RU2808276C2 - Orally dispersable vaccine containing virosomes - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: described solid unit dosage form for oral administration. The composition contains the following ingredients: lipid-based vesicles containing an immunogenic amount of at least one vaccine antigen; 5–20 wt.% of at least one cryolioprotectant; 25–40 wt.% of the substance forming the matrix; and 40–55 wt.% of the substance forming the structure. The following is presented: a method of inducing an immune response in a patient involving introduction of a standard dosage form into the human oral cavity. A method of obtaining a solid standard dosage form for oral administration is described. The liquid virosome composition is dosed into a preformed form. The virosome composition includes the following ingredients: lipid-based vesicles containing an immunogenic amount of at least one vaccine antigen; 1–5 wt.% of cryolioprotector; 4–8 wt.% of the substance forming the matrix; and 5–10 wt.% of the substance forming the structure. The dosed virosome composition is frozen at a temperature of -60°C to -90°C. The frozen virosome composition is annealed by keeping it at a temperature below -15°C for 3–9 hours. The hybridized virosome composition is lyophilized to form a dosage form. A method of preparing a solid unit dosage form for oral administration is also described. The liquid virosome composition is dosed into a preformed form. The virosome composition includes the following ingredients: 20–50 wt.% of virosome concentrate, where the virosome concentrate contains the following: virosomes containing an immunogenic amount of at least one vaccine antigen; 2–10 wt.% of cryolioprotector; and 60–200 mM of buffer system; 4–8 wt.% of the substance forming the matrix; and 5–10 wt.% of the substance forming the structure. The dosed virosome composition is frozen at a temperature of -60°C to -80°C. The frozen virosome composition is annealed by keeping it at a temperature below -15°C for 3–9 hours. The hybridized virosome composition is lyophilized to form a dosage form.

EFFECT: invention makes it possible to obtain virosome compositions for sublingual administration that have physical stability, integrity and stability of particles and antigens.

40 cl, 5 dwg, 17 tbl, 6 ex

Description

Link to related applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/772823, filed November 29, 2018, which is incorporated herein by reference in its entirety.

Field of invention

[0002] The present invention relates to a unit oral dosage form that induces mucosal immunization. More specifically, the present invention relates to a lyophilized orally dispersible vaccine containing virosomes. The main project presented in this application was created with funds received from the European Union's Horizon 2020 in accordance with the research and innovation program carried out under grant no. 646122.

Prerequisites for creating an invention

[0003] Vaccines are traditionally administered by intramuscular, intradermal, or subcutaneous injection. These injections can induce strong systemic immune responses, while the effectiveness for triggering mucosal immune responses varies and is often weak or undetectable, particularly for subunit vaccines. From the draining lymph nodes that dispose of the injected vaccine, antigen-specific cytotoxic T cells (CTL) and antibodies produced by B cells can migrate to various organs in the body, but their migration to various mucosal tissues (eg, genitals, intestines) , respiratory tract) is often limited or impossible due to inadequate homing of mucosal receptors and chemotaxis. However, the intranasal route, which is also considered a parenteral immunization route, can trigger effective mucosal immune responses in the respiratory tract, genital tract, and intestinal tract, which sometimes act in concert to make them more accessible if the vaccine is delivered to the mucosal area. Therefore, in some cases, such parenteral vaccines may provide protection against mucosal pathogens.

[0004] Because most pathogens enter the body through mucosal tissues (oral, respiratory, genital, and intestinal tracts), and many replicate only in mucosal tissues, mucosal vaccination may optimally induce first-line protection through induction of both innate immune response (eg, NK cells) and adaptive immune response (T and B cells) in the local and distal areas of the mucosa.

[0005] By continuously protecting the mucosa, such protection can occur immediately without delay in cell recruitment from peripheral sites, allowing transmission and infection events to be more effectively blocked at a very early stage before the pathogen has spread, and in some cases, reservoir formation.

[0006] Mucosal tissues and lymph nodes contain more than 90% immune cells. In addition, mucosal antibodies account for approximately 80% of all antibodies produced by the body. Thus, the local immune response produced by mucosal antibodies may act as a first-line defense against mucosal infections (eg, HIV-1, herpes viruses, rotaviruses, etc.) and their penetration through mucosal tissues into other organs (eg , in the case of HIV-1, hepatitis B, tuberculosis). In contrast, blood antibodies may act as a backup defense after pathogens have overcome mucosal defenses or act synergistically with mucosal antibodies and the mucus environment. Blood antibodies act primarily as an effective first-line defense to combat pathogens that enter directly into the bloodstream after mosquito bites (for example, Plasmodium falciparum, chikungunya virus, dengue virus, Zika virus, West Nile virus, yellow fever virus, etc.) .p.) or after accidental damage to the skin/mucous membrane (for example, by bacteria Staphylococcus aureus, Pseudomonas aeruginosa).

[ 0007] Transmucosal vaccine delivery (transmucosal following buccal, sublingual, intranasal, oral or vaginal administration) is of increasing interest as a means of inducing local and distal humoral immune responses as well as systemic immune responses. In addition, mucosal delivery of vaccine in solid dosage forms (eg, buccal/sublingual tablets, oral tablets or capsules, vaginal pads) may offer several advantages, such as the ability to provide mass immunization, patient compliance, and treatment regimens, ease of use, product stability during storage, possibility of autonomous use regardless of the cold chain. Additionally, mucosal delivery of the vaccine may be appropriate for patients who suffer from needle phobia, and the patient may self-administer the vaccine if given adequate instructions. The buccal/sublingual route has been used for many years to deliver drugs and small molecules into the bloodstream, but its use as a vehicle for mucosal vaccine delivery has received little consideration.

Brief description of the invention

[0008] Lipid-based vesicles can be used as drug, vaccine or adjuvant delivery systems and combinations thereof. Lipid-based vesicles may be composed of one or more natural and/or synthetic lipids forming a core structure (particle) and additional optional components (peptides, proteins, carbohydrates, nucleic acids, small molecules). Lipid-based vesicles can range in size from nanoparticles (approximately 20-200 nm) to submicron particles (approximately 200-800 nm) to micron particles (approximately 800 nm-10 μm).

[0009] Virosomes and liposomes are lipid-based vesicle systems. Virosomes are single-layered, while liposomes can be single-layered, double-layered or multi-layered. Virosomes are a type of subunit vaccine that can contain any envelope protein derived from viruses that is used as starting material for the formation of lipid-based virosome particles. These virosomes may be devoid of genetic material and are non-replicative and non-infectious, making them suitable and safe for enteral and parenteral immunization, provided they can be obtained in a stabilized form. Virosomes may contain: additional molecules, such as homologous molecules (from the same pathogenic source) or heterologous target molecules (derived from a pathogen different from the original viral material), in the form of peptides, proteins and/or carbohydrates; nucleic acids; adjuvants; specific lipids and/or small molecules (drugs). Like virosomes, liposomes can also be used as a carrier for the administration of pharmaceutical drugs, vaccines and adjuvants, but the lipid membrane does not contain any natural viral proteins. In some embodiments, the liposomes used herein may be proteoliposomes (ie, liposomes with proteins).

[0010] Ideally, stabilization either allows the vaccine to be stored independent of the cold chain, or allows the vaccine to be maintained at high and low temperature excursions beyond recommended cold chain conditions without impairing the biological activity of the product.

[0011] Applicants have created a lyophilized orally dispersible vaccine containing virosomes, and have developed a method for producing such a vaccine that can maintain the stability of virosomes (the physical stability of the particle structure and the chemical stability of target molecules). Product stability may be maintained when stored at ambient temperature (e.g., approximately 25°C) regardless of cold chain storage conditions, and may be maintained under occasional freezing conditions (e.g., between approximately -4°C and approximately -17°C). °C), as well as when exposed to high temperatures typical of warm countries (for example, from 35°C to 45°C). The lyophilized sublingual dosage forms described herein that contain a virosome vaccine can induce mucosal immunity and can also induce systemic immune responses. In particular, the combination of a liquid virosome concentrate composition and its buffer, a liquid base matrix composition for a solid dosage form (excluding virosome concentrate), and manufacturing conditions for preparing solid dosage forms in the form of lyophilized sublingual tablets can provide a solid vaccine form that has suitable physical properties for sublingual tablets while maintaining the virosome and structural integrity of the target molecule and storage stability under various environmental conditions. Administration of stable virosomal solid dosage forms may be a suitable method for mucosal immunization by sublingual administration.

[0012] Virosomes belong to the family of enveloped virus-like particles (VLPs) because they have lipid membranes containing viral membrane proteins derived from the original purified viruses. As VLPs, virosomes can closely mimic viral particle architecture, composition, antigen presentation on the membrane surface, and may or may not mimic the functional activity of the native viral envelope. Virosomes may contain reconstituted viral membranes, typically prepared by purifying solubilized membrane proteins and lipids from enveloped viruses with a solubilizing agent, then adding natural or synthetic lipids in the presence or absence of adjuvants, and removing said solubilizing agent from the mixture, resulting in lipid bilayer with proteins protruding from it. Antigens can be native proteins, as well as recombinant or synthetic proteins or peptides. Virosomes can also be prepared first and then modified by covalently or non-covalently modifying their membranes so that they contain adjuvants and other molecules. A characteristic feature of virosomes is that they can closely mimic the composition, surface architecture and functional activities of the native viral envelope. In some cases, if induction of CD8 ⁺ T cells is part of a vaccination strategy, a particularly important property of these virosomes is the retention of hemagglutinin (HA) receptor binding and their hybrid membrane activity. If the vaccination strategy focuses on inducing antigen-specific antibodies, then HA binding activity is not absolutely necessary, however, the presence of HA as an excipient is still important as it can provide assistance to T cells, in particular for small antigens and peptides. which may lack such properties. Although specific vaccines are mentioned herein, all vaccines that use virosomes as a delivery vehicle are within the scope of the present invention. Influenza virosome containing HIV antigens (P1 and rgp41) and TLR 7/8 adjuvant can be used as models of virosome concentrates for the vaccines disclosed herein. Influenza virosomes with the HIV Rgp41 antigen were shown to be able to induce immune responses in various animal studies and in a phase 1 clinical trial by intranasal and intramuscular administration. In such studies, virosome formulations are provided in liquid form, administered by a needle or as an intranasal liquid spray, and these formulations had limited stability when stored at 4°C due to chemical modifications of the active pharmaceutical ingredients (“APIs”). To prevent or reduce such chemical modifications of APIs often present in aquatic environments, the development of solid forms of vaccines with low moisture content has been identified as a promising approach.

[0013] Applicants have been able to overcome various problems in the production of solid vaccine in the form of lyophilized orally dispersible vaccines described in this application. In particular, influenza virus virosome concentrates, which contain virosomes carrying antigens and adjuvants, can be supplied as a suspension in a buffered saline solution. To obtain lyophilized vaccine dosage forms in the form of tablets, these tablets must have stable and porous structures during their manufacture, and these structures must be maintained during subsequent storage. The presence of buffer salts in virosome concentrates can pose a problem in the production of such a stable and porous structure. As such, Applicants have provided the appropriate amount of excipients in combination with appropriate production conditions to provide a stable dosage form that does not degrade or is only partially degraded during lyophilization.

[0014] In addition, the process of freezing, annealing and lyophilization can also lead to disruption of the integrity of the virosome particles. This virosome damage must be minimized so that a sufficient number of virosome particles are retained in the lyophilized product and that sufficient quantities can pass through the mucosal membrane upon sublingual administration when treated with immune cells to induce mucosal immune responses that may also be supported by systemic immune responses. Essentially, applicants have been able to balance the use of excipient and the lyophilization process to maintain both virosome immunogenicity (eg, intact virosomes with limited cluster presence) and beneficial properties of the dosage form (eg, oral disintegrating sublingual tablets).

[0015] In some embodiments, the oral solid vaccine dosage form includes lipid vesicles containing an immunogenic amount of at least one target molecule; 5-20 wt. % of at least one cryolioprotectant; 25-40 wt. % of the substance forming the matrix, and 40-55 wt. % of the substance forming the structure. In some embodiments, the lipid-based vesicles are virosomes or proteoliposomes. In some embodiments of the invention, the dosage form contains 10-15 wt. % of at least one cryolioprotectant. In some embodiments, the at least one cryolioprotectant contains trehalose. In some embodiments of the invention, the dosage form contains 33-37 wt. % of the substance forming the matrix. In some embodiments of the invention, the matrix substance contains gelatin. In some embodiments, the gelatin includes fish gelatin. In some embodiments, the fish gelatin is a high molecular weight fish gelatin. In some embodiments of the invention, the dosage form contains 45-50 wt.% structure-forming substance. In some embodiments of the invention, the structure-forming substance contains mannitol. In some embodiments, virosomes are derived from the membrane of influenza virus or other enveloped viruses. In some embodiments of the invention, at least one target molecule is present on the virosome. In some embodiments, the at least one target molecule comprises an antigen derived from the HIV-1 envelope. In some embodiments, the HIV-1 envelope-derived antigen comprises HIV-1 PI peptide and/or recombinant HIV-1 gp41. In some embodiments of the invention, the virosomes contain an adjuvant. In some embodiments of the invention, the dosage form facilitates the absorption of at least one target molecule in the oral cavity. In some embodiments of the invention, the dosage form disintegrates within 180 seconds after it is placed in the oral cavity. In some embodiments of the invention, the dosage form disintegrates within 90 seconds after it is placed in the oral cavity. In some embodiments, the dosage form disintegrates within 60 seconds of being placed in the oral cavity. In some embodiments, the dosage form disintegrates within 30 seconds of being placed in the oral cavity. In some embodiments of the invention, an immune response is induced when the dosage form is administered to a patient in the oral cavity. In some embodiments of the invention, placement into the oral cavity is accomplished by administering the dosage form onto or under the tongue or into the buccal or pharyngeal region.

[0016] In some embodiments, a method of inducing an immune response in a patient includes administering any of the dosage forms described above to the oral cavity of a person in need of an immune response. In some embodiments of the invention, administration to the oral cavity is accomplished by placing the dosage form on or under the tongue or in the buccal or pharyngeal region.

[0017] In some embodiments of the invention, a method of preparing an oral solid vaccine dosage form includes dosing a liquid virosome composition in a preformed form, where the virosome composition includes: lipid vesicles containing an immunogenic amount of at least one target molecule, 1-5 wt. % cryolioprotectant, 4-8 wt.% of the substance forming the matrix, and 5-10 wt.% of the substance forming the structure; freezing the dosed virosome composition at a temperature from -60°C to -90°C; annealing the frozen virosome composition by keeping it at a temperature below -15°C for 3-9 hours; and lyophilizing the hybridized virosome composition to form a dosage form. In some embodiments of the invention, the dosed virosome composition is frozen at a temperature of -60°C to -90°C for about 1-5 minutes. In some embodiments, lyophilization of the hybridized virosome composition includes a first step of keeping the hybridized virosome composition at a temperature of -10°C to -20°C for 20 to 28 hours and a second step of keeping the hybridized virosome composition at a temperature of -5°C to -5°C to -20°C. approximately -15°C for 14-22 hours. In some embodiments of the invention, lyophilization is carried out at a pressure of less than 600 mbar. In some embodiments, the virosome composition has a pH of approximately 6.5-8. In some embodiments of the invention, the cryolioprotectant contains trehalose. In some embodiments of the invention, the matrix substance contains gelatin. In some embodiments, the gelatin includes fish gelatin. In some embodiments, the fish gelatin is a high molecular weight fish gelatin. In some embodiments of the invention, the structure-forming substance contains mannitol. In some embodiments, the lipid vesicles are derived from influenza virus or respiratory syncytial virus. In some embodiments, the at least one target molecule comprises an antigen derived from the HIV-1 envelope. In some embodiments, the HIV-1 envelope-derived antigen comprises HIV-1 PI peptide or recombinant HIV-1 gp41. In some embodiments of the invention, the lipid-based vesicles include an adjuvant.

[0018] In some embodiments of the invention, a method of preparing an oral solid vaccine dosage form includes: dispensing a liquid virosome composition into a preformed form, wherein the virosome composition includes: (1) 20-50 wt.% virosome concentrate, wherein the virosome concentrate includes: virosomes containing an immunogenic amount of at least one target molecule; 2-10 wt. % cryolioprotector; and 60-200 mM buffer system; (2) 4-8 wt.% of the substance forming the matrix; and (3) 5-10 wt. % of substance forming the structure; freezing the dosed virosome composition at a temperature from -60°C to -90°C; annealing the frozen virosome composition by keeping it at a temperature below -15°C for 3-9 hours; lyophilization of the hybridized virosome composition to obtain a dosage form. In some embodiments of the invention, the buffer system contains HEPES sodium chloride.

[0019] Additional advantages will be readily apparent to those skilled in the art from the following detailed description. The examples and descriptions provided herein are to be considered illustrative and not limiting.

[0020] All publications, including patent documents, scientific articles and databases, mentioned in this application are incorporated herein by reference in their entirety and for all purposes as if each individual publication were separately incorporated by reference. If the definition set forth herein conflicts with or is otherwise inconsistent with the definitions set forth in patents, applications, published applications, and other publications which are incorporated herein by reference, then the definitions set forth in this application shall prevail, and not the definitions that are incorporated herein by reference.

Brief description of drawings

[0021] Representative embodiments of the invention are described with reference to the accompanying drawings, in which:

[0022] Figure 1 presents an illustrative flow diagram for the preparation of the vaccine dosage form disclosed herein.

[0023] Figure 2 shows a flow chart for the production of the vaccine dosage form disclosed herein, from the preparation of the matrix to the preparation of the finished sublingual tablets.

[0024] Figure 3A is a schematic representation of a fully moistened tablet.

[0025] Figure 3B is a schematic representation of tablets with dense lumps.

[0026] Figure 3C is a schematic representation of a tablet with a disintegrated matrix film to form a composition that forms on the surface of a lyophilized (coated) tablet.

[0027] Figure 4 is a photograph of immunoblots illustrating anti-P1 and anti-rg41 specific antibodies binding to virosome-P1 and virosome-rgp41 in reconstituted Zydis® sublingual tablets stored at 5°C, 25°C and 40°C for 1 and 3 months (assay described in Example 2).

[0028] Figure 5 illustrates the immunogenicity of PI and rg41 antigens in liquid virosome concentrates and lyophilized sublingual tablets containing virosomes and stored at 4°C and 40°C for a specified period of time.

Detailed Description of the Invention

[0029] This application describes pharmaceutical compositions of lipid-based vesicles (eg, virosomes or VLPs) and methods for preparing these pharmaceutical compositions. In particular, the present invention relates to lyophilized orally dispersible or disintegrating dosage forms that can maintain VLP stability (i.e., the structural integrity and chemical stability of the antigen), can be stored independently of cold chain storage conditions, and can also be stored under random freezing conditions, and also under the influence of high temperatures characteristic of warm countries. Dosage forms may also retain the physical and chemical properties of VLP, making it suitable for sublingual delivery to induce mucosal immunization.

[0030] Applicants have obtained and optimized the amount of matrix-forming substance and structure-forming substance, which make it possible to obtain a composition that allows the use of high loads of virosome concentrates containing buffer systems and cryolioprotectants. In combination with correction of the amount of excipient, the production parameters of dosage forms were optimized. In particular, annealing time was optimized to maximize mannitol crystallization, which imparts stability to the dosage form and minimizes damage to virosomes. In addition, lyophilization conditions were optimized to minimize damage to virosome particles as well as minimize structural degradation during lyophilization.

Lipid-based vesicle systems

[0031] Lipid-based vesicles can be used as drug, vaccine or adjuvant delivery systems and combinations thereof. Lipid-based vesicular systems may consist of one or more natural and/or synthetic lipids (e.g., phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, cholesterol, sphingolipids, and derivatives thereof) forming the vesicular membrane (particles), and additional optional components ( peptides, proteins, carbohydrates, small molecules). Lipid-based vesicles can range in size from nanoparticles (approximately 20-200 nm) to submicron particles (approximately 200-800 nm) to micron particles (approximately 800 nm-10 μm).

[0032] The lipid-based vesicle/particle may comprise a unilamellar, bilamellar or multilamellar lipid bilayer vesicle. In some embodiments, the lipid-based vesicles/particles may include a lipid bilayer containing lipids selected from natural and/or synthetic lipids. Such lipids can be used to further mimic the pathogenic membrane and lipid backbone microdomains to improve antigen membrane anchoring, antigen presentation, and/or folding for optimal epitope exposure. The lipid-based vesicle/particle may contain a membrane-anchored antigen and/or an adjuvant located on the surface of the particle, within the particle, or in a random orientation. The antigen and/or adjuvant may also be encapsulated within the lumen of the lipid-based vesicle/particle.

[0033] Virosomes are lipid-based vesicles assembled in vitro in a cell-free system to form enveloped VLPs, which belong to the category of subunit vaccines. The virosome lipid membranes used as carriers can be derived from any enveloped virus and therefore contain at least the native viral membrane proteins of the parent virus. These virosomes lack viral genetic material, cannot replicate, and are non-infectious, making them suitable and safe for systemic and mucosal immunization. In addition, virosomes may contain additional molecules, such as antigens (e.g., peptides, proteins, carbohydrates, nucleic acids), adjuvants, specific lipids, and/or small molecules (drugs), which may be anchored on the surface of the virosome and/or enclosed inside the lumen of the virosome.

[0034] Liposomes are also lipid-based vesicles that form a type of subunit vaccine that can be prepared as vesicles having at least one lipid bilayer that do not contain proteins derived from the natural viral membrane of enveloped viruses. Such lipid-based particles, assembled in vitro, do not contain genetic material and may be suitable for systemic and mucosal administration. In addition, liposomes may contain additional molecules, such as antigens (peptides, proteins and/or carbohydrates, nucleic acids), adjuvants, specific lipids and/or small molecules (drugs), which can be anchored on the surface of the liposomes and/or enclosed inside the lumen of liposomes. In some embodiments, the liposomes used in the present invention may be proteoliposomes (ie, liposomes with proteins).

[0035] Lipids used in the dosage forms described herein may be cationic lipids, glycolipids, phospholipids, glycerophospholipids, galactosylceramides, sphingolipids, cholesterol, and derivatives thereof. Phospholipids may include, but are not limited to, phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, cardiolipin and phosphatidylinositol with various fatty acyl compositions.

[0036] In addition, lipids can be selected from DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N, N,N-trimethylammonium chloride), DOTAP (N-[1-(2,3 -dioleoyloxy)propyl]-N,N,N-trimethylammonium), DODAC (N,N-dioleyl-N,N-dimethylammonium chloride), DDAB (didodecyldimethylammonium bromide) and stearylamine or other aliphatic amines and the like.

Virosomal drug

Virosome concentrate

[0037] Figure 1 is a flow diagram of a method 100 for preparing the vaccine dosage form described herein. At step 101, the liquid virosome concentrate may be mixed with the base matrix premix to form a liquid virosome composition suitable for lyophilization. Applicants have discovered that when a virosome concentrate composition is combined with a specific base matrix composition and prepared in combination with a number of formulation conditions optimized to retain sufficient quantities of virosomes with the desired particle characteristics, the biological activity of the vaccine can be maintained. The bioactivity of virosome particles may depend on the conformational integrity of the particle and the quality of the antigenic molecules that are associated with its unilamellar phospholipid membrane.

[0038] In some embodiments, the virosome concentrate may be a liquid virosome concentrate. The virosome concentrate includes at least one virosome population. The virosome population may be virosomes containing the drug or virosomes acting as a vaccine delivery vehicle containing vaccine antigens and viral proteins (eg, HA if the virosomes are derived from influenza virus).

[0039] In some embodiments, virosomes can be derived from influenza virus to produce influenza virus virosomes as enveloped VLPs that act as a carrier for heterologous vaccine antigens (e.g., HIV antigens anchored on influenza virus-derived virosomes), or 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 virus can be used to produce the corresponding RSV virus, Sendai virus, SFV virus, VSV -virosome or Sindbis virosome to present a homologous vaccine antigen (eg, native RSV antigens on RSV-derived virosomes). In some embodiments, virus-based virosomes can be derived from any enveloped virus. In some embodiments, virus-based virosomes can be derived from DNA viruses, including, but not limited to, herpesviruses, poxviruses, and hepadnaviruses. In some embodiments, virus-based virosomes can be derived from RNA viruses, including, but not limited to, flavivirus, togavirus, coronavirus, hepatitis D virus, orthomyxovirus, paramyxovirus, rhabdovirus, bunyavirus, and filovirus. In some embodiments, virus-based virosomes can be derived from retroviruses.

[0040] In some embodiments, an influenza virus virosome can be produced by various methods described in Influenza virosomes as vaccine adjuvant and carrier system; Moser C et al. Expert review, 779-791 (2013); WO 2004110486; WO 2004071492; WO 2007099446; WO 201603919; EP 2058002 and WO 2016039620, which are incorporated herein by reference in their entirety. In addition, various patents and other publications listed in the previously cited documents are also incorporated herein by reference in their entirety. As such, the present application is not limited to a particular method for producing virosomes. So, for example, the link above simply points to an example of a virosome preparation. This application applies to all lipid-based particles, such as, but not limited to, virosomes, VLPs and nanoparticle preparation, including antigen-loaded liposomes.

[0041] Although this section describes a liquid virosome concentrate, however, the virosome can be replaced by a liposome to form a liquid liposome concentrate. Thus, for example, components in a virosome concentrate (other than the virosomes themselves) may also be equally applicable to a liposome concentrate.

[0042] A virosome (eg, a virosome derived from an influenza virus) may have viral proteins on its surface. In some embodiments, the protein may be hemagglutinin (“HA”) and/or neuraminidase (“NA”). In some embodiments, the liquid virosome concentrate includes viral membrane proteins (eg, HA) present at concentrations determined by standard assays. In some embodiments, the viral membrane proteins comprise approximately 10-300 μg/ml, or approximately 5-150 μg/ml liquid virosome concentrate if influenza virus virosomes are used as the carrier of heterologous vaccine antigens. For influenza virosomes designed to induce CTL responses, the HA concentration may be in the range of about 150-800 μg/ml or about 75-400 μg/ml. In some embodiments of the invention, the concentration of viral membrane proteins may exceed the concentrations listed above by way of example.

[0043] The virosome concentrate may also include lipids. The lipids used in the virosome concentrate may be cationic lipids, glycolipids, phospholipids, glycerophospholipids, galactosylceramides, sphingolipids, cholesterol and derivatives thereof. Phospholipids may include, but are not limited to, phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, cardiolipin and phosphatidylinositol with various fatty acyl compositions. In addition, the lipids can be selected from DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP (N-[1-(2,3-dioleoyloxy) chloride) propyl]-N,N,N-trimethylammonium), DODAC (N,N-dioleyl-N,N-dimethylammonium chloride), DDAB (didodecyldimethylammonium bromide) and stearylamine or other aliphatic amines and the like. In some embodiments, the liquid virosome concentrate may include about 0.1-10 mg/ml, about 0.3-8 mg/ml, or about 0.5-5 mg/ml lipids.

[0044] Virosomes may contain target molecules (eg, vaccine antigens) in the presence or absence of adjuvants. Antigens may be soluble and may be entrapped within the virosome lumen or covalently or non-covalently anchored to virosomes, and may be peptides, proteins, polysaccharides, whole particles or partial fragments or extracts of bacterial cells, viral particles or nucleic acids, or they may be produced from parasites such as protozoa or worms that cause disease, or combinations thereof, or from any cells or cell lines of plants, animals or humans. Any antigen known to those skilled in the art may be suitable for use with virosomes, including commercially available virosomes, or may be prepared by purifying pathogen or cancer cell preparations or untransformed cells (native proteins), recombinantly expressed or synthesized in a standard industrial way. Methods for preparing suitable antigens for inclusion in virosomes are known to those skilled in the art, and any of these known methods can be used as described herein.

[0045] The target molecule(s) are included in the virosomes of the liquid virosome concentrate and in the resulting dosage forms described herein in an amount sufficient to render it immunogenic when administered in dosage form. An "immunogenic amount" is defined as an amount sufficient to stimulate the desired immune response. One skilled in the art can readily determine the immunogenic amount for a given disease or infection depending on, among other factors, the route of immunization and the age and weight of the patient to whom the dosage form will be administered.

[0046] In some embodiments, the liquid virosome concentrate may include approximately 1-2000 μg/ml of the target molecule. The target molecule may be a peptide, protein, carbohydrate, nucleic acid or small molecule, or a combination thereof. The target molecule may function as an antigen (eg, 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 one target molecule and about 1-1000 μg/ml of a second target molecule. After further processing into solid dosage forms, each tablet may contain from approximately 0.01 to 250 μg of each target molecule.

[0047] In more preferred embodiments of the invention, the liquid virosome concentrate may include about 25-500 μg/ml, 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 100-200 μg/ml, or about 200-400 μg/ml of at least one target molecule (eg, antigen). In some embodiments, the liquid virosome concentrate may 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 , approximately 25-200 μg/ml, approximately 50-250 μg/ml, approximately 75-225 μg/ml or approximately 100-200 μg/ml per target molecule and approximately 50-450 μg/ml, approximately 50-400 μg /ml, approximately 25-225 μg/ml, approximately 25-200 μg/ml, approximately 100-500 μg/ml, approximately 150-450 μg/ml, approximately 175-425 μg/ml or approximately 200-400 μg/ml second target molecule.

[0048] The dosage forms disclosed herein may be used to deliver therapeutic or prophylactic vaccines for the purpose of preventing or reducing symptoms associated with allergies or infection, tumor development and spread, pathogen transmission, cellular infection, pathogen load, following B cell (antibody) induction ) and/or corresponding T cell subpopulations (for example, Th1, Th2, Thl6, Thf, Tel, Tc2, Tc3, Treg, etc.), depending on the composition of the virosome compositions. For this purpose, target molecules can be formulated to provide protection against the following diseases, the list of which is not exhaustive, such as influenza, tuberculosis, meningitis, hepatitis, whooping cough, polio, tetanus, diphtheria, malaria, cholera, herpes , typhus, HIV/AIDS, measles, Lyme disease, traveler's diarrhea, hepatitis A, B and C, otitis media, dengue fever, rabies, parainfluenza, rubella, yellow fever, dysentery, Legionnaires' disease, toxoplasmosis, Q-fever, hemorrhagic fever, Argentine hemorrhagic fever, dental caries, Chagas disease, E. coli urinary tract infection, pneumococcal diseases, mumps, chikungunya virus disease, cancer, allergies and combinations thereof. In addition, the target molecules may provide protection against disease caused by the following non-exhaustive microorganisms, such as Vibrio spp., Salmonella spp., Bordetella spp., Haemophilus spp., Toxoplasmosis gondii, Cytomegalovirus, Chlamydia spp., Streptococcus spp., Norwalk virus , Escherischia coli, Helicobacter pylori, rotavirus, Neisseria gonorrhae, Neisseria meningiditis, adenovirus, Epstein-Barr virus, Japanese encephalitis virus, Pneumocystis carini, herpes simplex virus, Clostridia species, respiratory syncytial virus, Klebsiella species, Shigella species, Pseudomonas aeruginosa, parvovirus, Camylobacter spp., Rickettsia spp., varicella zoster virus, Yersinia spp., Ross River virus, J.C. virus, Rhodococcus equi, Moraxella catarrhalis, Borrelia burgdorferi, Pasteurella haemolytica, and combinations thereof. Additionally or alternatively, the target molecule may provide protection or treatment against allergies (ie, using virosomes containing allergens), cancer (eg, using tumor antigens, antibodies, anticancer drugs, nucleic acid) and other types of diseases.

[0049] The use of the present invention in veterinary medicine is also contemplated. Accordingly, the target molecules may provide protection against animal diseases specified in the following non-exhaustive list, such as coccidiosis, Newcastle disease, enzootic pneumonia, feline leukemia, atrophic rhinitis, erysipelas, foot and mouth disease, porcine diseases, pneumonia and other pathological conditions and other infections, affecting domestic pets and farm animals, and their combinations.

[0050] In some embodiments, the virosome contains 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 P1 peptide and/or recombinant HIV-1 gp41.

[0051] As stated above, liquid virosome concentrate virosomes may also contain an adjuvant or may be mixed with an adjuvant. Improving the immune response may require the inclusion of an adjuvant in virosomes and other subunit vaccines, which will lead to accelerated and enhanced antibody and T-cell production while maintaining immunological memory. To be effective, they preferably produce an immune response associated with the production of immunological memory that provides long-lasting protection against a particular disease. An adjuvant can also reduce the dose of antigen (provide a gentle dose) and broaden the spectrum of the desired immune response. Once exposed to antigens, the immune system can “remember” it, and when exposed to the antigen again, the immune response will be produced much faster. The effectiveness of an adjuvant in enhancing the immune response may be independent of the antigen with which it is combined, since the adjuvant itself can trigger nonspecific immune responses that can lead to autoimmune side effects if strong cell activation is achieved in the absence of the antigen. However, if the antigen and adjuvant physically bind to each other, then they can all migrate to the same site after injection, which will promote antigen-specific immune activation with lower nonspecific inflammatory responses. Suitable adjuvants include, but are not limited to: decoy-like receptor (TLR) agonists, inflammasome agonists, nucleotide-binding and oligomerization domain-like receptor (NOD) (NLR) agonists, and more particularly, non-toxic bacterial moieties; cholera toxin (and its detoxified forms and fractions); chitosan; heat labile E. coli toxin (and its detoxified forms and fractions); lactide/glycolide homopolymers and copolymers (PLA/GA); polyanhydride, e.g. trimellithylimido-L-tyrosine, DEAE-dextran, saponins complexed with membrane protein antigens (immunostimulating complexes - ISCOM), bacterial products such as lipopolysaccharide (LPS) and muramyl dipeptide (MDP), liposomes, cochleates, proteonoids, cytokines (interleukins, interferons), genetically engineered live microbial vectors, non-infectious pertussis mutant toxin, neurimidase/galactose oxidase, and attenuated bacterial and viral toxins derived from mutant strains, and combinations thereof. A suitable amount of adjuvant can be easily determined by one skilled in the art.

[0052] In some embodiments, the virosomes may contain the adjuvant 3M-052 (a TLR7/8 agonist available from 3M). In some embodiments, the liquid virosome concentrate may contain 3M-052 adjuvant in an amount of about 8-140 μg/ml, about 4-70 μg/ml, about 1-60 μg/ml, and from 0.01 to 16 μg per tablet .

[0053] In some embodiments, the liquid virosome concentrate may include at least two different virosome populations, each containing at least one antigen with or without adjuvant. In some embodiments, the two different virosome populations may have different antigens but the same adjuvant (eg, virosome-P1/3M-052 mixed with virosome-rgp41/3M-052). In some embodiments, the two different virosome populations may have different antigens and different adjuvants (eg, virosome-P1/adjuvant A mixed with virosome-rgp41/adjuvant B).

[0054] In some embodiments, the virosome concentrate may include at least two different antigens per virosome, with or without adjuvant (eg, a virosome containing P1 and rgp41 antigens with or without adjuvant).

[0055] The liquid virosome concentrate may also include a buffer system. In some embodiments of the invention, virosomes can be suspended in a buffer system. The buffer system can 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 desired pH of about 6-9, about 6.5-8, about 7-8, about 7.2-7.6, about 7.3-7.5, or approximately 7.4. In addition, the buffer system can stabilize virosomes if it is present in liquid form at a storage temperature of approximately 2-8°C.

[0056] Suitable buffer systems include, but are not limited to, HEPES-sodium chloride (HN) buffers, HEPES-sodium chloride-EDTA (HNE) buffers, phosphate buffer systems (PBS), or combinations thereof. In some embodiments, the buffer system may 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, approximately 185-200 mM, approximately 185-195 mM, or approximately 190-195 mM in virosome concentrate. If the buffer system is HEPES-sodium chloride in virosome concentrate, then the sodium chloride 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 approximately 140-145 mM in virosome concentrate. If the buffer system is HEPES-sodium chloride in viral concentrate, then HEPES 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, approximately 45-55 mM or approximately 48-52 mM in virosome concentrate.

[0057] The liquid virosome concentrate may also include at least one cryolioprotectant. Virosomes may be damaged during the freezing and/or lyophilization steps used to obtain the dosage forms described herein. For example, a cryolioprotectant may be included in a virosome concentrate to improve virosome preservation during freezing and/or lyophilization steps. Examples of cryolioprotectants 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 oligosaccharide), or combinations thereof. The cryolioprotectants used here may be inert to the vaccine composition. The liquid virosome concentrate may include approximately 1-20% w/w. approximately 1.5-10% w/w, approximately 2-10% w/w, approximately 4-10% w/w, approximately 2-9% w/w, approximately 2-5 % w/w, approximately 3-8% w/w, approximately 3.5-8% w/w, approximately 3.5-7% w/w, approximately 4-8% w/w ./mass. or approximately 5-7% w/w. cryolioprotector.

[0058] The virosome concentrate may be approximately 1-75% w/w. approximately 10-65% w/w approximately 15-60% w/w approximately 20-55% w/w approximately 20-50% w/w or approximately 25-50% w/w. virosome composition. In some embodiments, the virosome concentrate may be approximately 15-35% w/w. approximately 20-30% w/w approximately 23-27% w/w or approximately 25% w/w. virosome composition.

Composition of the main matrix

[0059] The composition of the base matrix forms the structure of the final dosage form. For example, the base matrix composition may include a matrix forming substance. The matrix substance may provide a network structure to the dosage form that imparts strength and resiliency during handling. Suitable matrix forming agents may include, but are not limited to, gelatin, starch, or combinations thereof. Additional matrix forming agents can be found in EP 2624815 B1, which is incorporated herein by reference in its entirety. The gelatin may be fish gelatin, bovine gelatin, porcine gelatin, or a combination thereof. Each of these gelatins may have different gelling properties. The extent to which a gelatin solution forms a gel may depend on the gelatin concentration and the temperature of the gelatin solution. Bovine gelatin solution tends to form a gel at temperatures below 18°C and can thus be considered a gelling gelatin. In contrast, fish gelatin can remain in solution at temperatures of at least 10° C. and thus can be considered a non-gelling gelatin. In some embodiments, the gelatin may be a low endotoxin content gelatin, such as gelatin derived from a specific source or prepared by a method described in Provisional Application No. 62/640394, which is incorporated herein by reference in its entirety. In some embodiments, the amount of matrix forming material in the virosome composition may be about 1-15% w/w, about 2-12% w/w, about 3-10% w/w. approximately 4-8% w/w approximately 4-6%, approximately 5-7% w/w. or approximately 6% w/w.

[0060] The temperature at which the virosome composition is dosed may be at least 10-18°C. Thus, for example, a composition that uses only bovine gelatin may not be dosed at these low temperatures. However, a combination of bovine gelatin and another type of gelatin (eg fish gelatin) can be used. Applicants have discovered that using fish gelatin, a lyophilized tablet with a strong matrix structure and a disintegration time of approximately 30-180 or 30-60 seconds can be prepared, which is desirable to ensure sufficient contact time with the oral mucosa. In addition, lyophilized dosage forms with good physical properties can be prepared using fish gelatin to produce drug formulations that have a high loading of soluble component, such as buffer salts, for example, in the amounts described herein.

[0061] In some embodiments, the fish gelatin may be high molecular weight fish gelatin, standard molecular weight fish gelatin, or combinations thereof. High molecular weight fish gelatin is defined as fish gelatin in which more than 50% of the molecular weight distribution exceeds 30,000 daltons. Standard molecular weight fish gelatin is defined as fish gelatin in which more than 50% of the molecular weight distribution is below 30,000 daltons.

[0062] In some embodiments, the matrix substance may also serve as an antigen stabilizer as well as a mucoadhesive. In addition, starch can also serve as an immune-stimulating filler.

[0063] The base matrix composition may also include a structure-forming substance. Suitable structure-forming agents may include sugars, including, but not limited to, mannitol, dextrose, lactose, galactose, cyclodextrin, or combinations thereof. Structure-forming substances can be used in lyophilization as an excipient if it crystallizes to provide structural stability to the lyophilized product. Soluble excipients such as buffer salts and trehalose in the virosome composition can inhibit its crystallization. To ensure crystallization, hybridization is usually carried out over an extended period of time. However, the presence of these soluble fillers may also cause the frozen product to melt during annealing. Thus, for example, applicants have established a balance between the amount of structure-forming substance, buffer salts and cryolioprotectant, and annealing conditions (ie, temperature and time). In some embodiments, the amount of structure-forming material in the virosome composition may be approximately 1-20% w/w. approximately 3-15% w/w approximately 4.5-10% w/w approximately 4.5-8% w/w approximately 5-10% w/w approximately 6-10% w/w approximately 7-9% w/w or approximately 8% w/w. Applicants have discovered that at builder values below 4.5% w/w, some microstructure disruption may occur during lyophilization, which will result in poor dispersion/disintegration of the lyophilized dosage form. For example, it has been found that more structure-forming material will minimize or eliminate microstructure disruption without any significant effect on the virosome.

[0064] In addition, the base matrix composition may also include a cryolioprotectant. Examples of cryolioprotectants 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 oligosaccharide), or combinations thereof. A cryolioprotectant can be used to protect the virosome from damage during subsequent freezing and lyophilization. However, the addition of a cryolioprotectant may cause destruction of the microstructure of the dosage form matrix during lyophilization. Thus, for example, a balance must be found to minimize microstructural degradation while maintaining sufficient virosome numbers to maintain virosome quality to induce an immune response. The amount of cryolioprotectant in the base matrix may be approximately 0.01-2% w/w, approximately 0.1-1.5% w/w, approximately 0.2-1% w/w. or approximately 0.25-0.75% w/w. Thus, for example, the total amount (hereinafter referred to as the “total amount”) of cryolioprotectant in the virosome composition (i.e., the liquid virosome concentrate plus the base matrix composition) may be about 0.5-6% w/w, about 0.5-6% w/w. 5% w/w, approximately 0.5-4.5% w/w, approximately 1-4.5% w/w, approximately 1.5-4.5% w/w , approximately 1.5-3% w/w, approximately 1.5-2.5, approximately 2-3% w/w, approximately 2.5% w/w. or approximately 2% w/w. Applicants have also discovered that at these levels, the cryolioprotectant can provide sufficient cryolioprotection without causing undesirable microstructure degradation during lyophilization.

[0065] In some embodiments of the invention, the base matrix composition may also include a mucoadhesive agent, such as a resin. Suitable gums include, but are not limited to, gum arabic, guar gum, agar, xanthan, gellan, carrageenan, curdling agent, konjac, locust bean, vellan, gum tragacanth, gum arabic, karaya gum, ghatti gum, pectins, dextran, glucomannan and alginates, or combinations thereof.

[0066] The base matrix composition may also contain additional pharmaceutically acceptable agents or excipients. Such additional pharmaceutically acceptable agents or excipients include, but are not limited to, sugars such as mannitol, dextrose and lactose, inorganic salts such as sodium chloride and aluminum silicates, mammalian gelatins, fish gelatin, modified starches, preservatives, antioxidants, surface active substances, viscosity enhancers, penetration enhancers, colorants, flavors, pH modifiers, sweeteners, taste-masking agents, and combinations thereof. Suitable coloring agents may include red, black and yellow iron oxides and FD&C dyes such as FD&C Blue No. 2 and FD&C Red No. 40, and combinations thereof. Suitable flavors may include mint, raspberry, licorice, orange, lemon, grapefruit, caramel, vanilla, cherry and grape flavors and combinations thereof. Suitable pH modifiers may include citric acid, tartaric acid, phosphoric acid, hydrochloric acid, maleic acid, sodium hydroxide (eg, 3% w/w sodium hydroxide solution), and combinations thereof. In some embodiments, the core matrix composition and/or virosome composition contains a pH modifier in an amount sufficient to maintain a desired pH of about 6-9, about 7-8, about 7.2-7.6, about 7.3-7 .5 or approximately 7.4. Suitable sweeteners may include aspartame, acesulfame K and thaumatin and combinations thereof. One skilled in the art can readily determine appropriate amounts of these various additional excipients, if desired.

[0067] The base matrix composition may also include a solvent. In some embodiments, the solvent may be water (eg, purified water). In some embodiments, everything else in the base matrix and/or virosome composition is a solvent.

[0068] The composition of the base matrix may be approximately 25-99% w/w. approximately 35-90% w/w approximately 40-85% w/w approximately 45-80% w/w or approximately 50-75% w/w. from the virosome composition. In some embodiments of the invention, the composition of the base matrix may be approximately 65-85% wt./mass. approximately 70-80% w/w approximately 73-77% w/w or approximately 75% wt./mass. from the virosome composition.

Preparation of a dosage form containing a virosome composition

[0069] As stated above, the liquid virosome concentrate is mixed with the base matrix composition to obtain a virosome composition in step 101, suitable for lyophilization. Figure 2 provides a more detailed description of the method for preparing the vaccine dosage form described herein. In some embodiments of the invention, the base matrix composition can be prepared by dissolving a matrix-forming substance and a structure-forming substance in a solvent to form a premix. For example, gelatin and mannitol can be dissolved in water as shown in step 201 in figure 2. The premix can be heated to about 40-−80°C, about 50-70°C, about 55-65°C, or to about 60°C and may be maintained for about 45-75 minutes, about 55-65 minutes, or about 60 minutes. As shown at step 202, the premix can be heated to 60°C and can be maintained for 1 hour. The premix can then be cooled to about 30-50°C, about 35-45°C, or about 40°C and sifted before cooling to about 10-20°C or about 15°C and can be maintained at this temperature for during the remaining time of this process. As shown in step 203, the premix can be cooled to 40°C and sieved. The premix can then be cooled to 15°C as shown in step 204.

[0070] A cryolioprotectant may then be added to the premix. For example, trehalose may be added to the premix as shown in step 205. The pH may then be adjusted to about 6-9, about 7-8, about 7.2-7.6, about 7.3 -7.5 or up to approximately 7.4 using a pH modifier. For example, the pH can be adjusted to 7.4 using sodium hydroxide solution as shown in step 206. After pH adjustment, liquid virosome concentrate can be added. After adding the liquid virosome concentrate, the pH can be adjusted again (at step 207) and, if necessary, adjusted to about 6.0-8.5, about 7-8, about 7.2-7.6, about to 7.3-7.5 or approximately 7.4 using an additional pH modifier. This mixture can be prepared to the required batch size with solvent (ie, to obtain a virosome composition). For example, if necessary, some water may be added to the mixture, as shown in step 208.

[0071] At step 102, as shown in Figure 1, the liquid virosome composition can be dispensed in a preformed form. As used herein, the term "metered" refers to the precipitation of a predetermined aliquot of a solution or suspension. As used herein, the term "preform" refers to any suitable container or vessel into which an aqueous solution or suspension can be deposited and in which lyophilization can subsequently be carried out. In some embodiments of the invention, the preformed form is a blister pack with one or more blister pockets. Predetermined aliquots of about 150-1000 mg or about 500 mg wet dose formulation weight (also referred to herein as formulation dose weight) of the virosome composition can be prepared in preformed forms. In some embodiments of the invention, the virosome composition can be dosed at about 10-20°C or about 15°C. Thus, for example, the virosome composition can be dosed at 15°C with a formulation weight of 500 mg, as shown in step 209.

[0072] At step 103 shown in Figure 1, the dosage virosome compositions can then be frozen in preformed forms. Dosed virosome compositions in preformed forms can be frozen by any method known to those skilled in the art. For example, the compositions can be passed through a cryogenic chamber (for example, through a liquid nitrogen tunnel). The temperature during freezing may 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 freezing time may be from about 1.5 to 5 minutes, about 2 to 4.5 minutes, about 2.5 to 4 minutes, about 3 to 4 minutes, about 3 to 3.5 minutes, or about 3.25 minutes. For example, the dosed virosome composition can be frozen at -70°C for 3 minutes and 15 seconds, as shown in step 210.

[0073] At step 104 shown in Figure 1, frozen unit doses in preformed forms can be collected and placed in a refrigerator at a temperature of less than about -25°C, about -20°C, about -15°C, about -10°C, approximately -5°C and annealed (that is, frozen) for a period of time sufficient to crystallize the substance forming the structure. Crystallization of the structure-forming material can result in frozen unit doses having structural strength suitable to prevent the frozen unit units from breaking during lyophilization. This may be important for the integrity of the virosome. The annealing time may be about 3-9 hours, about 4-8 hours, about 5-7 hours, or about 6 hours. For example, frozen unit doses can be annealed at temperatures below -15°C for approximately 3-9 hours, as shown in step 211.

[0074] After annealing, the hybridized frozen unit doses can be lyophilized at step 105 to form a dosage form. During the lyophilization process, water is sublimated from frozen unit doses. In some embodiments of the invention, frozen unit doses may be loaded onto freeze dryer shelves. In some embodiments, the lyophilizer may be pre-cooled to a temperature of about -15 to -35°C, about -20 to -30°C, or about -25°C. Once the hybridized frozen unit doses are placed in the lyophilizer, the lyophilization cycle can be started. In some embodiments, once the lyophilization cycle has begun, the vacuum may be released and the temperature on the shelf will increase. The lyophilizer can operate at low pressure (i.e., under vacuum). In some embodiments, the lyophilizer may be operated at a pressure of less than or equal to about 1000 mbar, about 900 mbar, about 800 mbar, about 700 mbar, about 600 mbar, about 500 mbar, or about 400 mbar.

[0075] Applicants have discovered that a two-step lyophilization cycle (step 212) can provide structural stability to the dosage form with minimal damage to the virosome in the dosage form. The two-step lyophilization cycle may include a first step of holding the frozen unit doses at a temperature of about -5°C to -25°C, about -10°C to -20°C, about -13°C to -17°C, or about -15°C for approximately 12-36 hours, approximately 18-30 hours, approximately 20-28 hours or approximately 24 hours. In addition, a two-stage lyophilization cycle may include a second stage that follows the first stage. The second stage may include holding the frozen unit doses at a temperature of about 0°C to about -20°C, about -5°C to about -15°C, about -8°C to about -12°C, or about -10 °C for approximately 6-30 hours, approximately 12-24 hours, approximately 14-22 hours or approximately 18 hours. At the end of the two-stage lyophilization cycle, the temperature of the lyophilizer can be raised to ambient temperature (ie, approximately 20-25°C or approximately 23°C).

[0076] In some embodiments of the invention, a two-step lyophilization method may include pre-cooling the lyophilizer to approximately -25°C, increasing the temperature of the lyophilizer for 2 hours to -15°C, maintaining the lyophilizer at -15°C for 24 hours, increasing temperature of the freeze dryer to -10°C for 2 hours, holding at -10°C for 18 hours, increasing the temperature to 0°C for 15 minutes and increasing the temperature to 23°C for 15 minutes in that order.

[0077] The lyophilized dosage forms may be removed from the lyophilizer and assessed for any defects (through quality testing as described below) at step 213. The dosage forms may then be placed in a storage cabinet at an air humidity of less than approximately 35% RH until until the dosage forms are sealed in preformed forms. The sealing process (step 214) can be carried out by wrapping them in sealed foil on preformed forms to produce blister packs of lyophilized dosage forms.

[0078] Water in lyophilized dosage forms can be removed by sublimation during lyophilization. Accordingly, the remainder of the virosome concentrate in the lyophilized dosage form, excluding the cryolioprotectants (i.e., virosomes, antigens, adjuvants and buffer system remaining in the lyophilized virosome concentrate), may be approximately 1-5 wt.%, approximately 2-4 wt.%, approximately 2.5-3.5 wt.%, approximately 2.6-3.4 wt.%, approximately 2.7-3.3 wt.%, approximately 2.8-3.2 wt. %, approximately 2.9-3.1 wt.% or approximately 3-3.1 wt.% of the dosage form.

[0079] As stated above, the target molecule(s) are included in the dosage forms described herein in an amount that is sufficient to impart immunogenicity when administered in the dosage form. One skilled in the art can readily determine the immunogenic amount for a given disease or infection based on, among other factors, the route of administration, age, and weight of the patient to whom the dosage form will be administered. In some embodiments, the solid dosage form may contain 0.01-250 μg of each target molecule (eg, HIV-1 P1 peptide and/or rgp41).

[0080] In some embodiments of the invention, at least one of the cryolioprotectants in the lyophilized dosage form may be approximately 5-20 wt. %, approximately 8-18 wt. %, approximately 10-15 wt. %, approximately 11-15 wt. % or approximately 12-15 wt. % dosage form. In some embodiments, at least one of the cryolioprotectants in the lyophilized dosage form may comprise about 1-5 wt%, about 1-4 wt%, or about 2-4 wt% of the dosage form.

[0081] In some embodiments of the invention, the amount of matrix forming substance in the dosage form may be approximately 20-50 wt. %, approximately 25-45 wt. %, approximately 25-40 wt. %, approximately 30-40 wt. %, approximately 33-37 wt. % or approximately 35-37 wt. %. In some embodiments of the invention, the amount of structure-forming substance in the dosage form may be approximately 27-65 wt. %, approximately 27-60 wt. %, approximately 40-55 wt. % or approximately 45-50 wt. %. In some embodiments of the invention, the residual amount of pH modifier in the lyophilized dose (eg, sodium hydroxide) may be approximately 0.01-0.08 wt. %.

[0082] The dosage forms of the invention are dissolving dosage forms and accordingly have the distinct advantage of faster disintegration time. The route of administration may be oral, vaginal or intranasal, although the preferred route of administration is oral (ie, sublingual and/or buccal). The dosage form, once placed in the oral cavity and contacted with saliva, may disintegrate over a period of about 1 to about 180 seconds, about 1 to about 120 seconds, about 1 to about 60 seconds, and preferably within about from 1 to about 30 seconds, more preferably for about 1 to about 10 seconds, and most preferably less than about 5 seconds.

Compositions, test methods and examples

[0083] As examples, a liquid virosome concentrate derived from influenza virus was used. The virosomes contained influenza virus HA as well as added antigens and adjuvants. Two virosome preparations were prepared, each containing one antigen derived from the HIV envelope glycoprotein. Liquid virosome concentrate is a mixture of two virosome preparations. The two antigens derived from HIV gp41 are the P1 peptide, which has the last 35 C-terminal ectodomain residues, and the truncated rgp41, which lacks cluster I and the last 21 C-terminal ectodomain residues. Adjuvant 3M-052 was either present or absent in any virosome preparation. Virosomes were suspended in HEPES-sodium chloride buffer containing 142.5 mM sodium chloride and 50 mM HEPES at pH 7.4. Additionally, trehalose (a crylioprotectant) has been tested at concentrations of 0-10% w/w. liquid virosome concentrate. The following Table 1 summarizes the target compositions of the liquid HIV-1 virosome concentrate used in some of our experiments, during which a 500 mg aliquot (formulated dose weight) of the aqueous virosome composition was measured and placed into the pockets of a preformed blister pack, followed by freezing and lyophilization. The weight of the prepared dose can range from 150 mg to 1000 mg, and the compositions of the liquid HIV-1 virosome concentrate can be adjusted to match the required target dose. It should be noted that the following target intervals may depend on the purpose and subsequent use, for example, for animal studies or for human studies. Thus, other desired concentrations may be used for other purposes.

Table 1

Example of lipid particle concentrates to produce a 500 mg weight prepared dose of Zydis® virosome composition Example of target molecules and excipients in lipid-based particle concentrates HIV Vaccine MYM-V202 (4 × concentrate for 25% liquid virosome loading in Zydis® virosome formulation) - Target molecule (P1 antigen): 50-450 μg/ml
- Target molecule (antigen (rgp41): 50-400 μg/ml
- HA Excipient: 10-160 µg/ml
- Adjuvant (eg 3M-052): 8-140 µg/ml
- Phospholipids: 0.5-5 mg/ml
- Sodium chloride: 50-150 mM
- HEPES 10-50 mM
- Trehalose: 4-10% w/w
- pH 6.5-8.0 HIV vaccine MYM-V202 (2 × concentrate for 50% liquid virosome loading in Zydis® virosome composition) - Target molecule (P1 antigen): 25-225 µg/ml
- Target molecule (antigen (rgp41): 25-200 μg/ml
- HA Excipient: 5-80 µg/ml
- Adjuvant (eg 3M-052): 4-70 mcg/ml
- Phospholipids: 0.5-5 mg/ml
- Sodium chloride: 50-150 mM
- HEPES 10-50 mM
- Trehalose: 2-5% w/w
- pH 6.5-8.0 Placebo vaccine VP02 (4 × concentrate for 25% liquid virosome loading in Zydis® final virosome formulation) - HA Excipient: 10-160 µg/ml
- Adjuvant (eg 3M-052): 8-140 µg/ml
- Phospholipids: 0.5-5 mg/ml
- Sodium chloride: 50-150 mM
- HEPES 10-50 mM
- Trehalose: 4-10% w/w
- pH 6.5-8.0

[0084] In some of our experiments, the target dose for HA and HIV-1 antigens for each tablet was 20 μg HA, 25 μg P1, and 50 μg rgp41. Producing these doses requires a high payload of liquid virosome concentrates in combination with a large wet weight of the prepared dose. Table 2 shows various loading combinations of liquid virosome concentrate (ranging from 25 to 50% w/w) with wet weight of the prepared dose (ranging from 250 to 1000 mg). The wet weight of the prepared dose represents the amount of aliquot of virosome composition that was measured per dose before lyophilization.

table 2

HA and antigen content in liquid virosome concentrate % Loading of liquid virosome concentrate as part of the main matrix Weight of prepared dose Target dose of virosome vaccine per tablet HA/P1/rpg41 80 µg/ml HA (with adjuvant 3M-052)
100 µg/ml P1
200 µg/ml rgp41 25% w/w 1000 mg 20 mcg /25 mcg /50 mcg 50% w/w 500 mg 20 mcg /25 mcg /50 mcg 160 µg/ml HA (with adjuvant 3M-052)
200 µg/ml P1
400 µg/ml rgp41 25% w/w 500 mg 20 mcg /25 mcg /50 mcg 50% w/w 250 mg 20 mcg /25 mcg /50 mcg

[0085] A 25% loading of liquid virosome concentrate can be added to the base matrix formulation. The weight of the prepared dose can be 500 mg. Table 3 shows the ranges of estimated NA and antigen content.

Table 3

Component Target composition in liquid concentrate Composition received for evaluation Composition of 25% loading composition Virosome labeled with antigens and adjuvants HA: 10-160 µg/ml
P1: 50-450 µg/ml
rgp41: 50-400 µg/ml
3M-052: 8-140 µg/ml HA: 70-160 µg/ml
P1: 40-100 µg/ml
rgp41: 70-230 µg/ml
3M-052: 16-65 µg/ml HA: 18-40 µg/ml
P1: 10-25 µg/ml
rgp41: 17.5-57.5 µg/ml
3M-052: 4-16.3 µg/ml Sodium chloride 50-150 mM 142.5 mM 35.625 mM HEPES 10-50 mM 50 mM 12.5 mM Trehalose 4-10% w/w 3.5-7% w/w 0.9-1.75% w/w

[0086] The presence of a buffer in an aqueous composition can lower the freezing point, making it difficult to freeze the composition and keep it frozen. In addition, destruction of the tablet matrix structure may also occur during lyophilization, since buffer salts may inhibit the crystallization of mannitol during annealing. Crystallization of mannitol is required to ensure the strength and integrity of the tablet matrix structure to prevent structure failure. However, mannitol crystallization can damage virosome particles during freezing, annealing, and lyophilization. A lower percentage loading of liquid virosome concentrate (eg, 25% loading) results in less such damage. A combination of a lower percentage of liquid virosome concentrate and a larger mass of the prepared dose may also be considered.

[0087] The high wet content of the formulated dose of virosome composition in combination with a high buffer formulation may also make it even more difficult to freeze and preserve the structure to minimize degradation during lyophilization. However, larger tablets (eg, with a 1000 mg dose of Massai) may cover a larger surface area and may potentially improve virosome passage. If a high wet weight of the prepared dose is required, it is preferable that the formulation have a low buffer content.

[0088] Table 4 (Composition 1) summarizes the aqueous compositions of the virosome preparation and the corresponding composition for the virosome tablet evaluated herein. The following compositions and tablets were prepared in accordance with the steps shown and described in figure 2. In addition, the frozen compositions were subjected to a two-step lyophilization process under vacuum at 500 mbar pressure, comprising: (a) pre-cooling to -25°C; (b) increase the temperature to -15°C for 2 hours; (c) holding at -15°C for 24 hours; (d) increase the temperature to -10°C for 2 hours; (e) holding at -10°C for 18 hours; (f) increase the temperature to 0°C for 15 minutes; (g) increase the temperature to 23°C for 15 minutes. The vacuum was then released and the pressure in the lyophilizer was brought back to atmospheric pressure.

[0089] The concentration of each ingredient (% w/w) is the amount before removing the water present in the liquid virosome concentrate, the sodium hydroxide solution and the water used to prepare the drug by sublimation during lyophilization. In addition, the table below indicates possible quantity ranges for each ingredient.

Table 4

Ingredient % wt./
wt. estimated interval % wt./
wt. compositions 1/2 Amount in mg for 500 mg wet weight of prepared dose Amount in mg after lyophilization Liquid virosome concentrate 25-50% 25% 125 mg ~2.5-2.6 mg* Trehalose 0.5% - 4.5% 2%** (general) 10 mg 10 mg** (total) Fish gelatin 4-6% 6% 30 mg 30 mg Mannitol 4.5-8% 4.5% / 8% 40 mg 40 mg Sodium hydroxide solution (e.g. 3% w/w) Ven. quantity up to pH 7.4 Ven. quantity up to pH 7.4 ~1.3 mg
(sufficient quantity up to pH 7.4) ~0.04 mg (water removed by sublimation) Water (to obtain an aqueous matrix mixture) Ven. quantity up to 100% Ven. quantity up to 100% ~293.7 mg Removed water Total weight of lyophilized vaccine tablet N/A N/A N/A ~82.64 mg

[0090] As stated above, the liquid virosome concentrate comprises virosomes suspended in a buffer system consisting of 142.5 mM NaCl and 50 mM HEPES with 5% wt./mass. trehalose. For a 25% loading of virosome composition with a prepared dose weight of 500 mg (i.e., 125 mg of liquid virosome concentrate), the amount of dry matter with water freeze-dried from virosomes, antigens, adjuvants, NaCl and HEPES (excluding trehalose) is approximately 2.5 -2.6 mg.

[0091] ** Means the total amount of trehalose in the virosome composition and in the dosage form. For example, such amount includes the amount of trehalose in the liquid virosome concentrate, as well as the amount of trehalose added from the base matrix.

Properties of lyophilized dosage forms:

[0092] Lyophilized dosage forms can be stable in their physical properties and virosome quality (particle size and antigen content) and can be stored regardless of cold chain storage conditions. In addition, lyophilized dosage forms may render virosomes resistant to accidental exposure to subfreezing storage conditions during storage or transportation.

Physical properties of the dosage form

[0093] Lyophilized tablets with acceptable physical properties were prepared. Physical properties of tablets include appearance, dispersion properties, disintegration time and moisture content.

[0094] The appearance of ten lyophilized tablets was tested. Each tablet was taken from a blister pack. Each tablet was visually inspected for defects in the surface and base of the tablet. The criterion is the good appearance of the lyophilized tablet and the absence of any defects on its surface. In addition, the tablets must be sufficiently durable to be removed from the blister pocket without being damaged.

[0095] Dispersion properties (in vitro test). A minimum of 5 tablets were tested. First, a beaker containing approximately 200 mL of purified water was prepared at 20 ± 0.5 °C. Each tablet was then removed from the blister pack and placed base down on the surface of the water. Time is defined as the time it takes for each tablet to become completely wet or dissociated. Wetting refers to the period of time during which the tablet becomes completely wet. Wetting of the tablet may occur in specific areas that eventually merge with each other, causing the entire tablet to become wet. The dispersion test is complete if the center of the tablet is a wet mass. Thus, the wetting time is determined from the moment the center of the tablet is wetted, since this part of the tablet is the thickest. Wetting time was recorded for each of the five tablets. The maximum time for each test was 60 seconds. Longer times may be recorded simply as times greater than 60 seconds. Dissociation time refers to the time required for the tablet to completely disintegrate. This time can be recorded after the tablet begins to disintegrate at the edges. The dissociation time was recorded for each of the five tablets. The maximum time for each test was 60 seconds. Longer times may be recorded as times greater than 60 seconds. Sometimes the tablet will not completely wet or completely dissociate in this time frame. Sometimes the tablet may have dense clumps; and sometimes, it may not be wetted on the surface at all. In addition, the entire tablet may be hard coated. It should be noted that when this occurs, this specification refers to "dense lumps" or "casing residue." The formation of “tight lumps” and/or “coating” may be an indication of microstructure breakdown during lyophilization. Figures 3A-C show a simplified side view and top view of the three possible undispersed states of the tablets as they appear in water. The photographs of Figures 3A-C show some representative tablets for the same categories. The criterion for passing the dispersibility test is that 5 tablets will be completely moistened and/or dissociated to form a palpable mass without dense lumps and/or coating in 60 seconds or less. In some embodiments, the dosage forms described herein can be completely moistened and/or dissociated to form a palpable mass free of hard lumps and/or coating in 60 seconds or less.

[0096] Disintegration time (in vitro test): Six tablets were used for this test. Six beakers were filled with purified water and placed in a water bath controlled at 37°C ± 0.5°C. Each tablet was then removed from the blister pack. A wire clamp was then carefully placed on each of the six tablets. It must be ensured that the clamp will grip the tablet without causing damage. The test was then performed as described in the Pharmacopoeia. An example of such a test is the USP (701) disintegration test. The maximum disintegration time was recorded for each tablet. The criterion for the disintegration time test is that the disintegration time should not exceed 60 seconds for each of the six tablets. In some embodiments, the dosage forms described herein may have a disintegration time of less than 60 seconds.

[0097] Moisture content. To determine the water content of the pellet, a Methron 831 Karl Fischer coulometer was used with a processor to feed the sample 744 into the oven (Metrohm, Herisau, Switzerland). The tablet was accurately weighed and placed in a glass container. The vessel was immediately sealed with a corrugated lid to ensure no moisture penetration. The sample vial was then placed into a 744 sample processor loaded into the oven and the temperature was set to 102°C. The evaporated moisture was titrated using Hydranal Coulometric AG Oven reagent to determine the amount of water released. The test was performed in triplicate and the average value was recorded. The criterion for assessing moisture content is the moisture content of the lyophilized dosage form being less than about 8%, preferably less than 6%, and more preferably less than 4%. In some embodiments, the dosage forms described herein may have a moisture content of less than about 8%, preferably less than 6%, and more preferably less than 4%.

Properties of virosomes:

[0098] Lyophilized dosage forms that contain virosomes can be sufficiently preserved in terms of the content of intact virosomes in the original liquid virosome population, their particle size, and the content of surface antigens necessary to impart virosome immunogenicity and immunological potency. The virosome structure can be destroyed by lyophilization carried out to obtain dosage forms. For example, the properties of virosome particles are assessed in a solution of a diluted lyophilized tablet. Virosomes can exist as intact individual particles and as clusters of various sizes, all of which are immunogenic, but are subject to different antigen/epitope exposure and have different abilities to cross the sublingual barrier. Virosomes can be characterized in terms of (a) average particle size and (b) degree of virosome retention (intact virosomes).

[0099] Estimation of particle size and virosome particle concentration (number of virosome particles per ml) was performed using the NTA method: Nanoparticle Pathway Assay (NTA; on a NanoSight NS300 instrument) is a sensitive method that can identify different particle sizes present in a solution in within 30-1000 nm. Particles in a sample solution can be individually tracked and simultaneously analyzed by direct observation. Nanoparticles move through Brownian motion due to the random movement of water molecules surrounding the particles. Small particles move faster than large particles. The Brownian motion of each particle is observed in real time via video, and NTA allows the Brownian motion to be analyzed to determine the particle size. The diffusion coefficient can be calculated by tracking the movement of each particle, and then the particle size can be calculated using the Stokes-Einstein equation. This "particle interaction" technique provides high-resolution results for particle size distribution and concentration (that is, the number of particles in a known volume of liquid). The target values for optimal detection used for on-instrument detection are summarized in Table 5 below.

Table 5

Quality indicator Result Conclusion PPF (Particles per dose) 50-100 The number of particles influences the achieved resolution and statistical accuracy of the resulting profiles Number of valid tracks >5000 Statistical accuracy of the obtained profiles for polydisperse samples S:N (Signal to Noise Ratio) High Improved detection of small or subtle particles SD (Standard Deviation) Low Less variable data gives a higher confidence interval when obtaining results Appearance in profile Good: non-Gaussian distribution, minimal shoulders at peaks The lower noise level observed for these data indicates pure populations with a certain particle size

[0100] The test sample must be in liquid form. To perform this test, the virosome vaccine is given in liquid form. For test samples of the vaccine compositions described herein, the mixture is supplied in liquid form, the frozen tablet is thawed to form a liquid form before testing, and the lyophilized dosage form is reconstituted with aqueous buffer to liquid form. The test sample should not be too concentrated. The liquid test sample can be further diluted with HN buffer if necessary to optimize detection. The dilution buffer may be of high purity and may be filtered through at least a 0.22 µm filter before use. The experimental parameters established for the NTA analysis are summarized below in Table 6.

Table 6

Parameter Conditions Sample dilution Depending on the content (can range from 1:100 to 1:8000 depending on the concentration of virosomes in the diluted test samples) Number of grips 5 Capture duration 60 seconds Adjustable temperature 25°С Viscosity 0.9 Sp

[0101] The samples were measured using NTA, as known to those skilled in the art. In some embodiments, the size range of virosome particles, including the fragments and clusters described herein, may be approximately 50-500 nm. In some embodiments, virosome particles that are intact virosomes may range from about 70-400 nm or about 70-200 nm (main peak). In some embodiments, the average diameter of the virosome particles may be about 70-200 nm, about 100-175 nm, and about 125-155 nm. For detection, the concentration of particles in the virosome population in the sample may be at least 10 ¹⁰ units/ml.

[0102] Quantitative assessment of virosome retention using flow cytometry. Flow cytometry was performed for this assessment. First, the original liquid virosome particles were labeled (control sample representing 100% of the starting material) by introducing the lipophilic dye Dil (long-chain diallylcarbocyanine) into the lipid bilayer of the virosomes (labeling with the Dil dye had no detectable effect on particle size). Next, the lyophilized tablet is reconstituted and the virosomes in the lyophilized tablet are labeled with Dil (test sample). The samples are then analyzed using an AMNIS imaging flow cytometer and a comparison is made between control samples (liquid virosomes before lyophilization) and test samples (lyophilized virosomes) to assess the degree of virosome retention after lyophilization by: (a) percentage of virosome recovery and (b) percentage of virosome clusters . In some embodiments, the percentage of virosome particles recovered as discrete particles may be about 20-50%, about 30-50%, or about 40-50% of the starting material. In some embodiments, the percentage of virosome clusters (doublets, triplets, or higher number forms) may be less than about 50%, about 25%, about 10%, or about 5%.

Content of hemagglutinin (HA), HIV-1 P1 antigen, HIV-1 antigens and adjuvant in the virosome

[0103] The content of influenza virus HA, HIV-1 antigens, and adjuvant in the virosome can be quantified by various methods. These results are presented below in Table 7.

Table 7

Quantified content Methods Influenza virus HA Immunoblot analysis
Light scattering (SRID) analysis
ELISA
HPLC analysis HIV-1 antigens Immunoblot analysis
HPLC analysis
ELISA Adjuvant Analysis by UV spectroscopy
HPLC analysis

[0104] Immunoblot assay method for HA, PI and rgp41: In this assay, the composition (liquid virosome concentrates or reconstituted lyophilized dosage forms containing virosomes) can be absorbed onto a nitrocellulose membrane and the antigens can be maintained in their native state due to the absence of heat , and the absence of denaturing or reducing agents. This assay identifies all antigens accessible to specific antibodies and indicates degradation or denaturation of the underlying antigen that disrupts or blocks access to a specific epitope. This assay also reveals whether specific excipients can prevent the antibody from binding to the antigen. To obtain lyophilized dosage forms for analysis (test samples), each tablet is dissolved in 0.5 ml of water (that is, the lyophilized tablet is diluted again to obtain the virosomal preparation composition before lyophilization). To obtain a positive control, a sample of liquid virosome concentrate is diluted with ultrapure water (4-fold). Ideally, the liquid virosome concentrate is the same batch used to obtain the dosage form test sample. Serial 2-fold dilutions of all test samples and positive controls are then made. Additional positive controls such as purified HA and Rgp41 and synthetic P1 can also be used. 1.5 µl of each sample and positive control are spotted onto a dry nitrocellulose membrane (increasing dilution: from left to right - undiluted, 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1 /128). The dry membrane is blocked with 1% (w/v) casein and incubated with a specific antibody solution, i.e., human monoclonal antibody (mab) 2F5 specific for the HIV-1 P1 antigen and rabbit anti-rgp41 antiserum specific for the antigen. HIV-1 rgp41. After incubation, the nitrocellulose membrane is washed. The specific antibodies bound to the fluorescently labeled second antibodies (anti-human or anti-rabbit) are then detected using a far-red fluorescent scanner, allowing simultaneous detection of 2 different fluorescent labels at 700 nm and 800 nm. The fluorescent signal for the raw data for each sample spot (from lowest to highest) is compared with the corresponding dilution of the spot used as a positive control (% of positive control). The arithmetic mean of the sample percentage is then calculated.

[0105] UV spectroscopic analysis for 3M-052: The adjuvant molecule 3M-052 has several peaks in the UV adsorption curve. Some of these peaks overlap with lipid and protein uptake. Analysis was performed using UV spectroscopy at 320 nm.

[0106] HPLC analysis for P1 and rgp41: HIV P1 and rgp41 antigens were separated by reverse phase high pressure liquid chromatography on a Cl8 column using a gradient of water and acetonitrile. Analysis was performed using UV spectroscopy at 280 nm, and peak areas were quantified using HPLC system software.

[0107] ELISA antibody endpoint titer: 96-well Maxisorp plate (Nunc flat bottom plate) and Polysorp plates were respectively coated at 4°C for 16 hours with 0.1 ml of rgp41 or P1 peptide (2 μg/ml) prepared in PBS pH 7.4. The plates were washed 3 times with PBS containing 0.05% v/v. Tween 20 (PBST), and then 1% BSA blocking solution 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 per well of nonimmune serum diluted 1/1000 or serial dilutions of immune sera (1/1000 to 1/64000) prepared in 0.1% BSA in PBST were added, and incubated for 2 hours at room temperature. The plates were washed three times with PBST and incubated for 2 hours at room temperature with HRP-conjugated goat anti-rat IgG antibody diluted 1:4000 in 0.1% BSA in PBST. The plates were washed again and then 0.1 ml o-phenylenediamine (OPD) colorimetric substrate was added and the reaction was stopped by adding 2M H ₂ SO ₄ followed by reading the plates at 492 nm.

Example 1: Use of a high level of mannitol (8% w/w) in combination with a low temperature lyophilization cycle to reduce microstructural degradation during lyophilization without compromising virosome integrity.

[0108] Mannitol is used in dosage forms to improve structural stability. Due to the presence of high levels of buffers and the addition of trehalose to protect virosome particles, application of mannitol at a typical level of 4.5% w/w. did not make it possible to ensure sufficient preservation of the structure during lyophilization. Thus, the microstructure is destroyed.

[0109] However, applicants have discovered that by using a combination of higher levels of mannitol and low temperature lyophilization cycles, a lyophilized tablet with a stronger structure can be obtained. This example presents comparison data for a composition containing 4.5% w/w. mannitol, with a composition containing 8% wt./mass. mannitol Both virosome compositions used 25% w/w. loading of liquid virosome concentrates MYM-201 lot 160125-1 supplied by Mymetics containing 70-80 µg/ml HA, 40-50 µg/ml P1, 70-80 µg/ml rgp41 (A/Brisbane/59/2007 (H1N1 ) in HN buffer, pH 7.4 (50 mM HEPES, 142.5 mM NaCl) In the virosome composition, the content of fish gelatin and pure trehalose was maintained at 6% w/w and 2% w/w. As shown in Figure 2, the compositions were dosed with the prepared dose weight in 500 mg aliquots at 15° C. in blister packs contained in pockets of aluminum trays.Trays containing the dosed aqueous vaccine mixture were frozen by passing the aluminum trays through a freezer set at -70°C for 3 minutes 15 seconds. Aluminum trays containing frozen products were collected and placed in a refrigerator at <-15°C° and hybridized for 6 hours in a frozen state before lyophilization. Then 2- staged FD cycle at -15°C for 24 hours and then at -10°C for 18 hours. Blister packs of lyophilized tablets were sealed in sachets and stored under ambient conditions.

[0110] The physical properties of the lyophilized tablets (appearance and dispersion time) were evaluated according to the tests described above. In addition, the size of the virosome particles in the lyophilized tablets was characterized according to the tests described above. The results are organized below in Table 8.

Table 8

Mannitol, 4.5% (Composition 1) Mannitol, 8% (Composition 2) Appearance: Good (n=182)
Dispersion time (n=8)
Wetting: > 60 sec. (2 coated tablets, 5 hard-clumped tablets
Dissociation: > 60 sec. (not completely dispersed)
NTA analysis
Lyophilized tablet: 136 nm (main peak)
% virosome particles (<200 nm): >50% Appearance: Good (n=184)
Dispersion time (n=8)
Wetting: < 28 sec.
Dissociation: <36 sec.
NTA analysis
Lyophilized tablet: 140 nm (main peak)
% virosome particles (< 200 nm): >50%

[0111] All tablets were in good appearance. Composition with 4.5% w/w mannitol has poor dispersion properties with coated and/or dense lumps observed in tablets due to microstructural breakdown. Increasing the mannitol in this composition to 8% w/w. promoted the crystallization of mannitol and improved the structure of the lyophilized tablet. Composition with 8% w/w mannitol had good dispersion properties and a soft and palpable mass. In combination with performing the lyophilization cycle at a low temperature, the virosome particles in the lyophilized tablet were not affected in any way. The size of virosome particles in the lyophilized tablets was generally comparable in all formulations.

Example 2: Stability data for lyophilized vaccine dosage forms (formulation 1) stored for 3 months under ICH conditions

[0112] Liquid virosome concentrates are generally stable at 2-8°C, but liquid virosome HIV vaccine has a limited shelf life of several months due to important chemical modifications occurring on the antigens and does not aggregate. By using a solid vaccine form with low moisture content, such modifications are expected to slow down and become minimal over time, thereby extending shelf life by >1 year. This example illustrates the stability of the virosome vaccine in the form of lyophilized tablet 1. To prepare the lyophilized vaccine tablets, MYM-201 liquid virosome concentrates, lot 170130-1, supplied by Mymetics, containing approximately 100 μg/ml HIV-1 P1, 230 μg, were purchased /ml rgp41 HIV-1, 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 (lot Z33787A101).

[0113] To prepare a vaccine tablet, a mixture of a liquid virosome composition is first prepared. This mixture contains 25% w/w. batches of liquid virosome concentrate MYM V202 170130-1, 6% w/w. fish gelatin, 4.5% w/w mannitol, 2% w/w (pure) trehalose, sodium hydroxide solution (sufficient amount) to pH 7.4 and purified water (sufficient amount) to 100% w/w.

[0114] The liquid virosome composition mixture was dosed at the prepared dose weight in 500 mg aliquots at 15°C in blister pockets contained in aluminum trays. Trays containing the dosed aqueous vaccine mixture were frozen by passing the aluminum trays through a freezer set at -70°C for 3 minutes 15 seconds. Aluminum trays containing frozen food were collected and placed in a refrigerator at <-15°C. A 2-stage FD cycle was then performed at -15°C for 24 hours and then at -10°C for 18 hours.

[0115] Blister packs of lyophilized tablets were sealed in sachets and stored for 3 months at 5°C, 25°C/60% RH and 40°C/75% RH. The results of the initial and 3-month tests are summarized in Table 9.

Table 9

Tests Initial 5°C /3 months 25°C at 60%RH /3 months 40°C at 75%RH /3 months Appearance Good Good Good Good Moisture content (%w/w) 5.22 5.20 5.76 4.83 Disintegration time (sec.) <13 <23 <41 <25 NTA: main peak (nm) 101 120 124 124 NTA: % virosome particles (<200 nm) >50% >50% >50% >50%

[0116] Stability data showed that tablet appearance was good and consistent across all batches. The moisture content was 5-6% w/w. with slight differences under different stability conditions over a 3-month storage period. Since this composition contains 4.5% w/w. mannitol, some destruction of the microstructure was observed during lyophilization, as indicated by a change in disintegration time. Regarding virosome particle size, DLS and NTA data showed no significant difference in virosome particle size between tablets stored under different stability conditions. The original liquid virosome concentrates had >50% particles <200 nm in size. The results showed that the <200 nm virosome isolated from the diluted tablet was essentially retained to the same order of magnitude.

[0117] Using semi-quantitative immunoblot assays, HIV-1 P1 and rgp41 antigens were monitored for degradation over a period of 3 months. All samples were previously diluted 2-fold. For this purpose, human mAb 2F5, specific for P1, and rabbit antiserum against rgp41 were used. The liquid virosome concentrate of batch MYM-V202 170130-1, which was used to obtain the vaccine batch, was diluted 8-fold and used as a positive control. Figure 4 shows a photograph of immunoblot analysis. For sample Z33787A101, tablets stored at 5°C showed no or only minimal reduction in rgp41 antigen signal after storage for 1 month and 3 months compared to the original sample (t=0). Similarly, there was only a minimal difference in signal intensity for tablets stored at 25°C for the rgp41 antigen, and a slight decrease for the P1 antigen. The difference for tablets stored at 40°C was more pronounced, although this difference may be within the 10-20% accuracy of the assay.

[0118] Quantification of fluorescence data for rgp41 and P1 is presented in Tables 10 and 11, respectively. The top of each table shows the raw data for the fluorescent signals for each spot (lowest to highest dilution) for the indicated sample being assessed for stability. The bottom of each table shows the values for each sample spot compared to the corresponding dilution of the positive control spot (as a % of the positive control). The bottom line corresponds to the arithmetic mean for all sample percentages. Obvious “outliers” of measurements were excluded from the calculation of the average.

Table 10

Table 11

[0119] For vaccine tablets lot Z33787A101, tablets stored at 5°C showed no or only minimal reduction in signal of rgp41 and P1 antigens after storage for 1 month and 3 months compared to the original sample (t=0). Similarly, there was only a minimal difference in signal intensity for tablets stored at 25°C for the rgp41 antigen, and a slight decrease for the P1 antigen. The difference for tablets stored at 40°C was more pronounced for the P1 and rgp41 antigens, although this difference may be within the limits of assay precision, especially since changes were observed again in serially diluted samples.

[0120] Analysis of 3M-052 was performed using UV spectroscopy at 320 nm for all vaccine tablets stored at various temperatures for more than 3 months. The values are presented below in Table 12.

Table 12

Time Storage conditions OD (320 nm) 0 5°C 0.116 1 month 5°C 0.114 1 month 25°C/60% RH 0.112 1 month 40°C/75% RH 0.110

[0121] No noticeable differences were observed between these samples and, therefore, the concentrations of 3M-052 were considered to be stable in all samples within the precision of this assay. Overall, HA, P1, rpg41 and 3M-052 were stable in lyophilized tablets under various storage conditions with little variation over time, as shown below in Table 13.

Table 13

Antigen and adjuvant detection in a stability study Liquid virosome concentrates Lyophilized tablets Changes for rgp41 over 3 months at 5°C (no degradation expected = reference) Not significant Not significant Changes for rgp41 over 3 months at 25°C (degradation) compared to changes at 5°C Absent Minimum reduction Changes for rgp41 over 3 months at 37-40°C (degradation) compared to changes at 5°C Absent Minimum reduction Changes for P1 over 3 months at 25°C (no decomposition expected = reference) Not significant Not significant Changes for P1 over 3 months at 25°C (decomposition) compared to changes at 5°C Absent Minimum reduction Changes for P1 over 3 months at 37-40°C (decomposition) compared to changes at 5°C Absent Minimum reduction Changes for 3M-052 over 3 months at 5°C (no degradation expected=reference) Not significant Not significant Changes for 3M-052 over 3 months at 37-40°C (decomposition) compared to changes at 5°C Not significant Not significant

Example 3: Stability data for lyophilized vaccine dosage forms (formulation 2) stored under ICH conditions

[0122] This example illustrates the stability of the virosome vaccine in lyophilized tablet form for Formulation 2. To prepare the lyophilized vaccine tablets, a batch of MYM-V202 liquid virosome concentrates was purchased, lot 17MYM002/F17255, supplied by Mymetics, containing approximately 121 μg/ml HIV P1 -1, 67 µg/ml rgp41 HIV-1, 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 (lot MYM-212, 1690747).

[0123] To prepare the vaccine tablet, a mixture of liquid virosome composition was first prepared. This mixture contains 25% w/w. batches of liquid virosome concentrate MYM V202 17MYM002/F17255, 6% w/w. fish gelatin, 8% w/w mannitol, 2% w/w (pure) trehalose, sodium hydroxide solution (sufficient amount) to pH 7.4 and purified water (sufficient amount) to 100% w/w.

[0124] The liquid virosome composition mixture was dosed at the prepared dose weight in 500 mg aliquots at 15°C in blister packs contained in blister pockets of aluminum trays. Trays containing the dosed aqueous vaccine mixture were frozen by passing the trays through a freezer set at -70°C for 3 minutes 15 seconds. Aluminum trays containing frozen food were collected and placed in a refrigerator at <-15°C. A 2-stage FD cycle was then performed at -15°C for 24 hours and then at -10°C for 18 hours.

[0125] Blister packs of lyophilized tablets were sealed in sachets and stored for 3 months at 5°C, 25°C/60% RH and 40°C/75% RH. The results of the initial and 6-month tests are summarized below in Table 14.

Table 14

Test Appearance Time of disintegration Moisture contents Elementary Good 22 seconds 3.6% 1 month at 5°C Good 8 seconds 3.8% 1 month at 25°C/60% RH Good 16 seconds 3.7% 1 month at 40°C/75% RH Good 14 seconds 3.6% 3 months at 5°C Good 12 seconds 3.7% 3 months at 25°C/60% RH Good 10 Seconds 3.7% 3 months at 40°C/75% RH Good 13 seconds 3.9% 6 months at 5°C Good 8 seconds 3.9% 6 months at 25°C/60% RH Good 10 Seconds 3.8% 6 months at 40°C/75% RH Good 13 seconds 4.0%

[0126] Stability data showed that tablet appearance was good and consistent across all batches. The moisture content was 3.6-4.0% w/w. with slight differences between different stability conditions during the 6-month storage period. Since this composition contains 8% wt./mass. mannitol, less microstructure destruction was observed during lyophilization, as indicated by shorter and more stable disintegration times.

[0127] Stability of antigens (HIV-1 antigen P1 and rgp41) for degradation was monitored over a period of 3 months using HPLC analysis. For MYM V202 liquid vaccine (starting material), P1 content was found to decrease by 4% and 7% when stored at 2-8°C (under refrigerated storage conditions) for 1 month and 3 months, respectively. For rgp41, this content was reduced by 16% and 25% after 1 and 3 months, respectively, under refrigerated storage conditions. When the liquid virosome was stored at 25°C and 40°C, P1 and rgp41 were no longer detectable after 1 month and 3 months.

[0128] The results of evaluating the antigen content of the lyophilized tablet during storage are presented in Table 15 and Table 16 for the P1 antigen and the rgp41 antigen, respectively. In lyophilized tablet form, the P1 antigen remained fairly stable with no observable decline after 3 months at 2-8°C (cold chain conditions), and also remained unchanged during 1 and 3 months of non-cold chain storage. For the rgp41 antigen, a decrease of approximately 5%, 10% and 20% was observed after 1 month at 2-8°C, 25°C and 40°C, respectively. When stored for 3 months, reductions of approximately 13%, 15% and 19% were observed, respectively. Taking into account the accuracy of the HPLC method, the observed difference in concentration reduction of less than 15% is not significant. In addition, the observed gradual loss or reduction in antigen levels is due to chemical modifications rather than increased degradation due to structure degradation, loss of amino acids, or aggregation. Meanwhile, chemical modification(s) in a given epitope can potentially alter its recognition, reducing or increasing the level of antibodies binding to that region while other regions remain equally well recognized.

[0129] At time 0, P1 and rgp41 antigens have a similar migration profile on SDS-PAGE and are still recognized by specific monoclonal antibodies in sublingual tablets, and serum antibodies against P1 and rgp41 still react to various P1 and gp41 peptides carrying key epitopes. These analyzes suggest that, in general, P1 and rgp41 retain most of their antigenicity and immunogenicity during the tableting process (data not shown).

Table 15

Time Contents P1 (lyophilized tablet) µg/ml mcg/tablet % decrease/increase Storage at 2-8 ºC (average 5ºC) (cold chain conditions) Start (T0) 24.7 12.35 Not significant 1 month (T1) 28.6 14.3 + 15.8 (no loss) 3 months (T3) 27.6 13.8 + 11.7 (no loss) Storage at 25ºC/60%RH 1 month (T1) 27.9 13.95 + 13.0 (no loss) 3 months (T3) 27.5 13.75 + 11.13 (no loss) Storage at 40ºC/75%RH 1 month (T1) 25.8 12.9 + 4.5 (no loss) 3 months (T3) 24.0 12.0 -2.8 (loss)

Table 16

Time Contents of rgp41(lyophilized tablet) µg/ml mcg/tablet % reduction Storage at 2-8 ºC (average 5ºC) (cold chain conditions) Start (T0) 13.5 6.75 Not significant 1 month (T1) 12.8 6.4 -5.2 3 months (T3) 11.8 5.9 -12.6 Storage at 25ºC/60%RH 1 month (T1) 12.1 6.05 -10.4 3 months (T3) 11.5 5.75 -14.8 Storage at 40ºC/75%RH 1 month (T1) 10.8 5.4 -20.0 3 months (T3) 10.9 5.45 -19.3

Example 4: Stability of lyophilized tablets stored under sub-zero storage conditions.

[0130] Data from Example 4 demonstrated the stability of the physical properties of virosome vaccine tablets and virosome particles when stored at subfreezing temperatures. A mixture of liquid virosome composition containing 25% wt./mass. loading of liquid virosome concentrate MYM-201, lot 160125-1, supplied by Mymetics (containing 70-80 µg/ml HA, 40-50 µg/ml P1, 70-80 µg/ml rgp41 (A/Brisbane/59/2007 (H1N1 ) in HN buffer pH 7.4 (50 mM HEPES, 142.5 mM NaCl), 6% w/w fish gelatin, 8% w/w mannitol and 2% w/w (neat) trehalose was dosed at a 500 mg compounded dose weight and lyophilized.The formulations were dosed at a compounded dose weight in 500 mg aliquots at 15°C in blister pockets contained in aluminum trays. The trays containing the dosed aqueous vaccine mixture were frozen by passing aluminum trays through a freezer set at -70°C for 3 minutes 15 seconds.Aluminum trays containing frozen food were collected and placed in a refrigerator at <-15°C, and annealed for 6 hours in the frozen form before lyophilization.Then a 2-step FD cycle was carried out at -15°C for 24 hours and then at -10°C for 18 hours. Blister packs of lyophilized tablets were hermetically sealed in sachets and stored in a refrigerator at -15°C for 1 week. A corresponding set of blister packs of tablets were hermetically sealed in a sachet and stored under ambient conditions for the same periods of time. These tablets were evaluated for appearance, dispersion properties (wetting time and dissociation time) and particle size distribution of virosome particles as described above in accordance with the test methods.

[0131] It was found that all tablets had good appearance after storage under both conditions. The data showed that storage at subzero temperatures had little effect on tablet dispersion properties (wetting and dissociation times) (Table 17) and on the particle size distribution of virosome particles (Table 18).

Table 17

Conditions Wetting time (sec.) Dissociation time (sec.) Storage at ambient temperature <31 <37 Storage below zero <32 <35

Table 18

Conditions NTA: Main peak for particle size (nm) NTA: % virosome particles (<200nm) 1 week at ambient temperature after hydrolysis 130 59.9 1 week below zero after hydrolysis 132 60.6

Example 5: Evaluation of virosome particles in reconstituted lyophilized tablets using flow cytometry

[0132] Estimates of the number of virosomes preserved in lyophilized tablet samples obtained by the developed method are systematized below. The values presented in Table 19 were derived from AMNIS flow cytometry data by assessing events in the focal zone corresponding to virosome gating.

Table 19

Vaccine composition Percentage of virosomes obtained at the end of the production process Percentage of clusters (doublets, triplets and larger forms) Liquid vaccine 100% <5% Lyophilized tablet (after reconstitution) 24-40% 10-25%

[0133] Data showed that 24-40% of the original virosome is retained in the lyophilized tablets, of which 10 to 25% of these virosomes are found in clusters, mainly in the form of doublets and triplets.

Example 6. Evaluation of the immunogenicity of P1 and rgp41 antigens in a liquid virosome composition and in diluted lyophilized sublingual tablets containing a virosome.

[0134] Liquid virosome concentrates (MYM-V202 liquid vaccine) and lyophilized sublingual tablets containing the virosome of Example 3 were stored at 4°C and 40°C for three months to assess immunogenicity. After storage for 1 month, samples of liquid vaccine and sublingual tablets, which had been stored at 40°C, were placed in a storage cabinet for immunogenicity assessment. After storage for 3 months, samples of liquid vaccine and sublingual tablets, which were stored at 4°C and 40°C, were collected for immunogenicity assessment.

[0135] Wistar rats (n=10 per group), 50% of each sex, were used to assess immunogenicity. Rats were immunized on days 0, 28, and 56. The liquid vaccine contained 3.9 μg P1, 2.2 μg rgp41, and 1.3 μg 3M-052 TLR7/8 (adjuvant) per 0.1 ml and was used for subcutaneous injection. For sublingual tablets, an adequate amount of sublingual tablets was dissolved in sterile water to provide approximately 3 μg of P1, 1.7 μg of rgp41, and 1 μg of 3M-052 TLR7/8 (adjuvant) per 0.1 mL for administration by subcutaneous injection. . Nonimmune sera were collected on day 0, and immune sera were collected on day 65 to determine final antibody titers in the sera of each animal and in the pooled sera.

[0136] Figure 5 shows the immunogenicity of P1 and rgp41 in liquid vaccine and sublingual tablets stored at different temperatures. The adjuvanted liquid vaccine formulation MYM-V202 containing P1 and rgp41 antigens was thermosensitive and served as a benchmark for comparison with the immunogenicity of a sublingual tablet vaccine with improved thermostability. The black line represents vaccines stored for 3 months at 4°C; the black dotted line corresponds to vaccines stored for 1 month at 40°C; and the gray line corresponds to vaccines stored for 3 months at 40°C. The data obtained indicate the preservation of the immunogenicity of antigens for lyophilized tablets. Endpoint antibody titers are also indicated in each panel. The end point titer corresponds to the last serum dilution that gives an OD value that is more than 2 times the nonimmune background value.

Additional definitions

[0137] Unless otherwise specified, all terms known to those skilled in the art, designations and other technical and scientific terms or terminology used in this application have their common meaning known to those skilled in the art that relates to the claimed subject matter inventions. In some cases, terms with generally accepted meanings are defined herein for clarity and/or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a material difference from definitions substantially known to those skilled in the art.

[0138] The term "about" as used herein includes (and describes) changes that relate to those values or parameters per se. Thus, for example, the expression "about X" includes the description of "X". In addition, the words “less than,” “more than,” “maximum,” “minimum,” “less than or equal to,” “greater than or equal to,” or other similar expressions followed by the words “magnitude” or “parameter ” means each quantity or parameter mentioned in a number of such quantities or parameters. Thus, for example, indicating 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 approximately 20 mM.

[0139] The articles “a” and “an” and “the” used here, when used with singular nouns, may also refer to plural nouns unless the context of the description indicates otherwise. It should also be noted that as used herein, the phrase “and/or” covers any and all possible combinations of one or more of the related elements listed herein. In addition, it should be noted that the terms “includes,” “including,” “contains,” and/or “comprising,” when used herein, indicate the presence of certain features, integers, steps, procedures, elements, components, and /or units, but does not exclude the presence or addition of one or more other features, integers, stages, procedures, elements, components, units and/or groups thereof.

[0140] This application discloses several numerical ranges presented in the text and drawings. Disclosed numeric ranges, by their nature, mean any intervals or values within disclosed numeric ranges, including the endpoints, even though the precise boundaries of those ranges are not stated verbatim in this specification, since such disclosure may be applied in practice to all specified numeric ranges. intervals.

[0141] The above description enables one skilled in the art to make and apply the present invention in accordance with specific purposes and requirements. Preferred embodiments of the invention may be subject to various modifications that will be apparent to one skilled in the art, and the general principles defined herein may be applied to other embodiments and purposes within the spirit and scope of the invention. Thus, the present invention is not limited to the embodiments set forth herein, but is intended to be within the broadest scope of the principles and features disclosed herein.

Claims (62)

1. Solid standard dosage form for oral administration, containing: lipid-based vesicles containing an immunogenic amount of at least one vaccine antigen; 5-20 wt.% of at least one cryolioprotectant; 25-40 wt.% of the substance forming the matrix; And 40-55 wt.% of the substance forming the structure. 2. Unit dosage form according to claim 1, wherein the lipid-based vesicles are virosomes or proteoliposomes. 3. Standard dosage form according to any one of paragraphs. 1, 2, where the dosage form contains 10-15 wt.% of at least one cryolioprotector. 4. Standard dosage form according to any one of paragraphs. 1-3, where at least one cryolioprotectant contains trehalose. 5. Standard dosage form according to any one of paragraphs. 1-4, where the dosage form contains 33-37 wt.% of the substance forming the matrix. 6. Standard dosage form according to any one of paragraphs. 1-5, where the substance forming the matrix contains gelatin. 7. Unit dosage form according to claim 6, where the gelatin includes fish gelatin. 8. Standard dosage form according to claim 7, where the fish gelatin is a high molecular weight fish gelatin. 9. Standard dosage form according to any one of paragraphs. 1-8, where the dosage form contains 45-50 wt.% of the substance that forms the structure. 10. Standard dosage form according to any one of paragraphs. 1-9, where the structure-forming substance contains mannitol. 11. Standard dosage form according to any one of paragraphs. 1-10, where the virosomes are derived from the membrane of influenza virus or other enveloped viruses. 12. Standard dosage form according to any one of paragraphs. 1-11, wherein at least one vaccine antigen is present on the virosome. 13. Standard dosage form according to any one of paragraphs. 1-12, wherein the at least one vaccine antigen comprises an antigen derived from the HIV-1 envelope. 14. The unit dosage form of claim 13, wherein the HIV-1 envelope-derived antigen comprises HIV-1 P1 peptide and/or recombinant HIV-1 gp41. 15. Standard dosage form according to any one of paragraphs. 1-14, where the virosomes contain an adjuvant. 16. Standard dosage form according to any one of paragraphs. 1-15, wherein the dosage form promotes the uptake of at least one vaccine antigen in the oral cavity. 17. Standard dosage form according to claim 16, where the dosage form decomposes within 180 seconds after its introduction into the oral cavity. 18. Standard dosage form according to claim 16, where the dosage form decomposes within 90 seconds after its introduction into the oral cavity. 19. Standard dosage form according to claim 16, where the dosage form decomposes within 60 seconds after its introduction into the oral cavity. 20. Standard dosage form according to claim 16, where the dosage form decomposes within 30 seconds after its introduction into the oral cavity. 21. Standard dosage form according to claim 16, where an immune response is induced when it is administered to a patient in the oral cavity. 22. Unit dosage form according to claim 21, where the introduction into the oral cavity is placement on the tongue or sublingually or in the buccal or pharyngeal region. 23. A method for inducing an immune response in a patient, including administering a standard dosage form according to any one of paragraphs. 1-22 into the oral cavity of a person in need of an immune response. 24. The method according to claim 23, where the introduction into the oral cavity is placement on the tongue or under the tongue or in the buccal or pharyngeal region. 25. A method for producing a solid standard dosage form for oral administration, including: dispensing a liquid virosome composition into a preformed form, wherein the virosome composition includes: lipid-based vesicles containing an immunogenic amount of at least one vaccine antigen; 1-5 wt.% cryolioprotector; 4-8 wt.% of the substance forming the matrix; And 5-10 wt.% of the substance forming the structure; freezing the dosed virosome composition at a temperature from -60°C to -90°C; annealing the frozen virosome composition by keeping it at a temperature below -15°C for 3-9 hours; And lyophilization of the hybridized virosome composition to form a dosage form. 26. The method according to claim 25, where the dosed virosome composition is frozen at a temperature of -60°C to -90°C for approximately 1-5 minutes. 27. Method according to any one of paragraphs. 25, 26, wherein lyophilization of the hybridized virosome composition includes a first step of keeping the hybridized virosome composition at a temperature of -10°C to -20°C for 20-28 hours and a second step of keeping the hybridized virosome composition at a temperature of -5°C to approximately -15°C for 14-22 hours. 28. Method according to any one of paragraphs. 25-27, where lyophilization is carried out under a pressure of less than 600 mbar. 29. Method according to any one of paragraphs. 25-28, wherein the virosome composition has a pH of approximately 6.5-8. 30. Method according to any one of paragraphs. 25-29, where the cryolioprotectant includes trehalose. 31. Method according to any one of paragraphs. 25-30, where the substance forming the matrix contains gelatin. 32. The method according to claim 31, where the gelatin includes fish gelatin. 33. The method according to claim 32, wherein the fish gelatin is a high molecular weight fish gelatin. 34. Method according to any one of paragraphs. 25-33, wherein the structure-forming agent includes mannitol. 35. Method according to any one of paragraphs. 25-34, wherein the lipid-based vesicles are derived from influenza virus or respiratory syncytial virus. 36. Method according to any one of paragraphs. 25-35, wherein the at least one vaccine antigen comprises an antigen derived from the HIV-1 envelope. 37. The method of claim 36, wherein the HIV-1 envelope-derived antigen comprises HIV-1 P1 peptide and/or recombinant HIV-1 gp41. 38. Method according to any one of paragraphs. 25-37, wherein the lipid-based vesicles contain an adjuvant. 39. A method for producing a solid standard dosage form for oral administration, including: dispensing a liquid virosome composition into a preformed form, wherein the virosome composition includes: 20-50 wt.% virosome concentrate, where the virosome concentrate contains: virosomes containing an immunogenic amount of at least one vaccine antigen; 2-10 wt.% cryolioprotector; And 60-200 mM buffer system; 4-8 wt.% of the substance forming the matrix; And 5-10 wt.% of the substance forming the structure; freezing the dosed virosome composition at a temperature from -60°C to -80°C; annealing the frozen virosome composition by keeping it at a temperature below -15°C for 3-9 hours; And lyophilization of the hybridized virosome composition to form a dosage form. 40. The method according to claim 39, where the buffer system contains HEPES-sodium chloride.