JP2011514337A - Sugar vitrified virus-like particles (VLP) - Google Patents

Abstract

The present invention relates to compositions and methods for stabilizing vaccines.
The present invention discloses and claims a VLP associated with a sugar glass to enhance the overall stability of virus-like particles (VLPs). These VLP formulations worldwide reduce the cost of VLP vaccines and expand their distribution and distribution. The present invention also claims a method of manufacturing the above formulation and a method of delivery to the patient.
[Selection figure] None

Description

  The present invention relates to the stabilization of VLPs by associating virus-like particles (VLPs) with sugar glass.

Most of the vaccines are generally ^recommended, 35 ^{o F~46} o F require storage temperature of (2 ° C. to 8 ° C.), the freezing temperature and the temperature (about 22 ° C. to 25 ° C.) above room temperature Should not be exposed. The introduction of the chickenpox vaccine in 1995 and the more recent introduction of the attenuated influenza vaccine (LAIV) has increased the complexity of vaccine storage. Both varicella vaccine and LAIV must be stored in a continuous frozen state <5 ^° F. (−15 ° C.) without a freeze-thaw cycle.

Recently, examples of inappropriate vaccine storage have been reported. It is more usual for approximately 17% to 37% of suppliers to expose the vaccine to an inappropriate storage temperature and keep the refrigerator temperature too cool rather than too warm. They freezing ^{temperatures,} there is a possibility to lower the 35 ^o F~46 o F vaccine efficacy that must be stored at (2 ° C. to 8 ° C.) irreversibly. Certain freezing-sensitive vaccines contain an aluminum adjuvant that precipitates when exposed to freezing temperatures. This can cause a loss of adjuvant efficacy and vaccine efficacy. The physical changes after exposure to freezing temperatures are not always apparent, and no visible signs of freezing are necessary to cause a reduction in vaccine efficacy. The efficacy of most vaccines can be adversely affected by storage temperatures that are too warm, but this effect is generally more gradual, predictable, and smaller than impairment due to too cold temperatures. In contrast, varicella vaccine and LAIV need to be stored in a continuous frozen state and lose efficacy when stored beyond their recommended temperature range.

  Virus-like particles (VLPs) are useful as vaccines against diseases (eg, influenza). Virus-like particles (VLPs) are very similar to virions, but do not contain viral genomic material (ie, viral genomic RNA). VLPs are therefore essentially non-replicating, which makes them safe for administration in the form of immunogenic compositions (eg vaccines). In addition, VLPs are capable of expressing envelope glycoproteins on the surface of VLPs, which is their major physiological form. Furthermore, since VLPs resemble intact virions and are multivalent particle structures, VLPs may be more effective than soluble envelope antigens in inducing neutralizing antibodies against envelope glycoproteins. Furthermore, unlike many recombinant vaccine approaches, VLPs can be administered repeatedly to a vaccinated host. However, like varicella vaccine and LAIV, VLPs are sensitive to temperature changes and must be kept in a controlled environment. An ideal VLP formulation should withstand high temperatures (above about 25 ° C.) and low temperatures (below about 2 ° C.) to facilitate delivery.

  Current methods to avoid temperature related vaccine degradation are inadequate. For example, attenuated vaccines (and certain non-live vaccines) are often lyophilized because of their inherent instability. These lyophilized products are reconstituted with diluent just prior to administration. Because lyophilization is a time consuming and volume limiting process of vaccine production, lyophilized vaccines are generally provided in multi-dose vials. Some global guidelines require that unused vaccines in multi-dose vials be discarded within 6 hours after reconstitution due to potential contamination and loss of efficacy. This causes vaccine wear and can cause a loss of more than 50% of the amount of vaccine delivered.

  In the public health field, a common approach to freeze protection is to strengthen and optimize the cold chain. Disadvantages of this approach include the costs associated with expanding, updating and improving cold chain equipment, monitoring that equipment, and training equipment users. Furthermore, while cold chain improvements minimize damage from freezing, such improvements will not eliminate the occurrence of freezing damage. Furthermore, cold chain improvements will not mitigate freezing outside the cold chain in colder regions.

  There remains a need for compositions and methods for stabilizing temperature sensitive vaccines, particularly compositions and methods for stabilizing vaccines.

  The present invention includes a composition comprising at least one virus-like particle (VLP) and a sugar glass. In one embodiment, the sugar glass consists of monosaccharides and / or disaccharides. In another embodiment, the monosaccharide is selected from the group consisting of glucose, sorbitol, galactose, mannose, and mannitol. In another embodiment, the disaccharide is selected from the group consisting of trehalose, maltose, maltotriose, lactose, lactulose, and sucrose. In another embodiment, the VLP and the sugar glass are powders. In another embodiment, the powder is produced by spray drying, freeze drying, pulverizing, or a combination thereof. In another embodiment, the powder has an average particle size between about 0.1 nm to about 100 microns. In another embodiment, the powder is suspended in a non-aqueous solvent that does not dissolve the sugar glass. In another embodiment, the non-aqueous solvent is selected from the group consisting of triacetin, isopropyl myristate, medium chain triglycerides, short chain, medium chain, long chain mono, di, triglycerides, aliphatic and aromatic alcohols, or combinations thereof. Is done. In another embodiment, the powder is administered to animals by oral, inhalation, intradermal, intranasal, intramuscular, intraperitoneal, intravenous, subcutaneous, or mucosal (eg, sublingual or buccal). In another embodiment, the VLP comprises at least one viral protein. In another embodiment, the viral protein is from influenza, RSV, and / or VZV. In another embodiment, the VLP has increased stability when compared to a VLP without a sugar glass.

  The present invention also includes a composition comprising at least one VLP and a sugar glass, the VLP having increased stability when compared to the composition without a sugar glass. In another embodiment, the increased stability is increased thermal stability. In another embodiment, the sugar glass consists of monosaccharides or disaccharides. In another embodiment, the monosaccharide is selected from the group consisting of glucose, sorbitol, galactose, mannose, and mannitol. In another embodiment, the disaccharide is selected from the group consisting of trehalose, maltose, maltotriose, lactose, lactulose, and sucrose. In another embodiment, the stabilized sugar glass VLP is a powder.

  The present invention also includes a dry powder formulation comprising VLP and sugar glass. In one embodiment, the sugar glass consists of a monosaccharide or a disaccharide. In another embodiment, the monosaccharide is selected from the group consisting of glucose, sorbitol, galactose, mannose, and mannitol. In another embodiment, the disaccharide is selected from the group consisting of trehalose, maltose, maltotriose, lactose, lactulose, and sucrose. In another embodiment, the dry powder is produced by spray drying, pulverization, or a combination thereof. In another embodiment, the powder is administered to an animal orally, by inhalation, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously or subcutaneously. In another embodiment, the powder is administered to an animal by inhalation using an inhaler and / or subcutaneously using a jet of high pressure air.

  The present invention also includes a method of delivering a sugar glass stabilized VLP comprising reconstitution of solid sugar vitrified VLP in a solvent and administering the reconstituted VLP to an animal. In one embodiment, the powder is administered to an animal orally, by inhalation, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously or subcutaneously.

  The present invention also includes a method of delivering a sugar glass stabilized VLP comprising administering solid sugar vitrified VLP by inhalation and / or infusion.

It is a figure which shows the SDS PAGE and Western blot of VLP before VLP vitrification. It is a figure which shows the SDS PAGE and Western blot of VLP after vitrifying VLP. FIG. 2 shows SDS PAGE and Western blot of VLPs before and after vitrification of VLPs.

  As used herein, the term “adjuvant” is used to increase, alter, or modify the resulting immune response when used in combination with a specific immunogen (eg, VLP) in the formulation. Is a compound. Modification of the immune response includes enhancing or spreading the specificity of one or both of the antibody and the cellular immune response. Altering the immune response may also mean reducing or suppressing certain antigen-specific immune responses.

  The term “ambient” temperature or condition as used herein is for any given time in a given environment. Generally, the ambient room temperature is about 22 ° C., and ambient atmospheric pressure and humidity are easily measured and should vary depending on the season, weather conditions, altitude, etc.

  As used herein, the term “buffer” refers to a buffer solution that resists changes in pH by the action of its acid-base conjugate components. In general, the pH of the buffer should be selected to stabilize the chosen active agent and should be ascertainable by one skilled in the art. Certain proteins may be stable over a wider range of pH, such as acidic pH, but generally the pH of the buffer should be within the physiological pH range. Accordingly, preferred pH ranges are from about 1 to about 10, with about 3 to about 8, about 6.0 to about 8.0, about 7.0 to about 7.4, and about 7.0 to about 7.2 being preferred. included. Suitable buffers include a pH 7.2 phosphate buffer and a pH 7.0 citrate buffer. As those skilled in the art are aware, there are numerous suitable buffers that can be used. Suitable buffers include, but are not limited to, amino acids, potassium phosphate, sodium phosphate, sodium acetate, histidine-HCl, sodium citrate, sodium succinate, bicarbonate and ammonium carbonate. In general, the buffer is used at a molar concentration of about 1 mM to about 2 M, preferably about 2 mM to about 1 M, particularly preferably about 10 mM to about 0.5 M, especially 25 to 50 mM.

  The term “effective dose” as used herein generally refers to inducing immunity and / or preventing and / or ameliorating an infection or alleviating at least one symptom of an infection. Therefore, and / or to increase the efficacy of another dose of VLP, it refers to the amount of VLP sufficient. An effective dose may refer to the amount of VLP sufficient to delay or minimize the signs of infection. An effective dose may also refer to the amount of VLP that provides a therapeutic benefit in the treatment or treatment of an infection. Further, an effective dose is that amount of the VLP of the present invention alone or in combination with other therapies that provides a therapeutic benefit in the treatment or treatment of infection. An effective dose may also be an amount sufficient to enhance the subject's (eg, human) 's own immune response to subsequent exposure to an infectious agent. The level of immunity can be monitored, for example, by plaque neutralization, complement binding, enzyme immunosorbent, or microneutralization assays, for example by measuring the amount of neutralizing secreted antibodies and / or neutralizing serum antibodies. In the case of a vaccine, an “effective dose” is an amount that prevents disease and / or reduces the severity of symptoms.

  The term “excipient” as used herein generally refers to increasing the stability of a therapeutic agent during vitrification (eg, by spray freezing) and for subsequent long-term storage. Refers to the compound or substance being added. Suitable excipients, for example, are not turbid or polymerized when contacted with water, are essentially harmless when administered to patients, and interact significantly with therapeutic agents to alter their biological activity Can not be chemicals. Suitable excipients are described below. They include, but are not limited to, proteins such as human and bovine serum albumin, gelatin, carbohydrates, sugar alcohols such as glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol, propylene glycol, pluronic, surfactants As well as combinations thereof. Excipients can be multifunctional components of the solutions or suspensions of the present invention.

  The term “effective amount” as used herein refers to the amount of VLP that is necessary or sufficient to achieve a desired biological effect. An effective amount of the composition should be that which achieves the selected result, and such amount can be determined by routine work by one skilled in the art. For example, an effective amount for preventing, treating and / or ameliorating an infection is necessary to cause the generation of an immune system by causing the generation of an antigen-specific immune response when exposed to a VLP of the invention. Can be in any amount. The term is also synonymous with “sufficient amount”.

  As used herein, the term “infectious agent” refers to a microorganism that causes an infection in a vertebrate. Generally these microorganisms are viruses, bacteria, parasites and / or fungi.

As used herein, the term “glass” or “glass state” or “glassy matrix” refers to a liquid with significantly reduced flow capacity. That is, it is a liquid with a very high viscosity in the range of 10 ¹⁰ to 10 ¹⁴ Pascal-seconds. It can be regarded as a metastable amorphous system in which the molecule has vibrational motion but has very slow (almost unmeasurable) rotational and translational components. As a metastable system, it is stable for long periods when stored at temperatures well below the glass transition temperature. Because these glasses are not in thermodynamic equilibrium, glasses stored at or near the glass transition temperature loosen until equilibrium and lose their high viscosity. The resulting rubbery or syrupy fluid liquid is often chemically and structurally unstable. Glass can be obtained by many different routes, but no matter which route is taken, it appears to be physically and structurally identical. The method used to obtain a glassy matrix for the purposes of the present invention is generally a solvent sublimation and / or evaporation technique.

  The term “glass transition temperature” as used herein is represented by the symbol Tg and is the temperature at which the composition changes from a glassy or glassy state to a syrup or rubbery state. Generally, Tg is determined using differential scanning calorimetry (DSC), and is typically taken as the temperature at which the onset of change in the heat capacity (Cp) of the composition occurs when the transition is scanned. The definition of Tg is always at the discretion and there is no international agreement. This Tg can be defined as the onset of transition, midpoint, or end point, and for the purposes of the present invention we will use the onset of change in Cp when using DSC and DER. “Formation of Glasses from Liquids and Biopolymers” by CA Angell: Science, 267, 1924-1935 (Mar. 31, 1995) and Jan P. Wolanczyk “Differential Scanning Calorimetry Analysis of Glass Transitions”: Cryo-Letters , 10, 73-76 (1989). For detailed mathematical processing, see “Nature of the Glass Transition and the Glassy State” by Gibbs and DiMarzio: Journal of Chemical Physics, 28, NO. 3, 373-383 (March, 1958). These articles are incorporated herein by reference.

  As used herein, the term “stable” formulation or composition refers to a bioactive agent (eg, VLP) therein that has physical and / or chemical stability and / or biology upon storage. It continues to have an intrinsic activity. Various analytical techniques for measuring stability are available in the art, such as Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A., Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature for a selected time period. Using trend analysis, the expected shelf life can be estimated before the material is actually stored for that period.

  As used herein, the term “vaccine” includes a form of VLP that can be administered to a vertebrate and is sufficient to prevent and / or ameliorate infection and / or infection. Refers to a formulation that elicits a protective immune response sufficient to alleviate at least one symptom of and / or to enhance the efficacy of another dose of VLP. When introduced into a host, the vaccine includes, but is not limited to, production of antibodies and / or cytotoxins and / or activation of cytotoxic T cells, antigen presenting cells, helper T cells, dendritic cells. Immune responses and / or other cellular responses can be elicited. In one embodiment, the VLP vaccine is accompanied by and / or stabilized with a sugar glass.

  The term “virus-like particle” (VLP), as used herein, refers to a structure that resembles a virus in at least one attribute but has not been demonstrated to be infectious. The virus-like particles according to the invention do not have genetic information encoding the proteins of those virus-like particles. In general, virus-like particles lack the viral genome and are therefore non-infectious. Furthermore, virus-like particles can often be produced in large quantities by heterologous expression and can be easily purified.

  Solution vaccines or drugs that can be injected as such are inherently unstable. Methods to address this instability are known in the art (eg lyophilization). However, inaccurate reconstitution or dried vaccines or drugs can result in wrong doses or contaminated solutions, and freezing and / or thawing of vaccines or drugs can also cause wrong administration. As it is, it is inconvenient and inherently dangerous. Thus, there is a need for new and improved vaccine formulations that make vaccine delivery easier and safer, reduce cold chain dependency and / or increase vaccine thermal stability and / or reduce the number of immune interventions Exists. Such vaccine formulations should make vaccine distribution around the world more efficient and economical.

  One of the fundamental principles in improving vaccine formulations is to obtain a stable antigen in the dry state. The most commonly used method for preparing solid proteinaceous drugs is freeze-drying (lyophilization). However, during lyophilization, the proteinaceous drug is subjected to freezing and drying stress, which can result in loss of activity. Therefore, a protective agent is required to prevent the harmful effects of lyophilization.

  It is known that carbohydrates can protect various types of drug ingredients such as proteins and vaccines during freezing, drying, and storage. When properly dried, the proteinaceous drug is incorporated into a matrix consisting of carbohydrates (sugar glass) in an amorphous glassy state. The stabilizing effect of these sugar glasses has been explained by the formation of a matrix that greatly reduces diffusion and molecular mobility and acts as a physical barrier between particles and molecules. Both the lack of mobility and physical barrier provided by this glass matrix prevent agglomeration and degradation of the dried material (Amorij, JP et al. (2007) Vaccine, 25, 6447-6457).

  In order to produce a sugar glass, the sugar must be dried at a temperature below the glass transition temperature of the carbohydrate, eg sugar. Below this glass transition temperature, the physical properties of the amorphous material change in a manner similar to the physical properties of the solid phase (glass state), and above that the amorphous material is liquid (rubber Temperature). The glass transition temperature Tg of the material is a temperature below which there is little relative mobility of molecules. Tg is generally applicable to fully or incomplete amorphous phases such as glass and plastic. Above Tg, the secondary bonds between polymer chains, i.e. non-covalent bonds, become weaker than thermal motion, and the polymer becomes rubbery and can be elastically deformed without breaking.

  Sugar glass is made when the sugar mixture is dried at a temperature below its Tg temperature. As the sugar solution containing the active molecule is dried, it can crystallize or become a supersaturated syrup when the solubility limit of the sugar is reached. The ability of sugars to resist crystallization is a very important property of a good stabilizer. Trehalose is well known to make glass (Green J L. and Angel CA A. Phase relations and vitrification in saccharide water solutions and the trehalose anomaly. J. Phys. Chem. 93 2880-2882 (1989)). Is not the only one. Upon further drying, the gradual syrup solidifies and turns into glass at a low residual water content. Unknowingly, the active molecule changes from a solution in water to a solid solution in a dried sugar glass. Chemical diffusion is negligible in the glass, so the chemical reaction is virtually stopped. Since denaturation is a chemical change, it cannot occur in glass and the molecule stabilizes.

  Accordingly, the present invention includes a composition comprising at least one associated virus-like particle (VLP) and a sugar glass. In one embodiment, the VLP is covered with the sugar glass. In one embodiment, the VLP associates with the sugar glass. In one embodiment, the sugar glass consists of monosaccharides and / or disaccharides. In another embodiment, the monosaccharide is selected from the group consisting of glucose, sorbitol, galactose, mannose, and mannitol. In another embodiment, the disaccharide is selected from the group consisting of trehalose, maltose, maltotriose, lactose, lactulose, and sucrose. Other sugars include galactose, mannose, xylose, sorbose, lactose, lactose, raffinose, maltodextrins, meletitol, maltose, fructose, arabinose, xylose, ribose, rhamnose, xylitol, erythritol, threitol, gluconate, and / or The thing similar to these is mentioned. This suspension or solution can also contain, for example, polymers such as starch, starch derivatives, carboxymethyl starch, hydroxyethyl starch (HES), and / or dextran.

  In another embodiment of the invention, when the VLP is associated with a sugar glass, the VLP is stable at ambient temperature. Preferably, the sugar is one that does not crystallize at freezing temperatures that would destabilize the VLP in a glassy formulation. The amount of sugar used in this suspension or solution will vary depending on the nature of the VLP, the type of sugar and the intended use. In general, however, the final concentration of sugar is between about 1 wt% and 40 wt%, more preferably between about 1 wt% and 20 wt%. In one embodiment, the suspension or solution contains about 60 mg / ml trehalose. In another embodiment, the suspension or solution contains about 50 mg / ml mannitol. In another embodiment, the suspension or solution comprises about 30 mg / ml trehalose and about 25 mg / ml mannitol. In another embodiment, the suspension or solution comprises about 30 mg / ml trehalose and about 30 mg / ml mannitol.

  In one embodiment, the VLP and sugar glass composition may be a powder. In another embodiment, the powder is produced by spray drying, freeze drying, pulverizing, or a combination thereof. In another embodiment, the powder has an average particle size between about 0.1 nm to about 100 microns. Spray drying and freeze drying are well known in the art (see US Pat. Nos. 6,372,258, 7,258,873, and 5,230,162, all of which are Which is incorporated herein by reference in its entirety)).

  The formulation of the present invention comprises virus-like particles (VLP) and sugar glass. Said VLP comprises at least one viral nucleoprotein (eg influenza M1, retrovirus gag, RSV M, Newcastle disease M etc.) and at least one viral surface coat protein (eg influenza HA and / or NA, HIV, gp120, RSV F, Newcastle disease F). A chimeric VLP is a VLP having at least two proteins in which one protein can drive VLP formation (eg, a matrix protein) and the other protein is derived from a heterologous infectious agent. is there. In one embodiment, the infectious agent protein can have an antigenic variation of the same protein. In another embodiment, the infectious agent protein is from an unrelated factor. In another embodiment, the chimeric VLP comprises a chimeric protein (fusion protein) comprising the antigen portion of one protein fused to the transmembrane and / or cytoplasmic region of another (heterologous) protein (February 2007). See U.S. Provisional Patent Application No. 60 / 902,337 filed 21 days and U.S. Provisional Patent Application No. 60 / 970,592 filed Sep. 7, 2007 (both of which are incorporated herein in their entirety for all purposes) Incorporated herein by reference)).

  These infectious agents can be viruses, bacteria, and / or parasites. Proteins that can be expressed on the surface of VLPs can be obtained from viruses, bacteria, and / or parasites. These proteins obtained from viruses, bacteria, and / or parasites are responsible for immune responses (cells and / or body fluids) in said vertebrates that will prevent, treat, address and / or ameliorate infections in vertebrates. Of) can be triggered.

  Non-limiting examples of viruses from which the infectious agent protein can be obtained include influenza (A and B, such as HA and / or NA), coronavirus (such as SARS), hepatitis virus A, B, C, D, and E3, human immunodeficiency virus (HIV), herpes virus 1, 2, 6, and 7, cytomegalovirus, varicella-zoster, papilloma virus, Epstein-Barr virus, parainfluenza virus, adenovirus, banyavirus (eg hantavirus) ), Coxsackie virus, picoma virus, rotavirus, rhinovirus, rubella virus, mumps virus, measles virus, rubella virus, poliovirus (polymorphism), adenovirus (polymorphism), parainfluenza virus (polymorphism) ), Triinflue The (various types), ship fever virus, western and eastern equine encephalomyelitis, Japanese encephalitis, fowlpox, rabies virus, slow brain virus, chicken sarcoma virus, papovaviridae, parvoviridae, Picomaviridae, Poxviridae (such as smallpox or complete vesicles), Reoviridae (eg rotavirus), Retroviridae (HTL V-I, HTL V-II, Lentivirus), Togaviridae (eg rubivirus) , Newcastle disease virus, West Nile virus, tick-borne encephalitis, yellow fever, chikungunya virus, and dengue virus (all serotypes).

  In another embodiment, the virus-derived specific protein is HA and / or NA from influenza virus (including birds), S protein from coronavirus, gp160, gp140, and / or gp41 from HIV, chickenpox It can include gp I to IV from herpes zoster and Vp, E and M precursors / M from yellow fever virus, dengue fever (all serotypes), or any flavivirus. Also included is any protein derived from a virus capable of eliciting an immune response (cells and / or body fluids) in said vertebrate capable of preventing, treating, addressing and / or ameliorating an infection in a vertebrate It is. In one embodiment, the VLP comprises at least one influenza protein. In another embodiment, the influenza protein is HA or NA. In another embodiment, the VLP comprises at least one RSV protein. In another embodiment, the RSV protein is RSV M, F, and / or G. In another embodiment, the VLP comprises a VZV protein. In another embodiment, the VZV protein is gE and / or at least one tegument protein.

  Non-limiting examples of bacteria from which the infectious agent protein can be obtained include B. perutussis, Leptospira pomona, S. paratyphi A and B, Diphtheria (C. diphtheriae), tetanus (C. tetani), Clostridium botulinum (C. botulinum), C. perfringens, C. C. feseri and other gas gangrene bacteria, B. anthracis, P. P. pestis, P. p. P. multocida, Neisseria meningitidis, N. gonorrheae, Hemophilus influenzae, Actinomyces (e.g., Norcaldia), Acinetobacter, Neisseria (e.g. Bacillus anthrasis), Bacteroides fragilis (eg Bacteroides fragilis), Blastomycosis, Borideterra, Borrelia (eg Borrelia burgdorferi), Brucella, Campylobacter, Chlamydia , Coccidioides, Corynebacterium (eg, Corynebacterium diphtheriae), E. coli (eg, Enterotoxigenic E. coli and Enterohemorrhagic E. coli), intestines Internal bacteria (eg, Enterobacter aeroge nes)), Enterobacteriaceae (Klebsiella, Salmonella (eg, Salmonella typhi, Salmonella enteritidis)), Serratia, Yersinia, Shigella, swine genus, hemophilus ( For example, Haemophilus influenza type B), Helicobacter genus, Legionella genus (eg Legionella pneumophila), Leptospira genus, Listeria genus (eg Listeria monocytogenes), Mycoplasma genus Genus Mycobacterium (eg Mycobacterium leprae and Mycobacterium tuberculosis), Vibrio (eg Vibrio cholerae), Pasteurellaceae, Proteus, Pseudomonas (eg Pseudomonas aeruginosa) (Pseudomonas aeruginosa )), Rickettsiaceae, Spirocheta (e.g., Treponema spp., Leptospira spp., Borrelia spp.). ), Shigella spp. Neisseria meningitidis, Streptococcus pneumoniae and Streptococcus (eg, Streptococcus pneumoniae and Group A, B, and C Streptococcus), ureaplasma, Treponema pollidum, Staphylococcus aureus), Pasteurella haemolytica, Corynebacterium diphtheriae toxoid, meningococcal polysaccharide, Bordetella pertusis, Streptococcus pneumoniae, Clostridium tetani, and Clostridium tetani Mycobacterium bovis.

  Non-limiting examples of parasites from which the infectious agent protein can be obtained include leishmaniasis (Leishmania tropica mexicana), tropical Leishmania tropica, forest type Leishmania major, Ethiopian Leishmania (Leishmania aethiopica), Mucosal Leishmania (Leishmania braziliensis), Donovan leishmania (Leishmania donovani), Infant Leishmania infantum (Leishmania chagasi), Trypanosoma disease (Gambian brusei trypanosomiasis) (Trypanosoma brucei gambiense), Rhodesia brucei trypanosoma disease (Trypanosoma brucei rhodesiense), toxoplasmosis (Toxoplasma gondii), schistosomiasis (Schistosoma haematobium), Schistosoma haematobium (Schistosoma haematobium) Schistosoma japonicum, Schistosoma mansoni, Schistosoma mekongi, Schistosoma intercalatum, malaria (Plasmodium virax), Plasmodium falciparium ), Plasmodium malariae, and Plasmodium ovale), amebiasis (Entamoeba histolytica), babesiosis (Babesiosis microti), cryptosporidiosis (bovine) For Cryptosporidium parvum), binuclear amebiasis (Dientamoeba fragilis), giardiasis (Giardia lamblia), helminthiasis, and trichomoniasis (Trichomonas vaginalis) It is a factor that works as a cause. The list is meant to be exemplary and is in no way meant to limit the invention to these specific bacterial, viral, or parasitic organisms.

  Another embodiment of the invention comprises a composition comprising at least one VLP in a sugar glass, said VLP having increased stability when compared to a VLP not in a sugar glass. In one embodiment, the increased stability is increased thermal stability and increased stability at ambient temperature. For example, if the VLP does not show a significant increase in aggregation, precipitation, and / or denaturation upon visual inspection of color and / or clarity as measured by UV scattering or by size exclusion chromatography or any biological assay The VLPs are “stable” in the pharmaceutical composition. A “stable” formulation or composition is one in which the bioactive substance therein essentially retains its physical and / or chemical stability and / or biological activity upon storage. Various analytical techniques for measuring stability are described below and are available in the art, and are also described in, for example, Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc. , New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature for a selected time period. Using trend analysis, the expected shelf life can be estimated before the material is actually stored for that period. For example, the composition can be stable at room temperature (about 25 ° C.) for at least 3 months and / or can be stable at about 2-8 ° C. for at least one year. Furthermore, the composition can be stable after freezing (eg, up to -70 ° C.) and thawing of the composition. In another embodiment, the sugar glass consists of monosaccharides or disaccharides. In another embodiment, the monosaccharide is selected from the group consisting of glucose, sorbitol, galactose, mannose, and mannitol. In another embodiment, the disaccharide is selected from the group consisting of trehalose, maltose, maltotriose, lactose, lactulose, and sucrose. In another embodiment, the stabilized sugar glass VLP is a powder.

  The VLP-related sugar glass composition can have additional exceptions that help stabilize the VLP. Accordingly, the present invention includes compositions such as suspensions or solutions of VLPs with polymer additives, amino acid additives, and / or surfactants to help increase stability. These suspensions or solutions may contain other components (ie excipients) such as buffers, carriers, preservatives, colloidal stabilizers, fillers, diluents, lubricants, amphiphiles, and / or stabilizers. Can be included. For example, a polymer can be included in the suspension or solution of the method, for example, to provide protective and structural advantages. The linear or branched chain of the polymer can give, for example, strong structural strength to the particle composition of the present invention. The polymer can be applied to the outside of the powder particles of the present invention as a protective film and / or a sustained-release film. Many polymers such as polyvinyl pyrrolidone, polyethylene glycol, polyamino acids such as poly L-lysine can significantly increase the rate of reconstitution in aqueous solution. Examples of the polymer protective agent in the method of the present invention include starch and starch derivatives such as oxidized starch, carboxymethyl starch, and hydroxyethyl starch (HES). Others include hydrolyzed gelatin, non-hydrolyzed gelatin, ovalbumin, collagen, chondroitin sulfate, sialated polysaccharide, actin, myosin, microtubules, dynein, kinetin, human serum albumin, and / or similar Things. Other excipients can also be included in the formulation. For example, amino acids such as arginine and methionine can be a component of formulations and compositions. These amino acids can serve, for example, as zwitterions that protect charged groups during surface processing and as storage vessels that prevent non-specific binding of bioactive substances. These amino acids can increase the stability of the composition, for example, by removing oxidative substances, by removing deamidating substances, and by stabilizing protein conformation. In another example, glycerol can be included in the formulations of the present invention, eg, to act as a plasticizer in a powder particle composition. For example, EDTA can be included in the composition to reduce aggregation of the formulation components and / or to remove metal ions that can initiate a free radical destructive chemical reaction.

  The VLP and sugar glass compositions of the present invention can also include, for example, a surfactant that can coexist with the particular bioactive agent involved. Surfactants can increase the solubility of other formulation ingredients to avoid aggregation or precipitation at higher concentrations. A surface-active substance can reduce the surface tension of a suspension or solution, for example, so that the bioactive substance does not denature at the gas-liquid interface and / or can form finer droplets during spraying. it can. The suspension or solution according to the present invention is between about 0.001-5%, preferably between about 0.05-1% or about 0.2% nonionic surfactant, ionic surfactant Agents, or combinations thereof.

  Buffers can be added to the formulations of the present invention to provide a stable pH suitable for example for the formulations of the methods of the present invention and the compositions of the present invention. Common buffers of the present invention include, for example, amino acids, potassium phosphate, sodium phosphate, sodium acetate, sodium citrate, histidine, glycine, sodium succinate, ammonium bicarbonate, and / or carbonate. These buffers can be adjusted to suitable acid and salt forms to provide pH stability, for example, in the range of about pH 3.0 to about pH 10.0, about pH 4.0 to about pH 8.0. A near neutral pH such as pH 7.2 is preferred for many compositions. In one embodiment, the VLP-related sugar glass comprises a pH 7.2 phosphate buffer with 0.5 M NaCl.

  In another embodiment, the VLP and sugar glass composition is a powder suspended in a non-aqueous solvent that does not dissolve the sugar glass. In another embodiment, the non-aqueous solvent is triacetin, isopropyl myristate, medium chain triglycerides, short chain, medium chain, and / or long chain monoglycerides, dimonoglycerides, and trimonoglycerides, aliphatic and aromatic alcohols, hydrofluoro Selected from the group consisting of ethers, perfluoroethers, hydrofluoroamines, perfluoroamines, hydrofluorothioethers, perfluorothioethers, and hydrofluoropolyethers or combinations thereof (see WO 2005/099669). Is hereby incorporated by reference)). In another embodiment, the powder is administered to an animal orally, by inhalation, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously.

  In another embodiment, the powder is produced by spray drying, milling, or a combination thereof. In another embodiment, the powder has an average particle size between about 0.1 nm to about 100 microns. In another embodiment, the powder is suspended in a non-aqueous solvent that does not dissolve the sugar glass. In another embodiment, the powder is administered to an animal orally, by inhalation, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously. These VLPs can have proteins from the aforementioned bacteria, viruses, fungi, parasites. In one embodiment, the VLP comprises at least one influenza protein. In another embodiment, the influenza protein is HA or NA. In another embodiment, the VLP comprises at least one RSV protein. In another embodiment, the VLP comprises a VZV protein. In another embodiment, the VZV protein is gE. In another embodiment, the composition further comprises an adjuvant.

  Another embodiment of the present invention comprises a dry powder formulation comprising VLPs in a sugar glass. In one embodiment, the sugar glass consists of a monosaccharide or a disaccharide. In another embodiment, the monosaccharide is selected from the group consisting of glucose, sorbitol, galactose, mannose, and mannitol. In another embodiment, the disaccharide is selected from the group consisting of trehalose, maltose, maltotriose, lactose, lactulose, and sucrose. In another embodiment, the dry powder is produced by spray drying, freeze drying, pulverizing, or a combination thereof. In another embodiment, the powder has an average particle size between about 0.1 nm to about 100 microns. In another embodiment, the powder is administered to an animal orally, by inhalation, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously. In another embodiment, the powder is reconstituted in a solvent prior to administration. In another embodiment, the powder is administered to an animal by inhalation using an inhaler and / or subcutaneously using a jet of high pressure air. This composition comprises any of the aforementioned excipients.

  Another embodiment of the present invention provides a method for delivering a sugar glass stabilized VLP comprising reconstitution of solid sugar vitrified VLP in a solvent and administering the reconstituted VLP to an animal. Including. In one embodiment, the VLP is administered to an animal orally, by inhalation, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously.

  Another embodiment of the invention includes a method of delivering a sugar glass stabilized VLP comprising administering solid sugar vitrified VLPs by inhalation and / or infusion. In one embodiment, the infusion is administered by injection of high pressure air.

  Another embodiment of the present invention includes a method for increasing the thermal stability of a VLP comprising formulating the VLP in a sugar glass. In one embodiment, the sugar glass consists of monosaccharides and / or disaccharides. In another embodiment, the monosaccharide is selected from the group consisting of glucose, sorbitol, galactose, mannose, and mannitol. In another embodiment, the disaccharide is selected from the group consisting of trehalose, maltose, maltotriose, lactose, lactulose, and sucrose. In another embodiment, the sugar glass comprising VLP is a powder. In another embodiment, the powder is produced by spray drying, freeze drying, pulverizing, or a combination thereof. In another embodiment, the powder has an average particle size between about 0.1 nm to about 100 microns. In another embodiment, the powder is suspended in an organic solvent that does not dissolve the sugar glass. In one embodiment, the VLP comprises at least one influenza protein. In another embodiment, the influenza protein is HA or NA. In another embodiment, the VLP comprises at least one RSV protein. In another embodiment, the VLP comprises a VZV protein.

As is well known in the art, the immunogenicity of a particular composition can be enhanced by the use of non-specific promoters of the immune response known as adjuvants. Adjuvants have been used experimentally to promote increased generalized immunity against unknown antigens (eg, US Pat. No. 4,877,611). Immunization protocols have used adjuvants for many years to stimulate responses, and adjuvants are therefore well known to those skilled in the art. Certain adjuvants affect the method of antigen presentation. For example, the immune response increases when protein antigens are precipitated by alum. Antigen emulsification also extends the antigen presentation period. "A Compendium of Vaccine Adjuvants and Excipients (2 nd Edition)" scope of the present invention comprise any adjuvant described in (its entirety of which is incorporated herein by reference for all purposes) such as Vogel It is thought that it is in. In one embodiment, the VLP complexed with the sugar glass formulation composition comprises at least one adjuvant.

  Examples of adjuvants include complete Freund's adjuvant (non-specific promoter of immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant. Other adjuvants include GMCSP, BCG, aluminum hydroxide, MDP compounds such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). A 2% squalene / tween 80 emulsion containing RIBI, MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS) containing three components extracted from bacteria is also contemplated. MF-59, Novasomes (Novasomes®), MHC antigens can also be used.

  In one embodiment of the invention, the adjuvant has about 2-10 bilayers arranged in a generally spherical shell separated by an aqueous layer surrounding a large amorphous central cavity without lipid bilayers. Paucilamellar lipid vesicle. The capsular thin film lipid vesicles can act as non-specific stimulators, as carriers for antigens, as carriers for additional adjuvants, and as a combination thereof to stimulate several paths of the immune response. it can. For example, when a vaccine is prepared by mixing an antigen with a pre-formed vesicle so that the antigen remains outside the vesicle, the capsular thin film lipid vesicle acts as a non-specific immune promoter. By encapsulating the antigen within the central cavity of the endoplasmic reticulum, the endoplasmic reticulum serves as both an immunostimulant and a carrier for the antigen. In another embodiment, the endoplasmic reticulum is made primarily from non-phospholipid vesicles. In another embodiment, the endoplasmic reticulum is Novasomes. These novasomes (Novasomes®) are capsular thin film non-phospholipid vesicles ranging from about 100 nm to about 500 nm. These include Brij 72, cholesterol, oleic acid, and squalene. Novasomes have been shown to be effective adjuvants against influenza antigens (US Pat. Nos. 5,629,021, 6,387,373, and 4,911,928). No. (these are hereby incorporated by reference in their entirety for all purposes).

  In one aspect, the adjuvant effect is achieved by the use of a drug such as alum used in an about 0.05 to about 0.1% solution in phosphate buffered saline. Alternatively, VLPs can be made as a mixture with a synthetic polymer of sugar (Carbopol®) used as an approximately 0.25% solution. Certain adjuvants, such as certain organic molecules obtained from bacteria, act on the host, not the antigen. An example is muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP)), a bacterial peptidoglycan. In other embodiments, hemocyanin and hemoerythrin can also be used with the VLPs of the present invention. Although other molluscs and arthropod hemocyanins and hemerythrin can also be used, in some embodiments, the use of hemocyanin from mussel (KLH) is preferred.

  Various polysaccharide adjuvants can also be used. For example, the use of various pneumococcal polysaccharide adjuvants in mouse antibody responses has been described (Yin et al., 1989). Doses that produce an optimal response or do not cause suppression should be used as described (Yin et al., 1989). Polyamine varieties of polysaccharides such as chitin and chitosan, including deacetylated chitin, are particularly preferred. Another embodiment describes the use of a lipophilic disaccharide tripeptide derivative of muramyl dipeptide in artificial liposomes formed from phosphatidylcholine and phosphatidylglycerol.

  Amphiphiles and surface active substances such as saponins and derivatives such as QS21 (Cambridge Biotech) form yet another group of adjuvants used with the VLPs of the present invention. Nonionic block copolymer surfactants (Rabinovich et al., 1994) can also be used. Oligonucleotides are another useful group of adjuvants (Yamamoto et al., 1988). Quil A and lentinene are other adjuvants that can be used in some embodiments of the present invention.

  Another group of adjuvants are detoxified endotoxins, such as the purified detoxified endotoxin of US Pat. No. 4,866,034. These purified detoxified endotoxins are effective in producing an adjuvant response in vertebrates. Of course, detoxified endotoxins can also be combined with other adjuvants to prepare multi-adjuvant formulations. As described in US Pat. No. 4,435,386, for example, the combined use of detoxified endotoxin and dihalic acid trehalose is specifically contemplated. Combinations of detoxified endotoxin with dihalic acid trehalose and endotoxin glycolipids (US Pat. No. 4,505,899) are also contemplated, US Pat. Nos. 4,436,727, 4,436,728, and The combination of detoxified endotoxin with the cell wall skeleton (CWS) as described in US Pat. No. 4,505,900, or the combination of CWS and dihalic acid trehalose is also the same. As described in US Pat. No. 4,520,019, a combination of simply CWS and trehalose dimycolate without detoxified endotoxin would also be useful.

  Those skilled in the art will know the various types of adjuvants that can be conjugated to vaccines according to the present invention, among others, alkyl lysophospholipids (ALP), BCG and biotin (including biotinylated derivatives). Is mentioned. Some adjuvants specifically contemplated for use are gram cell derived teichoic acids. They include lipoteichoic acid (LTA), ribitol teichoic acid (RTA), and glycerol teichoic acid (GTA). The active forms of their synthetic counterparts can also be used in connection with the present invention (Takada et al., 1995).

  For example, if it is desired to produce antibodies or subsequently obtain activated T cells, various adjuvants can be further used in other vertebrates, even those that are not generally used in humans. Toxicity or other adverse effects that may result from either adjuvants or cells, such as may occur using non-irradiated tumor cells, are irrelevant in such circumstances.

  Another way of eliciting an immune response can be achieved by formulating the VLPs of the invention together with an “immune stimulant”. These are internal chemical messengers (cytokines) to increase the immune system's response. Immunostimulatory agents include, but are not limited to, various cytokines, lymphokines, and chemokines that have immunostimulatory, immune-enhancing, and pro-inflammatory effects, such as interleukins (eg, IL-1, IL-2) , IL-3, IL-4, IL-12, and IL-13); growth factors such as granulocyte macrophage (GM) colony stimulating factor (CGF); and other immunostimulatory molecules such as macrophage inflammatory factor, Flt3 Ligands, B7.1 and B7.2, etc. The immunostimulatory molecule can be administered in the same formulation as the influenza VLP or can be administered separately, the protein or an expression vector encoding the protein. Any of these can be administered to produce an immunostimulatory effect.

  Where appropriate, the sugar glass-related VLPs of the invention can be administered to animals, for example. The VLP and sugar glass composition of the present invention may comprise influenza, HIV, RSV, VZV VLPs.

  The VLP and sugar glass composition can be administered to a patient by topical application. For example, the powder particles can be directly kneaded into an ointment, a topical carrier, a pressurized liquid, a gas propellant, and / or a penetrant for application to the patient's skin. Alternatively, the powder particles can be reconstituted, for example in an aqueous solvent, and then mixed with other components prior to application.

  The VLP and sugar glass composition can be administered by inhalation. Dry powder particles having an aerodynamic diameter of less than about 10 μm can be inhaled into the lung for pulmonary administration. Optionally, powder particles having an aerodynamic diameter of about 20 μm or more can also be administered intranasally or into the upper respiratory tract, in which case they move from the air stream onto the patient's mucosa by inertial impact. Alternatively, the powder particles can be reconstituted as an aqueous mist into a suspension or solution for inhalation administration.

  The VLP and sugar glass composition can be administered by infusion. The powder particles can be administered directly into the patient's skin, for example using a jet of high pressure air. More generally, the powder particles can be reconstituted with a sterile aqueous buffer, for example, for injection by a hollow injection needle. Such infusion can be appropriate, for example, intramuscular, intravenous, subcutaneous, intrathecal, intraperitoneal, and the like. The powder particles of the present invention are suitable for dosage and handling considerations, for example, from less than about 0.1 ng / ml to less than about 1 mg / ml to about 500 mg / ml, or from about 5 mg / ml to about 400 mg / ml. It can be reconstituted into a solution or suspension at bioactive substance concentrations up to The reconstituted powder particles can be further diluted, for example, for multiple vaccinations, for administration by IV infusion.

  Appropriate doses of these VLP substances (“therapeutically effective amount”) are, for example, the condition being treated, the severity and course of the condition, whether the administration of the bioactive substance is for prophylactic or therapeutic purposes, It will depend on the medical history, the patient's medical history and its response to the bioactive substance, the type of bioactive substance used, and the discretion of the attending physician. These VLPs are suitably administered to the patient once or over a series of doses and can be administered to the patient at any time. These VLPs can be administered as the sole treatment or in conjunction with other drugs or therapies that help treat the condition in question.

  As a general suggestion, the therapeutically effective amount of VLP administered is in the range of about 0.00001 to about 50 mg / kg of patient body weight, whether only once or more than once, Typical ranges of proteins used are, for example, less than about 0.01 ng / kg to about 20 mg / kg, more preferably about 0.1 mg / kg to about 15 mg / kg for daily administration (with antigenic protein on the VLP). Will be measured). However, other dosing schedules are also effective. The progress of this therapy is easily monitored by conventional techniques.

  The present invention also encompasses methods for increasing the “shelf life” or storage stability of VLPs stored at elevated temperatures. Increased storage stability can be determined by restoring biological activity in accelerated aging experiments. The dry particle composition produced by the method of the present invention can be stored at any suitable temperature. Preferably these compositions are stored at about 0 ° C to about 80 ° C. More preferably, these compositions are stored at about 20 ° C to about 60 ° C. Most preferably these compositions are stored at ambient temperature.

Example 1: Lyophilization of VLPs H5N1 clade 2 influenza VLPs were co-pending US patent application Ser. No. 11 / 582,540 filed Oct. 18, 2006, which is incorporated herein by reference in its entirety. ) And purified according to the method described in). VLPs were purified and suspended in phosphate buffered saline pH 7.2 with the desired concentration of NaCl in a PETG bottle with the desired concentration of sugar. The prescription is described in Table 1.

  Next, 1.2 ml of this formulation (aqueous suspension) was aliquoted into 2 ml USP Type I glass bottles. The efficacy of these formulations was in the range of 40-80 μg HA / ml. Efficacy was measured using the quantitative simple radial immunodiffusion (SRID) method. Lyo-stopper (13 mm, gray chlorobutyl) was placed on the jar and all these formulations were vitrified by lyophilization according to the parameters outlined in Table 2 using a 2 square foot Hull 2FS48C lyophilizer. It was used for.

  After vitrification, the material in each bottle was reconstituted with 1.2 ml of sterile water for injection (WFI) and then analyzed for VLP efficacy and stability (after lyophilization).

Example 2: VLP analysis after vitrification After reconstitution, studies were performed for simple radial immune diffusion (SRID), total protein analysis, particle size analysis, HA and NA activity. Total protein was measured by the BCA method (using a commercial kit using bicinchoninic acid). The particle size was measured using a Malvern zeta sizer (dynamic light scattering principle). The HA activity was measured by the hemagglutination method using Turkey RBC, and the NA activity was measured by the fluorometric method using Munana reagent. In addition, SDS PAGE and Western blot analysis were performed. Tables 3 to 7 and FIG. 1 summarize the results of these analyses.

  FIGS. 1A and B compare VLPs containing HA and NA before and after vitrification by SDS and Western blot. After VLP vitrification as shown, there is no difference in the amount of HA and NA as shown in the gels. Vitrification therefore does not reduce or destroy VLPs or antigens expressed on VLPs.

  These results indicate that the control, Cryo-II, and Cryo-V formulations most effectively maintained the physical, chemical, and biological properties of VLPs upon lyophilization. The protective effect (control) in the absence of sugar was surprising. However, the advantages of sugar glass can be clarified from a study of stability at higher temperatures.

Example 3: Stability of VLPs at elevated temperatures The recommended storage temperature for aqueous suspensions containing VLPs is 4-8 ° C. Lyophilized bottles (control, Cryo-II, and Cryo-V, 5 or 12 weeks) were stored at 25 ° C. and 50 ° C. to determine the protective effect of sugar on VLP stability. The VLPs were analyzed using the same method as above. The results are summarized in the table below (FD = lyophilized).

  There appeared to be no significant difference between the control and sugar vitrification product data up to 5 weeks at 25 ° C., but there was a significant decrease in potency (SRID value) at 12 weeks for the control. At 50 ° C., the control formulation completely lost its potency within 5 weeks and showed aggregation and loss of bioactivity (as indicated by HA titer concentration).

  Sugar vitrification formulations (Cryo-II and Cryo-V) maintained their potency, particle size, and HA titer concentration at 25 ° C. for up to 3 months. At 50 ° C., about 13% and 45% potency loss was observed for Cryo-II and Cryo-V, respectively, while particle size and HA titer concentration remained unchanged. Trehalose appeared to preserve VLP integrity over the trehalose + mannitol combination.

Example 4: Spray drying of VLPs H5N1 clade 2 influenza VLPs were co-pending US patent application Ser. No. 11 / 582,540 filed Oct. 18, 2006, which is incorporated herein by reference in its entirety. ) And purified according to the method described in).

  A placebo was prepared with a 6 w / v% solution of trehalose by dissolving 24 g of trehalose in 400 ml of phosphate buffered saline (pH 7.2) using 0.15 M NaCl. The solution was sterilized by passing through a 0.22 μ membrane filter.

  VLPs were recovered (potency of about 45 μg HA / ml) in phosphate buffered saline, pH 7.2 with 0.15 M NaCl in PETG bottles. Trehalose was then added to make up a 6 w / v% solution. This solution was gently mixed (handshaking) until all the trehalose was dissolved.

  A spray dryer, ie SD-Micro (Niro) equipped with a glass chamber (2.4 / 3/4 mm Morprene tube with “Y” shape) was then prepared and operated under the following conditions. Compressed air was used as the drying air. Table 11 shows the conditions of each test.

  After reconstitution, investigations were performed on the simple radioimmune diffusion (SRID) method, HA titer concentration, particle size, and NAA (as before). Furthermore, SDS PAGE blot analysis was performed. The results are summarized in Table 12 and on the gel of FIG.

  These data show that the actual potency (the potency obtained in units of μg HA / mg of dry powder after spray drying) is the theoretical potency (substance input, eg amount of VLP, trehalose, about 5-10% moisture on drying) And potency estimated by calculation based on recovery over 95%), and thus retains the original properties of VLP after spray drying. In addition, SDS PAGE and Western blot gel (FIG. 1A) showed no band shift or broadening before and after lyophilization in any of these formulations, and the three major proteins (HA, NA, and M1) were intact. It shows that it is a state.

  All publications, patents, and patent applications in this specification are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated and incorporated by reference. .

  The foregoing detailed description has been provided for clarity of understanding only, and several modifications will become apparent to those skilled in the art, but unnecessary limitations should not be inferred therefrom. Any publication provided that any of the information provided herein is prior art or pertains to the presently claimed invention, or is specifically or implicitly referenced. Is not admitted to be prior art.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

  Although the invention has been described in connection with specific embodiments thereof, it is to be understood that the invention is amenable to further modifications and that this application is generally in accordance with the principles of the invention and as described below. Deviations from the disclosure of the invention, i.e., as applicable to the essential features previously described herein, as within the well-known or customary manner within the relevant art of the present invention, It should also be understood that it encompasses any variation, application or modification of the invention including deviations from the scope of the appended claims.

Claims (55)

  A composition comprising at least one virus-like particle (VLP) and a sugar glass.   The composition according to claim 1, wherein the sugar glass is composed of a monosaccharide and / or a disaccharide.   The composition of claim 2, wherein the monosaccharide is selected from the group consisting of glucose, sorbitol, galactose, mannose, and mannitol.   The composition of claim 2, wherein the disaccharide is selected from the group consisting of trehalose, maltose, maltotriose, lactose, lactulose, and sucrose.   The composition of claim 1, wherein the VLP and sugar glass are powders.   6. The composition of claim 5, wherein the powder is produced by spray drying, freeze drying, pulverizing, or a combination thereof.   7. The composition of claim 6, wherein the powder has an average particle size between about 0.1 nm to about 100 microns.   6. The composition of claim 5, wherein the powder is suspended in a non-aqueous solvent that does not dissolve the sugar glass.   The non-aqueous solvent is selected from the group consisting of triacetin, isopropyl myristate, medium chain triglycerides, short / medium / long chain mono / di / triglycerides, aliphatic and aromatic alcohols, or combinations thereof; The composition according to claim 8.   6. The powder of claim 5, wherein the powder is administered to an animal orally, by inhalation, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, subcutaneously, or mucosally (eg sublingual or buccal). Composition.   The composition of claim 1, wherein the VLP comprises at least one influenza protein.   12. The composition of claim 11, wherein the influenza protein is HA or NA.   The composition of claim 1, wherein the VLP comprises at least one RSV protein.   The composition of claim 1, wherein the VLP comprises at least one VZV protein.   A composition comprising at least one VLP and a sugar glass, wherein the VLP has increased stability when compared to a composition without sugar glass.   The composition of claim 15, wherein the increased stability is increased thermal stability.   The composition according to claim 15, wherein the sugar glass comprises a monosaccharide or a disaccharide.   18. The composition of claim 17, wherein the monosaccharide is selected from the group consisting of glucose, sorbitol, galactose, mannose, and mannitol.   18. The composition of claim 17, wherein the disaccharide is selected from the group consisting of trehalose, maltose, maltotriose, lactose, lactulose, and sucrose.   16. The composition of claim 15, wherein the stabilized sugar glass VLP is a powder.   21. The composition of claim 20, wherein the powder is produced by spray drying, milling, or a combination thereof.   24. The composition of claim 21, wherein the powder has an average particle size between about 0.1 nm and about 100 microns.   21. The composition of claim 20, wherein the powder is suspended in a non-aqueous solvent that does not dissolve the sugar glass.   21. The composition of claim 20, wherein the powder is administered to an animal orally, by inhalation, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously.   16. The composition of claim 15, wherein the VLP comprises at least one influenza protein.   26. The composition of claim 25, wherein the influenza protein is HA or NA.   16. The composition of claim 15, wherein the VLP comprises at least one RSV protein.   16. The composition of claim 15, wherein the VLP comprises at least one VZV protein.   16. The composition of claim 15, wherein the composition further comprises an adjuvant.   A dry powder formulation comprising VLP and sugar glass.   The dry powder formulation according to claim 30, wherein the sugar glass is composed of a monosaccharide or a disaccharide.   31. The dry powder formulation of claim 30, wherein the monosaccharide is selected from the group consisting of glucose, sorbitol, galactose, mannose, and mannitol.   32. The dry powder formulation of claim 31, wherein the disaccharide is selected from the group consisting of trehalose, maltose, maltotriose, lactose, lactulose, and sucrose.   32. The dry powder formulation of claim 30, wherein the dry powder is produced by spray drying, pulverizing, or a combination thereof.   35. The dry powder formulation of claim 34, wherein the powder has an average particle size between about 0.1 nm to about 100 microns.   32. The dry powder formulation of claim 30, wherein the powder is administered to animals by oral, inhalation, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously.   40. The dry powder formulation of claim 36, wherein the powder is reconstituted in a solvent prior to administration.   37. The dry powder formulation of claim 36, wherein the powder is administered to an animal by inhalation using an inhaler and / or subcutaneously using a jet of high pressure air.   A method of delivering a sugar glass stabilized VLP comprising reconstituting a solid sugar vitrified VLP in a solvent and administering the reconstituted VLP to an animal.   40. The method of claim 39, wherein the powder is administered to an animal orally, by inhalation, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously.   A method for delivering sugar glass stabilized VLPs comprising administering solid sugar vitrified VLPs by inhalation and / or infusion.   42. The method of claim 41, wherein the infusion is administered by injection of high pressure air.   A method for increasing the thermal stability of a VLP, comprising formulating said VLP in a sugar glass.   44. The method of claim 43, wherein the sugar glass consists of monosaccharides and / or disaccharides.   45. The method of claim 44, wherein the monosaccharide is selected from the group consisting of glucose, sorbitol, galactose, mannose, and mannitol.   43. The sugar glass stabilized VLP of claim 42, wherein the disaccharide is selected from the group consisting of trehalose, maltose, maltotriose, lactose, lactulose, and sucrose.   44. The method of claim 43, wherein the sugar glass comprising VLP is a powder.   48. The method of claim 47, wherein the powder is produced by spray drying, milling, or a combination thereof.   49. The method of claim 48, wherein the powder has an average particle size between about 0.1 nm and about 100 microns.   48. The method of claim 47, wherein the powder is suspended in a non-aqueous solvent that does not dissolve the sugar glass.   44. The method of claim 43, wherein the VLP comprises at least one influenza protein.   52. The method of claim 51, wherein the influenza protein is HA or NA.   44. The method of claim 43, wherein the VLP comprises at least one RSV protein.   44. The method of claim 43, wherein the VLP comprises a VZV protein.   The composition comprises at least one excipient selected from the group consisting of a buffer, carrier, preservative, colloidal stabilizer, filler, diluent, lubricant, amphiphile, and stabilizer. The composition of claim 1.

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