Lipid nanocarrier vaccine

Abstract

The present disclosure relates to a lipid nanoparticle that is a carrier for an antigen. The present disclosure also relates to an immunogenic composition, said immunogenic composition comprising said antigen. The immunogenic composition may be a vaccine composition. The disclosure further relates to methods and uses of the vectors and immunogenic compositions.

Description

Technical Field

[0001] The present disclosure relates to a lipid nanoparticle and uses thereof, wherein the lipid nanoparticle is a carrier of an antigen. Background Art

[0002] Vaccine delivery is an extensive area of ​​research to develop new materials or carrier systems for the effective therapeutic delivery of antigens. Carrier systems for subunit vaccines are needed to overcome various challenges associated with the properties of the antigens being delivered, including but not limited to poor solubility, low bioavailability, shortened half-life, lack of selectivity, poor cellular interactions, and toxicity.

[0003] Many different delivery methods have been tested, including polymeric delivery vehicles, conjugates, and core-shell particles. A suitable delivery method must be able to circulate systemically for an appropriate period of time, be able to protect the antigen during that time, and be amenable to a suitable fusion or other delivery event into the cell of interest.

[0004] Tuberculosis (TB), an infectious disease caused by the pathogen Mycobacterium tuberculosis (MTB), remains one of the top 10 causes of death worldwide and is expected to cause 1.2 million deaths in 2019. The continued emergence of drug-resistant TB and the slow development of new treatments are expected to lead to an increase in TB-related morbidity and mortality worldwide.

[0005] The vaccine Mycobacterium bovis (Mycobacterium bovis) BCG (BCG) of current selection is the vaccine that was launched nearly 100 years ago and is the most widely used vaccine in the world. However, although the BCG vaccine effectively prevents children's serious disseminated TB, it cannot prevent lung disease, which is the main cause of TB mortality. One of the main causes of BCG failure is that it cannot produce lung-specific immunity and even systemic immunity reduces during adolescence.

[0006] As part of a vaccine composition, it is necessary to provide a vehicle for delivering the antigenic agent that is likely to result in the generation of an appropriate immune response.

[0007] Any reference to background art herein is not to be construed as an admission that such art constitutes common general knowledge in Australia or elsewhere. Summary of the invention

[0008] According to a first aspect of the present disclosure, a non-lamellar lyotropic liquid crystal phase carrier is provided, wherein the non-lamellar lyotropic liquid crystal phase carrier comprises one or more lipids forming the carrier and an antigen associated with the carrier.

[0009] In a second aspect of the present disclosure, an immunogenic composition is provided, comprising the non-lamellar lyotropic liquid crystal phase carrier of the first aspect and a pharmaceutically acceptable carrier, diluent and / or excipient.

[0010] In embodiments, the immunogenic composition is a vaccine.

[0011] In certain embodiments, the immunogenic composition contains no or substantially no adjuvant that is not a component of the non-lamellar lyotropic liquid crystal phase carrier itself.

[0012] In a third aspect of the present disclosure, a method for delivering an antigen to a cell is provided, the method comprising the step of contacting the cell with the non-lamellar lyotropic liquid crystal phase carrier of the first aspect or the immunogenic composition of the second aspect.

[0013] In a fourth aspect of the present disclosure, a method of inducing an immune response in a subject is provided, the method comprising the step of administering to the subject an effective amount of the non-lamellar lyotropic liquid crystal phase carrier of the first aspect or the immunogenic composition of the second aspect.

[0014] In the fifth aspect of the present disclosure, a method for preventing, treating or ameliorating an infection, disease, disorder or condition is provided, the method comprising the step of administering a therapeutically effective amount of the non-lamellar lyotropic liquid crystal phase carrier of the first aspect or the immunogenic composition of the second aspect to a subject in need thereof, so as to prevent, treat or ameliorate the infection, disease, disorder or condition.

[0015] In a sixth aspect of the present disclosure, a non-lamellar lyotropic liquid crystal phase carrier of the first aspect or an immunogenic composition of the second aspect is provided, which is used to prevent, treat or improve an infection, disease, disorder or condition.

[0016] In the seventh aspect of the present disclosure, there is provided a use of the non-lamellar lyotropic liquid crystal phase carrier of the first aspect or the immunogenic composition of the second aspect in the preparation of a medicament for preventing, treating or ameliorating an infection, disease, disorder or condition.

[0017] The various features and embodiments of the present disclosure mentioned in the above-mentioned sections are applicable to other sections after necessary modifications where appropriate. Therefore, the features specified in one section can be combined with the features specified in other sections where appropriate.

[0018] Additional features and advantages of the present disclosure will become apparent from the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order that the present invention may be easily understood and put into practice, preferred embodiments will now be described by way of example with reference to the accompanying drawings, in which:

[0020] Figure 1 (A) Lattice parameters of monoolein (MO) based cubes after addition of trehalose 6,6'-dimycolate (TDM) (0.4-10 mol%) in PBS at 25 °C. Different phases were identified as follows: the original bicontinuous cubic (Q II P ) and diamond bicontinuous cubic (Q II D ). (B) Representative 1D SAXS plots of intensity versus q for MO cubes and MO-TDM cubes (0.4-10 mol%).

[0021] Figure 2 Representative cryo-TEM images and fast Fourier transforms of (A) MO-cubes, (B) MO-TDM (1 mol%) cubes. The total lipid concentration of all cubes was 50 mg / mL. Scale bar = 200 nm.

[0022] Figure 3 Hela and THP-1 cell viability after 72 hours of incubation in the presence of 20 μg / ml MO and MO-TDM (1 mol%) cubes. Cell viability was calculated as a percentage (%) of the control. Percent (%) cell viability data are presented as the mean ± standard deviation (SD) of two independent experiments analyzed in duplicate for each sample.

[0023] Figure 4 Solid-state X-ray diffraction patterns of MO, TDM, and MO-TDM (1 mol %), collected from drop-cast films on glass coverslips.

[0024] Figure 5 Differential Scanning Calorimetry (DSC) Analysis of MO, TDM and MO-TDM (1 mol%) Mixtures

[0025] Figure 6 FTIR spectra of MO, TDM and MO-TDM mixture (1 mol%) (700-4000 cm -1 )analyze

[0026] Figure 7.(a) Proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α) were measured after MTB infection and simultaneous stimulation with MO, TDM, and MO-TDM (1 mol%). (b) Macrophages were incubated with MO, TDM, and MO-TDM (1 mol%) for three days before MTB infection. Proinflammatory cytokines IL-6 and TNF-α were measured 24 hours after infection. (c and d) MTB load in macrophages pre-stimulated or simultaneously stimulated with MO, TDM, and MO-TDM (1 mol%), as measured by colony forming unit (CFU) assay over a 7-day infection period. Data represent the mean of three independent experiments performed in duplicate. Bars and error bars represent the mean and SD, respectively. Statistical analysis was performed using paired two-tailed Student's t-test / one-way ANOVA, with post hoc analysis of *p≤0.05, **p≤0.005, ***p≤0.0005, ****p≤0.0001. ns indicates not significant.

[0027] Figure 8 .Monocytes were trained for 18 hours with medium only (as negative control) or with MO, TDM or MO-TDM (1 mol%), and then rested for five days. On the sixth day after restimulation with BCG, (a) the concentrations of IL-6 and TNF-α cytokines were measured, and (b) the enrichment of H3K4me3 on the IL-6 and TNF-α promoters was measured by using chromatin immunoprecipitation (ChIP)-quantitative polymerase chain reaction (qPCR). Data represent the mean of three independent experiments performed in duplicate. Bars and error bars represent the mean and SD, respectively. Statistical analysis was performed using paired two-tailed Student's t-test / one-way ANOVA, with post hoc analysis *p≤0.05, **p≤0.005, ***p≤0.0005, ****p≤0.0001. ns indicates not significant.

[0028] Fig. 9.The IL-2 level secreted by Mycobacterium tuberculosis Ag85B-specific T cells during co-culture with MO, TDM, MO-TDM (1 mol%) and untreated macrophages at different times after infection represents its antigen presentation capacity. During simultaneous treatment (a), macrophages were incubated with MO, TDM and MO-TDM (1 mol%) at the beginning of infection, while during pretreatment (b), the macrophages were incubated with MO, TDM and MO-TDM for three days before MTB infection. The data shown are macrophages from a single donor, but represent three separate donors. The bars and error bars represent the mean and SD, respectively. Statistical analysis was performed using paired two-tailed Student's t-test / one-way variance and post hoc analysis. *p≤0.05, **p≤0.005, ***p≤0.0005, ****p≤0.0001. ns indicates not significant.

[0029] Fig.10 A. Mathematical model description and calibration. (a) Schematic diagram showing the key system interactions and variables of the model. The model contains two compartments: plasma and lymph, which communicate through antigen presenting cells (APCs) and antibodies. Antigen is injected intraperitoneally into the plasma compartment, and MTB is introduced as an intravenous bolus, and the innate and adaptive responses to antigen and MTB are simulated. Antigen and MTB are not present in the plasma compartment at the same time, but are depicted as such only to demonstrate all possible interactions occurring in the model. In fact, MTB is introduced into the system only when all antigens are cleared, i.e., 60 days after antigen injection. (b) The innate immune response (i.e., IL-6 and TNF-α-induced MTB neutralization) component of the model is calibrated using in vitro data after MTB is exposed to macrophages, pre-incubated (Pre; top row) or simultaneously (Con; bottom row) with (TDM, MO or MO-TDM (1 mol%)) or without (no) antigen. The insets in panels ii and iii (top row) show the magnified area demarcated by the dashed box to highlight the kinetics of the cytokines over the culture period (2 days). (c) The antigen presentation process was calibrated using in vitro data involving IL-2 secretion from effector CD4+ T cells when antigen was presented by unprimed (None) or primed (TDM, MO or MO-TDM (1 mol%)) macrophages. The in vitro data (markers) used for the above calibration are as follows Figure 8 and 9 shown.

[0030] Fig.10B. Model-based in vivo predictions. In vitro-in vivo extrapolation of the calibrated model shows the innate and adaptive immune responses of mice to antigen injected intraperitoneally on day 0, followed by re-challenge with MTB on day 60. The concentration kinetics of (a) antigen, (b) MTB, (c) IL-6, (d) TNF-α, and (h) antibodies in the plasma compartment are shown up to 120 days, with the inset highlighting the kinetics during the first 48-72 hours. The concentration kinetics of (e) effector CD4+ T cells, (f) IL-2, and (g) plasma cells in the lymphoid compartment are shown up to 120 days.

[0031] Fig.11 .OVA IgG antibody response of mice immunized twice with cube-OVA, modified cube-OVA, PSNP-OVA 50nm or PSNP-OVA 500nm. Serum was harvested from the final blood of mice immunized twice, and antigen-specific OVA IgG antibodies were assessed using ELISA. Starting from a 1:200 dilution, serum was serially diluted 1:2. The data shown are antibody responses measured as optical density readings for each dilution, and the mean value for each group was calculated. DETAILED DESCRIPTION

[0032] The present disclosure is based, at least in part, on the recognition that non-lamellar lyotropic liquid crystal phase carriers ('carriers') may be particularly suitable for delivering antigens as part of subunit vaccine compositions and are surprisingly effective in generating an immune response based on the effective delivery of the antigen of interest. One or more of the chemical and / or physical properties and / or structure of the non-lamellar lyotropic liquid crystal phase carrier, such as the nature of the constituent lipids, the internal or mean spontaneous curvature, the charge, the loading efficiency, and the microrheology, can advantageously provide a carrier that is capable of one or more of the following: (i) improving the encapsulation and / or solubility of the antigen; (ii) protecting the antigen from damage or binding that would otherwise occur and inactivate or reduce the activity of the antigen; (iii) reducing the toxicity of the antigen compared to administration of the free antigen; and (iv) improving the observed immunogenicity of the delivered antigen compared to delivery of the free antigen. The formation of non-lamellar lyotropic liquid crystal phase carriers will allow for a wide range of uses in medical applications including immunization, where the carriers can be tailored to improve the delivery of specific antigens.

[0033] In certain instances, the antigen may be suitable for generating an immune response, such as an adaptive immune response, to TB infection. Mycobacterial components that may be used in new tuberculosis vaccines remain largely unknown. Undeveloped tuberculosis vaccine candidates are mycolic acids, or the cord factor trehalose 6,6' dimycolate (TDM), lipid components abundant in the TB cell wall that are known to strongly stimulate host inflammatory responses and granuloma formation. Although TDM is one of the oldest and best studied virulence factors of TB, its high toxicity and low water solubility severely limit its development as a possible subunit vaccine.

[0034] It is shown herein that non-lamellar lyotropic liquid crystal phase carriers capable of delivering such antigens to a host can be designed. The design of the carrier herein can particularly, but not exclusively, facilitate its delivery of an active form of the antigen to promote the generation of an immune response. Further, when administered to a subject, the toxicity of the antigen can be reduced, and not only the innate immune response that can be elicited by the carrier itself, but also an adaptive response is observed, indicating successful delivery to the cell.

[0035] Inducing the desired immune response also requires that the antigen be delivered to professional antigen presenting cells and activate these cells. Delivery systems such as carriers and immunopotentiators jointly determine the magnitude and quality of the innate immune response and the uptake and processing of antigens by antigen presenting cells. The non-lamellar lyotropic liquid crystal phase carrier disclosed herein shows a high surface area to volume ratio, which provides several advantages, including increased bioavailability, dose proportionality and reduced toxicity relative to a single antigen. The structure of the non-lamellar lyotropic liquid crystal phase carrier also enables antigens of different compositions and physical properties to be encapsulated.

[0036] It has been shown in the literature that the addition of hydrophobic components to certain non-lamellar lyotropic liquid crystal phase carriers such as cubes can negatively affect the internal nanostructure of the carrier by changing the intrinsic curvature of the lipid bilayer (Strachan et al. Australian Journal of Chemistry 2020, 73, pp. 1042-1050; Freire et al. Journal of Colloid and Interface Science 2021, 596, pp. 352-363). Based on this knowledge, it is expected that large or long-chain hydrophobic components will therefore have a significant effect in such carriers. Surprisingly, the examples herein demonstrate that even very long chain (C-80-90) fatty acid chains associated with TDM do not negatively affect the carrier nanostructure and allow effective delivery.

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0038] In this patent specification, the terms 'comprises', 'comprising', 'includes', 'including' or similar terms are intended to mean a non-exclusive inclusion, such that a method or composition comprising a list of elements includes not only those elements but may also include other elements that are not listed.

[0039] “Consisting of is meant to include and be limited to whatever follows the phrase “consisting of.” Thus, the phrase “consisting of indicates that the listed elements are required or necessary, and other elements may not be present.

[0040] "Consisting essentially of" means including any elements listed after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action specified in the present disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or necessary, but other elements are optional and may be present or absent depending on whether they affect the activity or action of the listed elements.

[0041] As used herein, the term "non-lamellar lyotropic liquid crystal phase carrier" refers to a self-assembled non-lamellar liquid crystal phase formed by at least one amphiphilic molecule to obtain a two-dimensional and / or three-dimensional mesophase structure capable of carrying antigens. It is shown herein that non-lamellar lyotropic liquid crystal phase carriers promote immune responses by delivering active forms of antigens to the host system. The terms "lipid carrier", "non-lamellar lyotropic liquid crystal phase carrier", "non-lamellar LLC carrier", "lyotropic liquid crystal (LLC) lipid carrier" and "carrier" are used interchangeably herein. As is known in the art, such carriers do not include liposomes. The term non-lamellar refers to a lyotropic liquid crystal phase or lipid carrier or particle that is not a liposome (or L α Liquid crystal phases are defined as a phase of a substance that exhibits properties intermediate between those of a conventional liquid and those of a solid crystal. There are different types of liquid crystal phases that can be distinguished based on their different optical properties and other properties known in the art.

[0042] In an embodiment, the non-lamellar lyotropic liquid crystal phase carrier of the present invention comprises only liquid crystal. That is, the non-lamellar lyotropic liquid crystal phase carrier of the present invention does not comprise any solid lipid component. Therefore, the non-lamellar lyotropic liquid crystal phase carrier of the present invention is not a solid lipid nanocarrier (SLN).

[0043] In embodiments, the term "non-lamellar lyotropic liquid crystal phase carrier" may be used to cover only cubic, hexagonal, and sponge morphologies. Although the "sponge phase" or "sponge particles" (L3) are considered to have no long-range order and do not exhibit the same properties as the inverse bicontinuous cubic phase (Q II ) has a comparable crystalline periodicity, but it is generally regarded as a "molten" QII cubic phase and is therefore included as particles of the first aspect. Therefore, the short-range ordered sponge phase is explicitly regarded as being within the scope of this term. In an embodiment, the term "non-lamellar lyotropic liquid crystal phase carrier" can be used to include one or more phases selected from the group consisting of: hexagonal phases (positive phases and negative phases), cubic phases (positive discrete phases, negative discrete phases, negative bicontinuous phases - including original phases, gyroscopic phases and diamond phases - and negative discontinuous phases) and other 'intermediate phases', including ribbon, network or non-cubic 'sponge' bicontinuous phases.

[0044] As used herein, the terms "amphiphile," "amphiphilic," and "amphiphilic lipid" refer to compounds that contain both hydrophilic and hydrophobic portions and can be used as lipids to form the non-lamellar lyotropic liquid crystal phase carriers described herein. Typically, such compounds will have a hydrophilic head group and a hydrophobic tail. Suitable examples include fatty acids and a range of lipid molecules.

[0045] The term "pharmaceutically acceptable salt" as used herein refers to a salt of one or more active agents that is toxicologically safe for systemic or local administration, such as a salt prepared from a pharmaceutically acceptable non-toxic base or acid including an inorganic or organic base and an inorganic or organic acid. Pharmaceutically acceptable salts can be selected from the group consisting of alkali metals and alkaline earth metals, ammonium, aluminum, iron, amines, glucosamine, chlorides, sulfates, sulfonates, bisulfates, nitrates, citrates, tartrates, bitartrates, phosphates, carbonates, bicarbonates, malate, maleate, naphthylsulfonate, fumarate, succinate, acetate, benzoate, terephthalate, palmitate, piperazine, pectinate, and S-methylmethionine salts, etc.

[0046] According to a first aspect of the present invention, a non-lamellar lyotropic liquid crystal phase carrier is provided, wherein the non-lamellar lyotropic liquid crystal phase carrier comprises one or more lipids forming the carrier and an antigen associated with the carrier.

[0047] Carrier

[0048] In an embodiment, the non-lamellar lyotropic liquid crystal phase carrier of the first aspect is formed by self-assembly of the one or more lipids, in an embodiment, the one or more lipids can be amphiphilic lipids. It should be understood that suitable amphiphilic lipids will self-assemble in the presence of an aqueous solution such as water or an aqueous buffer solution to form a lyotropic liquid crystal structure exhibiting a non-lamellar mesophase.

[0049] In an embodiment, the non-lamellar lyotropic liquid crystal phase carrier of the first aspect comprises at least one amphiphilic lipid, at least two amphiphilic lipids, at least three amphiphilic lipids, at least four amphiphilic lipids, at least five amphiphilic lipids, at least six amphiphilic lipids or at least seven amphiphilic lipids.

[0050] In an embodiment, the non-lamellar lyotropic liquid crystal phase carrier of the first aspect may consist of or consist essentially of one, two, three, four or five amphiphilic lipids. In this context, the expression "consisting essentially of" will be understood to mean that the lyotropic liquid crystal phase carrier consists of one, two, three, four or five amphiphilic lipids forming the carrier, and does not consist of any other lipids forming the carrier.

[0051] In an embodiment, the non-lamellar lyotropic liquid crystal phase carrier is formed by self-assembly of one or more amphiphilic lipids in the presence of an antigen. It should be understood that the antigen can be associated with the carrier in a variety of ways, such as attachment, incorporation or encapsulation in the carrier, and the final method will depend on the nature of the antigen and the mode of delivery of the antigen by the carrier. For example, in certain embodiments, it may be appropriate to focus on the attachment of the antigen to most of the particle surface. Typically, however, the carrier will be formed in the presence of an antigen so that, in addition to any incidental surface-bound antigen, the antigen is incorporated into the lipid bilayer or internal channels and folds of the carrier.

[0052] In an embodiment, the non-lamellar lyotropic liquid crystal phase carrier may be a colloidal carrier, the particle size of which is less than 10 micrometers.

[0053] In an embodiment, the particle size of the carrier of the first aspect may be between about 10 microns and about 40 nanometers. Preferably, the particle size is between about 5 microns and about 50 nanometers, more preferably between about 1 micron and about 50 nanometers, even more preferably between about 800 nanometers and about 50 nanometers, still more preferably between about 600 nanometers and about 50 nanometers, even more preferably between about 500 nanometers and about 50 nanometers, or between about 400 nanometers and about 50 nanometers, or between about 5 microns and about 80 nanometers, more preferably between about 1 micron and about 80 nanometers, even more preferably between about 800 nanometers and about 80 nanometers, still more preferably between about 600 nanometers and about 50 nanometers. Preferably, the particle of the first aspect can be between about 600 nanometers and about 80 nanometers, even more preferably between about 500 nanometers and about 80 nanometers, or between about 400 nanometers and about 80 nanometers, or between about 5 micrometers and about 100 nanometers, more preferably between about 1 micrometer and about 100 nanometers, even more preferably between about 800 nanometers and about 100 nanometers, still more preferably between about 600 nanometers and about 100 nanometers, even more preferably between about 500 nanometers and about 100 nanometers, or between about 400 nanometers and about 100 nanometers. Therefore, the particles of the first aspect can be used as nanocarriers of the one or more active agents in the embodiments of the above-mentioned particle size ranges.

[0054] In an embodiment, the non-lamellar lyotropic liquid crystal phase vehicle has a bulk phase selected from the group consisting of a cubic phase, a hexagonal phase, and a sponge phase, including normal and reverse / inverse phases of each, as appropriate.

[0055] These carrier matrices have a range of advantages in delivering antigens compared to their lamellar analogs such as liposomes. Their lipid composition allows them to fuse more easily with the outer membrane of appropriate cells, and non-lamellar lyotropic liquid crystal phase carriers such as cubic ones have the ability to encapsulate and release a range of antigens due to their high internal surface area and amphiphilic nature. Such carrier matrices can also protect the structural integrity of the encapsulated antigen from enzymatic degradation and can reduce the innate toxicity of the antigen, allowing for appropriate use as subunit vaccines.

[0056] In an embodiment, the non-lamellar lyotropic liquid crystal phase particles are particles selected from the group consisting of: hexagonal phase (forward phase and reverse phase), cubic phase (forward discrete phase, reverse discrete phase, reverse bicontinuous phase - including pristine phase, gyroscopic phase and diamond phase - and reverse discontinuous phase) and other 'intermediate phases' including ribbon, network or non-cubic 'sponge' bicontinuous phases.

[0057] In an embodiment, the non-lamellar lyotropic liquid crystal phase carrier of the first aspect may be a cube or a hexagon.

[0058] In an embodiment, the non-lamellar lyotropic liquid crystal phase vehicle is cubic.

[0059] In an embodiment, the non-lamellar lyotropic liquid crystal phase carrier is hexagonal.

[0060] Preferably, the cube is a bicontinuous cubic phase (V1) or an anti-bicontinuous cubic phase (V2) cube. The anti-bicontinuous cubic phase (V2) cube is particularly preferred. Those skilled in the art will understand that V2 is a general term for various cubic phases. V2 may also be referred to as V II or Q II .Q II Q inside II D (Pn3m), Q II P (Im3m), QII G (Ia3d).

[0061] Reverse (inverse) phase carriers may be preferred because they provide a complex series of internal channels that can accommodate one or more active agents and, in some cases, allow for better controlled release profiles.

[0062] The cubic phase structure within the cube provides a lipid bilayer motif that is repeatedly wrapped on a three-fold periodic minimum surface. The increased surface curvature of the lipid membrane within these carriers of the first aspect can help promote the fusion of the bilayer when in contact with other self-assembly systems including lipid membranes. Therefore, in the carriers of the present disclosure, high curvature values ​​are preferred. Due to its high internal surface area and amphipathic properties, the cube has the ability to encapsulate and release a variety of antigens.

[0063] In the case of lipids used to form the carrier of the first aspect, the critical packing parameter (CPP) of the lipid can be used to rationalize the average and Gaussian curvatures, which are properties of the particles formed and therefore indicate the properties of the formed or forming mesophase, and allow consideration of the suitability of the resulting non-lamellar lyotropic liquid crystal phase carrier as an antigen carrier. The CPP is related to the average and Gaussian curvatures by the following equation:

[0064]

[0065] in:

[0066] lc is the effective length of the hydrocarbon chain

[0067] H is the mean curvature

[0068] K is the Gaussian curvature

[0069] The molecular geometry of the relevant amphiphilic lipids used to form the particles of the present disclosure should satisfy the above equation to yield a CPP>1.

[0070] The same method can be used for more than one amphiphilic lipid. For multiple amphiphiles, an amphiphile with an intrinsic CPP less than 1 can be included in a composition with a second amphiphile CPP greater than 1, so that the average CPP is greater than 1. Second additives that also contribute to the increase in curvature to achieve a CPP greater than 1 include small hydrophobic molecules and polymers that interact with the amphiphilic head group. In contrast, curvature can be reduced by including an amphiphile with a CPP less than 1, a high molecular weight PEG, a strong chaotropic agent, a charged head group, and a solvent with a LogP between -1.5 and 0.

[0071] Therefore, non-lamellar lyotropic liquid crystal phase carriers can be classified according to their interfacial curvature, which can be calculated by methods known in the art. In general, the curvature of the anti-lyotropic phase increases in the order of lamellar < bicontinuous cubic < hexagonal < micellar cubic.

[0072] In embodiments, the critical packing parameter (CPP) of said one or more amphiphilic lipids is about or greater than 1.0.

[0073] In an embodiment, the CPP of the one or more amphiphilic lipids is between about 1.0 and about 3.0, preferably between about 1.0 and about 2.5, more preferably between about 1.0 and about 2.0, even more preferably between about 1.0 and about 1.75, and still more preferably between about 1.0 and about 1.5.

[0074] It will be appreciated that when a plurality of lipids are incorporated into the vector of the first aspect, then the CPP values ​​of all the individual lipids mentioned above become references to the average CPP value. That is, when the LLC particles contain more than one amphiphile (lipid), the average CPP may be defined as the molar average of all the CPP values ​​of the constituent amphiphile lipids. The average CPP value may be selected from the values ​​provided above.

[0075] In an embodiment, the average CPP value of the non-lamellar lyotropic liquid crystal phase carrier is between about 1.0 and about 3.0, preferably between about 1.0 and about 2.5, more preferably between about 1.0 and about 2.0, even more preferably between about 1.0 and about 1.75, and still more preferably between about 1.0 and about 1.5.

[0076] CPP is calculated as follows: v / a0l c ; where l c is the effective length of the amphiphile (lipid) chain; a0 is the effective surfactant head group area (determined by the balance of interchain attractive and head group repulsive interactions); and v is the average volume occupied by the amphiphile molecules.

[0077] Similarly, the spontaneous extension value corresponds to the spontaneous curvature of the non-lamellar LLC particles. When the two lipids have corresponding spontaneous extension energies, then the fusion between them becomes more energetically favorable. Therefore, it is believed that the particles of the first aspect having the following extension values ​​will be more likely to undergo the desired fusion event with the biological membrane.

[0078] The choice of amphiphilic lipid will significantly affect extension and can be determined based on, for example, the choice of hydrophobes that enhance chain extension, including the use of unsaturated hydrophobes such as myristyl, pentadecenyl, oleyl, anti-oleyl, oleyl, arachidenyl, docosene and / or isoprenoid-type hydrophobes such as 3,7,11-trimethyl-dodecyl, 5,9,13-trimethyltetradecyl, 3,7,11,15-tetramethyl-hexadecyl, 5,9,13,17-tetramethyloctadecyl. Non-limiting examples of such lipids include ME, MP, MM, MV, MO, ML and MR as known in the art.

[0079] The energy cost per unit surface area due to stretching can be approximated as

[0080]

[0081] where κ is the tensile modulus of the monolayer, It is self-development and extension.

[0082]

[0083] κ t is the stretch modulus of the monolayer and t is the tilt vector.

[0084] Total energy cost

[0085]

[0086] In an embodiment, the internal curvature-induced stretching of the non-lamellar lyotropic liquid crystal phase carrier Less than -0.05nm -1 .

[0087] In an embodiment, the internal curvature-induced stretching of the non-lamellar lyotropic liquid crystal phase carrier Less than -0.10nm -1 .

[0088] In an embodiment, the internal curvature-induced stretching of the non-lamellar lyotropic liquid crystal phase carrier Less than -0.15nm -1 .

[0089] In an embodiment, the internal curvature-induced stretching of the non-lamellar lyotropic liquid crystal phase carrier Less than -0.20nm -1.

[0090] In an embodiment, the internal curvature-induced stretching of the non-lamellar lyotropic liquid crystal phase carrier Less than -0.25nm -1 .

[0091] In an embodiment, the internal curvature-induced stretching of the non-lamellar lyotropic liquid crystal phase carrier Between about -0.05nm -1 to about -0.95nm -1 Between, or between about -0.05nm -1 to about -0.85nm -1 Between, or between about -0.05nm -1 to about -0.75nm -1 Between, or between about -0.05nm -1 to about -0.65nm -1 Between, or between about -0.05nm -1 to about -0.55nm -1 Between, or between about -0.05nm -1 to about -0.40nm -1 Between, or between about -0.10nm -1 to about -0.95nm -1 Between, or between about -0.10nm -1 to about -0.85nm -1 Between, or between about -0.10nm -1 to about -0.75nm -1 Between, or between about -0.10nm -1 to about -0.65nm -1 Between, or between about -0.10nm -1 to about -0.55nm -1 Between, or between about -0.10nm -1 to about -0.40nm -1 Between, or between about -0.15nm -1 to about -0.75nm -1 Between, or between about -0.15nm -1 to about -0.65nm -1 Between, or between about -0.15nm -1 to about -0.55nm -1 Between, or between about -0.15nm -1 to about -0.40nm -1 Between, or between about -0.20nm -1 to about -0.75nm -1 Between, or between about -0.20nm-1 to about -0.65nm -1 Between, or between about -0.20nm -1 to about -0.55nm -1 Between, or between about -0.25nm -1 to about -0.75nm -1 Between, or between about -0.25nm -1 to about -0.65nm -1 Between, or between about -0.25nm -1 to about -0.55nm -1 between.

[0092] Further, in light of this disclosure, one skilled in the art will be able to use the following equation to determine the appropriate level of curvature to provide the benefits described herein:

[0093] c 0T =xc 0i +(1-x)c 0j

[0094] where c 0T is the total curvature, x is the lipid fraction i, c 0i is the curvature of lipid i, c 0j is the curvature of lipid j, and (1-x) is the composition fraction of lipid j. For additional combinations, the equation is expanded to include lipid k; where the sum of the compositions equals 1.

[0095] In an embodiment, the lattice parameter of the non-lamellar lyotropic liquid crystal phase carrier is between about to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between or between to about between.

[0096] For example, the lattice parameters associated with sponge particles, which typically have larger lattice parameters than cubes or hexagons, can be swollen to a range of up to More common values ​​for the swelling phase can be The swelling lattice parameters have certain design rules, including that the head group can contain electrostatic charges, and these electrostatic charges can be negatively charged (such as PG and PS phospholipids) or positively charged (such as DOTAP (1,2-dioleoyl-3-trimethylammonium propane) and DODMAC (dimethyldioctadecyl ammonium chloride)). The head group can contain a hydrating agent with multiple hydroxyl groups (such as DGMO and OG). The hydrophobic region can contain cholesterol or other hardeners to stabilize the membrane and / or can contain amphiphiles that promote reduced membrane curvature (such as PC and PE phospholipids). Lipid-PEG polymers (such as DOPE-PEG and MO-PEG) can be used in combination with charged lipids to swell the water channels. Further, when nanoparticle dispersions are required, block copolymers (such as Pluronic F127, F108 and polysorbate 80) can be used as stabilizers, although they may not have a direct effect on the swelling of the water channels.

[0097] The one or more amphiphilic lipids forming the carrier of the first aspect can be selected from lipids known in the art, particularly lipids forming cubes and hexagons. The selection of one or more suitable amphiphilic lipids can be carried out based on certain requirements understood in the art. For example, lipids can be selected from lipids that adopt type II lyotropic liquid crystal phases at ambient temperature and physiological temperature. Parameters that can be suitable for selecting suitable lipids include (i) about hydrophobic components: 1. The temperature should be higher than the chain melting temperature so that there are molten chains; and 2. In a carbon chain consisting of at least 14 carbons, at least one cis unsaturated bond should be present at least in the middle position along the skeleton; or 3. The carbon skeleton should contain at least 12 carbons, 3 of which are secondary carbons with methyl branches; and 4. The molecular weight of the hydrophobic molecule should be at least greater than 200amu, and (ii) about the head group: 5. The head group should contain at least three functional groups (e.g., hydroxyl groups) with minimal hydrophilicity; 6. The head group should be able to form a head group-water hydrogen bond network; and 7. The head group area should be small relative to the space occupied by the hydrophobic molecule. As a guide, this is exemplified by the MO lipid used in the examples of the present disclosure, as the MO lipid: meets criteria 1, 2, and 4 for a hydrophobic molecule; and meets criteria 5, 6, and 7 for a head group. It will be appreciated that many other lipids are available that suitably meet these criteria, and that the lipids may be selected based on criteria of known or readily determined values.

[0098] Guidance may be found in one or more of the following publications, each of which is incorporated herein by reference). Incorporated herein by reference in its entirety: (i) T. Kaasgaard and CJ Drummond "Ordered 2D and 3D Nanostructured Amphiphile Self-Assembly Materials Stable in Excess Solvent," Phys. Chem. Chem. Phys. 2006, 8, pp. 4957-4975.(ii) C. Fong, T. Le and C. J. Drummond, “Lyotropic Liquid Crystal Engineering-Ordered Nanostructured Small Molecule Amphiphile Self-Assembly Materials by Design,” Chem. Soc. Rev., 2012, 41, pp. 1297-1322; (iii) L. van't Hag, S. L. Gras, C. E. Conn and C. J. Drummond, “Lyotropic liquid crystal engineering moving beyond binary compositional space-Ordered nanostructured amphiphile self-assembly materials by design,” Chem. Soc. Rev., 2017, 46, pp. 2705-2731.; and (iv) S. Sarkar, N. Tran, M. Drummond, “Lysotropic liquid crystal engineering moving beyond binary compositional space-Ordered nanostructured amphiphile self-assembly materials by design,” Chem. Soc. Rev., 2017, 46, pp. 2705-2731. H. Rashid, T. C. Le, I. Yarovsky, C. E. Conn, and C. J. Drummond. “Toward cell membrane biomimetic lipidic cubic phases: a high-throughput exploration of lipid compositional space.” ACS Applied Biomaterials, 2019, 2, pp. 182-195.

[0099] Polyhydroxyl (glycolipid) and polyether (polyethylene oxide) form two of the largest categories in type II forming head groups. Non-limiting examples of head group motifs include alcohol, fatty acid, monoacylglycerol, MAG, 2-MAG, glycerate, glyceryl ether, ethylene oxide, amide, monoethanolamide, diethanolamide, serinolamide, methyl propylene glycol amide, ethyl propylene glycol amide, urea, urea alcohol, biuret, biuretol, urea, endogenous cannabinoids (cannabinoids, virodhamine, 2-glycerol, dopamine, 2-glyceryl ether) and glycolipid. Examples include phospholipids, such as DMPC and DMPE.

[0100] In an embodiment, the one or more amphiphilic lipids may be selected from the group consisting of ethylene oxide-based amphiphiles, monoacylglycerol-based amphiphiles, glycolipid-based amphiphiles, phosphatidylethanolamine-based amphiphiles and urea-based amphiphiles, and derivatives or analogs thereof.

[0101] Oxirane amphiphiles may include C 12 (EO)2, C 12 (EO)4, C 12 (EO)5 and C 12 (EO)6 and dialkyl ethylene oxide amphiphilic molecules. Monoacylglycerols can include myristolein, monoolein, monovaccenin and monoerucin. Amphiphilic molecules similar to monoacylglycerols can be suitable and include oleyl glyceride, phytane glyceride, glyceryl monooleyl ether, glyceryl phytane ether, phytantriol and monononadecenoin. Possibly suitable glycolipids with sugar moieties include monosubstituted glycolipids: β-Mal3(Phyt)2, β-Glc(Phyt), β-Xyl(Phyt), β-Glc-(TMO)2, β-Mal2(Phyt)2, and β-Glc(Phyt)2; and disubstituted unbranched glycolipids: 1,2-diacyl-(β-D-glucopyranosyl)-sn-glycerol; 1,2-dialkyl-(β-D-glucopyranosyl)-sn-glycerol; 1,3-diacyl-(β-D-glucopyranosyl)-sn-glycerol; 1,3-dialkyl-(β-D-glucopyranosyl)-sn-glycerol. Phosphatidylethanolamine amphiphiles may include dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE). Urea amphiphiles may include dodecyl urea (DU), octadecyl urea (ODU), oleyl urea (OU), oleyl biuret (OBU), linoleyl urea (LU), phytanyl urea (PU), hexahydrofarnesyl urea (HFU).

[0102] In an embodiment, the one or more amphiphilic lipids are selected from the group consisting of 1-monoolein, 2-monoolein, citrem, oleyl lactate, oleamide, monoelaidin, linoleic acid, elaidic acid, monopalmitolein, monolinolein, phytantriol, diolein, triolein, dioleoylglycerol, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), NN-dioleoyl-N,N-dimethylammonium chloride (DODAC) , dioctadecyl ammonium chloride (DOAC), dioctadecyl dimethyl ammonium chloride (DODMAC) or dioctadecyl dimethyl ammonium bromide (DODAB), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-phosphatidylglycerol (DOPG), oleic acid (OA), lyso-1-hydroxy-2-oleoyl-sn-glycero-3-phosphocholine (PC), 1,2-dioleoyl-sn-glycero-3-dihexyl-phosphocholine (DOPC), vitamin E tocopherol, vitamin E (tocopherol) acetate, phytanyl monoethanolamide, farnesyl monoethanolamide, oleyl monoethanolamide, linoleyl monoethanolamide and linolenoyl monoethanolamide.

[0103] The single-chain amphiphilic lipid can be selected from the group consisting of: saturated fatty acids C7-C16, oleic acid, elaidic acid, linoleic acid, sodium oleate / gadolinium, oleamide, 1-glyceryl monooleyl ether (GME), GMO, 2-MO, oleyl lactate, monoglyceryl citrate, diglyceryl monooleate (DGMO), lyso(1-oleoyl)-phosphatidyl-choline, (Z)-octadec-9-enylferrocene, N-dodecyl-caprolactam (C12), vitamin K1, ubiquinone-10 (coenzyme Q10), vitamin E, vitamin E acetate, vitamin A palmitate, α-tocopheryl PEO1000 succinate (vitamin E TPGS), PEG2000-MO, PEG-PT, PEO x - Stearate (x=40-100), polysorbate 80.

[0104] The amphiphilic lipid having multiple alkyl chains can be selected from the group consisting of: didodecyldimethylammonium bromide (DDAB); bis(rapeseed ethyl)dimethylammonium chloride (DEEDAC); dioctadecyl(dimethyl)ammonium chloride (DODMAC), dioctadecylammonium chloride (DOAC) or dioctadecyldimethylammonium bromide (DODAB); dioleylglycerol; dioleoyl-glycerol (DOG), EDTA-bis-oleoyl; EDTA-bis-phytyl; 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); 1,2-dioleoyl-phosphatidic acid (DOPA); 1,2-dioleoyl-phosphatidylglycerol (DOPG), 1,2-distearoyl-phosphatidylglycerol (DSPG); 1 ,2-dioleoyl-phosphatidylethanolamine (DOPE), 1,2-distearoyl-glycero-3-phosphoethanolamine (DSPE); 1,2-dioleoyl-phosphatidylcholine (DOPC); 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS); 1,2-dipalmitoylphosphatidylserine (DPPS); DSPE-mPEG350, 750, 2000 (X = 7, 16 or 45); DSPE-PEG2000, 3400, 5000; DMPE-mPEG550; (C18)2DTPA (Gd), cardiolipin, cyclodextrin derivatives (βCD-nC 10 ).

[0105] In any of the embodiments herein, the amphiphilic lipid can be glyceryl monooleate and / or phytantriol.

[0106] In any of the embodiments herein, the carrier of the present disclosure may comprise a combination of MO or phytantriol and one or more of cholesterol, DLPC, DSPC, DPPE, DPPS, DOPS, DPPC, DMPC, DMPS, and DLPS.

[0107] It is known that monoacylglycerols form inverted phases over large regions of their phase diagrams, with monoolein being the most prominent. The formation of inverted phases is favored by the kinks introduced by the cis double bonds. Longer acyl chains increase the volume of the hydrophobic chains and make monoolein more wedge-shaped and shift toward the Type 2 phase in the spectrum of the mesophase. If the double bond is closer to the end of the lipid, the double bond will weaken its effect and make it less wedge-shaped. It is expected that acyl chain extension will drive further mesophase formation toward the Type 2 phase, and on this basis, it is not surprising that the H2 phase becomes the dominant phase with this change.

[0108] In an embodiment, the lipids forming the non-lamellar lyotropic liquid crystal phase vehicle of the first aspect may consist essentially of glyceryl monooleate.

[0109] In an embodiment, the lipids forming the non-lamellar lyotropic liquid crystal phase vehicle of the first aspect may essentially comprise glyceryl monooleate and / or phytantriol.

[0110] In an embodiment, the lipids forming the non-lamellar lyotropic liquid crystal phase vehicle of the first aspect may essentially comprise monoolein and / or phytantriol and DOPE.

[0111] In an embodiment, the lipids forming the non-lamellar lyotropic liquid crystal phase vehicle of the first aspect may essentially comprise glyceryl monooleate and / or phytantriol and DOTAP.

[0112] In an embodiment, the lipids forming the non-lamellar lyotropic liquid crystal phase vehicle of the first aspect may essentially comprise monoolein and / or phytantriol and TOAB.

[0113] In an embodiment, the lipids forming the non-lamellar lyotropic liquid crystal phase vehicle of the first aspect may essentially comprise glyceryl monooleate and / or phytantriol and oleic acid.

[0114] In an embodiment, the lipids forming the non-lamellar lyotropic liquid crystal phase vehicle of the first aspect may essentially comprise monoolein and / or phytantriol and DOPE and DOTAP.

[0115] In embodiments, where the non-lamellar lyotropic liquid crystal phase carrier of the first aspect comprises DOPE, it may be present in an amount of 10 mol % to 40 mol %.

[0116] In embodiments, where the non-lamellar lyotropic liquid crystal phase carrier of the first aspect comprises DOTAP, it may be present in an amount of 0.5 mol % to 5 mol % or 0.5 mol % to 4 mol %.

[0117] In an embodiment, the non-lamellar lyotropic liquid crystal phase vehicle of the first aspect comprises glyceryl monooleate (80-99.9 mol %) and glyceryl trioleate (0.1-20 mol %).

[0118] In an embodiment, the non-lamellar lyotropic liquid crystal phase carrier of the first aspect comprises glyceryl monooleate (80-99.9 mol %) and vitamin E (0.1-20 mol %).

[0119] In an embodiment, the non-lamellar lyotropic liquid crystal phase vehicle of the first aspect comprises glyceryl monooleate (80-99.9 mol %) and DOPE (0.1-20 mol %).

[0120] In an embodiment, the non-lamellar lyotropic liquid crystal phase vehicle of the first aspect comprises glyceryl monooleate (95-99.9 mol %) and DOTAP (0.1-5 mol %).

[0121] In an embodiment, the non-lamellar lyotropic liquid crystal phase vehicle of the first aspect comprises glyceryl monooleate (95-99.9 mol %) and DODAB (0.1-5 mol %).

[0122] Stabilizer

[0123] In an embodiment, the non-lamellar lyotropic liquid crystal phase carrier may further comprise at least one stabilizer. The stabilizer is selected from stabilizers known in the art and can be used to provide steric stabilization of the carrier and / or reduce flocculation of the carrier.

[0124] In embodiments, the stabilizer is a poloxamer or a modified version thereof.

[0125] In embodiments, the stabilizer is a surfactant or a modified version thereof.

[0126] In embodiments, the stabilizer is a PEGylated lipid stabilizer or a modified version thereof.One skilled in the art will understand that reference to a PEGylated lipid is a lipid that has been modified with polyethylene glycol (PEG).

[0127] In an embodiment, stabilizer is selected from PEG-PPO-PEG triblock copolymer and nonionic block copolymer surfactant and PEO copolymerized with charged part.Poloxamer 407 and Pluronic 127 can be suitable examples of stabilizer, and can be incorporated into any embodiment in the embodiment of the first aspect described herein.PEO copolymerized with (3-acrylamidopropyl) trimethylammonium chloride or similar charged part can also be suitable.PEGylated lipid stabilizer is also suitable, including but not limited to PEG2000-MO, PEG-PT, DSPE-PEG (2000) amine, 18:0PEG2000 PE and DSPE-PEG (5000) amine.Many other such stabilizers are known in the art.

[0128] Other possible useful PEGylated lipids include but are not limited to PEG-modified phosphatidylethanolamine, PEG-modified phosphoric acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol and PEG-modified dialkylglycerol. In an embodiment, PEGylated lipids include PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE lipids and combinations thereof.

[0129] In embodiments, the nature of the stabilizer is selected based on the nature of the lipid, and the selection of these components and the understanding of their compatibility can be based on information known in the art.

[0130] The many steric stabilizers reported so far can be divided into four groups: (i) amphiphilic block copolymers (i.e., Poloxamer TM), (ii) PEGylated lipids, (iii) customized lipid-copolymers, and (iv) alternative steric stabilizers (e.g., bile salts, proteins). Ideally, the selected stabilizer will prevent the aggregation of particles by providing an electrostatic or more commonly a steric barrier between particles in proximity. Stabilizers that can best function in lipid particles of the present disclosure share similar properties, including (i) due to an asymmetric amphiphilic polymer structure with a larger hydrophilic domain, which is generally highly hydrophilic with a high HLB (hydrophilic-lipophilic balance) value. Importantly, the hydrophilic portion of the molecule is not surrounded by a hydrophobic region. High HLB can be achieved by using longer PEG chains or multiple PEG chains; (ii) the presence of hydrogen bond acceptors and the absence of hydrogen bond donors; and (iii) electrical neutrality. Those skilled in the art can select an appropriate stabilizer based on this. In addition, the following journal articles describe key aspects of stabilizers that may be suitable for use with the lipid particles of the present disclosure and are hereby incorporated by reference in their entirety: (i) JYT Chong, X. Mulet, BJ Boyd and CJ Drummond; "Steric Stabilizers for Cubic Phase Lyotropic Liquid Crystal Nanodispersions (Cubosomes)", Advances in Planar Lipid Bilayers and Liposomes, Vol. 21, Chapter 5, (2015) pp. 131-187, ISSN 1554-4516, Elsevier; (ii) J. Zhai, B. Fan, SHThang, CJ Drummond "Novel amphiphilic block copolymers for the formation of stimuli-responsive non-lamellar lipid nanoparticles" non-lamellar lipid nanoparticles)" "Molecules", 2021, 26, 3648-3664.; (iii) J. Zhai, R. Suryadinata, B. Luan, N. Tran, TMHinton, J. Ratcliffe, X. Hao and CJDrummond "Amphiphilic brush polymers produced by the RAFT polymerisation method stabilise and reduce the cell toxicity of lipidlyotropic liquid crystalline nanoparticles" Faraday Discussions, 2016, 191, 545-563.; Faraday Discussion 191 on Nanoparticles with Morphological and Functional Anisotropy; (iv) J. Zhai, T. J. Hinton, L. J. Waddington, C. Fong, N. Tran, X. Mulet, C. J. Drummond and B. W. Muir "Lipid-PEG Conjugates Sterically Stabilise and Reduce the Toxicity of Phytantriol-Based Lyotropic Liquid Crystalline Nanoparticles" Phytantriol-Based Lyotropic Liquid Crystalline Nanoparticles” Langmuir, 2015, 31, 10871–10880. (v) JYT Chong, X. Mulet, D. Keddie, LJ Waddington, S. T. Mudie, BJ Boyd and CJ Drummond “Novel Steric Stabilisers for Lyotropic Liquid Crystalline Nanoparticles: Pegylated Phytanyl Copolymers” Langmuir, 2015, 31, 2615–2629. (vi) JYT Chong, X. Mulet, A. Postma, DJ Keddie, LJ Waddington, BJ Boyd and CJDrummond "Novel RAFT Amphiphile Brush Copolymer Steric Stabilisers for Cubosomes: Poly(octadecylacrylate)-block-poly(polyethylene glycol methyl ether acrylate)" Soft Matter, 2014, 10, 6666-6676. (vii) A. Tilley, C. J. Drummond and B. J. Boyd "Disposition and Association of the Steric Stabiliser Pluronic F127 in Lyotropic Liquid Crystalline Nanostructured Particle Dispersions" J. Colloid and Interface Science Science, 2013, 392, 288-296. (viii) JYT Chong, X. Mulet, LJ Waddington, BJ Boyd and CJ Drummond "High Throughput Discovery of Novel Steric Stabilisers for Cubic Lyotropic Liquid Crystal Nanoparticle Dispersions" Langmuir, 2012, 28, 9223-9232. (ix) JYT Chong, X. Mulet, LJ Waddington, BJ Boyd and CJ Drummond "High Throughput Discovery of Novel Steric Stabilisers for Cubic Lyotropic Liquid Crystal Nanoparticle Dispersions" Langmuir, 2012, 28, 9223-9232.Drummond "Steric Stabilisation of Cubic Lyotropic Liquid Crystalline Nanoparticles: High Throughput Evaluation of Triblock Polyethylene Oxide-Polypropylene Oxide-Polyethylene Oxide Copolymers". Soft Matter, 2011, 7, 4768-4777.

[0131] In embodiments, a stabilizer is present during formation of the carrier of the first aspect.

[0132] In embodiments, the stabilizer is present at 1 wt % to 20 wt % or 5 wt % to 20 wt % of the carrier.

[0133] In an embodiment, the stabilizer is present in an amount of 6 wt % to 18 wt %.

[0134] In an embodiment, the stabilizer is present in an amount of 7 wt % to 16 wt %.

[0135] In an embodiment, the stabilizer is present in an amount of 8 wt % to 14 wt %.

[0136] Cationic and / or ionizable lipids

[0137] In an embodiment, the non-lamellar lyotropic liquid crystal phase carrier may further comprise at least one additional cation and / or ionizable lipid, such as one or more cations and / or ionizable lipids comprising cyclic or non-cyclic amines. The at least one additional cation and / or ionizable lipid may be selected from those known in the art. The at least one additional cation and / or ionizable lipid may be appropriately selected so that it does not destroy the structure of the non-lamellar lyotropic liquid crystal phase carrier.

[0138] Ionizable lipids are a class of lipid molecules that remain neutral at physiological pH, but are protonated at low pH, making them positively charged. As known to those skilled in the art, a variety of ionizable lipids have been developed and are commercially available.

[0139] Without wishing to limit the scope of applicable ionizable lipids, such lipids will typically have an amino-containing head group that can be protonated at acidic pH values. The pKa value of the ionizable lipid may be between 5.5 and 7.2, preferably between 5.9 and 6.8. The ionizable lipid also typically has at least one lipid chain, but preferably has two or more such tails, and it has been demonstrated that branches in at least one tail provide desired properties.

[0140] In an embodiment, the ionizable lipids may be selected from those described in WO2017 / 218704, WO2018 / 078053, WO2015 / 199952, WO2018 / 081480, WO2017 / 117528, WO2018 / 081638, WO2018 / 107026, WO2019 / 089828, WO2020 / 081938 and WO2021 / 030701, which are incorporated herein by reference in their entirety.

[0141] In certain embodiments of the present disclosure, the ionizable lipid may be an amino lipid having a structure of formula (I):

[0142] Cyc-LR(I)

[0143] in:

[0144] Cyc is a nitrogen heterocycle or a heteroaryl;

[0145] L is an amido linker; and

[0146] R is for C 10 To C 44 Carbon chain.

[0147] In an embodiment, the amino lipid has the structure of Formula (Ib):

[0148]

[0149] in:

[0150] Cyc is a 5- or 6-membered nitrogen heterocyclic group or heteroaryl group, the nitrogen heterocyclic group or heteroaryl group is optionally selected from the group consisting of pyridyl, pyrimidinyl, piperidinyl, piperazinyl, morpholinyl and pyrazinyl;

[0151] L is an amido linker;

[0152] R is for C 10 To C 44 Carbon chain, optionally C 12 To C 24 alkyl or alkenyl, which is optionally interrupted by one or more heteroatoms; and

[0153] n is an integer of 1 to 6.

[0154] In an embodiment, R is selected from the group consisting of oleyl, linoleoyl, linolenoyl, phytanoyl and farnesoyl.

[0155] In an embodiment, the amino lipid is selected from the group consisting of:

[0156]

[0157] wherein R is as defined above for formula (I) or (Ib).

[0158] In certain embodiments, the cationic and / or ionizable lipids can be selected from the non-limiting group consisting of: 3-(docosylamino)-N1,N1,4-triazolyl-1-piperazineethylamine (KL10), N1-[2-(docosylamino)ethyl]-N1,N4,N4-triazolyl-1,4-piperazinediethylamine (KL22), 14,25-tricosyl-15,18,21,24-tetraaza-triazodecane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl -[1,3]-dioxolane (DLin-K-DMA), 4-(dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-triacontriacontria-6,9,28,31-tetraene-19-methyl ester (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,1 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2R)), (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan -1-amine (octyl-CLinDMA (2S)), ((4-hydroxybutyl) azadiyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate)) (ALC-0315), 8-[(2-hydroxyethyl) [6-oxo-6-(undecyloxy) hexyl] amino] -octanoic acid, 1-octyl nonanoate, 4-hydroxybutyl) azadiyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate)), 8-[2-hydroxyethyl-(6-oxo-6-(undecyloxyhexyl) amino] octanoic acid heptadecan-9-yl ester (SM-102) and dioleoyl-3-trimethylammonium propane (DOTAP).

[0159] In embodiments, cationic and / or ionizable lipids are present during formation of the vector of the first aspect.

[0160] The amount of the cation / ionizable lipid in the carrier can be appropriately selected according to, for example, specific application, lipid composition, other carrier components such as the amount of auxiliary lipids or polymers and the desired properties of the carrier. Typically, cations and / or ionizable lipids can exist in 0.5wt% to 10wt% of the carrier.

[0161] In an embodiment, the cationic lipid is present at 0.5 wt% to 5 wt% of the vector. Advantageously, such amounts can be used to maintain high stability of the vector when using highly efficient cationic and / or ionizable lipids that exhibit effective transfection or delivery capabilities, and / or for applications where lower charge density or reduced toxicity is desired.

[0162] In an embodiment, the cationic lipid is present at 0.5 wt% to 10 wt% of the carrier. Advantageously, such amounts can be used to enhance the interaction between the cationic and / or ionizable lipid and the antigen, which can result in improved complex formation and cellular uptake.

[0163] antigen

[0164] In embodiments, the antigen is selected from the group consisting of: proteins, glycoproteins, peptides, glycopeptides, polysaccharides, lipids, glycolipids, lipoproteins, lipopeptides, and nucleic acids. In embodiments, the antigen is selected from the group consisting of: proteins, glycoproteins, peptides, glycopeptides, polysaccharides, lipids, glycolipids, lipoproteins, and lipopeptides. In embodiments, the antigen is selected from the group consisting of: proteins, glycoproteins, peptides, polysaccharides, lipids, and glycolipids. In embodiments, the antigen protein antigen is selected from the group consisting of: antigenic proteins, peptides, glycoproteins, or glycolipids.

[0165] When the antigen is an antigenic protein, peptide, glycoprotein or glycolipid, it may be derived from a pathogenic bacterial, viral or fungal organism or a cancer cell. Such organisms may include, but are not limited to, Streptococcus species, Candida species, Brucella species, Salmonella species, Shigella species, Pseudomonas species, Bordetella species, Clostridium species, Norwalk virus, Bacillus anthracis, Mycobacterium tuberculosis, human immunodeficiency virus (HIV), Chlamydia species, human papillomavirus, Japanese encephalitis virus, influenza virus, Paramyxovirus species, herpes virus, cytomegalovirus, Varicella-Zoster virus, Epstein-Barr virus, hepatitis virus, Plasmodium species, species), Trichomonas species, sexually transmitted disease pathogens, viral encephalitis pathogens, protozoan disease pathogens, fungal disease pathogens, prion disease pathogens, and bacterial disease pathogens.

[0166] In an embodiment, the antigen is hydrophobic or at least comprises at least one hydrophobic chain, such as one, two, three, four, five or six hydrophobic chains. In this context, "hydrophobic" will be understood to mean that the antigen has an overall hydrophobic property. Further, the terms "hydrophobic chain", "aliphatic chain" and "lipophilic chain" are used interchangeably herein. The hydrophobic chain is capable of associating with or embedding in a lipid bilayer.

[0167] Examples of suitable groups that can constitute a hydrophobic chain include, but are not limited to, fatty acids (lipids) including mycolic acids, glycerolipids, glycerophospholipids, sphingolipids, steroids including sterols, terpenes and terpenoids, glycolipids, polyketides, and poly (hydrophobic amino acids) (pHAA). In embodiments, the antigen comprises one or more hydrophobic chains independently selected from fatty acids (lipids) including mycolic acids, glycerolipids, glycerophospholipids, sphingolipids, steroids including sterols, terpenes and terpenoids, glycolipids, and polyketides. In embodiments, the antigen comprises one or more hydrophobic chains independently selected from fatty acids (lipids), glycerolipids, glycerophospholipids, and sphingolipids.

[0168] In an embodiment, one or more of the hydrophobic chains include or may be saturated or unsaturated hydrocarbon chains, and optionally oxidized derivatives thereof. The hydrocarbon chain may be straight or branched, and may contain a carbocycle in the chain, such as cyclopropane. Each such hydrocarbon chain may independently contain at least 10, 20, 30, 40 or 50 carbon atoms in the chain. Each such minimum total carbon chain number may be combined with each of the upper limits of 100, 90, 80, 70 or 60 carbon atoms in the chain. In an embodiment, one or more of the hydrophobic chains comprises or has 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 30 to 100, 30 to 90, 30 to 80, 30 to 70, 30 to 60, 40 to 100, 40 to 90, 40 to 80, 40 to 70, 40 to 60, 50 to 100, 50 to 90, 50 to 80, 50 to 70 or 50 to 60 carbon atoms in the chain. Each hydrocarbon chain can independently be saturated or unsaturated. Each hydrocarbon chain can independently be straight or branched and can include carbocyclic rings such as cyclopropane in the chain. Each hydrocarbon chain can independently include one, two, three or four oxygen interruptions in the chain. Each hydrocarbon chain may be independently substituted with one, two, three or four oxygen-containing functional groups, such as hydroxyl, methoxy and keto groups.

[0169] In an embodiment, one or more of the hydrophobic chains comprise a carbocycle which may be saturated or unsaturated, and include polycyclic carbocycles, and include fused, bridged, and spirocyclic systems. Examples of suitable carbocycles include, but are not limited to, monocyclic carbocycles, such as cyclopropane, and polycyclic carbocycles, such as steroids including sterols such as cholesterol. In an embodiment, one or more of the hydrophobic chains comprise or consist of a carbocycle and a hydrocarbon chain.

[0170] In an embodiment, the total number of carbon atoms combined in all such hydrophobic chains can be at least 20, 30, 40, 50, 60, 70 or 80 carbon atoms. Each such minimum total carbon chain number can be combined with each of the upper limits of 200, 150 or 100 carbon atoms. In an embodiment, the total number of carbon atoms in the hydrophobic chains of the combined antigens is about 20 to about 200 carbon atoms, about 20 to about 150 carbon atoms, about 20 to about 100 carbon atoms, about 30 to about 200 carbon atoms, about 30 to about 150 carbon atoms, about 30 to about 100 carbon atoms, about 40 to about 200 carbon atoms, about 40 to about 150 carbon atoms, about 40 to about 100 carbon atoms, about 50 to about 20 In some embodiments, the present invention relates to a moiety having at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms, at least 0 carbon atoms

[0171] In other embodiments, one or more of the hydrophobic chains comprise a hydrophobic peptide, such as a poly(hydrophobic amino acid) (pHAA), such as poly(Phe), poly(Leu), poly(Val), etc. The hydrophobicity of such chains can be measured by methods known in the art, such as the hydropathicity index, which quantifies the hydrophobicity or hydrophilicity of amino acid residues in a protein sequence. It should be understood that such methods can also be used to measure the hydrophobicity of amino acid-containing antigens, such as lipoproteins and lipoproteins.

[0172] When the antigen is hydrophobic or comprises at least one hydrophobic chain, it can be associated with or embedded in the lipid bilayer forming the wall or channel of the carrier. It has been surprisingly found that the incorporation of antigens, even with multiple hydrophobic chains of significant length, such as those of trehalose dimycolate (cord factor or TDM), does not negatively affect the architecture of the carrier and still allows successful delivery of the antigen.

[0173] In embodiments, the antigen is hydrophilic.

[0174] In embodiments, the antigen is contained within the lipid layer of the non-lamellar lyotropic liquid crystal phase vehicle.

[0175] In embodiments, the antigen is contained within the aqueous channels of the non-lamellar lyotropic liquid crystal phase carrier.

[0176] In embodiments, the antigen is a subunit vaccine antigen.

[0177] In an embodiment, relative to the total lipid in the content of the non-lamellar lyotropic liquid crystal phase carrier, the antigen is present in about 0.1mol% to about 20mol%. In some instances, the antigen can be present in about 0.1mol% to about 10mol%, about 0.1mol% to about 9mol%, about 0.1mol% to about 8mol%, about 0.1mol% to about 7mol%, about 0.1mol% to about 6mol%, about 0.1mol% to about 5mol%, about 0.1mol% to about 4mol%, about 0.1mol% to about 3mol%, about 0.1mol% to about 2mol%, about 0.5mol% to about 10mol%, about 0.5mol% to about 9mol%, about 0.5mol% to about 8mol%, about 0.5mol% to about 7mol%, about 0.5mol% to about 6mol%, about 0.5mol% to about 5mol%, about 0.5mol% to about 4mol%, about 0.5mol% to about 3mol% or about 0.5mol% to about 2mol%.

[0178] In embodiments, the antigen is present at 0.5 mol % to 5 mol % relative to the total lipids in the contents of the non-lamellar lyotropic liquid crystal phase carrier.

[0179] In an embodiment, the antigen is present at about 1 mol % relative to the total lipids in the contents of the non-lamellar lyotropic liquid crystal phase carrier.

[0180] Antigens can be selected from pathogenic antigens, tumor antigens, allergic antigens or autoimmune self-antigens. In an embodiment, the antigen is a pathogenic antigen. Such pathogenic antigens can be antigens derived from pathogenic organisms, specifically bacteria, viruses or protozoa (multicellular) pathogenic organisms, which cause an immune response in mammalian subjects such as humans. Pathogenic antigens can be surface or cell surface expressed antigens, such as proteins or parts or fragments thereof, at least partially located on the surface of viruses or bacteria or protozoa organisms.

[0181] Pathogenic antigens of interest may include antigens derived from one or more of the following: Acinetobacter baumannii, Anaplasma genus, Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostoma duodenale, Arcanobacterium haemolyticum, Ascaris lumbricoides, Aspergillus genus, Astroviridae, Babesia genus, Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus, Blastocystshominis, Blastomyces dermatitidis. dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus, Brugia malayi, Bunyaviridae family, Burkholderia cepacia and other Burkholderia species, Burkholderia mallei, Burkholderia pseudomallei, Caliciviridae family, Campylobacter genus, Candida albicans, Candida spp, Chlamydia trachomatis trachomatis), Chlamydophilapneumoniae, Chlamydophila psittaci, QD prions, Clonorchis sinensis, Clostridium botulinumbotulinum), Clostridium difficile, Clostridium perfringens, Clostridium perfringens, Clostridium spp, Clostridium tetani, Coccidioides spp, coronavirus, Corynebacterium diphtheriae, Coxiella burnetii, Crimean-Congo haemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus, cytomegalovirus (CMV), dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4), Dientamoeba fragilis, Ebola virus (EBOV), Echinococcus spp. genus, Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia genus, Entamoeba histolytica, Enterococcus genus, Enterovirus genus, Enteroviruses, mainly Coxsackie A virus and enterovirus 71 (EV71), Epidermophyton spp., Epstein-Barr virus (EBV), Escherichia coli 0157:H7, O111, and O104:H4, Fasciola hepatica and Fasciola gigantica, FFI prions, Filarioidea superfamily, Flaviviruses, Francisella tularensis, tularensis), Fusobacterium genus, Geotrichum candidum, Giardia intestinalis, Gnathostoma spp., GSS prions, Guanarito virusvirus), Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, Henipavirus (Hendra virus, Nipah virus), Hepatitis A virus, Hepatitis B virus (HBV), Hepatitis C virus (HCV), Hepatitis D virus, Hepatitis E virus, Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Histoplasma capsulatum, HIV (human immunodeficiency virus), Hortaea werneckii, Human bocavirus (HBoV), Human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7), Human metapneumovirus (hMPV), Human papillomavirus (HPV), Human parainfluenza virus (HPIV), Japanese encephalitis virus, JC virus, Junin virus (Junin virus), virus), Kingella kingae, Klebsiella granulomatis, Kurupiron, Lassa virus, Legionella pneumophila, Leishmania genus, Leptospira genus, Listeria monocytogenes, Lymphocytic choriomeningitis virus (LCMV), Machupovirus, Malassezia spp., Marburg virus, Measlesvirus, Metagonimus yokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV), Mumps virus virus), Mycobacterium leprae and Mycobacterium lepromatosis, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Naegleria fowleri, Necatoramericanus), Neisseria gonorrhoeae, Neisseria meningitidis, Nocardiaasteroides, Nocardia spp, Onchocerca volvulus, Orientia tsutsugamushi, Orthomyxoviridae family (influenza), Paracoccidioides brasiliensis, Paragonimus spp, Paragonimus westermani, Parvovirus B19, Pasteurella genus, Plasmodium genus, Pneumocystis jirovecii jirovecii), poliovirus, rabies virus, respiratory syncytial virus (RSV), Rhinovirus, rhinoviruses, Rhodococcus spp, Rickettsia akari, Rickettsia genus, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi, Rift Valley fever virus, Rotavirus, Rubella virus, Sabia virus, Salmonella genus, Sarcoptes scabiei, SARS coronavirus, Schistosoma genus, Shigella genus, Sin Nombre virus virus), Hantavirus, Sphingomonas spp, Sporothrix schenckii, Staphylococcus genus, Staphylococcus spp, Streptococcus agalactiae, Streptococcus pneumoniaepneumoniae, Streptococcus pyogenes, Strongyloides stercoralis, Taeniagenus, Taenia solium, Tick-borne encephalitis virus (TBEV), Toxocara canis or Toxocara cati, Toxoplasma gondii, Treponemapallidum, Trichinella spiralis, Trichomonas vaginalis, Trichophyton spp, Trichuris trichiura, Trypanosoma brucei, Trypanosoma cruzi, Ureaplasma urealyticum, Varicella zoster virus (VZV), Variola major major or Variola minor, vCJD prion, Venezuelan equine encephalitis virus, Vibriocholerae, West Nile virus, Western equine encephalitis virus, Wuchereria bancrofti, yellow fever virus, Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis.

[0182] In certain embodiments, the relevant antigens can be derived from a pathogen selected from the group consisting of severe acute respiratory syndrome (SARS), severe acute respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-1 and SARS-CoV-2), influenza virus, respiratory syncytial virus (RSV), herpes simplex virus (HSV), human papillomavirus (HPV), human immunodeficiency virus (HIV), Plasmodium, Staphylococcus aureus, dengue virus, Chlamydia trachomatis, cytomegalovirus (CMV), hepatitis B virus (HBV), Mycobacterium tuberculosis, rabies virus, and yellow fever virus.

[0183] In some embodiments, the relevant pathogenic antigens can be selected from: outer membrane protein A OmpA, biofilm-associated protein Bap, transport protein MucK (Acinetobacter baumannii, Acinetobacter infection); variable surface glycoprotein VSG, microtubule-associated protein MAPP15, trans-sialidase TSA (Trypanosoma brucei, African sleeping sickness (African trypanosomiasis)); HIV p24 antigen, HIV envelope protein (Gpl20, Gp41, Gpl60), polyprotein GAG, negative factor protein Nef, transcription transactivator Tat (HIV (human immunodeficiency virus), AIDS (acquired immunodeficiency syndrome)); galactose-inhibitory adhesion protein GIAP, 29kDa antigen Eh29, Gal / GalNAc lectin, protein CRT, 125kDa immunodominant antigen, protein M17, adhesin ADH112, protein STIRP (entamoeba histolytica, amoebiasis); major surface proteins 1-5 (MSP1a, MSP1b, MSP2, MSP3, MS P4, MSP5), type IV secretion system proteins (VirB2, VirB7, VirB11, VirD4) (Anaplasma, Anaplasmosis); protective antigen PA, edema factor EF, lethal factor LF, S layer homologous protein SLH (Bacillus anthracis, Anthrax); acranolysin, phospholipase D, collagen binding protein CbpA (Bacillus hemolyticus, Bacillus hemolyticus infection); nucleocapsid protein NP, glycoprotein precursor GPC, glycoprotein GP1, glycoprotein GP2 (Junín virus, Argentine hemorrhagic fever fever); chitin-protein layer protein, 14kDa surface antigen A14, major sperm protein MSP, MSP polymerization-organizing protein MPOP, MSP fiber protein 2MFP2, MSP polymerization-activated kinase MPAK, ABA-1-like protein ALB, protein ABA-1, epidermal CUT-1 (human roundworm, Ascariasis); 41kDa allergen Asp v13, allergen Aspf3, major conidia surface protein rod A, protease Pep1p, GPI anchor protein Ge11p, GPI anchor protein Crf1p (Aspergillosis); family VP26 protein, VP29 protein (Astroviridae, astrovirus infection); rod-associated protein 1RAP-1, merozoite surface antigen MSA-1, MSA-2 (a1, a2, b, c), 12D3, 11C5, 21B4, P29, variant erythrocyte surface antigen VESA1, apical membrane antigen 1AMA-1 (Babesia, Babesiosis);Hemolysin, enterotoxin C, PXO1-51, glycolate oxidase, ABC-transporter, penicillin-binding protein, zinc transporter family protein, pseudouridine synthase Rsu, plasmid replication protein RepX, oligoendopeptidase F, prophage membrane protein, protein HemK, flagellar antigen H, 28.5-kDa cell surface antigen (Bacillus cereus, Bacillus cereus infection); large T antigen LT, small T antigen, capsid protein VP1, capsid protein VP2 (BK virus, BK virus infection); 29kDa-protein, caspase-3-like antigen, glycoprotein (Blastocystis hominis, Blastomycosis hominis infection); yeast surface adhesin WI-1 (Blastomycosis dermatitidis, blastomycosis); nucleoprotein N, polymerase L, matrix protein Z, glycoprotein GP (Machupo virus, Bolivian hemorrhagic fever); outer surface protein A OspA, outer surface protein OspB, outer surface protein OspC, decorin binding protein A DbpA, decorin binding protein B DbpB, flagellar filament 41kDa core protein F1a, basic membrane protein A precursor BmpA (immunodominant antigen P39), outer surface 22kDa lipoprotein precursor (antigen IPLA7), variable surface lipoprotein v1sE (Borrelia, Borrelia infection); botulinum neurotoxins BoNT / A1, BoNT / A2, BoNT / A3, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, recombinant botulinum toxin F Hc domain FHc (Clostridium botulinum, botulism (and infant botulism)); nucleocapsid, glycoprotein precursor (Sabiya virus, Brazilian hemorrhagic fever (Brazilian hemorrhagic fever) fever); copper / zinc superoxide dismutase SodC, bacterial ferritin Bfr, ​​50S ribosomal protein RpIL, protein Omp31 containing OmpA-like transmembrane domain, immunogenic 39-kDa protein M5 P39, zinc ABC transporter periplasmic zinc-binding protein znuA, periplasmic immunogenic protein Bp26, 30S ribosomal protein S12 RpsL, glyceraldehyde-3-phosphate dehydrogenase Gap, 25-kDa outer membrane immunogenic protein precursor Omp25, invasion protein B1a1B, trigger factor Tig, molecular chaperone DnaK, putative peptidyl-prolyl cis-trans isomerase SurA, lipoprotein Omp19, outer membrane protein MotY Omp16, conserved outer membrane protein D15, malate dehydrogenase Mdh, type IV secretion system component (T4SS) VirJ, lipoprotein of unknown function BAB1_0187 (Brucella spp., Brucellosis);ABC transporter family members (LolC, OppA and PotF), putative lipoprotein release system transmembrane proteins LolC / E, flagellar protein FliC, Burkholderia intracellular motility A BimA, bacterial elongation factor-Tu EF-Tu, 17kDa OmpA-like protein, boaA-encoded protein, boaB-encoded protein (Burkholderia cepacia and other Burkholderia species, Burkholderia infection); Mycobacterial acyltransferase Ag85A, heat shock protein Hsp65, protein TB10.4, 19kDa antigen, protein PstS3, heat shock protein Hsp70 (Mycobacterium ulcerans, Buruli ulcer (Buruli ulcer); Norovirus major and minor capsid proteins VP1 and VP2, Zarovirus capsid protein VP1, protein Vp3, genomic polyprotein (Caliviridae, Calicivirus infection (Norovirus and Zarovirus)); major outer membrane protein PorA, flagellin FlaA, surface antigen CjaA, fibronectin binding protein CadF, aspartate / glutamate binding ABC transporter Peb1A, protein FspA1, protein FspA2 (Campylobacter, Campylobacteriosis); glycolytic enzyme enolase, secreted aspartyl protease SAP1-10, glycophosphatidylinositol (GPI)-linked cell wall protein, protein Hyr1, complement receptor 3-related protein CR3-RP, adhesin A1s3p, heat shock protein 90kDa hsp90, cell surface hydrophobic protein CSH (usually Candida albicans and other Candida species, Candidiasis); 17-kDa antigen, protein P26, trimeric autotransporting adhesin TAA, Bartonella adhesin ABadA, variably expressed outer membrane protein Vomps, protein Pap3, protein HbpA, envelope-associated protease HtrA, protein OMP89, protein GroEL, protein La1B, protein OMP43, dihydrolipoamide succinyltransferase SucB (Bartonella henselae, cat-scratch disease disease); amastigote surface protein-2, amastigote specific surface protein SSP4, cruzipain, trans-sialidase TS, trypanosome surface glycoprotein TSA-1, complement regulatory protein CRP-10, protein G4, protein G2, paraaxonal rod protein PAR2, paraflagellar rod component Pari, mucin-associated surface protein MPSP (Trypanosoma cruzi, Chagas Disease)(American trypanosomiasis)); envelope glycoproteins (gB, gC, gE, gH, gl, gK, gL), (Varicella zoster virus (VZV), chickenpox);Major outer membrane protein MOMP, possible outer membrane protein PMPC, outer membrane complex protein B OmcB, heat shock protein Hsp60 HSP10, protein IncA, protein from type III secretion system, ribonucleotide reductase small chain protein NrdB, plasmid protein Pgp3, Chlamydia outer membrane protein N CopN, antigen CT521, antigen CT425, antigen CT043, antigen TC0052, antigen TC0189, antigen TC0582, antigen TC0660, antigen TC0726, antigen TC0816, antigen TC0828 (Chlamydia trachomatis, Chlamydia); low calcium response protein E LCrE, Chlamydia outer protein N CopN, serine / threonine protein kinase PknD, acyl-carrier-protein S-malonyltransferase FabD, single-stranded DNA binding protein Ssb, major outer membrane protein MOMP, outer membrane protein 2 Omp2, polymorphic membrane protein family (Pmp1, Pmp2, Pmp3, Pmp4, Pmp5, Pmp6, Pmp7, Pmp8, Pmp9, Pmp10, Pmp11, Pmp12, Pmp13, Pmp14, Pmp15, Pmp16, Pmp17, Pmp18, Pmp19, Pmp20, Pmp21), (Chlamydophila pneumoniae, Chlamydophila pneumoniae infection); cholera toxin B CTB, toxin co-regulatory pilin ATcpA, toxin co-regulatory pilin TcpF, toxin co-regulatory pilin biosynthesis protein FTcpF, cholera toxin subunit A, cholera toxin subunit B, heat-stable enterotoxin ST, mannose-sensitive hemagglutinin MSHA, outer membrane protein U porin ompU, porin B protein, polymorphic membrane protein D (Vibrio cholerae, cholera); propionyl-CoA carboxylase PCC, 14-3-3 protein, inhibin, cysteine ​​protease, glutathione transferase, gelsolin, cathepsin L protease CatL, envelope protein 20.8kDa TP20.8, envelope protein 31.8kDa TP31.8, lysophosphatidic acid phosphatase LPAP, (Clonorchiasis sinensis, Clonorchiasis sinensis); surface protein SLPs, glutamate dehydrogenase antigen GDH, toxin A, toxin B, cysteine ​​protease Cwp84, cysteine ​​protease Cwp13, cysteine ​​protease Cwp19, cell wall protein CwpV, flagellin FliC, flagellin FliD (Clostridium difficile, Clostridium difficile infection); rhinovirus: capsid protein VP1, VP2, VP3, VP4; coronavirus: spike protein S, envelope protein E, membrane protein M, nucleocapsid protein N (including rhinovirus and coronavirus, common cold (acute viral nasopharyngitis; acute rhinitis)); prion protein Prp (CJD prion, Creutzfeldt-Jakob disease (Creutzfeldt-Jakob disease, CJD));Envelope protein Gc, envelope protein Gn, nucleocapsid protein (Crimean-Congo hemorrhagic fever virus, Crimean-Congo hemorrhagic fever (CCHF)); virulence-related DEAD-box RNA helicase VAD1, galactomannan-protein GalXM, glucuronoxymannan GXM, mannoprotein MP (Cryptococcus neoformans, cryptococcosis); acidic ribosomal protein P2 CpP2, mucin antigens Muc1, Muc2, Muc3, Muc4, Muc5, Muc6, Muc7, surface adhesion protein CP20, surface adhesion protein CP23, surface protein CP12, surface protein CP21, surface protein CP40, surface protein CP60, surface protein CP15, surface-associated glycopeptide gp40, surface-associated glycopeptide gp15, oocyst wall protein AB, profibrillar protein PRF, apyrase (Cryptosporidiosis); fatty acid and retinol binding Protein-1FAR-1, tissue inhibitor of metalloproteinase TIMP (TMP), cysteine ​​protease ACEY-1, cysteine ​​protease ACCP-1, surface antigen Ac-16, secretory protein 2ASP-2, metalloproteinase 1MTP-1, aspartyl protease inhibitor API-1, surface-associated antigen SAA-1, adult-specific secretory factor Xa serine protease inhibitor anticoagulant AP, cathepsin D-like aspartic protease ARR-1 (usually Brazilian hookworm; various other parasites, cutaneous larva migrans (Cutaneous larva migrans, CLM); cathepsin L-like protease, 53 / 25-kDa antigen, 8-kDa family member, cysticercoid protein TsAg5 with marginal trypsin-like activity, hexapod protein TSOL18, hexapod protein TSOL45-1A, lactate dehydrogenase A LDHA, lactate dehydrogenase B LDHB (hook tapeworm, cysticercosis); pp65 antigen, membrane protein pp15, capsid proximal envelope protein pp150, protein M45, DNA polymerase UL54, helicase UL105, glycoprotein gM, glycoprotein gN, glycoprotein H, glycoprotein B gB, protein UL83, protein UL94, protein UL99 (cytomegalovirus (CMV), cytomegalovirus infection); capsid protein C, premembrane protein prM, membrane protein M, envelope protein E (domain I, domain II, domain II), protein NS1, protein NS2A, protein NS2B, protein NS3, protein NS4A, protein 2K, protein NS4B, protein NS5 (dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4) - flavivirus, dengue fever);39kDa protein (Dienucleate amoeba fragilis, dientamoebiasis); diphtheria toxin precursor Tox, diphtheria toxin DT, pilus protein specific sortase SrtA, axial pilus protein SpaA, tip pilus protein SpaC, minor pilus protein SpaB, surface-associated protein DIP1281 (Corynebacterium diphtheriae, diphtheria); nucleoprotein NP, minor matrix protein VP24, major matrix protein VP40, transcription activator VP30, polymerase cofactor VP35, RNA polymerase L (Ebola virus (EBOV), Ebola hemorrhagic fever); prion protein (vQD prion, variant Heteromorphic Creutzfeldt-Jakob disease (vCJD, nvCJD); UvrABC system protein B, protein F1p1, protein F1p2, protein F1p3, protein TadA, hemoglobin receptor HgbA, outer membrane protein TdhA, protein CpsRA, regulatory protein CpxR, protein SapA, 18kDa antigen, outer membrane protein NcaA, protein LspA, protein LspA1, protein LspA2, protein LspB, outer membrane component DsrA, lectin DltA, lipoprotein Hip, major outer membrane protein OMP, outer membrane protein OmpA2 (Haemophilus ducreyi, Chancroid); aspartyl protease 1Pepl, phospholipase B PLB, α-mannosidase 1AMN1, glucosyltransferase GEL1, urease URE, peroxisomal matrix protein Pmp1, proline-rich antigen Pra, human T-cell response protein TcrP (Coccidioides immitis and Coccidioides posadasii, Coccidioidomycosis); allergen Tri r2, heat shock protein 60Hsp60, fungal actin Act, antigen Tri r2, antigen Tri r4, antigen Tri t1, protein IV, glycerol-3-phosphate dehydrogenase Gpdl, osmotic sensor HwSholA, osmotic sensor HwSholB, histidine kinase HwHhk7B, allergen Mala s 1, allergen Mala s 11, thioredoxin Trx Mala s 13, allergen Mala f, allergen Mala s (usually Trichophyton, Epidermophyton, Malassezia, Wernicke's Exophiala, Dermatophytosis);Protein EG95, protein EG10, protein EG18, protein EgA31, protein EM18, antigen EPC1, antigen B, antigen 5, protein P29, protein 14-3-3, 8-kDa protein, myophilin, heat shock protein 20HSP20, glycoprotein GP-89, fatty acid binding protein FAPB (Echinococcus, echinococcosis); major surface protein 2MSP2, major surface protein 4MSP4, MSP variant SGV1, MSP variant SGV2, outer membrane protein OMP, outer membrane protein 19OMP-19, major antigen protein MAPI, major antigen protein MAP1-2, major antigen protein MAP1B, major antigen protein MAP1-3, Erum2510 encoded protein, protein GroEL, protein GroES, 30-kDA major outer membrane protein, GE 100-kDa protein, GE 130-kDa protein, GE160-kDa protein (Ehrlichia, Ehrlichiosis); secreted antigen SagA, SagA-like proteins SalA and SalB, collagen adhesin Scm, surface proteins Fms1 (EbpA(fm)), Fms5 (EbpB(fm)), Fms9 (EpbC(fm)) and Fms10, protein EbpC(fm), 96kDa immunoprotective glycoprotein G1 (Enterococcus, enterococcal infection); polymerase 3D, viral capsid protein VP1, viral capsid protein VP2, viral capsid protein VP3, viral capsid protein VP4, protease 2A, protease 3C (Enterovirus, enterovirus infection); outer membrane protein OM, 60kDa outer membrane protein, cell surface antigen OmpA , cell surface antigen OmpB (sca5), 134kDa outer membrane protein, 31kDa outer membrane protein, 29.5kDa outer membrane protein, cell surface protein SCA4, cell surface protein Adr1 (RP827), cell surface protein Adr2 (RP828), cell surface protein SCA1, invasion protein invA, cell division protein fts, secretory protein sec family, virulence protein virB, tlyA, tlyC, small protein-like protein Pip, preprotein translocase SecA, 120-kDa surface protein antigen SPA, 138kD complex antigen, major 100-kD protein (protein I), cytoplasmic protein D, protective surface protein antigen SPA (Corickettsia prowazekii, Epidemictyphus);Epstein-Barr nuclear antigens (EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA leader protein (EBNA-LP)), latent membrane proteins (LMP-1, LMP-2A, LMP-2B), early antigen EBV-EA, membrane antigen EBV-MA, viral capsid antigen EBV-VCA, alkaline nuclease EBV-AN, glycoprotein H, glycoprotein gp350, glycoprotein gpIIO, glycoprotein gp42 , glycoprotein gHgL, glycoprotein gB (Epstein-Barr virus (EBV), Epstein-Barr virus infectious mononucleosis); capsid protein VP2, capsid protein VP1, major protein NS1 (parvovirus B19, erythema infectiosum (fifth disease)); pp65 antigen, glycoprotein 105, major capsid protein, envelope glycoprotein H, protein U51 (human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7), Exanthem infantum subitum); thioredoxin glutathione reductase TGR, cathepsins L1 and L2, Kunitz-type protein KTM, leucine aminopeptidase LAP, cysteine ​​protease Fas2, saponin-like protein-2SAP-2, thioredoxin peroxidase TPx, Prx-1, Prx-2, cathepsin 1 cysteine ​​protease CL3, protease cathepsin L CL1, phosphoglycerate kinase PGK, 27-kDa secreted protein, 60-kDa protein HSP35-α, glutathione transferase GST, 28.5-kDa envelope antigen 28.5 kDa TA, cathepsin B3 protease CatB3, type I cysteine ​​protease stefin-1, cathepsin L5, cathepsin Llg and cathepsin B, fatty acid binding protein FABP, leucine amino polypeptide LAP (Fasciolosis and Fasciolosis gigantea, Fasciolosis); prion protein (FFI prion, fatal familial insomnia (FFI));Venom allergen homolog-like protein VAL-1, abundant larval transcript ALT-1, abundant larval transcript ALT-2, thioredoxin peroxidase TPX, wasp allergen homolog VAH, thioredoxin peroxidase 2 TPX-2, antigenic protein SXP (peptide N, N1, N2 and N3), activation-associated protein-1 ASP-1, thioredoxin TRX, transglutaminase BmTGA, glutathione-S-transferase GST, myosin, wasp allergen homolog VAH, 175 kDa collagenase, glyceraldehyde-3-phosphate dehydrogenase GAPDH, cuticular collagen Col-4, secreted larval acidic protein SLAP, chitinase CHI-1, maltose binding protein MBP, glycolytic enzyme fructose-1,6-bisphosphate aldolase Fba, tropomyosin TMY-1, nematode-specific gene product OvB20, onchocystatin CPI-2, Cox-2 (Filarioidea, Filariasis); Phospholipase C PLC, Heat-labile enterotoxin B, Iota toxin component lb, Protein CPE1281, Pyruvate ferredoxin oxidoreductase, Elongation factor G EF-G, Aerolysin O Pfo, glyceraldehyde-3-phosphate dehydrogenase GapC, fructose bisphosphate aldolase Alf2, Clostridium perfringens enterotoxin CPE, alpha toxin AT, alpha toxin ATd, epsilon toxin ETd, protein HP, macrocytotoxin TpeL, endo-β-N-acetylglucosaminidase Naglu, phosphoglycerate mutase Pgm (Clostridium perfringens, Clostridium perfringens food poisoning); leukotoxin IktA, adhesion FadA, outer membrane protein RadD, high molecular weight arginine binding protein (Fusobacterium, Fusobacterium infection); Clostridium species, gas gangrene (clostridial myonecrosis); lipase A, lipase B, peroxidase Decl (Geotrichum candidum, Geotrichosis); prion protein (GSS prion, Gerstmann-Strauss-Shahn syndrome (GS S)); cyst wall protein CWP1, CWP2, CWP3, variant surface protein VSP, VSP1, VSP2, VSP3, VSP4, VSP5, VSP6, 56kDa antigen, pyruvate ferredoxin oxidoreductase PFOR, alcohol dehydrogenase EADHE, α-Giardia, α-8 Giardia, α-1 Giardia and β-Giardia, cysteine ​​protease, glutathione-S-transferase GST, arginine deaminase ADI, fructose-1,6-bisphosphate aldolase FBA, Giardia trophozoite antigen GTA (GTA1, GTA2), ornithine carboxyltransferase OCT, striped fiber-subcrystalline protein-like protein SALP, uridine phosphorylation protein UPL, α-tubulin, β-tubulin (Giardia intestinalis, Giardiasis);Cyclophilin CyP, 24kDa third-stage larval protein GS24, excretion-secretion products ESPs (40, 80, 120 and 208kDa) (Gnathostomaspinigerum and Gnathostoma hispidum, Gnathostomiasis); fimbriae proteins, minor fimbriae protein-related subunit pilC, major fimbriae protein subunits and variants pilE, pilS, phase variant protein porA, porin B PorB, protein TraD, Neisseria outer membrane antigen H.8, 70kDa antigen, major outer membrane protein PI, outer membrane proteins PIA and PIB, W antigen, surface protein A NspA, transferrin binding protein TbpA, transferrin binding protein TbpB, PBP2, mtrR encoding protein, ponA encoding protein, membrane permease FbpBC, FbpABC protein system, LbpAB protein, outer membrane protein ompA, outer membrane transport protein FetA, iron inhibition regulator MpeR (Neisseria gonorrhoeae, gonorrhea); outer membrane protein C ompC, outer membrane protein K17 OmpK17 (Klebsiella granulomatosis, granuloma inguinale (Donovan disease)); fibronectin binding protein Sfb, fibronectin / fibrinogen binding protein FBP54, fibronectin binding protein FbaA, M protein type 1 Emml, M protein type 6 Emm6, immunoglobulin binding protein 35Sib35, surface protein R28 Spr28, superoxide dismutase SOD, C5a peptidase ScpA, antigen I / II Agl / II, adhesin AspA, G-related α2-macroglobulin binding protein GRAB, surface fibrillin M5 (Streptococcus pyogenes, group A streptococcal infection); C protein beta antigen, arginine deaminase protein, adhesin BibA, 105kDA protein BPS, surface antigen C, surface antigen R, surface antigen X, trypsin-resistant protein Rl, trypsin-resistant protein R3, trypsin-resistant protein R4, surface immunogenic protein Sip, surface protein Rib, leucine-rich repeat protein LrrG, serine-rich repeat protein Srr-2, C protein α-antigen Bca, β antigen Bag, surface antigen ε, α-like protein ALP1, α-like protein ALP5 surface antigen δ, α-like protein ALP2, α-like protease ALP3, α-like protein ALP4, Cβ protein Bac (Streptococcus agalactiae, group B streptococcal infection);Transferrin binding protein 2Tbp2, phosphatase P4, outer membrane protein P6, peptidoglycan-associated lipoprotein Pal, protein D, protein E, adhesion and permeation protein Hap, outer membrane protein 26Omp26, outer membrane protein P5 (filament protein), outer membrane protein D15, outer membrane protein OmpP2, 5'-nucleotidase NucA, outer membrane protein PI, outer membrane protein P2, outer membrane lipoprotein Pep, lipoprotein E, outer membrane protein P4, fucoidan kinase FucK, [Cu,Zn]-superoxide dismutase SodC, protease HtrA, protein 0145, α-galactosylceramide (Haemophilus influenzae, Haemophilus influenzae infection); protease 2A , protease 3C (enterovirus, mainly coxsackie A virus and enterovirus 71 (EV71), hand, foot and mouth disease (HFMD)); RNA polymerase L, protein L, glycoprotein Gn, glycoprotein Gc, nucleocapsid protein S, envelope glycoprotein Gl, nucleoprotein NP, protein N, polyprotein M (Sin Nombre virus, Hantavirus, Hantavirus pulmonary syndrome (HPS)); heat shock protein HspA, heat shock protein HspB, citrate synthase GltA, protein UreB, heat shock protein Hsp60, neutrophil activation protein NAP, catalase KatA, vacuolating cytotoxin VacA, urease α urea, urease β UreB, protein Cpn lO, protein groES, heat shock protein HsplO, protein MopB, cytotoxicity-related 10kDa protein CAG, 36kDa antigen, β-lactamase HcpA, β-lactamase HcpB (Helicobacter pylori, Helicobacter pylori infection); integral membrane protein, aggregation-prone protein, O-antigen, toxin-antigen Stx2B, toxin-antigen StxlB, adhesion-antigen fragment Int28, protein EspA, protein EspB, tight adhesion (Intimin), protein Tir, protein IntC300, protein Eae (Escherichia coli O157:H7, O111 and O104:H4, hemolytic urinary tract infection); Hepatitis B virus (HBV) infection); hepatitis B surface antigen HBsAg, hepatitis B core antigen HbcAg, polymerase, protein Hbx, preS2 surface protein, surface protein L, large S protein; envelope glycoprotein El gp32gp35, envelope glycoprotein E2 NS1 gp68 gp70, capsid protein C, core protein core, polyprotein, antigen G, protein NS3, protein NS5A, (hepatitis C virus, hepatitis C);Hepatitis D antigen (large), hepatitis D antigen (small) (hepatitis D virus, hepatitis D); capsid protein E2 (hepatitis E virus, hepatitis E);Glycoprotein L UL1, uracil-DNA glycosidase UL2, protein UL3, protein UL4, DNA replication protein UL5, portal protein UL6, virion maturation protein UL7, DNA helicase UL8, replication origin binding protein UL9, glycoprotein M UL10, protein UL11, alkaline exonuclease UL12, serine-threonine protein kinase UL13, envelope protein UL14, terminase UL15, envelope protein UL16, protein UL17, capsid protein VP23 UL18, major capsid protein VP5 UL19, membrane protein UL20, envelope protein UL21, glycoprotein H (UL22), thymidine kinase UL23, protein UL24, protein UL25, capsid protein P40 (UL26, VP24, VP22A), glycoprotein B (UL27), ICP18.5 protein (UL28), major DNA binding protein ICP8 (UL29), DNA polymerase UL30, nuclear matrix protein UL31, envelope glycoprotein UL32, protein UL33, inner nuclear membrane protein UL34, capsid protein VP26 (UL35), Large envelope protein UL36, capsid assembly protein UL37, VP19C protein (UL38), ribonucleotide reductase (large subunit) UL39, ribonucleotide reductase (small subunit) UL40, envelope protein / virion host shutoff VHS protein (UL41), DNA polymerase processing factor UL42, membrane protein UL43, glycoprotein C (UL44), membrane protein UL45, envelope protein VP11 / 12 (UL46), envelope protein VP13 / 14 (UL47), virus particle maturation protein VP16 (UL48 , α-TIF), envelope protein UL49, dUTP diphosphatase UL50, envelope molecule UL51, DNA helicase / primase complex protein UL52, glycoprotein K (UL53), transcriptional regulatory protein IE63 (ICP27, UL54), protein UL55, protein UL56, viral replication protein ICP22 (IE68, US1), protein US2, serine / threonine protein kinase US3, glycoprotein G (US4), glycoprotein J (US5), glycoprotein D (US6), glycoprotein I (US7), glycoprotein protein E (US8), envelope protein US9, capsid / envelope protein US10, Vmw21 protein (US11), ICP47 protein (IE12, US12), major transcription activator ICP4 (IE175, RSI), E3 ubiquitin ligase ICPO (IE110), latency-associated protein 1 LRP1, latency-associated protein 2 LRP2, neurovirulence factor RL1 (ICP34.5), latency-associated transcript LAT (herpes simplex virus 1 and 2 (HSV-1 and HSV-2), herpes simplex);Heat shock protein Hsp60, cell surface protein H1C, type IV dipeptidyl peptidase DppIV, M antigen, 70kDa protein, 17kDa histone-like protein (capsular histoplasma, histoplasmosis); fatty acid and retinol binding protein-1FAR-1, metalloproteinase tissue inhibitor TIMP (TMP), cysteine ​​protease ACEY-1, cysteine ​​protease ACCP-1, surface antigen Ac-16, secretory protein 2ASP-2, metalloproteinase 1MTP-1, aspartate protease inhibitor API-1, surface-associated antigen SAA-1, surface-associated antigen SAA-2, adult-specific secretory factor Xa, serine protease inhibitor anticoagulant AP, histidine protease inhibitor ... Cathepsin D-like aspartase ARR-1, glutathione S-transferase GST, polymerase L (human parainfluenza virus (HPIV), human parainfluenza virus infection); hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), M1 protein, M2 protein, NS1 protein, NS2 protein (NEP protein: nuclear export protein), PA protein, PB1 protein (polymerase basic 1 protein), PB1-F2 protein and PB2 protein (Orthomyxoviridae, influenza virus (influenza)); protein E, protein M, capsid protein C (Japanese encephalitis virus, Japanese encephalitis); RTX toxin, type IV pili, major pili subunit PilA, regulatory transcription factors PilS and PilR, protein sigma 54. Outer membrane protein (Kingella pneumophila, Kingella pneumophila infection); prion protein (Kuru prion, Kuru disease); matrix protein Z, glycoprotein GP (Lassa virus, Lassa fever); peptidoglycan-associated lipoprotein PAL, 60kDa chaperone protein Cpn60 (groEL, HspB), type IV pilus protein PilE, outer membrane protein MIP, major outer membrane protein MompS, zinc metalloproteinase MSP (Legionella pneumophila, Legionnaires' disease (Legionnaires' disease, Pontiac fever)); P4 nuclease, protein WD, ribonucleotide reductase M2, surface membrane glycoprotein Pg46, cysteine ​​protein white enzyme CP, glucose-regulated protein 78GRP-78, stage-specific S antigen-like protein A2, ATPase F1, β-tubulin, heat shock protein 70Hsp70, KMP-11, glycoprotein GP63, protein BT1, nucleoside hydrolase NH, cell surface protein Bl, ribosomal protein Pl-like protein PI, sterol 24-c-methyltransferase SMT, LACK protein, histone HI, SPB1 protein, thiol-specific antioxidant TSA, protein antigen STI1, signal peptidase SP, histone H2B, surface antigen PSA-2, cysteine ​​protease b Cpb (Leishmania, Leishmaniasis);Major membrane protein I, serine-rich antigen-45kDa, 10kDa chaperone protein GroES, HSP kDa antigen, amino-oxononanoic acid synthase AONS, protein recombinase A RecA, acetyl / propionyl-CoA carboxylase α, alanine racemase, 60 kDa chaperone 2, ESAT-6-like protein EcxB (L-ESAT-6), protein Lsr2, protein ML0276, heparin-binding hemagglutinin HBHA, heat shock protein 65 Hsp65, mycPl or ML0041 encoded protein, htrA2 or ML0176 encoded protein, htrA4 or ML2659 encoded protein, gcp or ML0379 encoded protein, clpC or ML0235 encoded protein (Mycobacterium leprae and disseminated Mycobacterium leprae, leprosy); outer membrane protein LipL32, membrane protein LIC10258, membrane protein LP30, membrane protein LIC12238, Ompa-like protein Lsa66, surface protein L igA, surface protein LigB, major outer membrane protein OmpLl, outer membrane protein LipL41, protein LigAni, surface protein LcpA, adhesion protein LipL53, outer membrane protein UpL32, surface protein Lsa63, flagellar protein FlaBl, membrane lipoprotein LipL21, membrane protein pL40, leptospiral surface adhesin Lsa27, outer membrane protein OmpL36, outer membrane protein OmpL37, outer membrane protein OmpL47, outer membrane protein OmpL54, acyltransferase LpxA (Leptospira, leptospirosis); listeriolysin O precursor Hly (LLO), invasion-associated protein lap (P60), listeriolysin regulatory protein PrfA, zinc metalloproteinase Mp1, phosphatidylinositol-specific phospholipase C PLC (PlcA, PlcB), O-acetyltransferase Oat, ABC transporter permease Im.G_1771, adhesion protein LAP, LAP receptor Hsp60, adhesin LapB, hemolysin listeriolysin OLLO, protein ActA, internalin A InIA, protein InIB (Listeria monocytogenes, listeriosis); protein Cox-2 (W. bancrofti and Brugia malayi, lymphatic filariasis (elephantiasis)); matrix protein Z, nucleoprotein N (lymphocytic choriomeningitis virus (LCMV), lymphocytic choriomeningitis);Thrombospondin-related anonymous protein TRAP, SSP2 spore surface protein 2, apical membrane antigen 1AMA1, rod membrane antigen RMA1, acidic alkaline repeat antigen ABRA, cell-traversing protein PF, protein Pvs25, merozoite surface protein 1MSP-1, merozoite surface protein 2MSP-2, ring-infected erythrocyte surface antigen RESA liver stage antigen 3LSA-3, protein Eba-175, serine repeat antigen 5SERA-5, circumsporozoite protein CS, merozoite membrane surface protein 3MSP3, merozoite surface protein 8MSP8, enolase PF10, hepatocyte erythrocyte protein 17kDaHEP17, erythrocyte membrane protein 1EMP1, protein Kβ merozoite surface protein 4 / 5MSP 4 / 5, heat shock protein Hsp90, glutamate-rich protein GLURP, merozoite surface protein 4MSP-4, STARP protein, circumsporozoite protein-associated antigen precursor CRA (Plasmodium spp., malaria); nuclear protein N, membrane-associated protein VP24, minor nuclear protein VP30, polymerase cofactor VP35, matrix protein VP40, envelope glycoprotein GP (Marburg virus, Marburg hemorrhagic fever (MHF)); hemagglutinin glycoprotein H; ABC transporter family members (LolC, OppA and PotF), boaB-encoded protein (Burkettsia pseudomallei, melioidosis (Whitmore's disease)); factor H binding protein fHbp, adhesin NadA, protein NhbA, repressor FarR (meningococcus aureus, men ... Neisseria, meningococcal disease); 66 kDa protein, 22 kDa protein (commonly in Yokogawa, metagonomiasis); polar tube protein (34, 75 and 170 kDa in Glugea, 35, 55 and 150 kDa in Encephalitozoon), kinesin-associated protein, RNA polymerase II largest subunit, integral membrane protein YIPA-like, anti-silencing protein 1, heat shock transcription factor HSF, protein kinase, thymidine kinase, NOP-2-like nucleolar protein (Microsporidia, microsporidiosis); CASP8 and FADD-like apoptosis regulator, glutathione peroxidase GPX1, RNA helicase NPH-II NPH2, Poly(A) polymerase catalytic subunit PAPL, major envelope protein P43K, early transcription factor 70kDa subunit VETFS, early transcription factor 82kDa subunit VETFL, metalloendopeptidase G1 type, nucleoside triphosphatase I NPH1, replication protein A28-like MC134L, RNA polymerase 7kDa subunit RP07 (molluscum contagiosum virus (MCV), molluscum contagiosum (MC)); matrix protein M, phosphoprotein P / V, small hydrophobic protein SH, nucleoprotein N, protein V, fusion glycoprotein F, hemagglutinin-neuraminidase HN, RNA polymerase L (mumps virus, mumps);Outer membrane protein OM, cell surface antigen OmpA, cell surface antigen OmpB (sca5), cell surface protein SCA4, cell surface protein SCA1, cytoplasmic protein D, crystalline surface layer protein SLP, protective surface protein antigen SPA (typhus Rickettsia, Typhimurium (endemic typhus)); adhesin PI, adhesion P30, protein pll6, protein P40, cytoskeleton protein HMW1, cytoskeleton protein HMW2, cytoskeleton protease HMW3, MPN152 encoding protein, MPN426 encoding protein, MPN456 encoding protein, MPN-500 encoding protein (Mycoplasma pneumonia, Mycoplasma pneumonia); NocA, iron-dependent regulatory protein, VapA, VapD, VapF, VapG, caseinolytic protease, 43-kDa protein associated with filament tip, P24 protein, P61 protein, 15-kDa protein, 56-kDa protein (commonly Nocardia asteroides and other Nocardia species, Nocardia); 43-kDa secretory glycoprotein, glycoprotein gpO, glycoprotein gp75, antigen Pb27, antigen Pb40, heat shock protein Hsp65, heat shock protein Hsp70, heat shock polymer Hsp90, protein P10, triosephosphate isomerase TPI, N-acetyl-glucosamine binding lectin paracoccin, 28-kDa protein Pb28 (Paracidioides brasiliensis, paracoccidioidomycosis (South American blastomycosis)); 28-kDa cruzipain-like cysteine ​​protease Pw28CCP (generally Paragonimus westermani and other lung fluke species, paragonimiasis); outer membrane protein OmpH, outer membrane protein Omp28, protein PM1539, protein PM0355, protein PM1417, repair protein MutL, protein BcbC, protein PM0305, formate dehydrogenase-N, protein PM0698, protein PM1422, DNA gyrase, lipoprotein PlpE, adhesion protein Cp39, heme acquisition system receptor HasR, 39kDa capsule protein, iron-regulated OMP IROMP, outer membrane protein OmpA87, fimbriae protein Ptf, fimbriae subunit protein PtfA, transferrin binding protein Tbpl, esterase MesA, Pasteurella multocida toxin PMT, adhesion protein Cp39 (Pasteurella, Pasteurella disease); "filamentous hemagglutinin FhaB, adenylate cyclase CyaA, pertussis toxin subunit 4 precursor PtxD, pertussis toxin precursor Prn, toxin subunit 1 PtxA, protein Cpn60, protein brkA, pertussis virus toxin subunit 2 precursor PtxB, pertussis toxin subunit 3 precursor PtxC, pertussis toxin subunit 5 precursor PtxE, pertussis toxin Prn, protein Fim2, protein Fim3;" (Bordetella pertussis, whooping cough);"Fl capsule antigen, virulence-associated V antigen, secreted effector protein LcrV, V antigen, outer membrane protease Pla, secreted effector protein YopD, putative secreted protein tyrosine phosphatase YopH, needle complex major subunit YscF, protein kinase YopO, putative autotransporter YapF, inner membrane ABC transporter YbtQ (Irp7), putative sugar binding protein YPO0612, heat shock protein 90HtpG, putative sulfatase protein YdeN, outer membrane lipoprotein carrier protein LolA, secretion chaperone YerA, putative lipoprotein YPO0420, Hemolysin activating protein HpmB, plague / yersin outer membrane receptor Psn, secreted effector protein YopE, secreted effector protein YopF, secreted effector protein YopK, outer membrane protein YopN, outer membrane protein YopM, coagulase / plasmin precursor Pla; (Yersinia pestis, plague); protein PhpA, surface adhesin PsaA, pneumococcal hemolysin Ply, ATP-dependent protease CIp, lipoic acid-protein ligase LplA, cell wall surface anchor protein psrP, sortase SrtA, glutamyl-tRNA synthetase GltX, choline binding protein A CbpA, pneumococcal surface protein A PspA, pneumococcal surface protein C PspC, 6-phosphogluconate dehydrogenase Gnd, iron-binding protein PiaA, murein hydrolase LytB, protein LytC, proteinase Al (Streptococcus pneumoniae, pneumococcal infection); major surface protein B, kexin-like proteinase KEX1, protein A12, 55 kDa antigen P55, major surface glycoprotein Msg (Pneumocystis jiroveci, Pneumocystis pneumonia (PCP)); protein Nfa1, exendin-3, secretory lipase, cathepsin B-like protease, cysteine ​​protease, cathepsin, peroxidase, protein Cry1Ac (usually Naegleria fowleri, primary amebic meningoencephalitis (PAM)); unknown protein (agnoprotein), large T antigen, small T antigen, major capsid protein VP1, minor capsid protein Vp2 (JC virus, progressive multifocal leukoencephalopathy); hypocalcemia response protein E LCrE, Chlamydial outer protein N CopN, serine / threonine protein kinase PknD, acyl-carrier-protein S-malonyltransferase FabD, single-stranded DNA binding protein Ssb, major outer membrane protein MOMP, outer membrane protein 2 Omp2, family of polymorphic membrane proteins (Pmp1, Pmp2, Pmp3, Pmp4, Pmp5, Pmp6, Pmp7, Pmp8, Pmp9, Pmp10, Pmp11, Pmp12, Pmp13, Pmp14, Pmp15, Pmp16, Pmp17, Pmp18, Pmp19, Pmp20, Pmp21) ( Chlamydia psittaci, Chlamydophila psittaci);Outer membrane protein PI, heat shock protein B HspB, peptide ABC transporter, GTP binding protein, protein IcmB, ribonuclease R, phosphatase SixA, protein DsbD, outer membrane protein TolC, DNA binding protein PhoB, ATPase DotB, heat shock protein B HspB, membrane protein Coml, 28kDa protein, DNA-3-methyladenine glycosidase I, outer membrane protein OmpH, outer membrane protein AdaA, glycine cleavage system T protein (Coxiella burnetii, Q fever); nucleoprotein N, large structural protein L; matrix protein M, matrix protein M2-1, matrix protein M2-2, major surface glycoprotein G, non-structural protein 1NS1, non-structural protein 2NS2 (respiratory syncytial virus (RSV), respiratory syncytial virus infection); outer membrane protein OM, cell surface antigen OmpA, cell surface antigen OmpB (sca5), cell surface protein SCA4, cell Cell surface protein SCA1, protein PS120, cytoplasmic protein D, protective surface protein antigen SPA (Rickettsia, Rickettsial infection); cytoplasmic protein D (small strain Rickettsia, Rickettsialpox); envelope glycoprotein GP, ​​nonstructural protein NSS (Rift Valley fever virus, Rift Valley fever (RVF)); cytoplasmic protein D (Rickettsia rickettsii, Rocky Mountain spotted fever (RMSF)); nonstructural protein 6 NS6, nonstructural protein 2 NS2, intermediate capsid protein VP6, inner capsid protein VP2, nonstructural protein 3 NS3, RNA-guided RNA polymerase L, protein VP3, nonstructural molecule protein 1 NS 1. Nonstructural gene protein 5NS5, outer capsid glycoprotein VP7, nonstructural glycoprotein 4NS4, outer capsid protein VP4; (rotavirus, rotavirus infection); polyprotein P200, glycoprotein E1, glycoprotein E2, protein NS2, capsid protein C (rubella virus, rubella); chaperone protein GroEL (MopA), inositol phosphate phosphatase SopB, heat shock protein HsIU, chaperone protein DnaJ, protein TviB, protein IroN, flagellar protein FliC, invasion protein SipC, glycoprotein gp43, outer membrane protein LamB, outer membrane protein PagC, outer membrane protein TolC, outer membrane protein N mpC, outer membrane protein FadL, transport protein SadA, transferase WgaP, effector proteins SifA, SteC, SseL, SseJ and SseF (Salmonella, salmonellosis); protein 14, nonstructural protein NS7b, nonstructural protein NS8a, protein 9b, protein 3a, nucleoprotein N, nonstructural molecule protein NS3b, nonstructural gene protein NS6, protein 7a, nonstructural cell protein NS8b, membrane protein M, envelope small membrane protein EsM, replicase polyprotein 1a, spike glycoprotein S, replicase polyprotein lab; SARS coronavirus, SARS (severe acute respiratory syndrome);Serine protease, atypical scabies antigen 1ASA1, glutathione S-transferase GST, cysteine ​​protease, serine protease, apolipoprotein (human scabies, scabies); glutathione S-transferase GST, paramyosin, hemoglobin SM32, major egg antigen, 14kDa fatty acid binding protein Sml4, major larval surface antigen P37, 22,6kDa membrane antigen, calpain CANP, triosephosphate isomerase Tim, surface protein 9B, outer capsid protein VP2 , 23kDa integral membrane protein Sm23, Cu / Zn-superoxide dismutase, glycoprotein Gp, myosin (Schistosomiasis, Schistosomiasis or Bilharziosis); 60kDa chaperone protein, 56kDa type-specific antigen, pyruvate phosphate dikinase, 4-hydroxybenzoate octopentenyltransferase (Orientia tsutsugamushi, Typhus); dehydrogenase GuaB, invasion protein Spa32, invasin IpaA, invasin IpaB , invasin IpaC, invasin IpaD, invasin IpaH, invasin IpaJ (Shigella, Shigella (Bacillus dysenteriae)); protein P53, virion protein US10 homolog, transcription regulator IE63, transcription transactivator IE62, protease P33, alpha trans-inducing factor 74kDa protein, deoxyuridine 5'-triphosphate nucleotide hydrolase, transcription transactivator IE4, membrane protein UL43 homolog, nuclear protein UL3 homolog, nuclear protein UL4 Homologs, replication origin binding protein, membrane protein 2, phosphoprotein 32, protein 57, DNA polymerase processivity factor, portal protein 54, DNA primase, envelope protein UL14 homolog, envelope protein UL21 homolog, envelope protein UL55 homolog, tripartite terminase subunit UL33 homolog, tripartite terminase subunit UL15 homolog, capsid binding protein 44, virion packaging protein 43 (Varicella zoster virus (VZV), Shingles or Herpes zoster); truncated 3-beta-hydroxy-5-enosteroid dehydrogenase homolog, virosome membrane protein A13, protein A19, protein A31, truncated protein A35 homolog, protein A37.5 homolog, protein A47, protein A49, protein A51, semaphorin-like protein A43, serine protease inhibitor 1, serine protease inhibitor 2, serine protease inhibitor 3, protein A6, protein B15, protein CI, protein C5, protein C6, protein F7, protein F8, protein F9, protein F11, protein F14, protein F15, protein F16 (Variola major or minor, Smallpox or Variola); adhesin / glycoprotein gp70, proteases (Sporotrichosis schenckii, sporotrichosis);Heme-iron binding protein IsdB, collagen adhesion protein Cna, clotting factor A ClfA, protein MecA, fibronectin binding protein A FnbA, enterotoxin type A EntA, enterotoxin type B EntB, enterotoxin type C EntC1, enterotoxin type C EntC2, enterotoxin type D EntD, enterotoxin type E EntE, toxic shock syndrome toxin-1 TSST-1, staphylococcal kinase, penicillin binding protein 2a PBP2a (MecA), secretory antigen SssA (Staphylococcus, staphylococcal food poisoning); heme-iron binding protein IsdB, collagen adhesion protein Cna, clotting factor AClfA, protein MecA, fibronectin binding protein A FnbA, enterotoxin A type EntA, enterotoxin B type EntB, enterotoxin C type EntC1, enterotoxin C type EntC2, enterotoxin D type EntD, enterotoxin E type EntE, toxic shock syndrome toxin-1 TSST-1, staphylococcal kinase, penicillin binding protein 2a PBP2a (MecA), secretory antigen SssA (Staphylococcus such as Staphylococcus aureus, staphylococcal infection); antigen Ss-IR, antigen NIE, strongylastacin, Na+-K+ ATPase Sseat-6, tropomyosin SsTmy-1, protein LEC-5, 41 kDa antigen P5, 41-kDa larval protein, 31-kDa larval protein, 28-kDa larval protein (stercoralis, strongyloidiasis); glycerol phosphodiester phosphodiesterase GlpQ (Gpd), outer membrane protein TmpB, protein Tp92, antigen TpF1, repeat protein Tpr, repeat protein FTprF, repeat protein G TprG, repeat protein I Tpr1, repeat protein J TprJ, repeat protein K TprK, syphilis Treponema pallidum membrane protein ATmpA, lipoprotein, 15 kDa Tpp15, 47 kDa membrane antigen, microferritin TpF1, adhesin Tp0751, lipoprotein TP0136, protein TpN17, protein TpN47, outer membrane protein TP0136, outer membrane protein TP0155, outer membrane protein TP0326, outer membrane protein TP0483, outer membrane protein TP0956 (Treponema pallidum, syphilis); cathepsin L-like protease, 53 / 25-kDa antigen, 8 kDa family member, cysticercosis protein TsAg5 with marginal trypsin-like activity, hexapod protein TSOL18, hexapod protein TSOL45-1A, lactate dehydrogenase A LDHA, lactate dehydrogenase B LDHB (Taenia, taeniasis); tetanus toxin TetX, tetanus toxin C TTC, 140 kDa S-layer protein, flavoprotein β-subunit CT3, phospholipase (lecithinase), phosphocarrier protein HPr (Clostridium tetani, Tetanus or Lockjaw);Capsid protein C (tick-borne encephalitis virus (TBEV), tick-borne encephalitis); 58-kDa antigen, 68-kDa antigen, Toxoplasma gondii larval excretory-secretory antigen TES, 32-kDa glycoprotein, glycoprotein TES-70, glycoprotein GP31, excretory-secretory antigen TcES-57, peri-intestinal fluid antigen Pe, soluble extract antigen Ex, excretory / secretory larval antigen ES, antigen TES-120, polyprotein allergen TBA-1, cathepsin L-like cysteine ​​protease c-cpl-1, 26-kDa protein (Toxocara canis or Toxoplasma gondii gondii, toxoplasmosis (ocular larva migrans (OLM) and visceral larva migrans (VLM)); microneme proteins (MIC1, MIC2, MIC3, MIC4, MIC5, MIC6, MIC7, MIC8), rod protein Rop2, rod protein (Rop1, Rop2, Rop3, Rop4, Rop5, Rop6, Rop7, Rop16, Rjop17), protein SR1, surface antigen P22, major antigen p24, major surface antigen p30, dense granule protein (GRA1, GRA2, GRA3, GRA4, GRA5, GRA6, GRA7, GRA8, GRA9, GRA10), 28kDa antigen, surface antigen SAG1, SAG2-related antigen, nucleoside triphosphatase 1, nucleoside triphosphatase 2, Stt3 protein, protein containing HesB-like domain, rhomboid protease 5, toxomepsin 1 (Toxoplasma gondii, toxoplasmosis); 43kDa secretory glycoprotein, 53kDa secretory glycoprotein, paramyosin, antigen Ts21, antigen Ts87, antigen p46000, TSL-1 antigen, caveolin-1CAV-1, 49kDa neonatal larval antigen, sphingolipid activator protein homolog, serine protease, serine protease inhibitor, 45-kDa glycoprotein Gp45 (Trichinella spiralis, trichinosis); Myb-like transcription factors (Myb1, Myb2, Myb3), adhesion protein AP23, adhesion protein AP33, adhesion protein AP33-3, adhesion protein AP51, adhesion protein AP65, adhesion protein Protein AP65-1, α-actin, kinesin-related protein, teneurin, 62kDa protease, subtilisin-like serine protease SUB1, cysteine ​​protease gene 3CP3, α-enolase Enol, cysteine ​​protease CP30, heat shock proteins (Hsp70, Hsp60), immunogenic protein P270, (Trichomonas vaginalis, trichomoniasis); β-tubulin, 47-kDa protein, secretory leukocyte-like protease-1 SLP-1, 50-kDa protein TT50, 17kDa antigen, 43 / 47kDa protein (Trichuris trichuris, trichuriasis (infection));Protein ESAT-6 (EsxA), 10kDa filtrate antigen EsxB, secretory antigen 85-BFBPB, fibronectin binding protein A FbpA (Ag85A), serine protease PepA, PPE family protein PPE18, fibronectin binding protein D FbpD, immunogenic protein MPT64, secretory protein MPT51, catalase peroxidase TKATG, periplasmic phosphate-binding lipoprotein PSTS3 (PBP-3, Phos-1), iron-regulated heparin-binding hemagglutinin Hbha, PPE family protein PPE14, PPE family protein PPE68, protein Mtb72F, protein Apa, immunogenic protein MPT63, periplasmic phosphate-binding lipoprotein PSTS1 (PBP-1), molecular chaperone DnaK, cell surface lipoprotein Mpt83, lipoprotein P23, phosphate transport system permease protein pstA, 14kDa antigen, fibronectin binding protein C FbpCl, alanine dehydrogenase TB43, glutamine synthetase 1, ESX-1 protein, protein CFP10, TB10.4 protein, protein MPT83, protein MTB12, protein MTB8, Rpf-like protein, protein MTB32, protein MTB39, crystal protein, heat shock protein HSP65, protein PST-S (usually Mycobacterium tuberculosis, tuberculosis); outer membrane protein FobA, outer membrane protein FobB, intracellular growth site IglCl, intracellular growth site IglC2, transaminase Wbtl, chaperone protein GroEL, 17kDa major membrane protein TUL4, lipoprotein LpnA, chitinase family 18 protein, isocitrate dehydrogenase, Nif3 family protein, type IV fimbriae glycosylation protein, outer membrane protein tolC, FAD binding family protein, type IV fimbriae protein polymer outer membrane protein, two-component sensor protein KdpD, chaperone Protein DnaK, protein TolQ (Bacillus tularensis, tularemia); MB antigen, urease, protein GyrA, protein GyrB, protein ParC, protein ParE, lipid-associated membrane protein LAMP, thymidine kinase TK, phospholipase PL-A1, phospholipase PL-A2, phospholipase PL-C, surface-expressed 96-kDa antigen; (Ureaplasma urealyticum, Ureaplasma urealyticum infection); non-structural polyprotein, structural polyprotein, capsid protein CP, protein El, protein E2, protein E3, protease PI, protease P2, protease P3 (Venezuelan equine encephalitis virus, Venezuelan equine encephalitis); protease NS3, protein AS2B, protein NS4A, protein NS4B, protein NS5 (yellow fever virus, yellow fever, West Nile virus, West Nile fever); capsid protein CP, protein El, protein E2, protein E3, protease P2 (Western equine encephalitis virus, Western equine encephalitis);Putative Yop-targeted protein YobB, effector protein YopD, effector protein YopE, protein YopH, effector protein YopJ, protein translocation protein YopK, effector protein YopT, protein YpkA, flagellar biosynthesis protein FlhA, peptidase M48, potassium efflux system KefA, transcriptional regulator RovA, adhesin Ifp, translocation protein LcrV, protein PcrV, invadin Inv, outer membrane protein OmpF-like porin, adhesin Yad A, protein kinase C, phospholipase CI, protein PsaA, mannosyltransferase-like protein WbyK, protein YscU, antigen YPMa (Yersinia pseudotuberculosis infection, Yersinia pseudotuberculosis infection); effector protein YopB, 60kDa chaperone protein, protein WbcP, tyrosine protein phosphatase YopH, protein YopQ, enterotoxin, galactoside permease, reductase NrdE, protein YasN, invadin Inv, adhesin YadA, outer membrane porin F OmpF, protein UspA1, protein EibA, protein Hia, cell surface protein Ail, chaperone SycD, protein LcrD, protein LcrG, protein LcrV, protein SycE, protein YopE, regulatory protein TyeA, protein YopM, protein YopN, protein YopO, protein YopT, protein YopD, protease ClpP, protein MyfA, protein FilA, protein PsaA (Yersinia enterocolitica, yersiniosis) and lipopolysaccharide (LPS). ;

[0184] The relevant pathogenic antigens can also be selected from lipid-based microbial antigens, such as antigens that can bind to cluster of differentiation 1 (CD1) molecules. Examples of such antigens include, but are not limited to: diacylglycerol glycolipids, such as α-glucosyl diacylglycerol (αGlc-DAG-s2) (Streptococcus pneumoniae), α-galactosyl diacylglycerol (αGalDAG) (Borrelia burgdorferi) and α-galactosyl diacylglycerol (BbGL-2c and BbGL-2f) (Borrelia burgdorferi); glycosphingolipids, such as α-galactosylceramide (αGalCer), α-galactosylceramide fragilis (αGalCer) and α-galactosylceramide fragilis (αGalCer). Bf)(Bacteroides fragilis), Agelasphin-9b (Agelas spp.) and α-galactomannuronosylceramide (GalA-GSL) (Sphingomonas); phospholipids such as phosphatidylglycerol (PG) (Listeria monocytogenes) and phosphatidylglycerol (PI) (Listeria monocytogenes); mycoketides such as Phosphomycoketide (PM) (Mycobacterium tuberculosis) and Mannosyl-phosphomycoketide (MPM) (Mycobacterium tuberculosis); cholesterol glucosides such as αCAG (Helicobacter pylori); diacylated sulfoglycolipids such as diacylated sulfoglycolipid (Ac2SGL) (Mycobacterium tuberculosis); mycolate esters such as glucose monomycolate (GMM) (Mycobacterium tuberculosis) ), glycerol monomycolate (Mycobacterium tuberculosis), mycolic acid (methoxy) (Mycobacterium tuberculosis), glucose monomycolate-C54 (GMM-C54) (Nocardia) and glucose monomycolate-C32 (GMM-C32) (Rhodococcus); lipopeptides such as dideoxymycobactin (Mycobacterium tuberculosis) and mycobactin lipopeptide (Mycobacterium); lipopeptide phosphopolysaccharides such as EhPIa (Entamoeba histolytica) and EhPIb (Entamoeba histolytica); lipophosphopolysaccharides such as phosphatidylinositol mannoside (PIM) (Mycobacterium tuberculosis) and lipoarabinomannan (LAM) (Mycobacterium tuberculosis); and mycobacterial acylglycolipids such as trehalose 6,6' dimycolate (TDM).

[0185] In embodiments where the infectious disease is influenza, the antigen can be hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), nonstructural protein 1 (NS1), nonstructural protein 2 (NS2), nuclear export protein (NEP), polymerase acidic protein (PA), polymerase basic protein PB1, PB1-F2, or polymerase basic protein 2 (PB2) from influenza virus or a fragment or variant thereof.

[0186] In certain examples, the antigen may be an antigen against one or more of cancer, malaria, tuberculosis, Campylobacter, influenza, rabies, RSV, pneumococcus, and HBV.

[0187] In some instances, the antigen can be selected from the group consisting of: BCG cell wall extract, RTS, S / AS01 (recombinant / mixed protein subunits), circumsporozoite protein (from Plasmodium) and HBsAg (hepatitis B virus), BCG subunit vaccine candidate + Mtb proteins and lipids, MTBVAC, GamTBvac, H56:IC31, ID93 / GLA-SE, recombinant flagellin subunit vaccine, KRAS antigen, hemagglutinin, G glycoprotein [NY-ESO-1, tyrosinase, MAGE-A3, TPTE], PCV-7, PCV-10, PCV-13 and HBsAg (also known as Australian antigen).

[0188] In embodiments, the antigen is mycolic acid (or cord factor trehalose 6,6' dimycolate).

[0189] It will be appreciated that the antigen of the non-lamellar lyotropic liquid crystal phase carrier of the first aspect may be appropriately selected depending on the infection, disease, disorder or condition to be treated in the subject, including infections, diseases, disorders and conditions as described herein.

[0190] In a second aspect of the present disclosure, an immunogenic composition is provided, comprising the non-lamellar lyotropic liquid crystal phase carrier of the first aspect and a pharmaceutically acceptable carrier, diluent and / or excipient.

[0191] As used herein, the term "immunogenicity" will be understood to mean that the composition induces or produces an immune response. Suitably, the immune response is a protective immune response. A "protective immune response" means an immune response sufficient to prevent or at least alleviate the severity or symptoms of, for example, an infection by a pathogenic organism. As used herein, "eliciting an immune response" or "inducing an immune response" indicates the ability or potential of an immunogenic composition to induce or produce an immune response to an antigen after being administered to a subject. As used herein, "immunize" and "immunization" refer to the administration of an immunogenic composition to induce or enhance a protective immune response to an antigen.

[0192] In certain examples, the immune response is or comprises a T cell-mediated immune response (ie, a cell-mediated immune response) and / or a B cell-mediated immune response (ie, a humoral immune response).

[0193] In embodiments, the immunogenic composition is a vaccine.

[0194] In certain embodiments, the immunogenic composition contains no or substantially no adjuvant that is not a component of the non-lamellar lyotropic liquid crystal phase carrier itself.

[0195] In certain examples of immunogenic compositions, the components of the non-lamellar lyotropic liquid crystal phase carrier other than the antigen do not induce or substantially induce or produce an immune response. That is, only the antigen non-lamellar lyotropic liquid crystal phase carrier induces or produces an immune response. Advantageously, as shown in the examples, the non-lamellar lyotropic liquid crystal phase carrier can be used alone as a carrier for presenting an antigen without itself triggering an immune response.

[0196] It should be understood that the selection of pharmaceutically acceptable carriers, diluents and / or excipients will depend at least in part on the mode of administration of the formulation. By way of example only, the composition can be in the form of a tablet, capsule, caplet, powder, injectable liquid, suppository, sustained release formulation, osmotic pump formulation, or any other form that is effective and safe for administration.

[0197] Preferably, the immunogenic composition is a liquid dispersion of the vector of the first aspect. The liquid dispersion may be an aqueous dispersion.

[0198] The immunogenic compositions of the present invention can be formulated into solid, semisolid, liquid or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols. Typical routes of administration of such immunogenic compositions include, but are not limited to, oral, topical, transdermal, inhaled, parenteral, sublingual, buccal, rectal, vaginal and intranasal. The term parenteral as used herein includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques. The immunogenic composition administered to the subject may be in the form of one or more dosage units, wherein, for example, a tablet or injectable liquid volume may be a single dosage unit. The actual method of preparing such dosage forms is known or obvious to those skilled in the art; for example, see Remington: The Science and Practice of Pharmacy, 20th edition (Philadelphia College of Pharmacy and Science, 2000).

[0199] The immunogenic composition can be used for parenteral, topical, oral or topical administration, intramuscular administration, aerosol administration or transdermal administration for prophylactic or therapeutic treatment. In one embodiment, the immunogenic composition of the second aspect is parenteral, such as intramuscular, subcutaneous or intravenous administration. In some embodiments, the immunogenic composition of the second aspect is intramuscular administration.

[0200] The formulation of the carrier of the first aspect to be applied will vary according to the route of administration and the formulation selected (e.g., solution, emulsion, capsule). The suitable immunogenic composition comprising the carrier of the first aspect to be applied can be prepared in a physiologically acceptable carrier. For solution or emulsion, suitable pharmaceutical carriers include, for example, aqueous solutions or alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles may include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. A variety of suitable aqueous vehicles are known to those skilled in the art, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), glucose solutions and glycine. Intravenous vehicles may include various additives, preservatives or liquids, nutrition or electrolyte supplements (generally referring to "Remington's Pharmaceutical Science", 16th edition, Mack, ed. 1980). The immunogenic composition may optionally contain pharmaceutically acceptable auxiliary substances required to approach physiological conditions, such as pH regulators and buffers and toxicity regulators, etc., for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate. According to lyophilization and reconstitution techniques known in the art, the carrier of the first aspect can be stored in a liquid phase, or can be lyophilized for storage and reconstituted in a suitable carrier before use.

[0201] When the immunogenic composition of the second aspect is a vaccine composition, the liquid carrier can be water, usually pyrogen-free water; isotonic saline or buffer (aqueous solution), such as phosphate, citrate and other buffer solutions. For the injection of the vaccine composition, water or preferably a buffer, more preferably an aqueous buffer, can be used, the buffer containing a sodium salt, preferably at least 50mM of a sodium salt, a calcium salt, preferably at least 0.01mM of a calcium salt and optionally a potassium salt, such as at least 3mM of a potassium salt. In an embodiment, the sodium salt, calcium salt and optionally potassium salt may be present as its chloride, iodide or bromide, or in the form of its hydroxide, carbonate, bicarbonate or sulfate, etc. The non-limiting examples of sodium salts include, for example, NaCl, NaI, NaBr, Na2CO3, NaHCO3, Na2SO4, the examples of optional potassium salts include, for example, KCl, KI, KBr, K2CO3, KHCO3, K2SO4, and the examples of calcium salts include, for example, CaCl2, CaI2, CaBr2, CaCO3, CaSO4, Ca(OH)2. In addition, the organic anions of the above-mentioned cations may be contained in the buffer. In certain embodiments, the buffer suitable for injection purposes may contain a salt selected from sodium chloride (NaCl), calcium chloride (CaCl2) and optionally potassium chloride (KCl), wherein in addition to chloride, other anions may be present. In an embodiment, the salt in the injection buffer is present in a concentration of at least 50mM sodium chloride (NaCl), at least 3mM potassium chloride (KCl) and at least 0.01mM calcium chloride (CaCl2). With reference to a specific reference culture medium, the injection buffer may be hypertonic, isotonic or hypotonic.

[0202] In some embodiments of the vaccine composition, one or more compatible solid or liquid fillers or diluents or encapsulating compounds suitable for administration to humans may be employed. Pharmaceutically acceptable carriers, fillers, and diluents will have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to subjects. Some examples of compounds that can be used as pharmaceutically acceptable carriers, fillers, or components thereof are sugars, such as, for example, lactose, glucose, trehalose, and sucrose; starches, such as, for example, corn starch or potato starch; glucose; cellulose and its derivatives, such as, for example, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solid glidants, such as stearic acid, magnesium stearate; calcium sulfate; vegetable oils, such as, for example, peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and oil from the genus Theobroma; polyols, such as, for example, polypropylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; and alginic acid.

[0203] When the immunogenic composition of the second aspect is a vaccine composition, it may further comprise one or more pharmaceutically acceptable adjuvants to enhance the immunostimulatory properties of the composition. The adjuvant may be any compound suitable for supporting the administration and delivery of the composition, and may induce or increase the immune response of the innate immune system, i.e., a nonspecific immune response.

[0204] Such adjuvants may be selected from any adjuvant known to the skilled person and adapted to the specific properties of the vaccine composition, i.e. for inducing a suitable immune response in a mammal. In an embodiment, the adjuvant may be selected from the group consisting of: (Squalene-water emulsion), TDM, MDP, muramyl dipeptide, Pluronic, alum solution, aluminum hydroxide, ADJUMER TM (polyphosphazene); aluminum phosphate gel; dextran from algae; algammulin; aluminum hydroxide gel (alum); high protein adsorption aluminum hydroxide gel; low viscosity aluminum hydroxide gel; AF or SPT (squalane emulsion (5%), Tween 80 (0.2%), Pluronic L121 (1.25%), phosphate buffered saline, pH 7.4); AVRIDINE TM (propylene diamine); BAYR1005 TM (N-(2-deoxy-2-L-leucylamino-bD-glucopyranosyl)-N-octadecyl-dodecanoyl-amide hydrogen acetate); CALCITRIOL TM (1-α,25-dihydroxy-vitamin D3); calcium phosphate gel; CAP TM (calcium phosphate nanoparticles); cholera holotoxin, cholera-toxin-A1-protein-AD fragment fusion protein, subunit B of cholera toxin; CRL 1005 (block copolymer P1205); liposomes containing cytokines; DDA (dimethyldioctadecyl ammonium bromide); DHEA (dehydroepiandrosterone); DMPC (dimyristoylphosphatidylcholine); DMPG (dimyristoylphosphatidylglycerol); DOC / alum complex (deoxycholic acid sodium salt); Freund's complete adjuvant; Freund's incomplete adjuvant; gamma inulin; Gerbu adjuvant (a mixture of: i) N-acetylglucosamine-(P1-4)-N-acetylmuramoyl -L-alanyl-D-glutamine (GMDP), ii) dimethyldioctadecyl ammonium chloride (DDA), iii) zinc-L-proline salt complex (ZnPro-8); GM-CSF); GMDP (N-acetylglucosaminyl-(b1-4)-N-acetylmuramoyl-L-alanyl-D-isoglutamine); imiquimod (1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine); ImmTher TM(N-acetylglucosaminyl-N-acetylmuramoyl-L-Ala-D-isoGlu-L-Ala-dipalmitin); DRV (immunoliposomes prepared from dehydration-rehydration vesicles); interferon-γ; interleukin-1β; interleukin-2; interleukin-7; interleukin-12; ISCOMS TM ;ISCOPREP 7.0.3. TM ; Liposome; LOXORIBINE TM (7-allyl-8-oxoguanosine); LT oral adjuvant (Escherichia coli labile enterotoxin-protoxin); microspheres and microparticles of any composition; MONTANIDE ISA 51 TM (Purified incomplete Freund's adjuvant); MONTANIDE ISA720 TM (metabolizable oil adjuvant); MPL TM (3-Q-deacylated-4'-monophosphoryl lipid A); MTP-PE and MTP-PE liposomes (N-acetyl-L-alanine-D-isoglutamine-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy))-acetamide, monosodium salt); MURAMETIDE TM (Nac-Mur-L-Ala-D-Gln-OCH3); MURAPALMITINE TM and D-MURAPALMITINE TM (Nac-Mur-L-Thr-D-isoGIn-sn-glycerol palmitoyl); NAGO (neuraminidase-galactose oxidase); nanospheres or nanoparticles of any composition; NISV (nonionic surfactant vesicles); PLEURAN TM (β-glucan); PLGA, PGA and PLA (homopolymers and copolymers of lactic and glycolic acid; microspheres / nanospheres); PLURONIC L121 TM ;PMMA(polymethyl methacrylate);PODDS TM (protein-like microspheres); poly(vinyl carbamate derivatives); poly-rA:poly-rU (poly(A-poly(URI)) complex); polysorbate 80 (Tween 80); helical protein (Avanti Polar Lipids, Inc., Alabaster, AL); STIMULON TM (QS-21); Quil-A (Quil-A saponin); S-28463 (4-amino-otec-dimethyl-2-ethoxymethyl-1H-imidazo[4,5c]quinoline-1-ethanol); SAF-1 TM("Syntex adjuvant formulations"); Sendai proteoliposomes and lipid matrices containing Sendai; Span-85 (sorbitan trioleate); Specol (an emulsion of Marcol 52, Span 85, and Tween 85); squalene or (2,6,10,15,19,23-hexamethyltetracosane and 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosane); octadecyl tyrosine (octadecyl tyrosine hydrochloride); (N-acetylglucosamine-N-acetylmuramoyl-L-Ala-D-isoGlu-L-Ala-dipalmitoylacrylamide); Threonyl-MDP (Termurtide TM or [thrl]-MDP; N-acetylmuramoyl-L-threonyl-D-isoglutamine); Ty particles (Ty-VLP or virus-like particles); Walter-Reed liposomes (liposomes containing lipid A adsorbed on aluminum hydroxide) and lipopeptides, including Pam3Cys, specifically aluminum salts such as Adju-phos, Alhydrogel, Rehydragel; emulsions, including CFA, SAF, IFA, MF59, Provax, TiterMax, Montanide, Vaxfectin; copolymers, including Optivax (CRL1005), L121, poloxamer 4010, etc.; liposomes, including stealth liposomes, spirochetes, including BIORAL; plant-derived adjuvants, including QS21, Quil A, Iscomatrix, ISCOM; adjuvants suitable for costimulation, including tomatine, biopolymers, including PLG, PMM, inulin; microbial adjuvants, including Romurtide, DETOX, MPL, CWS, mannose, CpG polynucleotide sequences, CpG7909, ligands of human TLR 1-10, ligands of mouse TLR 1-13, ISS-1018, IC31, Imidazoquinoline, Ampligen, Ribi529, IMOxine, IRIV, VLP, cholera toxin, heat-labile toxin, Pam3Cys, flagellin, GPI anchor, LNFPIII / Lewis X, antimicrobial peptides, UC-1V150, RSV fusion protein, cdiGMP; and adjuvants suitable as antagonists, including CGRP neuropeptide. In a preferred embodiment, the adjuvant can be an oil-in-water emulsion adjuvant Especially if the vaccine is a flu shot.

[0205] However, in embodiments, the immunogenic composition of the second aspect may not require a separate adjuvant, as the vector of the first aspect itself can induce a suitable innate immune response.

[0206] In some instances, the immunogenic composition further comprises a cell targeting ligand. For this reason, the lipid nanoparticles or vesicles of the immunogenic composition can target receptors on antigen presenting cells (APC), for example, by placing the ligand of the cell receptor of the APC on the surface of the particle (for example, a mannose base part or a complement protein such as C3d). For such examples, the immunogenic composition additionally comprises a cell targeting ligand located at or on the surface of the lipid nanoparticle. The cell targeting ligand promotes that the immunogenic composition is delivered to immune cells such as APC. In some specific instances, immune cells are APCs such as dendritic cells and / or macrophages. In some other instances, immune cells comprise mannose receptors or C-lectin type receptors on their cell surfaces.

[0207] In a third aspect of the present disclosure, a method for delivering an antigen to a cell is provided, the method comprising the step of contacting the cell with the non-lamellar lyotropic liquid crystal phase carrier of the first aspect or the immunogenic composition of the second aspect.

[0208] In a fourth aspect of the present disclosure, a method of inducing an immune response in a subject is provided, the method comprising the step of administering to the subject an effective amount of the non-lamellar lyotropic liquid crystal phase carrier of the first aspect or the immunogenic composition of the second aspect.

[0209] Suitable regimens for the administration of the immunogenic compositions disclosed herein are known in the art. The above compositions can be administered in a manner compatible with the dosage formulation and in an effective amount. The dosage administered to the patient should be sufficient to produce a beneficial response to the patient within an appropriate time period. The amount of the medicament to be administered may depend on the subject to be treated, including its age, sex, weight and general health, which factors will depend on the physician's judgment.

[0210] In some cases, only one dose of the immunogenic composition is required, but in some cases, such as in cases of more severe immunodeficiency, a second, third or fourth dose may be given. Following the initial vaccination, the subject may receive one or more booster immunizations at appropriate intervals.

[0211] The immunogenic compositions disclosed herein can be administered as a single dose. Alternatively, the immunogenic compositions disclosed herein can be administered in multiple dosage regimens. In one example, multiple dosage regimens consist of a series of two doses separated by about 1 month to about 2 months.

[0212] In another example, the multiple dose regimen consists of a series of two doses separated by about 1 month or a series of two doses separated by about 2 months.

[0213] In the fifth aspect of the present disclosure, a method for preventing, treating or ameliorating an infection, disease, disorder or condition is provided, the method comprising the step of administering a therapeutically effective amount of the non-lamellar lyotropic liquid crystal phase carrier of the first aspect or the immunogenic composition of the second aspect to a subject in need thereof, so as to prevent, treat or ameliorate the infection, disease, disorder or condition.

[0214] In a sixth aspect of the present disclosure, a non-lamellar lyotropic liquid crystal phase carrier of the first aspect or an immunogenic composition of the second aspect is provided, which is used to prevent, treat or improve an infection, disease, disorder or condition.

[0215] In the seventh aspect of the present disclosure, there is provided a use of the non-lamellar lyotropic liquid crystal phase carrier of the first aspect or the immunogenic composition of the second aspect in the preparation of a medicament for preventing, treating or ameliorating an infection, disease, disorder or condition.

[0216] It will be appreciated that the infection, disease, disorder or condition to be treated will depend on the antigen of the non-lamellar lyotropic liquid crystalline phase carrier of the first aspect or the immunogenic composition of the second aspect.

[0217] In embodiments, the infection, disease, disorder or condition is a bacterial, protozoan, viral or fungal infection, including infections described herein or caused by organisms described herein. In embodiments, the infection, disease, disorder or condition is a bacterial or viral infection. In embodiments, the bacterial infection is tuberculosis (TB). In embodiments, the viral infection is influenza.

[0218] In an embodiment, the infection, disease, disorder or condition is a bacterial infection. In an embodiment, the infection, disease, disorder or condition is tuberculosis (TB).

[0219] In an embodiment, the infection, disease, disorder or condition is caused by pathogenic bacteria, protozoa, viruses or fungal organisms, including those described herein. In an embodiment, the infection, disease, disorder or condition is caused by a pathogenic organism selected from severe acute respiratory syndrome (SARS), severe acute respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-1 and SARS-CoV-2), influenza virus, respiratory syncytial virus (RSV), herpes simplex virus (HSV), human papillomavirus (HPV), human immunodeficiency virus (HIV), Plasmodium, Staphylococcus aureus, dengue virus, Chlamydia trachomatis, cytomegalovirus (CMV), hepatitis B virus (HBV), Mycobacterium tuberculosis, rabies virus and yellow fever virus.

[0220] In an embodiment, infection, disease, illness or condition is caused by pathogenic bacteria or viral organisms. In an embodiment, bacterial organisms are selected from Staphylococcus aureus, Chlamydia trachomatis and Mycobacterium tuberculosis. In certain embodiments, bacterial organisms are Mycobacterium tuberculosis. In an embodiment, viral organisms are selected from severe acute respiratory syndrome (SARS), severe acute respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-1 and SARS-CoV-2), influenza virus, respiratory syncytial virus (RSV), herpes simplex virus (HSV), human papillomavirus (HPV), human immunodeficiency virus (HIV), dengue virus, cytomegalovirus (CMV), hepatitis B virus (HBV), rabies virus and yellow fever virus.

[0221] In an embodiment, the infection, disease, disorder or condition is caused by a pathogenic bacterial organism. In an embodiment, the infection, disease, disorder or condition is caused by Mycobacterium tuberculosis (MTB).

[0222] In embodiments, the infection, disease, disorder or condition is cancer.

[0223] In embodiments, the infection, disease, disorder or condition is selected from cancer, malaria, tuberculosis, Campylobacter, influenza, rabies, RSV, pneumococcus and HBV.

[0224] In embodiments where the antigen is a mycolic acid (or cord factor trehalose 6,6' dimycolate, TDM), then the infection, disease, disorder or condition is tuberculosis (TB) and / or is caused by Mycobacterium tuberculosis (MTB).

[0225] The following comments may apply to any one or more of the third to seventh aspects, as appropriate.

[0226] After formulation, the compositions of the present disclosure will be administered in a manner compatible with the dosage formulation and in a therapeutically / preventively effective amount. The dosage range of the administration of the carrier of the first aspect is a dosage range large enough to produce the desired effect. In one embodiment, the composition comprises an effective amount of encapsulated or associated antigens. In one embodiment, the composition comprises a therapeutically effective amount of antigens. In another embodiment, the composition comprises a preventively effective amount of antigens.

[0227] The dosage should not be too large to avoid causing adverse side effects. Generally, the dosage will vary with the patient's age, condition, gender and degree of disease, and can be determined by a person skilled in the art. In the event of any complications, the dosage can be adjusted by a separate physician.

[0228] Methods for preparing the above-described compounds and compositions are further described herein and / or known in the art.

[0229] As generally used herein, the term "administering" or "administration" and the like describe the introduction of a relevant vector or immunogenic composition into a mammal, such as by a specific route or vehicle. The route of administration may include topical, parenteral and enteral, including oral, buccal, sublingual, nasal, anal, gastrointestinal, subcutaneous, intramuscular and intradermal routes of administration, but is not limited thereto.

[0230] "Treat," "treatment," or "treating," or "ameliorating" means administering to a subject a relevant vector or immunogenic composition to at least ameliorate, reduce or suppress existing signs or symptoms of a disease, disorder or condition experienced by the subject to an extent that such a condition is improved according to clinically acceptable standard medical criteria.

[0231] "Prevent," "prevention," or "preventing" means the prophylactic administration of the relevant vector or immunogenic composition to a subject who does not exhibit signs or symptoms of a disease, disorder, or condition but is expected or predicted to exhibit such signs or symptoms in the absence of prevention. Prophylactic treatment may at least alleviate or partially ameliorate the expected symptoms or signs.

[0232] As used herein, "effective amount" or "therapeutically effective amount" refers to the amount of the relevant vector or immunogenic composition administered that is sufficient to prevent the occurrence of symptoms of the condition being treated, or to stop the worsening of symptoms, or to treat and alleviate symptoms, or at least reduce the severity of symptoms. The effective amount will vary with the patient's age, sex, weight, etc. in a manner known to those skilled in the art. The appropriate dose or dosage regimen can be determined by routine experimentation or based on current treatment regimens for delivering one or more active agents via the particles of the first aspect.

[0233] As used herein, the term "subject" or "individual" or "patient" may refer to any subject for which therapy is desired, specifically a vertebrate subject, and even more specifically a mammalian subject. Suitable vertebrates include, but are not limited to, primates, avian, livestock animals (e.g., sheep, cows, horses, donkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g., cats, dogs), and captive wild animals (e.g., foxes, deer, wild dogs). Preferred subjects are those who need treatment for diseases, disorders, or conditions as described herein. However, it should be understood that the aforementioned terms do not mean that symptoms must be present.

[0234] In one embodiment, the subject is a human who has been vaccinated against a pathogenic organism.

[0235] The human patient can be a toddler (approximately 12 to 24 months) or a young child (approximately 2 to 5 years old). The compositions disclosed herein are also suitable for use in older children, adolescents, and adults (e.g., 18 to 45 years, 18 to 50 years, 18 to 55 years, 18 to 60 years, or 18 to 65 years).

[0236] In one example, the human patient is an elderly person. For example, the patient can be 50 years old or older. In another example, the patient is 55 years old or older. In another example, the patient is 60 years old or older. In another example, the patient is 65 years old or older. In another example, the patient is 70 years old or older.

[0237] Patients treated with the immunogenic compositions disclosed herein may be immunocompromised.

[0238] In some examples, the immunogenic composition can be administered together with a vaccine against another antigen, such as influenza.

[0239] The optimal amounts of the components of a particular vaccine can be determined by standard studies involving observation of appropriate immune responses in subjects. For example, the dosage for human vaccination is determined by extrapolation from animal studies to human data. In another embodiment, the dosage is determined empirically.

[0240] Those skilled in the art will appreciate that, without departing from the broad general scope of the present disclosure, various changes and / or modifications may be made to the above-described embodiments. Therefore, the present embodiments should be considered in all aspects as illustrative and not restrictive.

[0241] Examples

[0242] Example 1 - Particle Formation

[0243] Glyceryl monooleate (MO) was obtained from Nu-check-Prep, Inc (Minnesota, USA) with a purity greater than 99% as determined by gas-liquid chromatography. Mycolic acid, ethanol, and Pluronic F127 were purchased from Sigma-Aldrich (NSW, Australia).

[0244] The dry powder of MO was dissolved in ethanol to prepare a 200 mg / mL stock solution. TDM powder was dissolved in a solution of ethanol and chloroform (1:1 ratio) with a 5 mg / mL stock solution. MO-TDM complexes were prepared by mixing the exact stock solutions in a desired ratio in a glass vial. In order to evaporate the solvent, the lipid mixture was kept in a vacuum oven overnight. The appropriate mol% of each lipid was calculated using the following equation:

[0245]

[0246] The stock solution (0.5wt%) of Pluronic F127 was prepared in PBS, and 1ml of this polymer solution was added to 50mg of the dry lipid mixture, which corresponds to 10% (w / w) of total lipids. All samples were dispersed on a high-throughput platform using an automatic multi-probe sonicator. The dispersed nanoparticles of 80 μL (from 50mg / mL stock solution) were dispensed into 96-well microplates, sealed with a microplate sealer, and maintained at room temperature for further characterization.

[0247] Example 2 - Particle Morphology Characteristics

[0248] All nanocarrier structures were studied using small and wide angle X-ray scattering (SAXS / WAXS) beamlines at the Australian Synchrotron. (12.0keV) X-ray wavelength, flux 10 13 photons / second. The achievable q range is (where the scattering vector q = 4πsin(θ) / λ, where λ represents the wavelength and θ represents the scattering angle). The scattering images were further integrated using the IDL-based ScatterBrain software (Kirby, NM et al. Journal of Applied Crystallography. 2013, 46, pp. 1670-1680). The nanoparticle samples were loaded in standard polystyrene 96-well plates and positioned in a high-throughput plate holder positioned 1 meter from the Decree-Pilatus 1-M detector that recorded the two-dimensional X-ray diffraction images (exposure time of 1 second). Temperature control was performed using a circulating water bath as previously described (Mulet, X. et al. Acc Chem Res. 2013, 46, pp. 1497-1505).

[0249] The SAXS data were analyzed using the IDL-based AXCESS software developed by Imperial College London (Seddon, J. et al. Philos. Trans. Royal Soc. A-Math Phys Eng Sci. 2006, 364, pp. 2635-2655). The lattice parameters (LP) and different phases of each nanoformulation were also calculated using the AXCESS software. Silver behenate was used in the software. 1D plots of intensity versus q (scattering vector) were generated by transforming the 2D diffraction images obtained from the machine. Based on the relative peak positions, three cubic phases Q II P , Q II D and Q II G . 'a', which represents the lattice parameter, is calculated from the 'q value' of each peak using the following equation:

[0250]

[0251] where h, k and l are Miller indices. Each data point was run in duplicate from two different sets of samples for SAXS analysis.

[0252] For pure MO and MO mixtures doped with TDM in concentrations ranging from 0.4 mol% to 10 mol% (relative to MO), the structures of the bicontinuous cubic phases were characterized using SAXS ( Figure 1 ). The adopted phases and associated lattice parameters are plotted as a function of TDM concentration in Figure 1 A. Figure 1 A representative 1D SAXS plot of intensity versus q is provided in B. Pure MO cubes were prepared at 25 °C using the original cubic (Q II P ) symmetry, with typical lattice parameters of about ( Figure 1 A), which is consistent with the literature (Sarkar, S. et al., ACS Appl Mater Interfaces 13, pp. 2336-2345, (2021)). Figure 1 Q is observed in B II P The √2, √4, and √6 reflections of the cubic phase are observed. As the lattice parameters level off, the Q II PThe lattice parameter of the phase decreases. This indicates that TDM is at least partially dissolved in the MO bilayer in this concentration range. For TDM concentrations ≤ 1 mol%, the Bragg peak remains quite intense, sharp, and well-defined, indicating the preservation of long-range order within the cubic phase. At TDM concentrations ≥ 2 mol%, the Bragg peak becomes broadened and the intensity decreases significantly, indicating that the encapsulated TDM destroys the Q phase to some extent. II P Long-range order. The coexistence of Q II D The √2 and √3 peaks of the phase. II D The lattice parameter of the phase is 92.4 at 2 mol% and slightly decreases to The Bonnet ratio predicts the theoretical ratio of the lattice parameters of two cubic phases that coexist at equilibrium and is II P / Q II D is 1.576 (Larsson; Current Opinion in Colloid & Interface Science 9, pp. 365-369). At 2, 2.5 and 3 mol% TDM, the lattice parameter ratio Q II P / Q II D At 5 mol% TDM, a single Q II P phase, whose lattice parameters are similar to those at 1 mol% TDM. However, by 10% TDM, the system undergoes a shift from Q II P Stage to H II This suggests that including this high level of TDM within the MO promotes the phase transition to a more curved phase.

[0253] Example 3 - Particle Size Characteristics

[0254] XRD patterns of solid powder TDM (1 mg / mL), MO 1 mg / mL, and a mixture of MO (99 mol%)-TDM (1 mol%), where the samples were prepared by drop casting (i.e., forming a thin solid film by dropping a solution onto a flat surface and then evaporating the solution) of these materials on separate cover glasses and allowing the solvent to evaporate. XRD patterns were obtained using a Cu Kα radiation source. Data were recorded on a Bruker AXSD8 advance wide-angle X-ray diffractometer.

[0255] Differential Scanning Calorimetry (DSC)

[0256] In DSC, the different heat flows to the aluminum pan carrying the sample and the empty reference pan are monitored by area thermocouples, and a preheated purge gas is used to provide additional baseline stability. In this study, 3 mg of dry powders of MO, TDM and MO-TDM (1%) mixture (solid state) were weighed into an aluminum DSC pan and the pan was hermetically sealed. Calorimetric analysis was performed on a Mettler Toledo instrument at a heating rate of 2°C / min in a temperature range of 4°C to 60°C under a nitrogen atmosphere. Instrument software was used for sample data acquisition and analysis.

[0257] Fourier-transform infrared spectroscopy (FTIR)

[0258] FTIR spectra of TDM (1 mg / mL), MO 1 mg / mL, and a mixture of MO (99 mol%)-TDM (1 mol%) in solid samples were recorded using a PerkinElmer D100 spectrophotometer in diffuse reflectance sampling accessory mode with a resolution of 4 cm -1 For the MO-TDM (1 mol%) mixture, a combination of XRD, DSC and FT-IR was used to characterize the extent of lipid mixing between MO and TDM.

[0259] X-ray diffraction (XRD)

[0260] Figure 4 XRD patterns of powder forms of TDM, MO and MO-TDM (1 mol%) are shown. The samples were prepared by drop casting as described above. The many sharp peaks in the XRD pattern of pure MO are consistent with the formation of a highly ordered crystalline structure upon evaporation of the solvent. The known lattice parameters of the crystalline lamellar phase formed are 5.64° at 2θ. The first Bragg peak observed at is the (003) reflection. The H bonding between the carboxylic acid groups in the MO head group promotes the formation of a crystalline structure.

[0261] In contrast, no Bragg peaks consistent with an amorphous structure were observed in the XRD pattern of TDM. The mixed system consisting of MO-TDM (1 mol%) showed an XRD pattern similar to that of pure MO. However, the sharp Bragg peaks in the low-angle region have shifted to higher angles, which is consistent with a decrease in the lamellar d-spacing and indicates at least partial mixing of the two lipids.

[0262] Differential Scanning Calorimetry (DSC)

[0263] DSC scans of pure MO, TDM and MO-TDM (1 mol%) were Figure 6 presented in . MO shows a sharp endothermic peak at 33.5°C corresponding to its melting temperature. In contrast, the DSC scan of TDM does not show any sharp phase transitions, which is due to its amorphous nature. However, the DSC scan of TDM shows two thermal transitions at 34 and 53°C. The relatively high transition temperature of TDM (53°C) is due to the melting of the mycolic acid ester chains, and the methyl ester is usually encountered at a lower temperature (34°C). This is consistent with previous studies that reported the melting transitions of dehydrated pure MO and TDM to be 33-35°C and 53-65°C, respectively. The DSC scan of MO-TDM (1 mol%) shows a single sharp peak near the melting transition of MO, but shifts to 32.25°C and is slightly broadened. The small drop in the melting point (33.5 to 32.25°C) indicates mixing between MO and TDM lipids, despite the very long chains of TDM.

[0264] Fourier-transform infrared spectroscopy (FTIR)

[0265] Both TDM and MO contain carboxylic acid groups in their head group regions, and therefore both molecules are likely to exhibit intermolecular hydrogen bonding. FTIR spectroscopy can provide information on the nature of hydrogen bonding interactions between molecules. FTIR spectral data for MO, TDM, and a MO-TDM mixture (1 mol%) are presented in Figure 5 MO shows a peak at 3401cm -1 A strong broad peak centered at -OH is characteristic of the -OH stretching frequency. The broad nature of this peak indicates intermolecular hydrogen bonding between the hydroxyl groups of the MO. TDM does not show any peak characteristic of this OH stretching frequency, indicating that there is no intermolecular hydrogen bonding between the TDM molecules. In this case, steric hindrance due to the extremely long hydrocarbon chains may inhibit the formation of hydrogen bonds. In the case of the MO-TDM (1 mol%) self-assembled structure, a strong and broad band due to the -OH stretching frequency was observed to shift slightly; a significant decrease in the intensity of this peak was also observed. This suggests that the encapsulated TDM has affected the MO intermolecular H-bonding network, providing further evidence of mixing between the two moieties. In addition, in the case of the methylene symmetry (2925cm- 1 To 2933cm- 1 ) and antisymmetric (2855cm- 1 To 2857cm- 1 ) stretching frequency. In addition, the change in its peak width indicates the stacking of TDM hydrocarbon chains within the MO hydrocarbon framework.

[0266] Cryo-Transmission Electron Microscopy (Cryo-TEM)

[0267] Use FEI VITROBOT to prepare cryogenic-TEM samples. The humidity and temperature on the device are set to 65% and 22°C, respectively. 2 μL (50 mg / mL) of nanoparticle solution is added to the top of the C- plane holey carbon grid and allowed to dry for 30 seconds and blotted for 2 seconds. Then, the grid loaded with the sample is immediately immersed in the liquid ethane solution. The treated carbon grid is stored in a liquid nitrogen container until it is brought to a cryogenic-holder (model 626) for further imaging. Transmission electron microscope (FEITecnai 12) is typically operated at 120 kV and used for sample imaging. A defocus level of 1.5-2 μm is used for each sample imaged at -190°C. At <900-1000nm -2 Under electron flow, the magnification range was between 35 and 100 k. ImageJ (NIH) software was used for fast Fourier transform (FFT) analysis of each sample image.

[0268] Nanocarrier size measurement

[0269] The Malvern Zetasizer Nano ZS (ZEN3600) instrument was used to measure the hydrodynamic diameter, polydispersity index and zeta potential of the nanoformulations. 1 mL of Milli-Q H2O (18.2 MΩcm) was added to 20 μL (50 mg / mL) of nanoformulation. These diluted nanoformulation solutions were transferred to a capillary cell (disposable) for particle size analysis. Sample measurements were counted at 25°C with a refractive index of 1.33 by 12-13 triplicate runs. The zeta potential value was obtained by converting the electrostatic mobility number using the Helmholtz-Smoluchowski equation for each sample.

[0270] The size, morphology and surface charge of MO cubes and MO-TDM cubes (1 mol%) were characterized using cryo-TEM and DLS. Representative cryo-TEM images of particles of MO and MO-TDM (1 mol%) are shown in Figure 2 Presented in.

[0271] In Figure 2A, an ordered typical cubic structure of MO cubes is observed. From the optical diffraction pattern of the cryo-TEM image (e.g. Figure 2 The fast Fourier transform (FFT) obtained from the Q II P Phase, d 200 The distance between the planes is visible, with a spacing of 7.2 nm, corresponding to a calculated lattice parameter of 14.4 nm. This value is in good agreement with the corresponding lattice parameter measured by SAXS analysis (14.5 nm). Figure 2In B, after adding 1 mol% TDM, ordered cubes are still observed, as well as some aggregated particles. This is consistent with the DLS results, which indicate that after encapsulating 1 mol% TDM, the particle size increases and the polydispersity index (PDI) increases. The lattice parameter value calculated by FFT is 6.4 nm, consistent with SAXS analysis (12.7 nm).

[0272] The zeta potential of pure MO cubic particles was measured to be -12.4 mV (Table 1). After adding 1 mol% TDM, the zeta potential became slightly more negative (-6.2 mV).

[0273] Table 1: Mean hydrodynamic diameter (MDD), polydispersity index (PDI) and zeta potential (n=2SD) of MO and MO-TDM (1 mol%) cubes.

[0274] sample MDD(nm) PDI Zeta potential (mV) MO 210±3 0.1 -12.4±1 MO-TDM (1 mol%) 289±2 0.3 -6.2±2

[0275] Example 4

[0276] THP-1, DU145 and Hela cell lines were purchased from ATCC, phosphate buffered saline was purchased from Thermo Fisher Scientific, and MTS assay kit (Promega CellTiter 96 aqueous solution) was purchased from Promega.

[0277] Cell viability assessment using Hela and THP-1 cell lines

[0278] Hela and THP-1 monocytic cell lines were obtained from ATTCC, Australia. Using the MTS assay kit, cell viability was determined by the number of live cells corresponding to the activity of mitochondrial succinate dehydrogenase. Cells (Hela and THP-1) were seeded in 96-well plates at a cell density of 1x 104 cells / mL (200 μL). RPMI culture medium was used for cell culture and supplemented with a mixture of fetal bovine serum (FBS; 10%) and streptomycin (100 μg / mL) and penicillin (100U / mL). Cells were cultured at 37°C under a 5% CO2 environment for 24 hours. The culture medium was replaced with fresh culture medium after 24 hours of incubation. Cell confluence was observed at 72 hours. Under an MO of 20 μg / mL, MO-TDM (1 mol%) lipid nanoparticles were added to the cells and incubated for 72 hours. The cell culture medium was decanted and the treated cells were washed twice with PBS solution. 100 μL of fresh culture medium was added to the treated cells. 20 μL of MTS solution of MTS was added to each well and incubated at 37°C with 5% CO2 for 4 hours. SpectraMax molecular device (microplate reader) was used for absorbance measurement at 490 nm. The MTS absorbance value of untreated control cells was set to 100%, and the absorbance value of nanoparticle-treated cells was expressed as a percentage (%) of control cell viability. The cell viability experiment was performed twice, and each sample was saved in triplicate.

[0279] The cytotoxicity of MO and MO-TDM (1 mol%) cubes was evaluated using the MTS assay against Hela cells (widely used in toxicity studies) and THP-1 (human monocyte) cell lines at a total lipid concentration of 20 μg / mL (the concentration used in subsequent immunostimulatory experiments) for up to 72 h of incubation ( Figure 3 ). Stimulated THP-1 cells (macrophages) are the primary infection site for MTB infection in humans. MO cubes were non-toxic to the Hela cell line (>90% cell viability), consistent with previous studies. Similar >90% cell viability was observed for MO cubes against the THP-1 cell line. Addition of 1 mol% TDM did not affect toxicity against the THP-1 cell line. At the same lipid concentration (20 μg / mL), cell viability of MO-TDM (1 mol%) cubes was slightly decreased relative to the Hela cell line. Although it was noted that there were significant differences in the measured toxicity due to the use of different cell lines and assay methods, many studies have investigated the in vitro and in vivo toxicity of MO-based cubes.

[0280] Example 5

[0281] Bacterial culture

[0282] Mtb (H37Rv) and BCG (Pasteur) were obtained from the American Type Culture Collection (ATCC). Mtb and BCG strains were grown to mid-log phase (OD 600nm = 0.6-0.8) in Middlebrook 7H9 broth (Difco, Becton Dickinson) supplemented with 0.05% (v / v) Tween 80 and Middlebrook AODC enrichment (Becton Dickinson) and re-inoculated in fresh medium 7 days before infection as previously described (Vermeulen; Lipid Res 58, pp. 709-718). Bacterial cultures were washed three times in PBS and sonicated for 60 seconds at 4 watts using a sonicator (60 sonicator, Fisher Scientific) before infection of monocyte-derived macrophages to prepare a uniform single-cell bacterial suspension.

[0283] Peripheral blood mononuclear cells and monocyte isolation

[0284] Obtain buffy coat from healthy donors.Peripheral blood mononuclear cells were isolated by diluting blood in pyrogen-free phosphate buffered saline (PBS) and using differential density centrifugation on Ficoll-Paque.The interphase layer was separated and the cells were washed with cold PBS.Plastic adhesion was used to further separate and culture cells, and characterized by flow cytometry according to the literature scheme.More than 90% of the adherent cells were monocytes, as determined by CD14 expression by flow cytometry (BD Biosciences).Monocytes were grown in Iscov's Dulbecco's modified medium (IMDM) containing 10% FBS for 6 days, collected at 37°C for 5 minutes with 0.05% trypsin / 1mM EDTA treatment (Life Technologies (Life Technologies)), and resuspended with fresh culture medium (IMDM+10% fetal bovine serum) for subsequent experiments in 6-well plates.

[0285] Training immunization experiments with human monocytes

[0286] Human PBMC (4x 106 cells / well) are cultivated in flat 6-well plates. After washing with PBS, monocytes are incubated with only culture medium for 18 hours, as negative control, or incubated with TDM or MO or MO-TDM (1mol%). After incubation, cells are washed once with PBS, and further incubated for 6 days in culture medium containing 10% serum, and culture medium is replaced once at the 3rd day. Stimulate cells again with IMDM (culture medium control) or 10ng / mL LPS at the 6th day. After 24 hours, supernatant is collected and stored at -20°C until cytokine measurement, and cells are processed for chromatin immunoprecipitation (CHIP) qPCR.

[0287] Cytokine measurement

[0288] Quantification of cytokines produced by PBMCs was performed by enzyme-linked immunosorbent assay (ELISA) using commercially available kits (Biolegend for human IL-6 and TNF-α). The supernatant was first filtered through a 0.22 μm filter (EMD Millipore, MA, USA) and then the cytokine levels were titrated according to the manufacturer's protocol.

[0289] Chromatin immunoprecipitation (ChIP) qPCR

[0290] After training the immune experiment, human monocytes were fixed with 1% formaldehyde at a concentration of approximately 1 million cells / mL. The fixed cell preparation was sonicated (60 ultrasonic crusher, Fisher Scientific) using 11 cycles of 30 seconds on and 30 seconds off. A total of 25 μg of chromatin was incubated with a dilution buffer containing a protease inhibitor cocktail and 2 μg of H3K4me3 (active motif, catalog number 39016) antibody and rotated overnight at 4°C. Protein A / G magnetic beads were washed in a dilution buffer containing 0.15% SDS, 75ng / μL single-stranded herring sperm DNA and 0.1% BSA, added to the chromatin / antibody mixture, and rotated at 4°C for 60 minutes. The magnetic beads were washed at 4°C with low salt, high salt and LiCl washing buffer. After washing, the chromatin was eluted for 20 minutes using 200 μL elution buffer. The supernatant, 4.8 μL 5M NaCl and 2 μl RNase A were collected and incubated overnight at 65 ° C. After this. The samples were incubated with 2 μL proteinase K at 65 ° C for 4 hours. Finally, the DNA samples were purified using the QIAGEN MinElute PCR purification kit, eluted in 20 μL elution buffer, and qPCR analysis was performed. The samples were analyzed using the % input method, in which myoglobin was used as a negative control and H2B was used as a positive control for H3K4me3. Primers used for analysis: myoglobin; H2B; TNF-α and IL-6 were reported in earlier studies (Blok; European Journal of Clinical Microbiology & Infectious Diseases 38, 449-456).

[0291] Mtb growth assay in macrophages

[0292] At different time points after MTB infection with or without MO-TDM (1 mol%), TDM and MO, macrophages were lysed with 0.05% SDS. Lysates were serially diluted 10-fold in PBS using 7H11 Middlebrook agar plates (Difco Laboratories, Surrey, UK). Plates were incubated at 37°C for 3 weeks before colony forming units (CFU) were counted. Data are expressed as log 10-CFU / million macrophages.

[0293] Ex vivo MTB Ag85B antigen presentation to CD4 T cells

[0294] Briefly, MΦ infected with M. tuberculosis were washed 4 hours after infection and covered with F9A6-CD4 T cell hybridoma, which recognizes the Ag85B epitope in the context of human HLA-DR1. IL-2 secretion by hybridoma T cells or other cytokines secreted by MΦ infected with M. tuberculosis were determined using a sandwich ELISA kit (Baijin Bio).

[0295] MO-TDM (1 mol%) nanoparticles induce robust pro-inflammatory cytokine responses in macrophages

[0296] Although the immunostimulatory effects of TDM have been studied, its role in cubic nanoparticle formulations is unknown. In order to demonstrate that cubic formulations of TDM (1 mol%) containing MO (99 mol%) can induce altered immune responses compared to native TDM, the major proinflammatory cytokines IL-6 and TNF-α were examined after stimulating untreated macrophages with MO, TDM, and MO-TDM (1 mol%). Both TNF-α and IL-6 are known to be key cytokines involved in the production of protective innate immune responses against MTB infection. Since macrophages are the main host cells for MTB infection, these cytokines were examined in MTB-infected macrophages after pre-stimulation and simultaneous stimulation with MO, TDM, and MO-TDM (1 mol%). In the pre-stimulation experiment, macrophages were treated with MO, TDM, and MO-TDM (1 mol%) 3 days before infection, while in the simultaneous stimulation, they were treated with TDM and MO-TDM (1 mol%) at the beginning of infection. During simultaneous stimulation, a significant increase in the secretion of both IL-6 and TNF-α was observed in macrophages treated with MO-TDM (1 mol %) compared to untreated, MO- or TDM-treated macrophages ( Figure 7 a). On the other hand, in pre-stimulated macrophages, not only MO-TDM (1 mol%) but also MO alone was able to induce increased secretion of both IL-6 and TNF-α compared with untreated and TDM-treated macrophages ( Figure 7 b). However, the MO-TDM (1 mol%) preparation had a more significant stimulatory effect on IL-6 and TNF-α secretion compared to MO-treated macrophages. In order to further examine whether the increase in the pro-inflammatory immune response of macrophages induced by MO-TDM (1 mol%) can also affect the survival rate of MTB in macrophages, the viability of the pathogen was examined in a time-dependent manner during simultaneous stimulation and pre-stimulation with MO, TDM and MO-TDM (1 mol%). Macrophages treated with nanocarriers (MO-TDM (1 mol%)) showed a significant reduction of more than 1 log10 CFU after 7 days of infection in simultaneously stimulated and pre-stimulated macrophages ( Figure 7c and 7d). Thus, similar to the increase in inflammatory cytokine responses, MO-treated macrophages also induced significant killing of M. tuberculosis in macrophages during pre-stimulation as well as during simultaneous stimulation. Overall, these results indicate that the MO-TDM (1 mol%) nanocarrier formulation induced a better inflammatory immune response, which was also associated with its enhanced anti-mycobacterial effect in macrophages.

[0297] MO-TDM (1 mol%) induces trained immunity in macrophages

[0298] Although it is known that TNF-α secretion in macrophages increases after TDM activation, the further multiple induction of MO-TDM (1mol%) formulations is significant. Even more interesting is the observation of the effect of MO-TDM (1mol%) formulations on IL-6 secretion by macrophages, which has not been reported in any early studies on TDM formulations to date. Paired stimulation of TNF-α and IL-6 together can be associated with a trained immune phenotype in macrophages, indicating that MO-TDM (1mol%) can induce cultured immunity in macrophages. In order to evaluate the strength of the trained immune response of MO-TDM (1mol%) cubes, an in vitro model of primary human monocytes (PBMCs) was used (Bekkering; "Clinical and Vaccine Immunology (Clin Vaccine Immunol)" 23,926-933). In brief, human PBMC-derived adherent primary monocytes were primed for 18 hours with culture medium only as a negative control or MO or TDM or MO-TDM (1mol%), and then rested for five days. On day 6, monocytes were stimulated with lipopolysaccharide (LPS) and the concentrations of IL-6 and TNF-α were measured. The same cells were used to study the enrichment of IL-6 and TNF-α promoters by H3K4me3 histone modification. A consistent induction of TNF-α and IL-6 in human PBMCs was observed in response to MO-TDM (1 mol%) acid cubes, a result that was significantly higher than that observed in control-exposed monocytes ( Figure 8 a).

[0299] In the literature, the ability of traditional BCG methods to induce trained immunity is associated with changes in epigenetic markers that increase the expression of pro-inflammatory genes. H3K4me3 modification plays an important role in BCG-induced trained immunity, as increased trimethylation of histone H3 at lysine 4 is associated with increased transcription of pro-inflammatory cytokine genes, representing a mechanism responsible for the long-term regulation of monocyte-derived cytokines. Therefore, it was of interest whether the enhanced induction of TNF-α and IL-6 expression triggered by MO-TDM (1 mol%) cubes compared to other controls was epigenetically mediated. The activating histone methylation mark H3K4me3 present in the promoters of TNF-α and IL-6 was quantified using a chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) assay. It was observed that MO-TDM (1 mol%) cubes induced a higher enrichment of H3K4me3 on the promoters of TNF-α and IL-6 compared to other controls, suggesting a stronger epigenetic training of monocytes ( Figure 8 b) Increased abundance of activated epigenetic marks in MO-TDM (1 mol %) cubes after restimulation with LPS on day 6.

[0300] Epigenetic reprogramming of macrophages at the level of histone methylation and acetylation was associated with high expression of genes involved in glucose metabolism, as well as the reacquisition of pro-inflammatory properties of macrophages and M1 repolarization. This immune process has been found to be effective in the treatment of various infectious diseases and cancers. This has been shown to be associated with increased survival of tumor-bearing mice and cancer patients. Therefore, induced trained immunity of engineered MO-TDM (1 mol%) cubes may be a fundamental mechanism for controlling tuberculosis infection in macrophages.

[0301] MO-TDM (1 mol%) stimulated macrophages improve MHC-II antigen presentation to CD4 T cells

[0302] Although the micellar form of TDM is known to induce antibody-mediated immune responses, it was found in previous studies to have an inhibitory effect on cell-mediated immunity (Welsh; Tuberculosis 93, S3-S9). By inhibiting phagosome-lysosome fusion in macrophages, TDM not only protects Mycobacterium tuberculosis from intracellular killing, but also reduces T cell priming due to reduced processing of mycobacterial antigens in the lysosomal compartment. Since increased killing of intracellular MTB was observed when macrophages were stimulated with cubic MO-TDM (1 mol%) preparations, this indicates that lysosomal processing of its antigens may be increased. Without wishing to be bound by theory, it is believed that the MO-TDM (1 mol%) carrier can better prime T cells due to enhanced antigen processing and presentation of bacterial peptides. An in vitro antigen presentation assay using CD4 hybridoma T cells that recognize specific epitopes of Ag85B was used to determine the priming of T cells by macrophages stimulated with MO, TDM, and MO-TDM (1 mol%). The secretion level of IL-2 by hybridoma T cells upon stimulation with MO, TDM and MO-TDM (1 mol%) and without stimulation of M. tuberculosis infected macrophages was monitored in a time-dependent manner ( Fig. 9 During simultaneous activation, antigen presentation by T cells, as indicated by secreted IL-2 levels, increased in macrophages stimulated with MO, TDM, and MO-TDM (1 mol %) until day 3, but decreased dramatically on day 5 post-infection ( Fig. 9 a). From day 3 to day 5, MO-TDM (1 mol%) significantly reduced the reduction in antigen presentation compared to TDM and MO stimulated macrophages, indicating that MO-TDM (1 mol%) treated macrophages were more beneficial for sustained antigen processing and presentation. Although relatively less obvious, similar observations were also observed in macrophages pre-stimulated with MO, TDM, and MO-TDM (1 mol%), where MO-TDM (1 mol%) exhibited more sustained antigen presentation compared to TDM and MO treated macrophages ( Fig. 9 b). Since CD4 T cells process and present antigens via the MHC-II pathway, which requires degradation of bacteria / its proteins via the lysosomal pathway, the increased antigen presentation by MO-TDM (1 mol%) treated macrophages indicated that cubic nanocarriers with MO-TDM could overcome the inhibitory effect of TDM on the maturation of MTB containing phagosomes, which resulted in their more efficient delivery to the lysosomal compartment.

[0303] Example 6

[0304] In vivo prediction of MO-TDM (1 mol%) vaccine efficacy using mathematical models

[0305] A mechanistic mathematical model was developed to simulate and study the innate and adaptive immune responses to administered antigens (MO, TDM, and MO-TDM (1 mol%)) and MTB in vivo. The model is based on previous simulation work involving the study of immune responses to infectious agents, supporting preclinical development of vaccines delivered to the lung, optimizing vaccine dosing regimens, and quantifying the in vivo pharmacokinetics of nanoparticle-based drug delivery systems. The in vitro experimental data generated above (such as Figure 8 and 9 The minimalist model presented here integrates key processes related to antigen pharmacokinetics, cytokine-mediated innate immune responses, and antigen-presenting cell (APC)-induced adaptive immune responses involving CD4+ T cells and antibodies, followed by temporally separate injections of antigen and MTB into virtual mice.

[0306] Specifically, the model contains two compartments, the plasma and lymphatic compartments, which communicate through APCs and antibodies ( Fig.10 A(a)). The antigen is injected intraperitoneally and absorbed into the systemic circulation (i.e., plasma compartment) according to first-order kinetics, where it is cleared (renal and / or hepatobiliary excretion) or processed by naive APCs, such as PBMCs. The interaction of the antigen with the APCs in the plasma compartment activates the latter and they begin to secrete proinflammatory cytokines (IL-6 and TNF-α) to neutralize live MTB (if any) and participate in components of adaptive immunity. After activation, the APCs migrate to the lymphoid compartment and interact with naive CD4+T cells and B cells to convert them into effector cell forms, which causes the former to produce IL-2 after conversion into plasma cells, and the latter to produce antibodies. It is worth noting that IL-6 promotes the activation of naive CD4+T cells, while IL-2 promotes the proliferation of effector CD4+T cells ( Fig.10 A (curved blue arrow in a). In addition, IL-2 induces the conversion of activated B cells into antibody-secreting plasma cells that migrate to the plasma compartment to neutralize live MTB, if any. Note that the same sequence of events occurs when APCs interact with MTB.

[0307] Model parameters specific to mice are either known a priori from the literature or are derived from model responses to the host’s innate immune response (IL-6 and TNF-α-induced Mtb death; Figure 8 ), and antigen presentation to CD4+ T cells ( Fig. 9) were estimated by nonlinear least squares regression of in vitro data associated with . In order to properly calibrate the model, the protocol used in the in vitro experiments was closely replicated with a simplified form of the model, i.e., equations 2-4 for the characterization of the innate immune response, and a combination of equations 5 and 6 for the characterization of antigen presentation (see Methods below for details).

[0308] exist Fig.10 In A(b)(i-iv), the graphs show the model fit of cytokine dynamics (squares, IL-6; triangles, TNF-α) in response to macrophages (i.e., APCs) pre-incubated with antigen (MO, TDM, MO-TDM (1 mol%)) (days 0-2) and then exposed to MTB (day 2). In addition, the effect of cytokines on the dynamics of the MTB population in the culture medium is also shown (circles). In contrast, the bottom row of graphs ( Fig.10 A(b)(v-viii) shows the cytokine and MTB dynamics when antigen and MTB are simultaneously applied to the culture medium containing macrophages (day 0). The numerical solution of the ODE system (Equations 2-4) that replicates the above scheme and represents the interaction between antigen, macrophage, cytokine and MTB is fitted to the experimental data under two culture conditions at the same time. From the Pearson correlation analysis, it is observed that the fit of equations 2-4 to MTB and cytokine dynamics is very consistent with the in vitro data (R>0.9, P<0.0001), thus providing confidence in the parameter values ​​estimated by the model.

[0309] As observed in the pre-incubation case ( Fig.10 A(b)(i-iv)), cytokine production by macrophages in response to antigen was highest when incubated with MO-TDM (1 mol%) ( Fig.10 A(b)(iv)), followed by MO( Fig.10 A(b)(iii)), and then TDM ( Fig.10 A(b)(ii)), as observed by the kinetics during the first two days in culture without MTB. As expected, the Michaelis-Menten constant k thus obtained Ag (indicating the potency of antigen in triggering macrophages to produce cytokines) was the lowest for MO-TDM (1 mol%), indicating that MO-TDM (1 mol%) antigen had the highest potency. Ag Located between TDM and MO-TDM.

[0310] When MTB was introduced into the culture on day 2 ( Fig.10A(b), (i-iv)), cytokine levels continued to follow the same trend as before, resulting in a more rapid decline in the MTB population in the MO-TDM (1 mol%) condition compared to the no antigen condition, followed by MO and TDM. This may indicate that antigen-induced priming of macrophages results in an increase in cytokine production after MTB exposure, which may result in an increase in cytokine-induced MTB inhibition and is most effective for MO-TDM (1 mol%). A similar trend was observed in the case of simultaneous administration ( Fig.10 A(b)(v-viii)), where simultaneous administration resulted in almost the same enhancement of antigen-induced cytokine production and MTB decline as preincubation, possibly indicating that the presence of antigen enhances the macrophage response to MTB.

[0311] Furthermore, antigen presentation by macrophages to CD4+ T cells was characterized to obtain kinetic parameters associated with IL-2 production by effector CD4+ T cells after antigen presentation was completed. Fig.10 As shown in A(c), the model fit (Eq. 9) is in good agreement with the experimental data, and the most efficient antigen presentation was observed for macrophages primed with MO-TDM (1 mol %).

[0312] Model predictions

[0313] After calibrating the model with experimental data and estimating unknown parameters, the complete model was used to study the innate and adaptive immune responses to intraperitoneal administration of antigen in mice (day 0), followed by re-challenge with MTB on day 60. The plasma pharmacokinetics of antigen after intraperitoneal injection showed comparable behavior between small molecule TDM and nanoparticle antigen (MO, MO-TDM (1 mol%)), which is expected due to the high renal excretion of the former and high hepatobiliary clearance of the latter, mainly driven by their respective sizes ( Fig.10 B(a, embedded graph)).

[0314] In response to antigen injection on day 0, innate responses in the form of IL-6 and TNF-α cytokines predominated in the MO-TDM condition (1 mol%), persisting up to about 2 days after injection, but were less pronounced for the other two antigens ( Fig.10 B(c) and (d), inset). As a result, IL-6 induced activation of CD4+ T cells through antigen presentation, which was stronger in the case of MO-TDM (1 mol%), but weaker in the case of TDM, and forced to be absent in the case of empty cube (MO). Note: Adaptive immunity against MTB can only be generated by vaccines carrying MTB-specific antigens (e.g., MO-TDM (1 mol%) or TDM, but not MO).

[0315] Furthermore, model simulations predicted that, in vivo, vaccination with MO-TDM (1 mol%) antigen would result in the generation of MTB-specific, IL-2-secreting effector CD4+ T cells ( Fig.10 B), due to the autocrine signaling of IL-2 ( Fig.10 B(f)), which can be self-sustaining and enable the conversion of active B cells into antibody-secreting plasma cells ( Fig.10 B(g)). Therefore, upon exposure to MTB (e.g., day 60 in the current simulation, Fig.10 B(b)), the plasma compartment already contains MTB-specific effector CD4+ T cells ( Fig.10 B(e)) and antibodies ( Fig.10 B(h)), the latter of which is readily used for MTB neutralization, thereby reducing the bacterial load by about 5 orders of magnitude within about 10 days of infection ( Fig.10 B(b)). In contrast, vaccination with TDM did not generate significant effector CD4+ T cells and antibodies, mainly due to its poor efficacy in inducing antigen presentation ( Fig.10 A(c)). Thus, unlike MO-TDM (1 mol%), the TDM vaccine acts similarly to the empty cube (MO) or no vaccine case and is driven primarily by freshly emerged innate and adaptive immune responses to MTB infection, i.e., no significant memory of previous exposure to antigen is retained in the system. Thus, it takes up to about 10 times longer to produce the same amount of MTB load reduction compared to the MO-TDM (1 mol%) case.

[0316] It should be emphasized that the model developed here is a simplified representation of a highly complex and dynamic immune response involving many participants. However, the main purpose of the model is to show the relative response of the immune system to the various antigens studied in this study, and therefore the application of the same model to each antigen proves its applicability in illustrating the insights obtained from numerical simulations. Additionally, in the absence of in vivo data, in the pharmacokinetic component of the model, all antigens are assumed to be one-compartment pharmacokinetics; therefore, future improvements may be necessary to more accurately represent pharmacokinetics, especially including the physicochemical properties of the antigen (e.g., size, zeta potential, shape, solubility, permeability, lipophilicity).

[0317] In summary, the experiments discussed herein demonstrate the ability of MO-TDM cubes to induce robust innate and adaptive responses in vitro. Existing mathematical models of immune responses to antigens further predict the efficacy of MO-TDM to induce potent innate and humoral immune responses that are protective against simulated MTB rechallenge in vivo.

[0318] Mathematical model equation

[0319] As described below, the model is formulated as a system of ordinary differential equations (ODEs) that describes the dynamics of MTB burden in response to key innate and adaptive immune variables induced during exposure to antigen and MTB.

[0320] Antigen concentration in plasma (Ag(t))

[0321] For in vivo simulations, antigen administration was simulated via the intraperitoneal route, such that the antigen was delivered at a rate k abs is absorbed into the systemic circulation at a rate k ex Cleared from the systemic circulation.

[0322]

[0323] where Ag0 is the administered dose of antigen and V p is the volume of the plasma compartment. Note that to simulate in vitro experiments, a constant level of antigen in the culture medium (=1) was assumed.

[0324] MTB density in plasma (M(t))

[0325] The immune response to MTB infection involves both innate and adaptive components. The roles of IL-6, TNF-α, and antibodies in neutralizing MTB are included in the current model.

[0326]

[0327] Where γ is the growth rate of MTB, δ cy is the mortality of MTB induced by pro-inflammatory cytokines (IL-6 and TNF-α); k cy is the Michaelis constant of the cytokine (IL-6, TNF-α, IL-2) that induces its biological effect; IL6(t) and TNFα(t) are the plasma concentrations of IL-6 and TNF-α, respectively; δ Ab is the mortality rate of MTB by antibody-mediated phagocytosis; Ab(t) is the plasma concentration of antibody; M0 is the MTB load introduced into the plasma at day i after the initial dose of antigen given as a vaccine on day 0, at which the immune response to MTB re-challenge was studied.

[0328] IL-6 concentration in plasma (IL6(t))

[0329] The production of cytokines by macrophages is regulated by antigen or Mtb concentration in plasma. This process was simulated using Michaelis-Menten kinetics.

[0330]

[0331] Among them, p IL6 and δ IL6are the production and degradation rates of IL-6, respectively; k Ag and k M are the Michaelis constants for the effects of antigen and MTB on cytokine production by macrophages, respectively.

[0332] TNF-α concentration in plasma (TNFα(t))

[0333] The plasma TNF-α concentration was measured.

[0334]

[0335] where p T and δ TNFα are the production and degradation rates of TNF-α, respectively.

[0336] The concentration of effector CD4+ T cells in the lymphoid compartment (CD4 * (t))

[0337] The population of effector CD4+T cells is controlled by the activation of naive CD4+T cells after interaction with active APCs mediated by IL-6 (the first term of the equation). After activation, CD4+T cells proliferate, and this process is promoted by IL-2 (the second term of the equation). Note that since active APCs are not explicitly simulated, antigen and MTB concentrations are used as substitutes for active APCs in the first term of the following equation. In addition, the population of effector CD4+T cells is limited by carrying capacity to avoid unrealistic overpopulation due to proliferation.

[0338]

[0339] Among them, T cD4 is the conversion rate of naive CD4+ T cells into their effector form; CD40 is the baseline population density of naive CD4+ T cells; p CD4 is the production rate of effector CD4 + T cells, IL2(t) is the concentration of IL-2 in the lymphoid compartment; is the carrying capacity of effector CD4+ T cells, and δ CD4 is the death rate of effector CD4+ T cells.

[0340] IL-2 concentration in lymphoid compartment (IL2(t))

[0341] Upon interaction with antigen or MTB, effector CD4+ T cells secrete IL-2 as a primary process, which also undergoes degradation as a primary process.

[0342]

[0343] Among them, p IL2 and δ IL2 are the production and degradation rates of IL-2, respectively.

[0344] Plasma cell concentration in the lymphoid compartment (Pl(t))

[0345] The process of conversion of naive B cells into their effector forms and subsequent differentiation of the latter into plasma cells (under the influence of effector CD4+ T cells and IL-2) has been simplified to capture the kinetics of plasma cell population density by explicitly introducing only the second step in the model, which is assumed to be the rate-limiting step.

[0346]

[0347] Where T Pl and δ Pl are the transformation rate and death rate of plasma cells, respectively.

[0348] Antibody concentration in plasma (Ab(t))

[0349] Antibody concentration kinetics depend on antibody production by plasma cells and clearance from the body.

[0350]

[0351] where p Ab and Cl are the production and clearance rates of the antibody, respectively. It is the body's ability to carry antibodies.

[0352] Mathematical model calibration

[0353] Nonlinear least squares regression was performed to fit the model to the in vitro data (eg Fig.10 ), thereby calibrating the parameters associated with cytokine-induced MTB death and APC presentation of antigen to CD4+ T cells. For the first part of the model calibration, Equations 2-4 and Fig.10 The data shown in A are fitted, but given the in vitro nature of the experiment, antibody-induced Mtb death is neglected in equation 2. For the second part, the following equation is Fig.10 The data given in B are fitted to estimate the unknown parameters:

[0354]

[0355] The above equation was obtained by combining equations 5 and 6, while only the transformation and death processes were included in equation 5 and their closed-form solutions were obtained in equation 6 for substitution. Pearson correlation was performed to evaluate the goodness of fit of the model to the experimental data. All analyses were performed in MATLAB R2018a

[0356] Example 7 - In vivo immunogenicity experiment

[0357] Immunogenicity experiments were performed in mice using ovalbumin (OVA) as a model antigen. The following nanoparticle carriers were evaluated, with a polystyrene nanoparticle carrier used as a positive control:

[0358] - Cubes; Composition: Monoolein (MO) and Pluronic F127 in Milli Q water (10% w / w relative to total lipid mass)

[0359] - Cationic cubes (or modified cubes); composition: dioleoyl-3-trimethylammonium propane (DOTAP, 1 mol%), monoolein (MO) and Pluronic F127 in Milli Q water (10% w / w relative to total lipid mass)

[0360] - Polystyrene nanoparticles with an average particle size of 50 nm or 500 nm (PSNP 50nm and PSNP 500nm, respectively).

[0361] The following groups of mice (4 mice per group) were evaluated: naive, cuboidal-OVA, modified cuboidal-OVA, PSNP-OVA 50nm, and PSNP-OVA 500nm. Experimental timeline: Day -1: pre-bleeding; Day 0: first immunization, intradermal at the base of the tail; Day 13: first bleed; Day 14: second immunization, intradermal at the base of the tail; Day 28: blood and spleen were picked and collected for ELISA and ELISpot assays. Each mouse was injected with 50 μg of antigen.

[0362] ELISpot results - IFNg-OVA: Splenocytes from immunized mice were co-cultured with whole OVA, CD4 T cell epitope OVA helper cells, and CD8 T cell epitope SIINFEKL, and IFNg cytokine responses were assessed by ELISpot assay. PSNP-OVA 50nm and 500nm induced high responses to all antigens. PSNP-OVA is a known inducer of antigen-specific IFNg secretion by T cells, especially 50nm PSNPs, so high responses to all antigens are expected. Compared to background, cubic-OVA and modified cubic-OVA showed no induction of IFNg secretion.

[0363] ELISpot results - IL-4OVA: Splenocytes from immunized mice were co-cultured with whole OVA and CD4 T cell epitope OVA helper cells and IL-4 cytokine responses were assessed by ELISpot assay. PSNP-OVA 50nm and 500nm were shown to induce variable responses to all antigens, with PSNP-OVA 50nm inducing moderate to high responses to OVA helper cells. Cubic-OVA and modified cubic-OVA were shown to not elicit IL-4 secretion compared to background.

[0364] ELISA final bleed results: Serum was harvested from the final bleed of mice immunized twice and assessed for antigen-specific OVA IgG antibodies using an ELISA assay. Sera were serially diluted 1:2 starting at a 1:200 dilution. Fig.11 The antibody response measured as optical density readings for each dilution is shown, and the mean for each group was calculated. Naive mice had low OVA-specific antibody responses, while cuboidal-OVA, modified cuboidal-OVA, and PSNP-OVA groups all showed induction of OVA-specific antibody responses.

[0365] The results of ELISpot and ELISA assays indicated that cuboidal-OVA and modified cuboidal-OVA could potentially elicit a B cell-mediated immune response. Specifically, these formulations did not show induction of IFNg or IL-4 secretion in the ELISpot assay, but showed induction of OVA-specific antibody responses in the ELISA assay.

[0366] This mechanism was further investigated in IgG subtype-specific ELISA assays. Immunoglobulin G (IgG) antibodies are secreted by B cells. The following subclasses of IgG were evaluated using the protocol described above: IgG1, IgG2a, IgG2b, and IgG3. Results: IgG1: Cube-OVA, modified cube-OVA, and PSNP-OVA groups all showed induction of OVA-specific IgG1 antibody responses; IgG2a: All formulations showed induction of low OVA-specific antibody responses; IgG2b: Compared with cube-OVA and PSNP-OVA500nm, modified cube-OVA and PSNP-OVA 50nm showed induction of higher OVA-specific IgG2b antibody responses; IgG3: All formulations showed induction of low OVA-specific antibody responses. In all assays, the OVA-specific antibody responses of naive mice were lower.

[0367] The above results indicate that cuboidal-OVA and modified cuboidal-OVA can induce B cell-mediated immune responses. The OVA-specific antibody response also indicates that it is the OVA antigen that increases the immune response, where cuboidal and modified cuboidal only serve as carriers for presenting antigens and do not induce immune responses themselves.

Claims

1. A non-lamellar lyotropic liquid crystal phase carrier comprising one or more lipids forming the carrier and an antigen associated with the carrier.

2. The vector of claim 1, wherein the one or more lipids are or comprise one or more amphipathic lipids.

3. The carrier according to claim 2, wherein the one or more amphiphilic lipids are selected from the group consisting of: 1-monoolein, 2-monoolein, citrem, oleyl lactate, oleamide, monoelaidin, linoleic acid, elaidic acid, monopalmitolein, monolinolein, phytantriol, diolein, triolein, dioleoylglycerol, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), NN-dioleoyl-N,N-dimethyl chloride ammonium (DODAC), dioctadecyl ammonium chloride (DOAC), dioctadecyl dimethyl ammonium chloride (DODMAC) or dioctadecyl dimethyl ammonium bromide (DODAB), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-phosphatidylglycerol (DOPG), oleic acid (OA), lyso-1-hydroxy-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-dihexyl-phosphocholine (DOPC), vitamin E tocopherol, vitamin E (tocopherol) acetate, phytanyl monoethanolamide, farnesyl monoethanolamide, oleyl monoethanolamide, linoleyl monoethanolamide and linolenoyl monoethanolamide.

4. The vector according to claim 3, wherein the one or more amphiphilic lipids are glyceryl monooleate (MO) and / or phytantriol.

5. A carrier according to claim 3 or claim 4, wherein the carrier comprises a combination of MO or phytantriol and one or more of cholesterol, DLPC, DSPC, DPPE, DPPS, DOPS, DPPC, DMPC, DMPS and DLPS.

6. The support according to any one of the preceding claims, wherein the non-lamellar lyotropic liquid crystal phase support is cubic or hexagonal.

7. The carrier of any of the preceding claims, wherein the particle size of the non-lamellar lyotropic liquid crystal phase carrier is between about 10 microns and about 40 nanometers.

8. The carrier according to any one of the preceding claims, wherein the non-lamellar lyotropic liquid crystal phase carrier further comprises at least one stabilizer.

9. The carrier of claim 8, wherein the at least one stabilizer is one or more of a poloxamer, a surfactant, or a PEGylated lipid.

10. A carrier according to claim 8 or claim 9, wherein the at least one stabiliser is present in an amount of 1 wt% to 20 wt% of the carrier.

11. The carrier according to any one of the preceding claims, wherein the antigen is encapsulated within the carrier.

12. The vector according to any one of the preceding claims, wherein the antigen is selected from the group consisting of proteins, glycoproteins, peptides, glycopeptides, polysaccharides, lipids, glycolipids, lipoproteins and lipopeptides.

13. The carrier according to any one of the preceding claims, wherein the antigen is hydrophobic or comprises at least one hydrophobic chain.

14. The vector according to claim 13, wherein the at least one hydrophobic chain is capable of associating with or embedding in a lipid bilayer.

15. A vector according to any one of the preceding claims, wherein the antigen comprises one or more hydrophobic chains independently selected from fatty acids (lipids), glycerolipids, glycerophospholipids, sphingolipids, steroids, terpenes and terpenoids, glycolipids, polyketides and poly (hydrophobic amino acids).

16. The carrier of any of the preceding claims, wherein the antigen is present at about 0.1 mol% to about 10 mol%, about 0.1 mol% to about 9 mol%, about 0.1 mol% to about 8 mol%, about 0.1 mol% to about 7 mol%, about 0.1 mol% to about 6 mol%, about 0.1 mol% to about 5 mol%, about 0.1 mol% to about 4 mol%, about 0.1 mol% to about 3 mol%, about 0.1 mol% to about 2 mol%, about 0.5 mol% to about 10 mol%, about 0.5 mol% to about 9 mol%, about 0.5 mol% to about 8 mol%, about 0.5 mol% to about 7 mol%, about 0.5 mol% to about 6 mol%, about 0.5 mol% to about 5 mol%, about 0.5 mol% to about 4 mol%, about 0.5 mol% to about 3 mol%, or about 0.5 mol% to about 2 mol%.

17. The vector according to any one of the preceding claims, wherein the antigen is the cord factor trehalose 6,6'-dimycolate (TDM).

18. An immunogenic composition comprising the non-lamellar lyotropic liquid crystal phase carrier according to any one of claims 1 to 17 and a pharmaceutically acceptable carrier, diluent and / or excipient.

19. The immunogenic composition of claim 18, wherein the immunogenic composition is a vaccine composition.

20. The immunogenic composition of claim 18 or claim 19, wherein the immunogenic composition comprises no or substantially no adjuvant which is not a component of the non-lamellar lyotropic liquid crystal phase carrier.

21. A method of delivering an antigen to a cell, the method comprising the step of contacting the cell with the non-lamellar lyotropic liquid crystal phase carrier according to any one of claims 1 to 17 or the immunogenic composition according to any one of claims 18 to 20.

22. A method of inducing an immune response in a subject, the method comprising the step of administering to the subject an effective amount of the non-lamellar lyotropic liquid crystal phase carrier according to any one of claims 1 to 17 or the immunogenic composition according to any one of claims 18 to 20.

23. A method for preventing, treating or ameliorating an infection, disease, disorder or condition, the method comprising administering to a subject in need thereof a therapeutically effective amount of the non-lamellar lyotropic liquid crystal phase carrier according to any one of claims 1 to 17 or the immunogenic composition according to any one of claims 18 to 20, thereby preventing, treating or ameliorating the infection, disease, disorder or condition.

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