Procedures for preparing vesicles and formulations produced from them

专利摘要:

公开号:ES2573427T9
申请号:ES10797727.4T
申请日:2010-07-06
公开日:2017-03-17
发明作者:David E. Anderson；Andrei Ogrel
申请人:Variation Biotechnologies Inc；
IPC主号:

专利说明:

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Procedures for preparing vesicles and formulations produced from them Cross-reference to related requests
This application claims priority under 35 U.S.C. § 119 (e) for the provisional US request. UU. with serial number 61 / 223,196, filed on July 6, 2009 and the provisional US request. UU. with serial number 61 / 256,912, filed on October 30, 2009.
Background
Vesicles were first described in the 1960s as a model of cell membranes (see Bangham et al., J. Mol. Biol. 13: 238-252, 1965). Vesicles have found several applications in the administration of drugs with small molecules, vaccine adjuvance, genetic transfer and diagnostic imaging (for example, see Liposome Technology, 3rd edition, edited by Gregory Gregoriadis, Informa Healthcare, 2006 and Liposomes: A Practical Approach (The Practical Approach Series, 264), 2nd edition, edited by Vladimir Torchilin and Volkmar Weissig, Oxford University Press, USA, 2003).
Several procedures for preparing vesicles have been described (for example, see references cited above and Walde and Ichikawa, Biomol. Eng., 18: 143-177, 2001). However, there is still a need in the art to obtain procedures that can be used to trap substances within vesicles.
A procedure that has been described in the art is the so-called 3-stage melting process. Initially, vesicle-forming lipids melt at high temperatures (eg, 120 ° C). An emulsion is created in a second stage by adding an aqueous buffer (eg, bicarbonate buffer) to the molten lipids. Finally, the substance to be trapped is homogenized with the components of the emulsion at a reduced temperature (for example, 50 ° C) before lyophilization. Alternatively, the emulsion vesicles are lyophilized and then reconstituted in the presence of the substance to be trapped.
Although procedures such as this may be very suitable for trapping substances that can withstand high temperatures and / or small molecules that can diffuse rapidly in empty vesicles, it has been found that they are not suitable for trapping the types of antigens (e.g., polypeptides , viruses, etc.) that are commonly involved in vaccines. In particular, it has been discovered that these procedures produce low entrapment efficiencies and can dramatically reduce the activity of the underlying antigen (for example, as measured by immune responses). Therefore, there is a need in the art to obtain processes for preparing vesicles that can trap antigens while minimizing the impact on the activity of the antigen.
Mann et al, Vaccine 22: 2425-2429, 2004 discloses the preparation of vesicles by melting lipids at 140 °, to which deoxycholate is added, followed by a formulation with influenza hemagglutinin.
Summary
The present invention provides a method according to claim 1.
Brief description of the drawing
Figure 1 compares the average particle sizes for two vesicle formulations that were prepared using the 3-stage melt process of Example 1 and the inverted 2-stage melt procedure of Example 2. The formulations were lyophilized and then rehydrated into presence of a buffer containing 2 | jg of inactivated hepatitis A antigen. Vesfcula size, which is a good stability marker, was measured using a Mastersizer immediately after hydration and 2, 4 and 6 hours after this.
Figure 2 shows the immune response caused by vesicles containing the hepatitis A antigen. Empty vesicles were prepared using the 3-stage melt procedure of Example 1 and the inverted 2-stage melt procedure of Example 2. The lyophilized cells were lyophilized. Formulations were then rehydrated in the presence of a buffer containing 2 jg of inactivated hepatitis A antigen. The mice were immunized orally 3 times on days 0, 14 and 28, and the sera were tested for reactivity 14 days after the last vaccination. Each symbol represents that serum endpoint assessment of an individual animal.
Figure 3 shows that the content of bile salt in the vesicles affects the maturation of immature dendritic cells as evidenced by flow cytometna. The maturation of immature dendritic cells was measured by flow cytometin using anti-MHC II and anti-CD86 antibodies. Mature CDs were defined as double positive for both antibodies. Immature dendritic cells were treated with lipid vesicles of non-ionic surfactant (VTNI) prepared as in steps 1 and 2 of Example 2 (without the subsequent addition of antigen) and with or without two different molar proportions of bile acid with respect to total lipid (0.1 and 0.5). As a positive control, immature dendritic cells were treated with TNF-a alone.
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Figure 4 compares the 31P NMR spectrum of exemplary vesicles prepared by the procedures of Example 1 and 2 without the addition of any antigen. All spectra were obtained at 25 ° C.
Definitions
Throughout this disclosure, several terms are defined as defined in the following paragraphs.
As used herein, the term "antigen" refers to a substance that contains one or more epftopos (linear, conformational or both) that can be recognized by an antibody. In certain embodiments, an antigen can be a virus, a polypeptide, a polynucleotide, a polysaccharide, etc. The term "antigen" indicates both subunit antigens, (that is, antigens that are separated and differentiated from a whole organism with which the antigen in nature is associated), as well as bacteria, viruses, fungi, parasites or other dead microbes. , dimmed or inactivated. In certain embodiments, an antigen can be an "immunogen."
As used herein, the term "trap" refers to any type of physical association between a substance and a vesicle, for example, encapsulation, adhesion (to the inner or outer wall of the vesicle) or inclusion in the wall. with or without extrusion of the substance. The term is used interchangeably with the terms "load" and "contain".
As used herein, the terms "immune response" refer to a response elicited in an animal. An immune response may refer to cellular immunity, humoral immunity or may involve both. An immune response may also be limited to a part of the immune system. For example, in certain embodiments, an immunogenic formulation may induce an increase in the IFNy response. In certain embodiments, an immunogenic formulation can induce a mucosal IgA response (for example, as measured in nasal and / or rectal washes). In certain embodiments, an immunogenic formulation can induce a systemic IgG response (for example, as measured in serum).
As used herein, the term "immunogen" means that it can produce an immune response in an animal host against an entity other than the host (for example, a hepatitis A virus or hepatitis B virus). In certain embodiments, this immune response forms the basis of protective immunity caused by a vaccine against a specific infectious organism (for example, a hepatitis A virus or a hepatitis B virus). An "immunogen" is an immunogenic substance (for example, a molecule).
As used herein, the terms "therapeutically effective amount" refer to the amount sufficient to show a significant benefit in a patient being treated. The therapeutically effective amount of an immunogenic formulation may vary depending on factors such as the desired biological assessment criteria, the nature of the formulation, the route of administration, health, height and / or age of the patient being treated, etc.
As used herein, the term "polypeptide" refers to a protein (ie, a chain of at least two amino acids linked together by peptide bonds). In some embodiments, the polypeptides may include non-amino acid residues (for example, they may be glycoproteins, proteoglycans, lipoprotems, etc.) and / or they may be processed or modified otherwise. Those skilled in the art will appreciate that a "protein" may be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or it may be a portion thereof. Those skilled in the art will appreciate that a protein may sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. The polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid or analog modifications known in the art. Useful modifications include, for example, acetylation, amidation, etc., terminal. In some embodiments, the polypeptides may comprise natural amino acids, unnatural amino acids, synthetic amino acids and combinations thereof.
As used herein, the term "polysaccharide" refers to a carbohydrate polymer. The polymer can include natural carbohydrates (for example, arabinose, lyxose, ribose, xylose, ribulose, xylulose, alose, altrose, galactose, glucose, gulose, idosa, mannose, talose, fructose, psychosa, sorbose, tagatose, manheptulose, sedoheptulose, octolose and sialose) and / or modified carbohydrates (for example, 2'-fluororibose, 2'-deoxyribose and hexose). Exemplary polysaccharides include starch, glycogen, dextran, cellulose, etc.
As used herein, the term "polynucleotide" refers to a nucleotide polymer. The polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, citidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine and deoxycytidine), nucleoside analogs (for example, 2-aminoadenosine, 2-thiotimidine, inosine, pyrrolopyrimidine methyladenosine, 5-methylcytidine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynyl cytidine, C5-methylcytidine, 7-desazaadenosine, 7-desazaguanosine, 8-oxoadenosine, 8-oxoadenosine, 8-oxoadenosine, 8-oxoadenosine, 8-oxoadenosine, 8-oxoadenosine, 8 -methylguanine, 4-acetylcytidine, 5- (carboxyhydroxymethyl) uridine, dihydrouridine, methylpseudouridine, 1-methyladenosine, 1-methylguanosine, N6-methyladenosine and 2-thiocytidine), chemically modified bases, biologically modified bases, for example, methylated bases intercalated bases, carbohydrates
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modified (for example, 2-fluororibose, ribose, 2'-deoxyribose, 2'-O-methylcytidine, arabinose and hexose), or modified phosphate groups (for example, phosphorothioates and 5'-N-phosphoramidite bonds).
As used herein, the term "small molecule therapeutic drug" refers to a non-polymeric therapeutic molecule that may contain several carbon-carbon bonds and has a molecular weight of less than about 1500 Da (for example, less than about 1000 Da, less than about 500 Da or less than about 200 Da). A small molecule therapeutic drug can be synthesized in a laboratory (for example, by combinatorial synthesis, using a genomanipulated microorganism, etc.) or it can be found in nature (for example, a natural product). In general, a small molecule therapeutic drug can modify, inhibit, activate or otherwise affect a biological event. For example, small molecule therapeutic drugs may include, but are not limited to, anti-AIDS substances, anti-cancer substances, antibiotics, anti-diabetic substances, immunosuppressants, antiviral substances, enzyme inhibitors, neurotoxins, opioids, hypnotics, antihistammics, lubricants, tranquilizers, anticonvulsants. , muscle relaxants and antiparkinsonian, antispasmodic and muscle contracting substances including channel blockers, myotics and anticholinergics, antiglaucoma compounds, antiparasitic and / or antiprotozoal compounds, modulators of cell extracellular matrix interactions including cell growth inhibitors and anti-adhesion molecules, agents vasodilators, inhibitors of the synthesis of DNA, RNA or proteins, antihypertensives, analgesics, antipyretics, steroidal and non-steroidal anti-inflammatory agents, antiangiogenic factors, antisecretory factors, anticoagulants and / or antithromatic agents boticos, local anesthetics, ophthalmic, prostaglandins, antidepressants, antipsychotic substances, antiemetics and imaging agents. A more complete list of small exemplary molecules suitable for use in the procedures of the present disclosure can be found in Pharmaceutical Substances: Syntheses, Patents, Applications, edited by Axel Kleemann and Jurgen Engel, Thieme Medical Publishing, 1999; Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals, edited by Susan Budavari et al., CRC Press, 1996, and United States Pharmacopeia-25 / National formulary-20, published by the United States Pharmacopeial Convention, Inc., 2001 Preferably, although not necessarily, the small molecule is one that has already been considered safe and effective for use by the appropriate government agency or agency. For example, drugs for use in humans listed by the FDA at 21 C.F.R. §§ 330.5, 331 to 361, and 440 to 460, and veterinary drugs listed by the FDA at 21 C.F.R. §§ 500 to 589, all are considered acceptable for use in accordance with the procedures of this disclosure.
As used herein, the term "treat" (or "treating", "treated", "treatment", etc.) refers to the administration of a formulation to a patient who has a disease, a symptom of a disease or a predisposition towards a disease, in order to alleviate, calm, modify, improve, overcome or influence the disease, a symptom or symptoms of the disease, or the predisposition towards the disease. In certain embodiments, the term "treat" refers to the vaccination of a patient.
Detailed description of some embodiments
I. Procedures for preparing vesicles
The present disclosure provides procedures for preparing vesicles. In general, the vesicles have an aqueous compartment enclosed by one or more bilayers that include lipids, optionally with other molecules. For example, as discussed in more detail below, in some embodiments, the vesicles of the present disclosure comprise transport-enhancing molecules (eg, bile salts) that facilitate the transport of lipids through mucous membranes.
In one aspect, the present disclosure provides methods for preparing vesicles that include the steps of providing a molten mixture of vesicle-forming lipids and then adding the molten mixture to an aqueous solution comprising an antigen so that the antigen-containing vesicles are formed. . In some embodiments, the aqueous solution comprising an antigen is controlled by temperature. In some embodiments, the aqueous solution comprising an antigen is maintained at a temperature of less than about 50 ° C during the addition stage (for example, less than about 40 ° C, less than about 30 ° C, etc. .). In some embodiments, the aqueous solution comprising an antigen is maintained in a temperature range between about 25 ° C and about 50 ° C. In some embodiments, the aqueous solution comprising an antigen is maintained at room temperature.
It should be understood that a molten mixture of vesicle-forming lipids can be obtained in any way, for example, the lipids are melted to form a molten mixture. In some embodiments, the fluids melt in a temperature range between 120 ° C and 150 ° C (for example, between 120 ° C and 125 ° C, between 120 ° C and 130 ° C, between 120 ° C and 140 ° C, between 130 ° C and 140 ° C, between 135 ° C and 145 ° C, or between 140 ° C and 145 ° C). In some embodiments, lipids are melted at approximately 120 ° C. In some embodiments, lipids are melted at approximately 125 ° C. In some embodiments, lipids are melted at approximately 130 ° C. In some embodiments, lipids melt at approximately 135 ° C. In some embodiments, lipids are melted at approximately 140 ° C. In some embodiments, lipids are melted at approximately 145 ° C. In some embodiments, lipids are melted at approximately 150 ° C.
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In another aspect, the present disclosure provides methods for preparing vesicles that include the steps of providing a lyophilized lipid product and rehydrating the lyophilized lipid product with an aqueous solution comprising an antigen so that vesicles containing antigen are formed. The lyophilized lipid product is prepared by melting the vesicle-forming lipids to produce a molten lipid mixture and then lyophilize the molten lipid mixture.
Without wishing to commit to any theona, it is believed that by adding an aqueous solution of antigens to the lyophilized lipid product, vesicles are formed in the presence of the antigen. This may explain the high entrapment efficiencies observed. Additionally, the procedures of the present disclosure avoid exposing the antigen to organic solvents and high temperatures. Without wishing to be limited by any theona, this may explain the high activity (ie, antigenicity and / or immunogenicity) of the antigens trapped in the resulting formulations.
Vesicle forming lipids
Lipids are organic molecules that, in general, are insoluble in water, but soluble in apolar organic substances (for example, ether, chloroform, acetone, benzene, etc.). Fatty acids are a class of lipids that include an acid moiety attached to a saturated or unsaturated hydrocarbon chain. Specific examples include lauric acid, palmic acid, stearic acid, arachidic acid, palmitoleic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, etc. The alkali metal salts of fatty acids are typically more water soluble than the acids themselves. Fatty acids and their salts that include hydrocarbon chains with eight or more carbons often have amphiphilic properties due to the presence of both hydrophilic (head) and hydrophobic (tail) regions in the same molecule. Non-ionic lipids that include polar head groups may also have amphiphilic properties (i.e., surfactants). The fatty acid triesters with glycerol (1,2,3-trihydroxypropane) make up another class of lipids known as triglycerides that are commonly found in animal fats and vegetable oils. The esters of fatty acids with long chain monohydric alcohols form another class of lipids found in waxes. Phospholipids are also another class of lipids. They resemble triglycerides in that they are derived from glycerol or sphingosine ester or amide with fatty acids and phosphoric acid. The resulting phosphatidic acid phosphate moiety can be further esterified with ethanolamine, choline or serine in the phospholipid itself. It should be understood that the procedures can be used with any lipid that may form vesicles including any of the lipids described in the prior art (for example, in Liposome Technology, 3rd edition, edited by Gregory Gregoriadis, Informa Healthcare, 2006 and Liposomes : A Practical Approach (The Practical Approach Series, 264), 2nd edition, edited by Vladimir Torchilin and Volkmar Weissig, Oxford University Press, USA, 2003).
In some embodiments, the vesicle-forming lipid is a phospholipid. Any natural or synthetic phospholipid can be used. Without limitation, examples of specific phospholipids are La- (diestearoyl) lecithin, La- (diapalmitoyl) lecithin, La-phosphatidic acid, La- (dilauroyl) -phosphatidic acid, La (dimiristoyl) phosphatidic acid, La (dioleoyl) phosphatic acid , DL-a (dipalmitoyl) phosphatidic, La (diestearoyl) phosphatidic, and the various types of La-phosphatidylcholines prepared from brain, liver, egg yolk, heart, soy and the like, or synthetically, and salts of the same.
In some embodiments, the vesicle-forming lipid is a non-ionic surfactant. Non-ionic surfactant vesicles are referred to herein as "VTNI". Without limitation, examples of suitable non-ionic surfactants include glycerol-based ester-bonded surfactants. Said glycerol esters may comprise one of two upper aliphatic acyl groups, for example, which contain at least ten carbon atoms in each acyl moiety. Surfactants based on said glycerol esters may comprise more than one unit of glycerol, for example, up to 5 units of glycerol. Monoesters of glycerol can be used, for example, those that contain a C12-C20 alkenophyl or alkanoplole residue, for example, caproflo, lauroflo, miristefflo, palmitoflo, oleflo or stearcfflo. An exemplary nonionic surfactant is 1-monopalmitoyl glycerol.
In some embodiments, ether-bonded surfactants can also be used as a non-ionic surfactant. For example, ether-bonded surfactants based on glycerol or a glycol having a lower aliphatic glycol of up to 4 carbon atoms, such as ethylene glycol, are suitable. Surfactants based on said glycols may comprise more than one glycol unit, for example, up to 5 units of (for example, diglycol glycol ether and / or polyoxyethylene-3-lanolic ether). Ether or glycerol monoethers can be used, including those containing a C12-C20 alkenyl or alkanyl moiety, for example, capryl, lauryl, myristyl, cetyl, oleflo or stearyl. The ethylene oxide condensation products that can be used include those disclosed in PCT Publication No. WO88 / 06882 (for example, amine surfactants and higher aliphatic polyoxyethylene ether). Exemplary ether-bonded surfactants include glycerol 1-monocephalic ether and diglycol glycol ether.
Other components
In some embodiments, the vesicles may contain other lipid and non-lipid components, provided that they do not prevent vesicle formation. It should be understood that these components can be mixed together with vesicle-forming lipids and / or can be mixed together with the antigen (s).
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In some embodiments, it has been found that it may be advantageous to mix these components together with the vesicle-forming lipids.
In some embodiments, the vesicles may include a transport enhancer molecule that facilitates the transport of Kpids through the mucous membranes. As described in US Pat. UU. No. 5,876,721, a variety of molecules can be used as transport enhancers. For example, cholesterol derivatives can be used in which the C23 carbon atom of the side chain carries a carboxylic acid, and / or derivatives thereof, as transport enhancers. Such derivatives include, but are not limited to, "bile acids" colic acid and chenodeoxycholic acid, their conjugation products with glycine or taurine such as glycolic and taurocholic acid, derivatives that include deoxycholic and ursodeoxycholic acid, and salts of each of these acids. VTNIs that also include a bile acid or salt are referred to herein as "bilosomas." In some embodiments, transport enhancers include acyloxylated amino acids, such as acylcarnitines and salts thereof. For example, acylcarnitine containing C6-20 alkenoflo and alkanophilic moieties, such as palmitoylcarnitine, can be used as transport enhancers. As used herein, it is intended that the term acyloxylated amino acid cover primary, secondary and tertiary amino acids as well as a, p and and amino acids. Acylcarnitines are examples of and acyloxylated amino acids. It should be understood that the vesicles may comprise more than one type of transport enhancer, for example, one or more different bile salts and one or more acylcarnitines. The transport enhancer (s), if present, typically comprise between 40 and 400% by weight of the vesicle-forming lipid (for example, between 60 and 100% by weight or between 70 and 90% by weight). In some embodiments, the transport enhancer (s), if present, will comprise between 1 and 40% by weight of the vesicle-forming lipid (for example, between 1 and 20% by weight, between 1 and 25% by weight, between 1 and 30% by weight, between 1 and 35% by weight, between 2 and 25% by weight, between 2 and 30% by weight or between 2 and 35% by weight).
In certain embodiments, the vesicles may lack a transport enhancing molecule. In some embodiments, the vesicles may lack a "bile acid" such as colic acid and chenodeoxycholic acid, their conjugation products with glycine or taurine such as glycolic and taurocholic acid, derivatives that include deoxycholic and ursodeoxycholic acid, and salts of Each of these acids. In some embodiments, the vesicles may lack acyloxylated amino acids, such as acylcarnitines and salts thereof, and palmitoylcarnitines.
In some embodiments, the vesicles may include an ionic surfactant, for example, to cause the vesicles to carry a negative charge. For example, this can help stabilize the vesicles and provide effective dispersion. Without limitation, for this purpose acid materials such as higher alkanoic and alkenoic acids (e.g., palmetic acid, oleic acid) or other compounds containing acidic groups including phosphates such as dialkyl phosphates (e.g., di-diethyl phosphate, can be used) or phosphatidic acid or phosphatidylserine) and sulfate monosteres such as higher alkyl sulfates (eg, cetyl sulfate). The ionic surfactant (s), if present, will typically comprise between 1 and 25% by weight of the vesicle-forming lipid. For example, between 2 and 20% by weight or between 5 and 15% by weight. In some embodiments, the ionic surfactant (s), if present, will comprise between 1 and 50% by weight of the vesicle forming lipid (for example, between 1 and 35% by weight , between 5 and 40% by weight, between 10 and 40% by weight, between 15 and 40% by weight, between 20 and 40% by weight, or between 20 and 35% by weight weight).
In some embodiments, the vesicles may include an appropriate hydrophobic material of greater molecular mass that facilitates the formation of bilayers (such as a steroid, for example, a sterol such as cholesterol). In some embodiments, the presence of the steroid can help form the bilayer on which the physical properties of the vesicle depend. The steroid, if present, will typically comprise between 20 and 120% by weight of the vesicle-forming lipid. For example, between 25 and 90% by weight or between 35 and 75% by weight. In some embodiments, the steroid, if present, will comprise between 25 and 95% by weight, between 25 and 105% by weight, between 35 and 95% by weight, or between 35 and 35%. 105% by weight of vesicle-forming lipid.
In some embodiments, a lioprotector may be included in any solution or mixture before lyophilization. Exemplary lioprotectors include sucrose, trehalose, polyethylene glycol (PEG), dimethyl succinate buffer (DMS), bovine serum albumin (BSA), mannitol and dextran.
In some embodiments, the vesicles of the present disclosure are bilosomas that also include an ionic surfactant or a steroid. In some embodiments, bilosomas may include an ionic surfactant and a steroid.
In some embodiments, the vesicles of the present disclosure are nonionic surfactant vesicles (VTNI) that lack a transport enhancing molecule and also include an ionic surfactant or a steroid. In some embodiments, the vesicles may lack a "bile acid" such as colic acid and chenodeoxycholic acid, their conjugation products with glycine or taurine such as glycolic and taurocholic acid, derivatives that include deoxycholic and ursodeoxycholic acid, and salts of Each of these acids. In some embodiments, the vesicles may lack acyloxylated amino acids, such as acylcarnitines and salts thereof, and palmitoylcarnitines. In some embodiments, the VTNI may lack a molecule
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transport enhancer (for example, any of the molecules mentioned above) and include an ionic surfactant and a steroid.
Freeze drying
As discussed above and then, in some embodiments, the methods of the present disclosure include a lyophilization step (either of a molten lipid mixture or a formulation of vesicles containing antigen). Freeze drying is an established procedure to enhance the long-term stability of products. It is believed that the enhancement of physical and chemical stability is achieved by preventing degradation and hydrolysis. Lyophilization involves freezing the preparation in question and then reducing the surrounding pressure (and optionally heating the preparation) to allow the frozen solvent (s) to sublimate directly from the gas-solid phase (i.e. drying phase ). In certain embodiments, the drying phase is divided into primary and secondary drying phases.
The freezing phase can be carried out by placing the preparation in a container (for example, a flask, Eppendorf tube, etc.) and optionally rotating the container in a bath which is cooled by mechanical refrigeration (for example, using dry ice and methanol, liquid nitrogen, etc.). In some embodiments, the freezing stage involves cooling the preparation to a temperature that is below the eutectic point of the preparation. Since the eutectic point occurs at the lowest temperature at which the solid and liquid phase of the preparation can coexist, keeping the material at a temperature below this point ensures that sublimation, instead of evaporation, will occur in subsequent steps.
The drying phase (or the primary drying phase when two drying phases are used) involves reducing the pressure and optionally heating the preparation to a point where the solvent (s) can be sublimed. This drying phase typically removes the mayone from the solvent (s) of the preparation. It will be appreciated that the freezing and drying phases are not necessarily distinct phases but can be combined in any way. For example, in certain embodiments, the freezing and drying phases can overlap.
Optionally, a secondary drying phase can be used to remove the residual solvent (s) adsorbed during the freezing phase. Without wishing to commit to any theona, this phase involves raising the temperature to break any physicochemical interaction that has formed between the solvent molecules and the frozen preparation. Once the drying phase is complete, the ford can be broken with an inert gas (for example, nitrogen or helium) before the freeze dried product is optionally sealed.
Rehydration
As discussed above, in some embodiments, the methods of the present disclosure include a step of rehydrating a lyophilized lipid product to form vesicles containing antigen. This is achieved by mixing the lyophilized lipid product with an aqueous solution comprising an antigen. In some embodiments, this involves adding the aqueous solution to the lyophilized lipid product.
In some embodiments, the vesicles containing antigen contain at least about 10% of the antigen added in the rehydration stage. In some embodiments, the vesicles containing antigen contain at least about 20% of the antigen added in the rehydration stage. In some embodiments, the vesicles containing antigen contain at least about 30% of the antigen added in the rehydration stage. In some embodiments, the vesicles containing antigen contain at least about 40% of the antigen added in the rehydration stage. In some embodiments, the vesicles containing antigen contain at least about 50% of the antigen added in the rehydration stage. In some embodiments, the vesicles containing antigen contain at least about 60% of the antigen added in the rehydration stage. In some embodiments, the vesicles containing antigen contain at least about 70% of the antigen added in the rehydration stage. In some embodiments, the vesicles containing antigen contain at least about 80% of the antigen added in the rehydration stage. In some embodiments, the vesicles containing antigen contain at least about 90% of the antigen added in the rehydration stage.
In some embodiments, the aqueous solution includes a buffer. The buffer typically used depends on the nature of the antigen or antigens in the aqueous solution. For example, without limitation, a PCB buffer, a Na2HPO ^ NaH2PO4 buffer, a PBS buffer, a bicin buffer, a Tris buffer, an HEPES buffer, a MOPS buffer, etc. can be used. The PCB buffer is produced by mixing sodium propionate, sodium cacodylate, and bis-Tris propane in the 2: 1: 2 molar ratios. Varying the amount of Hcl added allows buffering over a pH range of 4-9. In some embodiments, a carbonate buffer can be used.
In some embodiments, a formulation of vesicles containing antigen prepared by any of the above-mentioned procedures can be lyophilized for future use, and subsequently rehydrated (for example, with water or an aqueous buffer) before use. In some embodiments, an adjuvant can be added during this rehydration step (for example, by inclusion in sterile water or aqueous buffer). In some embodiments, a formulation of vesicles containing antigen can be stored at -80 ° C
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before lyophilization. In some embodiments, a lyophilized formulation can be stored in a temperature range between -20 ° C and 10 ° C (for example, between -5 ° C and 10 ° C, between 0 ° C and 5 ° C or between 2 ° C and 8 ° C).
Vesicle size and processing
It will be appreciated that a vesicle formulation will typically include a mixture of vesicles with a range of sizes. It should be understood that the diameter values listed below correspond to the most frequent diameter within the mixture. In some embodiments,> 90% of the vesicles in a formulation will have a diameter that is within 50% of the most frequent value (for example, 1000 ± 500 nm). In some embodiments, the distribution may be narrower, for example,> 90% of the vesicles in a formulation may have a diameter that is within 40, 30, 20, 10 or 5% of the most frequent value. In some embodiments, sonication or ultrasonication can be used to facilitate vesicle formation and / or to modify the size of vesicle particle. In some embodiments, filtration, dialysis and / or centrifugation can be used to adjust the distribution of vesicle size.
In general, the vesicles produced in accordance with the procedures of the present disclosure may be of any size. In some embodiments, the formulations may include vesicles with a diameter in the range of about 150 nm to about 15 pm, for example, from about 800 nm to about 1.5 pm. In certain embodiments, the vesicles may have a diameter that is greater than 10 pm, for example, from about 15 pm to about 25 pm. In certain embodiments, the vesicles may have a diameter in the range of about 2 pm to about 10 pm, for example, from about 1 pm to about 4 pm. In certain embodiments, the vesicles may have a diameter that is less than 150 nm, for example, from about 50 nm to about 100 nm.
Antigens
In general, it should be understood that any antigen or antigens can be trapped using a method of the present disclosure. As previously analyzed, the antigen or antigens can be associated with vesicles in any way. In some embodiments, the antigen or antigens may be present in the aqueous core of the vesicles. However, depending on its hydrophobicity, an antigen may also be partially or completely associated with a bilayer. In general, it should also be understood that, in some embodiments, a vesicle formulation may include amounts of one or more antigens that are not associated with vesicles.
In some embodiments, the procedures of the present disclosure may be used to trap one or more of the antigens included in a vaccine. Table 1 is a non-limiting list of suitable vaccines.
Table 1
 Vaccine  Disease
 BioThrax®  Carbuncle
 DTaP (Daptacel®, Infanrix®, Tripedia®)  Diphtheria
 Td (Decavac®)  Diphtheria
 DT, TT  Diphtheria
 Tdap (Boostrix®, Adacel®)  Diphtheria
 DTaP / IPV / HepB (Pediarix®)  Diphtheria
 DTaP / Hib (TriHIBit®)  Diphtheria
 HepA (Havrix®, Vaqta®)  Hepatitis A
 HepA / HepB (Twinrix®)  Hepatitis A
 HepB (Engerix-B®, Recombivax HB®)  Hepatitis B
 HepB / Hib (Comvax)  Hepatitis B
 DTaP / IPV / HepB (Pediarix),  Hepatitis B
 HepA / HepB (Twinrix®)  Hepatitis B
 Hib (ActHIB®, HibTITER®, PedvaxHIB®)  HIB
 HepB / Hib (Comvax®)  HIB
 DTaP / Hib (TriHIBit®)  HIB
 PVH (Gardasil®)  PVH
 Flu (Fluarix®, Fluvirin®, Fluzone®, Flulaval®, FluMist®)  Seasonal flu
(continuation)
 Vaccine  Disease
 Influenza (Afluria®)  Seasonal flu
 Influenza (Agriflu®)  Seasonal flu
 Flu (Begrivac®)  Seasonal flu
 Influenza (Enzira®)  Seasonal flu
 Flu (Fluad®)  Seasonal flu
 Flu (Fluvax®)  Seasonal flu
 Flu (Fluviral, Fluviral S / F®)  Seasonal flu
 Influenza (Crippol®)  Seasonal flu
 Flu (Inflexal, Inflexal S, Inflexal V®)  Seasonal flu
 Influenza (Influvac®)  Seasonal flu
 Flu (Mastaflu®)  Seasonal flu
 Flu (Mutagrip®)  Seasonal flu
 Influenza (Optaflu®)  Seasonal flu
 Flu (Vaxigrip®)  Seasonal flu
 H1N1 pandemic flu (Arepanrix®)  H1N1 pandemic flu
 Pandemic H1N1 flu (Calvapan®)  H1N1 pandemic flu
 Pandemic H1N1 flu (Focetria®)  H1N1 pandemic flu
 Pandemic H1N1 (Influenza A (H1N1) 2009 Monovalent Vaccine®)  H1N1 pandemic flu
 H1N1 pandemic flu (Pandemrix®)  H1N1 pandemic flu
 JE (JE-Vax®)  Japanese encephalitis
 Lyme disease (LYMErix®)  Lyme's desease
 Measles (Attenuvax®)  Measles
 MMR (M-M-R II®)  Measles
 MMRV (ProQuad®)  Measles
 Mening conjugate (Menactra®)  Meningococcal
 Polysaccharic mening. (Menomune®)  Meningococcal
 Mumps (Mumpsvax®)  Mumps
 MMR (M-M-R II®)  Mumps
 MMRV (ProQuad®)  Mumps
 DTaP (Daptacel®, Infanrix®, Tripedia®)  Whooping cough
 Tdap (Boostrix®)  Whooping cough
 DTaP / IPV / HepB (Pediarix®)  Whooping cough
 DTaP / Hib (TriHIBit®)  Whooping cough
 Conjugate pneumo. (Prevnar®)  Pneumococcal
 Pneumo polysaccharide. (Pncumovax 23®)  Pneumococcal
 Polio (Ipol®)  Poliomyelitis
 DTaP / IPV / HepB (Pediarix®)  Poliomyelitis
 Rabies (BioRab®, Imovax Rabies®, RabAvert®)  Rage
 Rotavirus (RotaTeq®)  Rotavirus
 Rubella (Meruvax II®)  Rubella
 MMR (M-M-R II®)  Rubella
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 Vaccine  Disease
 MMRV (ProQuad®)  Rubella
 Zoster (zostavax®)  Zoster
 Vaccinia (Dryvax®)  Smallpox and soulic smallpox
 DTaP (Daptacel®, Infanrix®, Tripedia®)  Tetanus
 Td (Decavac®)  Tetanus
 DT, TT  Tetanus
 Tdap (Boostrix®)  Tetanus
 DTaP / IPV / HepB (Pediarix®)  Tetanus
 DTaP / Hib (TriHIBit®)  Tetanus
 BCG  Tuberculosis
 Anti-typhoid (Typhim Vi®)  Typhoid
 Oral typhoid (Vivotif Berna®)  Typhoid
 Chickenpox (Varivax®)  Chickenpox (Varicella)
 MMRV (ProQuad®)  Chickenpox (Varicella)
 Yellow Fever (YF-Vax®)  Yellow fever
In the following sections some exemplary antigens that can be used are analyzed.
Hepatitis A
Hepatitis A is a severe hepatopathy caused by the hepatitis A virus (HAV). The virus is found in the feces of people with hepatitis A. As shown in Table 1, several inactivated hepatitis A vaccines are currently authorized. For example, Havrix® is manufactured by GlaxoSmithKline Biologicals. U.S. Patent UU. No. 6,180,110 describes the attenuated HAV strain (HAV 4380) used in Havrix® that was originally derived from HAV strain HM175 (U.S. Patent No. 4,894,228). Havrix® contains a sterile suspension of HAV inactivated with formalin. The activity of the vmco antigen is referenced to a pattern using an ELISA and is expressed in terms of ELISA units (U). Each adult dose of 1 ml of the vaccine consists of 1440 U of vmco antigen, adsorbed in 0.5 mg of aluminum as aluminum hydroxide (alum). Havrix® (as with all other authorized hepatitis A vaccines) is supplied as a sterile suspension for intramuscular administration (i.m.). Although a dose of Havrix® provides at least short-term protection, a second recall dose is now recommended after six to twelve months to ensure long-term protection.
Another example of an inactivated hepatitis A vaccine, AIMMUGEN® has been authorized and marketed in Japan since 1994 by Kaketsuken. AIMMUGEN® contains a sterile suspension of HAV inactivated with formaldetndo. The recommended adult dose is 0.5 | jg i.m. at 0, 1 and 6 months.
As used herein, the term "HAV antigen" refers to any antigen that can stimulate a neutralizing antibody for HAV in humans. The HAV antigen may comprise live attenuated virus particles or inactivated attenuated virus particles or it may be, for example, a HAV capside or a HAV protease virus, which can be conveniently obtained by recombinant DNA technology.
In one aspect, the present disclosure provides methods for preparing immunogenic formulations that include an inactivated or attenuated hepatitis A virus (also called "hepatitis A vmco antigen" or "vmco antigen" herein). It will be appreciated that the methods can be used to prepare an inactivated hepatitis A virus. In general, these procedures will involve spreading a hepatitis A virus in a host cell, lysing the host cell to release the virus, isolating and then inactivating the vmco antigen. After removal of the cell culture medium, the cells are lysed to form a suspension. This suspension is purified through ultrafiltration and gel permeation chromatography procedures. Thereafter, the lysate treated with formalin is treated to ensure vmca inactivation (for example, see Andre et al., Prog. Med. Virol. 37: 72-95, 1990).
In the preparation of AIMMUGEN®, the hepatitis A virus strain KRM0003 (established from a natural HAV, which has been isolated from the feces of a patient with hepatitis A) is propagated in GL37 cells (a cell strain established for the production of vaccines from an original African green monkey kidney cell strain). GL37 cells are inoculated with VHA strain KRM0003 and the vmco antigen is collected, thoroughly purified and formally inactivated.
Another example of an inactivated hepatitis A virus that is commercially available but not an authorized vaccine is the hepatitis A antigen (HAV-ag) from Meridian Life Sciences. Like Havrix®, Meridian VHA-ag also derives from the hepatitis A virus strain HM175 but spreads in FRhK-4 cells (macaque fetal kidney of the
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India). After removal of the cell culture medium, the cells are lysed to form a suspension and the suspension is partially purified by gradient centrifugation and quenched by formalin treatment.
It will be appreciated that any strain of hepatitis A virus can be used, for example, without limitation any of the following strains that have been described in the art (and other non-human variants):
human hepatitis A virus Hu / Arizona / HAS-15/1979
Human hepatitis A virus Hu / Australia / HM175 / 1976
human hepatitis A virus Hu / China / H2 / 1982
human hepatitis A virus Hu / Costa Rica / CR326 / 1960
human hepatitis A virus Hu / France / CF-53/1979
human hepatitis A virus Hu / Georgia / GA76 / 1976
Human hepatitis A virus Hu / Germany / GBM / 1976
human hepatitis A virus Hu / Japon / HAJ85-1 / 1985
Human hepatitis A virus Hu / Los Angelos / LA / 1975
Human hepatitis A virus Hu / North Africa / MBB / 1978
human hepatitis A virus Hu / Norway / NOR-21/1998
Human hepatitis A virus Hu / Sierra Leone / SLF88 / 1988
MSM1 human hepatitis A virus
human hepatitis A virus Shanghai / LCDC-1/1984
In addition, although formalin and formaldelddo are commonly used to inactivate authorized inactivated hepatitis A vaccines, it should be understood that other techniques may be used, for example, chlorine treatment, exposure to high temperatures (the vmco antigen is inactivated above 85 ° C / 185 ° F), etc.
In certain embodiments, it may be advantageous to add additional steps to the traditional procedure for preparing an inactivated hepatitis A virus. For example, the US patent. UU. No. 6,991,929 describes including a stage of protease treatment (eg trypsin) after the virus has spread. It was discovered that this stage improves the removal of the host cell material and provides a purer vmca preparation.
Although all hepatitis A vaccines currently authorized include inactivated vmcos antigens, alternative vaccines including attenuated vmco antigen have also been described in the literature. In certain embodiments, an immunogenic formulation may comprise an attenuated viral antigen of this type. As is well known in the art, the advantage of an attenuated vaccine lies in the potential for greater immunogenicity resulting from its ability to replicate in vivo without causing a complete infection.
A procedure that has been used in the art to prepare attenuated hepatitis A viruses is the vmca adaptation that involves passing a vmca strain in series across multiple cell cultures. Over time, the strain mutates and then attenuated strains can be identified. In certain embodiments, the virus can be passed through different cell cultures. For example, researchers have generated attenuated hepatitis A virus by passing strain CR326 sixteen times in human diploid lung cell (MRCS) cultures (see Preboste et al., J. Med. Virol. 20: 165-175, 2005 ). A slightly more virulent strain was obtained by passing the same strain fifteen times in macaque kidney fetal cell cultures of India (FRhK6) plus eight times in MRCS cell cultures. An alternative attenuated hepatitis A vaccine has also been described which was prepared in this way from strain H2 (see European Patent No. 0413637 and Mao et al., Vaccine 15: 944-947, 1997).
In certain embodiments, it may be advantageous to perform one or more of the cell culture steps at a reduced temperature. For example, European Patent No. 0413637 describes including one or more stages of inoculation in which the temperature is reduced (for example, to 32-34 ° C instead of 35-36 ° C).
U.S. Patent UU. No. 6,180,110 describes an attenuated hepatitis A virus (HAV 4380) that grows in MRC-5 cells. The researchers identified mutations in HAV 4380 that appear to be associated with attenuation, comparing their genome with the genome of a more virulent strain. This allowed them to design mutant HAV strains with optimal characteristics for a candidate attenuated hepatitis A vaccine. It will be appreciated that this approach could be applied to any known attenuated hepatitis A virus and could be used to genetically modify the variants without the need for vmca adaptation.
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Hepatitis B
Hepatitis B virus (HBV) causes both acute and chronic infections. The broad spectrum of HBV infection varies from asymptomatic to acute symptomatic hepatitis; from a carrier state of hepatitis B surface antigen (HBsAg) inactive to liver cirrhosis and its complications during the chronic phase (Fattovich, J. Hepatol. 39: s50-58, 2003). HBV is transmitted in parenteral or mucosal exposure to HBsAg positive body fluids usually from people infected with HBV (Hilleman, Vaccine 21: 4626-4649, 2003).
Currently, there are two commercial vaccines used to prevent HBV infection, both of which are manufactured using recombinant technology. For example, Engerix-B ™ is a non-infectious recombinant DNA hepatitis B vaccine developed by GlaxoSmithKline Biologicals. It contains the purified HBV surface antigen obtained by culturing genetically modified Saccharomyces cerevisiae cells, which carry the HBV surface antigen gene.
As used herein, the term "hepatitis B surface antigen" or "HBsAg" refers to any HBsAg antigen or fragment thereof that exhibits the antigenicity of HBV surface antigen in humans.
Engerix-B ™ and other authorized hepatitis B vaccines, which are administered parenterally, have been successful in inducing a systemic immune response to HBV. However, antibodies produced as part of the systemic immune response cannot provide protection at the level of the mucosa, which is the main site of entry for the mayone of infectious agents including HBV. Therefore, there is a need in the art to obtain a vaccine for hepatitis B administered orally.
In one aspect, the present disclosure provides methods for preparing immunogenic formulations that include a hepatitis B virus surface antigen or a fragment thereof that exhibits the antigenicity of HBsAg. All known hepatitis B vaccines include a recombinant HBsAg. It should be understood that any one of these hepatitis B vaccines authorized as a source of antigen can be used in a process of the present disclosure to produce an immunogenic formulation.
In general, any procedure can be used to prepare a hepatitis B surface antigen. The preparation of HBsAg is well documented (for example, see Harford et al., Develop. Biol. Standard 54: 125, 1983 and Gregg et al. , Biotechnology 5: 479, 1987, among others). In general, recombinant aDn technology procedures that involve culturing genetically modified cells that carry the HBV surface antigen gene can be used. The expressed surface antigen is then purified and normally formulated as a suspension of the surface antigen adsorbed on aluminum hydroxide (for example, see Valenzuela et al., Proc. Natl. Acad. Sci. USA 80: 1-5, 1983 and McAleer et al., Nature 307: 178-180, 1984).
Flu
The flu is a common infectious disease of the respiratory system associated with the Ortomyxoviridae virus family. Influenza A and B are the two types of influenza viruses that cause a human epidemic. Influenza A viruses are also categorized into subtypes based on two surface antigens: hemagglutinin (HA) and neuraminidase (N). Gripo B viruses are not categorized into subtypes. Vaccination is recognized as the only most effective way to prevent or mitigate the flu for those who are at high risk of serious illness from influenza and related complications. The inoculation of antigen prepared from the inactivated influenza virus stimulates the production of specific antibodies. In general, protection was provided only against the virus strains from which the vaccine is prepared or from closely related strains.
Influenza vaccines, of all types, are usually trivalent vaccines. In general, they contain antigens derived from two strains of the influenza A virus and one strain of the influenza B virus. The influenza virus strains to be incorporated into influenza vaccines each season are determined by the World Organization of Health (WHO) in collaboration with national health administrations and vaccine manufacturers. It will be appreciated that any influenza virus strain may be used in accordance with the present disclosure, and that influenza virus strains will differ from year to year based on WHO recommendations.
Monovalent vaccines are also included, which can be useful, for example, in a pandemic situation. A monovalent pandemic influenza vaccine will most likely contain the influenza antigen of a single strain A. In some embodiments, influenza antigens are derived from pandemic influenza strains. For example, in some embodiments, influenza antigens are viral antigens of influenza A (H1N1 of porcine origin).
Worldwide, three types of inactivated vaccines are predominantly used to protect against the flu: complete virus vaccines, fractional virus vaccines that contain external and internal virus components, and subunit vaccines composed of only the external components of the virus ( hemagglutinin and neuraminidase). Without wishing to be limited by any theona, it is believed that the higher purity of subunit vaccines should make them less reactogenic and better tolerated. Conversely, it is believed that complete virus and fractionated virus vaccines contain more epftopos and are therefore more immunogenic.
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In some embodiments, influenza antigens are based on subunit vaccines. In general, subunit vaccines contain only the parts of the influenza virus that are needed for effective vaccination (for example, that cause a protective immune response). In some embodiments, subunit flu antigens are prepared from virus particles (eg, purification of particular virus components). In some embodiments, subunit influenza antigens are prepared by recombinant procedures (eg, cell culture expression). For example, the US patent. UU. No. 5,858,368 describes methods of preparing a recombinant influenza vaccine using recombinant DNA technology. The resulting trivalent influenza vaccine is based on a mixture of recombinant hemagglutinin antigens cloned from influenza viruses that have epidemic potential. Recombinant hemagglutinin antigens are non-cleaved, full-length glycoprotems, produced from baculovirus expression vectors in insect cells cultured and purified under conditions without denaturation. In some embodiments, subunit flu antigens are generated by synthetic procedures (eg, peptide synthesis). Subunit vaccines may contain purified surface antigens, hemagglutinin antigens and neuraminidase antigens prepared from selected strains determined by WHO. Without wishing to commit to any theona, it is believed that surface antigens, hemagglutinin antigens and neuraminidase antigens play a significant role in causing the production of virus neutralizing antibodies after vaccination.
In some embodiments, influenza antigens are fractionated virus antigens. Vaccines prepared using fractionated virus antigens typically contain a higher concentration of the more immunogenic portions of the virus (eg, hemagglutinin and neuraminidase), while the concentration of the less immunogenic viral proteins as well as the non-viral proteins present in eggs (used to produce the virus) or foreign agents in (for example, avian leukosis virus, other microorganisms and cell debris). In general, fractionated virus antigens are prepared by a physical procedure that involves breaking the vmca formula, in general with an organic detergent or solvent (eg, Triton X-100), and separating or purifying the vmcas proteins in different grades, such as by centrifugation in a sucrose gradient or passage of allantoic fluid in a chromatographic column. In some embodiments, the rupture and separation of vmcas particles is followed by dialysis or ultrafiltration. Fractional virus antigens normally contain the mayone of or all the structural protections of the virus but not necessarily in the same proportions that occur in the entire virus. Vmco fractionation procedures as well as suitable fractionation agents are known in the art (see, for example, U.S. Patent Publication No. 20090155309). In some embodiments, the final antigen concentration (for example, hemagglutinin and / or neuraminidase antigens) of the fractionated virus antigen is standardized using methods known in the art (eg, ELIsA).
In some embodiments, influenza antigens are complete virus antigens. It is believed that in non-sensitized individuals, vaccines prepared with complete virus antigens may be more immunogenic and induce a greater response of protective antibodies to a lower dose of antigens than other formulations (eg, fractionated virus or subunit antigens). However, influenza vaccines that include complete virus antigens can produce more side effects than other formulations.
The influenza virus antigens present in the immunogenic formulations described herein may be infectious, inactivated or attenuated.
In certain embodiments, an immunogenic formulation may comprise an inactivated vmco antigen. It will be appreciated that any procedure can be used to prepare an inactivated influenza virus antigen. WO 09/029695 describes exemplary procedures for producing a complete inactivated virus vaccine. In general, these procedures will involve spreading a flu virus in a host cell, optionally lysing the host cell to release the virus, then isolate and then inactivate the vmco antigen. A chemical treatment of the virus (for example, formalin, formaldehyde, among others) is commonly used to inactivate the virus for vaccine formulation. However, it should be understood that other techniques may be used, for example, chlorine treatment, exposure to high temperatures, etc. In these treatments, the outer shell of the virion is typically left intact while the replication function is disturbed. Non-replicating virus vaccines preferably contain more antigen than live virus vaccines that can be replicated in the host.
In certain embodiments, an immunogenic formulation may comprise a vmco attenuated antigen. As is well known in the art, an advantage of a vaccine prepared with an attenuated vmco antigen lies in the potential for increased immunogenicity resulting from its ability to replicate in vivo without causing a complete infection. Live virus vaccines that are prepared from attenuated strains preferably lack pathogenicity but can still be replicated in the host. A procedure that has been used in the art to prepare influenza vmcos antigens is the vmca adaptation that involves passing a vmca strain in series through multiple cell cultures. Over time, the strain mutates and then attenuated strains can be identified. In certain embodiments, the virus can be passed through different cell cultures. In certain embodiments, it may be advantageous to perform one or more of the cell culture steps at a reduced temperature.
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Currently, several influenza vaccines are authorized (see Table 1). For example, Fluzone®, which is an inactivated influenza vaccine with fractionated cells, is developed and manufactured by Sanofi Pasteur, Inc. and can be used in accordance with the present disclosure. Fluzone® contains a sterile suspension prepared from influenza viruses propagated in embryonated chicken eggs. Fluids containing viruses are collected and inactivated with formaldehyde. The influenza virus is concentrated and purified in a linear sucrose density gradient solution using a continuous flow centrifuge. Next, the virus is chemically broken using a non-ionic surfactant, octoxynol-9, (Triton® X-100) producing a fractionated vmco antigen. Then the fractionated virus is further purified by chemical means and suspended in isotonic sodium chloride solution buffered with sodium phosphate. Next, the Fluzone® vaccine is standardized according to the requirements for the flu season and is formulated to contain 45 | jg of hemagglutinin (HA) per 0.5 ml of dose, in the recommended ratio of 15 jg of HA each one, representative of the three prototype strains (for example, 2007-2008 vaccine prepared with strains A / Solomon Islands / 3/2006 (H1N1), A / Wisconsin / 67/2005 (H3N2) and B / Malaysia / 2506/2004) . The Fluzone® vaccine is formulated for intramuscular injection.
Another example of an authorized influenza vaccine that can be used in accordance with the present disclosure is Vaxigrip®, which is an inactivated influenza vaccine with fractionated cells also developed and manufactured by Sanofi Pasteur, Inc. Vaxigrip® is prepared in a similar manner to the indicated procedure. previously for Fluzone® and similarly formulated for intramuscular injection.
Yet another example of an authorized influenza vaccine that can be used in accordance with the present disclosure is Flumist®. Flumist® is a trivalent vaccine with live attenuated viruses for intranasal spray administration. Flu virus strains in Flumist® have three genetic mutations that result in temperature-restricted growth and an attenuated phenotype. The cumulative effect of antigenic properties and genetically modified influenza viruses is that they can replicate in the nasopharynx and induce protective immunity. To produce Flumist®, eggs without specific pathogen (SPF) are inoculated with each of the appropriate vmcas strains and incubated to allow vmca replication of the vaccine. The allantoic liquid is collected from these, mixed and then clarified by filtration. The virus is concentrated by ultracentrifugation and diluted with stabilization buffer to obtain the final concentrations of potassium phosphate and sucrose. Next, the collected viruses are sterile filtered to produce the monovalent masses. Subsequently, the monovalent masses of the three strains are mixed and diluted as required to achieve the desired potency with stabilizing buffers to produce the trivalent bulk vaccine. The bulk vaccine is then loaded directly into individual sprays for nasal administration. Each pre-filled refrigerated Flumist® sprayer contains a single dose of 0.2 ml. Each 0.2 ml dose contains 106.5 "7.5 FFU of live attenuated influenza virus regroups of each of the three appropriate vmcas strains.
As described above, several flu vaccines are currently authorized. It should be understood that any one or combination of these authorized influenza vaccines can be combined with a vesicle as described herein to produce an immunogenic formulation. For example, commercial Fluzone® and / or Vaxigrip® can be combined in this way to produce an active immunogenic formulation. In some embodiments, authorized flu vaccines are first purified (for example, to remove alum adjuvant or other reagents in the vaccine). In some embodiments, authorized influenza vaccines are not purified prior to formulation with a vesicle as described herein.
PCT Patent Application No. PCT / US09 / 47911 describes some other exemplary influenza antigens that may be used in the procedures and formulations of the present disclosure. Exemplary influenza antigens have also been described in U.S. Pat. UU. No. 7,527,800; 7,537,768; 7,514,086; 7,510,719; 7,494,659; 7,468,259; 7,399,840; 7,361,352; 7,316,813; 7,262,045; 7,244,435; 7,192,595; 7,052,701; 6,861,244; 6,743,900; 6,740,325; 6,635,246; 6,605,457; 6,534,065; 6,372,223; 6,344,354; 6,287,570; 6,136,606; 5,962,298; 5,948,410; and 5,919,480.
Other viruses
Hepatitis C virus (HCV) is now recognized as the leading cause of hepatitis neither A, nor B (NANB) associated with transfusion. HCV is a single-stranded positive sense RNA virus with similarities to flaviviruses and pestiviruses (Miller et al., Proc. Natl. Acad. Sci. 87: 2057, 1991 and Weiner et al., Virology 180: 842, 1990) . U.S. Patents UU. No. 7,348,011; 6,831,169; 6,538,123 and 6,235,888 describe all exemplary HCV antigens that can be used in a vaccine.
The human immunodeficiency retrovirus (HIV) is responsible for AIDS (acquired immunodeficiency syndrome), a disease in which the body's immune system fails to leave it vulnerable to opportunistic infections. U.S. Patents UU. No. 7,067,134; 7,063,849; 6,787,351; 6,706,859; 6,692,955; 6,653,130; 6,649,410; 6,541,003; 6,503,753; 6,500,623; 6,383,806; 6,090,392; 5,861,243; 5,817,318; and 4,983,387 describe all exemplary HIV antigens that can be used in a vaccine. Various HIV antigens are also disclosed in the US patent application publication. UU. No. 20090117141 and 20090081254.
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In certain embodiments, an immunogenic formulation that is prepared in accordance with the methods of the present disclosure may comprise an antigen that is heat labile. As used herein, the terms "thermolabile antigens" refer to an antigen that loses antigenic integrity when exposed to certain elevated temperatures. In some embodiments, the exposure of a thermo-labile antigen at elevated temperatures destroys more than 20% of the antigenic integrity of the antigen (for example, more than 30%, more than 40%, more than 50% or more ) measured in an antigen integrity test (for example, an ELISA) compared to the unhandled antigen. In certain embodiments, a thermo-labile antigen loses antigenic integrity at temperatures above 30 ° C (for example, above 35 ° C, above 40 ° C, above 45 ° C, or above 50 ° C). In some embodiments, the storage of a thermo-labile antigen at one of these elevated temperatures for more than 3 minutes (e.g., 5 minutes, 10 minutes, 15 minutes or more) destroys more than 20% of the antigenic integrity of the antigen. (for example, more than 30%, more than 40%, more than 50% or more) measured in an antigenic integrity test (for example, an ELISA) compared to the unhandled antigen. As discussed above, the methods of the present disclosure are particularly beneficial for thermolabile antigens because they can use a lower temperature of antigen solution, which allows for better preservation of antigenic integrity.
It should be understood that the present disclosure is not limited to antigens and that, in general, the procedures can be used to trap any substance whether antigenic or non-antigenic. Therefore, in some embodiments, the methods of the present disclosure may be used to trap one or more polypeptides, polynucleotides or polysaccharides that may or may not be antigenic. Specific classes of substances include, but are not limited to, adjuvants, enzymes, receptors, neurotransmitters, hormones, cytokines, cellular response modifiers such as growth factors and chemotactic factors, antibodies, haptens, toxins, interferons, ribozymes, agents antisense, plasmids, DNA and RNA. In some embodiments, the polypeptide may be an antibody or antibody fragment, for example, a humanized antibody. In some embodiments, these substances are thermolabile as they become degrading under the conditions referred to above in the context of the antigens.
Adjuvants
In certain embodiments, the methods of the present disclosure may also include a step of adding one or more adjuvants to a vesicle formulation. As is well known in the art, adjuvants are agents that enhance immune responses. Adjuvants are well known in the art (for example, see "Vaccine Design: The Subunit and Adjuvant Approach", Pharmaceutical Biotechnology, Volume 6, Eds. Powell and Newman, Plenum Press, New York and London, 1995). In some embodiments, an adjuvant can be added once the vesicle formulation (with trapped antigen) has been prepared. In some embodiments, an adjuvant can be added during the process of preparing vesicle formulations (for example, together with vesicle-forming lipids or other vesicle components, together with the antigen or with a dedicated stage).
In certain embodiments, an adjuvant is added before the antigen is added. In some embodiments, the adjuvant is fused together with vesicle-forming lipids. In some embodiments, a TLR-4 adjuvant (described below) is fused together with vesicle-forming lipids. In certain embodiments, an adjuvant is added after an antigen is added. In some embodiments, the adjuvant is added together with a lioprotector after an antigen is added. In some embodiments, a TLR-3 adjuvant (described below) is added together with a lioprotector after an antigen is added. In some embodiments, the lioprotector is sucrose.
Exemplary adjuvants include Freund's complete adjuvant (CFA), incomplete Freund's adjuvant (IFA), squalene, squalane and alum (aluminum hydroxide), which are materials well known in the art, and are commercially available from various sources. In certain embodiments, aluminum or calcium salts (for example, phosphate or hydroxide salts) can be used as adjuvants. Alum (aluminum hydroxide) has been used in many existing vaccines. Typically, about 40 to about 700 µg of aluminum is included per dose when i.m. is administered. For example, Havrix® includes 500 jg of aluminum per dose.
In various embodiments, oil in water emulsions or water in oil emulsions can also be used as adjuvants. For example, the oil phase may include squalene or squalene and a surfactant. In various embodiments, non-ionic surfactants such as the mono- and di-esters of C12-C24 fatty acids of sorbitan and manide can be used. Preferably, the oil phase comprises about 0.2 to about 15% by weight of the immunogenic formulation (for example, about 0.2 to 1%). PCT Publication No. WO 95/17210 describes exemplary emulsions.
The adjuvant named QS21 is an immunologically active saponin fraction that has adjuvant activity derived from the bark of the South American tree Quillaja Saponaria Molina, and the production procedures are disclosed in US Pat. UU. No. 5,057,540. Synthetic and semi-synthetic derivatives of Quillaja saponaria Molina saponins are also useful, such as those described in US Pat. UU. No. 5,977,081 and 6,080,725.
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TLRs are a family of homologous proteins to the Drosophila Toll receptor, which recognize molecular patterns associated with pathogens and, therefore, help the body to distinguish between own and non-own molecules. Common substances in pathogens vmcos are recognized by TLRs as molecular patterns associated with pathogens. For example, TLR-3 recognizes patterns in double-stranded RNA, TLR-4 recognizes patterns in lipopolysaccharides while TLR-7/8 recognizes patterns that contain adenosine in viral and bacterial RNA and DNA. When a TLR is triggered by said pattern recognition, a series of signaling events occurs that results in inflammation and activation of innate and adaptive immune responses. Several synthetic ligands containing the molecular patterns recognized by several TLRs are being developed as adjuvants and can be included in an immunogenic formulation as described herein.
For example, poly (riboinosmic acid: ribocytidyl) or poly (I: C) (available from InvivoGen of San Diego, CA) is a synthetic analogue of double stranded RNA (a molecular pattern associated with vmca infection) and an exemplary adjuvant that is a agonist for TLR-3 (for example, see Field et al., Proc. Natl. Acad. Sci. USA 58: 1004 (1967) and Levy et al., Proc. Natl. Acad. Sci. USA 62: 357 (1969 )). In some embodiments, poly (I: C) can be combined with other agents to improve stability (for example, reducing degradation through the activity of RNAse). For example, the US patent. UU. No. 3,952,097; 4,024,241 and 4,349,538 describe poly (I: C) complexes with poly-L-lysine. It has also been shown that the addition of poly-arginine to poly (I: C) reduces degradation through the activity of RNAse. Poly (IC: LC) is a synthetic double-stranded poly (I: C) stabilized with poly-L-lysine-carboxymethyl cellulose. U.S. Patent Publication UU. No. 20090041809 describes double-stranded nucleic acids with one or more of a blocked nucleic acid (ANB) nucleoside that can act as TLR-3 agonists. Those skilled in the art will be able to identify other adjuvants of suitable TLR-3 agonists.
The attenuated lipid A derivatives (DLA) such as monophosphoryl lipid A (MPL) and 3-desacyl monophosphoryl lipid A (3D-MPL) are exemplary adjuvants that are agonists for TLR-4. DLAs are molecules similar to lipid A that have been altered or constructed so that the molecule exhibits minor or different adverse effects from lipid A. These adverse effects include pyrogenicity, toxicity and local Schwarzman reactivity as assessed in the test of 50% lethal dose in chicken embryo (CELD50). MPL and 3D-MPL are described in U.S. Pat. UU. No. 4,436,727 and 4,912,094, respectively. MPL was originally derived from lipid A, a component of enterobacterial lipopolysaccharides (LPS), a potent but very toxic immune system modulator. 3D-MPL differs from MPL in that the acyl residue that is ester-linked to the reducing terminal glucosamine in position 3 has been selectively removed. It will be appreciated that MPL and 3D-MPL may include a mixture of various fatty acid substitution patterns, that is, heptaacyl, hexaacyl, pentaacyl, etc., with variable fatty acid chain length. Therefore, various forms of MPL and 3D-MPL, including mixtures thereof, are encompassed herein.
In some embodiments, these DLAs can be combined with trehalose dimicolate (TDM) and cell wall skeleton (CWS), for example, in a 2% squalene / Tween ™ 80 emulsion (for example, see patent GB No. 2122204). MPl is available from Avanti Polar Lipids, Inc. of Alabaster, aL as PhAD (phosphorylated hexaacyl disaccharide). Those skilled in the art will be able to identify other adjuvants of suitable TLR-4 agonists. For example, other lipopolysaccharides have been described in pCt Publication No. WO 98/01139; U.S. Patent UU. No. 6,005,099 and EP Patent No. 729473.
II. Vesicle Formulations
In another aspect, the present disclosure provides formula formulations containing antigen prepared using these procedures. In some embodiments, antigen-containing vesicle formulations have antigen entrapment levels that are higher than those obtainable using prior art procedures. In some embodiments, antigen-containing vesicle formulations have antigenic activity levels (i.e., antigenicity and / or immunogenicity) that are greater than those obtainable using prior art methods.
Immunogenic vesmula formulations are useful for treating many diseases in humans including adults and children. In general, however, they can be used with any animal. In certain embodiments, the procedures herein can be used for veterinary applications, for example, canine and feline applications. If desired, the procedures herein can also be used with farm animals, such as sheep, avian, bovine, swine and equine breeds.
In general, the immunogenic vesicle formulations described herein will be administered in such amounts and for a time such that it is necessary or sufficient to induce an immune response. Dosage regimens may consist of a single dose or a plurality of doses over a period of time. The exact amount of antigen to be administered may vary from one patient to another and may depend on several factors. Therefore, it will be appreciated that, in general, the precise dose used will be that determined by the prescribing physician and will depend not only on the weight of the patient and the route of administration, but also on the dosage frequency, the age of the patient and the severity of the symptoms and / or the risk of infection. In certain embodiments, the dose of antigen in an immunogenic formulation may vary from about 5 | jg to about 5 mg, for example, from about 100 jg to about 750 jg. Smaller doses of antigen can be
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sufficient when sublingual or oral administration is used, or in the presence of adjuvant. Higher doses may be more useful when administered orally, especially in the absence of adjuvants.
In general, the formulations can be administered to a patient by any route. In particular, the results in the examples demonstrate that the immunogenic formulations described herein can induce a protective response even when administered orally. It will be appreciated that the oral route is particularly desirable in view of the advantages of oral administration over any form of injection (ie, compliance, mass distribution, etc.). It will also be appreciated that the results are unexpected in view of the fact that the mayone of the vaccines (including all known hepatitis A vaccines) have so far been administered parenterally.
Therefore, in certain embodiments, the immunogenic formulations can be administered orally (including orally, sublingually, and by gastric lavage or other artificial feeding means). Such oral administration can be achieved using solid or liquid formulations, for example, in the form of tablets, capsules, multiparticles, gels, films, ovules, elixirs, solutions, suspensions, etc. In certain embodiments, when a liquid formulation is used, the formulation can be administered in conjunction with a basic formulation (eg, a bicarbonate solution) to neutralize the pH of the stomach. In certain embodiments, the basic formulation can be administered before and / or after the immunogenic formulation. In certain embodiments, the basic formulation can be combined with the immunogenic formulation before administration or taken at the same time as the immunogenic formulation.
Although oral administration is of special interest, it will be appreciated that, in certain embodiments, an immunogenic formulation can also be formulated for parenteral administration, for example, by injection. In such embodiments, the administration can be, for example, intravenous, intramuscular, intradermal or subcutaneous, or by means of infusion or needleless injection techniques. By such parenteral administration, immunogenic formulations can be prepared and maintained in conventional lyophilized formulations and reconstituted before administration with a pharmaceutically acceptable saline solution, such as a 0.9% saline solution. The pH of the injectable formulation can be adjusted, as is known in the art, with a pharmaceutically acceptable acid, such as methanesulfonic acid. Other acceptable vehicles and solvents that may be employed include the Ringer solution and USP. In addition, sterile fixed oils are conventionally used as solvent or suspension medium. For this purpose any soft fixed oil including synthetic mono or diglycerides can be used. In addition, fatty acids such as oleic acid are used in the preparation of injectable substances. Injectable formulations can be sterilized, for example, by filtration through a bacterial retention filter, or by incorporating sterilizing agents in the form of solid sterile formulations that can be dissolved or dispersed in sterile water or other sterile injectable medium before use.
Immunogenic formulations can also be administered intranasally or by inhalation and are conveniently administered in the form of an aerosol spray or dry powder inhaler presentation from a pressurized container, pump, sprayer, atomizer or nebulizer, with or without the use of a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to administer a dosed amount. The pressurized vessel, pump, sprayer, atomizer or nebulizer may contain a solution or suspension of the antibody, for example, using a mixture of ethanol and the propellant as a solvent, which may additionally contain a lubricant, for example, sorbitan trioleate. Capsules and cartridges (manufactured, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mixture of the immunogenic formulation and a suitable powder base such as lactose or starch.
Formulations for rectal administration are preferably suppositories that can be prepared by mixing the immunogenic formulation with suitable non-irritating excipients or vehicles such as cocoa butter, polyethylene glycol or a suppository wax that are solid at room temperature but liquid at body temperature and, therefore, therefore, they fuse in the rectal vault and release the antibodies. Retention enemas and rectal catheters can also be used as is known in the art. Viscosity enhancing vehicles such as hydroxypropyl cellulose are also certain vehicles of the disclosure for rectal administration as they facilitate retention of the formulation in the rectum. In general, the volume of the vehicle added to the formulation is selected to maximize the retention of the formulation. In particular, the volume should not be so large as to jeopardize the retention of the formulation administered in rectal vault.
Exemplary Formulations
In some embodiments, the present disclosure provides immunogenic formulations that include an antigen, a TLR-3 agonist adjuvant and a vesicle comprising a non-ionic surfactant and a transport enhancer that facilitates the transport of lipid-like molecules through of the mucous membranes. In some embodiments, these formulations can be administered orally. In some embodiments, the TLR-3 agonist adjuvant comprises poly (I: C). In some embodiments, the TLR-3 agonist adjuvant comprises poly (IC: LC). In some embodiments, the transport enhancer is a bile acid, a derivative thereof or a salt of any of these (eg, deoxycholate of
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sodium). In some embodiments, the nonionic surfactant is a glycerol ester (for example, 1-monopalmitoylglycerol). In some embodiments, the vesicle also comprises an ionic amphiphile (for example, di-methylphosphate). In some embodiments, the vesicle also comprises a steroid (eg, cholesterol). In some embodiments, the vesicles comprise 1-monopalmitoyl glycerol, dicetylphosphate, cholesterol and sodium deoxycholate.
In some embodiments, the present disclosure provides immunogenic formulations that include an amphegen, a TLR-3 agonist adjuvant and a vesicle comprising a non-ionic surfactant. In some embodiments, these formulations can be administered parenterally (for example, by intramuscular injection). In some embodiments, the TLR-3 agonist adjuvant comprises poly (I: C). In some embodiments, the TLR-3 agonist adjuvant comprises poly (IC: LC). In some embodiments, the nonionic surfactant is a glycerol ester (for example, 1-monopalmitoylglycerol). In some embodiments, the vesicle also comprises an ionic amphiphile (for example, di-methylphosphate). In some embodiments, the vesicle also comprises a steroid (eg, cholesterol). In some embodiments, the vesicles comprise 1-monopalmitoylglycerol, dicethylphosphate and cholesterol. In some embodiments, the vesicle may lack a transport enhancing molecule. In some embodiments, the vesicle may lack a "bile acid" such as colic acid and chenodeoxycholic acid, its conjugation products with glycine or taurine such as glycolic and taurocholic acid, derivatives that include deoxycholic and ursodeoxycholic acid, and salts of Each of these acids. In some embodiments, the vesicle may lack acyloxylated amino acids, such as acylcarnitines and salts thereof, and palmitoylcarnitines.
In some embodiments, the present disclosure provides immunogenic formulations that include an amphegen, a TLR-4 agonist adjuvant and a vesicle comprising a non-ionic surfactant and a transport enhancer that facilitates the transport of phypid-like molecules through of the mucous membranes. In some embodiments, these formulations can be administered orally. In some embodiments, the TLR-4 agonist adjuvant comprises fast monophosphoryl A or 3-desacyl fast monophosphoryl A. In some embodiments, the transport enhancer is a bile acid, a derivative thereof or a salt of any of these (for example, sodium deoxycholate). In some embodiments, the nonionic surfactant is a glycerol ester (for example, 1-monopalmitoylglycerol). In some embodiments, the vesicle also comprises an ionic amphiphile (for example, di-methylphosphate). In some embodiments, the vesicle also comprises a steroid (eg, cholesterol). In some embodiments, the vesicles comprise 1-monopalmitoyl glycerol, dicetylphosphate, cholesterol and sodium deoxycholate.
In some embodiments, the present disclosure provides immunogenic formulations that include an amphegen, a TLR-4 agonist adjuvant and a vesicle comprising a non-ionic surfactant. In some embodiments, these formulations can be administered parenterally (for example, by intramuscular injection). In some embodiments, the TLR-4 agonist adjuvant comprises fast monophosphoryl A or 3-desacyl monophosphoryl monophosphoryl A. In some embodiments, the non-ionic surfactant is a glycerol ester (eg, 1-monopalmitoylglycerol). In some embodiments, the vesicle also comprises an ionic amphiphile (for example, di-methylphosphate). In some embodiments, the vesicle also comprises a steroid (eg, cholesterol). In some embodiments, the vesicles comprise 1-monopalmitoylglycerol, dicethylphosphate and cholesterol. In some embodiments, the vesicle may lack a transport enhancing molecule. In some embodiments, the vesicle may lack a "bile acid" such as colic acid and chenodeoxycholic acid, its conjugation products with glycine or taurine such as glycolic and taurocholic acid, derivatives that include deoxycholic and ursodeoxycholic acid, and salts of Each of these acids. In some embodiments, the vesicle may lack acyloxylated amino acids, such as acylcarnitines and salts thereof, and palmitoylcarnitines.
In certain embodiments, the formulations of the present disclosure comprise vesicles that have a laminar structure (for example, a bilayer structure). In some embodiments, the formulations of the present disclosure are substantially devoid of non-lamellar structures (eg, micelles).
It will be appreciated that the physical characteristics (eg, laminar structure) of the vesicles present in the formulations described herein can be measured by any known procedure. For example, in some embodiments, the physical characteristics of vesicles can be measured by NMR of 31P at 25 ° C. In some embodiments, an anisotropic peak with a maximum high field around -2.5 ppm with a chemical displacement anisotropy of approximately 15 to 20 ppm is indicative of the presence of a laminar structure. In some embodiments, an isotropic peak observed in the 31P NMR spectra centered around 2.5 ppm is indicative of the presence of non-lamellar structures. In some embodiments, the 31P NMR spectra of a formulation of the present disclosure substantially lack an isotropic peak around 2.5 ppm. In some embodiments, if an isotropic peak around 2.5 ppm is present then it has an intensity (peak height) that is less than the intensity (peak height) of an anisotropic peak with a chemical displacement anisotropy of approximately 15 to 20 ppm and a maximum high field around -2.5 ppm. In some embodiments, if an isotropic peak around 2.5 ppm is present then it has an intensity (peak height) that is less than 50% of the intensity (peak height) of an anisotropic peak with an anisotropy of chemical displacement of approximately 15 to 20 ppm and a maximum field
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high around -2.5 ppm (for example, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 2% or less than one one %).
In some embodiments, the present disclosure provides any one of the formulations mentioned above in lyophilized form.
III. Kits
In yet another aspect, the present disclosure provides kits that include a lyophilized lipid product in a first container and an aqueous solution comprising an antigen (and optionally an adjuvant) in a second container. In some embodiments, the kit also includes instructions for mixing the contents of the two containers to produce vesicle formulations containing antigen.
As discussed above, the lyophilized lipid product is one that was previously prepared by melting vesicle-forming lipids to produce a molten lipid mixture and then lyophilizing the molten lipid mixture to produce the lyophilized lipid product.
In yet another aspect, the present disclosure provides kits that include any vesicle formulation containing lyophilized antigen of the present disclosure in a first container and an aqueous solution (optionally containing an adjuvant) in a second container. In some embodiments, the kit also includes instructions for mixing the contents of the two containers to rehydrate the vesicle formulation containing antigen.
In some embodiments, the kit may include additional components such as a syringe to inject the vesicle formulation containing antigen into a patient.
Examples
The following examples describe some of the exemplary ways of manufacturing and putting into practice certain formulations described herein. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the formulations and procedures described herein.
Example 1: Three-stage melting procedure to prepare vesicles
This example describes a three-stage melting process that was used to prepare some of the vesicles described in the subsequent examples.
In step 1, a 5: 4: 1 molar ratio of the following lipids was arranged: 1-monopalmitoylglycerol (MPG, 270 mg), cholesterol (CHO, 255 mg) and dicetyl phosphate (DCP, 90 mg) in a 50 ml flat bottom glass beaker, ensuring that no dust adheres to the wall of the glass beaker. Next, the lipid mixture was melted in an oil bath heated at 120 ° C for 10 minutes, with occasional stirring in the glass beaker covered with aluminum foil.
While maintaining the temperature of the molten lipid mixture at 120 ° C, an emulsion was created in step 2 by adding 10.9 ml of 25 mM bicarbonate buffer, pH 7.6 (preheated to 50 ° C). The emulsion was immediately homogenized for 2 minutes at 50 ° C (homogenization at 8000 rpm in water bath at 50 ° C). While still homogenizing, 1.1 ml of a 100 mM sodium deoxycholate solution (a "bile salt") in 25 mM bicarbonate buffer, pH 9.7 (preheated to 50 ° C) are added and homogenization continued for 8 minutes at 50 ° C.
In step 3, the antigen (for example, HAV antigen or HBV surface antigen) in a PBS solution of approximately pH 7.2 was added to the heated molten lipid mixture containing the bile salt.
In a variation of this procedure in 3 stages, the molten lipid mixture prepared with bile salt in step 2 was cooled to 30 ° C, incubated in an incubator / shaker (220 rpm) for 2 hours, frozen at -80 ° C, lyophilized and then reconstituted with the antigen solution in 100 mM phosphate buffer pH 8.5 before use.
Example 2: Inverted two-stage melting procedure to prepare vesicles
This example describes an inverted two stage melt process that was used to prepare some of the vesicles described in the subsequent examples.
In step 1, the same 5: 4: 1 molar ratio of lipids (MPG: CHO: DCP) was used; however, in this procedure, a 0.1-0.5 molar ratio of deoxycholic acid (a "bile acid") was also included and melted together with the lipids in an oil bath heated at 135 ° C for 10 minutes . In the procedure of Example 1, only an aqueous solution of bile salt was added in step 2 after converting the molten lipids into an emulsion.
In this phase, a stock solution of antigen was pre-incubated (for example, 4 ml of 25 µg / ml of HAV antigen solution diluted with 6 ml of PBS buffer, pH 7.11 or 1.25 ml of 1.0 mg / ml of HBV surface antigen solution diluted with 8.75 ml of PBS buffer, pH 7.2) for 5 minutes in a hot water bath (25 ° C to 50 ° C). In step 2, the resulting antigen stock solution was homogenized (at 8,000 rpm), molten lipid mixture 5 was added and homogenization was continued for another 10 minutes. The resulting homogenate was stirred for 2 hours at 220 rpm and 30 ° C. 10 ml of a 400 mM sucrose solution in PBS buffer was added to the stirred homogenate and the homogenate was further mixed in vortex for 30 seconds. This mixture was frozen at -80 ° C, lyophilized and then reconstituted in 100 mM phosphate buffer pH 8.5 before use.
In a 2-stage variation of this procedure, the bile acid / molten lipid solution co-prepared 10 prepared in step 1 to 30 ° C was cooled, incubated in an incubator / shaker (220 rpm) for 2 hours, frozen at -80 ° C, lyophilized and then reconstituted with the antigen solution in 100 mM phosphate buffer pH 8.5 before use.
Example 3: Analysis of the integrity of hepatitis B antigen
HBV surface antigen solutions were homogenized at 8,000 rpm at temperatures of 4 ° C, 25 ° C and 15 50 ° C. Table 2 below compares the percentage of resulting antigen measured by ELISA in relation to
unhandled antigen measured directly by ELISA. As shown, the exposure of HBsAg at 50 ° C involved in the 3-stage melt process of Example 1 destroyed more than 50% of the antigenic integrity of the antigen. The use of the inverted 2-stage melting process of the present disclosure allows the temperature of the buffer containing antigen to be substantially reduced (eg, at 25 ° C). The inverted 2-stage melting process, because it can use a lower temperature of antigen solution, allows for better preservation of the antigenicity of the protein subunit.
Table 2
 Antigen  Antigen solution temperature
 4 ° C  25 ° C 50 ° C
 HBsAg  67% 71% 38%
Example 4: Hepatitis B antigen entrapment analysis
25 This example describes experiments that were performed to measure hepatitis B surface antigen entrapment levels. Entrapment levels were measured using a ninhydrin test. The ninhydrin test is a colorimetric procedure to determine the concentration of a polypeptide in a sample. Substances containing amino groups react with the ninhydrin reagent to provide a purple-blue complex.
30 Hepatitis B surface antigen was trapped in vesicles using the procedures of Example 1 and 2. Two different proportions of bile acid (0.10 and 0.50) were tested using the procedure of Example 2. They were hydrolyzed Hepatitis B surface antigens trapped from the vesicles were neutralized, mixed with the ninhydrin reagent and then incubated at 110 ° C. Next, the solution was allowed to cool and the absorbance at 595 nm was measured. There is a linear relationship between absorbance at this wavelength and the
35 amount of polypeptide present in the original sample. Table 3 shows that high levels of antigen trapping (in this case HBV surface antigen) were achieved using the inverted 2-stage procedure of Example 2. Table 3 also suggests that trapping efficiency may be affected by the Bile acid content.
Table 3
 Antigen  Vesicle preparation procedure
 Cast in 3 stages Cast in 2 stages inverted
 Salt / bile acid  Proportion 0.17 of bile salt Proportion 0.50 of bile acid Proportion 0.10 of bile acid
 HBsAg  42% 56% 40%
Example 5: Physicochemical characterization of vesicle stability after rehydration
This example describes experiments that were performed to measure vesicle stability using dynamic light scattering. Particle size and size distribution were determined using a Malvern Zetasizer Instrument
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Nano ZS (ZEN3600) using triplicate readings and an equilibrium time of 2 minutes. 20 ml of vesteula sample was added to 980 ml of bicarbonate buffer pH 7.6, stirred in vortex, and then added to a polystyrene cuvette (Sarstedt 67,754). Statistical analysis was performed using Minitab vl4 with a 2-sample t-test at the 95% confidence level. The results obtained from the nanometric size analysis are shown in Figure 1. Prepared vesteulas were measured as described in Examples 1 and 2 using a Mastersizer immediately after rehydration in the presence of buffer containing 2 | jg of HAV antigen and 2, 4 and 6 hours after rehydration. As shown in Figure 1, the vesteulas prepared by the inverted 2-stage melt process of Example 2 were more stable (evaluated for size stability) over time than the vesteulas prepared by the 3-stage melt process of the example 1. Vesteula stability after hydration is a potentially important factor for vesteula formulations to be administered to a patient.
Example 6: Antibody response to hepatitis A antigen in the immunization of mice
This example describes the in vivo tests of certain immunogenic formulations in mice. Vesteulas were prepared as described in examples 1 and 2 and then rehydrated in the presence of a buffer containing 2 jg of HAV antigen. Female BALB / c mice (n = 4) were vaccinated three times by oral catheter with these veins containing antigen on days 0, 14 and 28 (equivalent to 2 jg HAV / dose antigen).
Serum samples were subsequently collected to evaluate the specific IgG values of hepatitis A induced by oral vaccination. Serum samples collected were tested 14 days after the last ELISA immunization against inactivated HAV antigen. As shown in Figure 2, oral vaccination of mice with the veins prepared by the inverted 2-stage melt procedure of Example 2 induced significantly higher systemic IgG (serum) responses against hepatitis A antigen than the vesteulas prepared by the 3-stage melting process of Example 1. Each symbol represents the serum endpoint value of an individual animal. These data demonstrate that the hydration of vadosulas prepared using the 2-stage melt procedure inverted with HAV antigen results in a better immunogenicity compared to the hydration of veins prepared using the 3-stage melt process.
Several researchers have shown that hepatitis A vaccines and currently authorized hepatitis B vaccines administered by intramuscular injection (i.m.) induce neutralizing IgG antibodies. It has been found that immunogenic hepatitis A formulations administered orally can systematically induce IgG antibodies (serum samples) and mucosal IgA antibodies (nasal wash samples). Since hepatitis A and hepatitis B infection occurs through mucosal surfaces, an IgA response (the characteristic feature of a mucosal immune response) may be more effective than a systemic IgG response. One could only expect systemic IgG responses if the immunogenic hepatitis A or hepatitis B formulations were to be administered by standard parenteral routes (for example, by i.m. injection).
Example 7: The bile salt content of the vesteulas affects the maturation of immature dendritic cells
It is now accepted that, in general, dendritic cells (CD) are important antigen presenting cells that play a role in establishing whether an antigen (e.g., HAV antigen) induces tolerance or a protective immune response in the intestine (Alpan et al., J. Immunol. 166 (8): 4843-4852, 2001). The activation of CDs, usually by inflammatory stimuli, promotes the expression of costimulatory molecules and the presentation of antigens in a way that allows the productive sensitization of T lymphocytes.
In summary, CD progenitors derived from bone marrow were isolated from undifferentiated BALB / c mice and cultured in the presence of interleukin 4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF) which gives rise to the differentiation for the immature CD phenotype (5 days). Subsequent treatment with tumor necrosis factor alpha (TNF-a) also differentiates immature CDs in mature dendritic cells. Immature CDs were incubated with non-ionic surfactant lipid veins (VTNI) prepared as in steps 1 and 2 of Example 2 (without the subsequent addition of antigen) with or without two different molar proportions of bile acid with respect to total lipid (0 , 1 and 0.5). As a positive control, immature CDs were treated with TNF-a alone. Immature CD maturation was measured by flow cytometry using anti-MHC II and anti-CD86 antibodies. Mature CDs were defined as double positive for both antibodies. As shown in Figure 3, VTNIs without bile acid did not significantly affect the maturation of immature CDs while VTNIs with bile acid increased the maturation of CDs. The results also suggest that this increase in maturation may be affected by bile acid content.
Example 8: Characterization of 31P NMR vesteulas
This example describes the 31P NMR characterization of certain exemplary vesteulas that were prepared according to the procedures of the present disclosure.
Vesteulas were prepared as described in examples 1 and 2 without the addition of any antigen. Lyophilized veins are reconstituted in sodium bicarbonate buffer (NaHCOa). The final lipid concentration was 50 mg / ml. 4 ml of suspended vesteulas were transferred in a 10 mm NMR tube and a few drops of D2O were added.
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The 31P NMR spectrum of vesicles prepared using the inverted 2-stage melt process is shown in Figure 4A. The asymmetric lmea shape, with a low field plateau and a high field peak and a chemical displacement anisotropy of approximately 20 ppm corresponds to DCP organized in a typical laminar structure.
The 31P NMR spectrum of vesicles prepared using the 3-stage melt process is shown in Figure 4B. An isotropic peak superimposed on the broad line and centered around 2.5 ppm was observed in the samples prepared by this procedure. The isotropic peak can probably be attributed to the presence of non-lamellar structures such as micelles, hexagonal phase, or very small size vesicles (nanotamano).
Example 9: Inverted two-stage melting procedure to prepare vesicles
This example describes an exemplary inverted two-stage melt process that can be used to prepare vesicles. A 5: 4: 1 molar ratio of lipids (5,575 g of MPG, 5,218 g of cHo, and 1,845 g of DCP) is arranged in a 250 ml flat bottom glass beaker, ensuring that nothing adheres of dust on the wall of the glass beaker. In certain embodiments, when bilosomas are manufactured, bile acid is added at this stage, for example, a 0.5 molar ratio of deoxycholic acid (0.662 g of deoxycholic acid).
Using a clamp to hold the beaker containing lipids and bile acid, the beaker is covered with an aluminum foil and the lipids are allowed to melt in an oil bath heated to 140 ° C to 145 ° C , with occasional agitation in the beaker.
In this phase, the antigen stock solution is prepared by mixing the antigen and concentrated phosphate buffer (5.174 g of Na2HPO4 and 1.179 g of NaH2PO4 in 15 ml of WFI sterile water). The antigen stock solution is homogenized at 8000 rpm in a sterile 1 l SS container. The molten lipids (with or without bile acid) are quickly transferred into the SS vessel by means of a sterilized glass funnel while the solution is homogenized. The mixture is homogenized for 10 minutes at 8000 rpm. The resulting suspension is transferred in a 1 l sterile bottle and stirred for 1-2 hours at 220 rpm and 30 ° to 35 ° C.
In certain embodiments, the resulting suspension is divided into two equal volumes (225 ml each) and the adjuvant poly (IC: LC) is added as follows.
For the first group, poly (IC: LC) in 400 mM sucrose solution is prepared by mixing 22.5 ml of a poly (IC: LC) suspension (45 mg of poly (IC: LC) at 2 mg / ml) and 202.5 ml of 400 mM sucrose solution in 100 mM phosphate buffer. The resulting suspension is added to the first volume of 225 ml of the antigen / vesmula suspension and stirred for 5 minutes at 220 rpm and 30 ° to 35 ° C.
For the second group, poly (IC: LC) in 400 mM sucrose solution is prepared by mixing 7.5 ml of a suspension of poly (IC: LC) (15 mg of poly (IC: LC) at 2 mg / ml) and 217.5 ml of 400 mM sucrose solution in 100 mM phosphate buffer.
The resulting suspension is added to the second volume of 225 ml of the antigen / vesmula suspension and stirred for 5 minutes at 220 rpm and 30 ° to 35 ° C.
The samples can then be frozen at -80 ° C overnight. In certain embodiments, the samples are subsequently lyophilized and stored at 4 ° C.
Example 10: Inverted two-stage melting procedure to prepare vesicles
This example describes another exemplary inverted two stage melt process that can be used to prepare vesicles. A 5: 4: 1 molar ratio of lipids (496 g of 1-monopalmitoyl glycerol (MPG), 496 g of cholesterol (CHO) and 164 g of dicetyl phosphate (DCP)) is placed in a glass beaker of flat bottom, making sure that no dust adheres to the wall of the glass beaker. A TLR-4 agonist is fused together with the lipids (for example, 12 mg of PHAD ™ (phosphorylated hexaacyl disaccharide from Avanti Polar Lipids)). The beaker is fixed and covered with an aluminum foil and the lipids are melted in a hot oil bath at 120-125 ° C with occasional agitation in the beaker.
In this phase, the antigen stock solution is prepared by mixing the antigen and a concentrated phosphate buffer (5.980 g of Na2HPO4 and 1.363 g of NaH2PO4 in 20 ml of sterile water). The stock antigen solution is homogenized at 8,000 rpm at 30-35 ° C, and the molten lipids with TLR-4 agonist are quickly transferred to the beaker and transferred while the solution is homogenized. The mixture is homogenized at 8,000 rpm continuously for 10 minutes at 30-35 ° C. The resulting lipid-antigen suspension is stirred for 1-2 hours at 220,610 rpm at 30-35 ° C.
In some embodiments, sucrose solution in water can be added to the vesmula / antigen solution and stirred for 5 minutes at 220610 rpm at 30-35 ° C.
The samples can then be frozen at -80 ° C overnight. In certain embodiments, the samples are subsequently lyophilized and stored at 4 ° C.
Incorporation by reference
The content of any reference referred to in this document is incorporated herein by reference in its entirety.
Other embodiments
It is intended that the specification and examples be considered only as copies. Other embodiments will be apparent to those skilled in the art from the consideration of the specification or the implementation of the procedures, formulations and kits disclosed herein.
10 In particular, although the above analysis has focused on trapping amphibians, it should be understood that, in general, procedures can be used to trap any substance whether antigenic or non-antigenic. Therefore, in some embodiments, the methods of the present disclosure may be used to trap one or more polypeptides, polynucleotides or polysaccharides that may or may not be antigenic. Specific classes of substances include, but are not limited to, adjuvants, enzymes, receptors, neurotransmitters, hormones, cytokines, cellular response modifiers such as growth factors and chemotactic factors, antibodies, haptens, toxins, interferons, ribozymes, antisense agents, plasmids, DNA and RNA. In some embodiments, the polypeptide may be an antibody or antibody fragment, for example, a humanized antibody. Table 4 provides a non-limiting list of exemplary substances that can be trapped using the procedures of the present disclosure.
20 Table 4
 Substance  Reference drug
 interferon gamma-lb  Actimmune®
 alteplase  Activase® / Cathflo®
 antihemofflic factor  Advate
 human albumin  Albutein®
 laronidase  Aldurazyme®
 interferon alfa-n3  Alferon N®
 human antihemophilic factor  Alfanate®
 human coagulation factor IX filtered with virus  AlfaNine® SD
 alefacept  Amevive®
 bivalirudin  Angiomax®
 darbepoetin alfa  Aranesp ™
 bevacizumab  Avastin ™
 interferon beta-la  Avonex®
 coagulation factor IX  BeneFIX ™
 interferon beta-lb  Betaseron®
 tositumomab  Bexxar®
 antihemofflic factor  Bioclate ™
 human growth hormone  BioTropin ™
 botulmic toxin type A  Botox®
 alemtuzumab  Campath®
 Substance  Reference drug
 acritumomab; technetium-99 marked  CEA-Scan®
 alglucerase  Ceredase®
 imiglucerase  Cerezyme®
 Crotalidae polyvalent immune Fab  CroFab ™
 Fab of antidigoxamic antibodies  DigiFab ™
 rasburicase  ELITEK®
 etanercept  Enbrel®
 epoetin alfa  Epogen®
 cetuximab  Erbitux ™
 beta algasidase  Fabrazyme®
 urofolitropin  Fertinex ™
 beta follitropin  Follistim ™
 teriparatide  Forteo®
 human somatropin  Genotropin®
 glucagon  GlucaGen®
 follitropin alfa  Gonal-F®
 antihemofflic factor  Helixate®
 factor XIII  Hemophil®
 insulin  Humalog®
 human complex antihemophilic factor / von Willebrand factor  Humate-P®
 somatotropin  Humatrope®
 adalimumab  Humira ™
 human insulin  Humulina®
 recombinant human hyaluronidase  Hilenex ™
 interferon alfacon-1  Infergen®
 eptifibatide  Integrilin ™
 interferon alfa  Intron A®
 palifermina  Kepivance
 anakinra  Kineret ™
 antihemofflic factor  Kogenate®FS
 Insulin glargine  Lantus®
 granulocyte-macrophage colony stimulating factor  Leukine®
 lutropin alfa, injectable  Luveris
 ranibizumab  Lucentis®
 Substance  Reference drug
 gemtuzumab ozogamycin  Milotarg ™
 galsulfase  Naglazyme ™
 nesiritida  Natrecor®
 pegfilgrastim  Neulasta ™
 oprelvecina  Neumega®
 filgrastim  Neupogen®
 fanolesomab  NeutroSpec ™
 somatropin  Norditropin® / Norditropin NordiFlex®
 insulin; zinc suspension  Novolin L®
 insulin; isophane suspension  Novolin N®
 insulin regular  Novolin R®
 insulin  Novolin®
 clotting factor Vila  NovoSeven®
 somatropin  Nutropin®
 intravenous immunoglobulin  Octagam®
 Pegylated L-asparaginase  Oncaspar®
 abatacept  Orencia ™
 muromomab-CD3  Ortoclonc OKT3®
 human chorionic gonadotropin  Ovidrel®
 pegylated interferon alfa-2a  Pegasys®
 pegylated interferon alfa-2b  PEG-Intron ™
 abarelix  Plenaxis ™
 epoetin alfa  Procrit®
 aldesleucine  Proleukin, IL-2®
 somatrem  Protropin®
 dornasa alfa  Pulmozyme®
 efalizumab  Raptiva ™
 interferon beta-la  Rebif®
 antihemofflic factor  Recombinate®
 rAHF / antihemophilic factor  ReFacto®
 lepirudin  Refludin®
 infliximab  Remicade®
 abciximab  ReoPro ™
 reteplasa  Retavase ™
 Substance  Reference drug
 rituximab  Rituxan ™
 interferon alfa-2a  Roferon-A®
 somatropin  Saizen®
 synthetic swine secretin  SecreFlo ™
 basiliximab  Simulect®
 eculizumab  Soliris®
 pegvisomant  Somavert®
 palivizumab  Synagis ™
 thyrotropin alfa  Thyrogen®
 tenecteplase  TNKase ™
 natalizumab  Tysabri®
 interferon alfa-n1  Wellferon®
 drotrecogin alfa  Xigris ™
 omalizumab  Xolair®
 daclizumab  Zenapax®
 ibritumomab tiuxetan  Zevalin ™
 somatotropin  Zorbtive ™ (Serostim®)
In addition, although it is considered that the methods of the present disclosure are particularly applicable to thermolabile substances that are sensitive to their chemical and / or physical environment (for example, biological substances such as microbes, polypeptides, polynucleotides, polysaccharides, etc.) should be Understand that, in some embodiments, the procedures can also be used to trap more stable substances including traditional small molecule therapeutic products.

权利要求:
Claims (20)
[1]
5
10
fifteen
twenty
25
30
35
40
Four. Five
1. A procedure comprising:
provide a molten mixture of vesicle-forming Ifpids; Y
adding the molten mixture to an aqueous solution comprising an antigen so that vesicles containing antigen are formed.
[2]
2. The method of claim 1, wherein the vesicle-forming ipsides are phospho Kpids, or non-ionic surfactants, for example 1-monopalmitoylglycerol.
[3]
3. The method of claim 1 or 2, wherein the mixture of vesicle-forming lipids further comprises:
an ionic surfactant such as dicethyl phosphate, phosphatidic acid or phosphatidylserine; or a steroid, such as cholesterol.
[4]
4. The method of any one of claims 1-3, wherein the aqueous solution comprises a lioprotector such as sucrose.
[5]
5. The method of any one of claims 1-4, further comprising lyophilizing the antigen containing vesicles.
[6]
6. The method of claim 5, further comprising rehydrating the antigen-containing vesicles after they have been lyophilized.
[7]
7. The method of any one of claims 1-6, wherein the mixture of vesicle-forming lipids comprises an adjuvant such as a TLR-4 agonist, preferably an attenuated lipid A derivative, such as a monophosphoryl derived from lipid A, or a 3-alkyl monophosphoryl derived from lipid A.
[8]
8. The method of any one of claims 1-6, further comprising adding an adjuvant, such as a TLR-3 agonist, preferably with a lioprotector, after the vesicles containing antigen are formed,
wherein the agonist of TLR-3 is preferably polybiboinosmic acid: polybibocitidyl, optionally stabilized with carboxymethylcellulose poly-L-lysine; Y
wherein the lioprotector is preferably sucrose, trehalose, polyethylene glycol (PEG), dimethyl succinate buffer (DMS), bovine serum albumin (BSA), mannitol or dextran.
[9]
9. The method of any one of claims 1-8, wherein the aqueous solution comprising an antigen is controlled by temperature.
[10]
10. The method of claim 9, wherein the aqueous solution comprising an antigen is maintained at a temperature of less than about 50 ° C, for example, less than about 40 ° C or less than about 30 ° C, for the formation of vesicles containing antigen.
[11]
11. The method of any one of claims 1-10, wherein the molten lipid mixture further comprises a transport enhancer that facilitates the transport of lipids through the mucous membranes.
[12]
12. The method of any one of claims 1-10, wherein the molten lipid mixture lacks a transport enhancer that facilitates the transport of lipids through mucous membranes.
[13]
13. The method of any one of claims 1-12, wherein the antigen is a virus, such as an attenuated virus or an inactivated virus.
[14]
14. The method of claim 13, wherein the virus is hepatitis A or influenza.
[15]
15. The method of any one of claims 1-12, wherein the antigen is selected from the group consisting of a polypeptide, a polynucleotide and a polysaccharide.
[16]
16. The method of claim 15, wherein the polypeptide is a vmco polypeptide.
[17]
17. The method of claim 16, wherein the vmco polypeptide is a hepatitis B polypeptide, a hepatitis C polypeptide, an HIV polypeptide or a flu polypeptide.
[18]
18. The method of claim 17, wherein the hepatitis B polypeptide is HBsAg.
[19]
19. The method of any one of claims 1-18, wherein the antigen is heat labile.
Particle size distribution (^ m)
image 1
Time after hydration (hours)
3-stage fade procedure
Inverted 2 stage fade procedure
Figure 1
Inverted endpoint values
Antibody response to hepatitis A antigen vmco
image2
Figure 2
CD86-PE
NISV
 NISV + NISV +
 (0.1x BA)  (0.5x BA) (+) Control - TNFa
image3
Figure 3
* $
(TO). Inverted fade
ifi
/ V
[5 ]
5 . /
*
vv
^ VV ^ s / VAju
ppm V.)

 10.0  5.0 0.0 -5.0 -10.0 -15.0
image4
Figure 4

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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US22319609P| true| 2009-07-06|2009-07-06|

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US223196P|2009-07-06|

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US25691209P| true| 2009-10-30|2009-10-30|

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US256912P|2009-10-30|

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PCT/US2010/041078|WO2011005769A1|2009-07-06|2010-07-06|Methods for preparing vesicles and formulations produced therefrom|

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