FR2538253A1 - Polypeptide fraction with an average molecular weight of 75,000, which induces protective antibodies to malaria parasites, and immunogenic compositions containing them - Google Patents

Abstract

The invention relates to a polypeptide fraction which induces protective antibodies to malaria parasites, which polypeptide fraction exhibits the following characteristics: it possesses an average molecular mass of the order of 75,000; it is recognised by antibodies to Plasmodium falciparum and by monoclonal or polyclonal antibodies produced against itself; it can be identified by metabolic incorporation of labelled amino acids such as [<35>S]methionine; it induces, especially in monkeys, and more particularly in the monkey Saimiri sciureus, active antibodies to malaria parasites, more particularly to Plasmodium falciparum or parasites which exhibit the same essential biological characteristics. This polypeptide fraction can be used in the production of vaccinal compositions against malaria.

Description

Polypeptide fraction of average molecular weight 75,000 inducing protective antibodies against malaria parasites and immunogenic compositions containing them.

The invention relates to polypeptide fractions inducing protective antibodies against malaria parasites, as well as to immunogenic compositions containing them, capable of leading to the production of vaccines for humans and animals. It also relates to the specific antibodies induced in vivo by these fractions.

The polypeptide fractions according to the invention exhibit the following characteristics - they have an average molecular mass of
around 75,000 (measurable in a migra
electrophoretic tion and by comparison with migra
tions under similar conditions of peptides mar
qués, in particular with fluorescein isothyocyanate, and
of known molecular weights, more particularly
Human IgG, bovine serum albumin (BSA) and ltoval-
chicken bumine); - they are recognized by active antibodies against
of Plasmodium falciparum and by monoclonal antibodies
nal or polyclonal formed against themselves ;; - they can be highlighted by 11 incorpora-
metabolic tion of labeled amino acids, for example
35S methionine; - they are inducers in particular in monkeys, and more
particularly the monkey SADIXRI SCIUREUS, anti
bodies active against malaria parasites, plus
particularly Plasmodium falciparum or para
sites with the same biological characteristics
essential gics.

In particular, the polypeptide fractions according to the invention are recognized by sera or immunoglobulin fractions originating from animals, in particular from SAIMIRI SCIFEUS monkeys, resistant to the parasite and capable of rendering animals sensitive to the parasite resistant.

Plasmodium falciparum is well known to specialists. The schizonts and merozoites of this parasite have already been used to produce experimental vaccines against malaria, in particular in monkeys (MITCHELL et al, (1977) Lancet, i, 1335 and SIDDIQUI,
WA (1977) Science. 197, 388 (1977)). This last article entitled "An effective immunization of experimental monkeys against the human malaria parasite, Plasmodium falciparum" can be considered as demonstrating a significant character with respect to the human biological results likely to be observed in monkeys. .

The same is true of the results capable of being observed with the polypeptide fractions of the invention. These were obtained from a strain of Plasmodium falciparum which was deposited in the National Collection of Micro Cultures.
Bodies of the INSTITUT PASTEUR of Paris (CNCM) under the nO FUPC I-212 on December 23, 1982.

The expression “polypeptide” should be understood to mean any peptide or protein constituent, in particular of the protein or glycoprotein type, such as those liable to originate from Plasmodium falciparum or from parasites which are derived therefrom.

The invention also relates to immunogenic preparations containing said fractions in association with a vehicle of the type used in vaccine compositions, these immunogenic preparations being capable of leading to the production of effective vaccines.
cures against malaria, more particularly of the type
that induced by Plasmodium falciparum or
related parasites.

The invention also relates to a method of obtaining
tion of such immunogenic fractions. This process consis
te to treat a preparation obtained previously from
Plasmodium falciparum with a detergent solution,
such as Sodium Dodenyl Sulfate (SDS) or that known as
the trade name TRITON x 100, or the like, suscep
tible to induce the dissolution of most of the
cell structures and protein constituents
of the parasite, to recover and separate from this
middle those of the polypeptides which present a mol weight
mean cular of the order of 75.00th daltons, which are suscepti
able to react with antibodies (sera or fractions
immunoglobulins) originating from animals resistant to
Plasmodium falciparum and, where appropriate, be labeled
by methionine '' S.

Additional features of the invent
tion will appear again in the course of the description which
follows from an example of the production of polypepti fractions
diques according to the invention, from a culture of para
sites, and biological properties of these fractions.

Culture of parasites and preparation.

The parasites are cultured on human red blood cells of group Ae in RPMI 1640 medium <FLOB10) supplemented with 10% human serum of the same group, under an atmosphere containing 1% 02, 3% C02, 96% -N2 (% by volume ).

The cultures are synchronized at the "rings" stage, as defined in the Science article 1976, 1932 673g by treatment with sorbitol (LAMBROS 1979, vol. 55, p. 418
J. of Parasitology). They are harvested 40 hours later, when the parasites reach the stage of mature schizonts, with 8 nuclei on average.

The red blood cells are washed by centrifugation in RPMI 1640 and lysed by the action of Saponin at 0.025% in RPs% by weight), in the presence of protease inhibitors "PMSF", "TtCK" (SIGMA) 10-3 M, for 10 minutes at 370C.

The free parasites are purified by centrifugation in a discontinuous gradient in PBS comprising 40 to 70 e of PERCOLL (P-llARt: iACIA), for 30 minutes at 40C. The 40-70% interface is harvested by pipetting and washed two to three times in PBS.

Preparation of semi-purified proteins
The parasites are treated with 5 to 10 volumes of buffer for electrophoresis, containing 6% by weight of
SDS and 10% ss-mercapto-ethanol (or an analogous agent capable of cleaving disulfide bonds in proteins and linearizing polypeptide chains), twice for 5 minutes at 1000 C.

The extracts are centrifuged for 5 minutes at 15,000 g, the supernatants (containing more than 90% of the total proteins initially contained in the parasites) are subjected to electrophoresis (for 4 hours at 200 V) in gel containing 10% by weight / volume of acrylamide in an aqueous Tris buffer (at pH 8-9) containing 0.2% SDS (w / v).

After electrophoresis, bands of gels are cut at the levels corresponding to the molecular weights 75,000 + 7,500 daltons marked on the gel by simultaneous electrophoresis of peptides labeled with fluorescein isothyocyanate (human IgG, BSA and ovalbumin).

The polypeptides are eluted from the gel bands by electrophoresis in SDS-free buffer, and harvested in a dialysis bag. The protein concentration is determined by colorimetric assay of the proteins (“Biorad Assay”, test marketed by BIORAD and described by SPECTOR, Analytical Biochemistry, 1978, 86, 142).

The preparations are analyzed by polyacrylamide-SDS gel electrophoresis and revealed by staining with silver nitrate. We can thus separate again - 3 major bands in the molecular weight zone
72,000, 76,000, 80,000 daltons, - 1 minor band in the molecular weight zone
90,000 daltons.

Minor degradation products of this 75,000 band are seen in the 33,000 and 45,000 daltons zones.

The biological properties of this fraction, whether it is the whole fraction of average molecular weight 75,000 (or 75,000 + 5,000) or the more purified protein fractions which can be isolated from the last bands mentioned above, can be put. prominently as described below in connection with the overall fraction.

SAIMIRI SCIUREUS Monkey Immunization Trial
The immunization results likely to be observed in these primates are particularly representative. of what could be obtained in a similar way in humans. These animals can be made even more susceptible to parasitic infection by splenectomy: the infection develops rapidly in splenectomy monkeys. It is manifested by significant parasitemia (approximately 50% of their red blood cells may be parasitized).

Splenectomized and malarious animals die after
lasting around 10 days.

The following tests were carried out on splenectomized animals divided respectively into two groups of 5 and 10 monkeys.

The animals of the first group are inoculated three times with each time a dose of 100 μg of the preparation diluted in 0.5 ml of PBS emulsified with an equal volume of Freund's adjuvant, complete for the first Latin inocu, incomplete for both. following. These inoculations are given three weeks apart, in the form of subcutaneous injections.

The monkeys of the second group (controls) receive only adjuvants.

Three weeks after the third immunization,
the animals are infected with 50 x 106 parasitized red blood cells, intravenously. The parasitaemia is then checked every day for three weeks.

A significant delay in parasite multiplication is immediately observed in those animals which have been immunized. 15 days later, the proportion of parasitized red blood cells in the immunized animals is less than 10%, while in the control group 5 days after the last injection the parasitaemia is more than 25 <0.

These results are highly significant, taking into account the splenectomized nature of the animals. Similar results can be obtained with further purified fractions.

The peptide fractions according to the invention first of all constitute particularly advantageous biological reagents in that they are active in vivo. They can be used as a reference for measuring the in vivo activity of other immunogenic preparations obtained from parasites responsible for various forms of malaria.

The invention still more particularly relates to the vaccinating compositions containing said polypeptide fractions in association, as already indicated, with the various pharmaceutical vehicles and / or adjuvants commonly used in vaccine compositions. Preferably, the invention relates to injectable solutions of said polypeptide fractions.
The polypeptide fractions according to the invention can also be used for the manufacture of antibodies more specific with regard to Plasmodium falciparum than the sera or purified fractions of immunoglobulins capable of being obtained from animals which have become resistant. for the manufacture of these more specific antibodies, recourse will be had to the technique of manufacturing cell hybrids or hybridomas, then comprising the following essential steps (capable of being implemented according to techniques known today) - fusion of myeloma cells and of splé cells
mice or rats, or even monkeys, having at least
previously immunized with the peptide fractions
above-defined, - selection among the hybridomas formed of those which secrete
try monoclonal antibodies active against the aforesaid
polypeptide fractions.

These more specific antibodies, particularly these monoclonal antibodies, can then in turn be used, for example to constitute affinity chromatography columns. Such a column is for example obtained by fixing these monoclonal antibodies on the resin marketed by PHAStACIA under the trademark SEPHARO
SE by the well-known cyanogen bromide method.

These affinity chromatography columns can then in turn be used to carry out the separation of immunogenic polypeptide fractions, whether from solutions obtained from preparations of parasites treated with dissolving agents, of the kind which have been mentioned above, or from fractions already concentrated and for which a state of greater purity is desired.

As goes without saying and as it follows moreover from the foregoing, the invention is in no way limited to those of its modes of application and of embodiments which have been more especially considered; on the contrary, it embraces all of them. variants, in particular those in which the immunogenic fractions of average molecular weight of 75,000 are obtained from parasites distinct from Plasmodium falciparum, but which possess the essential biological properties thereof. The invention also extends to those of the polypeptide fractions which can be obtained from parasites which can be considered as derivatives or even mutants of.
Plasmodium falciparum In particular are included in the framework
claims all immunogenic polypeptides having
an average molecular weight of the order of 75,000, which are
recognized by protective antibodies, obtained from
animals resistant to the strain of Plasmodium falciparum
filed with the CNCM under number FUPC I-212 on December 23, 1982,
and more particularly protective antibodies
more specific likely to be obtained from
animals immunized with the molecular weight polypeptides
average weight of around 75,000 from the same
strain or from hybridomas formed in the condi
tions which have also been indicated, by putting into
vre, for the immunization of cell donor animals.
splenic cells required, said polypeptides derived from
said same strain.

In general, it is possible, from a given preparation of parasites, to determine those of the protein constituents capable of inducing in vivo the production of protective antibodies by proceeding as follows.

We start with a culture of the parasite studied, previously carried out in a culture medium containing a radioactive marker specific for the protein constituents of the parasites in question, such as methionine 355
The parasitic constituents are then collected and processed as described above with regard to
Plasmodium falciparum, but with a view to dissolving most of the cellular and protein constituents of the cho-isi parasite, except that the detergent TRITON X100 is used instead of SDS. TRITON X100 indeed solubilizes antigenic proteins without profoundly altering their structure, so that they can still be recognized by antibodies. The following series of operations are then carried out.

First series of oDerations
a) Electrophoresis is carried out on a sample of all the protein constituents of the parasites. A whole series of blue bands visualized by autoradiography is obtained. By comparison with proteins whose molecular weights are known, the successions of molecular weights are determined, relative to the migrations carried out concomitantly by peptides of known molecular weights.

b) Starting from a separate sample of said protein constituents (this is always the labeled preparation), it is reacted with the antibodies from a monkey resistant to the parasite; the whole is then reacted with protein A from Staphylococcus aureus (commercial preparation). The precipitate formed, which contains a protein A - antibody - radioactive antigen complex, is separated by centrifugation.

After washing the precipitate, it is redissolved in the presence of SDS. This dissociates the antigen from the antibody and from the protein A; The assembly is resubmitted to electrophoresis and the marked bands are identified, in a smaller number than in the previous case, as well as their respective molecular weights.

Second series of operations
All of steps a) and b) above are repeated, but this time using antibodies from a monkey susceptible to the parasite.
By comparing the bands obtained on the gels after the reactions with the antibodies from resistant animals, on the one hand, and those obtained after reaction with the antibodies from sensitive animals, on the other hand, those of the bands which are not 'were obtained only at the end of step b) of the first series of operations and their molecular weight regions.

These bands contain protein constituents preferentially recognized by sera from resistant animals and, therefore, the antigens specifically recognized by protective antibodies.

Third series of operations
Finally, a new culture of the same parasite is carried out, but in the absence of the radioactive label, and a new electrophoretic separation of its protein constituents is carried out under the same conditions as in the first and second series of operations.

The capacity of the constituents which have migrated at the same distances as those which were recognized by the antibodies at the end of the second series of operations, to induce in vivo the production of protective antibodies, is then tested, and those which are collected. of these constituents which have induced a positive immunological reaction, for example under the same conditions as those mentioned above with regard to the active protein constituents of Plasmodium falciparum.

The latter therefore fall within the scope of the claims or constitute equivalents of the protein constituents which have more particularly been claimed.

Claims (8)

1 - Polypeptide fraction inducing protective antibodies against malaria parasites which have the following characteristics - they have an average molecular mass of the order of 75,000; - they are recognized by antibodies directed against Plasmodium falciparum and by monoclonal or potclonal antibodies formed against themselves - they can be demonstrated by metabolic incorporation of labeled amino acids, for example methionine 35; - They induce in particular in the monkey, and more particularly the SAIMIRI SCIUREUS monkey, of active antibodies against malaria parasites, more particularly Plasmodium falciparum or parasites which have the same essential biological characteristics. 2 - Polypeptide fraction according to claim 1, characterized in that it has a molecular weight of the order of 75,000 - 5,000. 3 - Polypeptide fraction as it can be isolated from the fraction according to claim 1 or claim 2, characterized by one of the following average molecular weights 72,000, 76,000, 80,000 or 90,000 daltons. 4 - Process for obtaining the polypeptide fractions according to claim 1 or claim 2, consisting in treating a preparation obtained beforehand of Plasmodium falciparum with a solution of a detergent such as sodium dodecyl sulfate (SDS) cucelui known as the trade designation TRITO4 'X100, or analogy, capable of inducing the dissolution of the major part of the cellular structures and of the protein constituents of the parasite, of recovering and separating from this medium those of the polypeptides which have an average molecular weight of around 75,000, which are likely to react with antibodies (sera or immunoglobulin fractions) originating from animals resistant to Plasmodium falciparum and, where appropriate, to be labeled with methionine 355 5 - Polypeptide fraction according to any one of claims 1 to 4, characterized in that it is recognized by the specific antibodies previously formed against the immunogenic peptides con trimes to any one of these claims and as obtained from of the strain deposited with the CNCM all n0FUPCI-212. 6 - Immunogenic preparation combining a fraction according to any one of claims 1 to 4 with a vehicle or a pharmaceutical excipient of the type of those used for the constitution of vaccines. 7 - Preparation of antibodies specific to the polypeptide fractions according to any one of claims 1 to 4. 8 - Application for diagnostic purposes of the fraction according to one of claims 1 to 3.