FR2696186A1 - Mycoplasma strains. Cytopathogenic fermentans and in vitro diagnostic and immunization methods using such strains. - Google Patents

Abstract

The invention relates to mycoplasmas also having cytopathogenicity specific to said mycoplasmas capable of infecting specifically human CD4 lymphocytes and inducing in the patient carrying them an immunodeficiency syndrome characteristic of AIDS, even in the absence of HIV. They may be used particularly in measuring the sensitivity of the CD4 lymphocytes to mycoplasmas capable of inducing an immunodeficiency syndrome similar to the AIDS type.

Description

NYCOPLABMA.FERMENTAN8 STRAINS CYTOPATHOGEN AND
IN VITRO DIAGNOSIS AND IMMUNIZATION PROCESSES
IMPLEMENTING SUCH STRAINS
AIDS (acquired immunodeficiency syndrome) is generally associated with infection by a virus
HIV.

 However, the possible role of certain mycoplasmas as co-factors of AIDS has been recently considered.

Several species of mycoplasma have been isolated from asymptomatic patients infected with
HIV and also with AIDS.

They are Mycoplasma fermentans, incognitus strain of Lo (3) and strain No. 8 deposited at the
National Collection of Cultures of Microorganisms (CNCM) of the Institut Pasteur in Paris, May 22, 1990, under the number I-949 (4) and also of Mycoplasma pirum.

 The weakness of humoral immunity to mycoplasmas in other types of conditions in which they may already have been involved made it difficult to diagnose mycoplasma infections or separate conditions that could be involved in vitro correlated with mycoplasma infection.

But the observation - then new for
M.fermentans- that the life cycle of the mycoplasma passes through an extremely compact form called "mycosphere", has already been reported in patent No. 9209626 filed on August 03, 1992.

 These mycospheres, whose average diameter is of the order of 140-160 nm, are formed by successive strangulations of the filamentous forms, which are the forms of growth of the mycoplasma.

 These mycospheres, at least those of M. fermentans, have been found to have a certain degree of stability even in acidic medium, contrary to what has been observed for other non-human mycoplasmas which have already given rise to 1 observation of "mycospheres".

 In addition, mycoplasmas in this form are more easily recognized by sera taken from patients infected with these mycoplasmas, hence the method more particularly described in the above patent, for the detection of anti-M antibodies. fermentans in a biological fluid, in particular a serum, originating from a patient possibly carrying the infection, a process which, in one of its preferred forms of implementation, included bringing this biological fluid into contact with mycoplasmas , in particular M. anti-fermentans in the form of mycospheres, and the detection, in the event of infection actually present, of an agglutination reaction of these mycospheres by anti-mycoplasma antibodies.

 Agglutination can be detected in negative staining with an electron microscope. It can also more practically be detected with the naked eye by agglutination of microbeads on which the mycospheres will have been fixed.

A process for producing mycospheres of
M.fermentans described in the aforementioned patent included the cultivation of such a strain of mycoplasma in SP4 medium (13) and the aging of these same cultures in SP4 medium, until most of the mycoplasma present is transformed into mycospheres.

SP4 medium is prepared, in particular as follows, from a "base medium" itself constituted from the following components, sold by the company DIFCO - DIFCO "PPL Broth": 1 g - DIFCO peptone: 1.6 g - DIFCO tryptone: 3 g
The basic medium is itself obtained by adding pyrolized water until a volume of 200 ml is obtained, the pH of which is finally adjusted to 7.8.

 The "base medium" thus obtained is sterilized by autoclaving 15 'at 120 "C.

The complete medium (SP4) is obtained by adding to the 200 ml of the base medium - 2.7 ml of an ampicillin solution dosed at 66 mg / ml - 30 ml of a yeast extract "Yeast extract GIBCO" - 15 ml of a medium itself obtained from
CMRL 1066 (GIBCO) supplemented with glutamine at a rate of 3 mg / l of fluid and without bicarbonate - 3 ml of a 50% glucose solution - 0.6 ml of a solution of phenol red (1% ) - 50 ml of fetal calf serum (GIBCO).

 The complete medium is then filtered on a membrane (0.22 μm mesh). The filtrate can be stored at 4 C for 2 weeks.

 The detection made possible by the invention of patent No. 9209626 filed on August 03, 1992 of the presence of anti-mycoplasma antibodies in biological samples, in particular sera, from subjects infected with mycoplasmas, confirmed a correlation. which had already been done in a large number of patients, of the coexistence of infections with both mycoplasmas and HIV. It opened a new way of screening at a very early stage of a possible HIV infection in subjects at high risk. In addition, the detection of an infection by mycoplasmas, in particular of the M. fermentans type, under the conditions mentioned above, preceded the detection of an seropositivity by the conventional tests implementing the realization of an immunological contact between the biological sample under study and an antigen or a composition of antigens characteristic of HIV.

 The present invention is based on the discovery that certain strains of mycoplasma also had a cytopathogenicity specific to these mycoplasmas, capable of specifically infecting human CD4 lymphocytes and of inducing in the patient who is carrying them an immunodeficiency syndrome characteristic of AIDS. , even in the absence of HIV.

 The invention relates more particularly to a category of strains of this type, a method for cultivating them, their applications to different forms of in vitro diagnosis, in particular of the sensitivity of lymphocytes to infection, and to the production of immunogenic compositions based on such strains.

These strains naturally present the characteristics of mycoplasmas: for a general documentation relating to mycoplasmas, one can refer to chapter 10 entitled "The
Mycoplasmas "from the book titled Bergey Manual, or the chapter titled" The molliculites
Mycoplasmas and ureaplasmas "by JG Tully and S.

Razin, in the book "Practical handbook of Microbiology" edited by William M. O'Leary, 1989, edited by CRC Press, Inc., Boca Raton, Florida, USA.

 They more particularly have the characteristics of infecting CD4 lymphocytes, of replicating therein and, in particular in the absence of CD8 lymphocytes, of producing complete lysis thereof, in particular in the space of 3 days.

 Some of these strains are capable of forming mycospheres, stable in an acidified medium, in particular with lactic acid, by culture in an SP4 medium.

Among these strains are some with the characteristic profile, even in PCR (Polymerase chain
Reaction: English abbreviation for "Polymerase chain reaction), from M.fermentans, these strains being able to penetrate the cytoplasm of lymphocytes
Human CD4 cells maintained in an RPMI medium 16-40, and to infect them, such a result not however being obtained with CD4 lymphocytes of chimpanzees under the same conditions.

 A strain characteristic of the invention (M.DEB) was the subject of a deposit with the CNCM under the number I-1265, on September 11, 1992.

A strain of such a microorganism has been isolated from a patient with clinical and immunological signs of AIDS, although all serological testing for HIV-1 and HIV-2 infection has been successful. revealed negative (tests carried out by the so-called
Western blot). These tests were carried out on lymphocytes purified from blood samples taken under heparin and centrifuged on a density gradient marketed under the brand Ficoll by the company Pharmacia, at 400 g for 30 min. No reverse transcriptase activity, no p24 / 25 type antigen could be detected in the supernatants of a culture of lymphocytes purified from the above gradient, activated by phytohemagglutinin (PHA) and cultured in a medium containing 1 'interleukin 2 even after a month of culture or co-culture. In addition, DNA sequence detection assays in
DNAs previously taken from these lymphocytes by PCR involving sequences coding for env, qaq of
HIV-1 and HIV-2 were all negative.

On the other hand, mycoplasmas could be detected in the cell suspension, in particular when the procedure was carried out as follows
1 ml of the cell suspension is labeled with 20 l of tritiated uracil for 18 hours. The supernatant is sedimented at density balance in a 70-20% sucrose gradient, 1 h at 45,000 rpm in a SW56 rotor (PBS buffer + 2% fetal calf serum).

 The radioactivity of each fraction is determined after precipitation with 5% trichloroacetic acid. The mycoplasmas form a band of density varying from 1.18 to 1.24.

 The mycoplasmas recovered from this density band were then propagated on the SP4 medium.

They have given rise to very rapid development.

In this medium which contains arginine and glucose, the change in pH was indicated by the change from red to yellow of a phenol red occurring 24 hours after the inoculation of this medium with the supernatant of lymphocytes in a report 1/100 single.

 The infectious titer expressed by the color change units in the SP4 medium was found to be of the order of 1012 / ml.

In PCR tests using primers characteristic of M. fermentans, pirum and pneumoniae, it was possible to observe a strong amplification band with M. fermentans. This amplification band had the capacity to hybridize in a Southern blot assay (Southern blot) after digestion with the enzyme EcoR1 with a ribosomal DNA probe from M.capricolum, which contained sequences conserved in all prokaryotic organisms. These tests were carried out more particularly under the following conditions
An amplification band was observed for
M.fermentans, with DNA extracted from a bone marrow sample, according to the following method: the bone marrow sample was subjected to a Ficoll gradient. At the end of the gradient, were sampled
- the band of lymphoid cells,
- the "plasma"
- a band composed of "debris" having sedimented
in the upper part of the "plasma".

 DNA was extracted from each of these fractions, as well as from a sample of urine, and lymphocytes and peripheral blood plasma (cytophoresis).

The amplification band was observed with the DNA coming from the "debris" obtained after the centrifugation of the bone marrow in the gradient.
Ficoll. This is probably due to the fact that at this low sedimentation rate (2,000 rpm), the intracellular mycoplasmas remained at the top of the gradient.

PCR conditions:
PCR was performed with DNA (1 g) from each sample, dissolved in 10 mM tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgC12, 100 mg / ml gelatin, 0.4 pM of each primer (J. Skov. Jensen et al, 1991) 200 μM of each dNTP and 1.5 units of Taq DNA polymerase. The final volume is 50 pl.

The mixture is covered with 50 Al of mineral oil, the samples were denatured at 95 ° C. for 3 min, then amplified for 35 cycles. Each cycle is composed of
1) denaturation at 94 C for 1 min,
2) hybridization at 55 "C for 1 min,
3) extension to 72 C for 2 min and 30 sec.

 17A1 of the reaction product were analyzed by electrophoresis on 1% agarose gel. Transfer using the Southern technique and hybridization with an internal probe.

 Primers and probes are defined in Wang et al, 1992 (M.fermentans) and Jensen et al, 1991 (M.genitalium). For M.pirum (cf FR 9209486).

The profile obtained in RFLP of N. fermentans (DEB) was compared with that of M. fermentans strain No. 8, as well as that of other mycoplasmas, after hybridization with the ribosomal probe pMCS (Amikam, D., S. Rarin, & BR <
G. Glaser, 1982, "Ribosomal RNA genes in mycoplasmas
Nucleic Acids Res. 10, 4215-4222). The pMC5 probe comes from the DNA of M.capricolum. Profile of
M. fermentans DEB is very close to that of
M.fermentans strain No. 8 The DNA was hydrolyzed by EcoR1 (Boehringer) for 16 h at 37 ° C., subjected to electrophoresis on 1% agarose gel, transferred by the Southern technique and hybridized with mPC5 (previously labeled with 32P by nick-Translation (Amersham kit).

 It follows from the above tests that the strain according to the invention had characteristics specific to M. fermentans. In addition, after culture in SP4 medium, the strain studied was found to exhibit the characteristic development cycle which had already been observed with other strains.

M. fermentans.

 Under stress conditions, the "plasmodial" (tube) forms of the growing strain transform into mycospheres or small dense units, substantially spherical, with diameters of about 140-160mM. This transformation can be observed in glass tubes and in an SP4 medium depleted first, then enriched in lactic acid. In practice when 10 units of color change (of the mycoplasma) are inoculated in 1 ml of SP4 medium, the complete transformation of the mycoplasmas into dense individualized spheres is observed approximately 3 days later. When these spheres are brought back into fresh medium they "germinate" by forming tubes having a diameter of then of 75 nm leading finally to the plasmodial forms. The same spheres - or mycospheres - are observed inside T4 lymphocytes when they are infected with mycoplasmas of this type when they enter the cytoplasm.

 The same cytoplasms were detected in white cells obtained by cytophoresis, in marrow cells taken under heparin and in the patient's urine. The white blood cells and the marrow lymphocytes were separated on Ficoll gradients, as already mentioned above.

 As in the previous case, characteristic signals of M. fermentans were found in DNA extracted from white cells and marrow cells.

 Electron microscopy tests have revealed, after negative staining in ammonium molitate, characteristic plasmodial forms of mycoplasmas in the lymphocytes in the surrounding marrow cells and in the urine with dense mycospheres. Dense mycospheres have also been found in scratches caused in the lining of the patient's cheek.

 These trials therefore clearly establish an infection of this patient with M.fermentans. It has also been hypothesized very seriously that this strain of M. fermentans could be the cause of the patient's immunodeficiency.

 In fact, this strain of M.fermentans has been shown to be able to infect several cultures of activated T lymphocytes from various blood donors. In some cases, the lymphocyte subpopulations have been purified by contacting plastic surfaces coated with a monoclonal antibody specifically recognizing CD8 lymphocytes.

 As already indicated above, the major cytopathic effect has been observed in cultures of T cells depleted in T8 cells. The cytopathic effect mainly affected T4 cells. In the absence of T8 cells, 100% of the cells were lysed in 3 days, the first effects of lysis having been observed from the second day of infection. T8 cells were affected to a lesser degree.

In contrast, an infection of T4 cells of chimpanzees similarly infected, in the absence of cells
T8, were not infected in the same way, in the presence of the uracyl marker.

 The electron microscope examination of infected lymphocytes led to the following observation, in human T4 lymphocytes and chimpanzee T4 lymphocytes. In the case of human lymphocytes, numerous mycospheres have been observed in the cytoplasms of cells undergoing lysis. On the contrary, in preparations of chimpanzee lymphocytes, the presence of mycoplasmas could only be observed outside the plasma membranes. These tests tend to establish that the cytopathic effect of the mycoplasma strain of human cells must be linked to their capacity for intracellular penetration into human lymphocytes.

 The invention also relates to the applications of these cytopathogenic mycoplasmas. In particular, they can be used in a method for measuring the in vitro sensitivity of a patient to infection by M. fermentans, this method comprising the infection of the lymphocytes of this patient by the cytopathogenic mycoplasma and observation, especially under the microscope of the delay in which this cytopathogenic effect manifests. The death of cells is, for example, identified by their permeability to blue trypan.

 In a variant of this application, applied to healthy lymphocytes, the method according to the invention allows the evaluation of the protective effect of drugs, in particular antibiotics, against mycoplasmas.

 Similarly, these mycoplasmas, in particular in the form of mycospheres, can they be used in tests for the diagnosis of the presence of M.fermentans antibodies in a biological fluid, in particular a serum, as has been recalled, more particularly in relation with the M. fermentans No. 8 strain.

 In its most general form, the method according to the invention for the in vitro diagnosis in a biological fluid of a previous infection with mycoplasmas is characterized by bringing this biological fluid into contact in an appropriate nutritive medium with mycoplasmas under conditions allowing an interaction between these mycoplasmas and antibodies recognizing these mycoplasmas and possibly present in this biological fluid.

 In one of the forms of implementation of this method, the in vitro diagnosis is based on the demonstration of a neutralizing action of these antibodies against said mycoplasmas, when these are brought into contact with the biological fluid studied within an appropriate culture medium. The visualization of the neutralizing action can be done by any suitable means.

 Advantageously, use may be made of the manifestations of a reaction indicator capable of being affected by one of the products released during the development of the mycoplasma, for example a pH change indicator making it possible to detect the production of lactic acid, when the culture medium used contains glucose and this undergoes fermentation by the mycoplasma during its development.

 Thus the presence of anti-mycoplasma antibodies in the sample studied will manifest itself by inhibiting the development of the microorganism. On the contrary, we will observe a development of mycoplasmas, consequently an acidification of the medium, in the absence of antibodies in the sample.

 The methods of in vitro diagnosis of a mycoplasma infection are easier and more sensitive to implement when, like M.

fermentans, the mycoplasma studied can be isolated and produced as mycospheres. These then give rise to more sensitive agglutination reactions, when they are brought into contact with biological samples, in particular blood sera containing anti-mycoplasma antibodies.

 The invention therefore also relates to kits for the diagnosis of an infection due to mycoplasmas, the main reagent of these kits then consisting of a mycoplasma, in particular in the form of mycospheres, or a mycoplasma antigen.

 In a preferred form of kit, at least when the mycoplasma studied can be produced and isolated in the form of mycospheres, the main reagent consists of a conjugate between this mycoplasma and inert support particles allowing the performance of agglutination reaction, or even of haemagglutination.

 To demonstrate a mycoplasma infection involving the serum neutralizing properties of the antibodies possibly present in the biological samples or samples to be studied, the kits of the genus in question contain as princeps constituents - mycoplasmas in lyophilized or frozen form likely to be re-cultured - the appropriate culture media suitable for receiving both the mycoplasmas and biological samples or samples to be studied - the indicator or indicators of the possible growth of the mycoplasma culture.

 The invention also relates to kits comprising, in addition to the main reagents mentioned above, synthetic or natural antigens capable of being recognized by antibodies characteristic of HIV, as soon as the biological sample or sample contains them.

 The invention also relates to anti-mycoplasma vaccine compositions essentially characterized by attenuated or killed forms of mycospheres of mycoplasmas, in particular of M.

fermentans, or proteins or peptides derived from these mycospheres as well as from their fragments, these can be obtained by enzymatic hydrolysis, for example with the V8 protein, or by chemical synthesis or by expression of nucleotide sequences corresponding to the peptide sequences of the above fragments by genetic engineering techniques. Fragments of proteins or peptides derived from the above mycospheres are naturally characterized by their ability to be recognized by the same antibodies as the mycospheres themselves.

 The invention therefore provides a vaccine intended to prevent AIDS due to cytopathic mycoplasmas. Such a vaccine can also be used as a complementary vaccine making it possible to delay, if not to halt, the development of AIDS in subjects who are seropositive or at high risk of developing it. This vaccination takes on its full value as soon as it is recognized that annihilation in the subject of mycoplasmas can also lead to a slowing down of the viral activity of HIV in the seropositive or potentially seropositive subject.

 Finally, the invention relates to seroneutralizing antibodies capable of being induced by mycoplasmas according to the invention and to their use in immunotherapy, in order to stop an infection due to such mycoplasmas.

Figures 1 and 2 correspond to the photographs which are attached. The captions for these photos are as follows
Photo n-1: PCR result with primers
specific to N. fermentans (Wang et al,
1992)
Track 1: H20
Lane 2: witness "Lyse"
Lane 3: PBME
Lane 4: marrow lymphoid cells
Lane 5: marrow "debris"
Lane 6: urine
Lane 7: plasma
Lane 8: lysis (witness)
Lane 9: M. fermentans (100 / g)
Photo n-2: RFLP For M.fermentans DEB and n S,
M. genitalium, M. pirum and M. hominis. DNA has
been hydrolyzed with EcoRI; then hybridized with
pME5

Claims (12)

R ~ E VENDICATION8  1. Mycoplasma strain also having a cytopathogenicity specific to these mycoplasmas, capable of specifically infecting lymphocytes Human CD4 and induce in the patient who is carrying it an immunodeficiency syndrome characteristic of AIDS, even in the absence of HIV.  2. Mycoplasma strain according to claim 1, capable of infecting CD4 lymphocytes and of replicating, in the absence of CD8 lymphocytes, and of producing complete lysis of these CD4 lymphocytes, in particular in the space of 3 days.  3. Strain according to claim 1 or claim 2, characterized in that it is of the Mycoplasma type. fermentans.  4. Strain according to claim 3, characterized in that it was the subject of a deposit with the CNCM under the number I-1265, on September 11, 1992.  5. Strain according to any one of claims 1 to 4, characterized in that it is in the form of mycospheres having an average diameter of the order of 140-160 nm.  6. Method for measuring the in vitro sensitivity of a patient to an infection by M. fermentans, this method comprising the infection of the lymphocytes of this patient with the cytopathic mycoplasm according to any one of claims 1 to 5, and l observation, in particular under the microscope, of the time within which this cytopathogenic effect manifests.  7. Method according to claim 6, characterized in that it is applied to the evaluation of the protective effect of drugs, in particular antibiotics, against mycoplasmas.  8. A method of in vitro diagnosis in a biological fluid of a previous infection with mycoplasmas, characterized by bringing this biological fluid into contact in an appropriate nutritive medium with mycoplasmas according to any one of claims 1 to 5, under conditions allowing an interaction between these mycoplasmas and antibodies recognizing these mycoplasmas and possibly present in this biological fluid.  9. Method according to claim 8, characterized in that the in vitro diagnosis is based on the demonstration of a neutralizing action of these antibodies against said mycoplasmas, when these are brought into contact with the biological fluid studied within an appropriate culture medium, the neutralizing action being able to be visualized by any suitable means, in particular the use of the manifestations of a reaction indicator capable of being affected by one of the products released on the occasion of the development of the mycoplasma, for example an indicator of a change in pH making it possible to detect the production of lactic acid, when the culture medium used contains glucose and this latter undergoes a fermentation by the mycoplasma during its development.  10. Method according to claim 8, characterized in that the mycoplasmas are in the state of mycospheres.  11. Anti-mycoplasma vaccines essentially characterized by attenuated or killed forms of mycospheres of mycoplasmas, according to any one of claims 1 to 5, or proteins or peptides derived from mycospheres of these mycoplasmas or fragments of these proteins or peptides.  12. Antibodies specific for mycoplasmas according to any one of claims 1 to 5.