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Title:  Process for the in vitro culture of different stages of tissular parasites
United States Patent: 
7,317,094
Issued: 
January 8, 2008

Inventors: 
Lemesre; Jean-Loup (Saint Martin de Londres, FR) Assignee:  Institut Francais de Recherche Scientifique pour le Developpement en Cooperation (Orstom) (Paris, FR)
Appl. No.:  10/260,497
Filed: 
October 1, 2002


 

Outsourcing Guide


Abstract

The process of the invention comprises the implementation of axenic conditions, with use of a liquid single-phase culture medium. For obtaining the amastigote forms, this medium is buffered at a pH of 5.5 to 6.5 and has an osmolarity of at least 400 milliosmoles/kg of liquid, and in particular 400 to 550 milliosmoles/kg of liquid. For obtaining promastigote forms, this medium is buffered at a pH of 7 to 7.5 and has an osmolarity of at least 300 milliosmoles/kg of liquid. This process allows the adaptation and culture in vitro of different stages of tissular parasites, such as leishmanias and T. cruzi or also hematoprotozoa.

Description of the Invention

A subject of the invention relates to a process for the in vitro culture of different stages of the developmental cycle of a parasite. It also relates to the parasitic forms obtained and their biological uses.

By culture is meant, in the description which follows and the claims, both the adaptation of the parasitic form by successive passages in a given medium, and complete differentiation when it occurs for the adaptation and the culture itself of the parasitic form.

It is known that parasites constitute a real plague causing, by the intermediary of vectors, the infection of millions of people and animals.

Thus, leishmaniases, which are widespread throughout the world, are caused by protozoa of the Leishmania genus which are usually transmitted by a sand fly, Phlebotomus. Leishmaniases of the Old World and those of the New World are usually distinguished according to their geographical localization. They have very diverse clinical forms which differ significantly by their seriousness and their effect on health. Cutaneous, mucocutaneous (attacking nasal, buccal mucous membrane and those of the ears) and visceral leishmaniases are distinguished.

As another parasite having a devastating effect, there can also be mentioned Trypanosoma cruzi, responsible for Chaga's disease. In South America, it causes the infection of millions of people. Over 150 species of wild and domestic animals can be counted which can serve as hosts to the parasite which is transmitted to man by a bug, namely a Triatoma, which feeds on blood.

The infection can pass unnoticed for several years until the trypanosomas attack the nervous system, the heart or the digestive system.

The development cycle of many parasites includes various parasitic stages. That of Leishmania, for example, includes two stages having important differences at the structural, morphological, biochemical, immunological and physiological level, namely in a vector insect, a flagellated form, called promastigote, which multiplies by fission before acquiring its infectious form, for the mammalian host, also called metacyclic, in a mammalian host, a non-flagellated stage, called amastigote, which exclusively parasitizes mononuclear phagocyte cells.

The differentiation into amastigotes occurs after attachment and penetration of the promastigotes into the monocytes. But only the amastigote forms persist and multiply inside the phagolysosome of the macrophages of the infected host.

In T. cruzi, this cycle includes three different parasitic stages, an epimastigote form, multiplication form of the vector insect, and the amastigote and trypomastigote forms, which are present in the infected host.

At present, most of the research carried out on the diagnosis of these parasitoses and the development of vaccines is conducted on the promastigote form whose production in culture is easy and quick.

Now, the only form present in the infected host is the amastigote form which, by persisting throughout the infection, triggers the immune response and participates in the development of the pathology. The danger of systematically extrapolating the experimental results obtained with the cultured promastigote forms within the scope of immuno-prophylactic, diagnostic or therapeutic studies aimed at the amastigote forms will be well understood. in a vector insect, a flagellated form, called promastigote, which multiplies by scissiparity before acquiring its infectious form, for the mammalian host, also called metacyclic,

In order to respond to this problem, various authors have been interested in obtaining amastigote forms. Thus the obtaining of amastigotes from tissues of experimentally infected animals or from cultures of infected macrophages have been reported. But this involves long and tedious isolation methods, which moreover lead to a limited number of parasites being obtained, which are often degenerated and which are incapable of multiplying and of surviving for longer than 2 to 3 days.

Furthermore, such amastigote forms are contaminated with cells, fragments and molecules derived from the macrophages, tissues or plasma of the host, designated hereafter by "cellular contaminants", limiting or making impossible the realization of many studies.

Culturing processes under axenic conditions, that is to say in the absence of any cellular contaminant, have been proposed for some species of leishmanias and for T. cruzi, but they do not allow an abundant and continual source of amastigote forms to be made available, and do not appear to be generally applicable to a large number of species and to different germs.

An aim of the invention is to resolve the above disadvantages and to provide more satisfactory experimental models by producing the desired parasitic stages in specific culture media, of totally defined simplified composition.

The invention relates in particular to providing a process generally applicable to a large number of species of a given parasite, allowing homogeneous populations of a given parasitic stage to be produced in a continuous manner and in unlimited quantities.

It relates more particularly to a process allowing these stages to be obtained in a form which is free from any cellular and seric contaminant, having the characteristic of not containing any macromolecules. By macromolecules is meant, in the description and the claims which follow, non-dialyzable molecules with a cut-off threshold of 3 kDa, that is to say having an apparent molecular weight of greater than 3 kDa (for example seric protein such as albumin).

It also relates to the in vitro production of the complete development cycle of parasites under axenic and aseric conditions.

According to another aspect, the invention relates to the new parasitic forms obtained, corresponding to the different stages of the parasitic cycle and, for each stage, to the different phases of their growth.

According to another aspect, the invention relates to the applications of the parasitic stages obtained and of the products produced or isolated from these stages, in particular in the domain of the diagnosis of parasitoses, immunoprophylaxis and screening of drug activities.

The process according to the invention, for the in vitro culture of different stages of tissular parasites, such as leishmania, T. cruzi, or also hematoprotozoa is characterized in that it is carried out in an axenic and aseric, liquid single-phase culture medium, free from macromolecules (non-dialyzable at a cut-off threshold of 3 kDa) and that for obtaining amastigote forms, this medium is buffered at a pH of 5.5 to 6.5 and has an osmolarity of at least 400 milliosmoles/kg of liquid, and in particular from 400 to 550 milliosmoles/kg of liquid, or that, for obtaining promastigote forms, this medium is buffered at a pH of 7 to 7.5 and has an osmolarity of at least 300 milliosmoles/kg of liquid, and in particular from 300 to 380 milliosmoles/kg of liquid.

The pH value of these media, within the range indicated above, ensures that the culture conditions are strictly controlled.

In the case of the culture of amastigotes, when the pH is greater than 6.5, a tendency towards the retransformation of the amastigotes into promastigotes is in fact noted and when it is less than 5.5, a poor growth is observed.

According to a preferred method of the invention, for obtaining amastigote forms, a culture medium is used containing a base medium, produced essentially from: at least one culture medium for insect cells which has added to it inorganic salts, of Hanks' salts type, products which are sources of amino acids, such as L-glutamine and soja bean extracts, sugars, such as D-glucose.

As soja bean extact, there can advantageously be used that marketed under the Trade mark trypto casein soja .RTM.. The culture medium for insect cells is advantageously the medium 199 H marketed by GIBCO.

Different compositions of this 199 H.RTM. medium are given in the GIBCO BRL catalogue page 48, 1992 edition.

A specially preferred composition for the production of base media carries the reference 042-01181 on page 48 of this catalogue, 1992 edition. The 199H M medium is more especially used, to which NaHCO.sub.3 and L-glutamine are added.

This medium, to which the above compounds are added, is advantageously buffered, for example with a buffer such as HEPES.

A preferred composition of the base medium contains several culture media for insect cells.

A base medium of this type results from the addition to the 199 H medium as marketed by Gibco, to which the compounds mentioned above have been added, of another medium such as the modified 199 H medium as marketed by Flow. A composition of this medium is given in the Flow Laboratories catalogue, 1992 under the reference 14230-54. Before adding it to the initial preparation, this latter modified 199 H medium is subjected to a thermal treatment.

The base media defined above are new and, as such, are also a subject of the invention. They can be stored for several months at -20.degree. C.

In order to avoid oxidation of the parasitic stages, this base medium has added to it, at the time of use, anti-oxidizing agents such as hemin, which also has the advantage of constituting a source of iron and agents with a reducing effect such as reduced glutathionee. Vitamins are also advantageously added. A suitable mixture of vitamins includes biotin, calcium D pantothenate, choline chloride, folic acid, inositol, nicotinamide, para-aminobenzoic acid, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, some of which are used and advantageously all of which are used.

The resultant culture media are advantageously characterized in that they are free from nucleotides as additives. They can be stored at +4.degree. C. for about two weeks without alteration to their properties.

The use of the resultant culture media, as illustrated by the examples given hereafter, allows amastigote forms to be produced which are capable of mass multiplica vitro, in a continuous manner.

The axenic and aseric media have the advantage of being free from all macromolecules and in particular from those present in foetal calf serum and/or originating from the host cells, which can mask other constituents (seric and cellular proteins).

These media are particularly suitable for the amastigote forms of tissular protozoa, such as cutaneous or muco-cutaneous or visceral leishmanias, or various clones of T. cruzi, or also Plasmodium or Babesia.

As species of leishmanias, there can be mentioned L. mexicana, L. amazonensis, and L. braziliensis or also L. major, L. quvanensis and L. oanamensis.

Other preferred culture media, particularly suitable for the culture of the amastigote forms of visceral leishmanias, such as L. donovani, L. infantun and L. chagasi, contain, in addition, sulphurous compounds. These are in particular sulphurated amino acids, such as cysteine, and notably the L form, and/or nutritive products such as bathocuproine sulphonic acid.

For the differentiation of promastigote forms into amastigote forms, in the case of Leishmania, the cellular culture medium represents, relative to the final medium, about 8% to 15% (V/V), notably of the order of 10%, the amino acids, or products which are sources of these amino acids, such as soja trypto casein.RTM. and L-glutamine, are present at a rate of about 4% to 8% (W/V), notably about 5% to 6%, the supply of sugar, notably as glucose, is advantageously carried out at a rate of about 2% to 4% (W/V), notably 2% to 3%, the anti-oxidizing agents such as hemin, at a rate of 0.0002% to 0.0015% (W/V), notably of the order of 0.0005%, and glutathione at a rate of 0.01% to 0.05%, notably of the order of 0.025% and the vitamin solution (100.times.) at a rate of 1% to 5% (V/V), notably of the order of 2%.

The sulphurous compounds, when they are used, in particular L-cysteine, are used at a rate of about 0.25 to 0.50% (W/V), notably of the order of 0.3%, and bathocuproine sulphonic acid is used at a rate of about 0.004 to (W/V), notably of the order of 0.005%.

For the culture of the promastigote stages, a culture medium is advantageously used which is produced from a medium suitable for cell culture, such as RPMI 1640 medium, to which are added amino acids such as L-glutamine and a buffer to adjust the pH to a value of 7 to 7.5, this medium also having added to it another medium suitable for cell culture, in particular 199H M medium, containing inorganic salts such as Hanks' salts, and anti-oxidizing agents, such as hemin.

This medium is therefore free from any seric contaminant and contains no macromolecule as proved by 10% polyacrylamide gel analysis.

The 199H M medium is used at a rate of about at least 2% (V/V), notably about 2 to 10% and bovine hemin is used at a rate of 0.0001 to 0.0015% (w/v), notably of the order of 0.0005%.

The simplicity of preparation of such a medium from products which are already being marketed will be observed. Moreover the absence of serum advantageously produces an inexpensive product.

In accordance with the invention, these culture media are used in a process for the continuous mass production of parasitic forms.

For the adaptation and continuous culture in vitro of amastigote forms, a suitable culture medium as defined above is inoculated with promastigotes at the end of the exponential phase, at a rate of 10.sup.6 to 10.sup.7 promastigotes/ml of medium.

The conditions for carrying out the adaptation and culture are advantageously chosen in such a way as to ensure a total transformation of the parasites in a reproducible manner.

The adaptation, then the culture, are carried out at temperatures of the order of 28.degree. C. to 36.degree. C., and notably around 32.degree. C. at a pH of 5.5 to 6.5. Usually a transformation of the promastigote forms into amastigote forms in excess of 90% is observed in 6 to 7 days. This transformation is for example total in 4 days for the Leishmania, after a number of passages which varies according to the species studied and which generally corresponds to 3 to 9 subcultures, and which decreases when the incubation temperature increases.

For the adaptation and continuous culture in vitro of so-called primary culture or short-term promastigote forms of parasites, such as Leishmania, directly obtained from the amastigote forms, inoculation with the amastigote forms as obtained above is carried out, at a rate of 10.sup.6 to 10.sup.7 amastigote forms/ml of medium. Amastigote forms at the end of the exponential phase are advantageously used. The culture is carried out at a temperature close to ambient temperature but preferably not exceeding 28.degree. C. at a pH of 7 to 7.5 in a culture medium as defined above for the development of promastigote forms.

When the use intended for these parasitic stages does not require both axenic and aseric conditions, without the presence of macromolecules, to be used, it is possible within the scope o f the invention to carry out the culture in a purely axenic medium, therefore in the presence of serum and also in the presence of macromolecules. For example RPMI medium which has foetal calf serum added to it can be used.

The short-term promastigote forms thus obtained can be used in the inoculation stage mentioned above for the purposes of the differentiation into amastigotes.

The transformation is very rapid and total, for example Leishmania in 4 days, after 2 to 5 subcultures, according to the species.

The implementation of the provisions of the invention allows standardized and reproducible cultures of the various forms corresponding to the various parasitic stages to be obtained, which are free from any cellular contaminant and from macromolecules and capable of multiplying in vitro.

The provisions which precede have been described more praticlarly relative to the promastigote forms and the amastigote forms of Leishmania, whether cutaneous, muco-cutaneous or also visceral Leismania, but also apply to the parasitic stages of T. cruzi or other hematoprotozoa such as Plasmodium and Babesia.

These cultures can be kept for several months, even several years for many species, in particular for Leishmania, whether they are cutaneous, mucocutaneous or visceral, or for T. cruzi.

The parasitic stages are capable of undergoing long-term cultivation, that is to say over more than 50 passages in in vitro cultures, and short-term cultivation, that is to say recently transformed from promastigote forms or from amastigote forms (earlier form of the cycle) of less than 10 passages.

The invention also supplies the means for producing a complete parasitic cycle in vitro. It will advantageously be noted that this complete cycle can be carried out in less than 15 days.

An embodiment of a process, according to the invention, for the production of stages corresponding to the developmental cycle of a parasite such as Leishmania is characterized in that it is used under the axenic and aseric conditions defined above, in the absence of macromolecules and in that it comprises: inoculation of a suitable culture medium, as defined above, with short-term or long-term promastigote forms, so as to obtain the differentiation into amastigote forms, recovery of the amastigote forms produced, their inoculation and their culture as indicated above so as to obtain the differentiation into promastigote forms, the cycle being repeated in its entirety, or partially, if desired, and indefinitely.

According to another embodiment, the short-term forms are cultivated under purely axenic conditions, or axenic and aseric conditions in the presence of macromolecules.

Via the process of the invention, it is therefore possible to obtain in vitro the various parasitic stages much more quickly and easily than by the in vivo techniques currently used which involve experimental infections which are sometimes difficult to bring about. These amastigote or promastigote parasitic forms are free from any cellular contaminant and are capable of multiplying in vitro. Thus means are available which allow the abundant, and even unlimited, obtaining of the parasitic stages recently, or not, differentiated from the earlier stage, in particular of Leishmania and those of T. cruzi (epimastigotes, metacyclic and bloodstream trypomastigotes and amastigotes).

Also a subject of the invention is the parasitic forms of the developmental cycle of a tissular protozoan, such as Leishmania or T. cruzi, as obtained by implementing the culture process defined above.

These forms are characterized in that they are free from cellular contaminants, in particular tissular macrophage and plasmatic contaminants accompanying the intracellular parasitic forms isolated from cell cultures or from tissues of experimentally-infected animals, as well as any seric contaminant, while being endowed with infective power in vitro and in vivo as observed on the intracellular forms when these are habitually infective, and their morphological, biochemical and immunological characteristics, they are presented in the form of a homogeneous population relative to the age in culture and the state of differentiation for a given stage of the developmental cycle, this population, originating from a standardized culture, being capable of multiplying in vitro in a continuous manner.

These amastigote forms are also characterized in that they possess an enzymatic activity, more particularly a peptidase activity which is qualitatively more complex than those of the promastigotes, and quantitatively different, more particularly at the level of the cysteine-protease activities, as set out in the examples.

The invention relates more particularly to the amastigote forms of both anthroponotic and zoonotic, or anthropozoonotic Leishmania.

They can be the amastigote forms of cutaneous or muco-cutaneous Leishmania. Amongst these, there can be mentioned L. mexicana, L. amazonensis, L. braziliensis, L. cuyanensis and L. panamensis. They can also be the amastigotes of visceral Leishmania such as L. chagasi, L. donovani or L. infantum.

Other amastigote forms according to the invention are those of various clones of T. cruzi.

The invention also relates to the short-term promastigote forms as defined above.

They can be populations directly transformed from amastigote forms which have an infective power similar to that of promastigotes recently isolated from an infected host.

Each of the parasitic stages, promastigote or amastigote, has different growth phases during multiplication in vitro, nameiy a latent phase, and exponential phase and a stationary phase which correspond to the preparation of the division, to an intense multiplication, then to a non-division stage respectively.

The invention advantageously allows forms corresponding to one of these phases to be obtained in a targeted manner and their properties and specific biochemical characteristics to be studied.

These forms are characterized in that they are free from cellular and seric contaminants, as well as molecules non-dialyzable at a cut-off threshold of 3 kDa.

They are therefore populations defined according to a well-determined phase of their growth.

The corresponding amastigote or short-term promastigote forms are particularly preferred.

The invention also relates to the total polypeptide extracts of these parasitic forms as obtained by lysis of the cells and recovery of the soluble or insoluble products. These extracts are also called total antigenic extracts in the examples. By these expressions "total polypeptide extracts" or "total antigenic extracts" is meant the products as obtained by lysis of the parasitic forms, whether they are of protein, lipid or saccharide nature.

They can be in particular total polypeptide extracts of short-term promastigote forms and quite particularly total polypeptide extracts of amastigote forms at different stages of their growth in vitro.

These extracts are characterized by their peptide profile as revealed in a standard manner using SDS-PAGE polyacrylamide gel, under reducing condition or not, or on polyacrylamide gel under non-denaturing condition, as described in the examples for certain species of Leishmania.

The invention also relates to the antigenic, protein, glucide or lipid fractions and determinants eluted or isolated from fractions of these extracts.

The antigenic fractions and determinants of these extracts recognized, according to an antigen-antibody type reaction, by sera of animals immunized with the total polypeptide extracts or sera of natural or experimental infections are particularly preferred with regard to the immunological applications which are a subject of the invention, and in particular the basic specific antigenic fractions and determinants.

Products of this type correspond to the antigens expressed on the surface of the amastigote forms and to the somatic antigens present at the level of the flagellar pocket or of vacuolar type as revealed by immunofluorescence techniques.

The purified or semi-purified molecules and the solutions specifically enriched with one or more of these molecules also come within the scope of the invention, originating from natural lyses.

Other products which are of great interest with regard to the biological applications according to the invention correspond to the excretion antigens as obtained from the culture supernatants conditioned by the promastigote forms or by the amastigote forms cultivated under the axenic and aseric conditions of the invention, originating from natural lyses.

The same goes for the differentiation antigens secreted during different ation according to the process of the invention of the promastigote forms into amastigote forms and that of the amastigote forms into promastigote forms (during the in vitro production of the cycle).

These antigenic products are recovered from the supernatants by simple concentration and dialysis. These supernatants therefore constitute a pre-enriched purified source of antigenic products.

The invention also relates to the immunization sera as obtained after administration of the antigenic extracts, fractions and molecules defined above according to the usual techniques, and the antibodies recovered from these sera.

It also relates to the infection sera obtained by the infection of animals with the infectious amastigote forms.

The antibody content of these sera varies according to the phase of the parasitic stage and is higher against the stationary phase of the amastigote forms.

The antibodies of the invention are characterized in that they recognize the specific peptides of amastigote or promastigote parasitic forms by producing an antigen-antibody type reaction.

Such antibodies include those only specifically recognizing the antigens of an amastigote or promastigote parasitic form, belonging to a homologous species, that is to say to the same species as that used for obtaining them, the recognized form being that against which they have been formed.

They can be for example antibodies formed against an amastigote form of Leishmania of a given species and which only specifically recognize the antigenic peptides of the amastigote forms of this species of Leishmania.

The antibodies of the invention also include those which in addition have a poor recognition of the other parasitic form of the species considered.

There can be mentioned for example the anti-amastigote antibodies recognizing, to a lesser degree, antigens of the promastigote forms of the same species.

Other antibodies also of the invention are in addition capable of recognizing, but to a lesser degree, the parasitic forms of a heterologous species, or of another genus such as T. cruzi. Antibodies of this type correspond for example to anti-amastigote antibodies of a species of Leishmania recognizing amastigote peptides of another species of Leishmania.

According to another aspect, the invention relates to the monoclonal antibodies as advantageously obtained according to the standard techniques of fusion of a cell line with the spleen or ganglion lymphocytes of an animal immunized by injection with a total peptide extract, an antigenic fraction or molecule as defined above by screening the supernatants of the hybridomas obtained, for example according to the Elisa or IFI technique so as to reveal the antibodies directed specifically against a parasitic form of a species, as well as the clones of hybridomas secreting these monoclonal antibodies.

These antibodies constitute tools for selectively separating or isolating specific antigens of species or of stages from a medium containing them and in particular from the total polypeptide extracts mentioned above by immuno-affinity techniques.

The reaction of the above immunosera with antigenic fractions and molecules originating from a given phase of the development of the parasitic form allows the identification and isolation of the specific antigens of this stage.

The possibility of the mass production, due to the invention, of the cultured amastigote forms makes it possible to extract the total and messenger RNA's and, from these, to create a cDNA library.

According to another aspect, the invention therefore relates to the total RNA's as recovered from parasitic cultures of amastigotes or promastigotes, and the corresponding m-RNA's and cDNA's.

By comparison with a cDNA library of corresponding promastigote forms, specific peptides of a given parasitic stage are then revealed and their synthesis is proceeded with if appropriate by genetic engineering.

The obtaining, in accordance with the invention, of in vitro and in vivo infecting extracellular amastigote forms, having morphological, biological and biochemical characteristics similar to those of the intracellular amastigote forms, opens up new and numerous applications in the domains of research and industry.

The parasitic forms of the invention are thus particularly useful as experimental models for carrying out a first screening in vitro of products which can be active for them in vivo.

The screening process of the invention comprises: putting the parasitic forms, more especially the amastigote forms as cultivated under axenic conditions and notably aseric conditions, and the promastigote forms as cultivated in a completely defined medium, in contact with the products to be tested, incorporation of nucleotides or amino acids labelled with a radioactive group, for example tritiated thymidine, in order to determine the activity of the products to be tested, or the carrying out of viability tests using for example a tetrazolium salt such as MTT.

In the stage of putting in contact, the parasitic forms are used at concentrations of 10.sup.6 parasite/ml and the activity of the medicaments is studied at increasing concentrations.

The products to be tested can advantageously be labelled, for example with a radioactive group, to determine the action mechanisms and the flow of the drugs.

Thus the invention provides a model allowing, if desired, the comparison of the in vitro activity of medicaments on the promastigote form and on the amastigote form at different phases of their growth for a given parasite, and the testing of this activity on the actual form which is found in the host.

It allows improved characterization of the medicamentous activity by revealing either a lytic effect (leishmanicidal or trypanocidal), or an inhibitory effect on the multiplication (leishmaniostatic or trypanostatic) of the product.

The use of these parasitic forms as experimental models also allows the chemical resistance of the parasitoses to be studied.

In fact it is known that at present the resistance to medicaments constitutes a significant problem. Study of the mechanisms producing this resistance, which can easily be achieved using the models of the invention, is therefore of great interest.

The invention also relates to a kit for screening products which can be used for the treatment of parasitoses, more especially leishmanias or Chagas' disease.

This kit comprises a support such as a multi-well plate containing the parasitic forms on which is it desired to test the activity of the product to be studied, some of these forms being used as controls, and reagents to determine the medicamentous activity of the product on the parasitic forms.

As indicated above, studies carried out up to now have not allowed parasitoses to be satisfactorily identified.

In man, or in animals and particularly in dogs, the diagnosis of leishmaniasis for example is most often determined either by isolating the parasite and identifying it (parasitological examination), or by detecting (specific circulating) antibodies in the serum (immunoserological tests).

The industrial culture of the amastigote forms under axenic or aseric conditions and in the absence of macromolecules makes available an abundant source of useful diagnostic tools.

In this way they allow the early detection, with a high degree of sensitivity and a high specificity, of circulating antibodies directed against the parasites.

The invention therefore relates to a method for the diagnosis of a parasitosis in man or animals, more especially of an infection caused by Leishmania or T. cruzi, or for their detection and identification in the vector insect, characterized in that it comprises: putting a biological sample originating from the patient or the animal to be examined in contact with an amastigote form from an axenic and aseric culture or a promastigote form from an aseric culture as defined above, or a total polypeptide extract of these amastigote or promastigote forms, or one or more antigens specific for this extract, purified or semi-purified, detection of the immunological complex.

The biological sample is more particularly a biological fluid such as blood, serum or urine.

When a purified or semi-purified antigen of a whole parasite is used, it is immobilized on a support.

Latex beads, Elisa plates or fluorescence slides are for example used.

The reaction can be revealed directly by macroscopic agglutination in the direct or indirect agglutination test by reacting a conjugated antibody with fluorescein (indirect immunofluorescence technique) or with an enzyme such as peroxidase or alkaline phosphatase (ELISA tests).

A positive reaction therefore allows the presence of antibodies circulating in the patient or animal examined to be diagnosed.

The invention also relates to a kit for implementing a method for diagnosing a parasitosis, as defined above, in man or in animals.

This kit is characterized in that it comprises: the antigenic reagents in immobilized form, namely the amastigote forms from axenic culture or axenic and aseric culture or the promastigote forms of defined culture, the total polypeptide extracts obtained from these forms or the antigens specific for these extracts with, if appropriate, a positive control, constituted by a serum of known titer, a negative control, as well as the buffers and reagents which can be used for revealing the immunological reaction.

The detection of circulating anti-parasite antibodies in a patient or an animal can be carried out by putting in competition with the antibody of the sample, a specific antibody of the antigen, in particular a monoclonal antibody. The antibody advantageously contains a label, for example a radioactive or enzymatic group.

As a variant, the diagnostic method is based on revealing the presence of the antigenic determinants of the parasitic forms (detection of circulating antigens).

In this variant, the biological sample originating from the man or animal is put in contact with specific antibodies directed against the antigens of the parasitic form, or fragments of these antibodies.

The detection of the immunological reaction is carried out for example using the same antibody but labelled. It is useful to note that the possibility provided by the invention of detecting circulating antigens, that is to say those which appear rapidly in the infected patient, allows early diagnosis of the disease to be carried out.

Antibodies directed against the promastigote or amastigote forms are used, in particular monoclonal antibodies, these forms originating from parasites of different species of leishmanias such as L. infantum or T. cruzi.

A corresponding diagnostic kit comprises: an appropriate solid phase serving as support for the immunological reaction, such as a microtitration plate, a preparation of antibodies according to the invention as defined above, or of fragments of these antibodies, immobilized on a support, a positive control, constituted by a serum of known titer, a negative control, as well as the buffers and reagents which can be used to carry out the immunological reaction and in particular the labelled homologous antibody.

According to another aspect, the diagnostic tools of the invention allow a differential diagnosis to be carried out between several parasitoses.

In fact both in man and in animals, for example rodents, cross reactions between T. cruzi, T. rangeli (trypanosome non-pathogenic to man) and visceral or cutaneous leishmanias are observed.

The study of the parasitic forms of the invention, of the total polypeptide extracts and of the specific antigens defined above has revealed their strong immunogenic properties.

The invention therefore also relates to their use as protective agents vis-a-vis parasitoses, more especially leishmaniases and Chagas' disease.

The vaccine compositions of the invention are characterized in that they are developed from amastigote forms or promastigote forms from axenic and aseric culture, in the absence of macromolecules, as defined above, at different phases of their growth, or from their constituents, in combination with a vehicle and/or an administration adjuvant.

The constituents of the amastigote or promastigote forms in question include the total polypeptide extracts obtained by lysis of these parasitic forms. They also include the antigenic fractions and the specific protective antigens isolated from the parasitic forms, but also from the culture supernatants conditioned by the parasites when they are cultivated in completely defined media.

The administration of these protection agents to man or animals allows them to be given an overall immunity against the parasitoses in which they occur in the natural infection process.

Their advantageous effect was especially revealed at the level of the immune response to cell mediation favouring the stimulation of the T lymphocytes secreting interleukin 2, and gamma interferon (TH.sub.1) or inhibiting the activation of the T cells secreting interleukins 4 and 5 (TH.sub.2).

These protection agents are advantageously in lyophilized form.

In the case of total polypeptide extracts, the vaccine compositions are administered by subcutaneous route at a rate of 100 to 1000 .mu.g in man and 100 to 500 .mu.g in dogs in the presence of adjuvants such as muramyldipeptide or saponin or in the presence of cytokine such as gamma interferon.

The excretion-secretion antigens of the culture supernatants metabolized by the amastigote forms of the invention offer an original strategy in the development of vaccines against parasitic diseases.

Their use for producing vaccines against canine or human visceral leishmaniases (L. infantum and L. chagasi) can in particular be emphasized. In fact they advantageously correspond to the forms present in the infected host.

The various experiments carried out have allowed their immunogenic properties and their protective effect for man or animals to be revealed.

To prepare vaccines from the said antigens, or exoantigens, one uses the dialyzed concentrated supernatants of amastigote cultures, or the cultures themselves containing the parasites and the supernatants, the parasites being killed for example by thermal treatment, or extracts or these solutions.

These products are used with adjuvants such as MDP or cytokines, such as gamma interferon.

A long-term or short-term immunization protocol can be used. The long-term protocol is for example carried out by injection of the vaccine preparation every three weeks, on days d=0, d=21 and d=42. For a short-term protocol, for example, two injections are given with a two week interval.

After the virulence test, for animals, it is verified that the hypersensitivity immunity to cell mediation has indeed been induced towards the activation of the TH1 cells secreting interleukin 2 and gamma interferon.

These analyses can also be carried out for man at the end of the immunization.
 

Claim 1 of 2 Claims

1. A hybridoma clone IVD6/F5, which was deposited in the Collection Nationale de Cultures de Microorganismes (CNCM) under registration number I-3768 on 24 May 2007.
 

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
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