WO1994003810A1

WO1994003810A1 - Method for detecting mycoplasmic infections

Info

Publication number: WO1994003810A1
Application number: PCT/FR1993/000787
Authority: WO
Inventors: Luc Montagnier; Charles Dauguet; Denise Guetard
Original assignee: Institut Pasteur
Priority date: 1992-08-03
Filing date: 1993-08-03
Publication date: 1994-02-17
Also published as: FR2694402A1; FR2694402B1

Abstract

The invention relates to a method for the in vitro diagnosis of a prior infection in a subject by mycoplasms, characterized by contacting a biological fluid taken from said subject with mycoplasms in a nutrient medium authorizing their development in conditions allowing an interaction between said mycoplasms, particularly Mycoplasma fermentans in the form of mycoplasms, and antibodies against said mycoplasms, optionally present in said biological fluid. A positive response may optionally be correlated with a prognosis of seropositivity, even AIDS.

Description

METHOD FOR THE DETECTION OF MYCOPLASM INFECTIONS

The importance of mycoplasma infections in animal medicine is recognized. This is less the case in humans, since few species have been recognized as pathogenic.

However, the possible role of certain mycoplasmas as co-factors of AIDS was recently considered by our team (1), following the work of Lo et al. (2) and from our own work.

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

These are Mycoplasma fermentans, Incognitus strain of Lo (3) and strain No. 8 (Montagnier, ref. 4) and also Mycoplasma pirum (Montagnier, ref. 5). A third species was recently isolated from the urine of AIDS patients by Lo and his team. This species is apparently new and has been given the name Mycoplasma penetrans (6).

In a study of 43 urine samples from 40 HIV-infected subjects, 10 (23%) were positive for Mycoplasma fermentans by the chain reaction or PCR (11), and no seronegative.

In another study, the DNA sequences of Mycoplasma fermentans were found in the blood of 6/55 (11%) HIV-positive patients, and in no individual at low risk of HIV infection (12). The three species of mycoplasma mentioned above have in common the properties of hydrolyzing arginine and fermenting glucose, and of being able to easily penetrate inside the cytoplasm of infected cells.

M. piru and M. penetrans have in common a differentiated end capable of attaching to the cell and probably of permitting penetration.

In vitro, these mycoplasmas are capable of considerably increasing the cytopathogenic power of HIV-1 and HIV-2 on T4 lymphocytes or derived tumor lines (7) (8) (9).

In addition, extracts from M. pirum can restore the multiplication potential of a strain of HIV-1 apparently free of its mycoplasma contamination (1).

Finally, an antibody directed against the adhesion site of M. genitalium (close to M. pirum) can inhibit the replication of certain strains of V H-1 (10) and HIV-2.

The weakness of humoral immunity against mycoplasmas in other types of conditions in which they may already have been involved (for general documentation on mycoplasmas, see Chapter 10 entitled "The Mycoplasmas" of the work entitled Bergey Manual, or in the chapter entitled "The molliculites Mycoplasmas and ureaplasmas" by JG Tully and S. Razin, in the work entitled "Practical handboo of Microbiology" edited by William M. O'Leary, 1989, edited by CRC Press, Inc., Boca Raton, Florida, USA) has not been an access route to date really explorable for the in vitro diagnosis of infections by mycoplasmas or of distinct affections likely to be correlated with an infection by mycoplasmas.

The present invention aims to determine the presence of anti-fermentan antibodies in serum and other biological fluids, but it can be applied to any other similar mycoplasma.

The invention follows from the observation - new for fermentans, even if a similar observation has been made in the past for certain non-human mycoplasmas - that the life cycle of the mycoplasma passes through an extremely compact form which we call here "mycosphere" .

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

It has been observed that these mycospheres, at least those of M. Fermentans, exhibit a certain degree of stability even in an acid medium, contrary to what has been observed for other mycoplasmas which have already given rise to the observation of " mycospheres ".

In addition, mycoplasmas in this form are more easily recognized by sera taken from patients infected with these mycoplasmas.

The possibility offered by the invention to allow the detection of anti-fermentan antibodies in a biological fluid, in particular a serum, has also opened the way, in particular to two forms of in vitro detection of infections by this type of mycoplasma. It turned out that the antibodies contained in the sera of infected patients and capable of recognizing M. anti-fermentans in the form of mycospheres, had seroneutralizing properties which could be used to inhibit the development of a corresponding mycoplasma inoculum brought into contact within 'an appropriate culture medium, the observation of a possible inhibition "signing" then the presence of antibodies of the genus in question in the biological fluid tested.

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 appropriate 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 acid production 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 use when, like M. fermentans, the mycoplasma studied can be isolated and produced in the form of 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.

It should be noted that in the patient's serum the titer of neutralizing antibodies is extremely low and does not exceed 1/20, which explains the difficulty in detecting said antibodies by conventional methods.

When these mycospheres can be produced and stored, they constitute a reagent of choice for the diagnosis of a mycoplasma infection.

As such, the mycospheres of M. fermentans, in particular the M. fermentans strain strain ⁰ 8, deposited at the CNCM under the number 1-949, on May 22, 1990, constitute new products, in particular for the production of kits in vitro detection of infections due to mycoplasmas. The process for the production of fermentan mycospheres (illustrated by an example presented below) involves the cultivation of such a strain of mycoplasmas in SP4 medium (13).

The invention also lies in obtaining these forms at will by allowing cultures to age in an SP4 medium.

The serum of patients infected with mycoplasma contains antibodies which agglutinate the mycospheres. 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 have been fixed.

In addition, a number of these sera have the power to neutralize the growth of the mycoplasma, particularly from the mycospheres. However, the antibody titer is low, 1/6 to 1/10, so it could not have been detected by other methods.

The 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 / 1 of fluid and without bicarbonate

- 3 ml of a 50% glucose solution

- 0.6 ml of a phenol red solution (1%)

- 50 ml of fetal calf serum (GIBCO).

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

One of the particular advantages of the mycospheres of mycoplasmas, in particular of M. fermentans, lies in their capacity to be fixed via covalent bonds, on immunologically inert supports, hence the possibility of visualizing the reactions between the antigens (in this case the mycospheres coupled to immunologically inert supports) and the antibodies possibly contained in the biological sample under study, thanks to agglutination or hemagglutination reactions.

Advantageously, the immunologically inert supports consist of micro-supports or microbeads in latex or any other suitable material (plastic, silicate, dextran ball, etc.).

The coupling by covalent bond of these mycospheres for micro-supports or microbeads can be carried out in any known manner. By way of example, it is possible to use the techniques described by Brandt et al or by Avraméas et al, which have been the subject of publications in articles. whose bibliographic references are provided below.

The detection made possible by the invention of the presence of anti-mycoplasma antibodies in biological samples, in particular sera, from subjects infected with mycoplasmas, also confirmed the correlation which had already been made in a large number of patients, the coexistence of both mycoplasma and HIV infections. The invention has been found to open up a new way of screening at a very early stage for possible HIV infection in high-risk subjects.

In particular, it is found in many cases that the detection of an infection with mycoplasmas, in particular of the M. fermentans type, under the conditions mentioned above, precedes the detection of an seropositivity by conventional tests using implements an immunological contact between the biological sample under study and an antigen or an antigen composition characteristic of HIV.

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. For the demonstration of an infection by mycoplasmas involving the seroneutralizing 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 which can be re-cultured; the appropriate culture media suitable for receiving both the mycoplasmas and the 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 their fragments; these can be obtained by enzymatic hydrolysis, for example with the V8 protein, or by chemical synthesis or also by expression of nucleotide sequences corresponding to the peptide sequences of the above fragments by genetic engineering techniques. The fragments of proteins or peptides derived from the above-mentioned mycospheres are naturally characterized by their ability to be recognized by the same antibodies as the mycospheres themselves.

The invention therefore provides 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.

Preferred embodiments of the invention and the results which have already been obtained by the implementation of the invention are indicated below, by way of nonlimiting examples.

I. Preparation of the mycospheres

The strain No. 8 (deposited in the National Collection of Microorganisms), isolated from an asymptomatic patient infected with HIV, is cultured in an SP4 medium (13), each passage receiving 1/50 of inoculu from a previous passage. (about 100 doses changing the color). The color change linked to glycolysis generally appears around day 5.

From this day, samples are examined daily with an electron microscope to estimate the degree of transformation into mycospheres.

When this change reaches 90%, the culture is stopped, the suspension is divided into 1 ml aliquots which are frozen at -135 ° C. The transformation into mycospheres is linked to the exhaustion of the medium and the drop in pH.

II. Agglutination by specific antibodies The sera are prepared from blood drawn from patients on dry tubes. They are stored at + 4 ° C until they are used.

For the immuno-agglutination observed by electron microscopy, they are diluted 1/5 in PBS and filtered through Millipore filters of 0.22 microns.

Mesch 200 copper grids are used, covered with a collodion film and a carbon film.

The grids are placed on the sera diluted for 10 minutes at laboratory temperature and absorbed on filter paper in order to remove the excess serum. They are deposited on a suspension of mycospheres for 30 minutes then are absorbed on filter paper and stained with 6% amomium molybdate in distilled water for 2 minutes.

These grids are examined under an electron microscope (for example "Jeol" JEM 1200 EXII) at an acceleration voltage of 80 Kv and a direct magnification of 50,000. The photos are taken on Kodak 6.5 X 9 film plans, ref. 4489.

If the serum has no agglutinating antibodies, it is observed that the mycospheres remain dispersed, or exist in small packages a few units. If the serum is agglutinating, large aggregates of several tens of mycospheres are observed. III. Antibody detection by agglutination of microbeads

A simpler and easier method to use in routine laboratory is to link the mycospheres by covalent bond (for example by the technique of Brandt et al, Biochemica Biophysica Acta 1975, 386, p 196-202 or the technique of Avraméas et al described in US patent n ° 4,925,921 issued on 5/15/90 or the technique described in PCT patent application n ° 82 / 00203 deposited on, 8/07/81) to microbeads of latex or any other suitable material (plastic, silicates, dextran beads, etc.).

These beads are then incubated with different dilutions of serum and the presence of a compact pellet, if the agglutination is positive, is observed either with the naked eye or with an optical microscope at low magnification.

Otherwise, the microbeads remain dispersed.

One can, from the greatest dilution of serum causing agglutination, calculate the titer of the antibodies. IV. Search for neutralizing antibodies

The sera of certain patients also contain antibodies capable of neutralizing the growth of M. fermentans in SP4 medium.

This growth results in an inhibition of the change in color from red to yellow of the SP4 medium. Indeed, this medium contains phenol red capable of turning yellow at an acid pH. The lactic acid produced by the fermentation of glucose by the mycoplasma will cause this acidification.

To perform this test, we proceed as follows:

- O, 2 ml of the serum to be tested are added to 1.1 ml of SP4 medium (ie 15.4%);

- the mixture is filtered successively on a 0.22 m Millipore filter, then 0.1 _/ . ; - narrow glass tubes (6 m / m in diameter) are used so as to observe the change of environment.

Each tube receives 50 μl of a suspension of M. fermentans N8 (a previous passage from a culture having turned yellow in SP4 medium, containing approximately 100 doses of turning yellow).

Control tubes are also used, one negative containing SP4 medium and 15% fetal calf serum, the other positive, like the previous one, but also receiving the suspension of M. fermentans.

Three days later, we look at the color changes of the tubes, compared to the positive control which turned yellow.

Dilutions of test sera can also be used to determine the titer of neutralizing antibodies.

A table giving the results obtained on 141 sera with this method is given below. Note that the presence of these antibodies means:

1 / that the person was infected with M. fermentans, probably up to a systemic infection since the presence of serum antibodies is rarely found in local urogenital mycoplasma infections;

2 / that the presence of neutralizing antibodies, even at a low level, means that the person has some protection against the mycoplasma. The titer observed, however, does not generally exceed 1/10 to 1/20; The compilation of the results shows a significant different response depending on the groups.

In a sample corresponding to a population with low exposure to HIV, no neutralizing serum was found M. fermentans, while under the same conditions, neutralizing antibodies against M. pneumoniae and M. pirum are quite frequently observed.

This corresponds to data already published in the literature which show that infection with M. fermentans is rare in the general population.

On the other hand, in a group of HIV positive people, almost a third have anti-fermentan antibodies. These antibodies exist at all stages of infection, even early, although they tend to disappear in the terminal phase of the disease, as was observed in a patient with sera available for 3 years.

On the other hand, no significant increase in the proportion of sera having anti-M antibodies was observed. pneumoniae or anti-M. pirum.

It should also be noted that several sera having neutralizing antibodies against M. fermentans came from patients, either having been infected with HIV by blood transfusion (one case), or by antihemophilic products (one case), or Africans (two cases).

This suggests that infection with M. fermentans may have been acquired through the bloodstream at the same time (or independently for hemophiliacs) as infection with HIV.

Particularly interesting are the so-called HIV-negative risk groups. In the group of hemophiliacs, who remained seronegative, but who had continuously received before 1985 concentrates of factor VIII potentially contaminated with HIV, and often presenting slight immune deficiencies, a significant proportion (43%) possessed neutralizing anti-fermentant antibodies, markedly more than the group of HIV-positive hemophiliacs (1/18). This difference would suggest that the presence of these antibodies would confer some protective power against HIV infection, at least in this type of population.

In individuals exposed to HIV through sexual transmission (especially partners who remain HIV-negative to HIV-positive people), here too a high proportion (38%) have neutralizing anti-fermentant antibodies.

In conclusion, these results suggest that M. fermentans infection may be a co-factor of HIV infection and AIDS. In the current state of our data, this co-factor role can be exclusive or non-exclusive: non-exclusive, if we admit that the serological data obtained by the new tests described reflect a real picture of the situation: some people infected with M. fermentans at the same time or not as with HIV, others not. In this case, other species of mycoplasma (M. penetrans, M. pirum) could play a role similar to M. fermentans. However, the serological data are not in favor of a particular role for M. pirum, which is also found in low-risk HIV negative people. exclusive, if we admit that the presence of antibodies - and especially neutralizers - is not constant in subjects infected with M. fermentans. However, other methods, in principle more sensitive, such as the search for the DNA of the mycoplasma by PCR do not give results significantly different from those given by serology.

Anyway, the serological detection of M. fermentans infection can bring a new parameter to the study of the evolution of seropositive people. It may be an unfavorable part of the prognosis for AIDS.

In addition, the results obtained in the seronegative risk groups, encourage the use of M. fermentans in an inactive form, or proteins derived from the mycoplasma, to protect against this mycoplasma from seronegative subjects exposed to HIV.

A vaccine complementary to that of HIV could thus be obtained from this mycoplasma or from fragments of vaccinating proteins or polypeptides, as defined above.

RESULT OF THE METABOLIC INHIBITION TEST OF THE

GROWTH OF M. FERMENTANS (N8) IN THE MIDDLE

SP4 BY 1/6 DILUTED HUMAN SERUMS

I. HIV negative, low risk 0/34 (<2.9%

II. HIV negative, high risk

(by sexual transmission) 5/13 (38%)

III. Hemophiliacs, HIV negative, high risk

(by concentrates until 1985) 6/14 (43%)

IV. Hemophiliacs, HIV positive 1/18 (5.5%)

V. HIV positive

(by sexual transmission) 20/62 (32%)

REPLACEMENT SHEET BIBLIOGRAPHY

1. Montagnier L.

A possible role of mycoplasmas as co-factors in

AIDS

Fifth Colloquium of the Hundred Guards, 1990

2. Lo S.-C. et al.

Identification of Mycoplasma incognitus infection in patients with AIDS: an immunohistochemical, in situ hybridization and ultrastructural study. Am J. Trop. Med. Hyg. , 41.601-616, 1989.

3. Saillard C., Carie P., Bové J.M .., Bébéar C., Lo S.-C, Shi J.W.-K., Wang R.Y.-H., Rose D.L. and Tully J.G.

Genetic and serology relatedness between • Mycoplasma fermentans strains and a mycoplasma recently identified in tissues of AIDS and non-AIDS patients. Res. Virol., 141.17-27, 1990.

4. CNCM filing

5. CNCM filing

6. Lo S.-C, Hayes M.M., Wang R. Y.-H., Pierce P.F., Kotani H. and Shih J.W.-K.

Newly discovered mycoplasma isolated from patients infected with HIV. Lancet, 388, 1415-1418, 1991.

7. Lemaître M., Guétard D., Hénin Y., Montagnier

L. and Zérial A. Protective activity of tetracycline analogs against the cytopathic effect of the human immunodeficiency viruses in CEM cells. Res. Virol., 141 _/ 5-16, 1990.

8. Lo S.-C, Tsai S., Benish J.R., Shih J.W.-K., Wear D.J. and Wong D.M.

REPLACEMENT SHEET Enhancement of HIV-l cytocidal effects in CD4 + lymphocytes by the AIDS-associated mycoplasma. Sciences, 251, 1074-1076, 1991.

9. Lemaître M., Hénin Y., Destouesse F., Ferrieux C., Montagnier L. and Blanchard A.

Role of mycoplasma infection in the cytopathic effect induced by hu an immunodeficiency virus type 1 in infected cell lines. Infect. Immun. , 0.742-748, 1992.

10. Montagnier L., Berneman D., Guétard D., Blanchard A., Chamaret S., Rame V., Van Rietschoten J., Mabrouk K. and Bahraoui E. Inhibition of infectivity of prototype HIV strains by antibodies to a mycoplasma peptide sequence. CR. Acad. Sci. Paris, 311, Series III, 425-430,

1990.

11. Dawson M.S., Wang R., Hayes M. et al. Detection and isolation of Mycoplasma fermentans from urine of patients with AIDS (abstract G-4).

In: Program and abstracts gênerai meeting of the American Society for Microbiology (Dallas). Washington DC. American Society for Microbiology, 1991.

12. Hawkins R.E., Rickman L.S., Vermund S.H. and Mitchell C.

Association of Mycoplasma and human immunodeficiency virus infection: detection of amplified Mycoplasma fermentans DNA in blood. J. of Infect. Diseases, 165, 581-585, 1992.

13. Whitcomb R.F.

Media culture for spiroplasmas. • Methods Mycoplasmol. , 1, 147-158, 1983.

REPLACEMENT SHEET

Claims

1. Method for the in vitro detection of antibodies resulting from a previous infection of a subject with mycoplasmas, characterized by bringing a biological fluid taken from this subject into contact with mycospheres obtained within a nutritive medium allowing the development of mycoplasmas into mycospheres under conditions allowing an interaction between these mycospheres and antibodies against mycosplas es, possibly present in this biological fluid.

2. Method according to claim 1, characterized by the demonstration of a neutralizing action of these antibodies against said mycoplasmas.

3. Method according to claim 2, characterized in that the above highlighted relies on the detection of a possible interaction between a product released into the culture medium by the mycoplasma during its development in the mycosphere and an indicator able to detect this product.

4. Method according to claim 1, characterized by the demonstration of agglutination, in particular hemagglutination, when the biological medium is a blood serum, of these mycospheres by said antibodies.

5. Method according to claim 4, characterized in that the mycospheres are covalently attached to immunologically inert microcarriers.

6. Method according to any one of claims l to 6, characterized in that the mycospheres come from mycoplasmas belonging to the species Mycoplasma fermentans.

7. Method according to any one of claims 1 to 6 applied to establishing a possible correlation with an AIDS prognosis, in particular in high-risk subjects, not yet seropositive.

8. A method of detecting neutralizing antibodies in a biological fluid, characterized in that said biological fluid is brought into contact with the mycoplasmas, within a nutritive medium allowing the development of these mycoplasmas in mycospheres, and in this that the capacity of said biological fluid to inhibit this development in mycospheres is measured.

9. Kit for the in vitro diagnosis of an infection by a mycoplasma in a sample or biological sample, in particular serum, characterized by the coexistence in this kit of the main reagent that constitutes a mycoplasma in the frozen or lyophilized state, suitable for being cultured, and components of a culture medium for these mycoplasmas.

10. Kit according to claim 9, characterized in that the mycoplasma, in particular M. fermentans, is in the state of mycospheres capable of being preserved in this state, in the frozen state, in particular in an SP4 medium.

11. Kit according to claim 9 or 10, characterized in that it contains a separate main reagent consisting of an antigen or composition of antigens capable of being recognized immunologically by the anti-HIV antibodies contained in the serum of a seropositive subject.

12. Mycospheres of M. fermentans.

13. A vaccine composition against mycoplasmas characterized in that the active principle consists of mycospheres of Mycoplasma fermentans or by fragments of proteins or polypeptides of M. fermentans recognized by the antibodies which also recognize M. fermentans.