ORAL DISPERSIBLE VACCINE COMPRISING VIROSOMES

Abstract

This description relates to oral vaccine dosage forms and processes for producing oral vaccine dosage forms. The dosage forms include lipid-based vesicles (e.g., virosomes, liposomes) that contain an immunogenic quantity of at least one vaccine target molecule, with or without an adjuvant. Specifically, the applicants have discovered a combination of the composition of liquid virosome concentrates, the composition of the base matrix for the solid dosage form formulation (excluding the virosome concentrate), and manufacturing conditions for the dosage forms that can produce a lyophilized sublingual dosage form that has physical robustness, particle integrity, and antigen stability.

Description

ORAL DISPERSIBLE VACCINE COMPRISING VIROSOMES FIELD OF INVENTION This description relates to an oral dosage form that induces mucosal immunization. More specifically, this description relates to an orally dispersible, lyophilized vaccine containing virosomes. The project resulting in this application has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 646122. BACKGROUND OF THE INVENTION Vaccines are traditionally administered by intramuscular, intradermal, or subcutaneous injections. These injections can generate strong systemic immune responses, while their efficacy in activating mucosal immune responses is variable and often weak or undetectable, particularly for subunit vaccines. From the draining lymph nodes that have processed the injected vaccine, antigen-specific cytotoxic T lymphocytes (CTLs) and antibodies produced by B cells can migrate to different organs in the body, but their migration to the various mucosal tissues (e.g., genital, intestinal, PC Lcnn / Lznz / E / YiAi) is limited. Ref. 316280 - 2 respiratory) is often limited or impossible due to inadequate echotaxy of mucosal recipients and chemotaxis. However, the intranasal route, which is also considered a parenteral immunization route, can activate positive mucosal immune responses in the respiratory, genital, and intestinal tracts, which share some interconnections and are more accessible if the vaccine is delivered at the mucosal site. Therefore, these parenteral vaccines may offer protection, in some cases, against mucosal pathogens. Because most pathogens enter the body through mucosal tissues (oral, respiratory, genital, and intestinal tracts) and many of them replicate only in mucosal tissues, mucosal vaccination can optimally induce a frontal line of defense by inducing both innate (e.g., NK cells) and adaptive (T and B lymphocytes) immune responses at local and distant mucosal sites. With resident mucosal defense, protection can be immediate without delay in the recruitment of cells from the periphery, which allows for more efficient interference in each of the early transmission events and infection events before pc Lcnn / Lznz / E / YiAi - 3 the dissemination of the pathogen and, in some cases, the establishment of reservoirs. Mucosal tissues and lymph nodes contain more than 90% of the body's immune cells. Furthermore, mucosal antibodies account for approximately 80% of the body's total antibody production. Thus, the local immune response by mucosal antibodies can act as a front-line defense against mucosal infections (e.g., HIV-1, herpesvirus, rotavirus, etc.) and in the entry through mucosal tissues to reach other organs (e.g., HIV-1, hepatitis B, tuberculosis). In contrast, antibodies in the blood can act as a backup defense once pathogens have crossed the mucosal defenses or in synergy with mucosal antibodies and the mucus environment.Antibodies in the blood primarily act as an efficient front line of defense against pathogens that directly enter the bloodstream, following mosquito bites (e.g., malaria, chikungunya infection, dengue, Zika, West Nile virus, yellow fever, etc.) or accidental skin / mucous membrane damage (e.g., Staphylococcus aureus, Pseudomonas aeruginosa). Mucosal vaccine delivery (via pc Lcnn / Lznz / E / YiAi) - 4 the buccal, sublingual, nasal, oral, or vaginal mucosa) has received increasing interest as a means to induce local and distant antibody responses, as well as a systemic immune response. Furthermore, mucosal vaccine delivery via solid dosage forms (e.g., buccal / sublingual tablets, oral tablets or capsules, vaginal inserts) may provide several advantages such as the potential for mass immunization, patient compliance, ease of use, product shelf-life stability, and the ability to be independent of a cold chain. Furthermore, mucosal vaccine delivery may be suitable for patients with needle injection phobia, and the patient may self-administer the vaccine with appropriate explanations.The buccal / sublingual route has been used for many years for the delivery of drugs and small molecules into the bloodstream, but its application as a means of mucosal delivery for vaccines has received little attention. SUMMARY OF THE INVENTION Lipid-based vesicles can be used as drugs, vaccines, or delivery system adjuvants, and combinations thereof. Lipid-based vesicles can consist of one or more lipids (e.g., Lcnn / Lznz / E / YiAi). - 5 natural and / or synthetic components that form the basic structure (particle) and additional optional components (peptides, proteins, carbohydrates, nucleic acids, small molecules). Lipid-based vesicles can range in size from nanoparticles (approximately 20 to 200 nm) to submicrons (approximately 200 to 800 nm) to microns (approximately 800 nm to 10 pm). Virosomes and liposomes belong to the lipid-based vesicle systems. Virosomes are unilamellar, and liposomes can be unilamellar, bilamellar, or multilamellar. Virosomes are a type of subunit vaccine that can contain any protein derived from enveloped viruses that serves as starting material for the formation of lipid-based virosome particles. These virosomes can be genetically devoid of protein, non-replicating, and non-infectious, making them suitable and safe for enteral and parenteral immunization, provided they can be formulated in a stabilized form.Virosomes may contain additional molecules, such as homologous (from the same origin as the pathogen) or heterologous (derived from a pathogen other than the initial viral material) target molecules in the form of peptides, proteins and / or carbohydrates, nucleic acids, adjuvants, specific lipids, and / or small molecules (drugs). This is similar to pc Lcnn / Lznz / E / YiAi. - 6. Virosomes and liposomes can also be used as a vehicle for the delivery of pharmaceutical drugs, vaccines, and adjuvants, but the lipid membrane does not contain any native viral proteins. In some embodiments, the liposomes used herein may be proteoliposomes (i.e., liposomes containing proteins). Ideally, stabilization should allow for cold-chain-independent vaccine storage or allow the vaccine to withstand high and low temperature excursions outside of recommended cold-chain conditions without impairing the product's bioactivity. Applicants have discovered a lyophilized orally dispersible vaccine containing virosomes and a process for making such a vaccine that can preserve the stability of the virosomes (physically for the particle structure and chemically for the target molecules). The product stability can be maintained during storage at room temperature (e.g., about 25°C), independent of cold chain storage conditions, and can also withstand accidental freezing conditions (e.g., about -4°C to about -17°C) as well as exposure to high temperatures present in hot countries (e.g., 35°C to 45°C). The lyophilized sublingual dosage forms described herein that pc Lcnn / Lznz / E / YiAi - 7 containing the virosome vaccine can induce mucosal immunity and can also induce systemic immune responses. Specifically, the combination of the composition of the liquid virosome concentrate and its buffer, the composition of the liquid base matrix for a solid dosage form (excluding the virosome concentrate), and the manufacturing conditions for generating solid dosage forms under lyophilization of sublingual tablets can generate a solid vaccine form that has physical attributes suitable for sublingual tablets, with preserved virosomes and target molecule integrity, endowing them with storage stability under different environmental conditions. Administering stable solid virosome dosage forms may be suitable for sublingual mucosal immunization. Virosomes belong to the family of enveloped virus-like particles (VLPs) because they have lipid membranes containing viral membrane proteins derived from the purified parent viruses. Like VLPs, virosomes can closely mimic the architecture, composition, and surface antigen presentation of virus particles with or without the functional activities of the native viral envelope. Virosomes can comprise Lcnn / Lznz / E / YiAi proteins. - 8 reconstituted viral membranes, generally obtained by purification of solubilized membrane proteins and lipids from enveloped viruses with a solubilizing agent, followed by the addition of natural or synthetic lipids and antigens, with or without adjuvants, and removal of the solubilizing agent from the mixture, resulting in the formation of lipid bilayers with the proteins protruding from them. The antigens can be native proteins as well as recombinant or synthetic proteins or peptides. Virosomes can also be formed first, and then modified by covalent or noncovalent modification of their membranes to 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, when CD8+ T cell induction is part of the vaccine strategy, a particularly important feature of virosomes is their continued binding to the hemagglutinin (HA) receptor, which has its membrane fusion activity. If the vaccine strategy focuses on the induction of antigen-specific antibodies, then HA fusion activity is not absolutely required, but the presence of HA as an excipient is still important, e.g., Lcnn / Lznz / E / YiAi. - 9 since it can provide help to T lymphocytes, particularly for small antigens and peptides that might lack these properties. Although specific vaccines are mentioned in this description, all vaccines that use virosomes as a delivery platform are contemplated by this description. HIV antigens containing influenza virosome (P1 and rgp41) and TLR 7 / 8 adjuvant can be used as virosome concentrate models for the vaccines described herein. Influenza virosomes with HIV rgp41 antigen have been shown to be capable of inducing immune responses in various animal studies and in a phase 1 clinical study via the intranasal and intramuscular routes.In these studies, virosome formulations were in liquid form administered by needle or nasal spray, which are susceptible to limited half-life stability at 4°C due to chemical modifications of the active pharmaceutical ingredients (APIs). To avoid or reduce the chemical modifications of APIs frequently present in aqueous environments, the development of solid vaccine forms with low moisture content has been identified as a promising solution. Applicants have been able to overcome various problems in developing a solid vaccine form. - 10 such as the lyophilized orally dispersible vaccines described herein. Specifically, influenza-based virosome concentrates containing the antigen-carrying virosomes and adjuvants can be supplied as a suspension in a buffered saline solution. To produce the lyophilized vaccine dosage forms, robust, porous structures within the tablets must be produced during manufacturing and must be sustained during subsequent storage. The presence of buffer salts in the virosome concentrates can present a challenge in producing this robust, porous structure. Thus, applicants discovered an appropriate amount of excipients along with manufacturing conditions to allow for the formation of a robust dosage form that does not collapse or partially collapses during the lyophilization process. Furthermore, freezing, reassociation, and the lyophilization process can also damage the integrity of virosome particles. This damage to virosomes is minimized so that sufficient virosome particles remain in the lyophilized product so that a sufficient amount of them can cross the sublingual mucosal membrane to be processed by immune cells to induce immune responses of the PC Lcnn / Lznz / E / YiAi - 11 mucosal tissues, which may also be supported by systemic immune responses. In this way, applicants have been able to balance the use of excipients and lyophilization processes to maintain both the immunogenicity of the virosome (e.g., intact virosomes with limited presence of clusters) and the properties of a good dosage form (e.g., orally disintegrating sublingual tablets). In some embodiments, a solid oral vaccine dosage form includes lipid-based vesicles comprising an immunogenic amount of at least one target molecule: 5 to 20% by weight of at least one cryo-lyoprotectant; 25 to 40% by weight of a matrix former; and 40 to 55% by weight of a scaffold former. In some embodiments, the lipid-based vesicles are virosomes or proteoliposomes. In some embodiments, the dosage form comprises 10 to 15% by weight of at least one cryo-lyoprotectant. In some embodiments, the at least one cryo-lyoprotectant comprises trehalose. In some embodiments, the dosage form comprises 33 to 37% by weight of the matrix former. In some embodiments, the matrix former comprises gelatin. In some embodiments, the gelatin comprises fish gelatin. In some embodiments, the fish gelatin is high molecular weight fish gelatin. In some embodiments, pc Lcnn / Lznz / E / YiAi- 12 the dosage form comprises 45 to 50% by weight of the structure former. In some embodiments, the structure former comprises mannitol. In some embodiments, the virosomes are derived from the membrane of influenza viruses or other enveloped viruses. In some embodiments, the at least one target molecule is present on the virosome. In some embodiments, the at least one target molecule comprises an HIV-1 envelope-derived antigen. In some embodiments, the HIV-1 envelope-derived antigen comprises HIV-1 P1 peptide and / or recombinant HIV-1 gp41. In some embodiments, the virosomes comprise adjuvant. In some embodiments, the dosage form facilitates uptake of at least one target molecule into the oral cavity. In some embodiments, the dosage form disintegrates within 180 seconds after being placed in the oral cavity.In some embodiments, the dosage form disintegrates within 90 seconds after being placed in the oral cavity. In some embodiments, the dosage form disintegrates within 60 seconds after being placed in the oral cavity. In some embodiments, the dosage form disintegrates within 30 seconds after being placed in the oral cavity. In some embodiments, an immune response is induced when administered to a patient by placement in the oral cavity. In some embodiments, Lcnn / Lznz / E / YiAi. - 13 modalities, placement in the oral cavity is placement on or under the tongue or in the buccal or pharyngeal region. In some embodiments, a method of inducing an immune response in a patient includes placing any of the above dosage forms into the oral cavity of a person in need of the immune response. In some embodiments, placement in the oral cavity is on or under the tongue or in the buccal or pharyngeal region. In some embodiments, a method of forming a solid oral vaccine dosage form includes dosing a liquid virosome formulation into a preformed mold, wherein the virosome formulation comprises: lipid-based vesicles comprising an immunogenic amount of at least one target molecule, 1 to 5% by weight of a cryo-lyoprotectant, 4 to 8% by weight of a matrix former, and 5 to 10% by weight of a structure former; freezing the dosed virosome formulation at a temperature of from -60°C to -90°C; annealing the frozen virosome formulation by holding it at a temperature of less than -15°C for 3 to 9 hours; and lyophilizing the annealed virosome formulation to form the dosage form. In some embodiments, the dosed virosome formulation is frozen at a temperature of from -60°C to -90°C. -90°C for a duration of about 1 to 5 minutes. In some embodiments, lyophilizing the reassociated virosome formulation comprises a first step of maintaining the reassociated virosome formulation at a temperature of 10°C to -20°C for 20 to 28 hours and a second step of maintaining the reassociated virosome formulation at a temperature of -5°C to about -15°C for 14 to 22 hours. In some embodiments, lyophilizing occurs at a pressure of less than 600 mbar. In some embodiments, the virosome formulation has a pH of about 6.5 to 8. In some embodiments, the cryo-lyoprotectant comprises trehalose. In some embodiments, the matrix former comprises gelatin. In some embodiments, the gelatin comprises fish gelatin. In some embodiments, the fish gelatin is high molecular weight fish gelatin. In some embodiments, the structure former comprises mannitol.In some embodiments, the lipid-based vesicles are derived from influenza virus or respiratory syncytial virus. In some embodiments, the at least one target molecule comprises an HIV-1 envelope-derived antigen. In some embodiments, the HIV-1 envelope-derived antigen comprises HIV-1 P1 peptide or recombinant HIV-1 gp41. In some embodiments, the lipid-based vesicles comprise adjuvant. In some embodiments, a method of forming pc Lcnn / Lznz / E / YiAi - 15 a solid oral vaccine dosage form includes: dosing a liquid virosome formulation into a preformed mold, wherein the virosome formulation comprises: (1) 20 to 50% by weight of a virosome concentrate, wherein the virosome concentrate comprises: virosomes comprising an immunogenic amount of at least one target molecule; 2 to 10% by weight of cryo-lyoprotectant; and 60 to 200 mM of a buffer system; (2) 4 to 8% by weight of a matrix former; and (3) 5 to 10% by weight of a structure former; freezing the dosed virosome formulation at a temperature of -60°C to -90°C; annealing the frozen virosome formulation by holding it at a temperature of less than -15°C for 3 to 9 hours; lyophilizing the annealed virosome formulation to form the dosage form. In some embodiments, the buffer system comprises HEPES-sodium chloride. Additional advantages will be readily apparent to those skilled in the art from the following detailed description. The examples and descriptions herein should also be considered illustrative and not limiting. All publications, including patent documents, scientific articles and databases referenced in this application are incorporated by reference in their entirety for all purposes in the same pc Lcnn / Lznz / E / YiAi - 16 to the extent that each individual publication were incorporated individually by reference. If a definition set forth herein conflicts with or otherwise conflicts with a definition set forth in any patent, application, published application, or other publication that is incorporated by reference herein, the definition set forth herein shall govern over the definition that is incorporated herein by reference. BRIEF DESCRIPTION OF THE FIGURES Exemplary modalities are described with reference to the attached figures, in which: Figure 1 illustrates a general flow diagram for producing a vaccine dosage form described herein. Figure 2 illustrates a flow diagram from a matrix formulation for final sublingual tablets, to the production of a vaccine dosage form described herein. Figure 3A is a schematic representation with an image of a fully moistened tablet. Figure 3B is a schematic representation with an image of a tablet with hard lumps. Figure 3C is a schematic representation with an image of a tablet with a PC matrix film Lcnn / Lznz / E / YiAi - 17 collapsed formulation that forms on the surface of the lyophilized tablet (coating). Figure 4 is a photograph of immunoblots showing binding of anti-PI and anti-rgp41 specific antibody to PI-virosomes and rgp41-virosomes from reconstituted ZydisMR sublingual tablets stored at 5°C, 25°C and 40°C for 1 and 3 months (the analysis of Example 2 described herein). Figure 5 illustrates the immunogenicity of P1 and rgp41 antigens from liquid virosome concentrates and lyophilized sublingual tablets containing virosomes, both stored at 4°C and 40°C over time. DETAILED DESCRIPTION OF THE INVENTION Described herein are pharmaceutical compositions for lipid-based vesicles (e.g., virosomes or VLPs) and methods of preparing these pharmaceutical compositions. Specifically, the present disclosure relates to lyophilized orally dispersible or disintegrating dosage forms that can retain VLP stability (i.e., the structural integrity and chemical stability of the antigen), that can be stored independent of cold chain storage conditions, and that also withstand accidental freezing conditions as well as exposure to high temperatures present in PC countries. - 18 tempered. The dosage forms can also retain the physical and chemical attributes of the VLPs, making them suitable for sublingual delivery to induce mucosal immunity. Applicants have been able to formulate and optimize the amount of matrix former and structure former to achieve a formulation that can address the use of highly loaded buffer systems containing virosome concentrates and cryo-lyoprotectants. In conjunction with excipient adjustments, the manufacturing parameters of the dosage forms are optimized. Specifically, the reassociation time is optimized to maximize mannitol crystallization, which imparts robustness to the dosage form and minimizes damage to the virosome. Furthermore, the lyophilization conditions are optimized to minimize damage to the virosome particles and minimize structural collapse during lyophilization. Lipid-based vesicle systems Lipid-based vesicles can be used as drug, vaccine, or adjuvant delivery systems and combinations thereof. Lipid-based vesicle systems can consist of one or more natural and / or synthetic lipids (e.g., phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, cholesterol, etc.) - 19 sphingolipids and their derivatives), forming the vesicle membrane (particle) and additional optional components (peptides, proteins, carbohydrates, small molecules). Lipid-based vesicles range in size from nanoparticles (approximately 20–200 nm) to submicrometers (approximately 200–800 nm) to micrometers (approximately 800 nm–10 pm). A lipid-based vesicle / particle may comprise a unilamellar, bilamellar, or multilamellar lipid bilayer vesicle. In some embodiments, a lipid-based vesicle / particle may include a lipid bilayer comprising lipids selected from natural and / or synthetic lipids. These lipids may be used to better mimic the lipid-based membrane of pathogens and microdomains in order to improve membrane and antigen anchoring, antigen presentation, and / or folding for optimal epitope exposure. The lipid-based vesicle / particle may harbor membrane-anchored antigen and / or adjuvant exposed on the surface of the particle or pointing toward the interior of the particle or having a random orientation. The antigen and / or adjuvant may also be encapsulated within the lumen of the lipid-based vesicle / particle. Virosomes are lipid-based vesicles assembled in vitro, in a pc-free manner Lcnn / Lznz / E / YiAi system. - 20 cells, forming enveloped VLPs that belong to the subunit vaccine category. The lipid membranes of virosomes as carriers can be derived from any enveloped virus and, consequently, contain at least native viral membrane proteins from the initiating virus. These virosomes lack viral genetic material, cannot replicate, and are non-infectious, making them suitable and harmless for systemic and mucosal immunization. In addition, virosomes can contain additional molecules such as antigens (e.g., peptides, proteins, carbohydrates, nucleic acids), adjuvants, specific lipids, and / or small molecules (drugs), which can be anchored to the surface of the virosomes and / or entrapped within the virosome lumen. Liposomes are also lipid-based vesicles that form a type of subunit vaccine. They can be formed as vesicles with at least one lipid bilayer, which does not contain proteins derived from the natural viral membrane of enveloped viruses. These lipid-based particles are also assembled in vitro and lack genetic material. They may be suitable for systemic or mucosal application. In addition, liposomes may contain additional molecules such as antigens (peptides, proteins and / or carbohydrates, nucleic acids), such as Lcnn / Lznz / E / YiAi. - 21 adjuvants, specific lipids and / or small molecules (drugs), which can be anchored to the surface of the liposomes and / or entrapped within the lumen of the liposomes. In some embodiments, the liposomes used herein can be proteoliposomes (i.e., liposomes with proteins). The lipids used in the dosage forms described herein may belong to cationic lipids, glycolipids, phospholipids, glycerophospholipids, galactosylceramide, sphingolipids, cholesterol and derivatives thereof. Phospholipids may include, but are not limited to, phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, cardiolipin and phosphatidylinositol with varying fatty acyl compositions. Furthermore, the lipids may be selected from DOTMA (N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP (N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride), DODAC (N,N-dioleoyl-N,N-dimethylammonium chloride), DDAB (didodecyldimethylammonium bromide), and stearylamine or other aliphatic amines and the like. Virosome formulation Virosome concentrate Figure 1 illustrates a flowchart for a Lcnn / Lznz / E / YiAi pc - 22 method 100 for the production of a vaccine dosage form described herein. In step 101, a liquid virosome concentrate can be mixed with a base matrix formulation premix to form a liquid virosome formulation suitable for lyophilization. Applicants have found that when a virosome concentrate composition is combined with a specific composition of a base matrix formulation and prepared in conjunction with a set of manufacturing conditions optimized to retain sufficient virosomes with required particle characteristics, vaccine bioactivity can be maintained. The bioactivity of the virosome particles can be dependent upon the conformational integrity of the particle and the quality of the antigenic molecules that are associated with its unilamellar phospholipid membrane. In some embodiments, the virosome concentrate may be a liquid virosome concentrate. The virosome concentrate includes at least one population of virosomes. A population of virosomes may be virosomes containing a given drug, or virosomes acting as a vaccine delivery vehicle harboring vaccine antigens and virus-derived proteins (e.g., HA if the virosomes are derived from influenza viruses). In some embodiments, virosomes can be pc Lcnn / Lznz / E / YiAi - 23 be derived from an influenza virus to generate influenza virosomes as enveloped VLPs that act as carriers for heterologous vaccine antigens (e.g., HIV antigens anchored on influenza-derived virosomes) or from other enveloped viruses 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 can be used to generate corresponding forms of RSV-virosomes, Sendai-virosomes, SFV-virosomes, VSV-virosomes, or Sindbis-virosome for the displayed homologous vaccine antigen (e.g., native RSV antigens on virosomes derived from RSV). In some embodiments, the virus-based virosomes can be derived from any enveloped virus.In some embodiments, the virus-based virosomes can be derived from DNA viruses, including, but not limited to, herpesviruses, poxviruses, and hepdnaviruses. In some embodiments, the virus-based virosomes can be derived from RNA viruses, including, but not limited to, flaviviruses, togaviruses, coronaviruses, hepatitis D, orthomyxoviruses, paramyxoviruses, rhabdoviruses, buniaviruses, and filoviruses. In some embodiments, the virus-based virosomes can be derived from retroviruses. cc Lonn / Lznz / E / YiAi - 24 In some embodiments, an influenza virus-based virosome can be formed according to the different processes described in Influenza virosomes as vaccine adjuvant and carrier system: Moser C. et al., Expert review, 779-791 (2013); WO2004110486; WO2004071492; WO2007099446; WO2016039619; EP2058002; and WO2016039620, all of which are incorporated herein by reference in their entireties. In addition, the various patents and other publications listed in the aforementioned references are also incorporated herein by reference in their entireties. Thus, this application is not limited to a specific process for virosome preparation. Thus, the reference described above is merely an example of virosome preparation. This application seeks all lipid-based particles such as, but not limited to, virosomes, VLPs, and nanoparticle preparations, including antigen-laden liposomes. Although this section describes a liquid virosome concentrate, the virosome can be substituted with a liposome to form a liquid liposome concentrate. In this way, the components in the virosome concentrate (in addition to the virosomes themselves) can also be applied to a liposome concentrate. The virosome (e.g. pc-derived virosome Lcnn / Lznz / E / YiAi - 25 influenza) 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 (e.g., HA) present in a concentration detectable by prior art analytical assays. In some embodiments, the viral membrane proteins are about 10 to 300 μg / ml or about 5 to 150 μg / ml of the liquid virosome concentrate, when influenza virosomes are used as carriers for heterologous viral antigens. For influenza virosomes designed to induce CTL responses, the concentration of HA may range from about 150 to 800 μg / ml or about 75 to 400 μg / ml. In some embodiments, the concentration of the viral membrane proteins may be greater than the exemplary concentrations listed above. The virosome concentrate may also include lipids. The lipids used in the virosome concentrate may be cationic lipids, glycolipids, phospholipids, glycerophospholipids, galactosylceramide, sphingolipids, cholesterol, and derivatives thereof. Phospholipids may include, in particular, phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, PCL Lcnn / Lznz / E / YiAi - 26 phosphatidylserine, phosphatidylglycerol, phosphatidic acid, cardiolipin, and phosphatidylinositol with varying fatty acyl compositions. In addition, the lipids can be selected from DOTMA (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride), DODAC (N,N-dioleoyl-N,N-dimethylammonium chloride), DDAB (didodecyldimethylammonium bromide), and stearylamine or other aliphatic amines and the like. In some embodiments, the liquid virosome concentrate can include about 0.1 to 10 mg / ml, about 0.3 to 8 mg / ml, or about 0.5 to 5 mg / ml of lipids. Virosomes may contain target molecules (e.g., vaccine antigens) with or without adjuvants. Antigens may be soluble and entrapped within the virosome lumen or covalently or non-covalently anchored onto the virosomes. Antigens may be peptides, proteins, polysaccharides, complete or partial fragments or extracts of bacterial cells, viral particles, or nucleic acids. Antigens may be derived from a parasite such as a protozoan or worm, which can cause disease, or combinations thereof. Antigens may be derived from any plant, animal, or human cell or cell line. Any antigen known in the art may be suitable for use with Lcnn / Lznz / E / YiAi antigens. - 27 virosomes, including those commercially available or prepared by purification of preparations from a pathogen or cancer cell or non-transformed cell (native proteins), which are expressed recombinantly or produced synthetically by standard manufacturing. Methods for generating antigens suitable for incorporation into virosomes are known in the art, and any of the known methods described herein may be used. One or more target molecules are included in the virosomes of the liquid virosome concentrate and subsequent dosage forms described herein in an amount sufficient to render them immunogenic when delivered in a dosage form. The immunogenic amount is defined as the amount appropriate to induce a desired immune response. One of skill in the art can readily determine the immunogenic amount for a given disease or infection based on, among other things, the route of immunization, the age and weight of the patient to whom the dosage form is to be administered. In some embodiments, the liquid virosome concentrate may include about 1 to 2000 µg / ml of the target molecule. The target molecule may be a peptide, a protein, a carbohydrate, a nucleic acid, or a peptide. - 28 a small molecule or a combination thereof. The target molecule may function as an antigen (e.g., a vaccine antigen), a drug, a diagnostic molecule, an analytical sensor, or a combination thereof. In some embodiments, the liquid virosome concentrate may include about 1 to 1000 μρ / ηA of one target molecule and about 1 to 1000 μg / ml of a second target molecule. After further processing into solid dosage forms, each tablet may contain about 0.01 to 250 μρ of each target molecule. In more preferred embodiments, the liquid virosome concentrate may include about 25 to 500, about 50 to 500 µg / ml, about 25 to 225 µρ / ηA, about 25 to 200 µg / ml, about 50 to 450 µg / ml, about 50 to 400, about 100 to 400 pg / ml, about 100 to 400 pg / ml, about 100 to 200 µg / ml, or about 200 to 400 µg / ml of at least one target molecule (e.g., antigen). In some embodiments, the liquid virosome concentrate may include about 1 to 500 μg / ml, about 25 to 500 μg / ml, about 50 to 450 μρ / ml, about 50 to 400 μρ / ml, about 25 to 250 μρ / ml, about 25 to 200 μg / ml, about 50 to 250 μg / ml, about 75 to 225 μg / ml, or about pc Lcnn / Lznz / E / YiAi - 29 100 to 200 pg / ml of 1 target molecule and approximately 50 to 450 pg / ml, approximately 50 to 400 μg / ml, approximately 25 to 225 μg / ml, approximately 25 to 200 μg / ml, approximately 100 to 500 μg / ml, approximately 150 to 450 μg / ml, approximately 175 to 425 μg / ml or approximately 200 to 400 μg / ml of a second target molecule. The dosage forms described herein can be used to deliver therapeutic or prophylactic vaccines to prevent or reduce symptoms related to allergies or infection, tumor development and dissemination, pathogen transmission, infection of cells, pathogen load after induction of B cells (antibodies) and / or relevant T cell subsets (e.g. Thl, Th2, Thl6, Thf, Tcl, Tc2, Tc3, Treg, others) which depend on the virosome formulations.For this purpose, the target molecules may provide protection against the following representative list of diseases which is not exhaustive: influenza, tuberculosis, meningitis, hepatitis, whooping cough, polio, tetanus, diphtheria, malaria, cholera, herpes, typhoid, HIV / AIDS, mumps, 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, caries, pc Lcnn / Lznz / E / YiAi. - 30 Chagas disease urinary tract infection caused by E. cali, pneumococcal disease, mumps, chikungunya, cancer, allergies and combinations thereof. In addition, the target molecules can provide protection against diseases caused by the following, non-exhaustive, list of causative organisms: Vibrio species, Salmonella species, Bordetella species, Haemophilus species, Toxoplasma gondii, Cytomegalovirus, Chlamydia species, Streptococcal species, Norwalk virus, Escherischia coli, Helicobacter pyiori, Rotavirus, Neisseria gonorrhae, Neisseria meningitidis, Adenovirus, Epstein-Barr virus, Japanese encephalitis virus, Pneumocystis carinii, Herpes simplex, Clostridia species, Respiratory syncytial virus, Klebsiella species, Shigella species, Pseudomonas aeruginosa, Parvovirus, Campylobacter species, Rickettsia species, Varicella zoster, Yersinia species, Ross River virus, JC virus, Rhodococcus equi, Moraxella catarrhalis, Borrelia burgdorferi, Pasteureiia haemolytica, and combinations thereof. In addition, or alternatively, the target molecule may provide protection or treatment against allergies (e.g., allergens containing virosomes), cancer (e.g., tumor antigens, antibodies, anticancer drugs, nucleic acids), and other types of conditions. Veterinary applications of this pc Lcnn / Lznz / E / YiAi - 31 descriptions are also contemplated. Accordingly, the target molecules may generate protection against the following non-exhaustive list of veterinary diseases: coccidiosis, Newcastle disease, enzootic pneumonia, feline leukemia, atrophic rhinitis, erysipelas, foot-and-mouth disease, swine pneumonia, and other disease conditions and infections affecting companion and farm animals, and combinations thereof. In some embodiments, the virosome contains a vaccine antigen in addition to the viral proteins present in the reconstituted membrane. In some embodiments, the vaccine antigen can be an HIV-1 P1 peptide and / or recombinant HIV-1 gp41. As stated above, the virosomes in the liquid virosome concentrate may also contain, or be mixed with, an adjuvant. Virosomes and other subunit vaccines may require an adjuvant to enhance the immune response, resulting in accelerated and increased production of antibodies and T cells, while also sustaining immunological memory. To be effective, it is preferable to have an immune response associated with the generation of a memory response that provides long-lasting protection from the specific disease. The adjuvant may also allow for the decrease in PC Lcnn / Lznz / E / YiAi - 32 doses of antigen (dose-sparing) and increase the range of the desired immune response. Once exposed to antigens, the immune system can remember them, and upon re-exposure, the immune response is much faster. The effectiveness of an adjuvant in increasing an immune response may be independent of the antigen with which it is combined, since the adjuvant itself can induce nonspecific immune responses and may generate autoimmune side effects if strong cellular activation is obtained in the absence of antigen. However, when the antigen and adjuvant are physically bound together, they can also co-migrate to the same site when injected, promoting antigen-specific immune activation with lesser nonspecific inflammatory responses.Suitable adjuvants include but are not limited to Toll-like receptor (TLR) agonists, inflammasome agonists, nucleotide oligomerization and binding domain (NOD) receptor (NLR) agonists, more specifically non-toxic bacterial fragments, cholera toxin (and detoxified forms and fractions thereof), guitosan, E. coli heat-labile toxin (and detoxified forms and fractions thereof), lactide / glycolide homopolymers and copolymers (PLA / GA, pc Lcnn / Lznz / E / YiAi). - 33), polyanhydride, for example trimellitylimido-L-tyrosine, DEAE-dextran, saponins complexed with membrane protein antigens (immunostimulating complexes-ISCOMS), bacterial products such as lipopolysaccharides (LPS) and muramyl dipeptide (MDP), liposomes, cochleates, proteinoids, cytokines (interleukins, interferons), genetically engineered live microbial vectors, non-infectious mutant Pertussis toxin, neurimidase / galactose oxidase and attenuated bacterial and viral toxins derived from mutant strains, and combinations thereof. A suitable amount of an adjuvant can be readily determined by a person of ordinary skill in the art. In some embodiments, the virosomes may harbor 3M-052 adjuvant (a TLR7 / 8 agonist supplied by 3M). In some embodiments, the liquid virosome concentrate may contain 3M-052 adjuvant in the range of about 8 to 140 μg / ml, about 4 to 70 μg / ml, about 1 to 60 μg / ml, and 0.01 to 16 μg per tablet. In some embodiments, the liquid virosome concentrate may include at least two different virosome populations, each harboring at least one pc Lcnn / Lznz / E / YiAi - 34 antigen with or without adjuvant. In some embodiments, these two different virosome populations may have different antigens, but the same adjuvant (e.g., Pl / 3M-052 virosome mixed with rgp41 / 3M-052 virosomes). In some embodiments, these two different virosome populations may have different antigens, but with different adjuvants (e.g., Pl / adjuvant A virosome mixed with rgp41 / adjuvant B virosomes). In some embodiments, the virosome concentrate may include at least two different antigens per virosome, with or without adjuvant (e.g., virosome harboring both P1 and rgp41 antigens, with or without adjuvant). The liquid virosome concentrate may also include a buffer system. In some embodiments, the virosomes may be suspended in the buffer system. The buffer system may maintain the physical integrity and chemical stability of the virosomes in the virosome concentrate. In some embodiments, the virosomes are suspended in a buffer system to maintain a target pH of about 6 to 9, about 6.5 to 8, about 7 to 8, about 7.2 to 7.6, about 7.3 to 7.5, or about 7.4. Additionally, the buffer system may stabilize the virosomes when they are in a liquid form. - 35 at a storage temperature of approximately 2 to 8°C. A suitable buffer system includes, but is 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 can be about 5 to 1000 mM, about 60 to 200 mM, about 100 to 300 mM, about 125 to 275 mM, about 150 to 250 mM, about 175 to 225 mM, about 180 to 210 mM, about 185 to 200 mM, about 185 to 195 mM, or about 190 to 195 mM in the virosome concentrate. If the buffer system is HEPES-sodium chloride in the virosome concentrate, the sodium chloride may be about 5 to 1000 mM; about 50 to 150 mM, about 125 to 175 mM, about 130 to 160 mM, about 130 to 150 mM, about 135 to 145 mM, or about 140 to 145 mM in the virosome concentrate.If the buffer system is HEPES-sodium chloride in the virosome concentrate, the HEPES may be about 1 to 200 mM, about 10 to 75 mM, about 10 to 50 mM, about 25 to 75 mM, about 30 to 70 mM, about 40 to 60 mM, about 45 to 55 mM, or about 48 to 52 mM in the virosome concentrate. cc Lonn / Lznz / E / YiAi - 36 The liquid virosome concentrate may also include at least one cryo-lyoprotectant. Virosomes may be damaged during the freezing and / or lyophilization steps in the production of dosage forms described herein. Thus, a cryo-lyoprotectant may be included in the virosome concentrate to improve preservation of the virosome during the freezing and / or lyophilization steps. Examples of cryo-lyoprotectants include, but are not limited to, polyols such as trehalose, sugars such as sucrose, and amino acids such as lysine, oligosaccharides such as inulin (a medium chain oligosaccharide), or combinations thereof. The cryo-lyoprotectants used may be inert so as to be suitable for formulation into vaccines. The liquid virosome concentrate may include about 1 to 20%, w / w, about 1.5 to 10% w / w, approximately 2 to 10% w / w, approximately 4 to 10% w / w, approximately 2 to 9% w / w, approximately 2 to 5% w / w, approximately 3 to 8% w / w, approximately 3.5 to 8% w / w, approximately 3.5 to 7% w / w, approximately 4 to 8% w / w or approximately 5 to 7% w / w of the cryo-lyoprotector. The virosome concentrate may be about 1 to 75% w / w, about 10 to 65% w / w, about 15 to 60% w / w, about 20 to 55% w / w, pc Lcnn / Lznz / E / YiAi - 37 about 20 to 50% w / w, or about 25 to 50% w / w of the virosome formulation. In some embodiments, the virosome concentrate may be about 15 to 35% w / w, about 20 to 30% w / w, about 23 to 27% w / w, or about 25% w / w of the virosome formulation. Base matrix formulation The base matrix formulation is what helps provide the structure of the final dosage form. Thus, the base matrix formulation may include a matrix former. The matrix former may provide a network structure to the dosage form that imparts strength and resilience during handling. Suitable matrix formers may include, but are not limited to, gelatin, starch, or combinations thereof. Additional matrix formers can be found in EP 2624815 Bl, which is incorporated herein by reference in its entirety. The gelatin may be fish gelatin, bovine gelatin, porcine gelatin, or combinations thereof. Each of the gelatins has different gelation characteristics. The extent to which a gelatin solution forms a gel may depend on the concentration of the gelatin and the temperature of the gelatin solution.A bovine gelatin solution tends to gel at temperatures below pc Lcnn / Lznz / E / YiAi. - 38 18°C and therefore may be considered a gelling gelatin. In contrast, fish gelatin may remain in solution at temperatures as low as 10°C and therefore may be considered a non-gelling gelatin. In some embodiments, the gelatin may be a low endotoxin gelatin such as one sourced or produced in accordance with the process described in Provisional Application No. 62 / 640,394, which is incorporated herein by reference in its entirety. In some embodiments, the amount of matrix former in the virosome formulation may be about 1 to 15% w / w, about 2 to 12%, about 3 to 10% w / w, about 4 to 8% w / w, about 4 to 6%, about 5 to 7% w / w, or about 6% w / w. The temperature at which the virosome formulation is dosed may be as low as 10 to 18°C. Thus, a formulation using only bovine gelatin may not be dosed at these low temperatures. However, a combination of bovine gelatin and another type of gelatin (e.g., fish gelatin) may be used. Applicants have discovered that fish gelatin can provide a lyophilized tablet with a robust matrix structure and a disintegration time of about 30 to 180 or 30 to 60 seconds that is desirable for inducing a disintegration time. - 39 sufficient contact with the oral mucosa. In addition, fish gelatin can provide lyophilized dosage forms with good physical attributes for formulation compositions containing a high loading of soluble components such as buffer salts, such as the amounts described herein. 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 is greater than 30,000 Daltons. Standard molecular weight fish gelatin is defined as fish gelatin in which more than 50% of the molecular weight distribution is below 30,000 Daltons. In some embodiments, the matrix former can also serve as a stabilizer for the antigens, as well as a mucoadhesive. In addition, the starch can also serve as an immune-stimulating excipient. The base matrix formulation may also include a structure former. Suitable structure formers may include sugars including, but not limited to, mannitol, dextrose, lactose, PCL, Lcnn / Lznz / E / YiAi - 40 galactose, cyclodextrin, or combinations thereof. The structure former can be used in lyophilization as a bulking agent as it crystallizes, providing structural strength to the lyophilized product. Soluble excipients such as buffer salts and trehalose in the virosome formulation can inhibit crystallization. Extended reassociation time is commonly used to allow for crystallization. However, the presence of these soluble excipients can also cause the frozen product to melt during reassociation. Thus, applicants have discovered a balance between the amount of structure former, buffer salts, and cryo-lyoprotectant and the reassociation conditions (i.e., temperature and time).In some embodiments, the amount of structure former in the virosome formulation can be about 1 to 20% w / w, about 3 to 15% w / w, about 4.5 to 10% w / w, about 4.5 to 8% w / w, about 5 to 10% w / w, about 6 to 10% w / w, about 7 to 9% w / w, or about 8% w / w. Applicants have discovered that at values below 4.5% w / w of the structure former, some microstructural collapse may occur during lyophilization resulting in poor dispersion / disintegration of the lyophilized dosage form. Thus, pc Lcnn / Lznz / E / YiAi. - 41 A higher amount of structure former can be found that minimizes or eliminates microstructural collapse without noticeably altering the virosome. In addition, the base matrix formulation may also include a cryo-lyoprotectant. Examples of cryo-lyoprotectants include, but are not limited to, polyols such as trehalose, sugars such as sucrose, and amino acids such as lysine, oligosaccharides such as inulin (a medium-chain oligosaccharide), or combinations thereof. A cryo-lyoprotectant may be used to protect the virosome from damage during subsequent freezing and lyophilization. However, the addition of a cryo-lyoprotectant may induce microstructural collapse of the dosage form matrix during lyophilization. Thus, a balance must be sought to minimize microstructural collapse while retaining a sufficient number of virosomes to maintain the virosome quality to induce an immune response. The amount of cryo-lyoprotectant in the base matrix formulation may be about 0.01 to 2% w / w, or about 0.1 to 1.5% w / w, approximately 0.2 to 1% w / w or approximately 0.25 to 0.75% w / w. Thus, the net amount (hereinafter (net)) of the cryo-lyoprotectant in the virosome formulation (i.e., the liquid virosome concentrate plus pc Lcnn / Lznz / E / YiAi. - 42 the base matrix formulation) may be about 0.5 to 6% w / w, about 0.5 to 5% w / w, about 0.5 to 4.5% w / w, about 1 to 4% w / w, about 1.5 to 4.5% w / w, about 1.5 to 3% w / w, about 1.5 to 2.5% w / w, about 2 to 3% w / w, about 2.5% w / w, or about 2% w / w. Applicants discovered that these amounts of cryoprotectant can provide sufficient cryo-lyoprotection without resulting in unacceptable microstructural collapse during lyophilization. In some embodiments, the base matrix formulation may also include a mucoadhesive such as a gum. Suitable gums include, but are not limited to, acacia, guar, agar, xanthan, gellan, carrageenan, curdlan, konjac, locust bean, welan, tragacanth, acacia, caraya, ghatti, pectins, dextran, glucomannan, and alginates or combinations thereof. The base matrix formulation may also contain additional pharmaceutically acceptable agents or excipients. These 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, gelatins of mammalian origin, fish gelatin, - 43 modified starches, preservatives, antioxidants, surfactants, viscosity improvers, permeability improvers, coloring agents, flavoring agents, 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 flavoring agents 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 (e.g., a 3% w / w sodium hydroxide solution), and combinations thereof.In some embodiments, the base matrix formulation and / or the virosome formulation has an amount of a pH modifier to maintain a target pH of about 6 to 9, about 7 to 8, about 7.2 to 7.6, about 7.3 to 7.5, or about 7.4. Suitable sweeteners may include aspartame, acesulfame K, and thaumatin, and combinations thereof. One of ordinary skill in the art will be readily able to determine suitable amounts of these various additional excipients, if desired. - 44 The base matrix formulation may also include a solvent. In some embodiments, the solvent may be water (e.g., purified water). In some embodiments, the remaining remainder of the base matrix formulation and / or the virosome formulation is the solvent. The base matrix formulation may be about 25 to 99% w / w, about 35 to 90% w / w, about 40 to 85% w / w, about 45 to 80% w / w, or about 50 to 75% w / w of the virosome formulation. In some embodiments, the base matrix formulation may be about 65 to 85% w / w, about 70 to 80% w / w, about 73 to 77% w / w, or about 75% w / w of the virosome formulation. Development of a dosage form comprising the formulation of virosomes As indicated above, a liquid virosome concentrate is mixed with a base matrix formulation to form a virosome formulation in step 101 suitable for the lyophilization process. Figure 2 provides a more detailed description of the process for forming a vaccine dosage form described herein. In some embodiments, the base matrix formulation can be prepared by dissolving a matrix former and a structure former in a solvent to form a premix. For example, gelatin and mannitol pc Lcnn / Lznz / E / YiAi - 45 can be dissolved in water as shown in step 201 of Figure 2. The premix can be heated to a temperature of about 40 to 80°C, about 50 to 70°C, about 55 to 65°C, or about 60°C and held for about 45 to 75 minutes, about 55 to 65 minutes, or about 60 minutes. As shown in step 202, the premix can be heated to 60°C and held for 1 hour. The premix can then be cooled to a temperature of about 30 to 50°C, about 35 to 45°C, or about 40°C and sieved before being further cooled to about 10 to 20°C or about 15°C and held at this temperature for the remainder of the process. As shown in step 203, the premix can be cooled to 40°C and sieved. The premix can then be cooled to a temperature of 15°C, as shown in step 204. Cryo-lyoprotectant is subsequently added to the premix. For example, trehalose may be added to the premix, as shown in step 205. Subsequently, the pH may be adjusted to about 6 to 9, about 7 to 8, about 7.2 to 7.6, about 7.3 to 7.5, or about 7.4 using a pH modifier. For example, the pH may be adjusted to 7.4 using a sodium hydroxide solution as shown in - 46 step 206. After the pH is adjusted, the liquid virosome concentrate may be added. After the liquid virosome concentrate is added, the pH is rechecked (step 207) and, if necessary, adjusted to about 6.0 to 8.5, about 7 to 8, about 7.2 to 7.6, about 7.3 to 7.5, or about 7.4 using an additional pH modifier. The mixture may be made up to a desired batch size with solvent (i.e., the virosome formulation). For example, an amount of water may be added to the mixture as needed, as shown in step 208. In step 102 of Figure 1, the liquid virosome formulation may be dispensed into a preformed mold. As used herein, dispensed refers to the deposition of a predetermined aliquot of solution or suspension. As used herein, preformed mold refers to any suitable container or compartment into which an aqueous solution or suspension may be deposited and within which it is subsequently lyophilized. In certain embodiments of the present disclosure, the preformed mold is a blister package with one or more blister receptacles. The predetermined aliquots in an amount of about 150 to 1000 mg or about 500 mg of fill dosage weight pc Lcnn / Lznz / E / YiAi - 47 Wet (also referred to as dosage fill weight) of the virosome formulation can be metered into the preformed molds. In some embodiments, the virosome formulation can be dosed at a temperature of about 10 to 20°C or about 15°C. For example, the virosome formulation can be dosed at 15°C with 500 mg of dosage fill weight, as shown in step 209. In step 103 of Figure 1, the dosed virosome formulations may then be frozen in the preformed molds. The dosed virosome formulations in the preformed molds may be frozen by any means known in the art. For example, the formulations may be passed through a cryogenic chamber (e.g., a liquid nitrogen tunnel). The temperature during lyophilization may be between 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 duration of freezing may vary 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, dosed virosome formulation can be frozen at a temperature of -70°C for 3 minutes and 15 seconds, - 48 as shown in stage 210. In step 104 of Figure 1, the frozen units in the preformed molds may be collected and placed in a freezer at a temperature less than about -25°C, about -20°C, about -15°C, about -10°C, or about -5°C and reassociated (i.e., kept frozen) for a period of time to crystallize the structure former. Crystallization of the structure former may provide the frozen units with the structural strength to prevent collapse of the frozen units during lyophilization. This may be critical for the integrity of the virosomes. The reassociation time may vary from about 3 to 9 hours, about 4 to 8 hours, about 5 to 7 hours, or about 6 hours. For example, the frozen units may be reassociated at less than -15°C for about 3 to 9 hours, as shown in step 211. After reassociation, the reassociated frozen units may be lyophilized in step 105 to form the dosage form. During the lyophilization process, water sublimates from the frozen units. In some embodiments, the frozen units may be loaded onto the racks of a freeze dryer. In some embodiments, Lcnn / Lznz / E / YiAi - In some embodiments, the freeze dryer may be pre-cooled to a temperature of about -15 to -35°C, about -20 to -30°C, or about -25°C. Once the reassociated frozen units are in the freeze dryer, the freeze-drying cycle may be initiated. In some embodiments, a vacuum may be drawn and the shelf temperature increased once the freeze-drying cycle has been initiated. The freeze-dryer may be operated at low pressure (i.e., vacuum). In some embodiments, the freeze dryer may operate at a pressure of about less than or equal to 1000 mbar, about 900 mbar, about 800 mbar, about 700 mbar, about 600 mbar, about 500 mbar, or about 400 mbar. Applicants have discovered that a two-stage lyophilization cycle (step 212) can provide structural robustness of the dosage form as well as minimal damage to the virosome in the dosage form. The two-stage lyophilization cycle may include a first step of maintaining the frozen units 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 about 12 to 36 hours, about 18 to 30 hours, about 20 to 28 hours, or about 24 hours. Additionally, the two-stage lyophilization cycle may include a second step following the first step. - 50 stage. The second stage may include maintaining the frozen units at about 0°C to -20°C, about -5°C to about -15°C, about -8°C to about -12°C, or about -10°C for about 6 to 30 hours, about 12 to 24 hours, about 14 to 22 hours, or about 18 hours. At the end of the two-stage freeze-drying cycle, the temperature of the freeze dryer may be increased to about room temperature (i.e., about 20 to 25°C or about 23°C). In some embodiments, the two-step freeze-drying process may include pre-cooling the freeze-dryer to a temperature of about -25°C, ramping the freeze-dryer down to -15°C for 2 hours, holding the freeze-dryer at -15°C for 24 hours, ramping the freeze-dryer down to -10°C for 2 hours, holding at -10°C for 18 hours, ramping to 0°C for 15 minutes, and ramping to 23°C for 15 minutes, in that order. The lyophilized dosage forms may be removed from the lyophilizer and inspected for any defects (quality inspection, as described below) in step 213. The dosage forms may then be placed in a storage cabinet at atmospheric humidity less than about 35% RH before pc Lcnn / Lznz / E / YiAi - 51 after the dosage forms are sealed in their preformed molds. The sealing process (step 214) may place a thin film over the preformed molds and provide ampoules of lyophilized dosage forms. Water in lyophilized dosage forms may be removed by sublimation during lyophilization. Accordingly, the remainder of the virosome concentrate in the lyophilized dosage form excluding cryo-lyoprotectants (i.e., the virosomes, antigens, adjuvants, and buffer system remaining in the lyophilized virosome concentrate) may be about 1 to 5% by weight, about 2 to 4% by weight, about 2.5 to 3.5% by weight, about 2.6 to 3.4% by weight, about 2.7 to 3.3% by weight, about 2.8 to 3.2% by weight, about 2.9 to 3.1% by weight, or about 3 to 3.1% by weight of the dosage form. As set forth above, one or more of the target molecules are included in the dosage forms described herein in an amount sufficient to render them immunogenic when supplied in a dosage form. A person skilled in the art will readily determine the immunogenic amount for a given disease or infection based on, pc Lcnn / Lznz / E / YiAi - 52, among other factors, the route of administration, the age, and the weight of the patient to whom the dosage form will be administered. In some embodiments, the solid dosage form may contain from 0.01 to 250 pg of each target molecule (e.g., HIV P1 peptide and / or rgp41). In some embodiments, at least one of the cryo-lyoprotectants in the lyophilized dosage form may be about 5 to 20% by weight, about 8 to 18% by weight, about 10 to 15% by weight, about 11 to 15% by weight, or about 12 to 15% by weight of the dosage form. In some embodiments, the at least one of the cryo-lyoprotectants in the lyophilized dosage form may be about 1 to 5% by weight, about 1 to 4% by weight, or about 2 to 4% by weight of the dosage form. In some embodiments, the amount of matrix former in the dosage form may be about 20 to 50% by weight, about 25 to 45% by weight, about 25 to 40% by weight, about 30 to 40% by weight, about 33 to 37% by weight, or about 35 to 37% by weight. In some embodiments, the amount of structure former in the dosage form may be about 27 to 65% by weight, - 53 about 27 to 60% by weight, about 40 to 55% by weight, or about 45 to 50% by weight. In some embodiments, the remainder of the pH modifier in the lyophilized dosage (e.g., sodium hydroxide) may be about 0.01 to 0.08% by weight. The dosage forms of the present disclosure are dissolving dosage forms and accordingly have the distinct advantage of a faster disintegration time. The route of administration may be oral, vaginal, or nasal, although oral (i.e., sublingual and / or buccal) is preferred. Once placed in the oral cavity and in contact with saliva, a dosage form can disintegrate within a period of about 1 to about 180 seconds, about 1 to about 120 seconds, about 1 to about 60 seconds, preferably within a period of about 1 to about 30 seconds, more preferably within about 1 to about 10 seconds, and most preferably in less than about 5 seconds. Formulations, test methods and examples For the examples, a liquid virosome concentrate prepared from influenza viruses was used. The virosomes contain influenza HA, as well as PC antigens Lcnn / Lznz / E / YiAi. - 54 and added adjuvants. Two virosome preparations were prepared, each containing a single antigen derived from an HIV envelope glycoprotein. The liquid virosome concentrate is a mixture of the two virosome preparations. The two HIV gp41-derived antigens are the P1 peptide, representing the last 35 residues of the C-terminal ectodomain, and the truncated rgp41, lacking group I and the last 21 residues of the C-terminal ectodomain. The adjuvant 3M-052 is either present or absent in either virosome preparation. Virosomes are suspended in HEPES-sodium chloride buffer containing 142.5 mM sodium chloride and 50 mM HEPES at pH 7.4. Additionally, trehalose (as a cryo-lyoprotectant) was tested in the range of 0 to 10% w / w of the liquid virosome concentrate.The following Table 1 summarizes the target compositions of the liquid HIV-1 virosome concentrate used for some of our experiments, during which a 500 mg aliquot (dosage fill weight) of the aqueous virosome formulation was dosed into preformed ampoule-type containers, followed by freezing and lyophilization. Dosage fill weights can vary from 150 mg to 1000 mg, and the compositions of the liquid HIV-1 virosome concentrate can be adjusted to meet the required target dose. Note that the following target ranges may depend on the purpose and subsequent use, e.g., - 55 for animal studies or for human studies. Thus, the other target concentrations may be useful for other purposes. cc Lonn / Lznz / E / YiAi TABLE 1 Example of lipid-based particle concentrates for a 500 mg dosage fill weight Zydis™ virosome formulation Exemplary target molecules and excipients in lipid-based particle concentrates HIV, MYM-V202 vaccine (4x concentrate to load 25% liquid virosome in Zydis™ virosome formulation) - target molecule (PL antigen): 50-450 pg / ml - target molecule (rgp41 antigen): 50-400 pg / ml - HA excipient: 10-160 pg / ml - adjuvant (e.g., 3M-052): 8-140 pg / ml - phospholipids: 0.5 to 5 mg / ml - sodium chloride: 10 to 150 mM - HEPES 10 to 50 mM - trehalose: 4-10% w / w - pH 6.5 to 8.0 HIV vaccine, MYM-V202 (2x concentrate to 50% liquid virosome loaded in Zydis™ virosome formulation) - target molecule (P1 antigen): 22-225 pg / ml - target molecule (rgp41 antigen): 25-200 pg / ml - HA excipient: 5-80 pg / ml - adjuvant (e.g., 3M-052): 4-70 pg / ml - phospholipids: 0.5 to 5 mg / ml - sodium chloride: 50 to 150 mM - HEPES 10 to 50 mM - trehalose: 2-5% w / w - pH 6.5 to 8.0 placebo vaccine VP02 (concentrated at 4x to load 25% liquid virosome in Zydis™ final virosome formulation) - excipient HA: 10-160 μg / ml - adjuvant (e.g., 3M-052): 8-140 pg / ml - phospholipids: 0.5 to 5 mg / ml - sodium chloride: 50 to 150 mM - HEPES 10 to 50 mM - trehalose: 4-10% w / w - pH 6.5 to 8.0. - 56 In some of our experiments, the target doses for HA and HIV-1 antigens per tablet were 20 µg of HA, 25 µg of P1, and 50 µg of rgp41). To obtain these doses, high payloads of liquid virosome concentrates in combination with high wet fill dose weight were required. Table 2 shows the various combinations of liquid virosome concentrate loading (varying from 25 to 50% w / w) with wet fill dosage weight (varying from 250 to 1000 mg). The wet fill of wet dosage is the aliquot amount of virosome formulation dosed per dose prior to lyophilization. cc Lonn / Lznz / E / YiAi TABLE 2 HA Liquid Virosome Concentrate and Antigen Content Liquid Virosome Concentrate % Loaded in Base Matrix Formulation Dosage Fill Weight Target Virosome Vaccine Dose per Tablet HA / P1 / rgp41 HA, 80 µg / ml (adjuvanted 3M052) P1, 100 pg / ml rgp41,200 pg / ml 25%, w / w 1000 mg 20 pg / 25 pg / 50 pg 50%, w / w 500 mg 20 pg / 25 pg / 50 pg HA, 160 pg / ml (adjuvanted 3M-052) P1, 200 pg / ml rgp41,400 pg / ml 25%, w / w 500 mg 20 pg / 25 pg / 50 pg 50%, w / w 250 mg 20 pg / 25 pg / 50 pg - 57 A 25% liquid filler can be added to the virosome concentrate's base matrix formulation, which can be dosed in a range of Ha and antigen contents evaluated. Table 3 shows the dosage weight of the 500 mg fill dose. TABLE 3 component target composition in liquid concentrate composition supplied for evaluation composition in a 25% target virosome loaded formulation with antigens and adjuvants HA: 10-160 pg / 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 The presence of buffer in the aqueous composition may lower the freezing point, and therefore make it difficult to freeze the formulation composition and maintain its frozen state. In addition, collapse of the tablet matrix structure may also occur during lyophilization since buffer salts may inhibit crystallization of mannitol during the reassociation process. Crystallization of mannitol is required to provide strength and structure to the tablet matrix to prevent structural collapse. However, crystallization of mannitol may damage the tablet matrix. - 58 virosome particles during freezing, reassociation, and lyophilization. A lower loading percentage of the liquid virosome concentrate (e.g., 25% loading) tends to reduce this impact. A combination of a lower loading percentage of the liquid virosome concentrate and a higher dosage fill weight can also be considered. A high wet-fill dosage weight of the virosome formulation combined with a formulation composition with a high buffer content may also be more difficult to freeze and maintain structure to minimize collapse during lyophilization. However, larger tablets (e.g., 1000 mg dosage fill weight) can cover larger surface areas and potentially improve virosome passage. When a high wet-fill dosage weight is required, a formulation composition with a low buffer content is preferred. Table 4 (formulation 1) summarizes the aqueous compositions of the virosome formulation and the corresponding composition of the virosome tablets evaluated herein. The following formulations and tablets were made according to the steps shown and described in Figure 2. In addition, the pc Lcnn / Lznz / E / YiAi formulations - 59 frozen cereals were subjected to a two-stage freeze-drying process under a vacuum of 500 mbar: (a) pre-cooling to 25°C; (b) ramp-up for 2 hours at -15°C; (c) holding @ -15°C for 24 hours; (d) ramp-up to -10°C for 2 hours; (e) holding @ -10°C for 18 hours; (f) ramp-up for 15 minutes at 0°C; (g) ramp-up for 15 minutes at 23°C. The vacuum was then released and the freeze-dryer returned to atmospheric pressure. The concentration of each ingredient (%, w / w) is the amount prior to removal of the water present in the liquid virosome concentrate, sodium hydroxide solution, and water used to prepare for sublimation during lyophilization. In addition, the following table includes quantities in which each ingredient may vary. TABLE 4 cc Lonn / Lznz / E / YiAi Ingredient % w / w Range Tested % w / w Formulation 1 / 2 Amount, in mg for 500 mg wet dosage fill weight Amount, in mg, post-lyophilized Liquid Virosome Concentrate 25-50% 25% 125 mg ~ 2.5-2.6 mg* Trehalose 0.5%-4.5% 2%** (net) 10 mg 10 mg* (net) 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) qs pH 7.4 qs pH 7.4 ~ 1.3 mg (qs pH 7.4) ~ 0.04 mg (water removed by sublimation) Water (for preparing aqueous matrix mixture) qs 100% qs 100% ~ 293.7 mg water removed Total weight of lyophilized vaccine tablet N / W / W / A ~ 82.64 mg pCLQnn / Lznz / E / Yi * As stated above, the liquid virosome concentrate includes virosomes suspended in a buffer system of 142.5 mM NaCl and 50 mM HEPES with 5% w / w trehalose. For a 25% loading of the virosome formulation in a 500 mg dosage fill weight (i.e., 125 mg of the liquid virosome concentrate), the estimated dry matter with sublimated water of the virosomes, antigens, adjuvants, NaCl, and HEPES (excluding trehalose) is approximately 2.5 to 2.6 mg. **This is the net amount of trehalose in the virosome formulation and dosage form. Thus, this amount includes trehalose from the liquid virosome concentrate, as well as added trehalose from the base matrix formulation. Properties of lyophilized dosage forms Lyophilized dosage forms may be - 61 stable in physical attributes and virosome quality (particle size characteristics and antigen content) and can be stored independently of cold chain storage conditions. Furthermore, lyophilized dosage forms can make virosomes resistant to accidental exposure to subzero storage conditions during storage or transport. Physical characteristics of the dosage form: Lyophilized tablets with acceptable physical characteristics were produced. The physical attributes of the tablets include appearance, dispersion characteristics, disintegration times, and moisture content. The appearance of 10 lyophilized tablets was tested. Each tablet was separated from its blister pack. A visual inspection of each tablet was performed for surface defects on the tablet surface and base. The criterion is that the lyophilized tablet must have a good appearance with no surface defects. In addition, the tablets must be strong enough to be separated from the blister pack without breaking. Dispersion characteristics (in vitro test): A minimum of 5 tablets were tested. First, Lcnn / Lznz / E / YiAi were - 62 Prepare a beaker containing approximately 200 ml of purified water at 20°C + 0.5°C. Each tablet is then removed from the blister pack and placed, base down, on the surface of the water. The time is recorded for the time at which each tablet becomes completely wetted or dissociates. The wetting time is recorded for the complete wetting of the unit. Wetting of each tablet may occur in patches, which eventually fuse together so that the entire unit is wetted. The dispersion test is considered complete when the center of the unit is a wetted mass. Thus, the wetting time is recorded from when the center of the unit has been wetted through to the thickest part of the unit. The wetting time is recorded for each of the five tablets. The maximum time for each test is 60 seconds.Longer times than this may be written simply as greater than 60 seconds. Dissociation = the time it takes for the unit to separate. This time can be taken when the unit begins to split into pieces at the edges. The dissociation time is recorded for each of the five tablets. The maximum time for each test is 60 seconds. Longer times than this may be written as greater than 60 seconds. Occasionally, the unit does not moisten completely. - 63 complete nor completely dissociated within this time limit. Sometimes, the unit may have hard lumps in it; other times, it may not have been wetted on the surface at all. Additionally, the entire unit may be covered in a hard coating. A note of this is made in the description if it occurs, mentioning hard lumps or the remaining coating, as appropriate. The formation of hard lumps and / or coating can be an indication of microstructural collapse during freeze-drying. Figures 3A through 3C show a simplified representation of the three possible undispersed states, with a side view and a top view of the units as they would appear in water. The photos in Figures 3A-3C show some representative units for the same categories.The criterion for the dispersion characteristics test is whether the 5 tablets completely moisten and / or dissociate into a palpable mass without the presence of hard lumps and coatings in 60 seconds or less. In some embodiments, the dosage forms described herein can completely moisten and / or dissociate into a palpable mass without the presence of hard lumps and / or coatings in 60 seconds or less. Disintegration time (in vitro test): Six tablets are used for this test. Six beakers are filled with purified water and placed in a container. - 64 a controlled mania bath at 37 ° C + 0.5 ° C. Each tablet is then removed from the blister pack. A wire clasp is carefully placed over each of the six tablets. It is ensured that the clasp holds the tablet without causing damage. After the test is performed as described in the Pharmacopoeia. An example of this test is the United States Pharmacopoeia disintegration test (701). For each tablet, the maximum disintegration time is recorded. The criterion for the disintegration time test is that the disintegration time should be no greater than 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. Moisture Content: A Methron 831 Karl Fischer Coulometer with a 744 Oven Sample Processor (Metrohm, Herisau, Switzerland) is used to determine the water content of the tablet. A tablet is accurately weighed and placed in a glass jar. The jar is tightly capped to ensure no moisture ingress. The sample jar is then placed in the 744 Oven Sample Processor, and the temperature is set to 102°C. The evaporated moisture is titrated using a Hydranal Coulometric AG oven reagent to quantify the amount of water released. The test is performed by pc Lcnn / Lznz / E / YiAi - 65 triplicate and the average is recorded. The criterion for moisture content is whether the lyophilized dosage form has a moisture content of less than about 8%, preferably less than 6%, and more preferably less than 4%. In some embodiments, the dosage forms described herein may have a moisture content of less than about 8%, preferably less than 6%, and more preferably less than 4%. Characteristics of the virosome Lyophilized dosage forms containing virosomes can be sufficiently preserved in terms of the proportion of intact virosomes from the initial liquid virosome population, their particle size, and contained surface antigens required for the immunogenicity and immunological benefits of virosomes. The virosome structure can be destroyed by the lyophilization process used for the production of the dosage forms. In this way, the virosome particulate characteristics are established from a solution of a reconstituted lyophilized tablet. Virosomes can exist as intact individual particles and as assemblies of different sizes; all are immunogenic, but have different antigen / epitope exposure and harbor a specific capacity. Lcnn / Lznz / E / YiAi - 66 different for crossing the sublingual barrier. Virosomes can be characterized in terms of: (a) mean particle size, and (b) the proportion of virosome preservation (intact virosomes). Virosome particle size and particle concentration (virosome particle counts per ml) determined using an NTA technique: Nanoparticle Tracking Analysis (NTA; using a NanoSight NS300 instrument) is a sensitive method that can identify different particle sizes in the 30–1000 nm range present in a solution. Particles in a sample solution can be individually tracked and simultaneously analyzed by direct observation. Nanoparticles move under Brownian motion due to the random motion of the surrounding water molecules. Small particles move faster than larger particles. The Brownian motion of each particle is followed in real time by video and NTA Brownian motion analysis is used to determine particle size.The diffusion coefficient can be calculated by tracking the motion of each particle, and then, by applying the Stokes-Einstein equation, the particle size can be calculated. This particle-by-particle methodology produces high-resolution results for the pc Lcnn / Lznz / E / YiAi distribution. - 67 particle size and concentration (i.e., the number of particles in a known volume of liquid). The target values for optimal detection for the detection instrument are summarized in the following table: 5 cc Lonn / Lznz / E / YiAi TABLE 5 Quality Indicator Objective Reasoning PPF (particles per frame) 50-100 Particle count affects the affordable resolution and statistical accuracy of generated profiles Number of valid tracks > 5000 Statistical accuracy of generated profiles for polydisperse samples S :N (signal to noise ratio) high Improved detection of small or minuscule particles Low SD (standard deviation) Fewer variable data provide greater confidence in results Good profile appearance: minimal non-Gaussian ridges on peaks Less noise present in the data, clear particle size populations The test sample may be in liquid form. For this work, the virosome vaccine is supplied in liquid form. For test samples from the - 68 vaccine formulations described herein, the mixture is supplied in liquid form, a frozen unit is reheated to liquid form prior to testing, and the lyophilized dosage form is reconstituted with buffer and water to form a liquid. The test sample should not be overly concentrated. A liquid test sample may be further diluted with HN buffer as appropriate to optimize detection. The dilution buffer may be of high purity and may be filtered through at least a 0.22 µm filter prior to use. The experimental parameters established for the NTA analysis are summarized in Table 6 below. cc Lonn / Lznz / E / YiAi TABLE 6 parameter adjustment sample dilution depends on the content (can vary from 1:100 to 1:8000, depending on the concentration of virosome beads in the diluted test samples) number of captures 5 capture duration 60 seconds temperature control 25°C viscosity 0.9 Cp Samples are measured by NTA, as is known in the art. In some embodiments, the range of virosome particles including fragments and clusters described herein may be about 50 to - 69,500 nm. In some embodiments, the virosome particles that are intact virosomes may be in the range of about 70 to 400 nm or about 70 to 200 nm (major peak). In some embodiments, the mean diameter of the virosome particles may be about 70 to 200 nm, about 100 to 175 nm, about 125 to 155 nm. For detection purposes, the concentration of particles of the virosome population in the sample may be at least 1010 counts / ml. Determining virosome preservation ratios using flow cytometry: Flow cytometry is used for this determination. First, the initial liquid virosome particles are labeled (reference sample representing 100% of the starting material) by inserting a lipophilic dye DiI (long-chain diallylcarbocyanine) into the lipid bilayer of the virosomes (labeled with DiI as a non-measurable effect on particle size). Second, the lyophilized tablet is reconstituted and the virosome in the lyophilized tablet is labeled with DiI (the test sample). Samples are analyzed using a flow cytometer and AMNIS imager and the events between the reference (liquid virosomes before lyophilization) and the test samples (lyophilized virosomes) are compared to estimate the virosome preservation ratios pc Lcnn / Lznz / E / YiAi - 70 post-lyophilization in terms of: (a) percent recovery of virosomes, and (b) percentages of virosome clusters. In some embodiments, the percent recovery of the virosome as single particles may be about 20 to 50%, about 30 to 50%, or about 40 to 50% of the starting material. In some embodiments, the percentage of virosome clusters (doublets, triplets, or higher number forms) may be about less than 505, about 25%, about 10%, or about 5%. Hemagglutinin (HA) content in virosome, HIV-1 P1 antigen, HIV-1 antigens and adjuvant Influenza HA, HIV-1 antigens, and virosome adjuvant contents can be quantified by various methods. These are listed in Table 7 below. cc Lonn / Lznz / E / YiAi TABLE 7 Content to be quantified Influenza HA methods Western blot assay SRID ELISA assay HPLC assay HIV-1 antigen Western blot assay HPLC ELISA assay adjuvant UV spectroscopy assay HPLC assay Immunoblot assay method for HA, - 71 P1 and rg41: In this assay, the formulation (liquid virosome concentrates or reconstituted lyophilized dosage forms containing virosomes) can be adsorbed onto a nitrocellulose membrane, and the antigens are maintained in their native state due to the absence of heating, denaturation, or reducing agents. The assay detects all antigens accessible to the specific antibodies and is indicative of major antigen degradation or denaturation that destroys or blocks access to specific epitopes. It also indicates whether specific excipients can prevent antibody binding to its antigen. To prepare the lyophilized dosage forms for analysis (test samples), each tablet is dissolved in 0.5 ml of water (i.e., the lyophilized tablet is reconstituted back to the virosome formulation composition prior to lyophilization).To prepare a positive control, a sample of liquid virosome concentrate is diluted with ultrapure water (a 4-fold dilution). The liquid virosome concentrate is ideally from the same lot as that used in the preparation of the dosage form test sample. Serial two-fold dilutions of all test samples and the positive control are prepared. Additional positive controls such as purified HA and rgp41, and synthetic P1, can also be used. A 1.5 μA volume of pc Lcnn / Lznz / E / YiAi. - 72 Each sample and the positive control are spotted onto a dried nitrocellulose membrane (with increasing dilution, from left to right - undiluted, 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, 1 / 128). The dried membrane is blocked with 1% (w / v) casein and incubated with a specific antibody solution - the human monoclonal antibody (mab) 2F5, specific for the HIV-1 P1 antigen, and rabbit anti-rgp41 serum for the HIV-1 rgp41 antigen. After incubation, the nitrocellulose membrane is washed. Specific antibodies bound to fluorescently labeled secondary antibodies (anti-human or anti-rabbit) are then detected using a far-red fluorescence scanner that allows simultaneous detection of two different fluorescent labels, at 700 nm and 800 nm.The raw fluorescence signal for each sample point (from lowest to highest) is compared to the respective dilution of the positive control point (as a % of the positive control). An arithmetic average of the sample percentage is calculated. UV spectroscopy assay for 3M-052: The 3M-052 adjuvant molecule has several UV absorption peaks. Some of these peaks overlap with the absorption of lipids and proteins. The determination is performed by UV spectroscopy at 320 nm. HPLC assay for P1 and rgp41: PC antigens Lcnn / Lznz / E / YiAi - HIV P1 and rgp41 are separated by reverse-phase high-pressure liquid chromatography on a C18 column using a water-to-acetonitrile gradient. Determination is performed by UV spectroscopy at 280 nm, and peak areas are quantified using the HPLC system software. Antibody titer endpoint ELISA: Maxisorp 96-well plate (Nunc-flat bottom) and Polysorp plates are coated, respectively, 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 are washed 3 times with PBS containing 0.05% v / v Tween 20 (PBST), then 1% BSA blocking solution prepared in PBST is added to each well and incubated for 2 hours at room temperature (rt). Plates are washed three times with PBST before adding 0.1 ml per well of pre-immune serum diluted 1 / 1000 or serial dilutions of immune serum (from 1 / 1000 to 1 / 64,000) prepared in 0.1% BSA in PBST and incubated for 2 hours at rt. Plates are washed three times with PBST and incubated for 2 hours at rt with goat anti-rat IgG-HRP antibody diluted 1:4000 in 0.1% BSA in PBST. Plates are washed again, before adding 0.1 ml of the colorimetric substrate o-phenylenediamine (OPD) is added and the reaction is stopped with 2M H2SO4, followed by plate reading at 492 nm. Example 1: Using high mannitol level (8%, w / w) in a pc manner Lcnn / Lznz / E / YiAi - 74 combined with a low-temperature freeze-drying cycle to reduce microstructural collapse during freeze-drying without damaging the integrity of the virosome Mannitol is used in dosage forms to increase structural robustness. Due to the presence of high levels of buffers and the addition of trehalose to protect the virosome particles, mannitol at a typical concentration of 4.5% w / w is unable to provide sufficient structural support during freeze-drying. Therefore, microstructural collapse occurs. However, the applicants discovered that using a combination of higher amounts of mannitol and low-temperature lyophilization cycles can produce a structurally more robust lyophilized tablet. This example shows data comparing a formulation containing 4.5% w / w mannitol with one containing 8% w / w mannitol. In both formulations, a 25% w / w loading of liquid virosome concentrates MYM-201 / batch 160125-1 (supplied by Mymetics containing 70 to 80 pg / ml HA, 40 to 50 qg / ml P1, 70 to 80 qg / ml rgp41 (A / Brisbane / 59 / 2007 (H1N1) in HN buffer, pH 7.4 (50 mM HEPES, 142.5 mM NaCl) is used in the virosome formulation. The amounts of fish gelatin and pure trehalose are maintained at 6% w / w and 2% w / w of the virosome formulation, respectively. pc Lcnn / Lznz / E / YiAi - 75 As illustrated in Figure 2, formulations are dosed with a 500 mg dosage fill weight aliquot at 15°C into aluminum tray blister receptacles. The trays containing the dosed aqueous vaccine mixture are frozen by passing the aluminum trays through a freezing chamber set at -70°C for 3 minutes 15 seconds. The aluminum trays containing the frozen products are collected and placed in a freezer at < -15°C and annealed for 6 hours, remaining frozen prior to lyophilization. A 2-step FD cycle using -15°C for 24 hours followed by -10°C for 18 hours thereafter is used. Blister packs of the lyophilized tablets are sealed in sachets and stored at ambient conditions. The physical characterization of the lyophilized tablets (appearance and dispersion time) was determined according to the tests explained above. In addition, the virosome particle size in the lyophilized tablets was characterized according to the tests indicated above. The results are summarized pc Lcnn / Lznz / E / YiAi in Table 8 below. cc Lonn / Lznz / E / YiAi TABLE 8 mannitol, 4.5% (formulation 1) mannitol 8% (formulation 2) appearance: good (n = 182) appearance: good (n = 184) dispersion time (n = 8) dispersion time (n = 8) • wetting: > 60 s (2 units-coating, 5 units-hard lumps) • dissociation: > 60 s (not • wetting: < 28 s • dissociation: < 36 s completely dispersed) NTA analysis • lyophilized tablet: 140 nm NTA analysis (main peak) • lyophilized tablet: 136 nm • % virosome particle (main peak) • % virosome particle (< 200 nm): > 50% (< 200 nm): > 50% All tablets look good. The 4.5% w / w mannitol formulation has poor dispersion behavior with coating and / or hard lumps observed in the tablets due to microstructural collapse. Increasing the formulation to 8% w / w, which promotes mannitol crystallization, improves the structure of the lyophilized tablet. The 8% w / w mannitol formulation has good dispersion behavior and provides a soft, palpable mass. In combination with a low-temperature lyophilization cycle, the mannitol particle - The virosome in the lyophilized tablet is not altered. The virosome particle size in the lyophilized tablets is generally comparable between the formulations. Example 2: Stability data of lyophilized vaccine dosage forms (formulation 1) stored for 3 months under ICH conditions Liquid virosome concentrates are generally stable at 2-8°C, but liquid HIV virosome vaccine has a limited shelf life of a few months due to extensive chemical modifications of the antigens, while aggregation does not occur. With the solid vaccine form with low moisture content, these modifications are expected to slow down and become minimal over time, extending the shelf life to >1 year. The stability of the virosome vaccine in the form of a lyophilized tablet formulation 1 is illustrated in this example. The liquid virosome concentrate, lot MYM V202, lot 170130-1 supplied by Mymetics which is constituted of approximately 100 µg / ml of HIV-1 P1, 230 µg / ml of HIV-1 rgp41, 130 µg / ml of HA (A / Brisbane / 59 / 2007 (H1N1), 65 µg / ml of 3M-052 adjuvant in HN buffer, pH 7.4 (50 mM HEPES, 142 mM NaCl).5 mM) with 7% w / w trehalose is supplied for the production of lyophilized vaccine tablets (batch Z33787A101). pc Lcnn / Lznz / E / YiAi - 78 To prepare the vaccine tablet, a liquid virosome formulation mixture is first prepared. It contains 25% w / w liquid virosome concretizer, MYM V202, lot 170130-1, 6% w / w fish gelatin, 4.5% w / w mannitol, 2% w / w (net) trehalose, sodium hydroxide solution (as sufficient) at pH 7.4 and purified water (as sufficient) to 100% w / w. The liquid virosome formulation mixture is dosed with a 500 mg dosage fill weight aliquot at 15°C into aluminum tray blister receptacles. The trays containing the dosed aqueous vaccine mixture are frozen by passing the trays through a freezing chamber set at -70°C for 3 minutes 15 seconds. The aluminum trays containing the frozen products are collected and placed in a freezer at < -15°C. A 2-step FU cycle using -15°C for 24 hours followed by -10°C for 18 hours is then used. Blister packs of lyophilized tablets are sealed in sachets and stored for 3 months at 5°C, 25°C / 60% relative humidity, and 40°C / 75% relative humidity. The results of the initial test and the 3-month test are summarized in Table 9 below. pc Lcnn / Lznz / E / YiAi TABLE 9 Initial test 5°C / 3 months 25°C @ 60% RH / 3 months 40°C @ 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 particle (< 200 nm): > 50% > 50% > 50% > 50% Stability data show that tablet appearance is good and consistent between batches. Moisture content is 5 to 6% w / w with little difference between different stability conditions over the 3-month storage period. Since this formulation contains 4.5% w / w mannitol, there is some microstructural collapse during lyophilization, as indicated by the variability in disintegration time. In terms of virosome particle size, DLS and NTA data show no discernible difference in particle size. - 80 virosome particles were found among tablets stored under the various stability conditions. Initial liquid virosome concentrates contained >50% of the <200 nm particle. The results show that the <200 nm virosome from the reconstituted unit was essentially preserved in a similar order of magnitude. Using semi-quantitative immunoblot analysis, HIV-1 antigens P1 and rgp41 were monitored for degradation over 3 months. All samples were pre-diluted twofold. The P1-specific human mAb 2F5 and rabbit anti-rgp41 serum were used for this purpose. The liquid virosome concentrate, lot MYM-V202, lot 170103-1, which was used to produce the vaccine batch, was diluted 8-fold and used as a positive control. Figure 4 shows a photograph of the immunoblot analysis. For sample Z33787A101, tablets stored at 5°C showed no or only a minimal decrease in rgp41 antigen signal after 1 month and 3 months of storage compared to the initial sample (t = 0). Similarly, there was only a minimal difference in signal intensity for the unit stored at 25°C for the rgp41 antigen and a slight decrease for the P1 antigen.The difference for units at 40°C is more pronounced, although it may still be within the test accuracy of 10 to 20%. pc Lcnn / Lznz / E / YiAi - 81 The quantitative evaluation of the fluorescence data for rgp41 and P1 is presented in Tables 10 and 11, respectively. The top part of each table shows the raw fluorescence signal for each spot (from lowest to highest dilution) for the indicated stability sample. The bottom part of each table shows the value for each sample spot compared to the respective dilution of the positive control spot pc Lcnn / Lznz / E / YiAi (as a % of the positive control). The bottom line shows the arithmetic average of all sample percentages. Various outliers were excluded from the average calculation. TABLE 10 ΜΪΜ -V2O2 Z33787AW1 to i 1 month (S'C 25 407^9x4 ú 446777x1 3403051 AI 76710 .5596? 74 3194193 32tx$i,S¿ 2 λ 2635034 2359183 3ÍÜS839 2159053 24SX939 1704534. 13S2173 3 / 09154 15 3342 ? 3448772 1556263 3O77&33 33 39732 308868^ 7S3S5Q 3056038 &75S17 7354.97 976990 52 3 83 9 S7SO9S 0321671 53 7094 S14 4S5&Í5 ASO 586 453385 4X7028 % of control 1.3 7, 119.5 101.3 133 ? 110.6 % of control 1P7.9 113.0 110.0 $ 79.6 % of control WS sa.a 47.6 $7.4 % of control 9a 69.0 irm.i 'W £ 'as % of control se, iM 119.9 95 & W9.9 119. 7 904 % of control 99.11 69 4 103.8 5^5. q 85.0 65.2 % of control 7&3| 56 í 7as 504 5S6 72^ 46.4 % of control 76.3 7518 67.3 72.2 S6L-S: 6^ % (middle) S3-2 97.3 96.0 74.0 r sao 6S5 TABLE 11 pc Lcnn / Lznz / E / YiAi >1 WM-V2S2 _________________J 1 month 3 months 5C Ζ5Χ 40X S'C 4GX 27S4S2S 14619SS 1245932 1471536 1233537 51547E 1351413 1572627 í xscwts- J6Qp470 723609 1255559 1358350 34 / 105 470287 5083 07 : 34535:5 997GSS S17270Í £1319.7 3S74S? j$90484 S5656O §79843; 3 ? <JG3 '3$ 882260 i 249520 317945 387210 S4075J. 6731<9 324733 339128 2S10551 149361 142741 SÍ1S53Í 342819 14®881 159835 11S0S8i 8616’7 28407 1 JOS® 6 $,$46 137920 ÍSSCtt 163997 19379 324010 3443® X> J · ·,Λ Í2 ¿ 49784 i % of control S? $ ^-1· 53.2 44.« 54.9 -43;® % of control 132 59.9 1>2.2 110.7 4573- 38.3 42.v 1 % of control 11 7.S S8.S 73.G: 118.1 73.2 41.7 7283 i % of control % control iñS.b 12 107.0 105.8 77 JQ i % control 143.3 73.1 2 67.2 85.0 9>S >.2. : % íptomediol S3S.X left. s 147.6' leía 112.4 36:.0 7S.YES For the Z33787A101 vaccine tablet, tablets stored at 5°C showed not only a minimal decrease in signal for rgp41 and P1 antigens after 1 month and 3 months of storage compared to the initial sample (t = 0). Similarly, there was only a minimal difference in signal intensity for the unit stored at 25°C for the rgp41 antigen, and a slight decrease for the P1 antigen. The difference for the 40°C units was more pronounced for both P1 and rgp41 antigens, although this is likely within the precision of the assay, especially since variations were observed within the serial dilution samples again. 3M-052 was determined by UV spectroscopy at 320 nm for all vaccine tablets stored at different temperatures for more than 3 months. The values are presented in Table 12 below. cc Lonn / Lznz / E / YiAi TABLE 12 Time point Storage condition DO (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 No discernible differences could be observed between these samples, and therefore, 3M-052 concentrations were considered to remain stable in all samples within the precision of this assay. Overall, HA, Pl, rpg41, and 3M-052 were stable in the lyophilized tablets stored under different storage conditions, with minor variations over time, as shown in Table 13 below. TABLE 13 Antigen and adjuvant detection during stability study Virosome concentrates lyophilized tablets Changes in rgp41 in 3 months at 5°C (unexpected degradation = reference) Not significant Not significant Changes in rgp41 in 3 months at 5°C (degradation) compared to 5°C Not performed Minimal decrease Changes in rgp41 in 3 months at 37-40°C (degradation) compared to 5°C Not performed Minimal decrease changes in P1 over 3 months at 5°C (unexpected degradation = reference) not significant not significant changes in P1 over 3 months at 25°C (degradation) compared to 5°C not realized minimal decrease changes in P1 over 3 months at 37-40°C (degradation) compared to 5°C not realized minimal decrease changes in 3M-052 over 3 months at 5°C (unexpected degradation = reference) not significant not significant changes in 3M-052 over 3 months at 37-40°C (degradation) compared to 5°C not significant not significant ccLQnn / Lznz / E / Yi Example 3: Stability data of lyophilized vaccine dosage forms (formulation 2) stored under ICH conditions This example illustrates the stability of the virosome vaccine in the form of a lyophilized tablet for formulation 2. The liquid virosome concentrate, lot MYM V202, lot 17MYM002 / F17255 supplied by Mymetics consists of approximately 121 µg / ml of HIV-1 P1, 67 µg / ml of HIV-1 rgp41, 41 µg / ml of HA (A / Brisbane / 59 / 2007 (H1N1), 39 µg / ml of 3M-052 adjuvant in HN buffer, pH 7.4 (50 mM HEPES, 142.5 mM NaCl) with 7% w / w trehalose which is used to manufacture the lyophilized vaccine tablets (lot MYM-212, lot 1690747). To prepare the vaccine tablet, a - 85 Liquid virosome formulation mix first. It contained 25% w / w liquid virosome concentrate lot MYM V202 batch 17MYM002 / F17255, 6% w / w fish gelatin, 8% w / w mannitol, 2% w / w (net) trehalose, sodium hydroxide solution (as sufficient) to pH 4, and purified water (as sufficient) to 100%, w / w. The liquid virosome formulation mixture was dosed with a 500 mg dosage fill weight aliquot at 15°C into aluminum tray blister receptacles. The trays containing the dosed aqueous vaccine mixture were frozen by passing the trays through a freezing chamber set at -70°C for 3 minutes 15 seconds. The aluminum trays containing the frozen products were collected and placed in a freezer at < -15°C. A 2-step FD cycle using -15°C for 24 hours followed by -10°C for 18 hours was subsequently used. Blister-type packages of lyophilized tablets were sealed in sachets and stored for 3 months at 5°C, 25°C / 60% relative humidity, and 40°C / 75% relative humidity. The results from the initial test and a 6-month trial are summarized in Table 14 below. pc Lcnn / Lznz / E / YiAi pc Lcnn / Lznz / E / YiAi TABLE 14 Test Appearance Disintegration Time Initial Moisture Content 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% Stability data show that the tablet appearance is good and consistent between batches. Moisture content was 3.6 to 4.0% w / w, with little difference between the different stability conditions over the 6-month storage period. Since this formulation contains 8% w / w mannitol, there was less microstructural collapse during lyophilization, as indicated by the shorter and more consistent disintegration times. - 87 The stability of antigen content (HIV-1 P1 antigen and rgp41) is monitored for degradation over 3 months using HPLC assay). For MYM V202 liquid vaccine (starting material), the P1 content was found to reduce by 4% and 7% when stored at 2 to 8°C (cold storage condition) for 1 month and 3 months, respectively. For rgp41, the content was reduced by 16% and 25% at 1 and 3 months, respectively, under cold storage condition. 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. The results of the antigen contents in the lyophilized tablet upon storage are presented in Table 15 and Table 16 for P1 antigen and rgp41 antigen, respectively. In the lyophilized tablet form, the P1 antigen remains very stable with no decline in content observed after 3 months at 2 to 8°C (cold chain condition) and also remains unchanged during 1 and 3 month storage outside the cold chain condition. For the rpg41 antigen, declines of approximately 5%, 10%, and 20% are observed after 1 month at 2 to 8°C, 25°C, and 40°C, respectively. At 3 month storage, the decline is approximately 13%, 15%, and 19%, respectively. Considering the precision of the pc Lcnn / Lznz / E / YiAi HPLC method, an observed difference in decline concentration of less than 15% is not significant.Furthermore, the observed gradual loss or decline of antigen is related to chemical modifications not due to advanced degradation involving structural cleavage, amino acid loss, or aggregation. Moreover, one or more chemical modifications to a given epitope can potentially alter its recognition or decrease or increase antibody binding to that region, while other regions remain equally well recognized. At T0, P1 and rgp41 antigens harbor a similar migration profile on SDS-PAGE and are still recognized by specific monoclonal antibodies once applied to sublingual tablets, and serum antibodies to P1 and rgp41 still react toward several key epitopes harbored by the P1 and rgp41 peptides. This analysis suggests that, overall, P1 and rgp41 have retained most of their antigenicity and immunogenicity during the manufacturing process (data not shown). pc Lcnn / Lznz / E / YiAi TABLE 15 Time point P1 content (lyophilized tablet) qg / ml qg / unit % reduction / increase Storage at 2 to 8°C (average, 5°C) (cold chain condition) Initial (T0) 24.7 12.35 Not applicable 1 month (TI) 28.6 14.3 +15.8 (no loss) 3 months (T3) 27.6 13.8 +11.7 (no loss) storage at 25°C / 60% RH 1 month (TI) 27.9 13.95 + 13.0 (no loss) 3 months (T3) 27.5 13.75 + 11.13 (no loss) storage at 40°C / 75% RH 1 month (TI) 25.8 12.9 + 4.5 (no loss) 3 months (T3) 24.0 12.0 -2.8 (loss) pCLQnn / Lznz / E / Yi TABLE 16 RGP41 content time point (lyophilized tablet) qg / ml qg / unit % decrease / increase storage at 2 to 8°C (average, 5°C) (cold chain condition) initial (T0) 13.5 6.75 not applicable 1 month (TI) 12.8 6.4 -5.2 3 months (T3) 11.8 5.9 -12.6 storage at 25°C / 60% RH 1 month (TI) 12.1 6.05 -10.4 3 months (T3) 11.5 5.75 -14.8 storage at 40°C / 75% RH 1 month (TI) 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 temperature storage conditions The data in Example 4 show the stability of the physical characteristics of virosome vaccine tablets and virosome particles when stored under sub-zero conditions. A liquid virosome formulation mixture containing 25% w / w loaded liquid virosome concentrate MYM-201 lot 160125-1 - 90 supplied by Mymetics (containing 70 to 80 μg / ml HA, 40 to 50 μg / ml P1, 70 to 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 (net) trehalose was dosed with a dosage fill weight of 500 mg and lyophilized. The formulations were dosed with an aliquot of 500 mg dosage fill weight at 15°C into blister packs of aluminum trays. The trays containing the dosed aqueous vaccine mixture were frozen by passing the aluminum trays through a refrigerated freezer. through a freezing chamber set at -70°C for a duration of 3 minutes 15 seconds. The aluminum trays containing the frozen products were collected and placed in a freezer at a temperature of < -15°C and annealed for 6 hours, keeping them frozen before lyophilization.A 2-stage FD cycle was subsequently used using 15°C for 24 hours followed by -10°C for 18 hours. Blister packs of lyophilized tablets were sealed in sachets and stored in a -15°C freezer for 1 week. A corresponding set of blister packs of tablets were sealed in sachets and stored at ambient conditions for the same duration. These tablets were determined for appearance and dispersion characteristics (pc time Lcnn / Lznz / E / YiAi. - 91 wetting and dissociation time) and virosome particle size distribution as described according to the above test methods. All units were found to have a good appearance after storage under both conditions. The data showed that storage at sub-zero temperatures had little effect on tablet dispersion characteristics (wetting and dissociation times) (Table 17) and virosome particle size distribution (Table 18). TABLE 17 condition wetting time (sec) dissociation time (sec) ambient storage < 31 < 37 sub-zero storage < 32 < 35 TABLE 18 NTA condition: main peak particle size (nm) NTA: % virosome particle (< 200 nm) 1 week ambient post-digestion 130 59.9 1 week sub-zero temperature, post-digestion 132 60.6 - 92 Example 5: Flow cytometric determination of virosome particles from reconstituted lyophilized tablets Estimates of virosome proportions preserved in lyophilized tablet samples from the development process are summarized below. The values provided in Table 19 are derived from AMNIS flow cytometry data using event measurements in the focus area corresponding to the virosome gates. pc Lcnn / Lznz / E / YiAi TABLE 19 Vaccine formulation percentage of virosome recovered since end of production percentage of clumps (doublets, triplets, higher forms) liquid vaccine 100% < 5% lyophilized tablet (reconstituted) 21 - 40% 10-25% The data show that 24 to 40% of the initial virosome is preserved in the lyophilized tablets, of which 10 to 25% of these virosomes are in clusters, mainly as doublets and triplets. Example 6: Immunogenicity evaluation of P1 and rgp41 antigens from liquid virosome formulation and reconstituted lyophilized sublingual tablets containing virosome Liquid virosome concentrates (MYM-V202 liquid vaccine) and lyophilized sublingual tablets containing - 93 virosome of Example 3 and placed in storage at 4 and 40°C for a period of three months for immunogenicity determination. After storage for 1 month, the samples of the liquid vaccine and sublingual tablets stored at -40°C were removed from the storage cabinet for immunogenicity determination. After storage for 3 months, the samples of the liquid vaccine and sublingual tablets stored at 4°C and 40°C were removed for immunogenicity determination. For immunogenicity determination, Wistar rats (n = 10 per group), 50% of each sex, were used. Rats were immunized on day 0, day 28, and day 56. The liquid vaccine contained 3.9 μg of Pl, 2.2 μq of rgp41, and 1.3 μg of 3M-052 TLR7 / 8 (adjuvant) in 0.1 ml and was used for subcutaneous injection. For sublingual tablets, an appropriate amount of sublingual tablet was dissolved in sterile water to obtain approximately 3 μρ of Pl, 1.7 μρ of rgp41, and 1 μρ of 3M-052 TLR7 / 8 (adjuvant) in 0.1 ml to be administered by subcutaneous injection. Preimmune sera were collected on day 0 and immune sera on day 65 for determination of endpoint antibody titers for each animal's sera and on the pooled serum. Figure 5 shows the immunogenicity of Pl and pc Lcnn / Lznz / E / YiAi - 94 rgp41 from the liquid vaccine and sublingual tablets stored at different temperatures. The MYM-V202-adjuvanted liquid vaccine formulation, which contains both P1 and rgp41 antigens, is temperature-sensitive and serves as a reference material for comparison with the immunogenicity of the sublingual tablet vaccine form to improve thermostability. The black line shows vaccines stored for 3 months at 4°C. The dashed black line shows vaccines stored for 1 month at 40°C; the gray line shows vaccines stored for 3 months at 40°C; these data demonstrate that the immunogenicity of the antigens for the lyophilized tablets is retained. Endpoint antibody titers are also indicated for each set. The endpoint titer corresponds to the last serum dilution generating an OD value >2-fold above preimmune background. Additional definitions Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the same meaning as would be ordinarily understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some instances, terms with commonly understood meanings include, but are not limited to, Lcnn / Lznz / E / YiAi - 95 are defined herein for clarity and / or ease of reference, and the inclusion of these definitions herein should not necessarily be construed as representing a substantial difference from what is generally understood in the field. Reference to the term "approximately" with respect to a value or parameter herein includes (and describes) variations that are related to that value or parameter itself. For example, the description "referencing approximately X" includes the description of X. Furthermore, reference to phrases such as less than, greater than, at most, at least, less than or equal to, greater than or equal to, or other similar phrases followed by a sequence of values or parameters is meant to apply the phrase to each value or parameter in the sequence of values or parameters. For example, a statement that a solution has a concentration of at least about 10 mM, about 15 mM, or about 20 mM means that the solution has a concentration of at least about 10 mM, at least about 15 mM, or at least about 20 mM. As used herein, the singular forms un, uno and el are intended to include the plural forms as well, unless the context pc Lcnn / Lznz / E / YiAi - 96 clearly indicates otherwise. It is also to be understood that the term and / or, as used herein, refers to and encompasses any and all possible combinations of one or more of the associated enumerated clauses. It is further understood that the terms include, including, comprising, and / or comprising, when used herein, specify the presence of established features, integers, stages, operations, elements, components, and / or units, but does not preclude the presence or addition of one or more other features, integers, stages, operations, elements, components, units, and / or groups thereof. This application describes various numerical ranges in the text and figures. The numerical ranges described inherently support any interval or value within the described numerical ranges, including the endpoints, although a precise range limitation is not specifically described in the specification because this description can be implemented using the numerical ranges described. The above description is presented to enable a person skilled in the art to develop and use it, and is provided in the context of a particular application and its requirements. Various modifications of the Lcnn / Lznz / E / YiAi PCs - 97 Preferred embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other application embodiments without departing from the spirit and scope of the disclosure. Thus, this disclosure is not intended to be limited to the embodiments shown, but rather to be consistent with the broadest scope consistent with the principles and features described herein. It is noted that in relation to this date, the best method known to the applicant to put the aforementioned invention into practice is the one that is clear from the present description of the invention.

Claims

Having described the invention as above, the following claims are claimed as property:

1. An oral solid vaccine dosage form, characterized in that it comprises: lipid-based vesicles comprising an immunogenic amount of at least one target molecule; 5 to 20% by weight of at least one cryo-lyoprotectant; 25 to 40% by weight of a matrix former; and 40 to 55% by weight of a structure former.

2. The dosage form according to claim 1, characterized in that the lipid-based vesicles are virosomes or proteoliposomes.

3. The dosage form according to any of claims 1 to 2, characterized in that it comprises 10 to 15% by weight of at least one cryoprotectant.

4. The dosage form according to any of claims 1 to 3, characterized in that at least one cryo-lyoprotectant comprises trehalose.

5. The dosage form in accordance with pc Lcnn / Lznz / E / YiAi - 99 any of claims 1 to 4, characterized in that it comprises 33 to 37% by weight of the matrix former.

6. The dosage form according to any of claims 1 to 5, characterized in that the matrix former comprises keltin.

7. The dosage form according to claim 6, characterized in that the gelatin comprises fish gelatin.

8. The dosage form according to claim 7, characterized in that the fish gelatin is high molecular weight fish gelatin.

9. The dosage form according to any of claims 1 to 8, characterized in that it comprises 45 to 50% by weight of the structure former.

10. The dosage form according to any of claims 1 to 9, characterized in that the structure former comprises mannitol.

11. The dosage form according to any of claims 1 to 10, characterized in that the virosomes are derived from the membrane of influenza virus or other enveloped viruses.

12. The dosage form according to any of claims 1 to 11, characterized in that at least one target molecule is present in pc Lcnn / Lznz / E / YiAi - 100 the virosome.

13. The dosage form according to any of claims 1 to 12, characterized in that at least one target molecule comprises an HIV-1 envelope-derived antigen.

14. The dosage form according to claim 13, characterized in that the HIV-1 envelope-derived antigen comprises HIV-1 P1 peptide and / or recombinant HIV-1 gp41.

15. The dosage form according to any of claims 1 to 14, characterized in that the virosomes comprise adjuvant.

16. The dosage form according to any of claims 1 to 15, characterized in that it facilitates the uptake in the oral cavity of at least one target molecule.

17. The dosage form according to claim 16, characterized in that it disintegrates within 180 seconds after being placed in the oral cavity.

18. The dosage form according to claim 16, characterized in that it disintegrates within 90 seconds after being placed in the oral cavity. pc Lcnn / Lznz / E / YiAi 19. The dosage form according to claim 16, characterized in that it disintegrates within 60 seconds after being placed in the oral cavity.

20. The dosage form according to claim 16, characterized in that it disintegrates within 30 seconds after being placed in the oral cavity.

21. The dosage form according to claim 16, characterized in that an immune response is induced when administered to a patient by placement in the oral cavity.

22. The dosage form according to claim 21, characterized in that the placement in the oral cavity is placement on or under the tongue or in the buccal or pharyngeal region.

23. A method for inducing an immune response in a patient, characterized in that it comprises placing the dosage form according to any of claims 1 to 22 in the oral cavity of a person in need of the immune response.

24. The method according to claim 23, characterized in that the placement in the oral cavity is placement on or under the tongue or in the buccal or pharyngeal region.

25. A method for forming a solid oral vaccine dosage form (pc Lcnn / Lznz / E / YiAi - 102), characterized in that it comprises: dosing a liquid virosome formulation into a preformed mold, wherein the virosome formulation comprises: lipid-based vesicles comprising an immunogenic amount of at least one target molecule; 1 to 5 wt% of a cryo-lyoprotectant; 4 to 8 wt% of a matrix former; and 5 to 10 wt% of a scaffold former; freezing the dosed virosome formulation at a temperature of -60°C to -90°C; reassociating the frozen virosome formulation by maintaining it at a temperature below -15°C for 3 to 9 hours; and lyophilizing the reassociated virosome formulation to form the dosage form.

26. The method according to claim 25, characterized in that the dosed virosome formulation is frozen at a temperature of -60°C to -90°C for a duration of approximately 1 to 5 minutes.

27. The method according to any of claims 25 to 26, characterized in that the lyophilization of the reassociated virosome formulation comprises a first step of maintaining the pc Lcnn / Lznz / E / YiAi - 103 reassociated virosome formulation at a temperature of -10°C to -20°C for 20 to 28 hours and a second step of maintaining the reassociated virosome formulation at a temperature of -5°C to approximately -15°C for 14 to 22 hours.

28. The method according to any of claims 25 to 27, characterized in that the freeze-drying is produced at a pressure of less than 600 mbar.

29. The method according to any of claims 25 to 28, characterized in that the virosome formulation has a pH of approximately 6.5 to 8.

30. The method according to any of claims 25 to 29, characterized in that the cryo-lyoprotectant comprises trehalose.

31. The method according to any of claims 25 to 30, characterized in that the matrix former comprises gelatin.

32. The method according to claim 31, characterized in that the gelatin comprises fish gelatin.

33. The method according to claim 32, characterized in that the fish gelatin is high molecular weight fish gelatin.

34. The method according to any of claims 25 to 33, characterized in that the pc Lcnn / Lznz / E / YiAi - 104 structure former comprises mannitol.

35. The method according to any of claims 25 to 34, characterized in that the lipid-based vesicles are derived from influenza virus or respiratory syncytial virus.

36. The method according to any of claims 25 to 35, characterized in that at least one target molecule comprises an HIV-1 envelope-derived antigen.

37. The method according to claim 36, characterized in that the HIV-1 envelope-derived antigen comprises HIV-1 P1 peptide and / or recombinant HIV-1 gp41.

38. The method according to any of claims 25 to 37, characterized in that the lipid-based vesicles comprise adjuvant.

39. A method for forming an oral solid vaccine dosage form, characterized in that it comprises: dispensing a liquid virosome formulation into a preformed mold, wherein the virosome formulation comprises: 20 to 50% by weight of a virosome concentrate, wherein the virosome concentrate comprises: virosomes comprising an immunogenic amount pc Lcnn / Lznz / E / YiAi - 105 of at least one target molecule; 2 to 10% by weight of a cryo-lyoprotectant; and 60 to 200 mM of a buffer system; 4 to 8% by weight of a matrix former; and 5 to 10% by weight of a scaffold former; freezing the dispensed virosome formulation at a temperature of -60°C to -80°C; reassociating the frozen virosome formulation by maintaining it at a temperature below -15°C for 3 to 9 hours; and lyophilize the reassociated virosome formulation to form the dosage form.

40. The method according to claim 39, characterized in that the buffer system comprises HEPES-sodium chloride.