Orally dispersible vaccines comprising virosomes

Abstract

The present disclosure relates to oral vaccine dosage forms and methods for producing oral vaccine dosage forms. The dosage forms include lipid-based vesicles (e.g., virosomes, liposomes) having an immunogenic amount of at least one vaccine target molecule, with or without an adjuvant. Specifically, the applicant has discovered a combination of a composition of a liquid virosome concentrate, a composition of a base matrix for solid dosage form formulation (excluding the virosome concentrate), and manufacturing conditions for the dosage form that can produce a freeze-dried sublingual dosage form with physical robustness, particle and antigenic integrity and stability.

Description

[0001] Citation of Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 772,823, filed on November 29, 2018, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to oral dosage forms that induce mucosal immunity. More specifically, the present disclosure relates to freeze-dried orally dispersible vaccines containing virosomes. The project leading to this application has been funded from the European Union's Horizon 2020 research and innovation project ( European Union's Horizon 2020 research and innovation program ) received funding under Grant Agreement No. 646122. Background Art

[0004] Vaccine is delivered by intramuscular, intradermal or subcutaneous injection traditionally.These injections can produce strong systemic immune response, and the effect for triggering mucosal immune response is variable, and often weak or undetectable, particularly for subunit vaccines.From the draining lymph nodes of processed injection vaccine, antigen-specific cytotoxic T cells (CTL) and antibodies produced by B cells can migrate to different organs in vivo, but due to homing mucosal receptors and chemotaxis deficiency, it is often limited or impossible to the migration of various mucosal tissues (such as reproductive, intestinal, respiratory).However, the intranasal approach also regarded as parenteral immune pathway can trigger good mucosal immune response in sharing some interconnected respiratory tracts, reproductive tracts and intestinal tracts, if vaccine is delivered at mucosal sites, then it is more easily obtained.Therefore, this type of parenteral vaccine can provide protection for mucosal pathogens in some cases.

[0005] Because most pathogens enter the body through mucosal tissues (oral, respiratory, genital, and intestinal), and many of them replicate only in mucosal tissues, mucosal vaccination can optimally induce a first line of defense by inducing both innate (e.g., NK cells) and adaptive (T and B cells) immune responses at local and distant mucosal sites.

[0006] With resident mucosal defenses, protection can be immediate without the delay of cell recruitment from the periphery, allowing for more effective interference with very early transmission and infection events before pathogens spread and, in some cases, reservoirs are established.

[0007] Mucosal tissue and lymph nodes contain more than 90% immune cells. In addition, mucosal antibodies represent about 80% of the body's total antibody production. Therefore, the local immune response by mucosal antibodies can serve as a first-line defense for mucosal infections (such as HIV-1, herpes virus, rotavirus, etc.), and cross mucosal tissue to enter to reach other organs (such as HIV-1, hepatitis B, tuberculosis). In contrast, once pathogens have crossed mucosal defense, blood antibodies can serve as backup defense, or synergize with mucosal antibodies and mucus environment. Blood antibodies mainly serve as an effective first-line defense, for responding to pathogens (such as malaria, chikungunya fever, dengue fever, Zika virus, West Nile virus, yellow fever, etc.) or accidental skin / mucosal damage (such as Staphylococcus aureus ( Staphylococcus aureus ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa )).

[0008] As a means of inducing local and distant antibody immune responses and systemic immune responses, mucosal vaccine delivery (via buccal, sublingual, nasal, oral or vaginal mucosa) has received increasing attention. In addition, mucosal vaccine delivery by solid dosage forms (such as buccal / sublingual tablets, oral tablets or capsules, vaginal inserts) can provide several advantages, such as the potential for large-scale immunization, patient compliance, ease of use, product shelf life stability, cold chain independence ability. In addition, mucosal vaccine delivery can be suitable for patients with needle injection phobia, and patients can self-administer vaccines in the case of full explanation. The buccal / sublingual route has been used for many years to deliver drugs and small molecules to the bloodstream, but its application as a mucosal delivery means for vaccines is rarely concerned. Summary of the invention

[0009] Vesicles based on lipids can be used as drug, vaccine or adjuvant delivery systems, and combinations thereof. Vesicles based on lipids can be composed of one or more natural and / or synthetic lipids and other optional components (peptides, proteins, carbohydrates, nucleic acids, small molecules) that form a basic structure (particle). The range of lipid-based vesicles can range from nanoparticles (approximately 20-200 nm) to submicron (approximately 200-800 nm) to micron (approximately 800 nm - 10 μm) scales.

[0010] Virosome and liposome belong to lipid-based vesicle system.Virosome is unilamellar, and liposome can be unilamellar, bilayer or multilayer.Virosome is a class of subunit vaccine, which can contain any envelope virus derived protein, which is used as the raw material for forming lipid-based virosome particles.These virosomes can lack genetic material, are non-replicative and non-infectious, which makes them suitable and safe for intestinal and parenteral immunity, provided that they can be formulated in stabilized form.Virosome may contain other molecules in the form of peptides, proteins and / or carbohydrates, such as homologous (same pathogen origin) or heterologous target molecules (derived from pathogens different from starting viral materials), nucleic acids, adjuvants, specific lipids and / or small molecules (drugs).Similar to virosome, liposome can also be used as a vehicle for pharmaceutical drugs, vaccines and adjuvant administration, but lipid membrane does not contain any native viral protein.In some embodiments, liposome used herein can be proteoliposome (that is, liposome with protein).

[0011] Ideally, stabilization would either allow for storage of the vaccine independent of the cold chain, or allow the vaccine to support high and low temperature excursions outside of recommended cold chain conditions without compromising the biological activity of the product.

[0012] The applicant has developed a freeze-dried orally dispersible vaccine containing a virion, and a method for preparing such a vaccine, which can preserve the stability of the virion (both physically stable for the particle structure and chemically stable for the target molecule). Product stability can be maintained during storage at ambient temperature (e.g., about 25°C), without relying on cold chain storage conditions, and can also support unexpected freezing conditions (e.g., about 4°C to about -17°C) and exposure to high temperatures (e.g., 35°C to 45°C) present in warm countries. The freeze-dried sublingual dosage form containing a virion vaccine disclosed herein can induce mucosal immunity and can also induce a systemic immune response. Specifically, a composition of a liquid virion concentrate and its buffer, a composition of a liquid base matrix for a solid dosage form (excluding a virion concentrate), and a combination of manufacturing conditions for a solid dosage form for generating a freeze-dried sublingual tablet can generate a solid vaccine form with appropriate physical properties for a sublingual tablet, with the integrity of the virions and target molecules preserved, and stability during storage under different environmental conditions. Administration of a stable virion solid dosage form can be suitable for sublingual mucosal immunization.

[0013] Virosomes belong to the family of enveloped virus-like particles ("VLPs") because they have a lipid membrane containing viral membrane proteins derived from the initial purified virus. As VLPs, virosomes can closely simulate the viral particle architecture, composition, antigenic membrane surface presentation, with or without the functional activity of the natural viral envelope. Virosomes may include the viral membrane generally obtained by the following reconstruction: the membrane proteins and lipids of solubilization are purified from the enveloped virus with a solubilizing agent, followed by the addition of natural or synthetic lipids and antigens, with or without adjuvants, and the solubilizing agent is removed from the mixture, resulting in the formation of a lipid bilayer with proteins protruding therefrom. Antigens can be natural proteins, as well as recombinant or synthetic proteins or peptides. Virosomes can also be formed first, and then modified by covalent or non-covalent modification of their membranes, to contain adjuvants and other molecules. The unique feature of virosomes is that they can closely simulate the composition, surface architecture and functional activity of the natural viral envelope. In some cases, when the induction of CD8+ T cells is a part of the vaccine strategy, the particularly important characteristic of the virosome is the retention of hemagglutinin (HA) receptor binding and its fusion membrane activity. If the vaccine strategy focuses on the induction of antigen-specific antibodies, HA fusion activity is not absolutely necessary, but the presence of HA as an excipient is still important because it can provide T cell assistance, especially for small antigens and peptides that may lack such properties. Although specific vaccines are mentioned in the present disclosure, all vaccines using virosomes as delivery platforms are covered by the present disclosure. Influenza virosomes containing HIV antigens (P1 and rgp41) and TLR 7 / 8 adjuvants can be used as model virosome concentrates for the vaccines disclosed herein. Influenza virosomes with HIV antigen rgp41 have been shown to be able to induce immune responses via intranasal and intramuscular routes in various animal studies and phase 1 clinical studies. In such studies, the virosome preparation is in a liquid form administered by needle or nasal liquid spray, which is prone to limited shelf life stability at 4°C due to chemical modification of the active pharmaceutical ingredient ("API"). In order to prevent or reduce such API chemical modifications that are often present in aqueous environments, the development of a solid vaccine form with a low moisture content has been identified as a promising method.

[0014] Applicants are able to overcome various problems in producing solid vaccine forms of orally dispersible vaccines as freeze-dried disclosed herein. Specifically, a flu-based virosome concentrate containing a virosome carrying an antigen and an adjuvant can be supplied as a suspension in a buffered saline solution. In order to produce freeze-dried vaccine dosage forms, a stable and porous structure in a tablet should be produced during the manufacturing process and maintained during subsequent storage. The presence of buffer salts in the virosome concentrate may present a challenge in manufacturing such a stable and porous structure. Therefore, the applicant has found that an appropriate amount of excipient is combined with manufacturing conditions to enable the formation of a stable dosage form that does not collapse or partially collapse during the freeze-drying process.

[0015] In addition, freezing, annealing and freeze drying processes may also damage the integrity of virion particles. This damage to virions should be minimized so that enough virion particles are retained in the freeze-dried product so that a sufficient amount of virion particles can cross the sublingual mucosa for being processed by immune cells to induce mucosal immune responses, which may also be supported by systemic immune responses. Therefore, the applicant can balance excipient use and freeze drying process to maintain the immunogenicity of virions (e.g., complete virions with limited clusters) and good dosage form properties (e.g., sublingual tablets that disintegrate orally).

[0016] In some embodiments, the oral solid vaccine dosage form includes a lipid-based vesicle comprising an immunogenic amount of at least one target molecule; 5-20% by weight of at least one cryo-lyophilized protective agent; 25-40% by weight of a matrix forming agent; and 40-55% by weight of a structure forming agent. In some embodiments, the lipid-based vesicle is a virosome or a lipoprotein. In some embodiments, the dosage form comprises 10-15% by weight of at least one cryo-lyophilized protective agent. In some embodiments, at least one cryo-lyophilized protective agent comprises trehalose. In some embodiments, the dosage form comprises 33-37% by weight of a matrix forming agent. In some embodiments, the matrix forming agent comprises gelatin. In some embodiments, the gelatin comprises fish gelatin. In some embodiments, the fish gelatin is a high molecular weight fish gelatin. In some embodiments, the dosage form comprises 45-50% by weight of a structure forming agent. In some embodiments, the structure forming agent comprises mannitol. In some embodiments, the virosome is derived from an influenza virus membrane or other enveloped virus. In some embodiments, at least one target molecule is present on the virosome. In some embodiments, at least one target molecule comprises an HIV-1 envelope-derived antigen. In some embodiments, the HIV-1 envelope-derived antigen comprises HIV-1 PI peptide and / or HIV-1 recombinant gp41. In some embodiments, the virion comprises an adjuvant. In some embodiments, the dosage form promotes oral intake of at least one target molecule. In some embodiments, the dosage form disintegrates within 180 seconds after being placed in the oral cavity. In some embodiments, the dosage form disintegrates within 90 seconds after being placed in the oral cavity. In some embodiments, the dosage form disintegrates within 60 seconds after being placed in the oral cavity. In some embodiments, the dosage form disintegrates within 30 seconds after being placed in the oral cavity. In some embodiments, when applied to a patient by being placed in the oral cavity, an immune response is induced. In some embodiments, being placed in the oral cavity is placed on the tongue or under the tongue or in the buccal or pharyngeal region.

[0017] In some embodiments, the method of inducing an immune response in a patient comprises placing any of the above dosage forms in the oral cavity of an individual in need of an immune response. In some embodiments, placing in the oral cavity is placing on or under the tongue or in the buccal or pharyngeal region.

[0018] In some embodiments, the method of forming an oral solid vaccine dosage form comprises dosing a liquid virosome preparation into a preformed mold, wherein the virosome preparation comprises: a lipid-based vesicle comprising an immunogenic amount of at least one target molecule, 1-5% by weight of a cryo-lyophilization protectant, 4-8% by weight of a matrix forming agent, and 5-10% by weight of a structure forming agent; freezing the dosing virosome preparation at a temperature of -60°C to -90°C; annealing the frozen virosome preparation by keeping it at a temperature below -15°C for 3-9 hours; and freeze-drying the annealed virosome preparation to form a dosage form. In some embodiments, the dosing virosome preparation is frozen at a temperature of -60°C to -90°C for a duration of about 1-5 minutes. In some embodiments, freeze drying the annealed virion preparation comprises: a first step of keeping the annealed virion preparation at a temperature of -10°C to -20°C for 20-28 hours, and a second step of keeping the annealed virion preparation at a temperature of -5°C to about -15°C for 14-22 hours. In some embodiments, freeze drying occurs at a pressure of less than 600 mbar. In some embodiments, the virion preparation has a pH of about 6.5-8. In some embodiments, the freeze-lyophilization protectant comprises trehalose. In some embodiments, the matrix forming agent comprises gelatin. In some embodiments, the gelatin comprises fish gelatin. In some embodiments, the fish gelatin is a high molecular weight fish gelatin. In some embodiments, the structure forming agent comprises mannitol. In some embodiments, the lipid-based vesicle is derived from influenza virus or respiratory syncytial virus. In some embodiments, at least one target molecule comprises an HIV-1 envelope-derived antigen. In some embodiments, the HIV-1 envelope-derived antigen comprises an HIV-1 PI peptide or HIV-1 recombinant gp41. In some embodiments, the lipid-based vesicles comprise an adjuvant.

[0019] In some embodiments, a method of forming an oral solid vaccine dosage form comprises: dosing a liquid virosome preparation into a preformed mold, wherein the virosome preparation comprises: (1) 20-50% by weight of a virosome concentrate, wherein the virosome concentrate comprises: virosomes comprising an immunogenic amount of at least one target molecule; 2-10% by weight of a cryo-lyophilization protectant; and 60-200 mM of a buffer system; (2) 4-8% by weight of a matrix forming agent; and (3) 5-10% by weight of a structure forming agent; freezing the dosing virosome preparation at a temperature of -60°C to -90°C; annealing the frozen virosome preparation by keeping it at a temperature below -15°C for 3-9 hours; freeze-drying the annealed virosome preparation to form a dosage form. In some embodiments, the buffer system comprises HEPES-sodium chloride.

[0020] Additional advantages will be apparent to those skilled in the art from the following detailed description.The examples and descriptions herein are to be considered illustrative in nature and not restrictive.

[0021] All publications, including patent documents, scientific papers, and databases, mentioned in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. If definitions set forth herein are contrary to or inconsistent with definitions set forth in patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth herein take precedence over those incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Exemplary embodiments are described with reference to the accompanying drawings, in which:

[0023] Figure 1 An overview of a flow chart for producing the vaccine dosage forms disclosed herein is shown.

[0024] Figure 2 A flow chart for producing the vaccine dosage forms disclosed herein, from matrix formulation to the final sublingual tablet, is shown.

[0025] Figure 3A is a schematic diagram of a picture with a completely wetted tablet.

[0026] Figure 3B is a schematic diagram with a picture of a tablet containing a hard mass.

[0027] Figure 3C is a schematic diagram with a picture of a tablet having a film (skin) of collapsed formulation matrix formed at the surface of a freeze-dried tablet.

[0028] Figure 4 are photographs of immunoblots showing the binding of anti-P1 and anti-rgp41 specific antibodies to virosome P1 and virosome rgp41 from reconstituted Zydis® sublingual tablets stored at 5°C, 25°C and 40°C for 1 month and 3 months (analysis of Example 2 disclosed herein).

[0029] Figure 5 The immunogenicity of the P1 and rgp41 antigens over time for a liquid virosome concentrate and a freeze-dried sublingual tablet containing the virosomes, both stored at 4°C and 40°C, is shown. DETAILED DESCRIPTION

[0030] Disclosed herein are pharmaceutical compositions of lipid-based vesicles (e.g., virions or VLPs), and methods for preparing these pharmaceutical compositions. Specifically, the disclosure relates to freeze-dried orally dispersible or disintegrating dosage forms that can preserve the stability (i.e., structural integrity and antigenic chemical stability) of VLPs, can be stored independently of cold chain storage conditions, and can also support unexpected freezing conditions and exposure to high temperatures present in warm countries. These dosage forms can also retain the physical and chemical properties of VLPs, making them suitable for sublingual delivery to induce mucosal immunity.

[0031] Applicants are able to formulate and optimize the amount of matrix forming agents and structure forming agents to have a formulation that can be used in high-loaded virion concentrates containing a buffer system and a freeze-freeze protection agent. Combined with the excipient adjustment, the manufacturing parameters of the dosage form are optimized. Specifically, the annealing time is optimized to maximize the mannitol crystallization, which imparts stability to the dosage form and minimizes virion damage. In addition, the freeze drying conditions are optimized to minimize damage to the virion particles and minimize structural collapse during freeze drying.

[0032] Lipid-based vesicle systems

[0033] Vesicles based on lipids can be used as medicine, vaccine or adjuvant delivery systems, and combinations thereof. Vesicle systems based on lipids can be composed of: one or more natural and / or synthetic lipids (e.g., phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, cholesterol, sphingolipids and derivatives thereof) forming vesicle membranes (particles) and other optional components (peptides, proteins, carbohydrates, small molecules). Vesicles based on lipids range from nanoparticles (about 20-200 nm) to submicron (about 200-800 nm) to micron (about 800 nm - 10 μm) scales.

[0034] Vesicle / particle based on lipid can comprise single layer, double layer or multilayer lipid bilayer vesicle.In some embodiments, vesicle / particle based on lipid can comprise lipid bilayer, and it comprises the lipid selected from natural and / or synthetic lipid.Such lipid can be used for simulating pathogen film and lipid raft microdomain better, so that improve antigen film anchoring, antigen presentation and / or folding, for best epitope exposure.Vesicle / particle based on lipid can have the surface of being exposed to particle or point to particle interior or have membrane anchored antigen and / or adjuvant of random orientation.Antigen and / or adjuvant also can be encapsulated in the cavity of vesicle / particle based on lipid.

[0035] Virion is the vesicle based on lipid that assembles in vitro in the mode of cell-free system, forms the envelope VLP that belongs to the subunit vaccine category.As the viral lipid membrane of carrier, can be derived from any envelope virus, and thus contain the natural viral membrane protein from initial virus at least.These virions lack hereditary virus material, can not replicate and are non-infectious, and this makes them suitable and safe for whole body and mucosal immunity.In addition, virion may contain other molecule, for example antigen (for example peptide, protein, carbohydrate, nucleic acid), adjuvant, specific lipid and / or small molecule (drug), it can be anchored in virion surface place and / or be trapped in virion cavity.

[0036] Liposome is also a lipid-based vesicle, forming a class of subunit vaccines, which can be formed as a vesicle with at least one lipid bilayer, which does not contain proteins derived from the natural viral membrane of enveloped viruses. Such lipid-based particles also assembled in vitro lack genetic material and can be suitable for systemic and mucosal applications. In addition, liposomes may contain other molecules, such as antigens (peptides, proteins and / or carbohydrates, nucleic acids), adjuvants, specific lipids and / or small molecules (drugs), which can be anchored at the liposome surface and / or trapped in the liposome cavity. In some embodiments, the liposome used herein can be a proteoliposome (that is, a liposome with protein).

[0037] The lipid used in the dosage form described herein can belong to cationic lipids, glycolipids, phospholipids, glycerophospholipids, galactosylceramide, sphingolipids, cholesterol and derivatives thereof. Phospholipids can include, but are not limited to, phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, cardiolipin and phosphatidylinositol with different fatty acyl groups.

[0038] In addition, the lipid can be selected from DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride, DODAC (N,N-dioleyl-N,N,-dimethylammonium chloride), DDAB (didodecyldimethylammonium bromide) and stearylamine or other fatty amines, etc.

[0039] Virus preparation

[0040] Virus Concentrate

[0041] Figure 1A flow chart for a method 100 for producing a vaccine dosage form disclosed herein is shown. In step 101, a liquid virosome concentrate can be mixed with a premix of a base matrix formulation to form a liquid virosome formulation suitable for freeze drying. Applicants have discovered that the biological activity of the vaccine can be maintained when the composition of the virosome concentrate is combined with a specific composition of the base matrix formulation and prepared in conjunction with a set of manufacturing conditions that are optimized to preserve sufficient virosomes with the desired particle properties. The biological activity of the virosome particles can depend on the conformational integrity of the particles, as well as the quality of the antigenic molecules associated with their monolayer phospholipid membrane.

[0042] In some embodiments, the virosome concentrate can be a liquid virosome concentrate. The virosome concentrate includes at least one virosome population. The virosome population can be virosomes containing a given drug, or virosomes with vaccine antigens and virus-derived proteins (e.g., HA if the virosomes are derived from influenza virus) that serve as vaccine delivery vehicles.

[0043] In some embodiments, virions can be derived from influenza virus, for generating influenza virions as envelope VLP, the envelope VLP serves as a carrier of heterologous vaccine antigens (for example, HIV antigens anchored on influenza-derived virions), or derived from another enveloped virus such as respiratory syncytial virus ("RSV"). In some embodiments, enveloped viruses such as RSV, Sendai virus, Semliki Forest virus (SFV), vesicular stomatitis virus (VSV) or Sindbis (Sindbis), can be used to generate corresponding RSV-virions, Sendai-virions, SFV-virions, VSV-virions or Sindbis-virions, for displaying homologous vaccine antigens (for example, derived from natural RSV antigens on RSV virions). In some embodiments, virions based on viruses can be derived from any enveloped virus. In some embodiments, virions based on viruses can be derived from DNA viruses, including but not limited to herpes virus, poxvirus (Poxyviruse) and hepatotropic DNA virus. In some embodiments, the viral-based virions can be derived from RNA viruses, including but not limited to flaviviruses, togaviruses, coronaviruses, hepatitis delta, orthomyxoviruses, paramyxoviruses, rhabdoviruses, bunyaviruses, and filoviruses. In some embodiments, the viral-based virions can be derived from retroviruses.

[0044] In some embodiments, influenza virus-based virosomes can be formed according to the various methods described below: Influenza virosomes as vaccine adjuvant and carrier system; Moser C. et al., Expert review, 779-791 (2013); WO2004110486; WO2004071492; WO2007099446; WO2016039619; EP2058002; and WO2016039620, the references are hereby incorporated herein by reference in their entirety. Additionally, the various patents and other publications listed in the previously cited references are also incorporated herein by reference in their entirety. Accordingly, the present application is not limited to a specific method for virion preparation. Thus, the above references are merely examples of virion preparation. The present application is applicable to all lipid-based particles, such as but not limited to virions, VLPs, and nanoparticle formulations, including liposomes having antigens.

[0045] Although this section describes a liquid virion concentrate, the virions can be replaced by liposomes to form a liquid liposome concentrate. Thus, the components in the virion concentrate (other than the virions themselves) can be similarly applied to the liposome concentrate.

[0046] Virions (such as influenza-derived virions) can have viral proteins on their surface. In some embodiments, the protein can be hemagglutinin (“HA”) and / or neuraminidase (“NA”). In some embodiments, the liquid virion concentrate includes viral membrane proteins (such as HA) present at a detectable concentration by prior art analytical assays. In some embodiments, when using influenza virions as a carrier for heterologous vaccine antigens, the viral membrane protein is in a liquid virion concentrate of about 10-300 μg / mL or about 5-150 μg / mL. For influenza virions designed to induce a CTL response, the HA concentration range can be about 150-800 µg / mL or about 75-400 µg / mL. In some embodiments, the concentration of the viral membrane protein can be greater than the exemplary concentrations listed above.

[0047] The virosome concentrate may also include lipids. The lipids used in the virosome concentrate may belong to cationic lipids, glycolipids, phospholipids, glycerophospholipids, galactosylceramide, sphingolipids, cholesterol and derivatives thereof. Phospholipids may particularly include phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, cardiolipin and phosphatidylinositol having different fatty acyl compositions. Additionally, the lipid may be selected from DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride, DODAC (N,N-dioleyl-N,N,-dimethylammonium chloride), DDAB (didodecyldimethylammonium bromide), and stearylamine or other fatty amines, among others. In some embodiments, the concentrate may include about 0.1-10 mg / mL, about 0.3-8 mg / mL, or about 0.5-5 mg / mL of lipid.

[0048] The virion can contain a target molecule (e.g., a vaccine antigen), with or without an adjuvant. Antigens can be dissolved and trapped in the virion cavity, or covalently or non-covalently anchored to the virion, and the antigen can be a peptide, protein, polysaccharide, complete or partial fragment or extract, viral particle or nucleic acid of a bacterial cell, or can be derived from a parasite causing a disease, such as a protozoan or worm or a combination thereof, or derived from any plant, animal or human cell or cell line. Any antigen known in the art can be suitable for use with the virion, including commercially available antigens, or prepared, recombinantly expressed, or synthesized by standard manufacturing of a preparation of a purified pathogen or cancer cell or non-transformed cell (natural protein). Methods for generating suitable antigens for incorporation into the virion are known in the art, and any known method can be used as disclosed herein.

[0049] The target molecule is included in the virosomes of the liquid virosome concentrate and the subsequent dosage forms disclosed herein in an amount sufficient to render it immunogenic when provided in the dosage form. An "immunogenic amount" is defined as an amount appropriate to induce the desired immune response. The immunogenic amount for a given disease or infection can be readily determined by one skilled in the art based on, inter alia, the route of immunization, the age and weight of the patient to whom the dosage form is to be administered.

[0050] In some embodiments, the liquid virosome concentrate may include about 1-2000 μg / mL of target molecules. The target molecule may be a peptide, protein, carbohydrate, nucleic acid or small molecule, or a combination thereof. The target molecule may serve as an antigen (e.g., a vaccine antigen), a drug, a diagnostic molecule, an analytical sensor, or a combination thereof. In some embodiments, the liquid virosome concentrate may include about 1-1000 μg / mL of a target molecule and about 1-1000 μg / mL of a second target molecule. After downstream processing into a solid dosage form, each tablet may contain about 0.01 to 250 μg of each target molecule.

[0051] In more preferred embodiments, the liquid virosome concentrate may include about 25-500, about 50-500 µg / mL, about 25-225 µg / mL, about 25-200 µg / mL, about 50-450 µg / mL, about 50-400, about 100-400 µg / mL, about 100-400 µg / mL, about 100-200 µg / mL, or about 200-400 µg / mL of at least one target molecule (e.g., antigen). In some embodiments, the liquid virosome concentrate can include about 1-500 µg / mL, about 25-500 µg / mL, about 50-450 µg / mL, about 50-400 µg / mL, about 25-250 µg / mL, about 25-200 µg / mL, about 50-250 µg / mL, about 75-225 µg / mL, or about 100-200 µg / mL of one target molecule and about 50-450 µg / mL, about 50-400 µg / mL, about 25-225 µg / mL, about 25-200 µg / mL, about 100-500 µg / mL, about 150-450 µg / mL, about 175-425 µg / mL, or about 200-400 µg / mL of a second target molecule.

[0052] The dosage forms disclosed herein can be used to deliver therapeutic or prophylactic vaccines to prevent or alleviate symptoms associated with allergy or infection, tumor development and spread, pathogen dissemination, cellular infection, pathogen load, following the induction of B cells (antibodies) and / or related T cell subsets (e.g., Th1, Th2, Th16, Thf, Tc1, Tc2, Tc3, Treg, etc.) depending on the virosome preparation. To this end, the target molecule can provide protection against the following representative list of diseases, which list is not exhaustive: influenza, tuberculosis, meningitis, hepatitis, whooping cough, polio, tetanus, diphtheria, malaria, cholera, herpes, typhoid, HIV / AIDS, measles, Lyme disease, traveler's diarrhea, hepatitis A, B and C, otitis media, dengue fever, rabies, parainfluenza, rubella, yellow fever, dysentery, legionellosis, toxoplasmosis, Q fever, hemorrhagic fever, Argentine hemorrhagic fever, dental caries, Chagas disease, urinary tract infection caused by E. coli, pneumococcal disease, mumps, chikungunya fever, cancer, allergies and combinations thereof. In addition, the target molecule can provide protection against diseases caused by the following non-exhaustive list of pathogenic organisms: Vibrio ( Vibrio ) species, Salmonella spp.( Salmonella Species, Bordetella Bordetella Species, Haemophilus Haemophilus Species, Toxoplasma gondii ( Toxoplasmosis gondii ), cytomegalovirus, Chlamydia spp. ( Chlamydia species, Streptococcal species, Norwalk virus, Escherichia coli Escherischia coli ), Helicobacter pylori ( Helicobacter pylori ), rotavirus, Neisseria gonorrhoeae ( Neisseria gonorrhae ), Neisseria meningitidis ( Neisseria meningiditis ), adenovirus, Epstein-Barr virus, Japanese encephalitis virus, Pneumocystis carini, herpes simplex, Clostridia species, respiratory syncytial virus, Klebsiella Klebsiella Species, Shigella spp. Shigella species, Pseudomonas aeruginosa, Parvovirus, Campylobacter spp. Camylobacter Species, Rickettsia spp. Rickettsia Species, Varicella zoster, Yersinia spp. Yersinia Species, Ross River virus, JC virus, Rhodococcus equi ( Rhodococcus equi ), Moraxella catarrhalis ( Moraxella catarrhalis ), Borrelia burgdorferi ( Borrelia burgdorferi ), Pasteurella haemolytica ( Pasteurella haemolytica) and combinations thereof. Additionally or alternatively, the target molecule can provide protection or treatment against allergies (i.e., virions containing allergens), cancer (e.g., tumor antigens, antibodies, anticancer drugs, nucleic acids), and other types of conditions.

[0053] Veterinary applications of the present disclosure are also contemplated. Accordingly, the target molecules can provide protection against the following non-exhaustive list of veterinary diseases: coccidiosis, Newcastle disease, enzootic pneumonia, feline leukemia, atrophic rhinitis, erysipelas, foot-and-mouth disease, swine pneumonia, and other disease conditions and other infections affecting companion animals and farm animals, and combinations thereof.

[0054] In some embodiments, the virions contain at least one vaccine antigen in addition to the viral proteins present in the reconstituted membrane. In some embodiments, the vaccine antigen may be HIV-1 PI peptide and / or HIV-1 recombinant gp41.

[0055] As mentioned above, the virion of liquid virion concentrate may also contain adjuvant or be mixed with adjuvant.Virosome and other subunit vaccines may need adjuvant to improve immune response, cause the acceleration and enhancement of antibodies and T cells, and also maintain immune memory.In order to be effective, preferably have the immune response relevant to the generation of memory response, and the memory response provides the long-acting protection from specific diseases.Adjuvant can also allow to reduce antigen dosage (dosage saving) and increase the width of required immune response.Once exposed to antigen, the immune system can "remember" it, and during re-exposure, immune response is much faster.The effectiveness of adjuvant enhanced immune response can not rely on the antigen it is combined with, because independent adjuvant can trigger nonspecific immune response, and if strong cell activation is realized in the absence of antigen, then autoimmune side effect may be caused.However, when antigen and adjuvant are physically linked together, they can all migrate to the same site after injection, which is conducive to antigen-specific immune activation, with lower nonspecific inflammatory response. Suitable adjuvants include, but are not limited to, Toll-like receptor (TLR) agonists, inflammasome agonists, nucleotide binding and oligomerization domain (NOD)-like receptor (NLR) agonists, more specifically, non-toxic bacterial fragments, cholera toxin (and detoxified forms and fractions thereof), chitosan, heat-labile toxin of Escherichia coli (and detoxified forms and fractions thereof), lactide / glycolide homopolymers and copolymers (PLA / GA), polyanhydrides such as trimellitylimido-L-tyrosine, DEAE-dextran, saponins complexed with membrane protein antigens (immunostimulating complexes-ISCOMS), bacterial products such as lipopolysaccharide (LPS) and muramyl dipeptide (MDP), liposomes, cochleates, proteinoids, cytokines (interleukins, interferons), genetically engineered live microbial carriers, non-infectious pertussis mutant toxins, ceramidase / galactose oxidase, and attenuated bacterial and viral toxins derived from mutant strains, and combinations thereof. The appropriate amount of adjuvant can be readily determined by one of ordinary skill in the art.

[0056] In some embodiments, the virosomes may have the adjuvant 3M-052 (TLR7 / 8 agonist supplied by 3M). In some embodiments, the liquid virosome concentrate may contain 3M-052 adjuvant in the range of about 8-140 µg / mL, about 4-70 µg / mL, about 1- 60 µg / mL, and 0.01 to 16 μg / tablet.

[0057] In some embodiments, the liquid virosome concentrate may include at least two different virosome populations, each population having at least one antigen, with or without an adjuvant. In some embodiments, the two different virosome populations may have different antigens but the same adjuvant (e.g., virosome-P1 / 3M-052 mixed with virosome-rgp41 / 3M-052). In some embodiments, the two different virosome populations may have different antigens but different adjuvants (e.g., virosome-P1 / adjuvant A mixed with virosome-rgp41 / adjuvant B).

[0058] In some embodiments, the virosome concentrate can include at least two different antigens / virosomes, with or without an adjuvant (eg, virosomes with both PI and rgp41 antigens, with or without an adjuvant).

[0059] The liquid virosome concentrate may further include a buffer system. In some embodiments, the virosomes may be suspended in a buffer system. The buffer system may maintain the physical integrity and chemical stability of the virosomes in the virosome concentrate. In some embodiments, the virosomes are suspended in a buffer system to maintain a target pH of about 6-9, about 6.5-8, about 7-8, about 7.2-7.6, about 7.3-7.5, or about 7.4. In addition, the buffer system may stabilize the virosomes when the virosomes are in liquid form at a storage temperature of about 2-8°C.

[0060] Suitable buffer systems include, but are not limited to, HEPES-sodium chloride (HN) buffer, HEPES-sodium chloride-EDTA (HNE) buffer, phosphate buffer system (PBS) or a combination thereof. In some embodiments, the buffer system in the virosome concentrate can be about 5-1000 mM, about 60-200 mM, about 100-300 mM, about 125-275 mM, about 150-250 mM, about 175-225 mM, about 180-210 mM, about 185-200 mM, about 185-195 mM, or about 190-195 mM. If the buffer system in the virosome concentrate is HEPES-sodium chloride, the sodium chloride in the virosome concentrate may be about 5-1000 mM, about 50-150 mM, about 125-175 mM, about 130-160 mM, about 130-150 mM, about 135-145 mM, or about 140-145 mM. If the buffer system in the virosome concentrate is HEPES-sodium chloride, the HEPES in the virosome concentrate may be about 1-200 mM, about 10-75 mM, about 10-50 mM, about 25-75 mM, about 30-70 mM, about 40-60 mM, about 45-55 mM, or about 48-52 mM.

[0061] The liquid virosome concentrate may also include at least one freeze-lyophilized protective agent. During the freezing and / or freeze-drying step of producing the dosage form disclosed herein, the virosome may be damaged. Therefore, a freeze-lyophilized protective agent may be included in the virosome concentrate to improve the preservation of the virosome during the freezing and / or freeze-drying step. Examples of freeze-lyophilized protective agents include, but are not limited to, polyols such as trehalose, sugars such as sucrose and amino acids such as lysine, oligosaccharides such as inulin (medium chain oligosaccharides) or combinations thereof. The freeze-lyophilized protective agent used may be inert, to be suitable for vaccine formulations. The liquid virosome concentrate may include about 1-20% w / w, about 1.5-10% w / w, about 2-10% w / w, about 4-10% w / w, about 2-9% w / w, about 2-5% w / w, about 3-8% w / w, about 3.5-8% w / w, about 3.5-7% w / w, about 4-8% w / w, or about 5-7% w / w of a cryo-lyoprotectant.

[0062] The virosome concentrate may be about 1-75% w / w, about 10-65% w / w, about 15-60% w / w, about 20-55% w / w, about 20-50% w / w, or about 25-50% w / w of the virosome preparation. In some embodiments, the virosome concentrate may be about 15-35% w / w, about 20-30% w / w, about 23-27% w / w, or about 25% w / w of the virosome preparation.

[0063] Basic matrix preparation

[0064] The basic matrix preparation helps provide the structure of the final dosage form. Therefore, the basic matrix preparation can include a matrix former. The matrix former can provide a network structure of the dosage form, which imparts strength and elasticity during processing. Suitable matrix formers can include, but are not limited to, gelatin, starch, or a combination thereof. Additional matrix formers can be found in EP 2624815 B1, which is incorporated herein by reference as a whole. Gelatin can be fish gelatin, bovine gelatin, porcine gelatin, or a combination thereof. Each gelatin can have different gelling properties. The degree to which a gelatin solution forms a gel can depend on the concentration of the gelatin and the temperature of the gelatin solution. Bovine gelatin solutions tend to gel at temperatures below 18°C, and can therefore be regarded as gelling gelatin. In contrast, fish gelatin can remain in solution at temperatures as low as 10°C, and can therefore be regarded as non-gelling gelatin. In some embodiments, gelatin can be low endotoxin gelatin, such as gelatin derived from or produced according to the method disclosed in provisional application No. 62 / 640,394, which is incorporated herein by reference as a whole. In some embodiments, the amount of matrix forming agent in the virosome formulation can be about 1-15% w / w, about 2-12, about 3-10% w / w, about 4-8% w / w, about 4-6%, about 5-7% w / w, or about 6% w / w.

[0065] The temperature at which the virosome formulation is dosed can be as low as 10-18°C. Therefore, formulations using bovine gelatin alone may not be dosed at these low temperatures. However, a combination of bovine gelatin and another type of gelatin (e.g., fish gelatin) can be used. Applicants have found that fish gelatin can provide freeze-dried tablets with a stable matrix structure and a disintegration time of about 30-180 or 30-60 seconds, which is desired to affect sufficient contact time with the oral mucosa. In addition, fish gelatin can provide a freeze-dried dosage form with good physical properties for a formulation composition containing a high load of soluble components such as buffer salts, such as the amounts disclosed herein.

[0066] In some embodiments, the fish gelatin can be high molecular weight fish gelatin, standard molecular weight fish gelatin, or a combination thereof. High molecular weight fish gelatin is defined as fish gelatin in which more than 50% of the molecular weight distribution is greater than 30,000 Daltons. Standard molecular weight fish gelatin is defined as fish gelatin in which more than 50% of the molecular weight distribution is less than 30,000 Daltons.

[0067] In some embodiments, the matrix forming agent may also serve as a stabilizer for the antigen as well as a mucoadhesive agent.In addition, starch may also serve as an immunostimulatory excipient.

[0068] The basic matrix preparation can also include a structure forming agent. Suitable structure forming agents can include sugars, including but not limited to mannitol, dextrose, lactose, galactose, cyclodextrin or a combination thereof. Structure forming agents can be used as fillers in freeze drying because they crystallize to provide structural stability to freeze-dried products. Soluble excipients such as buffer salts and trehalose in the virosome preparation can suppress its crystallization. Usually, the annealing time of extension is used to allow crystallization. However, the presence of these soluble excipients can also cause the melting of the frozen product during the annealing process. Therefore, the applicant has found a balance between the amount of structure forming agents, buffer salts and freeze-freeze protection agents and the annealing conditions (i.e., temperature and time). In some embodiments, the amount of structure forming agent in the virosome formulation can be about 1-20% w / w, about 3-15% w / w, about 4.5-10% w / w, about 4.5-8% w / w, about 5-10% w / w, about 6-10% w / w, about 7-9% w / w, or about 8% w / w. Applicants have found that at values ​​below 4.5% w / w structure forming agent, some microstructural collapse may occur during freeze drying, resulting in poor dispersion / disintegration of the freeze-dried dosage form. Thus, higher amounts of structure forming agent have been found to minimize or eliminate microstructural collapse without significantly affecting the virosome.

[0069] In addition, basic matrix preparation can also include freeze-freeze protection agent.The example of freeze-freeze protection agent includes but is not limited to polyol such as trehalose, sugar such as sucrose and amino acid such as lysine, oligosaccharide such as inulin (medium chain oligosaccharide) or its combination.Freeze-freeze protection agent can be used to protect virion from damage during subsequent freezing and freeze drying. However, the interpolation of freeze-freeze protection agent can induce the microstructure collapse of dosage form matrix during freeze drying. Therefore, balance should be achieved so that microstructure collapse is minimized, and sufficient number of virions are preserved simultaneously, to maintain the virion quality for inducing immune response.The amount of freeze-freeze protection agent in basic matrix preparation can be about 0.01-2% w / w, about 0.1-1.5% w / w, about 0.2-1% w / w or about 0.25-0.75% w / w. Thus, the net amount of cryo-lyoprotectant in the virosome preparation (i.e., liquid virosome concentrate plus base matrix preparation) (hereinafter "(net)") may be about 0.5-6% w / w, about 0.5-5% w / w, about 0.5-4.5% w / w, about 1-4.5% w / w, about 1.5-4.5% w / w, about 1.5-3% w / w, about 1.5-2.5, about 2-3% w / w, about 2.5% w / w, or about 2% w / w. Applicants have found that at these levels, the cryo / lyoprotectant can provide adequate cryo-lyoprotection without causing unacceptable microstructural collapse during freeze drying.

[0070] In some embodiments, the base matrix preparation can also include a mucoadhesive such as a gum. Suitable gums include, but are not limited to, gum arabic, guar gum, agar, xanthan gum, gellan gum, carrageenan, curdlan, konjac gum, locust bean gum, welan gum, tragacanth gum, gum arabic, karaya gum, ghatti gum, pectin, dextran, glucomannan and alginate or a combination thereof.

[0071] The basic matrix preparation can also contain other pharmaceutically acceptable reagent or excipient.Such other pharmaceutically acceptable reagent or excipient include but are not limited to sugar such as mannitol, dextrose and lactose, inorganic salts such as sodium chloride and aluminum silicate, gelatin, fish gelatin, modified starch, preservative, antioxidant, surfactant, viscosity enhancer, permeability enhancer, coloring agent, flavoring agent, pH adjusting agent, sweetener, taste masking agent and combination thereof.Suitable coloring agent can include red, black and yellow iron oxide, and FD&C dye such as FD&C blue No. 2 and FD&C red No. 40 and combination thereof.Suitable flavoring agent can include peppermint, raspberry, licorice, orange, lemon, grapefruit, caramel, vanilla, cherry and grape flavoring and these combinations. Suitable pH adjusters may include citric acid, tartaric acid, phosphoric acid, hydrochloric acid, maleic acid, sodium hydroxide (e.g., 3% w / w sodium hydroxide solution), and combinations thereof. In some embodiments, the base matrix formulation and / or virosome formulation has an amount of pH adjuster to maintain a target pH of about 6-9, about 7-8, about 7.2-7.6, about 7.3-7.5, or about 7.4. Suitable sweeteners may include aspartame, acesulfame potassium, and thaumatin, and combinations thereof. Suitable amounts of these various additional excipients may be readily determined by one of ordinary skill in the art, as needed.

[0072] The basic matrix preparation may also include a solvent. In some embodiments, the solvent may be water (e.g., purified water). In some embodiments, the remainder of the basic matrix preparation and / or the virosome preparation is a solvent.

[0073] The basic matrix preparation can be about 25-99% w / w, about 35-90% w / w, about 40-85% w / w, about 45-80% w / w, or about 50-75% w / w of the virosome preparation. In some embodiments, the basic matrix preparation can be about 65-85% w / w, about 70-80% w / w, about 73-77% w / w, or about 75% w / w of the virosome preparation.

[0074] Preparation of dosage forms comprising virosome preparations

[0075] As described above, in step 101, a liquid virosome concentrate is mixed with a base matrix preparation to form a virosome preparation suitable for a freeze-drying process. Figure 2 A more detailed description of the methods of forming the vaccine dosage forms disclosed herein is provided. In some embodiments, the base matrix formulation can be prepared by dissolving the matrix forming agent and the structure forming agent in a solvent to form a premix. For example, gelatin and mannitol can be dissolved in water, such as Figure 2 As shown in step 201 of . The premix can be heated to about 40-80°C, about 50-70°C, about 55-65°C, or about 60°C, and maintained for about 45-75 minutes, about 55-65 minutes, or about 60 minutes. As shown in step 202, the premix can be heated to 60°C and maintained for 1 hour. The premix can then be cooled to about 30-50°C, about 35-45°C, or about 40°C, and sieved, then further cooled to about 10-20°C or about 15°C, and maintained at this temperature throughout the remainder of the method. As shown in step 203, the premix can be cooled to 40°C and sieved. Next, as shown in step 204, the premix can be cooled to 15°C.

[0076] Next, a freeze-freeze protection agent can be added to the premix. For example, as shown in step 205, trehalose can be added to the premix. Subsequently, a pH adjusting agent can be used to adjust the pH to about 6-9, about 7-8, about 7.2-7.6, about 7.3-7.5 or about 7.4. For example, as shown in step 206, a sodium hydroxide solution can be used to adjust the pH to 7.4. After adjusting the pH, a liquid virosome concentrate can be added. After adding the liquid virosome concentrate, the pH (step 207) can be rechecked, and when necessary, another pH adjusting agent is used to adjust the pH to about 6.0-8.5, about 7-8, about 7.2-7.6, about 7.3-7.5 or about 7.4. This mixture can be supplemented to the required batch size with a solvent (i.e., a virosome preparation). For example, as shown in step 208, a desired amount of water can be added to the mixture.

[0077] exist Figure 1In step 102, the liquid virosome preparation can be quantitatively dosed into the preformed mold. As used herein, "dose" refers to the placement of a predetermined aliquot of a solution or suspension. As used herein, "preformed mold" refers to any suitable container or compartment, an aqueous solution or suspension can be placed therein, and then freeze-dried therein. In certain embodiments of the present disclosure, the preformed mold is a blister package with one or more blister bags. It can be measured into the preformed mold with a predetermined aliquot of a virosome preparation of about 150-1000 mg or about 500 mg wet fill dosing weight (also referred to as dosing fill weight). In some embodiments, the virosome preparation can be quantitatively dosed at about 10-20°C or about 15°C. For example, as shown in step 209, the virosome preparation can be quantitatively dosed at 15°C with a dosing fill weight of 500 mg.

[0078] exist Figure 1 In step 103, the quantitatively fed virion preparation can then be frozen in a preformed mold. The quantitatively fed virion preparation in the preformed mold can be frozen by any means known in the art. For example, the preparation can be passed through a cryogenic chamber (such as a liquid nitrogen tunnel). The temperature during the freezing process can be about -50 to -100°C, about -60 to -90°C, about -60 to -80°C, about -65 to -75°C or about -70°C. The scope of the freezing duration can be about 1.5-5 minutes, about 2-4.5 minutes, about 2.5-4 minutes, about 3-4 minutes, about 3-3.5 minutes or about 3.25 minutes. For example, as shown in step 210, the quantitatively fed virion preparation can be frozen at -70°C for 3 minutes and 15 seconds.

[0079] exist Figure 1 In step 104, the freezing unit in the preforming mold can be collected and placed in a refrigerator at a temperature lower than about -25°C, about -20°C, about -15°C, about -10°C, about -5°C, and annealed (i.e., frozen and kept) for a period of time to crystallize the structure forming agent. The structure forming agent crystallization can provide structural strength for the freezing unit to prevent the collapse of the freezing unit during freeze drying. This may be critical for virion integrity. The range of annealing time can be about 3-9 hours, about 4-8 hours, about 5-7 hours or about 6 hours. For example, as shown in step 211, the freezing unit can be annealed for about 3-9 hours at less than -15°C.

[0080] After annealing, the annealed freezing unit can be freeze dried in step 105 to form a dosage form. During the freeze drying process, water sublimates from the freezing unit. In some embodiments, the freezing unit can be loaded onto the shelf of a freeze dryer. In some embodiments, the freeze dryer can be precooled to about -15 to -35 ° C, about -20 to -30 ° C, or about -25 ° C. Once the annealed freezing unit is in the freeze dryer, the freeze drying cycle can be started. In some embodiments, once the freeze drying cycle is started, it is possible to evacuate and increase the shelf temperature. The freeze dryer can be operated at low pressure (i.e., vacuum). In some embodiments, the freeze dryer can be operated at a pressure of about less than or equal to 1000 mbar, about 900 mbar, about 800 mbar, about 700 mbar, about 600 mbar, about 500 mbar, or about 400 mbar.

[0081] Applicants have developed a two-step freeze drying cycle (step 212), which can achieve the structural stability of the dosage form and minimize the damage to the virion in the dosage form. The two-step freeze drying cycle can include a first step in which a freezing unit is kept for about 12-36 hours, about 18-30 hours, about 20-28 hours, or about 24 hours at about -5°C to -25°C, about -10°C to -20°C, about -13°C to -17°C, or about -15°C. In addition, the two-step freeze drying cycle can be included in the second step after the first step. The second step can include a freezing unit kept for about 6-30 hours, about 12-24 hours, about 14-22 hours, or about 18 hours at about 0°C to -20°C, about -5°C to about -15°C, about -8°C to about -12°C, or about -10°C. At the end of the two-step freeze drying cycle, the temperature of the freeze dryer can be raised to about ambient temperature (i.e., about 20-25°C or about 23°C).

[0082] In some embodiments, a two-stage freeze drying process may include precooling the freeze dryer to about -25°C, warming the freeze dryer to -15°C over 2 hours, maintaining the freeze dryer at -15°C for 24 hours, warming the freeze dryer to -10°C over 2 hours, maintaining at -10°C for 18 hours, warming to 0°C over 15 minutes, and then warming to 23°C over 15 minutes, in that order.

[0083] In step 213, the freeze-dried dosage form can be removed from the freeze dryer and inspected for any defects (quality inspection as described below). The dosage form can then be placed in a storage cabinet at an atmospheric humidity of less than about 35% RH, and the dosage form can then be sealed in its preformed mold. The sealing process (step 214) can place a cover foil on the preformed mold and provide a blister of the freeze-dried dosage form.

[0084] Water in the freeze-dried dosage form can be removed via sublimation during freeze drying. Accordingly, the remainder of the virosome concentrate excluding the freeze-lyoprotectant in the freeze-dried dosage form (i.e., the virosomes, antigens, adjuvants, and buffer systems present in the freeze-dried virosome concentrate) can be about 1-5% by weight, about 2-4% by weight, about 2.5-3.5% by weight, about 2.6-3.4% by weight, about 2.7-3.3% by weight, about 2.8-3.2% by weight, about 2.9-3.1% by weight, or about 3-3.1% by weight of the dosage form.

[0085] As described above, the target molecule is included in the dosage form disclosed herein in an amount sufficient to render it immunogenic when provided in the dosage form. One skilled in the art can readily determine the immunogenic amount for a given disease or infection, particularly based on the route of administration, the age and weight of the patient to whom the dosage form is to be administered. In some embodiments, the solid dosage form may contain 0.01-250 μg of each target molecule (e.g., HIV-1 P1 peptide and / or rgp41).

[0086] In some embodiments, at least one cryo-lyoprotectant in a freeze-dried dosage form can be about 5-20% by weight, about 8-18% by weight, about 10-15% by weight, about 11-15% by weight, or about 12-15% by weight of the dosage form. In some embodiments, at least one cryo-lyoprotectant in a freeze-dried dosage form can be about 1-5% by weight, about 1-4% by weight, or about 2-4% by weight of the dosage form.

[0087] In some embodiments, the amount of the matrix forming agent in the dosage form can be about 20-50 weight%, about 25-45 weight%, about 25-40 weight%, about 30-40 weight%, about 33-37 weight%, or about 35-37 weight%. In some embodiments, the amount of the structure forming agent in the dosage form can be about 27-65 weight%, about 27-60 weight%, about 40-55 weight%, or about 45-50 weight%. In some embodiments, the remainder of the pH adjusting agent (e.g., sodium hydroxide) in the freeze-dried dosage form can be about 0.01-0.08 weight%.

[0088] The dosage forms of the present disclosure are dissolving dosage forms and accordingly have the distinct advantage of faster disintegration times. The route of administration may be oral, vaginal or nasal, although oral (i.e., sublingual and / or buccal) is preferred. Once placed in the oral cavity and in contact with saliva, the dosage form may disintegrate in about 1 to about 180 seconds, about 1 to about 120 seconds, about 1 to about 60 seconds, preferably in about 1 to about 30 seconds, more preferably in about 1 to about 10 seconds, and most preferably in less than about 5 seconds.

[0089] Preparations, Test Methods and Examples

[0090] For example, a liquid virosome concentrate prepared from influenza virus was used. The virosome contained influenza HA and added antigens and adjuvants. Two virosome preparations were prepared, each containing a single antigen derived from HIV envelope glycoprotein. The liquid virosome concentrate was a mixture of two virosome preparations. The two HIV-gp41 derived antigens were the P1 peptide representing the last 35 C-terminal extracellular domain residues, and the truncated rgp41 lacking cluster I and the last 21 C-terminal extracellular domain residues. The adjuvant 3M-052 was present or absent in any virosome preparation. The virosome was suspended in a HEPES-sodium chloride buffer containing 142.5 mM sodium chloride and 50 mM HEPES at pH 7.4. In addition, trehalose (freeze-freeze-drying protective agent) in the range of 0-10% w / w liquid virosome concentrate was tested. Table 1 below summarizes the target composition of the HIV-1 liquid virosome concentrate used for some of our experiments, during which aliquots of 500 mg (dosing fill weight) of the aqueous virosome preparation were dosed into bags of preformed blisters, followed by freezing and freeze drying. The dosing fill weight range can be from 150 mg to 1000 mg, and the composition of the HIV-1 liquid virosome concentrate can be adjusted to meet the desired target dose. Please note that the target ranges described below can depend on the purpose and further use, such as for animal studies or human studies. Therefore, other target concentrations can be used for other purposes.

[0091] Table 1

[0092]

[0093] In some of our experiments, the target doses for HA and HIV-1 antigens per tablet were 20 µg HA, 25 µg P1, and 50 µg rgp41, respectively. To achieve these doses, a high payload of liquid virosome concentrate combined with a high wet fill dose weight was required. Table 2 shows various combinations of liquid virosome concentrate loading (ranging from 25-50% w / w) and wet fill dosing weight (ranging from 250 to 1000 mg). The wet fill dosing weight is the amount of aliquot of the virosome formulation metered for each dose prior to freeze drying.

[0094] Table 2

[0095]

[0096] A 25% loading of liquid virosome concentrate can be added to the base matrix formulation. The dosing fill dose weight can be 500 mg. Table 3 shows the range of HA and antigen content evaluated.

[0097] Table 3

[0098]

[0099] The presence of buffer in the aqueous composition can reduce the freezing point, therefore making it difficult to freeze the formulation composition and maintain its frozen state. In addition, because buffer salts can reduce the crystallization of mannitol during the annealing process, the collapse of the tablet matrix structure may also occur during freeze drying. The crystallization of mannitol is needed to provide strength and structure to the tablet matrix to prevent structural collapse. However, the crystallization of mannitol can damage virosome particles during freezing, annealing and freeze drying. The lower percentage load (e.g., 25% load) of liquid virosome concentrates causes this impact to be reduced. The combination of the lower percentage load of liquid virosome concentrates and the larger quantitative feed filling weight can also be considered.

[0100] The high wet fill dosing weight of the virosome formulation combined with the formulation composition having a high buffer content may also make it more difficult to freeze and maintain the structure to minimize collapse during freeze drying. However, larger tablets (e.g., 1000 mg dosing fill weight) can cover a larger surface area and can potentially improve virosome passage. When a high wet fill dosing weight is required, a formulation composition with a low buffer content is preferred.

[0101] Table 4 (Formulation 1) summarizes the aqueous composition of the virosome formulation and the corresponding composition of the tablet virosomes evaluated herein. Figure 2 The following formulations and tablets were prepared by the steps shown and described in . In addition, the frozen formulations were subjected to a two-step freeze drying process with a 500 mbar vacuum, with the following: (a) pre-cooling at -25°C; (b) warming to -15°C over 2 hours; (c) holding at -15°C for 24 hours; (d) warming to -10°C over 2 hours; (e) holding at -10°C for 18 hours; (f) warming to 0°C over 15 minutes; (g) warming to 23°C over 15 minutes. Next, the vacuum was released and the freeze dryer was returned to atmospheric pressure.

[0102] The concentration (% w / w) of each ingredient is the amount before the water present in the liquid virosome concentrate, the sodium hydroxide solution and the water used for preparation are removed by sublimation during lyophilization. In addition, the table below includes possible ranges of amounts for each ingredient.

[0103] Table 4

[0104]

[0105] *As described above, the liquid virosome concentrate includes virosomes suspended in a buffer system of 142.5 mM NaCl and 50 mM HEPES with 5% w / w trehalose. For a 25% loading virosome preparation (i.e. 125 mg liquid virosome concentrate) in a 500 mg dosing fill weight, the dry matter was estimated to be about 2.5-2.6 mg with sublimation of water from virosomes, antigen, adjuvant, NaCl and HEPES (excluding trehalose).

[0106] **This is the net amount of trehalose in the virosome preparation and dosage form. Therefore, this amount includes trehalose from the liquid virosome concentrate as well as trehalose added by the base matrix preparation.

[0107] Properties of freeze-dried dosage forms:

[0108] The freeze-dried dosage form can be stable in terms of physical attributes and virion quality (particle size characteristics and antigen content) and can be stored independently of cold chain storage conditions. In addition, the freeze-dried dosage form can allow the virion to be resistant to accidental exposure to sub-zero storage conditions during storage or transportation.

[0109] Physical characteristics of dosage form:

[0110] Freeze-dried tablets with acceptable physical properties were produced. The physical attributes of the tablets included appearance, dispersion characteristics, disintegration time and moisture content.

[0111] Ten freeze-dried tablets were tested for tablet appearance. Each tablet was taken out from the blister pack. A visual inspection was performed on each tablet for surface defects on the tablet surface and the matrix. The criterion was that the freeze-dried tablet should have a good appearance without surface defects. In addition, the tablet should have sufficient stability without cracking for its removability from the blister bag.

[0112] Dispersion properties (in vitro test): A minimum of 5 tablets were tested. First, a beaker containing approximately 200 mL of purified water at 20°C ± 0.5°C was prepared. Each tablet was then removed from the blister pack and placed with the tablet matrix facing down on the surface of the water. The time taken is the time taken for each tablet to be completely wetted or dissociated. Wetting lasts for the time taken for the unit to be completely wetted. Wetting of the tablet may occur in the form of small pieces that eventually merge together so that the entire unit is wetted. The dispersion test is considered complete when the center of the unit is a wet mass. Therefore, the wetting time is the time taken when the center of the unit has been fully wetted, as this is the thickest part of the unit. The wetting time was recorded for each of the five tablets. The maximum time for each test was 60 seconds. Times longer than this can simply be written as greater than 60 seconds. Dissociation = the time taken for the unit to disintegrate. This time can be taken when the unit begins to spread out at the edges. The dissociation time was recorded for each of the five tablets. The maximum time for each test was 60 seconds. Times longer than this can be written as greater than 60 seconds. Occasionally, the unit does not completely wet out or completely dissociate within this time limit. Sometimes, the unit may have hard lumps in it; other times, it may not wet on the surface at all. In addition, the entire unit may be covered with a hard skin. If this happens, this is noted in the description, with appropriate reference to "hard lumps" or "skin remains." The formation of "hard lumps" and / or "skin" can indicate microstructural collapse during freeze drying. Figure 3A -C shows a simplified representation of the three possible non-dispersed states, along with side and top views of the cell as they occur in water. Figure 3A The photo in -C shows some representative units of the same category. The criterion for the dispersion property test is whether 5 tablets can be completely wetted and / or dissociated into a palpable mass in 60 seconds or less without the presence of lumps and skins. In some embodiments, the dosage forms disclosed herein can be completely wetted and / or dissociated into a palpable mass in 60 seconds or less without the presence of lumps and / or skins.

[0113] Disintegration time (in vitro test): Six tablets are used for this test. Six beakers are filled with purified water and placed in a water bath controlled at 37°C ± 0.5°C. Each tablet is then taken out of the blister package. A wire clip is carefully placed on each of the six tablets. Make sure that the clip can clamp the tablet without causing damage. Next, the test is performed as described in the pharmacopoeia. An example of such a test is the United States Pharmacopeia (701) disintegration. The maximum disintegration time is recorded for each tablet. The criterion for the disintegration time test is that the disintegration time should not be more than 60 seconds for each of the six tablets. In some embodiments, the dosage form disclosed herein may have a disintegration time of less than 60 seconds.

[0114] Moisture content: A Methron 831 Karl Fischer Coulometer (Metrohm, Herisau, Switzerland) with a 744 Oven Sample Processor is used to determine the moisture content of the tablets. Accurately weigh the tablets and place them in a glass vial. Immediately crimp the vial shut to ensure that no moisture enters. The sample vial is then placed in a 744 Oven Sample Processor and the temperature is set to 102°C. The evaporated moisture is titrated using the Hydranal Coulometric AGOven reagent to quantify the amount of water released. The test is performed in triplicate and the average value is recorded. The criterion for moisture content is whether the freeze-dried dosage form has a moisture content of less than about 8%, preferably less than 6%, and more preferably less than 4%. In some embodiments, the dosage forms disclosed herein may have a moisture content of less than about 8%, preferably less than 6%, and more preferably less than 4%.

[0115] Virus characteristics:

[0116] The freeze-dried dosage form containing the virosomes can be fully preserved in terms of the proportion of intact virosomes from the starting liquid virosome population, its particle size, and the surface antigen content required for the immunogenicity and immunological benefits of the virosomes. The virosome structure can be destroyed by the freeze-drying method used to produce the dosage form. Therefore, the virosome particle characteristics are evaluated from the solution of the reconstituted freeze-dried tablet. The virosomes can exist as intact individual particles and clusters of different sizes, which are all immunogenic, but have different antigen / epitope exposures and have different abilities to pass through the sublingual barrier. The virosomes can be characterized according to (a) the average particle size and (b) the proportion of virosome preservation (intact virosomes).

[0117] Evaluation of virion particle size and particle concentration (counts / mL of virion particles) using NTA technology: Nanoparticle Tracking Analysis (NTA; using NanoSight NS300 instrument) is a sensitive method that can identify different particle sizes in the range of 30-1000 nm present in solution. The particles in the sample solution can be tracked individually and analyzed simultaneously by direct observation. Nanoparticles move under Brownian motion due to the random motion of water molecules around them. Small particles move faster than larger particles. The Brownian motion of each particle is tracked in real time via video, and NTA analyzes the Brownian motion to determine the particle size. The diffusion coefficient can be calculated by tracking the movement of each particle, and then the particle size is calculated by applying the Stokes-Einstein equation. This particle-by-particle method produces high-resolution results for particle size distribution and concentration (i.e., the number of particles in a known volume of liquid). The target values ​​for optimal detection of instrumental detection are summarized in Table 5 below.

[0118] Table 5

[0119]

[0120] The test sample must be in liquid form. For this work, the virosome vaccine is supplied in liquid form. For the test samples from the vaccine formulations disclosed herein, the mixture is provided in liquid form, the frozen unit is thawed to liquid form before testing, and the freeze-dried dosage form is reconstituted to liquid form with water buffer. The test sample should not be too concentrated. The liquid test sample can be further diluted with HN buffer as appropriate to optimize the detection. The dilution buffer can be of high purity and can be filtered at least through a 0.22 µm filter before use. The experimental parameter set for NTA analysis is summarized in Table 6 below.

[0121] Table 6

[0122] parameter set up Sample dilution Depends on the amount (depends on the concentration of virion counts in the diluted test sample, which can range from 1:100 to 1:8000) Number of captures 5 Capture duration 60 seconds Temperature control 25℃ Viscosity 0.9Cp

[0123] The sample is measured by NTA as known in the art. In some embodiments, the range of virosome particles disclosed herein including fragments and clusters can be about 50-500 nm. In some embodiments, the virosome particles that are intact virosomes can be in the range of about 70-400 nm or about 70-200 nm (main peak). In some embodiments, the average diameter of the virosome particles can be about 70-200 nm, about 100-175 nm, about 125-155 nm. For detection purposes, the particle concentration of the virosome population in the sample can be at least 10 10 Counts / mL.

[0124] Use the ratio of flow cytometry virion preservation evaluation: flow cytometry is used for this evaluation.First, by inserting lipophilic dye Dil (long-chain dialkyl carbocyanine (long-chain diallylcarbocyanin)) into the lipid bilayer of virion, mark the initial liquid virion particles (representing the reference sample of 100% raw material). (Marking with Dil does not have a measurable effect on granularity). Secondly, freeze-dried tablets are reconstructed, and the virion in the freeze-dried tablets is marked with Dil (test sample). Then use AMNIS imaging flow cytometer analysis sample, and compare the event between reference sample (liquid virion before freeze drying) and test sample (freeze drying virion), to assess the virion preservation ratio after freeze drying according to the following aspects: (a) the percentage of virion recovery and (b) the percentage of virion clusters. In some embodiments, the recovery percentage of the virion as a single particle can be about 20-50%, about 30-50% or about 40-50% of raw material. In some embodiments, the percentage of virion clusters (in the form of doublets, triplets, or a higher number) may be less than about 50%, about 25%, about 10%, or about 5%.

[0125] Contents of virion hemagglutinin (HA), HIV-1 antigen P1, HIV-1 antigen and adjuvant

[0126] The influenza HA, HIV-1 antigen and adjuvant content of the virions can be quantified by various methods. These are tabulated in Table 7 below.

[0127] Table 7

[0128]

[0129] Immunoblot assay for HA, P1 and rgp41: In this analysis, the preparation (liquid virosome concentrate or reconstituted freeze-dried dosage form containing virosomes) can be absorbed onto a nitrocellulose membrane, and the antigen is maintained in its native state due to the absence of a heating procedure, a denaturant or a reducing agent. This assay detects all antigens accessible to specific antibodies and indicates major antigen degradation or denaturation, which destroys or hinders access to specific epitopes. It also indicates whether a specific excipient can prevent the antibody from binding to its antigen. In order to prepare the freeze-dried dosage form (test sample) for analysis, each tablet is dissolved in 0.5 mL of water (i.e., the freeze-dried tablet is reconstituted back to the composition of the virosome preparation before freeze drying). In order to prepare a positive control, a sample of the liquid virosome concentrate is diluted with ultrapure water (dilution is 4 times). Ideally, the liquid virosome concentrate is the same batch as that used to prepare the dosage form test sample. Prepare serial 2-fold dilutions of all test samples and positive controls. Additional positive controls, such as purified HA and rgp41, and synthetic P1, can also be used. Spot 1.5 µL of each sample and positive control onto a dry nitrocellulose membrane (increasing dilutions from left to right – undiluted, 1 / 2、 1 / 4、 1 / 8、 1 / 16、 1 / 32、 1 / 64、 1 / 128 ). The dried membrane was blocked with 1% (w / v) casein and incubated with specific antibody solutions - human monoclonal antibody (mab) 2F5 specific for HIV-1 P1 antigen and rabbit anti-rgp41 serum for HIV-1 Rgp41 antigen. After incubation, the nitrocellulose membrane was washed. The specific antibodies bound to the fluorescently labeled secondary antibodies (anti-human antibodies or anti-rabbit antibodies) were then detected using a far-red fluorescence scanner that allows simultaneous detection of 2 different fluorescent labels at 700 nm and 800 nm. The fluorescence raw data signal of each sample spot (from lowest to highest) was compared with the respective dilution of the positive control spot (in % of the positive control). The arithmetic mean of the sample percentages was calculated.

[0130] UV spectrometry of 3M-052: The adjuvant molecule 3M-052 has several UV absorption peaks. Some of these peaks overlap with the absorption of lipids and proteins. The determination is done by UV spectroscopy at 320 nm.

[0131] HPLC Assays for P1 and rgp41: HIV antigens P1 and rgp41 were separated by reverse phase high pressure liquid chromatography on a C18 column using a water to acetonitrile gradient. Detection was accomplished by UV spectroscopy at 280 nm and peak areas were quantified by the HPLC system software.

[0132] ELISA endpoint antibody titer: Maxisorp 96-well plates (Nunc flat bottom) and Polysorp plates were coated with 0.1 mL of rgp41 or P1 peptide (2 µg / mL) prepared in PBS pH 7.4 for 16 hours at 4°C. The plates were washed three times with PBS with 0.05% v / v) Tween 20 (PBST), and then a blocking solution of 1% BSA prepared in PBST was added to each well and incubated for 2 hours at room temperature (RT). The plates were washed three times with PBST, and then 0.1 mL / well of pre-immune serum diluted 1 / 1000 or serial dilutions of immune serum prepared in 0.1% BSA in PBST (from 1 / 1000 to 1 / 64,000) were added and incubated for 2 hours at RT. The plate was washed 3 times with PBST and incubated with goat anti-rat IgG-HRP diluted 1:4000 in 0.1% BSA in PBST for 2 hours at RT. The plate was washed again, then 0.1 mL of the colorimetric substrate o-phenylenediamine (OPD) was added and the reaction was stopped with 2M H2SO4, followed by plate reading at 492 nm.

[0133] Example 1 : High mannitol levels (8% w / w) were used in conjunction with low-temperature freeze-drying cycles to reduce microstructural collapse during freeze-drying without compromising the integrity of the virions.

[0134] Mannitol was used in the dosage form to increase structural stability. Due to the presence of high levels of buffer and the addition of trehalose to protect the virosome particles, the use of mannitol at a typical level of 4.5% w / w did not provide sufficient structural support during freeze drying. Therefore, microstructural collapse occurred.

[0135] However, the applicant has discovered that by using a combination of higher levels of mannitol and a low temperature freeze drying cycle, a more structurally stable freeze dried tablet can be achieved. This example shows data comparing a formulation containing 4.5% w / w mannitol to a formulation containing 8% w / w mannitol. In both formulations, a 25% w / w loading of liquid virosome concentrate MYM-201 batch 160125-1 supplied by Mymetics was used in the virosome formulation, which contained HA 70-80 µg / ml, P1 40-50 µg / ml, rgp41 70-80 µg / ml (A / Brisbane / 59 / 2007 (H1N1) in HN buffer pH 7.4 (50 mM HEPES, 142.5 mM NaCl). Fish gelatin and neat trehalose levels were maintained at 6% w / w and 2% w / w of the virosome formulation, respectively. As Figure 2 As shown in, at 15 ° C, the formulation is dosed into blister bags of aluminum trays with 500 mg dosing fill weight aliquots. The tray containing the dosing aqueous vaccine mixture is frozen by passing the aluminum tray through a freezer set at -70 ° C for a duration of 3 minutes and 15 seconds. The aluminum tray containing the frozen product is collected and placed in a freezer at a temperature of <-15 ° C, and kept frozen annealed for 6 hours before lyophilization. Then a two-step FD cycle is used, which uses 24 hours at -15 ° C, followed by 18 hours at -10 ° C. The blisters of the freeze-dried tablets are sealed in pouches and stored under ambient conditions.

[0136] The physical properties (appearance and dispersion time) of the lyophilized tablets were evaluated according to the tests described above. In addition, the virion particle size in the lyophilized tablets was characterized according to the tests described above. The results are summarized in Table 8 below.

[0137] Table 8

[0138]

[0139] All tablets have good appearance. The 4.5% w / w mannitol preparation has weak dispersion behavior due to microstructure collapse, with visible crusting and / or lumps in the tablet. Increasing the preparation to 8% w / w (which promotes the crystallization of mannitol) improves the structure of the freeze-dried tablet. The preparation with 8% w / w mannitol has good dispersion behavior and provides soft and touchable agglomerates. Combined with the low-temperature freeze drying cycle, the virion particles in the freeze-dried tablet are not affected. The virion particle size in the freeze-dried tablet is generally comparable between preparations.

[0140] Example 2: Stability data of freeze-dried vaccine dosage form (Formulation 1) stored under ICH conditions for 3 months.

[0141] Liquid virosome concentrates are generally stable at 2-8°C, but liquid HIV virosome vaccines have a limited shelf life of several months without aggregation due to important chemical modifications that occur on the antigens. For solid vaccine formats with low moisture content, such modifications are expected to slow down and be minimized over time, extending the shelf life to > 1 year. The stability of virosome vaccines in the form of freeze-dried tablet formulation 1 is shown in this example. Liquid virosome concentrate batch MYM V202 lot 170130-1, containing approximately 100 µg / ml HIV-1 P1, 230 µg / ml HIV-1 rgp41, 130 µg / ml HA (A / Brisbane / 59 / 2007 (H1N1), 65 µg / ml adjuvant 3M-052 in HN buffer pH 7.4 (50 mM HEPES, 142.5 mM NaCl) with 7% w / w trehalose, was supplied by Mymetics for use in the manufacture of lyophilized vaccine tablets (lot Z33787A101).

[0142] To prepare the vaccine tablets, a liquid virosome preparation mixture was first prepared. It contained 25% w / w of liquid virosome concentrate batch MYM V202 batch number 170130-1, 6% w / w fish gelatin, 4.5% w / w mannitol, 2% w / w (neat) trehalose, sodium hydroxide solution to pH 7.4 (appropriate amount) and purified water to 100% w / w (appropriate amount).

[0143] At 15°C, the liquid virosome preparation mixture was dosed into blister bags in aluminum trays with 500 mg dosing fill weight aliquots. The trays containing the dosing aqueous vaccine mixture were frozen by passing the trays through a freezer set at -70°C for a duration of 3 minutes 15 seconds. The aluminum trays containing the frozen product were collected and placed in a freezer at a temperature of <-15°C. A two-step FD cycle was then used, which used 24 hours at -15°C, followed by 18 hours at -10°C.

[0144] Blisters of freeze-dried tablets were sealed in pouches and placed in 3 month storage at 5°C, 25°C / 60% RH and 40°C / 75 RH. The results at the initial test and the 3 month test are summarized in Table 9 below.

[0145] Table 9

[0146]

[0147] The stability data show that the tablet appearance is good and consistent between batches. The moisture content is 5-6% w / w, and there is little difference between different stability conditions through a 3-month storage period. Since the formulation contains 4.5% w / w mannitol, there are some microstructural collapses during freeze drying as indicated by the variability of the disintegration time. With regard to virion particle size, DLS and NTA data show that there are no distinguishable differences in the virion particle size between tablets stored under various stability conditions. The initial liquid virion concentrate has > 50% < 200 nm particles. The results show that the virions < 200 nm from the reconstitution unit are substantially preserved to a similar order of magnitude.

[0148] Use semi-quantitative immunoblot analysis, through 3 months monitoring antigen HIV-1 antigen P1 and rgp41 degradation.All samples are pre-diluted 2 times.For P1 specific human mAb 2F5 and rabbit anti-rgp41 serum are used for this purpose.The liquid virion concentrate batch MYM-V202 lot number 170130-1 that will be used to produce vaccine batch is diluted 8 times, and is used as positive control. Figure 4 Photographs of immunoblot analysis are shown. For sample Z33787A101, tablets stored at 5°C showed no or only minimal reduction in the rgp41 antigen signal after 1 month and 3 months of storage compared to the initial sample (t=0). Similarly, there was only minimal difference in signal intensity for the units stored at 25°C for the rgp41 antigen, and a slight reduction for the P1 antigen. The differences for the 40°C units were more pronounced, although this was probably still within the 10-20% accuracy of the assay.

[0149] Quantitative evaluation of the fluorescence data for rgp41 and P1 is presented in Table 10 and Table 11, respectively. The upper portion of each table shows the fluorescence raw data signal for each spot of the indicated stability sample (from the lowest dilution to the highest dilution). The lower portion of each table shows the value (in % of the positive control) of each sample spot compared to the respective dilution of the positive control spot. The bottom row shows the arithmetic mean of all sample percentages. Obvious outliers measured were excluded from the average calculation.

[0150] Table 10

[0151]

[0152] Table 11

[0153]

[0154] For the vaccine tablets of Z33787A101, tablets stored at 5°C showed no or only minimal reduction in the signal for the rgp41 and P1 antigens after 1 and 3 months of storage compared to the initial sample (t=0). Similarly, for the rgp41 antigen, there was only minimal difference in signal intensity for the units stored at 25°C, with a slight reduction for the P1 antigen. The differences for the 40°C units were more pronounced for both antigens P1 and rpg41, although this is likely still within the accuracy of the assay, especially since variations were again visible in the serial dilution samples.

[0155] For all vaccine tablets stored at different temperatures over 3 months, the determination of 3M-052 was done by UV spectroscopy at 320 nm. The values ​​are presented in Table 12 below.

[0156] Table 12

[0157] Time Storage conditions OD (320 nm) 0 5℃ 0.116 1 month 5℃ 0.114 1 month 25℃ / 60%RH 0.112 1 month 40℃ / 75%RH 0.110

[0158] No distinguishable differences were observed between these samples, and therefore, within the accuracy of the assay, 3M-052 concentrations were considered to remain stable in all samples. Overall, HA, P1, rpg41, and 3M-052 were stable in freeze-dried tablets stored under different storage conditions, with minor variations over time, as shown in Table 13 below.

[0159] Table 13

[0160] Antigen and adjuvant testing during stability studies Liquid virosome concentrate Freeze-dried tablets Changes in Rgp41 after 3 months at 5°C (no degradation expected = reference) Not significant Not significant Changes (degradation) of Rgp41 after 3 months at 25°C compared to 5°C Minimize Changes (degradation) of Rgp41 after 3 months at 37-40°C compared to 5°C Minimize Change in P1 after 3 months at 5°C (no degradation expected = reference) Not significant Not significant Change in P1 after 3 months at 25°C compared to 5°C (degradation) Minimize Changes in P1 after 3 months at 37-40°C compared to 5°C (degradation) Minimize Changes in 3M-052 after 3 months at 5°C (no degradation expected = reference) Not significant Not significant Changes (degradation) of 3M-052 after 3 months at 37-40°C compared to 5°C Not significant Not significant

[0161] Example 3 : Stability data of freeze-dried vaccine dosage form (Formulation 2) stored under ICH conditions.

[0162] In this example the stability of the virosome vaccine in freeze-dried tablet form is shown for Formulation 2. Liquid virosome concentrate batch MYM V202 lot 17MYM002 / F17255 supplied by Mymetics, containing approximately 121 µg / ml HIV-1 Pl, 67 µg / ml HIV-1 rgp41, 41 µg / ml HA (A / Brisbane / 59 / 2007 (H1N1), 39 µg / ml adjuvant 3M-052 in HN buffer pH 7.4 (50 mM HEPES, 142.5 mM NaCl) with 7% w / w trehalose, was used to make freeze-dried vaccine tablets (batch MYM-212 lot 1690747).

[0163] To prepare the vaccine tablets, a liquid virosome preparation mixture was first prepared. It contained 25% w / w of liquid virosome concentrate batch MYM V202 batch number 17MYM002 / F17255, 6% w / w fish gelatin, 8% w / w mannitol, 2% w / w (neat) trehalose, sodium hydroxide solution to pH 7.4 (q.s.) and purified water to 100% w / w (q.s.).

[0164] At 15°C, the liquid virosome preparation mixture was dosed into blister bags in aluminum trays with 500 mg dosing fill weight aliquots. The trays containing the dosing aqueous vaccine mixture were frozen by passing the trays through a freezer set at -70°C for a duration of 3 minutes 15 seconds. The aluminum trays containing the frozen product were collected and placed in a freezer at a temperature of <-15°C. A two-step FD cycle was then used, which used 24 hours at -15°C, followed by 18 hours at -10°C.

[0165] Blisters of freeze-dried tablets were sealed in pouches and placed in 3 month storage at 5°C, 25°C / 60% RH and 40°C / 75 RH. The results at the initial test and the 6 month test are summarized in Table 14 below.

[0166] Table 14

[0167] <![CDATA[ test ]]> <![CDATA[ Appearance ]]> <![CDATA[ Disintegration time ]]> <![CDATA[ Moisture content ]]> <![CDATA[ initial ]]> <![CDATA[ good ]]> <![CDATA[ 22 seconds ]]> <![CDATA[ 3.6% ]]> <![CDATA[ exist 5℃ 1 month ]]> <![CDATA[ good ]]> <![CDATA[ 8 seconds ]]> <![CDATA[ 3.8% ]]> <![CDATA[ In 2 5℃ / 60%RH 1 month ]]> <![CDATA[ good ]]> <![CDATA[ 16 seconds ]]> <![CDATA[ 3.7% ]]> <![CDATA[ In 40 ℃ / 75% RH 1 month ]]> <![CDATA[ good ]]> <![CDATA[ 14 seconds ]]> <![CDATA[ 3.6% ]]> <![CDATA[ exist 5℃ 3 months ]]> <![CDATA[ good ]]> <![CDATA[ 12 seconds ]]> <![CDATA[ 3.7% ]]> <![CDATA[ In 2 5℃ / 60%RH 3 months ]]> <![CDATA[ good ]]> <![CDATA[ 10 seconds ]]> <![CDATA[ 3.7% ]]> <![CDATA[ In 40 ℃ / 75% RH 3 months ]]> <![CDATA[ good ]]> <![CDATA[ 13 seconds ]]> <![CDATA[ 3.9% ]]> <![CDATA[ exist 5℃ 6 months ]]> <![CDATA[ good ]]> <![CDATA[ 8 seconds ]]> <![CDATA[ 3.9% ]]> <![CDATA[ In 2 5℃ / 60%RH 6 months ]]> <![CDATA[ good ]]> <![CDATA[ 10 seconds ]]> <![CDATA[ 3.8% ]]> <![CDATA[ In 40 ℃ / 75% RH 6 months ]]> <![CDATA[ good ]]> <![CDATA[ 13 seconds ]]> <![CDATA[ 4.0% ]]>

[0168] The stability data showed that the tablet appearance was good and consistent between batches. The moisture content was 3.6-4.0% w / w with little variation between different stability conditions over a 6-month shelf life. Since the formulation contained 8% w / w mannitol, there was less microstructural collapse during freeze drying as indicated by the shorter and more consistent disintegration times.

[0169] The stability of the antigen content (HIV-1 antigens P1 and rgp41) was monitored over 3 months of degradation using an HPLC assay. For the liquid vaccine MYM V202 (raw material), it was found that the P1 content decreased by 4% and 7% when stored at 2 - 8ºC (cold storage conditions) for 1 month and 3 months, respectively. For rgp41, the content decreased by 16% and 25% at 1 month and 3 months, respectively, under cold storage conditions. When the liquid virions were stored at 25ºC and 40ºC, P1 and rgp41 were no longer detected after 1 month and 3 months.

[0170] For antigen P1 and antigen rgp41, the results of antigen content of lyophilized tablets under storage are presented in Table 15 and Table 16, respectively. In the form of lyophilized tablets, the P1 antigen remains very stable, and no decrease in content is observed after 3 months at 2-8ºC (cold chain conditions), and it remains unaffected during 1 month and 3 months of storage outside cold chain conditions. For the rgp41 antigen, after 1 month at 2-8ºC, 25ºC and 40ºC, decreases of about 5%, 10% and 20% were observed, respectively. Under 3 months of storage, the decreases were about 13%, 15% and 19%, respectively. Considering the accuracy of the HPLC method, the observed decrease in concentration of less than 15% is not significant. In addition, the observed gradual antigen loss or decrease is related to chemical modification, rather than due to deep degradation, amino acid loss or aggregation with structural cleavage. At the same time, chemical modifications in a given epitope may potentially change its recognition, reduce or increase antibody binding to the region, while other regions remain equally well recognized.

[0171] At T0, P1 and rgp41 antigens had similar SDS-PAGE migration profiles and were still recognized by specific monoclonal antibodies when in sublingual tablets, and serum antibodies against P1 and rgp41 were still reactive against various P1 and gp41 peptides with key epitopes. These analyses suggest that, overall, P1 and rgp41 have preserved most of their antigenicity and immunogenicity during the manufacturing process (data not shown).

[0172] Table 15

[0173]

[0174] Table 16

[0175]

[0176] Example 4 : Stability of freeze-dried tablets stored under sub-zero temperature storage conditions.

[0177] The data in Example 4 show the stability of the physical properties of the virosome vaccine tablets and virosome particles when stored under sub-zero conditions. A liquid virosome formulation mixture containing a 25% w / w loading of liquid virosome concentrate MYM-201 batch 160125-1 supplied by Mymetics (containing HA 70-80 µg / ml, P1 40-50 µg / ml, rgp41 70-80 µg / ml (A / Brisbane / 59 / 2007 (H1N1), 6% w / w fish gelatin, 8% w / w mannitol and 2% w / w (neat) trehalose in HN buffer pH 7.4 (50 mM HEPES, 142.5 mM NaCl) was dosed with 500 mg dosing fill weight and freeze dried. The formulation was then freeze dried at 15°C. mg quantitative feeding fill weight aliquots are quantitatively fed into the blister bag of aluminum tray.By making the aluminum tray pass through the duration of 3 minutes and 15 seconds in the freezing chamber set at -70 ℃, the tray containing the aqueous vaccine mixture of quantitative feeding is frozen.Collect the aluminum tray containing frozen product, and be placed in the freezer at a temperature of <-15 ℃, and keep frozen annealing for 6 hours before lyophilization.Then use two-step FD circulation, it uses 24 hours at -15 ℃, followed by 18 hours at -10 ℃.The blister of freeze-dried tablets is sealed in a pouch, and is placed in a freezer at -15 ℃ and stored for 1 week.A corresponding group of tablet blisters is sealed in a pouch, and the same duration is placed under ambient conditions.As described according to the above test method, the appearance, dispersion characteristics (wetting time and dissociation time) and virion particle size distribution of these tablets are evaluated.

[0178] After storage under both conditions, all units were found to have good appearance. The data show that sub-zero storage has little effect on the dispersibility properties (wetting and dissociation time) (Table 17) and virosome particle size distribution (Table 18) of the tablets.

[0179] Table 17

[0180] condition Wetting time (seconds) Dissociation time (seconds) Environmental Storage <31 <37 Sub-zero storage <32 <35

[0181] Table 18

[0182]

[0183] Example 5 : Flow cytometric evaluation of virosome particles from reconstituted freeze-dried tablets.

[0184] Estimates of the proportion of virions preserved in freeze-dried tablet samples from process development are summarised below. The values ​​provided in Table 19 are derived from AMNIS flow cytometry data using measurements of events in the focal region corresponding to the virion gate.

[0185] Table 19

[0186] Vaccine preparations Percentage of virions recovered from production end Percentage of clusters (doublets, triplets, higher forms) Liquid vaccines 100% <5% Freeze-dried tablets (reconstituted 24 – 40% 10-25%

[0187] The data showed that 24-40% of the starting virions were preserved in the freeze-dried tablets, of which 10-25% of these virions were in clusters, mostly in doublets and triplets.

[0188] Example 6: Evaluation of the immunogenicity of P1 and rgp41 antigens from liquid virosome preparations and reconstituted freeze-dried sublingual tablets containing virosomes.

[0189] The liquid virosome concentrate (liquid vaccine MYM-V202) and the freeze-dried sublingual tablet containing the virosome of Example 3 were placed in storage at 4°C and 40°C for a period of three months for immunogenicity evaluation. After 1 month of storage, samples of the liquid vaccine and sublingual tablet stored at 40°C were taken out of the storage cabinet for immunogenicity evaluation. After 3 months of storage, samples of the liquid vaccine and sublingual tablet stored at 4°C and 40°C were taken out for immunogenicity evaluation.

[0190] For immunogenicity evaluation, 50% of Wistar rats (n=10 / group) of each sex were used. At the 0th day, the 28th day and the 56th day, rats were immunized. The liquid vaccine contained 3.9 μg P1, 2.2 μg rgp41 and 1.3 μg 3M-052 TLR7 / 8 (adjuvant) in 0.1 mL, and this was used for subcutaneous injection. For sublingual tablets, sufficient number of sublingual tablets were dissolved in sterile water to reach about 3 μg P1, 1.7 μg rgp41 and 1 μg 3M-052TLR7 / 8 (adjuvant) in 0.1 mL, to be used by subcutaneous injection. Pre-immune serum was collected at the 0th day, and immune serum was collected at the 65th day for determining the endpoint antibody titer in each animal serum and serum bank.

[0191] Figure 5The immunogenicity of P1 and rgp41 from liquid vaccines and sublingual tablets stored at different temperatures is shown. The liquid adjuvanted vaccine formulation MYM-V202 containing both P1 and rgp41 antigens is temperature sensitive and serves as a reference material for comparison with the immunogenicity of sublingual tablet vaccine forms with improved thermal stability. (Black line) shows the vaccine stored at 4°C for 3 months; (black dotted line) shows the vaccine stored at 40°C for 1 month; (gray line) shows the vaccine stored at 40°C for 3 months. The data show that the immunogenicity of the antigens of the freeze-dried tablets is retained. In each group, the endpoint antibody titer is also indicated. The endpoint titer corresponds to the last serum dilution, and the generated OD value is> 2 times the background before immunity.

[0192] Additional Definitions

[0193] Unless otherwise defined, all art terms, symbols, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by those of ordinary skill in the art to which the claimed subject matter belongs. In some cases, for clarity and / or ease of reference, terms with commonly understood meanings are defined herein, and the inclusion of such definitions herein should not be construed as representing a substantial difference from what is generally understood in the art.

[0194] References herein to "about" a value or parameter include (and describe) variations for the value or parameter itself. For example, a description referring to "about X" includes a description of "X". In addition, reference to a phrase "less than," "greater than," "at most," "at least," "less than or equal to," "greater than or equal to," or other similar phrases followed by a string of values ​​or parameters is intended to apply the phrase to each value or parameter in the string of values ​​or parameters. For example, a statement that a solution has a concentration of at least about 10 mM, about 15 mM, or about 20 mM means that the solution has a concentration of at least about 10 mM, at least about 15 mM, or at least about 20 mM.

[0195] As used herein, the singular forms "a", "an", and "the" are also intended to include the plural forms, unless the context clearly indicates otherwise. It should also be understood that, as used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items. It should be further understood that when used herein, the terms "includes", "including", "comprises", and / or "comprising" specify the presence of stated features, integers, steps, operations, elements, components, and / or units, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, units, and / or groups thereof.

[0196] The present application discloses several numerical ranges in the text and drawings. The disclosed numerical ranges inherently support any range or value within the disclosed numerical ranges, including the endpoints, even if the precise range limitations are not stated verbatim in the specification, because the present disclosure can be practiced within the entire disclosed numerical range.

[0197] The above description is presented to enable those skilled in the art to prepare and use the present disclosure, and is provided in the context of a specific application and its requirements. Various modifications to the preferred embodiments will be apparent to those skilled in the art, and the general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the present disclosure. Therefore, the present disclosure is not intended to be limited to the embodiments shown, but should be given the broadest scope consistent with the principles and features disclosed herein.

Claims

1. A freeze-dried oral solid vaccine dosage form, comprising: (1) Virosomes derived from influenza virus, the virosomes being freeze-dried from a liquid virosome preparation, the liquid virosome preparation comprising 25-50% by weight of a liquid virosome concentrate containing virosomes suspended in a buffer system comprising 130-150 mM NaCl and 40-60 mM HEPES; (2) 10-15% by weight of trehalose; (3) 33-37% by weight of fish gelatin; and (4) 45-50% by weight of mannitol.

2. The dosage form of claim 1, wherein the fish gelatin is high molecular weight fish gelatin.

3. The dosage form of claim 1, wherein the virosome comprises an adjuvant.

4. The dosage form of any one of claims 1 to 3, wherein the dosage form promotes oral uptake of at least one target molecule.

5. The dosage form of claim 4, wherein the dosage form disintegrates within 180 seconds after being placed in the oral cavity.

6. The dosage form of claim 5, wherein the dosage form disintegrates within 90 seconds after being placed in the oral cavity.

7. The dosage form of claim 6, wherein the dosage form disintegrates within 60 seconds after being placed in the oral cavity.

8. The dosage form of claim 7, wherein the dosage form disintegrates within 30 seconds after being placed in the oral cavity.

9. The dosage form of claim 4, wherein when administered to a patient by placement in the oral cavity, an immune response is induced.

10. The dosage form of claim 9, wherein placement in the oral cavity is placement on or under the tongue or in the buccal or pharyngeal region.

11. Use of a dosage form according to any one of claims 1 to 10 for the preparation of a vaccine for inducing an immune response in a patient, wherein the vaccine is placed in the oral cavity of an individual in need of an immune response.

12. The use of claim 11, wherein placement in the oral cavity is placement on or under the tongue or in the buccal or pharyngeal region.

13. A method for forming an oral solid vaccine dosage form, the method comprising: A liquid virosome preparation is quantitatively dosed into a preformed mold, wherein the virosome preparation comprises: (1) a 25-50% by weight virosome concentrate containing virosomes derived from influenza virus suspended in a buffer system comprising 130-150 mM NaCl and 40-60 mM HEPES; (2) 1-5% by weight of trehalose; (3) 4-8% by weight of fish gelatin; and (4) 5-10% by weight of mannitol; Freezing the quantitatively dosed virosome preparation at a temperature of -60°C to -90°C; Annealing the frozen virion preparation by keeping it at a temperature below -15°C for 3-9 hours; and The annealed virion preparation is freeze-dried to form a dosage form.

14. The method of claim 13, wherein the dosed virion preparation is frozen at a temperature of -60°C to -90°C for a duration of 1-5 minutes.

15. The method of claim 13, wherein freeze-drying the annealed virion preparation comprises: (1) maintaining the annealed virion preparation at a temperature of -10°C to -20°C for 20-28 hours, and (2) maintaining the annealed virion preparation at a temperature of -5°C to -15°C for 14-22 hours.

16. The method of any one of claims 13 to 15, wherein the freeze drying occurs at a pressure of less than 600 mbar.

17. The method of any one of claims 13-15, wherein the virosome preparation has a pH of 6.5-8.

18. The method of claim 17, wherein the fish gelatin is high molecular weight fish gelatin.

19. The method of any one of claims 13-15, wherein the virosomes comprise an adjuvant.

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