Title: Process for the in vitro
culture of different stages of tissular parasites
United States Patent: 7,317,094
Issued: January 8, 2008
Jean-Loup (Saint Martin de Londres, FR)
Institut Francais de Recherche Scientifique
pour le Developpement en Cooperation (Orstom) (Paris, FR)
Filed: October 1, 2002
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
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
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
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
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
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
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.
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
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
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
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
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
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.
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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.
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
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
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
These products are used with adjuvants such as MDP or cytokines, such as
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
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
These analyses can also be carried out for man at the end of the
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.
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