytes.

 The effectiveness of stimulation of viral activity by streptococcal fractions opens up an additional field of application for them, that of in vitro diagnosis of the effective presence of the LAV virus in the lymphocytes of an anti-LAV antibody carrier, even if it is only in the "latent" state or at the first stage of its incubation in the carrier, when the disease does not yet manifest itself by clinical symptoms.

 In particular, it is known that patients with AIDS or active pre-AIDS probably constitute only a minority proportion of the people in whom anti-LAV antibodies have been detected. However, it is not possible today to distinguish between asymptomatic carriers whose lymphocytes appear in fact to be completely free of the virus, or even of lysogenized DNA sequences corresponding to viral RNA, and those whose lymphocytes are infected and / or likely to be infected and who could, after a certain incubation period, develop ALS or AIDS. We cannot even distinguish in the last category of carriers, those in whom the virus has started to multiply.

 One of the aims of the present invention is therefore to provide the means making it possible to determine the level of viral activity in which the LAV virus is found in individuals suffering from pre-AIDS and thus allowing discrimination, among the abovementioned individuals, between those who more or less short term risk of developing AIDS disease and those who are not always threatened in the short term, or even not threatened at all. It is understandable that such discrimination will result in better monitoring of surveillance and treatment in the former and the elimination of a significant anxiety factor in the latter.

 It is within the framework of this application that the invention tends to take advantage of the highly stimulating power of streptococcal fractions. Consequently, the invention relates to a method of in vitro diagnosis of the presence in lymphocytes of a potential viral activity or on the contrary of the absence of this potential activity.

 The method according to the invention therefore comprises stages of culturing lymphocytes and more particularly T4 lymphocytes obtained from a biological fluid, in particular from blood obtained from the patient under diagnosis with a streptococcal fraction of the type indicated above and , after an incubation time sufficient to induce the manifestation of a reverse transcriptase activity (or of any other manifestation testifying to the activation or production by said lymphocytes of LAV virus), to detect said reverse transcriptase activity or the other appropriate suitable event.

 As in the case of virus production, the implementation of this diagnostic method preferably involves the presence of 1 to 5 μg / ml of streptococcal fractions, in particular of scarlet toxin per ml of culture media.

. The invention therefore takes advantage of the sti
particularly high mulant of streptococ fractions
cics to activate in vitro ion any latent viruses
present in the lymphocytes studied, or for
cause "delyzogenization" of any acids
nucleic acid derived from the LAV virus genome.

If necessary, it should be checked that
the virus produced does belong to the LAV family.

If necessary, it can be cultivated and lysed.
extracts can then be reacted with
reference anti-LAV antibody. Detection of a
immunological reaction will then sign the belonging of the
virus produced in the LAV family of viruses.

 The production of a reverse transcriptase activity therefore indicates an already progressive infection or, on the contrary, the latent nature of a potential viral infection. The absence of detection of reverse transcriptase activity or of any other manifestation which can be correlated with the presence of the virus or of any other similar manifestation, may then be likely to be correlating with the absence of virus and therefore lead to a favorable prognosis.

 It goes without saying that detection can be based on any other precondition or biological manifestation of the virus produced. As already indicated above, lysates of these viruses could possibly be deposited and fixed in a manner known per se on membranes or filters, in particular of cellulose, then brought into contact with labeled antibodies specific for LAV proteins or glycoproteins. The retention of the labeled antibody on the filter then makes it possible to conclude a positive diagnosis, as for the possibility that the patient from which the studied lymphocytes came is threatened with AIDS.

The possibilities of the invention can be better deduced from the following description of the conditions in which the effect of stimulation and amplification of the production of LAV virus has been demonstrated, in cultures of lymphocytes infected with the virus LAV. During this description, reference will be made to the drawing in which: FIG. 1 is representative of the results obtained
in LAV micro-culture trials on different
types of lymphocytes and in the presence of various
mitogenic preparations, - Figure 2 provides curves allowing the comparison
because of the stimulating effects of a preparation of
SM on the one hand and PHA on the other with regard to the
production of LAV virus by a suspension of cells
unfractionated and infected blood mononuclear cells
with LAV, - Figure 3 similarly represents the stimulating effects
streptococcal mitogens on the production of
LAV by different types of lymphocytes and - Figure 4 shows the influence on the production of
LAV of the different streptococcal fractions and
their mixture.

The streptococcal mitogens used in the following have been purified from the strain of
Streptococcus pyogenes, as described in J. Lab. Clin. Immunol. 2: 155-163 (1975) and
Immunol. letters 5: 317-322 (1982), except that the molecular sieve chosen for the purification of the toxin recoverable from the culture medium was different.

The fraction recovered after precipitation of the culture supernatants with a 50 and 80% saturated ammonium sulfate solution was filtered through a column (0.25 x 100 cm) of the gel for filtration sold under the brand name of
BIOGEL P-100 (Bio-Rad), balanced in a PBS buffer at pH 6.8, rather than dru gel for filtration marketed under the brand SEPHADEX G-100. The fraction corresponding to the volume eluted between 160 and 240 ml was collected and concentrated by ultrafiltration on a filtering membrane sold under the designation AMICON PM 10, this membrane allowing the passage of molecules having a molecular weight of less than approximately] 0,000. not diffusible through the filter thus constitute the preparation of purified streptococcal mitogens (MS) (20 mg of proteins per ml) which was used in the experiments which followed. This preparation was subjected to isoelectric preparative migration as described in the last aforementioned publication, which led to the isolation of three mitogenic fractions, <,, and, which after concentration contain 1.6, 1.5 and 0 respectively. , 5 mg protein per ml. These fractions were homogeneous, as could be determined by polyacrylamide gel electroporesis containing sodium dodecyl sulfate (SDS-
PAGE).

The spread of the LAV virus was carried out on total or fractionated T lymphocytes from peripheral blood of two healthy adult donors, who did not exhibit antibodies against the virus and whose lymphocytes did not spontaneously release the virus (as it can be determined by measuring the activity of the reverse transcriptase (RT) type or which did not express viral proteins. The peripheral blood mononular cells (CMP) were isolated by centrifugation on a density gradient known as FICOLL-HYPAQUE, stored and frozen at -196 C in liquid nitrogen, as described in Nature (London) 312: 767 Before use, the cells were thawed in a bath at 37 "C, then washed and incubated at 37" C in a culture medium for one hour, before being counted and adjusted to appropriate concentrations (Nature ( London) 312: 767-771). A suspension enriched in T lymphocytes was obtained after operations comprising exhaustion of the monocytes by adhesion to plastic, the formation of rosettes in the presence of sheep erythrocytes treated with aminoethylisothiouranium, then centrifugation on a FICOLL gradient.
HYPAQUE (Science 225: 59-63). On average, suspensions enriched in T cells contained more than 90% of T lymphocytes with 5 to 7% of contaminating cells whereas preparations of non-T cells (NT) included 5 at 6% T cells and a mixture of substantially equal number of B cells and monocytes. T4 lymphocytes and
Purified T8s (or CD4 + and CD8 + lymphocytes) were obtained by cell affinity chromatography, as described in Science 225: 59-63.

Viral infection of the target cells was carried out as described in the publication cited above, with some modifications. 5,106 cells were cultured at 37 "C in an atmosphere containing 5% of
CO2 in the presence, either of 1% of PHA-M (DIFCO), or of different concentrations of purified SM preparation or of the Y, t and & fractions in RPMI 1640 medium (GIBCO) to which 10% of calf serum had been added fetal. After three days, the cells which had sedimented were resuspended in a culture medium containing no mitogens but interleukin-2 (IL-2), anti-g interferon serum and polybrene (Science 220: 868 The preparation of LAV virus (RT titre of the supernatant: 50,000 cpm) was added to the medium. After three days, the cells were centrifuged in order to remove the non-adsorbed virus and resuspended in the culture medium. Every three days, the cells were centrifuged (sedimented) and re-cultured in the supernatant containing no cells under the conditions of the last publication mentioned above.

 Cell proliferation experiments in micro-culture of different lymphocyte preparations, carried out in the presence of MS (fig. 1) have shown that the T4 lymphocytes were preferentially stimulated compared to the T8 lymphocytes. This stimulation requires the presence of non-T helper cells.

 Comparison of the effects obtained with the preparation of MS, compared with those obtained with PHA on the production of LAV virus by a suspension of peripheral blood mononuclear cells infected with this virus (fig. 2) under the conditions recalled below , shows that a higher yield in RT activity is obtained in the presence of MS than of PHA. The activity in the presence of MS, as well as the optimal RT titer appear earlier than those obtained with PHA and decline less rapidly. The yields of RT, obtained from the supernatant of culture media of T4 lymphocytes and of T8 lymphocytes, are much higher - for cultures of T4 lymphocytes - in the presence of MS (approximately 10 times) than in its absence. Maximum activity is obtained, more than twice as high as in the case where the stimulation of these lymphocytes is carried out in the presence of PHA. No significant effect of these two mitogens on the production of LAV from T8 lymphocytes was observed. The RT activity is reduced by approximately 50% in the mixtures of T4 and T8 lymphocytes (5.106 for each class of cells) stimulated by the MS preparation in comparison with the T4 lymphocytes alone.

PBM, peripheral blood mononuclear cells, infected with LAV and stimulated by the purified fractions X, t and i or by their mixtures (fig. 4), also show good yields in RT activity, with no significant difference between these three mitogens. .

The foregoing observations, of which Figures 1 to 4 point out, were carried out as follows - Figure 1 relates to 1 1 proliferation test in
microculture in the presence of streptococcal mitogens.

The cells of each population (2 x 106 / ml) were
incubated alone or in combination (50 μl) in the wells
a microtiter plate. with 5 pg (optimal dose)
of a streptococcal mitogen preparation in
100 μl of culture medium at 37 ° C. in an atmosphere
containing 5% CO2 for six days before the measurement
of the incorporation of H-thymidine pCi added 8 hours
before harvesting the cells).
lay in the means (tests repeated three times) (A)
total mononuclear peripheral blood cells,
(B) enriched T cells, (C) CD4 + T cells, (D) cel
lules CD8 + T, (E) T + NT, (F) T + * (G) CD4 + + NT, *
(H) CD4 + + NT, (I) CD8 + + NT, (J) CD8 + + NT
(K) NT, (L) NT + T, (M) NT + CD4 + *, (N) NT + CD8 + *.

 (*) 20-Gy irradiated cells.

- Figure 2 shows the production of LAV measured by
sequential determination of transcriptase activity
reverse (RT) in mononu cell supernatants
peripheral keys not split by preparations
streptococcal mitogens at a concentration
optimum of 5 pg / ml (curve a) or by a preparation of
1% PHA-M (curve b).

- Figure 3 relates to the production of LAV in the
conditions indicated above by CD4 + T cells (cour
be c), CD8 + T cells (curve d) and mixtures of
CD4 + + CD8 + T cells (curve e), - respectively sti
mulated by streptococcal mitogens and by - PHA (curves e and f).

- Finally, Figure 4 relates to the production of LAV
by unfractionated mononuclear cells, by
2.5 pg (optimal dose) in the presence of streptococcal &alpha; (curve j), # (curve k), #
(curve 1) and the mixture of the three (curve m).

 The documents whose references follow should be considered as contributing, as necessary, to the description of the invention.

1. ALOUF JE et al (1977) Ann. Immunol. Inst. Pastor
] 28 C: 37-39.

2. ALOUF J.E. (1980) Pharmac. Therap. il: 661-717.

3. ABE, Y., et al (1980) Infect. Immun. 29: 814-818.

4. BARRE-SINOUSSI, et al, (1983), Science 220: 868-871.

5. BARSUMIAN EL, et al (1978), Infect. Immun. 22
681-688.

6. CAVAILLCN, J.-M., et al (1979), J. Lab. Clin. Immunol
2: 155-163.

7. CAVAILLON, J.-M., et al (1982), A. Immunol. Letts 5
317-322.

8. CAVAILLON, J.-M., et al (1982), Immunol. Letts 5
323-326.

9. CAVAILLON, J.-M., et al (1983), Cell Immunol. 76
200-206.

10..DALGLEISH, AG, et al (1984), Nature (London) 312
763-767.

 ll: FEORINO, P.M., et al (1984), Science 225: 69-72.

12. FOLKS, T., et al (1985), Proc. Natl. Acad. Sci. USA
82: 4539-4543.

13. GALLO, R.C., et al (1984), Science 224: 500-502.

14. GOTTLIEB, MS, et al (1981), N Engl. J. Med. 305
1425-1431.

15. KLATZMANN, D., et al (1983), Transplantation 35
332-338.

16. KLATZMANN, D., et al (1984), Nature (London) 312
767-771.

17. KLATZMANN, D., et al (1984), Science 225: 59-63.

18. KNOLL, H., et al, Zblt. Bakt. Hyg. A256
49-60.

19. LANE, HC, et al (1985), Ann. Rev. Immunol. 3
477-500.

20. MONTAGNIER, L., et al (1984), Science 225: 63-66.

21. MONTAGNIER, L., et al (1984), Cold Spring Harbor
Laboratory, New York.

22. PARISH, W.E., (1971), Clin. Allergy 1: 295-309.

23. POPOVIC, N., et al (1984), - Science 224: 497-500.

24. QUAGLIATA, F., et al (1982), Cell. Immunol. 68
146-154.

25. REGELMANN, WE, et al (1982), J. Immunol. 128
1631-1636.

26. WEISS, A., et al (1985), Ann. Rev. Med. 35: 545-562.

27. WONG-STAAL, F., et al (1985), Nature (London) 317
395-403.

Claims (9)

 1. Method for stimulating virus production LAV by cell populations containing cells carrying T4 receptors, when these populations are affected by the virus, characterized by the cultivation of these cell populations under conditions allowing their development in the presence of mitogenic fractions of streptococcal origin and by detection of reverse transcriptase activity after an adequate incubation time necessary for the production of the virus.  2. Method according to claim 1, characterized in that the concentration of the culture medium in streptococcal mitogens varies from 0.1 to 2 pg / 2.106 cells, preferably per ml of culture medium.  3. Method according to claim 1 or claim 2, characterized in that the culture is carried out in the presence of non-T helper cells, in particular from 5 to 7% of non-T helper cells per ml of culture medium.  4. Method according to any one of claims 1 to 3, characterized in that the streptococcal antigen used consists of the purified fraction.  5. Method according to any one of claims 1 to 3 characterized in that the mitogenic streptococcal fraction consists of the purified fraction.  6. Method according to any one of claims 1 to 5, characterized in that the streptococcal mitogenic fraction consists of the purified fraction,  7. Method according to any one of claims 1 to 3, characterized in that the mitogenic fractions used are formed by a mixture of the fractions  and X  8. Application of the method according to any one of claims 1 to 7 to the production of LAV virus by infectious cell populations initially healthy, previously infected with the virus.  9. Application of the method according to any one of claims 1 to 7 to the in vitro diagnosis of the existence or not of a potential threat or of the start of the course of a disease which may lead to AIDS, including the cultivation of lymphocytes obtained from this person in the presence of streptococcal fractions and the detection, after the time necessary for the incubation of the virus - possibly present, of the reverse transcriptase activity which would then be linked to it.