LU86736A1 - LIPOSOMES SENSITIZED TO ANTIGENIC MOLECULES OF INTRACELLULAR PARASITES, METHOD FOR THEIR PREPARATIONS AND THEIR APPLICATIONS AS DIAGNOSTIC MEANS AND AS IMMUNIZING AGENTS - Google Patents

Description

'* ._ · - / "* ·, BL-3984 / vdw

‘I V. GRAND DUCHY OF LUXEMBOURG

* ·: 'V'

Patent N ................................................ .............. Mr. Minister of January 15, 1987 of the Economy and the Middle Classes _ 3¾¾ Intellectual Property Service

Title issued -.................—— | gg | Luxembourg

Patent Application -! - ----------------- ("I. Application

Free University of Brmce 1 1 pg, 50 "y. F.P, Rnncoyalt_ (2). B - 1050 Brussels_____________ ing.cons. en propr lnd., 46 rue du Cimetière, Luxembourg_ (¾) acting as agents _ depositor) this 5 ^ .1 ^^^^ BylggLj ^ il - Aeuf-genfc ^, guatr £ ^ yliigt-sept- (4 ) at 15? _Ρ 0 hours, at the Ministry of the Economy and the Middle Classes, in Luxembourg: 1.1a this request for obtaining a patent of invention concerning: "Liposomes sensitized to antigenic molecules of {5) intracellular parasites "process for the preparations and their applications as means of diagnosis and as agents ....... of immunization" ________ 2. the description in language ........ French _______ of the invention in three copies; 3 .......................... // ........-............ ......... drawing boards, in triplicate; · 4. the receipt of the taxes paid to the Luxembourg Registration Office, _15rlrl5_82-; 5. the delegation of power, dated__________________________________________ on --- 6. the successor document (authorization); declaring), assuming responsibility for this declaration, that the inventor (s) is (are): (6) - ...... Mons leur Frana LEGROS, 313 Chaussée £ ..de ... ^. QQndaie .l ___ ________________________B - 1050 Bruxelles__________ - Monsieur ...... Jean-Marie .RUYSSCHAE.RT.f.-_. 7 ..-. av ........ da ..... Perce-Neige_ - ................................................. ^ __ B - 1640 Rhode-Saint-Genese _: ____ claims (nt) for the above patent application the priority of one (s) application (s) of (7) ............ ..................................... // ----------- --------: ------------ ____ filed in (8) _______ on (9) ................ ......... [J ------------------ under N ° (10) .... —LL ___________________-____ in the name of (11) __ ______IL__________ elect (elect) domicile for him / her and, appointed, for his / her proxy, in Luxembourg ---—....

46 ...... rue ...... du Cimetière _______________ '_ (12) is requesting the issue of a patent for the invention for the subject described and represented in the abovementioned appendices, with postponement of this issue a month. (13) the agent: ^^ 7-, ------ A · 4) MEYERS ^ Ernest 'Π. Deposit Minutes

The above application for a patent for invention has been filed with the Ministry of the Economy and the Middle Classes, Intellectual Property Service% L ^ xembourg, dated: January 15, 1987 (TW \ Pr. The Minister of the Economy and the Middle Classes, V .. Vu V, V. ψ I. pd

I ;, '- jg f The head of the intellectual property department, A 68007_X "4 ι · .Ι." „Νγ ^ __ -. . i EXPLANATIONS RELATING TO THE FORM & DEJ300T.

I (1) if applicable "Request for certificate of addition to the patent, on main patent application No ............ of ........” - (2) enter the last name, first name, profession.

j address of the applicant, when the applicant is an individual or company name, legal form, address of head office, when the applicant is a legal person -13) enter

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DESCRIPTIVE MEMORY filed in support of a PATENT INVENTION APPLICATION

in Luxembourg

- LIPOSOMES SENSITIZED TO ANTIGENIC MOLECULES OF INTRACELLULAR PARASITES, PROCESS FOR THE PREPARATIONS AND THEIR APPLICATIONS AS DIAGNOSTIC MEANS AND AS IMMUNIZING AGENTS

Free University of Brussels 50 av. F.D. Roosevelt B - 1050 Bruxelles 05 10 Subject of the invention

The present invention is based on the sensitization of liposomes to antigenic molecules of intracellular parasites and therefore opens new applications of such sensitized liposomes, as means of detection of Xg (lytic or non-lytic) against these infectious antigens in sera where · other body fluids (pleural, peritoneal, bronchoalveolar lavage) and as vaccination agents capable of eliciting immunity (cellular, but also humoral) against the infecting agent.

As a particular application illustrated by the present invention, the aim is more specifically to provide means of diagnosis and immunization for tuberculosis, but the invention is of course not limited to this application, as will appear. below.

Summary of the state of the art 30 --------------------------------

Numerous studies have been devoted to liposomes for various pharmacological applications, in particular as vectors of substances with medicinal activity.

Antigenic molecules, natural or haptic, have also been exposed on the surface of liposomes. These so-called sensitized liposomes have been used either for the detection of immunoglobulins in the sera Λ 2 of immunized animals or as immunity adjuvants capable of increasing humoral but also cellular immunization.

With regard to tuberculosis, there are 05 purified protein extracts from different Mycobacteria and more particularly from Mycobacterium tuberculoses var. hominis (H37Rv) = PPD H37Rv = Tuberculin H37Rv Tuberculins are capable of revealing preexisting cellular immunity in a man or an ani-10 poorly vaccinated or infected (intradermoreaction or delayed hypersensitivity reaction), but it is unable to immunize because it is not immunogenic.

Characteristic elements of the invention 15 --------------; -------------------------- The The invention relates specifically as new agents which can be used for the abovementioned purposes (immunoassay and vaccination) on liposomes sensitized to antigenic molecules specific for intracellular parasites.

These liposomes are preferably of the so-called "multilamellar" (MLV) type with or without cholesterol.

Preference is given, according to the invention, generally to multilamellar liposomes with choles-25erol having Egg Pc / Ch ratios between 4/1 and 4/4, the latter ratio or values close to it being entirely particularly preferred.

Also provided are methods of producing sensitized liposomes.

30 Two operating variants are available for this purpose:

It is possible to carry out a direct encapsulation of antigenic molecules or to bring the preformed liposome into contact with these molecules.

Both types of sensitized liposomes are able to bind immunoglobulins raised against antigenic molecules.

It is generally observed in the first case that more immunoglobulins are linked to the liposomes while the proteins 3, incorporated by exposure to preformed vesicles have a higher affinity for the corresponding antibodies. As a result, the type of sensitization of the liposomes to a mixture of im-05 munoglobulins corresponds to variable quantities of selected antigenic molecules exposed to the surface of the liposomes and to differences in the exposure and the affinity of these antigens. .

The repeated subcutaneous or intraperitoneal administration of the two types of liposomes gives rise to the appearance of a delayed hypersensitivity reaction, witness to cell type immunity, in the guinea pig.

The list of specific applications pertaining to the invention appears below in the description. 15

Detailed description of procedure with reference to the particular application of Mycobacterium tuberculosis Method for sensitizing liposomes 20 1. Formation of liposomes

Total lipid concentration at 10 or 20 mg / ml. Molar ratios 1) Egg Pc (or Egg Pc = 4 / Ch = 0) 2) Egg Pc / Ch 4; 1 25 4 s 2 4; 3 4: 4

MLV formation in sterile 0.15 M NaCl (= physiological water) with or without PPD.

30 Two awareness-raising methods are used s a. Direct encapsulation of PPD in liposomes. The liposomes are formed in a PPD solution at 0.75 mg / ml of 150 mM NaCl.

The preparation is left to stand for 30 35 min. at room temperature.

Then, 5 rinses in 150 mM NaCl.

b. Contact of preformed liposomes with PPD. The MLVs are formed in 150 mM NaCl, 4 ug. Laids at rest for 30 min., Centrifuged at 3500 g for 10 min., Brought into contact for 30 min with a PPD solution at 0.75 mg / ml of 150 mM NaCl and rinsed 5 times in the same solvent.

05 2. Radioimmunoassays using sensitized liposomes

Liposomes of different compositions and sensitized according to the two methods are exposed to a 10-year-old sheep tiserum raised against H37Rv, radioiodinated and unlabeled. The preparation and radioiodination of this an-tiserum is described in the Applicant's patent application filed the same day for s "Method for rapid detection of infections with intracellular germs, in particular Mycobacterium tuberculosis or Mycobacterium leprae and kit suitable for this purpose ”.

Competition curves are obtained between a quantity of 125χ antiserum and different dilutions of the unlabeled antiserum (from 1/10 to 1/10 000). The proportion of 20 radioiodic molecules attached to. liposomes-PPD and the dissociation constant of the molecules are measured.

The liposomal composition which fixes the greatest number of radioiodine molecules with the greatest affinity seems to be Egg Pc / Ch 4; 4 with direct encapsulation 25 of PPD.

5. Radioimmunoassays on sera humans.

Competitions were carried out between the radioiodinated sheep antiserum and different dilutions of human sera (1/10 to 1/10 000). No competition was observed for sera from non-tuberculosis patients. In contrast, 6 sera from tuberculosis patients contained Ig competing with antiserum-I. The presence of Ig in the sera of these patients was confirmed by ELISA test. Ditto for the absence of Ig in sera from non-tuberculosis patients. Liposomes used in this study: Egg Pc / Ch 4: 3; encap-

V

Direct sulation of PPD or exposure of PPD to preformed liposomes. This radioimmunoassay detects Ig in the sera of tuberculosis patients, but does not provide information on the nature of these Ig.

5 · * 4. Development of an immunoassay with fluorescent marker "Liposome Lysis Immune Test.

A fluorescent marker, calcein, was incorporated into the Egg Pc / Ch (4; 3) liposomes sensitized to PPD by direct encapsulation. As long as it is encapsulated, therefore "quenché", this marker does not fluoresce. If the liposome is lysed, the marker is released into the incubation medium and a fluorescence proportional to the extent of the liposomal lysis can be measured.

The lysis of the liposomes takes place if lytic IgGs have come to bind to the "· * antigens'exposed by the vesicles (here, antigens extracted from the PPD) in ·> 1.

10 presence of complement. Qualitatively, this immunoassay is the only one currently detecting lytic Ig.

False positives are excluded since liposomal lysis only takes place if the

Ig have attached to the antigens exposed by the vesicles. In addition, if the immunoassay mentioned in 3) requires 20 mg of lipids per competition point, the immune lysis test with calcein requires only 0.1 mg for the same measurement.

_ Liposomes made up of Egg PC and Ch __ (4; 3), containing calcein and sensitized to PPD according to the two methods indicated above (encapsulation and contacting), presented an immune lysis (calcein release and appearance of measurable fluorescence in the medium) in the presence of anti-BCG rabbit serum (Dakopatts) or anti Mycobacterium tuberculosis sheep serum and complement from rabbit serum. This reaction was not observed in the presence of a serum known to have no an.timycobactérien® nl antibody in the absence of complement.

These same PPD-sensitized liposomes were contacted with human sera from intradermal-negative tuberculosis and non-tuberculosis patients. The lyses measured (L.S.) after 30 minutes were compared with those found for liposomes not sensitized to PPD or to any other protein antigen (LNS). For each serum, the difference between the L.S. and LNS percentages was calculated (LILA). The results obtained are shown in the following table.

6

Tuberculosis patients Non-tuberculosis patients

LS LNS LILA LS LNS LILA

1 23.7 12.4 11.1 11 11 6.8 4.2 2 31 7 24 12 3.8 3.7 0.1 3 31 13 18 13 12.4 5.2 7.2 4 20 9, 5 10.5 14 11 3.6 7.4 5 14.7 10.6 4.1 15 7.7 2.6 5.1 6 39 14 25 16 3.8 1.3 2.5 7 57 15, 6 41.4 17 3 2 1 8 32 8 24 18 4.6 2.9 1.5 9 25 12 13 19 6 1 5 10 31 15.8 15.3 20 3 1 2 M 30.4 11.8 18 .7 6.6 3 3.6 ± SD ± 11 ± 2.8 ± 10 ± 3.5 ± 1.8 ± 2.4

S S NS NS

~ T ~ l ï S = statistically significant according to the student test NS = statistically not significant according to the student test 7

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l

From these results, it appears that the immune lysis of liposomes carrying PPD is significantly higher in sera from tuberculosis patients than in sera from non-tuberculosis patients. Likewise, the lysis of liposomes non-carriers of PPD is greater in sera from tuberculosis, although significantly lower than that of liposomes PPD. The serum of tuberculosis patients therefore contains antibodies directed against the components of PPD, but also agents capable of causing a lysis of liposomes but PPD greater than in non-tuberculosis patients. It can therefore be affirmed that, by the present invention, a test for immune lysis of liposomes sansibilisés à la PPD was able to demonstrate. antiseracobacterial antibodies in the serum of tuberculosis patients. We can also note the speed of this test (30 minutes).

This test makes it possible to diagnose all infectious diseases by demonstrating serum antibodies by means of liposomes carrying antigens of the infecting mi-20 organism.

8 05 5. In vivo liposome immunoreactivity test ”

It is known that liposomes sensitized by antigens are capable of playing a role in the cellular mechanisms of immunity in vivo, either by imitating the infectious agent (bilayer of phospholipid carrying immunogenic molecules of this agent) and by being captured by the local macrophages of the infected host, either by imitating those macrophages which have "processed" the infectious agent and which have exposed some of its antigenic molecules on their surface. In both cases, the antigenic molecules by which the liposome has been sensitized are presented to the lymphocytes, recognized by receptors and the mechanisms of cellular and humoral immunity can be stimulated. As such, the sensitized liposome can constitute the vehicle for a new type of vaccination, because it represents an almost natural model of presentation of antigenic molecules to the immune system. If this hypothesis is correct, the liposome-PPD must be able to induce a positive reaction of delayed hypersensitivity (positive skin reaction) 20 in animals previously sensitized by the B.C.G,

We therefore injected into the dermis liposomes sensitized to PPD (Egg Pc sensitized by “direct PPD encapsulation or by preformation followed by contacting; Egg Pc / Ch, 4 3, sensitized according to the same two methods) immune guinea pigs. The 4 types of liposomes cause an in-tradermoreaction whose intensity and evolution are different from those of non-encapsulated PPD (See in this regard, the draft article "Immunological activity of. Tuberculin sensitized liposomes", in particular p. 7 and Fig. 1 and 2, for the Egg Pc liposomes). In contrast, npn liposomes carrying PPD antigens do not elicit a positive intradermal reaction in the same animals (Idem, Fig. 1).

It can be concluded that the liposomes-PPDs deposited in the dermis are phagocytosed by macrophages and that the antigens of which these liposomes were carrying

r I

They are then presented to and call up sensitized lymphocytes, thereby inducing the onset of the erythema and induration characteristic of positive intradermoreaction.

This conclusion therefore allows the use of the liposome as a vehicle for vaccination.

s · * 9 r

6. Appearance of cellular immunity in guinea pigs under the effect of liposomes - PPD

Guinea pigs not infected and not sensitized by tuberculous mycobacteria (PPD negative skin reaction) received 3 biweekly injections, 05 of liposomes-PPD, either under the skin or in the peritoneal cavity (Egg Pc liposomes in Egg Pc / Ch, 4: 3, formed under the two conditions described above, see 1). Two weeks after the last injection (or 6 weeks after the first), an intradermoreaction was performed, with PPD alone, with liposomes-PPD, or with liposomes. Like co-1o bayes which would have been previously sensitized by the mycobacterium (vaccination with B.C.G., for example), these animals present a positive intradermal reaction to PPD or liposomes-PPD, but not to liposomes not carrying PPD. These experiments indicate that liposomes sensitized to PPD induce cell-like defense in guinea pigs. Recall that · / 15 PPD alone is incapable of eliciting the slightest appearance of immunity in an animal (or a man) which has not previously been brought into contact with the mycobacterium.

It can therefore be estimated that the liposome-PPD constitutes a safer vaccination agent than the current B.C.6 vaccine. where attenuated mycobacteria are administered, since the liposome-PPD contains only lipids (Egg Pc, Ch) and proteins extracted from Mycobacteria, to the exclusion of all mycobacteria and its genetic material.

The technique can be applied as much to the "Old Tuberculin", which counts the proteins of the bacteria as well as its surface polysaccharides and glycolipids, the character of which is general and aspecific immunogenicity, as well as to antigenic molecules, specific, of Mycobacterium tuberculosi: A liposome sensitized to the "Old Tuberculin" therefore imitates at best the phospholipid bilayer of the mycobacterial membrane carrying the proteins, polysaccharides and lipids of this intracellular infectious agent. By eliciting a reaction relating to cellular immunity, there is thus a display vaccine against the intracellular parasite that is Mycobacterium tuberculosis.

t On the other hand, it is known that 35 Mycobacterium leprae (leprosy) is very similar to M. tuberculosis. There is a lepromin, similar in composition to the Old Tuberculin. The formation of liposomes-lepromin is suitable as a means of immunization against leprosy, a disease for which there is, to date, no effective vaccine,

The technique of the invention also finds its application as a method of. 4q vaccination for conditions resulting from the intracellular parasites taken up 10 I. Mycoses 1. Cryptococcus neoformans: "Cryptococcosis - opportunistic germ - pulmonary entry point in the immunocompromised with generalization in 90% of severe meningitis 05 2, Candida albicans (mainly); Candidiasis - mainly opportunistic germ: diabetic patients, treated with corticosteroids, with broad spectrum antibiotics - first cutaneous or mucosal involvement (skin, mouth, esophagus, vagina, respiratory tract); possible spread to systemic level (preferred locations: kidneys, eyes, meninges, skin and myocardium) 3. Histoplasma capsulatum: Histoplasmosis (USA) - pulmonary involvement with important general signs 15 - possible systemic dissemination 4, Coccidioides immitis: Coccidioidomycosis - pulmonary involvement (cyst - abscess ...) II Bacteria 2o b Salmonella; Salmonellosis - gastroenteritis, secondary involvement; osteomyelitis, aneurysm 6. List ia monocytogenes: Listeriosis - sepsis - meningitis, serious condition in immunocompromised 25 7. Brucella (suis, melitensis, abortus); Brucellosis - generalized chronic granulomatous disease (spleen, bone, etc.) 8. Francisella tularensis; Tularemia 3q - cutaneous or pulmonary entry point, local abscess, regional lymphadenopathy III. Chlamydiae 9. Chlamydiae (Bedsoniae) Psittacosis - Pneumonia (rarely hepatitis, encephalitis) 35 IV.Rickettsiae 10. Rickettsiae (prowazekii..) Rickettsiosis - temperature, headache, rash

11. Coxiella burnetii Fever Q

40 -temperature, fever, pneumonia, hepatitis V. Mycobacterioses 12. Mycobacteria: - tuberculosis, bovis: tuberculosis • »π

In all cases, it should be noted that the same liposome sensitized by the antigens of a given parasite can be used both for vaccination and for detection of Ig (lytic or non-lytic) in human serum,

05 1 _> _ Vaccine and diagnosis for AIDS

The encapsulation of an AIDS virus extract (HTLV3) can be done using octylglucoside. The method of preparation below is given by way of non-limiting example.

The preparation of the liposomes is carried out here in two stages: a) preparation of monolamellar liposomes by the injection method b) fixation in the liposomal bilayer of the extract of HTLV3 by its molecules with hydrophobic component after formation of 'a glycoprotein - detergent complex and "thinning" of liposomes.

a) Preparation of the liposomes The lipids are dissolved in pctylglucoside 20 (octyl-ß-D-glucopyranoside) at 20 ° C. in a tris-HCl buffer pH = 7.2 containing 100 mM octylglucoside in order to obtain the following concentrations; 1.5 μM / ml Chol.

21 μM / ml Egg Pc.

25 0.375 μM of cholesterol and 3 m of Egg Pc are then mixed. The whole is then gently injected into 3 ml of phosphate buffer (TP) pH = 7.4 (NaCl = 0.137 M, KC1 = 8.7 mM, KH2P04 = 1.47 mM, Na2HP04 = 8.1 l mM) cooled to 4 ° C, then dialyzed against the same buffer (TP). The liposomes are then separated from the planar liquids by filtration on a Sephadex-G75 column previously equilibrated with the buffer (TP).

b) Fixation of the extract of HTLV34 on the liposomes The HTLV3 was extracted with octylglucoside (final concentration 7.5 mM). The liposomes are thinned with 1 mM octylglucoside. The two solutions are then mixed in proportions of 500 μg of HTLV3 // rM lipid extract.

40 This means that 0.375 μΜ of lipids are added to 137 μ g of HTLVS extract.

»En 12

The extract-detergent complex and the fluidized liposomes are dialyzed, first against a decreasing octylglucoside concentration gradient (from 7.5 to 0 mM) to form liposomes 05 carrying HTLV3 molecules, then against the tam pon (TP) containing 1 mM NaN3 ·

The liposomes thus obtained, sensitized to molecules extracted from the AIDS virus, were injected I.v. to mice. After 3 injections of 1Q this preparation, carried out 3 weeks prior, the presence of anti-HTLV3 antibodies has been demonstrated in their serum.

We can conclude that this technique is a means of AIDS vaccination and 15 'diagnosis of this disease, as in the case of tu berculose,

Claims (8)

1. Agents suitable as diagnostic means and as agents for the immunization of intracellular parasites, characterized in that they consist of liposomes sensitized to specific antigenic molecules of intracellular parasites. 2. Agents according to claim 1 characterized in that the liposomes are of the type called "multilamellai-res" with or without cholesterol. 10 3, Agents according to claim 2, characterized in that the liposomes are multilamel liposomes with cholesterol having Egg Pc / Ch ratios between 4: 1 and 4: 4. 4. Agents according to claim 3 characterized in that the. Egg Pc / Ch ratios are equal to 4: 4 or close to this value. 5. Process for the production of sensitized liposomes according to any one of claims 1 to 4, characterized in that a direct encapsulation of the antigenic molecule is carried out. 6. Process for the production of sensitized liposomes according to any one of claims 1 to 4, characterized in that the preformed liposome is brought into contact with the antigenic molecule. 7. Method according to any one of claims 5 or 6 characterized in that the antigenic molecule consists of tuberculin, Old tuberculin, lepromin or HTLV3. 8. Application of the agents according to any one of claims 1 to 4 as a means of diagnosing infections accompanied by the formation of specific immunoglobulins directed against viral, bacterial and mycotic agents, and as means of immunization (vaccination) for intracellular pathogens resulting in the development of cellular immunity.