MXPA97008226A - Vaccines that contain a saponine and an eastern - Google Patents

Abstract

The invention relates to a vaccine composition comprising an antigen, an immunologically active saponin fraction and an ester

Description

VACCINES CONTAINING A SAPONINE AND A STEROL The present invention to formulations of novel vaccines, to methods of their production and to their use in medicine. In particular, the present invention relates to vaccines containing an antigen, an immunologically active fraction derived from bark of Quillaja Saponaria Molina such as QS21 and a sterol. The immunologically active fractions having auxiliary activity derived from the bark of the South American tree Quillaja Saponaria Molina are known in the art. For example QS21 also known as QA21, a purified fraction of CLAR from the Quillaja Saponaria Molina tree and its method of production is described (as QA21) in the US patent. No. 5,057,540. Quillaja's Saponin is also described as an auxiliary by Scott and others, Int. Archs. Allergy Appl. Immun., 1985, 77, 409. However, the use of QS21 as an auxiliary is associated with certain disadvantages. For example, when QS21 is injected into a mammal given that a free molecule has been observed that necrosis, which, to put it another way, is localized tissue death, occurs at the site of injection. It has now surprisingly been found that injection site necrosis can be avoided by the use of formulations containing a combination of QS21 a sterol. Preferred sterols include β-sitosterol, stigmasterol, ergosterol, ergocalciferol and cholesterol. These sterols are well known in the art, for example, cholesterol is described in Merck I ndex, 1 1 to Ed. Page 341, as a sterol present in nature in animal fat. In a first aspect, the present invention therefore provides a vaccine composition comprising an antigen, an immunologically active saponin fraction and a sterol. Preferably the compositions of the invention contain the immunologically active saponin fraction in substantially pure form. Preferably the compositions of the invention contain QS21 in substantially pure form, ie, the QS21 is at least 90% pure, preferably at least 95% pure and more preferably at least 98% pure. Other immunologically active saponin fractions useful in compositions of the invention include QA 17 / QS 17. Compositions of the invention comprising QS21 and cholesterol showed to decrease reactogenicity when compared to compositions in which cholesterol is absent, while environment the auxiliary effect. In addition, it is known that QS21 degrades under basic conditions where the pH is about 7 or greater. An additional advantage of the present compositions is that the stability of QS21 to the base-mediated hydrolysis is increased in cholesterol-containing formulations. Preferred compositions of the invention are those that form a liposome structure. Compositions wherein the sterol / immunologically active saponin fraction forms an ISCOM structure also form an aspect of the invention.

The ratio of QS21: sterol will normally be in the order of 1: 100 to 1: 1 by weight to weight. Preferably excess sterol is present, the ratio of QS21: sterol being at least 1: 2 w / w. Normally for administration to humans, QS21 and sterol will be present in a vaccine in the range of about 1 μg to about 100 μg, preferably about 10 μg to about 50 μg per dose. Preferably the liposomes contain a neutral lipid, for example phosphatidylcholine, which is preferably non-crystalline at room temperature, for example, egg white phosphatidylcholine, dioleoyl phosphatidylcholine or dilauryl phosphatidylcholine. The liposomes also contain a charged liquid that increases the stability of the QS21 liposome structure for liposomes composed of saturated liquid. In these cases, the amount of liquid loaded is preferably 1-20% w / w, more preferably 5-10%. The ratio of sterol to phospholipid is 1-50% (ml / mol), more preferably 20-25%. Preferably the compositions of the invention contain MPL (3-deacylated monophosphoryl lipid A also known as 3S-MPL). The 3D-MPL of GB2 220 21 1 (Ribi) is known as a mixture of 3 types of De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains and is manufactured by Ribi I mmunochem, Montana. A preferred form is disclosed in International Patent Application 92/1 16556.

Suitable compositions of the invention are those in which the liposomes are initially prepared without MPL and MPL is then added, preferably as 100 nm particles. Therefore MPL is not contained within vesicle membrane (known as MPL outside). The compositions wherein MPL is contained within the membrane of vesicles (known as MPL in) also forms an aspect of the invention. The antigen may be contained within the vesicle membrane or contained outside of the vesicle membrane. Preferably the soluble antigens are outside and the hydrophobic or lipidated antigens are contained both inside and outside the membrane. Frequently the vaccines of the invention do not require any specific vehicle and are formulated in a pharmaceutically acceptable aqueous buffer solution and another. In some cases it may be advantageous that the vaccines of the present invention also contain alumina or may be present in an oil in water emulsion, or other suitable vehicle, such as for example, liposomes, microspheres or encapsulated antigen particles. Preferably the vaccine formulations will contain an antigen or antigenic composition capable of producing an immune response against a human or animal pathogen. The antigen or antigenic compositions known in the art can be used in the compositions of the invention, including polysaccharide antigens, antigens or antigenic compositions derived from HIV-1, (such as gp 120 or gp 160), any of the immunodeficiency virus. felines, human herpes viruses or animals, such as gD or derivatives thereof or immediate immediate protein such as HSV1 or HSV2 ICP27, cytomegalovirus (especially human) (such as gB or derivatives thereof), Varicella Zoster Virus (such as as gpl, II or III) or a hepatitic virus such as hepatitis virus for example Hepatitis B surface antigen or a derivative thereof, hepatitis A virus, hepatitis C virus and hepatitis B virus, or other viral pathogens, such as respiratory syncytial viruses (for example HSRV F and Ge proteins or immunogenic fragments thereof described in US Pat. No. 5,149,650 or chimeric polypeptides They contain immunogenic fragments of F and G proteins of HSRV, e.g., FG glycoprotein described in the U.S. Patent. 5,194,595), antigens derived from meningitis strains such as meningitis A, B and C, Sterptoccoccus pneumonia, human papilloma virus, Influenza virus, Hemophilus Influenza B (Hib), Epstein Barr Virus (EBV), or bacterial pathogen derivatives such as Salmonella, Neisseria, Borrrelia (for example OspA or OspB or derivatives thereof), or Chlamydia, or Bordetella for example P.69, Pt and FHA, or parasite derivatives such as plasmodium or toxoplasma. HSV glycoprotein D (gD) or derivatives thereof is a preferred vaccine antigen. It is located in the viral membrane and is also found in the cytoplasm of infected cells (Eisenberg R. J. et al, J. of Virol 1980 35 428-438). It comprises 393 amino acids including a signal peptide and has a molecular weight of approximately 60 kD. Of all the HSV envelope glycoproteins, this is probably the best characterized (Cohen et al J. Virology 60 157-166). I n vivo is known to play a central role in viral binding to cell membranes. In addition, glycoprotein D has been shown to be capable of producing neutralizing antibodies in vivo and protects animals from the lethal challenge. A truncated form of the gD molecule releases the C-terminal anchor region and can be produced in mammalian cells as a soluble protein that is exported into the cell culture supernatant. Such soluble forms of g D are preferred. The production of truncated forms of gD is described in EP 0 139 417. Preferably g D is derived from H SV-2. One embodiment of the invention is a truncated HSV-2 glycoprotein D of 308 amino acids comprising amino acids 1 to 306 of the glycoprotein present in nature with the addition of Asparagine and Glutamine at the C terminal end of the truncated protein is released from its membrane anchor region. This form of the protein includes the signal peptide that separates to allow the mature soluble 283 amino acid protein to be secreted from a host cell. In another aspect of the invention, Hepatitis B surface antigen is a preferred vaccine antigen. As used herein, the term "Hepatitis B surface antigen" or "H BsAg" includes any H BsAg antigen or fragment thereof exhibiting the antigenicity of the HBV surface antigen. It will be understood that in addition to the 226 amino acid sequence of the HBsAg antigen (see Tiollais et al., Nature, 317, 489 (1985) references therein) HBsAg as described herein, if desired, may contain all or part of the HBsAg antigen. it pre-S sequence as described in the above references in EP-A-0 278 904. In particular HBsAg may comprise a polypeptide comprising an amino acid sequence comprising residues of 12-52 followed by residues of 133-145 followed by residues 175-400 of the L protein of HBsAg relative to the open reading frame in a Hepatitis B virus of serotype of addition (this polypeptide is referred to as L *; see EP 0414 374). HBsAg within the scope of the invention may also include the pre-S1-preS2-S polypeptide described in EP 0198 474 (Endotronics) or close analogs thereof such as those described in EP 0 304 578 (Me Cormick and Jones ), HBsAg as described herein may be referred to as mutants, for example, the 'escape mutant' described in EP 0 304 578 (Me Cormick and Jones). HBsAg as described herein may also refer to mutants, for example, the 'escape mutant' described in WO 91/14703 or European Patent Application Number 0 511 855a1, especially HBsAg wherein the amino acid substitution in the position 145 is from glycine to arginine. Normally HBsAg will be in the form of particles. The particles may comprise, for example the S protein alone or they may be mixed particles, for example (L *, S) where L * is as defined above and S denotes the S protein of H BsAg. Said particle advantageously has the form in which it is expressed in yeast. The preparation of hepatitis B surface antigen S protein is well documented. See, for example, Harford et al. (1983) in Develop. Biol. Standard 54, p. 125, Gregg and others (1987) in Biotechnology. 5, page 479, EP 0 226 846, EP 0 299 108 and references herein. Formulations within the scope of the invention may also contain an antitumor antigen and be useful for immunotherapy treatment cancers. Vaccine preparation was generally described in New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland, E. U .A. 1978. Encapsulation within liposomes was described, for example, by Fullerton, Patent of E. U.A. 4,235,877. The conjugation of proteins to macromolecules was described, for example, by Likhite, Patent of E. U .A. 4,372,945 and by Armor et al., Patent of E. U.A. 4, 474,757.

The amount of protein in each vaccine dose is selected as an amount that induces an immunoprotective response without significant side effects, adverse in normal vaccines. Said amount will vary depending on which specific immunogen is used and how it is presented. Generally, it is expected that each dose will comprise from 1 to 1000 mcg of protein, preferably from 2 to 100 mcg, more preferably from 4 to 40 mcg. An optimal amount for a particular vaccine can be assessed by normal studies involving the observation of appropriate immune responses in the subjects. After an initial vaccination, subjects may receive one or several booster immunizations properly separated. The formulations of the present invention can be used for both prophylactic and therapeutic purposes. Consequently, in a further aspect, the invention thus provides the use of a vaccine of the invention for the treatment of human patients. The invention provides a method of treatment comprising administering an effective amount of a vaccine of the present invention to a patient. In particular, the invention provides a method for treating viral, bacterial, parasitic or cancer infections which comprises administering an effective amount of a vaccine of the present invention to a patient. The following examples and data illustrate the invention. EXAMPLES 1.1 Liposome Preparation Method: A mixture of lipids (such as phosphatidylcholine, either egg yolk or synthetic) and cholesterol in organic solvent, is dried under vacuum (or alternatively under a stream of inert gas). An aqueous solution (such as saline with pH regulated phosphate solution) is then added and the vessel is agitated until all the lipid is in suspension. This suspension then microfluidizes until the liposome size is reduced to 1 nm and then filtered with sterilization through a 0.2 μm filter. Extrusion or sound treatment could replace this step. Normally the cholesterol: phosphatidylcholine ratio of 1: 4 (w / w) and the aqueous solution is added to give a final cholesterol concentration of 5 to 50 mg / ml. If MPL in organic solution is added to the liquid in organic solution, the final liposomes contain MPL in the membrane (referred to as MPL inside). Liposomes have a defined size of 100 nm and are referred to as SUVs (for small unilamellar vesicles). If this solution is repeatedly frozen and thawed, the vesicles melt to form large multilamellar structures (MLV) of size ranging from 500 nm to 15 μm. The liposomes by themselves are stable over time and have no fusogenic capacity. 1.2 Formulation procedure: QS21 in aqueous solution is added to the liposomes. This mixture is then added to the antigenic solution which, if desired, may contain MPL in the form of 100 nm particles. 1.3 The lytic activity of QS21 is inhibited by liposomes containing cholesterol When QS21 is added to erythrocytes, they lyse them releasing hemoglobin. This lytic activity can also be measured using liposomes containing cholesterol in its membrane and a trapped fluorescent dye, carboxyfluorescein, as the liposomes are lysed the dye is released, which can be monitored by fluorescence spectroscopy. If the fluorescent liposomes do not contain cholesterol in their membrane, no lysis of the liposomes is observed. If the QS21 is incubated with liposomes containing cholesterol before adding it to erythrocytes, the lysis of erythrocytes decreases depending on the cholesterol to QS21 ratio. If a ratio of 1: 1 is used, no lytic activity is detected. If the liposomes do not contain cholesterol, the inhibition of lysis requires a hundredfold excess of phospholipid on QS21. The same is true using fluorescent liposomes to measure the lytic activity. In the following graph, the lithic activity of 4 μg of QS21 treated with liposomes lacking cholesterol (1 mg of egg yolk lecithin per ml) or containing cholesterol (1 mg lecithin, 500 μg of cholesterol per ml) was measured by fluorescence The data show that QS21 is associated in a specific way with cholesterol in a membrane, thus causing lysis (of fluorescent cells or liposomes). If QS21 is first associated with cholesterol in liposomes, it is not lytic towards cells or other liposomes. This requires a minimum ratio of 0.5: col. : QS21 (P / P). Cholesterol is insoluble in aqueous solutions and does not form a stable suspension. In the presence of phospholipids, cholesterol resides within the bilayer of phospholipids that can form a stable suspension of vesicles called liposomes. To avoid the requirement to add phospholipids, a soluble derivative was analyzed. The polyoxyethanil-cholesterol sebacate is soluble in water at 60 mg / ml however, even at an excess of 2000 times (w / w) on QS21, no reduction in the lytic activity of QS21 was detected. 1 .4 Increased stability of QS21 by liposomes containing cholesterol. QS21 is very susceptible to hydrolysis at a pH above 7. Hydrolysis can be monitored by measuring the decrease in peak corresponding to QS21 in reverse phase HPLC. For example, the following graph shows that a pH 9 and at a temperature of 37 ° C, 90% of QS21 is hydrolyzed within 16 hours. If the liposomes containing cholesterol are added to the QS21 at a ratio of 2: 1 (col: QS21 w / w) hydrolysis was not detected under the same conditions. If the ratio of 1: 1 is degraded 10% of QS21.

Incubation of 20 μg QS21 in the presence of SUV containing cholesterol of pH 9 for 16 hrs at 37 ° C μg present cholesterol It is concluded that when QS21 is associated with liposomes containing cholesterol, it becomes much less susceptible to base-mediated hydrolysis. The hydrolysis product is described as having no auxiliary activity when given parenterally, therefore vaccines containing QS21 have to be formulated at acid pH and kept at 4 ° C to maintain auxiliary composition. The use of liposomes can exceed this requirement. 1.5 Reactogenicity studies Mice were injected into muscle of the tibia with 5 μg QS21 (or digitonin) added to increase quantities of liposomes (expressed in terms of μg cholesterol). The lithic activity is expressed as μg QS21 equivalent, which means the amount of QS21 required to achieve the same hemolysis as the sample.

The reddish coloration, necrosis and toxicity in the muscle at the site of injection were visually classified after killing the mice.

The data show that when the lytic activity is abolished by the addition of liposomes containing cholesterol, the toxicity is also abolished after killing the mice. 1 .6 Intramuscular reactogenecity in rabbits U-values. l ./L The data show that the addition of liposomes containing cholesterol to the formulation significantly reduces the elevation in CPQ (creatinine phosphokinase) caused by QS21. Since the increase in CPQ is a measure of muscle damage, it indicates decreased muscle damage and is confirmed by histopathology. 1.7 Binding of the liposome-QS21 complex to alumina QS21 was incubated with neutral liposomes containing excess cholesterol as well as radioactive cholesterol and then incubated with alumina (AI (OH) 3) in PBS. Alone, neither the neutral liposomes nor QS21 bind to alumina in PBS, nor do the negatively charged liposomes. However, when they are together, QS21 and the neutral liposomes bind to alumina. The supernatant did not contain either QS21 (analyzed by orcinol test) or radioactive cholesterol.

This indicates that QS21 has bound to the liposomes and allows the combination of liposome-QS21 to bind to the alumina. This may arise from a negative charge that is imposed on the liposomes by QS21, or to an exposure of hydrophobic regions in the liposomes. These results also imply that QS21 does not remove cholesterol from the membrane. This indicates that the compositions of the invention can be used in alumina-based vaccines. 1.8 Comparison of QS21 / MPL liposomal and QS21 + MPL for antibody and induction of CMI SUV was prepared by extrusion (EYPC: col: MPL, 20: 5: 1). For M PL outside, the liposomes were prepared without MPL and MPL added as 100 nM particles. QS21 was added before the Col antigen: QS21 = 5: 1 (w / w) MLV were formed by freeze-thawing of SUV 3 times before the addition of antigen. To trap the antigen, antigen was added to the SUV before freezing-thawing and QS21 was added before freezing-thawing. The encapsulation of the antigen = 5% in, 95% out. Mice (balb / c for gD, B 10BR for RTSs) were injected twice into the pad of the leg. G D is the glycoprotein D of the Herpes Simplex virus. RTS is the Hepatitis B surface antigen (HbsAg) genetically modified to contain a sporozoite epitope of Plasmodiium falciparum m. titers of antibodies at least as good as QS21 + MPL, as well as induce IL2 a marker of cell-mediated immunity, while cooling reactogenicity 21.

The additional results of a second experiment comparing QS21 and QS21 in the presence of cholesterol (SUV) in balb / c mice with HSV gD as antigen are shown below: 1 .9 Comparison of gp120 plus MPL / QS21 liposomal with free MPL / QS21 Liposomes = SUV containing MPL on the Col membrane: QS21 = 6: 1 Response was tested two weeks after immunization After a second immunization: The data show that QS21 associated with liposomes containing cholesterol and MPL induces a Th 1 / Th0 response equal to MPL * QS21. At this cholesterol to QS21 ratio, QS21 is not toxic in rabbits (by CPK). In a second experiment the balb / c mice were immunized between the pads with gp120 in the presence of QS21 or QS21 + SUVs containing cholesterol. The activity of cytotoxic T lymphocytes in spleen cells was measured. This demonstrates that QS21 only induces CTL activity, and that QS21 in the presence of liposomes containing cholesterol induces CTL activity at least as well as, or better than, QS21 alone. 2. Vaccines 2.1 Formulation of particles HbsAg L *, S I laughed White effector Reí. Effector: white This demonstrates that QS21 only induces CTL activity and that QS21 in the presence of liposomes containing cholesterol induces CTL activity at least as good as, or better than, QS21 alone. 2. Vaccines 2.1 Formulation of particles HbsAg L *, S The particles HbsAgL *, S can be formulated as follows: 10μg of HbsAgL * particles, S / dose is incubated 1 hour at room temperature under agitation. The volume is adjusted using water for injection and a PBS solution and completed to a final volume of 70μl / dose with an aqueous solution of QS21 (10μg / dose). The pH is maintained at 7 ± 0.5. Similar formulations can be prepared using 1 and 50 μg of HbsAgL *, S and also using the HbsAg S antigen. These formulations can be tested in the Woodchuck surrogate therapeutic model using HBV Woodchuck antigens as a model. Woodchuck model DQ QS21 (ie QS21 / cholesterol or extinct QS21) can be tested in the Woodchuck therapeutic model where the animals are chronically infected with the virus. The Woodchuck hepatitis virus vaccine can be added mixed with QS21 as such or DQ and with or without MPL and can be administered to animals monthly for 6 months. The effectiveness of the vaccine can be assessed by suppressing viral DNA. 2.2 Model of Guinea pigs of India (HSV) 2.2.1 Prophylactic model Groups of 12 female guinea pigs of Harley Indian were injected intramuscularly on day 0 and day 28 with the following formulations: 1st experiment: gD 5 μg + QS21 50 μg + SUV containing 50 μg of cholesterol gD 5 μg + QS21 100 μg + SUV containing 100 μg of cholesterol gD 5 μg * QS21 50 μg + SUV containing 250 μg of cholesterol gD 5 μg * QS21 50 μg 2nd experiment : gD 5 μg + MPL 12.5 μg + QS21 12.5 μg + SUV containing 62.5 μg of cholesterol, or left untreated. The animals were mixed at 14 and 28 days after the second immunization and the sera were tested for their gD-specific ELISA antibody titers. The animals were then challenged intravaginally with the pfu of the HSV-2 MS strain. They were classified daily from day 4 to 12 for evaluation of primary herpetic lesions. The classification was as follows: Vaginal lesions: - bleeding = 0.5 - reddish coloration for 1 or 2 days without bleeding = 0.5 - reddish coloration and bleeding during a day = 1 - reddish coloration without bleeding lasting at least 3 days = 1 Vesicles Herpetic external: - < 4 small vesicles = 2 - > = 4 small vesicles or one large vesicle 4 > = 4 large lesions, 8 that fuse large lesions = 16 Fusion of large lesions in the entire external genital area = 32. This results in the following table: NJ CO Sum of the lesions is classified for days 4 to 12 after the infection (Animals without injury are not considered) Classification of injuries: without injury (0), vaginal lesions (0.5 or 1), external skin vesicles (2, 4, 8 or 16) ** primary infection index = SUM (Max Cías I) x (incidence%); with i = 0, 0.5, 1, 2, 4, 8 or 16.

The table and graph show that in the prophylactic model, a very high level of protection against primary disease was induced when immunized with gD / MPL / QS21 / SUV. Both the incidence of external injuries and the severity of lesions appeared highly reduced in the group of animals immunized with gD / MPL / QS21 / SUV. 2.2.2 Therapeutic Model In the therapeutic model, the female Hartley guinea pigs were first confronted with the pfu of the HSV-2 MS strain. Animals with herpetic lesions were randomly distributed to the groups of 16. On day 21 and day 43, they were immunized with one of the following formulations: - gD + MPL 50 μg + SUV containing 250 μg of cholesterol. - GD + AI (OH) 3 + MPL 50 μg, + S UV containing 250 μg of cholesterol or left untreated. They were monitored daily from day 22 to 75 to evaluate the current disease. The classification was as described for the prophylactic model. The results are shown in the following table and graph.

Sum of injury ratings for days 25 to 75 after infection. ** Total days when animals experienced recurrent injuries for days 22 to 75 after infection *** Number of frequency episodes for days 22 to 75 after infection. An episode is preceded and followed by a day without injury and characterized for at least two days with erythema c (classification = 0.5) or one day with external vesicle (classification> = 2) The immunotherapeutic treatment was performed on days 21 and 42. The results show that good levels of protection were also induced in the therapeutic model of infection with HSV-2. Immunization with gD / M PL / QS21 / SUV with or without Alumina had a noticeable effect on the mean severity of recurrent disease. The number and duration of the episode were also slightly reduced (see Table).

Claims (10)

1. CLAIMS 1. A vaccine composition comprising an antigen, an immunologically active saponin fraction and a sterol.
2. 2. A vaccine composition according to claim 1, wherein the immunologically active saponin fraction is QS21.
3. 3. A vaccine composition according to claim 1 or 2, wherein the sterol is cholesterol.
4. 4. A vaccine composition according to claim 2 or 3, wherein the ratio of QS21: sterol is 1: 100 to 1: 1.
5. 5. A vaccine composition according to any of claims 1 to 4, which further contains MPL.
6. 6. A vaccine composition as claimed herein comprising an antigen or antigenic composition derived from either Human Immunodeficiency Virus, Feline Immunodeficiency Virus, Varicella Zoster Virus, Herpes Simplex Virus type 1, Herpes Simplex Virus type 2 , human cytomegalovirus, hepatitis A, B, C, or E, Respiratory Syncytial virus, human papilloma virus, Influenza virus, Hib, meningitis virus, Salmonella, Neisseria, Borrelia, Chlamydia, Bordetella, Plasmodium or Toxoplasma.
7. 7. A vaccine according to any of claims 1 to 5, wherein the antigen is a tumor antigen.
8. 8. The use of the composition as defined in any one of claims 1 to 5, for the manufacture of a vaccine for the prophylactic treatment of viral, bacterial or parasitic infections.
9. 9. The use of the composition as defined in any of claims 1 to 5 for the manufacture of a vaccine for the immunotherapeutic treatment of viral, bacterial, parasitic or cancer infections.
10. 10. A method for treating a mammal suffering from or susceptible to a pathogenic infection comprising the administration of a safe and effective amount of a composition according to any of claims 1 to 5. 1 1. A method for treating a mammal suffering from cancer comprising the administration of a safe and effective amount according to claims 1 to 5. 12. A process for formulating a vaccine composition according to claim 1, comprising mixing a fraction of immunologically active saponin and cholesterol with an antigen or antigenic composition.