FORMATION OF AN IMMUNOSOME EXCLUSIVELY MADE OF VIRAL ANTIGENS RECONSTITUTED ON AN ARTIFICIAL MEMBRANE

Abstract: Current virus vaccines made of inactivated particles often induce undesirable local and general secondary reactions. Therefore there is a need for a vaccine that would not contain the substances responsible for these side effects. An immunosome has been constructed, highly immunogenic, which is exclusively made of the antigens attached onto the membrane of a preformed li­ posome. This immunosome is prepared by providing a quantity of preformed liposomes; providing an antigen viral sub-unit-detergent complex; treating the preformed liposomes to enable antigen sub-units to anchor onto the liposomes so as to form projections on the outer surface of said liposomes; and combining said antigen viral sub-­ unit-detergent complex with the liposomes obtained above so as to give said immunosomes. This technique recreates a structure that is almost undistinguishable from the virus particle. This immunosome is more im­ munogenic than the subunits used for its preparation.

Description: Folmption of an in @@@osome e\elusiyclyu ode of viral antigens reconstituted on an ertificial membrane This invention relates to a novel product which is exelusively made of viral antigens reconstituted on an artifieial membrane hnown as a liposume. The new product will hereinafter be ralled an immunosomeO It is understood that whenever it. will be used in the present specification and in the appended claims, the term "immunosome" will be used according to the above definition, The current influenza virus vaccines which are made of inactivated particles often induce undesirable local and general pyrogenic reactions. Therefore, there is a need for a vaccine that would not contain the substances responsible for these side effects, In this line of thought, AlTseida J. D. et al have done some work on a new product which thcy have called "virosome" The results have been published in an article entitled "Forma- tion of Virosomes from Influenza Subunits and Lipozomes", which appeared in the Lancet, November 8, pp. 899-901, 1975. The product called "virosome" was prepared by Almeida in the following manner : a mixture of lecitine and dicetylphosphate (9:1) is solubilized in chloroform, the organic solvent is evaporated under a flow of nitrogen ; to the multilamellar lipidic film obtained there is added a 3JB S buffer and the mixture is treated with ultrasonic waves during one hour and thirty minutes at a frequency of 50 HKz to produce closed vesicles, The viral subunits, iOeO hemagglutinines and neuraminidases are added to the suspension of liposomes and the mixture is treated with ultrasonic waves during 15 minutes to enable the integration of the subunits into the membrane. The results obtained are the following : there are viral subunits, in a small density, at the surface of some liposomes. The viral subunits seem to be anchored in the double sheet of phospholipidic material. No tests have been made to verify the hemagglutinating activity or the immunegenie poter of these substanees It could reasprably be assumed that the viral subunits are anchored both on the outside and on the inside of the liposome, speeielly in view of the particular manner of produeing the so-called virosomes. Later, other searchers have tried to rebuild proteinic irembranes of various viruses wrapped in liposomes. Among the most important are the following : DAVIS JP. P., BCCHER D.J. "Integration of Purified Influenza Viral Neuraminidase with Liposomes." American Society of Mierobiology, Annual Meeting Abstract T 114, 1980. HELENIUS A., FRIES E., KARTENBECK J. "Reconstitution of Semliki Forest Virus Mcmbrane." J. Cell BiolO 75 PP. 866-880, 1977. HSU M.C., SCHEIN A., CHOPPIN P.S. "Reconstitution of Membranes with Individual Paramyxovirus Glycoproteins and Phospholipid in cholate Solutions." Virol. 95, ppO 476-491, 1979. HUANG R.T.C., WAHN K, KLEXK H.-D., ROTT R. "Association of the Envelope Glycoproteins of Influenza Virus with Liposomes. A Model Study on Viral Envelope Asscmbl." Virol. 97, pp. 212-217, 1979. MILLER D.K., FEVER B.I., VANDEROEF R., UENARD J. "Reconstituted Go protein-lipid Vesicles from Vesicular Stomatitis Virus and their Inhibition of VSV infection, J. Cell. Biol. 84, pp. 421-429, 1980. PETRI W.A., WAGNER R.R. "Reconstitution into Liposomes of the Glycoproteins of Vesicular Stomatitis Virus by Detergent Dialysis." J. Biol. Chem. 254, 11, ppO 4313-4316, 19790 It will be noted that our approach was totally different from the one used by Almeida. The basic method applied by everybody essentially consisted in solubilizing phospholipids and proteins in a detergent, to mix the two components together and to rewove the detergent by dialysis. This procedure which consists in forming the liposo.lle in the presence of proteins noeessarily leads to the emprisonment of a large part of the proteins inside the liposome, which means that there is an important loss of immunogenic materials It is an object of the present invention to synthetize a product called immunosome, which is a highly immunogenic, and is exclusively made of antigens attached on the membrane of a preformed liposome. It is another object of the present invention to provide a method which is totally different from that of Almeida and other methods known in the art, It is another object of the present invention to provide for a process of producing imtnunosomes by leaving out the technique used by Almeida which is characterized by solubilizing the phospholipids in an organic solvent which must be evaporated to give a multilamellar structure, and using ultrasonic waves which generally contribute to the destruction of the nature of the proteins. It is another object of this invention to provide a method whereby the viral subunits are incorporated into the membranes by biochemical means, It is still another object of the present invention to provide a method which uses preformed liposomes whereby glycoproteins are inserted at the surface of the liposomes by biochemical means. It is well known that although the hemaglutinine and the neuraminidase induce immunity, when subunits HA (hemaglutinine) are in the presence of preformed liposomes, the incorporation into the phospholipidic double layer is carried out with an extremely low yield. This difficulty can be overcome by providing a method of preparing immunosomes, as defined above, which comprises : a) providing a quantity of preformed liposomes b) providing an antigen viral sub-unit-detergent complex c) treating the preformed liposomes to enable antigen sub-units to anchor onto the liposomes so as to form projoctions on the outer surfe of said liposomes ; and d) combining said antigen viral sub-unit-detergent complex with the liposomes obtained in step (c) so as to give said immunosomesO In accordance with a preferred embodiment according to the invention, the liposomes are prepared by solubilization of phospholipids in the presence of a detergent, followed by injection of the solution obtained in a buffer and dialysis. In accordance with another preferred embodiment according to the invention, the antigen viral sub-units are prepared by mixing a virus with a detergent to solubilize the membrane thereof, thereby giving a mixture of HA, neuraminidases, and a core formed of the nucleo-protein nnd proteins M, subjecting the mixture.to centrifugation to separate the nucleo proteins and the proteins M therefrom and to give the HA and neuraminidases, subjecting the iiA and neuraminidases to dialysis in an aqueous buffer solution and adding a detergent to give the protein detergent complex. In accordance with yet another preferred embodiment according to the invention, the viral sub-unit-detergent complex is added to liposomes which have been treated to become susceptible to anchor said sub-units and the mixture obtained is dialysed under a linearly decreasing gradient of detergent to give the immunosomes. If desired, when the gradient becomes zero, the suspension is dialysed in a PBS buffer. The immunosme obtained comprises a membrane made of phospholipid material and viral sub-units essentially distributed in the form of projections on the outer surface of the membrane, the immunosomes being substantially free of secondary effects when injected to a living being. Figure 1 is a graph shoving the immunogenicity of immunosomes produced by the method according to the invention, in comparison to viruscs and viral sub-units Figure 2 slows a group of immunosomes obtained by the method according to tie invention ; and Figure 3 is a.n enlarged reproduction of an immunosome according to the invention showing the viral subunits anchored on the outer surface of the membrane. It will be realized that whenever preformed liposomes are brought together with a solution of protein-detergent com- plex, the membrane of the liposomes has been treated to become so to speak more fluid such as by the addition of a small quantity of detergent. The dialysis which can be carried out on a linearly decreasing gradient of detergent enables an exchange of the proteins of the protein-detergent complex in the phospho lipidic layer of the liposome. It is therefore possible to artificially recreate a structure which is nearly identical to the viral particle, which possesses a very high hemagglutinating activity and which has been shown to be immunogenic. The invention will now be illustrated by means of the following example, EXAMPLE Formation of liposomes - Phospholipids : phosphatidyl choline, cholesterol, lysoleci- tine (8:1:0.5) are solubilized in Tris-HCl buffer 10 mM pH 7.4 which contsins ss-d-octylglucoside 30 mM. - The solution of phospholipids is injected in a PBS buffer (saline phosphate) and the solution of liposomes so obtained is deposited in a small column made of Sephadex G-100 # (hydrophilic insoluble molecular-sieve chromatographic medium, made by crosslinking Dextran). - The column is centrifuged at 600 g during 15 seconds. - The suspension of purified liposomes is exhaustively dialyzed in PBS buffer0 Formation of protein-detergent complex The HA and neuraminidases are in solution in PBS, concentration in proteins is adjusted to 700 pg/ml and to the solution of proteins, ss-d-octylglucoside is added to give a final conccnlration of 7.5 mM : the mixture is allowed to stand at 220C during tO minutes. Formation of immunosome To the suspension of liposomes, there is added ss -d-octylglucoside in solution in Tris buffer (hydroxymethyl minomethane) 10 mM pH 7.4 to give a final concentration of 1 nM : the mixture is allowed to stand at 220C during 10 minutes. To the suspension of liposomes made more fluid or in other words treated to become susceptible to anchor the viral subunits, there is added the protein-detergent complex and the integration of the glycoproteins in the phospholipidic double layer of the liposome is carried out by dialysis on a linearly decreasing gradient of detergent. - When the gradient becomes zero, the suspension of immunosomes is dialysed in a PBS buffer. This procedure was carried out with both the A/ strains of the England/864/75 (H3N2)X-49 and A/Bangkok/1/79 (H3N2)X 73 influenza virus Tests were made to determine the activities of the sub-units and of the immunosomeO Reference is made to the Table which follows and also Figure 1 of the drawings, It will be found that the immunosome according to the invention is much more active than the viral sub-units and nearly as active as the whole virus0 Referring to Figures 2 and 3 of the drawings, it will first of all be realized that the immunosomes can be obtained as a group of particles as shown in Figure 2. Referring to Figure 3, it is obvious that the sub-units are exclusively and highly concentrated on the outer surface of the membrane and that inside the membrane, there are no nucleic acids or other substances which can give secondary effects upon injection0 HA ACTIVITY OF SUB-UNITS AND OF IMMUNOSOME g protein Titer * Number of HA Sp@@ific Activity in 50 l units in 50 l U@A/mg protein Sub-units 19 212 4,096 215 500 Immunosome 6 220 1,048 576 175 762 000 * Average of 15 det@rminations.

Claims: C L A I M S 1. A method of preparing immunosomes which comprises : a) providing a quantity ofpreformed liposomes b) providing an antigen viral sub-unit-detergent complex ; c) treating the preformed liposomes to enable antigen sub-units to anchor onto the liposomes so as to form projections on the outer surface of said liposomes ; and d) eombining said antigen viral sub-unit-detergent complex with the liposomes obtained in step c) so as to give said immunosomes, characterized in that said preformed liposomes are prepared by solubilization of phospholipids in the presence of a detergent, followed by injection of solution obtained in a buffer and dialysis, 2. A method according to claim 1, which comprises solubilizing phospholipids consisting of a mixture of phospha tidyl choline, cholesterol and lysolecitine in a molar ratio of 8:1:0.5 in a tris-HCl buffer containing about 30mM of octylglucoside, injecting the solution obtained in a PBS buffer to form a suspension of liposomes and subjecting said suspension of liposomes to dialysis to give said preformed liposomes; 30 A method according to claim 2, which comprises treating liposomes by chromatography to give liposomes which are substantially homogeneous and which have a size varying between about 30 - 90 nm. 4. A method according to claims 1, 2 or 3, wherein said antigen viral sub-units are prepared by mixing a virus with a detergent to 503 ubilize membrane thereof thereby giving a mixture of HA, neuraminidasses, nucleoproteins and proteins M, subjecting said mixture to centrifugation to separate said nucleoproteins and said proteins M therefrom and to give said HA and neuralrinidases, subjecting said HA and neuraminidases to dialysis in an aqueous buffer solution and adding a detergent to give said complex, 5. A mothod according to claim 4, wherein said detergent is added in the form of a solution thereof in Tris buffer so as to give a final concentration of 7.5 m, and allowing the mixinre to stant at 22 C during about 10 minutes, thereby giving a protein-detergent complex susceptible to anchor in the membrane of the liposome. 6. A method according to claim 5, wherein said viral sub-unit-detergent complex is added to liposomes treated to become susceptible to anchor said sub-units and the mixture obtained is dialysed under a linearly decreasing gradient of detergent to give said immunosomes. 7. A method according to claim 6, erein when the gradient becomes zero, the suspension is dialysed in a PBS buffer, 8. A method according to claim 6, wherein said liposolnes treated to become susceptible to anchor said subunits are prepared by adding to a solution of preformed liposomes a solution of ss-d-octylglucoside in Tris-HCl buffer at p11 about 7.4 so as to give a final concentration of 1 mM, and allowing the mixture obtained to stand at 220C during about 10 minutes, 9. An immunosome comprising a membrane made of phospholipid material and viral sub-units essentially distributed in the form of projections on the outer surface of said membrane, said immunosome being prepared by a method according to claims 1 to 8 and being substantially free of secondary effects when injected to a living being.

Inventors: BOUDREAULT, ARMAND, NAUD, PIERRE, THIBODEAU, LISE
Applicants: INSTITUT ARMAND FRAPPIER
Application year: 1981
Main classification: A61K39
IPC codes: A61K9, A61K39, C12P1, A61P31