TLR4-TLR7 ligand formulations as vaccine adjuvants

Abstract

A method to enhance an immune response in a mammal, and a composition comprising liposomes, a TLR4 agonist and a TLR7 agonist, are provided.

Description

A "substituent group," as used herein, means a group selected from the following moieties: (A) -OH, -NH2, -SH, -ON, -CF3, -CCh, -NO2, oxo, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, substituted with at least one substituent selected from: (i) oxo, -OH, -NH2, -SH, -CN, -CFs, -CCI3, -NO2, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, substituted with at least one substituent selected from: (a) oxo, -OH, -NH2, -SH, -CN, -CF3, -CCh, -NO2, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, substituted with at least one substituent selected from: oxo, -OH, -NH2, -SH, -CN, -CF3, -CCh, -NO2, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, and unsubstituted heteroaryl, A "size-limited substituent" or" size-limited substituent group," as used herein, means a group selected from all of the substituents described above for a "substituent group," wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C20 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C4-Ce cycloalkyl, and eacn substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 4 to 8 membered heterocycloalkyl. A "lower substituent" or" lower substituent group," as used herein, means a group selected from all of the substituents described above for a "substituent group," wherein each substituted or unsubstituted alkyl is, for example, a substituted or unsubstituted Ci-Ca alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloaikyl is a substituted or unsubstituted C5-C7 cycloalkyl, and each substituted or unsubstituted heterocycloaikyl is a substituted or unsubstituted 5 to 7 membered heterocycloalkyl. In some embodiments, each substituted group described in the compounds herein is substituted with at least one substituent group. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloaikyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloaikylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene described in the compounds herein are substituted with at least one substituent group, in other embodiments, at least one or all of these groups are substituted with at least one sizelimited substituent group. In other embodiments, at least one or ail of these groups are substituted with at least one lower substituent group. in some embodiments, a compound as described herein may include multiple instances of a substituent, e.g., R5, R5A, R5B, R5C, RSA, R6B, R6C, R7, R7A, R7B, R7C , R8, RSA, R8B, and / or R8C. In such embodiments, each substituent may optional be different at each occurrence and be appropriately labeled to distinguish each group for greater clarity. For example, where each R5A is different, they may be referred to as e.g^R5^1, R5A-2, R5A-3, R5A-4, R5A-5. Similarly, where any of R5A, R58, Rsc, R6A, R88, R80, R?, R7A, R7B, R7C , R8, R8A, R8B, and / or R8C multiply occur, the definition of each occurrence of R5A, R5B, R5C, R8A, R6B, R6C, R7, R7A, R7B, R7C , R8, R8A, R8B, and / or R8C assumes the definition ofR5A, R5B, R5C, R6A, R6B, R6C, R7, R7A, R7B, R7C , R®, R8A, R8B, and / or R8C, respectively. In one aspect, there is provided a compound having formula (II): or a pharmaceutically acceptaole salt thereor, in formula (il), zl is an integer from 0 to 4, and z2 is an integer from 0 to 5, R5 is substituted or unsubstituted cycioaikyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, R8 is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, R7 is hydrogen, or substituted or unsubstituted alkyl, and R8 is independently halogen, -CN, -SH, -OH, -COOH, -NH2, -CONH2, nitro, -CF3, -CCh, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycioaikyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryi. In one embodiment, R5 is R5A-substituted or unsubstituted cycioaikyl, R5A substituted or unsubstituted heterocycloalkyl, R5A substituted or unsubstituted aryl, or R5A substituted or unsubstituted heteroaryl. R5A is independently halogen, -CN, -CF3, -CCh, -OH, -NH2, -SO2, -COOH, oxo, nitro, -SH, -CONHz, R5B-substituted or unsubstituted alkyl, R5B-substituted or unsubstituted heteroalkyl, R5B-substituted or unsubstituted cycioaikyl, R5B-substituted or unsubstituted heterocycloalkyl, R5B-substituted or unsubstituted aryl, or R5B-substituted or unsubstituted heteroaryl. R5S is independently halogen, -CN, -CF3, -CCI3, -OH, -NHz, -SO2, -COOH, oxo, nitro, -SH, -CONH2, R5C-substituted or unsubstituted alkyl, R6C-substituted or unsubstituted heteroalkyl, R5C-substituted or unsubstituted cycioaikyl, R5C-substituted or unsubstituted heterocycloalkyl, R5C-substituted or unsubstituted aryl, or R5C-substituted or unsubstituted heteroaryl. R5C is independently halogen, -CN, -CF3, -CCh, -OH, -NH2, -SO2, -COOH, oxo, nitro, -SH, -CONH2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycioaikyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryi. Further to this embodiment, R8 is R8A-substituted or unsubstituted alkyl, R6A substituted or unsubstituted heteroalkyl, R6A substituted or unsubstituted cycioaikyl, R6A substituted or unsubstituted heterocycloalkyl, R6A substituted or unsubstituted aryl, or R8A substituted or unsubstituted heteroaryi. R8A is independentiy halogen, -CN, -CF3, -CCh, -OH, -NH2, -SO2, -COOH, oxo, nitro, -SH, -CONH2, R6B-substituted or unsubstituted alkyl, R6B-substituted or unsubstituted heteroalkyl, R6B-substituted or unsubstituted cycioaikyl, R6B-substituted or unsubstituted heterocycloalkyl, R8B-substituted or unsubstituted aryl, or 10 RSB-substituted or unsubstituted heteroaryi, R8S is independentiy halogen, -CN, -CF3, -CCI3, -OH, -NHz, -SO2, -COOH, oxo, nitro, -SH, -CONH2, R6C-substituted or unsubstituted alkyl, R6C-substituted or unsubstituted heteroalkyl, R8C-substituted or unsubstituted cycioaikyl, R6C-substituted or unsubstituted heterocycloalkyl, R6C-substituted or unsubstituted aryl, or R6C-substituted or unsubstituted heteroaryi, R6C is independentiy halogen, -CN, -CF3, -CCh, -OH, -NHz, -SO2, -COOH, oxo, nitro, -SH, -CONHz, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cydoalKyl, unsubstituted neterocycloalkyl, unsubsntuted aryl, or unsubstituted heteroaryl. Further to this embodiment, R7 is hydrogen, or R7A-substituted or unsubstituted alkyl. R7A is independently halogen, -CN, -CF3, -CCh, -OH, -NHz, -SO?, -COOH, oxo, nitro, -SH, -CONHz, unsubstituted alkyl, unsubstituted heteroalkyi, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl. Further to this embodiment, R8 is independently halogen, -CN, -SH, -OH, -COOH, - NH2, -CONH2, nitro, -CF3, -CCh, R8A-substituted or unsubstituted alkyl, R8A-substituted or unsubstituted heteroalkyl, R8A substituted or unsubstituted cycloalkyl, R8A-substituted or unsubstituted heterocycloalkyl, R8A substituted or unsubstituted aryl, or R8A-substituted or unsubstituted heteroaryl. R8A is independently halogen, -CN, -CF3, -CCh, -OH, -NHz, -SO2, -COOH, oxo, nitro, -SH, -CONH2, R8B-substituted or unsubstituted alkyl, R8B-substituted or unsubstituted heteroalkyi, R8B-substituted or unsubstituted cycloalkyl, R8B-substituted or unsubstituted heterocycloalkyl, R8B-substituted or unsubstituted aryl, or R8B-substituted or unsubstituted heteroaryl. R8B is independently halogen, -CN, -CF3, -CCh, -OH, -NH2, -SO2, -COOH, oxo, nitro, -SH, -CONH2, R8C-substituted or unsubstituted alkyl, 84C-substituted or unsubstituted heteroalkyi, R8C-substituted or unsubstituted cycloalkyl, R8C-substituted or unsubstituted heterocycloalkyl, R8C-substituted or unsubstituted aryl, or R8C-substituted or unsubstituted heteroaryl. R8C is independently halogen, -CN, -CF3, -CCh, -OH, -NHz, -SO2, -COOH, oxo, nitro, -SH, -CONHz, unsubstituted alkyl, unsubstituted heteroalkyi, unsubstituted cycioalkyi, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl. In another aspect, there is provided a compound of formula (II) as disclosed above, provided, however, that: (i) the compound of formula (II) is not (Ila), wherein R° is p-fluorophenyl or p-methylphenyl; (ii) the compound is not wherein Rs is unsubstituted aryl, unsubstituted cyclohexyl, unsubstituted thiazole, or-CHz-furanyl; or (iii) R7 is not hydrogen. Further to any aspect disclosed above, in one embodiment, R5 is not substituted phenyl, in one embodiment, R5 is not p-fluorophenyl or p-methylphenyl. In one embodiment, the compound does not have the structure of formula (Ila) wherein Rs is substituted phenyl. In one embodiment, the compound does not have the structure of formula (Ila) wherein R6 is p-fluorophenyl or p-methylphenyl. Further to any aspect disclosed above, in one embodiment, R6 is not substituted or unsubstituted aryl, unsubstituted cyclohexyl, unsubstituted thiazole, or-CHz-furanyl. In one embodiment, the compound does not have the structure of formula (lib) wherein Rs is substituted or unsubstituted aryl, substituted or unsubstituted cyclohexyi, substituted or unsubstituted thiazole, or alkyl substituted with a substituted or unsubstituted furanyl. In one embodiment, R6 is not unsubstituted aryl, unsubstituted cyclohexyl, unsubstituted thiazole, or-CHz-furanyl. Further to any aspect disclosed above, in one embodiment R5 is substituted or unsubstituted cycloalkyl or substituted or unsubstituted aryl, in one embodiment, R5 is unsubstituted cycloalkyl or unsubstituted aryl. In one embodiment, R5 is substituted or unsubstituted Ce-Cs cycioaikyl or substituted or unsubstituted phenyl. In one embodiment, R5 is substituted or unsubstituted Cs, cycioaikyl or substituted or unsubstituted phenyl. in one embodiment, R5 is R5A-substituted or unsubstituted C6 cycioaikyl or R5A-substituted or unsubstituted phenyl, wherein R5A is a halogen, in one embodiment, R5 is R5A- substituted or unsubstituted phenyl, wherein R5A is a halogen. In one embodiment, R5 is R5A- substituted or unsubstituted phenyl, wherein R5A is a fluoro, in one embodiment, R5 is unsubstituted phenyl. Further to any aspect disclosed above, in one embodiment the compound does not have the structure of Formula (lb) wherein R6 is substituted or unsubstituted aryl, substituted or unsubstituted cyclohexyi, substituted or unsubstituted thiazole, or aikyi substituted with a substituted or unsubstituted furanyl. in one embodiment, R5 is substituted or unsubstituted C4-C12 cycioaikyl, substituted or unsubstituted C3-C12 aikyi, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In one embodiment, R6 is substituted or unsubstituted C4-C12 cycloalkyl, substituted or unsubstituted C4-C12 alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In one embodiment, R6 is substituted or unsubstituted C4-C12 cycloalkyl, substituted or unsubstituted C4-Ci2 branched alkyl, or substituted or unsubstituted phenyl. In one embodiment, R6 is R8A-substituted or unsubstituted C4-C12 cycloalkyl, R8A-substituted or unsubstituted C4-C12 branched alkyl, or R6A-substituted or unsubstituted phenyl, wherein R6A is halogen. In one embodiment, R6 is R8A-substituted or unsubstituted C4-C12 cycloalkyl, R8A- substituted or unsubstituted C4-C12 branched alkyl, or R8A-substituted or unsubstituted phenyl, wherein R6A is fluoro. In one embodiment, R6 is unsubstituted C4-C12 cycloalkyl, unsubstituted C4-C12 branched alkyl, or R6A-substituted or unsubstituted phenyl, wherein R6A is fluoro. In one embodiment, R6 is unsubstituted C6-C12 cycloalkyl, unsubstituted C4-C12 branched alkyl, or unsubstituted phenyl. In one embodiment, R8 is unsubstituted Ce-Cw cycloalkyl. In one embodiment, R8 is unsubstituted Cs-Cb cycloalkyl. In one embodiment, R8 is unsubstituted cyclohexyl. In one embodiment, R7 is hydrogen or substituted or unsubstituted alkyl. In one 30 embodiment, R7 is hydrogen or unsubstituted alkyl. In one embodiment, R7 is hydrogen or unsubstituted C1-C3 alkyl. In one embodiment, R7 is hydrogen, methyl or ethyl. In one embodiment, R3 is methyl. In one embodiment, R7 is ethyl. In one embodiment, R7 is hydrogen. In one embodiment, zl is 0, 1,2, 3, or 4. In one embodiment, zl is 0 or 1. In one embodiment, zl is 0, In one embodiment, zl is 1, In one embodiment, z2 is 0,1,2, 3, 4, or 5. In one embodiment, z2 is 1. In one embodiment, R8 is independently substituted or unsubstituted alkyl. In one embodiment, R8 independently is substituted alkyl. In one embodiment, R8 is independently unsubstituted alkyl. In one embodiment, R8 is independently substituted or unsubstituted heteroalkyl. In one embodiment, R8 is independently substituted heteroalkyl. In one embodiment, R6 is independently unsubstituted heteroalkyl. In one embodiment, R8 is independently substituted or unsubstituted aryl. In one embodiment, R8 is independently substituted or unsubstituted heteroaryl. For formula (lie) (above), R6 is substituted or unsubstituted alkyl, or substituted or unsubstituted cycloalkyl; and R7 is substituted or unsubstituted alkyl. In one embodiment, R6 is unsubstituted cycloalkyl, e.g., cyclohexyl, cycloheptyl or cyclooctyl. In one embodiment, R6 is unsubstituted alkyl, e.g., 3,3-dimethylbutyl. In one embodiment, R7 is unsubstituted alkyl. In one embodiment, R10 is an alkyl ester. In another aspect, there is provided a compound having formula (lid): For formula (lid), L2 is a linker, and B1 is a purine base or analog thereof. in one embodiment, L2 is a substituted or unsubstituted alkylene, ora substituted or unsubstituted heteroalkylene. In one embodiment, L2 includes a water soluble polymer. A “water soluble polymer" means a polymer which is sufficiently soluble in water under physiologic conditions of e.g., temperature, ionic concentration and the like, as known in the art, to be useful for the methods described herein. An exemplary water soluble polymer is polyethylene glycol. In one embodiment,thewater soluble polymer is -(0C2CH2)m- wherein m is 1 to 100. In one embodiment, L2 includes a cleavage element. A “cleavage element” is a chemical functionality which can undergo cleavage (e.g., hydrolysis) to release the compound, optionally including remnants of linker L2, and B1, optionally including remnants 20 of linker L2. Table 1 human IL-6a 100 IL-8rf Too" Compound 27 40 TLR4C 10G mouse IP-10* 100 41 TLR4® 100 37 38 N-cyclopentyl Routes and Formulations Administration of compositions having one or more antigens and one or more 10 15 adjuvants and optionally another active agent or administration of a composition having one or more antigens and a composition having one or more adjuvants, can be via any of suitable route of administration, particularly parenterally, for example, intravenously, intra-arterially, intraperitoneally, mtrathecally, mtraventricularly, intraurethrally, intrasternaily, intracranially, intramuscularly, or subcutaneously. Such administration may be as a single bolus injection, multiple injections, or as a short- or long-duration infusion, implantable devices (e.g., implantable infusion pumps) may also be employed for the periodic parenteral delivery overtime of equivalent or varying dosages of the particular formulation. For such parenteral administration, the compounds (a conjugate or other active agent) may be formulated as a sterile solution in water or another suitable solvent or mixture of solvents. The solution may contain other substances such as salts, sugars (particularly glucose or mannitol), to make the solution isotonic with blood, buffering agents such as acetic, citric, and / or phosphoric acids and their sodium salts, and preservatives. The compositions invention alone or in combination with other active agents can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the cnosen route of administration, e.g., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes. Thus, the compositions alone or in combination with another active agent, e.g,, an antigen, may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the composition optionally in combination with an active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of conjugate and optionally other active compound in such useful compositions is such that an effective dosage level will be obtained. The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the phospholipid conjugate optionally in combination with another active compound may be incorporated into sustained-release preparations and devices. The composition optionally in combination with another active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the antigen(s), and adjuvant(s) optionally in combination with another active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms, Ine pharmaceutical dosage forms suitable ror injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms during storage can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it may be useful to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating compound(s) in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, one method of preparation includes vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions. For topical administration, the antigen(s) and adjuvant(s) optionally in combination with another active compound may be applied in pure form, e.g., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid. Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol / glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and antimicrobial agents can be added to enhance the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers. Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user, in addition, in one embodiment, the invention provides various dosage formulations of the antigen(s) and adjuvant(s) optionally in combination with another active compound for inhalation delivery. For example, formulations may be designed for aerosol use in devices such as metered-dose inhalers, dry powder inhalers and nebulizers. Examples of useful dermatological compositions which can be used to deliver compounds to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508). Useful dosages can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949. The ability of an adjuvant to act as a TLR agonist may be determined using pharmacological models which are well known to the art, including the procedures disclosed by Lee et al,. Proc. Natl. Acad. Sci, USA, 100: 6646 (2003). Generally, the concentration of the phospholipid optionaiiy in combination with another active compound in a liquid composition, such as a lotion, will be from about 0.1-25 wt-%, e.g., from about 0.5-10 wt-%. The concentration in a semi-solid or solid composition such as a gel ora powder will be about 0.1-5 wt-%, e.g., about 0.5-2.5 wt-%. The active ingredient may be administered to achieve peak plasma concentrations of the active compound of from about 0.5 to about 75 pM, e.g,, about 1 to 50 pM, such as about 2 to about 30 pM. This may be achieved, for example, by the intravenous injection of a 0.05 to 5% solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 1-100 mg of the active ingredient. Desirable blood levels may be maintained by continuous infusion to provide about Q.Q1-5.0 mg / kg / hr or by intermittent infusions containing about 0.4-15 mg / kg of the active ingredient(s). The amount of the antigen(s) and adjuvant(s) optionally in combination with another active compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician. In general, however, a suitable dose will be in the range of from about 0.5 to about 100 mg / kg, e.g., from about 10 to about 75 mg / kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, for instance in the range of 6 to 90 mg / kg / day, e.g., in the range of 15 to 60 mg / kg / day. The antigen(s) and adjuvant(s) optionally in combination with another active compound may be conveniently administered in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form. Ine desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day- The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye. The dose, and perhaps the dose frequency, will also vary according to the age, body weight, condition, and response of the individual patient. In general, the total daily dose range for an active agent for the conditions described herein, may be from about 50 mg to about 5000 mg, in single or divided doses. In one embodiment, a daily dose range should be about 100 mg to about 4000 mg, e.g., about 1000-3000 mg, in single or divided doses, e.g., 750 mg every 6 hr of orally administered compound. This can achieve plasma levels of about 500-750 uM, which can be effective to kill cancer cells. In managing the patient, the therapy should be initiated at a lower dose and incd depending on the patient’s global response. A specific antigen includes an amino acid, a carbohydrate, a peptide, a protein, a nucleic acid, a lipid, a body substance, or a cell such as a microbe. A specific peptide has from 2 to about 20 amino acid residues. Another specific peptide has from 10 to about 20 amino acid residues. A specific antigen includes a carbohydrate. A specific antigen is a microbe. A specific microbe is a virus, bacteria, or fungi. Specific bacteria are Bacillus anthracis, Listeria monocytogenes, Francisella tularensis, Salmonella, or Staphylococcus- Specific Salmonella are S. typhimurium or S. enteritidis. Specific Staphylococcus include S. aureus. Specific viruses are RNA viruses, including RSV and influenza virus, a product of the RNA virus, or a DNA virus, including herpes virus. A specific DNA virus is hepatitis B virus. The invention includes compositions that include of a TLR4 agonist and TLR7 agonist phospholipid conjugate optionally in combination with other active agents that may or may not be antigens, e.g., ribavirin, mizoribine, and mycophenolate mofetil. Exemplary Embodiments In one embodiment, a method to enhance an immune response in a mammal is provided. In one embodiment, the method comprises administering to a mammal in need thereof a composition comprising an effective amount of a TLR4 agonist and a TLR7 agonist. In one embodiment, the composition is a liposomal composition, in one embodiment, the composition comprises liposomes comprising a TLR4 agonist and liposomes comprising a TLR7 agonist, in one embodiment, the composition comprises liposomes comprising a TLR4 agonist and a TLRr agonist. In one embodiment ine ILR4 agonist and a ILR7 agonist are administered simultaneously. In one embodiment, the TLR4 agonist has formula (II). In one embodiment, the TLR4 agonist comprises 1Z105, 2B182c, INI-2004, or CRX601. In one embodiment, the TRL4 agonist is not 1Z105. In one embodiment, the TLR7 agonist has formula (I). In one embodiment, the liposomes comprise PC, DOPC, orDSPC. In one embodiment, the liposomes comprise cholesterol. In one embodiment, the method further comprises administering one or more immunogens, in one embodiment, the immunogen is a microbial immunogen, e.g., one or more microbial proteins, glycoproteins, saccharides and / or lipopolysaccharides. In one embodiment, the microbe is a virus, such as influenza or varicella, or a bacteria. In one embodiment, the microbe is a parasite or fungus, in one embodiment, the liposomes comprise the one or more immunogens. In one embodiment, the composition comprises the one or more immunogens, in one embodiment, the mammal is a human. In one embodiment, the mammal is a rodent, equine, bovine, caprine, canine, feline, swine or ovine. In one embodiment, the amount of the TLR7 agonist is about 0.01 to 100 nmol, about 0.1 to 10 nmol, or about 100 nmol to about 1000 nmol. In one embodiment, the amount of the TLR4 agonist is about 2 to 20 umol, about 20 nmol to 2 umol, or about 2 umol to about 100 umol. In one embodiment, the ratio ofTLR7 to TLR4 agonist is about 1:10, 1:100, 1:200, 5:20, 5:100, or 5:200. In one embodiment, the composition is injected. In one embodiment, the liposomes comprise DOPC and cholesterol. In one embodiment, the immunogen is a cell, protein or spore. In one embodiment, the immunogen is administered before or after the composition. In one embodiment, the administration is effective to prevent a microbial infection. In one embodiment, the composition is intranasally administered. In one embodiment, the composition is intradermally administered. In one embodiment, a pharmaceutical formulation comprising liposomes, a TLR4 agonist and a TLR7 agonist is provided. In one embodiment, the liposomes comprise 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphochoime (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2 dioleoyl-sn-glycero-34phosphor-L-serine] (DOPS), 1,2-dio!eoyi-3-trimethylammonium-propane (18:1 DOTAP), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dioleoyl-sn-glycero-3- PE), 1,2-dipalmitoyi-sn-glycero-3-phosphoethanolamine-N-[methoxy(poiyethylene glycoi)-2000] (16:0 PEG-2000 PE), 1-oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl]-sn-glycero-3-phosphocholine (18:1-12:0 NBD PC), 1 -palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)ammo]lauroyl}-sn-glycero-3-phosphocholine (16:012:0 NBD PC), and mixtures thereof; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioieoyl-sn-glycero-3-phosphoethanoiamine (DOPE), cholesterol, ora mixture thereof, in one embodiment, the liposomes comprise DOPC, cholesterol or combinations thereof. In one embodiment, the amount of the TLR7 agonist is about 0.01 to 100 nmol, about 0.1 to 10 nmol, or about 100 nmol to about 1000 nmol, in one embodiment, the amount of the TLR4 agonist is about 2 nmol to 20 umoi, about 20 nmol to 2 umoi, or about 2 umoi to about 100 umoi. In one embodiment, the ratio of TLR7 to TLR4 agonist is about 1:10, 1:100, 1:200, 5:20, 5:100, or 5:200. In one embodiment, the TLR7 agonist comprises a compound of Formula (I), in one embodiment, formula (I) comprises ZNR / °\ o'^y^or11 wherein R11 and R12 are each independently a hydrogen or an acyl group, R13 is a negative charge ora hydrogen, and m is 1 to 8, wherein a wavy line indicates a position of bonding, wherein an absolute configuration at the carbon atom bearing OR12 is R, S, or any mixture thereof, in one embodiment, m is 1. in one embodiment, R’1 and R12 are each oleoyl groups. In one embodiment, the phospholipid of R3 comprises two carboxylic esters and each carboxylic ester includes one, two, three or four sites of unsaturation, epoxidation, hydroxylation, or a combination thereof. In one embodiment, the phospholipid of R3 comprises two carboxylic esters and the carboxylic esters of are the similar or different, in one embodiment, each carboxylic ester of the phospholipid is a C17 carboxylic ester with a site of unsaturation at C8-C9. in one embodiment, each carboxylic ester of the phospholipid is a C18 carboxylic ester with a site of unsaturation at C9-C1Q. in one embodiment, X2 is a bond ora chain having one to about 10 atoms in a chain wherein the atoms of the chain are selected from the group consisting of carbon, nitrogen, sulfur, and oxygen, wherein any carbon atom can be substituted with oxo, and wherein any sulfur atom can be substituted with one or two oxo groups, in one embodiment, X2 is C(O), 5 In one embodiment, R3 comprises dioleoylphosphatidyl ethanolamine (DOPE). In one embodiment, R3 is 1,2-dioieoyl-sn-glycero-3-phospho ethanolamine and X2 is C(O). In one embodiment, X1 is oxygen. In one embodiment, X1 is sulfur, or -NRC- where Rc is hydrogen, Ci-s alkyl or substituted Ci-s alkyl, where the alkyl substituents are hydroxy, Cs-ecycloalkyl, Ci-salkoxy, amino, cyano, or aryl. In one embodiment, X’ is -NH-, In one 10 embodiment, R1 and Rc taken together form a heterocyclic ring or a substituted heterocyclic ring. In one embodiment, R1 and Rc taken together form a substituted or unsubstituted morpholino, piperidino, pyrrolidino, orpiperazino ring, in one embodiment, R1 is a C1-C10 alkyl substituted with CI-6 alkoxy. In one embodiment, R1 is hydrogen, Chalky!, or substituted Chalky!. In one embodiment, R1 is hydrogen, 15 methyl, ethyl, propyl, butyl, hydroxyCi-4alkylene, or Ci-4alkoxyCi-4alkylene. in one embodiment, R1 is hydrogen, methyl, ethyl, methoxyethyl, or ethoxyethyl. In one embodiment,R2 is halogen or Cwalkyl, or R2 is absent, in one embodiment, R2 is chloro, bromo, methyl, or ethyl, orR2 is absent, in one embodiment, X1 is O, R1 is Ci.4alkoxy-ethyi, n is 0, X2 is carbonyl, and R3 is 1,2-dioleoyiphosphatidyl ethanolamine (DOPE). In 20 one embodiment, the compound of Formula (I) is: In one embodiment, the compound of Formula (I) is 0 In one embodiment, in formula (II), z2 is 1,2 or 3. In one embodiment, in formula (II), z1 is 1 or 2. In one embodiment, in formula (II), z1 is 0. In one embodiment, in formula (II), R5 is substituted or unsubstituted aryl or heteroaryl, e.g., unsubstituted C5 or C6 aryl, in one embodiment, in formula (II), R6 is substituted or unsubstituted cycloalkyl or heterocycloalkyl, e.g., a 5, 6 or 7 cycloalkyi. In one embodiment, in formula (II), R7 is substituted or unsubstituted alkyl, e.g., a C1 to C5 alkyl. In one embodiment, in formula (II), R8 is a substituted or unsubstituted aryl or heteroaryl, e.g., a 5, 6 or 7 heteroaryl such as furanyl, pyrrolyl or imidazolyl. The invention will be further described by the following non-limiting examples. Example 1 Adjuvant potency of liposome-formulated 2B182c, TLR4 agonist, and 1V270, TLR7 agonist The hposomai formulation of 2B182c (200nmol / injection) and 1V270 (1 nmol / injection) alone or the combination of 200nmol 2B182c and 1nmol 1V270 were prepared (Inimmune Corp, Missoula, MI), The adjuvant potency of liposome-formulated adjuvants was compared to the DMSO formulation (10% DMSO). The formulated adjuvants were tested using the same protocol. In brief, female BALB / c mice were immunized on days 0 and 21 with liposome-formulated 2B182c (200 nmol / injection) and / or 1V270 (1 nmol / injection) with inactivated influenza virus and sera were evaluated foranti-HA and anti-NA antibodies (igM, lgG1 and lgG2a) by ELISA. Inguinal lymph nodes were harvested and analyzed for B cell populations by FACS to see whether formulated agonists affect germinal center B cell and plasmablast (antigen secreting ceil) populations. TLR4 are located both on the cell surface and in the endosomal compartment. The signaling through the endosomal receptors inhibits NF-kB activation by LPS. Endosomal TLR4 activation triggers TRIF pathway activation, leading type 1 IFN release through IRF3 activation. Therefore, the adjuvant activity of 2B182c might be attenuated by liposomal formulation. Liposome-formulated 2B182c induces significantly higher anti-HA lgG2a, while liposomal 2B182c reduced HA and NA specific lgG1 in mice immunized with 2B182c alone or 2B182c plus 1V270, in comparison with DMSO formulated adjuvants (Fig. 19A). The liposomal formulation did not affect lgG2a levels in 2B182c and 2B182c plus 1V270 combined adjuvant (Fig. 19A). The decreased levels of IgGI by liposomal formulation attributed to Th1 skewing immune responses by liposome-formulated 2B182c and the 2B182 / 1V270 combined adjuvants (Fig. 19B). These data are consistent to the report that described intracellular delivery ofTLR4 ligand induces effective Th1 immune responses dependent to type 1 IFN dependent manner. After the antigen exposure, activated na'ive and memory B cells are expanded and maturated in the germinal center (GC). Maintenance of high antigen specific Ab titers required for long-term vaccine efficacy is correlated with the GC formation. Activated B ceils further form antigen-specific Ab-secreting cells (ASCs; piasmablasts and plasma cells), memory B cells and other subsets. Plasmablasts were induced after the seasonal influenza virus vaccination and peak sharply on day 7 post-vaccination. Frequency of plasmablast in peripheral blood after the vaccination with an inactivated virus correlates with the magnitude of protective hemagglutinin inhibition titles in humans . Thus, GC B cells and plasmablasts in the draining lymph nodes were examined. The number of germinal center B cells and plasmablasts were increased by the combination with liposomal 2B182c and 1V270 (Fig. 20). In summary, liposome-formulated TLR4 and TLR7 ligands adjuvant induced Th1 skewed immune responses and increased GC center B cells and plasmablasts. To evaluate the quality of B cell responses induced by the combined adjuvant in liposomal formulation, we are currently conducting BCR and TCR repertoire analyses of the lymph node ceils. Furthermore, the functional evaluation of vaccine adjuvant is evaluated by the live virus (homologous and heterologous challenge). Example 2 A combination of synthetic small molecule TLR4 and TLR7 agonists is a potent adjuvant for recombinant influenza virus hemagglutinin, inducing rapid and sustained immunity that is protective against influenza viruses in homologous, heterologous, and heterosubtypic murine challenge models. However, the TL.R4 agonist used in those studies was 1Z105, a first-generation lead synthetic TLR4 agonist in the pyrimidoindoie class that was optimized from hits identified in a high throughput screening campaign to discover adjuvants that act as innate immune receptor agonists. 1Z105 was found to have good immunoactivity in murine cells, but was devoid of significant activity in human cells. In more recent studies, a second-generation series of compounds that contained a C8-aryl substituent was more potent than 1Z105 in murine cells, but was also very active in human cells as well. Within this active group of C8-aryl derivatives, the C8-furan-2-yl derivative (2B182C) was selected for further study based on potency and favorable preliminary formulation data (Figures 34 and 36). The pyrimidoindoie 2B182C was evaluated in combination with 1V270 for comparison. A MPLA analog (MPLA-1), a potent 1 LR4 agonist, demonstrated good protection against homologous and heterologous flu challenge In vivo. The TLR7 agonist, 1V270, is a phospholipid conjugate of a known TLR7 agonist. Major advantages that are conferred by the phospholipid moiety of the agonist conjugate over the corresponding unconjugated agonist include greater potency and lack of local or systemic toxicity, often observed as cytokine syndromes. These favorable properties demonstrating efficacy and safety support the selection of 1V270 as the lead TLR7 agonist for combination adjuvant studies described in this technical proposal. As mentioned previously, the combined adjuvants comprising the TLR4 agonist 1Z105 and TLR7 agonist 1V270 induced broadly protective responses with influenzavirus vaccine. SAR studies yielded 2B182C which demonstrated higher agonistic potency than 1Z105 in THP-1 ceils and in human and murine primary ceils in vitro. The adjuvant potency of 2B182C was examined in vaccination models using inactivated influenza virus [A / Cahfornia / 04 / 09 (Cal / 09)] and compared to 1Z105. These studies were conducted using simple DMSO-water formulations of the TLR agonists. Combined adjuvant with TLR4- and TLR7-aqonist induces rapid and broad ly-protective immunej^snonsestojnfluenza^ To assess profile of protective immune responses against influenza virus infection induced by TLR4 / TLR7 agonist-combined adjuvant, mice were immunized with low dose (0.2 pg / injection) recombinant hemagglutinin (rHA) and humoral responses and protection against lethal virus challenge (Figures 34A-2D). The mice immunized with rHA with the combined adjuvant showed minimal body weight loss and higher survival rate (Figures 34B and 2C). The combined adjuvant and 1V270, TLR7 agonist, alone induced Th1 biased immune responses. To further examine whether TLR4 / TLR7 combined adjuvant provides crossprotection against heterotypic influenza virus challenge, we immunized mice with 20092010 Fluzone, containing B / Brisbane / 60 / 2008 (Victoria lineage), and challenged them with 25 mLD50 of a heterologous mouse-adapted virus B / Flonda / 04 / 2006 (Yamagata lineage). More than 90% mice were survived following vaccination of Fluzone adjuvanted with 1V270, alone or in combination with 1Z105 (Figures 34E-2G). These data indicated that 1V270, alone or in combination with 1Z105, induces rapid and cross-protective immunity to heterologous influenza viruses. Determination of doses forTLR4- and TLR7-aqomsts As mentioned, SAR study yielded 2B182C that exhibited higher potency in vitro in comparison with 1Z105 in human and mouse immune ceils. To examine whether the higher potency observed in vitro study is also reproducible in vivo, female Balb / c mice were intramuscularly (IM) immunized on days 0 and 21 with the TLR4 agonists (1Z105 or 2B182C, 40 or 200 nmol / injection) and 1V270 (phospholipid TLR7 agonist conjugate, 0.2 or 1 nmol / injection) with inactivated influenza virus (A / California / 04 / 2009 (H1N1) pdm09, Cat# NR-49450, BEI resources) (Fig, 34A). The sera were collected on day 28 and anti-hemagglutinin (HA) and anti- neuraminidase (NA) antibodies (IgM, IgGI and lgG2a) were determined by ELISA. 1V270, 1Z105 and 2B182C were dissolved in DMSO and diluted and the final concentration of DMSO was 10% used as a vehicle control. Data were pooled from four independent experiments showing similar results. Effects of TLR agonist single agents on antibody secretion As a single adjuvant, 0.2 nmol and 1 nmol 1V270, and 40 nmol and 200 nmol 2B182C or 1Z105 were compared (Figure 34B). Both TLR4 agonists,1Z105 and 2B182C, induced significantly higher levels of igG1 against HA and NA, Regarding lgG2a induction, both 0.2 and 1 nmol / injection 1V270 significantly increased anti-HA (p<0.05), while only 200 nmol / injection 2B182C, but not 1Z105, enhanced anti-NA Abs (p<0.01) (Figure 34B). 2B182C and 1Z105 induced similar levels of HA specific lgG2a. There were no differences in IgM response by any adjuvant treatments. These data support reports that TLR4 agonists increased IgG 1 production and TLR7 agonist was effective on igG2a secretion and 2B182C showed similar or modestly higher potency compared to 1Z105 in vivo. Effects of combination treatment with 2B182C and 1V270 on antibody secretion Next, the potency of the combined adjuvants was evaluated using the DMSO-water formulations. Both combined adjuvants of 1V270 with 1Z105 and 2B182C improved induction of IgGI against both HA and NA. 2B182C enhanced significantly higher IgGI compared to 1Z105 at both 40 and 200 nmol (p<0.05, Figures 35A and 35B). In lgG2a induction, 2B182C increased the levels of anti HA- and anti NA- Abs; however, 1Z105 failed in most cases (Figures 35C and 35D). The adjuvants showed minimal effects on IgM release (Figures 35E and 35F). To compare the antibody titers of all combinations tested, the average IgGI and lgG2a titers are plotted in Figure 36A. 200 nmol 2B182C plus 0.2 or 1 nmol 1V270 showed the highest inductions of both IgGI and lgG2a (Figure 36A). Further, to evaluate Th1 / Th2 immune balance, lgG2a: IgGI ratio was calculated in individual animals (Figure 36B). 1 nmol 1V270 significantly shifted the Th2-biased immune responses by 1Z105 or2B182C, indicating that 1V270 shifted immune responses to Th1 bias (Figure 36B). In summary, these results indicated that the combination of 200 nmol / injection 2B182C plus 1 nmol / injection 1V270 induced the highest quantity of IgGI and lgG2a and Th1-skewing immune responses are desirable for heterologous protection in the influenza virus infection. Thus, we selected this combination for further preciinical formulation. Preliminary data with a TLR4 agonist The in vivo evaluation for MPLA-2, a sulfate analog of MPLA, combinations and for ail lead TLR agonists in nanoparticle formulations are conducted. A potent TLR4 agonist was discovered during NIAID adjuvant discovery and development contracts where it demonstrated additive if not synergistic enhancement of influenza relevant cytokine production in vitro (in hPBMCs), enhancement of lgG2A antibody and Hi titers with 1V270 in vivo in mice and pigs. A major weakness of MPLA-1 as an adjuvant is its lack of chemical stability as it is prone hydrolysis in aqueous media. In a preliminary murine study of non-specific resistance, MPLA-2 protected mice from lethal influenza challenge better than an equivalent dose of MPLA-1 and thus, MPLA-2 represents a next generation TLR4 agonist. in support of the objectives outlined above the experiments detailed below will be carried out. Research Area 1: Formulation and analytical assay development for lead TLR agonist combinations Development of formulations of TLR4 / TLR7 combinations Task 1 A: Development of colloidally stable nanoparticle formulations of lead compounds alone and in combination Particulate delivery systems act as adjuvants through mimicking the size and shape of the viral and bacterial pathogens our immune systems evolved to recognize and combat via pattern recognition receptors (PRRs). Research over the past 30 years has brought about numerous nano and microparticle based systems that are biodegradable and suitable for vaccine antigen delivery. Their utility as vaccine delivery systems has been demonstrated in the literature with liposomes, virosomes, Iscoms, emulsions, virus-like-particies (VLPs), solid-iipid-nanoparticles (SLNs) and polylactic co-glycolic acid (PLGA) polymers, with examples of each type advancing to human clinical trials. The primary adjuvant mechanism of particulate delivery vehicles is thought to be enhanced uptake of particle incorporated or associated antigens by APCs. It is now well established that the addition of PAMPs to antigens facilitates a robust innate and adaptive immune response through ligation of TLRs and other PRRs leading to innate immune ceil activation. A number of PAMPs (bacterial lipoproteins, glycolipids, DNA and viral RNA etc.) have been identified and isolated from viral and bacterial pathogens. Many of these agonists are powerful adjuvants, but exert an unacceptable level of inflammation or have unfavorable physical / chemical characteristics for clinical development. In response, researchers have successfully produced synthetic analogs with improved safety and chemical profiles and many of these have been added to particulate delivery systems to enhance their pathogen mimicry through PRR ligation. Particulate delivery systems can also be used to improve the biodistribution kinetics of adjuvants in vivo and reduce adjuvant side effects without sacrificing adjuvant immunogenicity. The effective sublingual vaccine use of PEGylated liposomes with bilayer incorporated TLR4 agonist MPLA-1 has been shown in murine models of influenza. This formulation reduces the pyrogenicity ofMPLA-1 200-fold without any loss of adjuvant potency in vivo. This is analogous to the observed reduction of pyrogenicity of LPS when incorporated in liposomes versus aqueous dispersions. The same reduction of pyrogenicity is expected forTLR4 agonists. A number of different lipids and components for the nanoparticle / microparticle formation, API incorporation, API stability and colloidal stability were evaluated. A range or commercially available cationic (DDA, DOTAP, DC-cholesteroi), anionic (DPPG, PS, POPG) and neutral lipids (PC, DOPC, DSPC) are tested with the TLR4 and TLR7 agonists. Other formulations may employ PLGA, polycaprolactone, poly(propargyl methacrylate) or PLMA. Because particle size and charge have been shown to significantly influence nanoparticle uptake and processing by DCs, the impact of these variables to enhance delivery vehicle design for the quality characteristics listed above is explored. Small-scale liposomal formulations can be prepared using a thin-film method adapted for sterile serum vials to further reduce scale and waste. Briefly, this will be done by: 1. Adding APIs to the lipid and dissolving them in chloroform (a fluorescent marker may also be added at this step if desired, e.g. NBD, BODIPY, FITC, etc.) 2. Rotary evaporation at a set speed and vacuum to a dry thin-film 3. Rehydration with aqueous buffer (0.1 M phosphate, TRIS or HEPES) 4. Particle size reduction by bath sonication above the lipid transition temperature (Tm) with in process monitoring of particle size, polydispersity and surface charge (zeta potential) by dynamic light scattering (DLS) 5. 3 to 10 mL scale lots of lead formulations will be prepared using a Lipex extruder which improves particle size homogeneity (polydispersity index, PDI) over sonication methods. Task IB: Stability studies to assess colloidal and physical stability of formulations Formulation stability is needed for development of a successful commercial product as it impacts product storage, shipping and shelf life which all directly contribute to product cost. Formulations are demonstrated to be suitable as potential products, as well stability, particularly when selecting lead candidates to pursue further. Lead formulations are assessed for short term accelerated (25 and 40 °C) and long-term real time stability (2--8 °C and 25 °C) to ensure formulations chosen provide sufficient stability for potential product development (minimum of 12 months at preferred storage condition). Accurate quantitation of adjuvant incorporation into a nanoparticle delivery system is essential for proper dosing, vaccine efficacy and safety. SEC-HPLC and RP-HPLC methods for quantitation of TLR4 and TLR7 / 8 agonists incorporated into nanoparticles, including liposomes, were developed. RP-HPLC is effective for analysis of total agonist content present in a nanoparticle when the sample is dissolved with a water miscible organic solvent with sufficiently low background UV absorbance (methanol, tetrahydrofuran, etc.). Dissolution with organic solvent disrupts the nanoparticle and releases any incorporated or surface bound agonist for accurate quantitation by RP-HPLC against a 5-point standard curve. For quantitation of liposome incorporated (bilayer or aqueous core) agonist, a method capable of analyzing intact liposomes and the extra-liposomal aqueous phase is needed. A SEC-HPLC method able to quantitate “free” TLR agonist with UV detection at 296, 225, and 310 nm (for2B182C, MPLA-2, and 1V270, respectively) was employed, The 1SK gel SWxl series columns provide excellent size-basea resolution for nanoparticle formulations in the 30-200 nm range. The mobile phase used is the same as the buffer utilized for the liposome rehydration to maintain a constant osmotic potential between the extra liposomal fluid and the aqueous phase in the liposome core. This method qualified as a complementary method to the in vitro potency assay, which only detects aqueous unincorporated TLR4 agonist. A preliminary study was conducted using these analytical methods to assess liposomal formulations of2B182C and 1V270, each prepared alone and in combination (co-encapsuiated). The work flow was performed as follows: 1) Lead adjuvant formulation screening (pharmaceutically acceptable co-solvents, excipients, liposomes) on a 2 mL scale with target concentrations of 1 nmol 1V270 and 200 nmols for2B182C contained in 50 uL for IM injection use. 2) Perform basic analytical method development and analysis on lead formulations to ensure formulations meet quality criteria 3) Evaluate stability of preferred formulations by real-time and accelerated methods, adding appropriate excipients as stabilizers if necessary 4) rFC testing to ensure no endotoxin contamination of finished formulations. All liposomal formulations were prepared on a 2mL scale for compounds 2B182C and 1V270. Briefly, the following procedure was used to prepare the liposomes and the following compositions were evaluated: 2B182 with and without 1V270 using (DOPC / with and without cholesterol, 2:1, respectively). The concentration of DOPC tested was held constant at 40 mg / mL, which resulted in a cholesterol concentration of 10 mg / mL. The liposomes were produced following the lipid film rehydration method using 9:1 Chloroform:Methanol as solvent. The rehydration buffer initially used was 50mM NaPB, lOOmM NaCI, pH=6.1. The agonist concentrations tested were the target concentrations. Sonication at elevated temperature was used to reduce the liposome particle size. A summary of the analytical results is depicted in Table 4. SM!; X _________________________ Table 5. Analysis of preliminary liposomal formulations of2B182C and 1V270 demonstrating the thorough analytical characterization of lead formulas. Other ratios of the TLR agonists, other lipid components, and varying amounts of cholesterol for nanoparticie formation are evaluated. At least IQ different formulations are prepared and screened for suitability in the process under Task 2A and compared to the results we obtained using the simple DMSO - water formulations described above. Nanoparticles: TLR7 and TLR4 agonists are prepared as nanoparticie formulations (liposomes, SLNs, PLGA, emulsions, etc.). The final lead formulations are selected based on immunology, stability and manufacturing data. DOPC / cholesterol liposomal formulations appear to be very promising based on preliminary immunology and stability data . One of the challenges expected with the TLR4 and TLR7 agonists is the co-incorporation of both agonists in the same nanoparticie in a controlled and consistent manner. The ratio of agonists to each other is fixed once co-encapsulated, so any dose adjustment at that point alters both agonists together. Analytical Methods: All of the analytical methods described in Task IC have been used with our lipidated TLR-7 / 8 agonists and TLR4 agonists and we expect to further improve their specificity, linearity and range with additional optimization. The RP-HPLC methods for quantitation of adjuvant in TLR4 and TLR7 agonist formulations will be optimized for peak shape, LOD and LOQ. These same methods will be gradient- and column-optimized to achieve baseline resolution and optimal LOD / LOQ for any degradants detected from stability studies to permit accurate monitoring of product stability. Accurate quantitation of the nanoparticie incorporation percentage for each agonist of the TLR4 and TLR7 agonist combinations could prove challenging since SECHPLC separates based on hydrodynamic volume only. Liposomes and unincorporated agonist may have similar particle sizes, which would limit the utility of bEC-HPLC for incorporation determination. This is discussed oelow in alternative approaches. Alternative Approaches: If development of co-encapsulated TLR4 and TLR7 agonists proves to be too difficult due to inconsistent levels of agonists in the nanoparticies, our immunology data has shown that adjuvant synergy can still be achieved by simply admixing TLR4 agonist in liposomes with TLR7 agonist in liposomes. This approach has the potential to produce a simpler, reliable product whose analytical characterization would be made easier by reducing the likelihood for interference of the agonists' signals with one another. Another option is to explore other formulations for co-encapsulation such as nano-emulsions where 100% of the agonist is incorporated by default because the aqueous and oil phases are mixed into nano-droplets. Emulsions also have the advantage of forming a depot at the site of administration, which can further enhance immune response. As discussed in Research Area 2, co-encapsulated TLR4 and TLR7 agonists versus admixed are compared in vitro and in vivo to weigh the pros and cons of these approaches. An alternative approach to using SEC-HPLC for determination of agonist incorporation into nanoparticies would be high-speed density gradient centrifugation to pellet the nanoparticies and analyze the supernatant for unincorporated agonists using established RP-HPLC methods. Formulations in the target ratio range that have acceptable properties for advancement are subjected to in vivo studies, including immunization and virus challenge studies. Research Area 2: Establish the immunological biomarkers of protection from lethal influenza virus challenge by lead adjuvant formulations Defining reliable biomarkers is needed for successful development of safe and effective vaccines. Selection of vaccine candidates with a profile that effectively prevents the infection without any safety issues is essential for the vaccine development program. In a vaccine clinical trial, identification of biomarkers that predict antigenspecific adaptive immune response with minimal reactogenicity is required. In this project, biomarkers are identified in two steps, 1) Innate immune biomarkers induced by the formulated lead adjuvant with and without antigen, and 2) Biomarkers correlating to adaptive immune responses. Thus, in vitro and in vivo studies are performed to identify the biomarker candidates that correlate to biologic activities of both TLR4 and TLR7 / 8 ligands and that also relate to reactogenicity. Task2A: Combination formulations based on in vivo antibody production studies for immunoactivitv and reactogenicity The hallmark of protection from infectious disease through vaccination is the induction of effective antibody production. Combining TLR4 with TLR7 agonists resulted in significant increases in antigen-specific antibody titers. A trend toward Th1 biasing of the immune response was observed. The effectiveness of the formulated adjuvants and their combinations is compared to the simple DMSO-water preparations. Task 2A.1: immunization studies in mice for leaa com&o formulations Formulations of lead adjuvants will be evaluated in immunization studies alone and in combination at various ratios of TLR agonists in a similar manner as previously completed for the DMSO-water formulations. The levels of IgM, total IgG, and lgG1 and lgG2a specific for both HA and NA are assessed. One or more ratios of TLR agonists in combination are identified that provide the maximum titers of antigen-specific antibody. This formulation(s) will be advanced to challenge studies under Research Area 3. Task 2A.2: Evaluation of reactogenicity and toxicity of lead combo formulations in mice Since infectious disease vaccines are designed to be protective in populations of healthy individuals, vaccine safety must be of the highest priority among development goals. Therefore, appropriate experiments to evaluate toxicity and reactogenicity of the candidate formulations are conducted. In these experiments and in general, overt toxicity is closely evaluated as initial toxicity assessments. Signs of any distress in the mice (i.e. lack of grooming, mobility issues, conjunctives, abnormal behavior, responsiveness etc.) will be noted. In addition to the gross observations, toxicity measurements comprise complete blood count, serum chemistry assessments (AST, ALT, ALP, amylase, blood urea nitrogen, creatinine, total protein, glucose, potassium, calcium, sodium, total bilirubin) and necropsy assessments (spleen, liver, and kidney sections stained with hematoxylin and eosin). Furthermore, the injection site is evaluated for visible signs of inflammation and any other abnormal findings. Tissue at the injection site is also evaluated histologically as a part of the necropsy assessments. These studies are summarized in Table 6 below. Task 2B: Identification of immune markers that can predict protective adaptive immune responses As mentioned, identification of biomarkers that predict antigen-specific adaptive immune response with minimal reactogenicity facilitate clinical trials design and methods, Task2B.1: Innate immune response signatures (cytokines, chemokines) Immune cell recruitment to the local vaccine administration site by chemokines is essential to recruit antigen presenting cells (APC) and influence induction of subsequent adaptive immune responses. However, the site of injection, i.e. muscle tissue, contains relatively few immune ceils and therefore effective adjuvants must induce recruitment of immune cells to the local site. TLR4, unlike TLR7 / 8, is abundantly expressed on non-immune cells, able to express sufficient chemokines to recruit the inflammatory cells. Following TLR stimulation, it is difficult to distinguish inflammatory responses from adjuvant effects because recruitment of APCs usually accompanies inflammatory cells. These complex cascades of immune activation cannot be studied in in vitro assays alone. Hence, panels of markers are selected from the above in vitro experiments in the samples obtained from in vivo studies in mice. The lead adjuvant formulations are administered intramuscularly (IM) to mice, and sera will be collected on days 1,3 and 7 after injection to examine levels of systemic cytokines / chemoKines. As mentioned in 1 ask 2A.2 above for local muscle tissue, expression of cytokines / chemokines and co-stimulatory molecule genes will be examined by qPCR or NanoString assays. Immune cell infiltration is assessed by histologic examination of the selected samples with hematoxylin-eosin staining and immunohistochemical staining. Splenocytes or PBMCs are used to evaluate the expression of co-stimulatory molecules assessed by flow cytometry. The draining lymph nodes are collected at the indicated time points and pooled in each experimental group and analyzed for immune cell populations and expression of chemokine receptors, and costimulatory molecules. A summary of the study design is shown in Table 6. Note that “Group 5: The combined adjuvant with antigen" group, could include combinations of different ratios of TLR4 and TLR7 agonists as necessary to provide desired profiles of cytokine / chemokine induction. Innate immune signatures that show biologic activities of both TLR4 and TLR7 ligands, and that also relate to reactogenicity, are selected. Table 6 L two <4 AaRy      R?     nwe Deva 4 a a Group 2.            oyhaww Gro^p 3 Adjuvam <Tt R4       olone Group 4 Aa^ovarR; 1 LR? hpmW'              i Group 5 The twjbwd       vath anugan Rroap A FRA approved        0" 9 ME RA J hoy A oM R,yy ? 1 Ravia IM                                             ' Evaluate Day * 3. F aRar JMunjemn - rw H™ per each tone pram Task2B.2: Adaptive immune response signatures. The experiments to assess adaptive immune responses are conducted in conjunction with Task 2A.1 above. Biomarker candidates that satisfy the following criteria are identified: 1) detected in peripheral blood, 2) driven by mechanism of actions of each TLR ligand and correlating their biological effect, 3) predicting long-term antigen-specific antibody induction and broad protection, 4) predicting reactogenicity. Outcomes and alternative approaches One or more ratios of TLR agonists in combination provide maximum titers of antigen-specific antibody. Moreover, the use of combinations of the two classes of TLR ligands results in a shift in the adaptive immune response toward a Th1-biased response compared to the use of a TLR4 agonist alone. Thus, the Th1 / Th2 response ratio likely increases. This Th1 bias may favor the broadening of the response to include heterologous virus protection. As for toxicity, systemic and oral administration of TLR7 / 8 ligands of the imidazoqumohne class have shown severe side effects comprising flu-like symptoms, nausea and lymphopenia with high levels of serum TNFa and IL-1 p. This may also be true for the oxoadenine class, of which 1V270 is a member. However, most of these undesirable side effects can be avoided by employing the usual local route of administration for vaccinations, IM. Moreover, the TLR7 / 8 ligand was prepared by conjugation to lipid moieties as well as by customizing the formulation, and successfully reduced the systemic cytokine release while maintaining the adjuvant activity. Thus, because of the low systemic exposure to inflammatory cytokines, there will likwely be little or no reactogenicity associated with the lead formulated combinations. Research Area 3: Selection of formulation(s) and immunization -- virus challenge studies in mice (Inimmune) Based on results of the formulation studies including stability (Task IB), immunoactivity (Task2A.1), and reactogenicity profile (Task2A.2), the leading formulated combination, along with a backup combination, are selected for the preclinical immumzation / virus challenge studies in mice. The virus antigens used for the studies may be selected from either recombinant vaccine antigens or inactivated whole viruses that have been used in licensed commercial vaccines, such as A / Victoria / 3 / 75(H3N2), A / Michigan / 45 / 2015 (HI N1) pdm09-like virus and A / Hong Kong / 4801 / 2014 (H3N2)-like virus. Task 3A: Selection of lead combo formulation(s) Lead selection criteria is based on: 1) stability of formulated combinations, 2) ratios of TLR agonists that provide desiredantigen-specific antibody levels, and 3) low reactogenicity profile, both local and systemic. Specific studies related to these criteria are summarized in Table 7. Following selection of a lead formulated combination and a backup lead combination, evaluation of the selections in immunization / virus challenge models in mice will be carried out (Task 3B). Table 7. Summary of Measurements Task 3B: immunization / virus challenge studies with lead formulations Task 3B.1: Determination of minimum protective dose for virus challenge studies Because inactivated influenza virus contains innate immune receptor ligands (PAMPS), a certain low level of protection might be expected following immunization of mice with sufficient antigen alone. Therefore, a study to determine the minimum protective dose, if any, with inactivated virus is conducted. The minimum protective dose of antigen is that dose that provides only partial protection (below 30% survival) upon subsequent challenge with matched strain of active virus. This strategy allows for a range of activity to be observed with the selected lead formulated adjuvant combinations. In addition, the amount of challenge virus can also be confirmed that results in complete mortality for non-immunized mice, typically a dose of about 5 LD50. Task 3B.2: Homologous virus protection study Following the antigen dose range finding study, a mouse model is used to evaluate the immunogenicity of the lead adjuvant combinations along with homologous influenza vaccine antigens. The primary determinants of success are: 1) durable influenzaspecific igG2a and lgG1 in the sera, 2) protection from lethal influenza virus challenge, 3) low reactogemcity, and 4) induction of multifunctional CD4+ versus CD8+ T cells as assessed by intracellular IFNy / TNFa staining. Secondary endpoints include weight gain / loss and a scoring of disease severity through the monitoring of the observable clinical symptoms (ruffled fur, hunched posture and labored breathing) following vaccination or influenza virus challenge. General in vivo methods Immunologic evaluation: Mice (male and female) are vaccinated (adjuvant + flu antigen such as A / Victoria / 3 / 75(H3N2)) one or two times via IM administration with 21 days between the primary and secondary vaccinations (Figure 12), Cell-mediated immunity (CMI) is evaluated in a subset of 4 mice per group by measuring Th1 / Th2 cytokine induction in splenocyte cultures (assayed by ELISA) and multifunctional CD4+ and GD8+ T-ceil responses (assayed by FACS, 10-color intracellular cytokine staining). Further, tetramer staining and cell surface phenotyping are performed to determine the frequency of influenza-specific memory CD4+ and GD8+ T cells. Flu specific humoral responses are measured in serum (lgG1 and lgG2a) and HI titers are used to measure functional antibody titers. Vaccinated and control mice are challenged with 5 LD50 of A / HK / 68(H3N2) and assessed for survival, weight gain / loss and a scoring of disease severity for 21 days. Reactogenicity in these murine studies is measured by weight loss and symptom scores and evaluation of injection site infiltrates. A p value difference of <0,05 is considered significant. Analysis of variance (ANOVA) and Tukey ANOVA is performed on all data to demonstrate robust statistical significance. Task3B.3: Heterologous virus protection study Following the homologous protection study, the same study design is used to evaluate the lead adjuvant combinations in a mouse model of heterologous or heterosubtypic protection. Mice are immunized as described above (Task 3B.2) but are challenged with an influenza virus strain of a different HA / NA type (e g., A / Puerto Rico / 8 / 1934 (H1N1)). Protection observed in such a challenge model would suggest a broadening of antigen-specific response to include antigens common to both strains, such as the stalk region of the HA protein. To confirm such broadening, a study of the B cell receptor (BCR) and T cell receptor (TCR) sequences is conducted. Outcomes and alternative approaches As mentioned previously, increasing numbers of literature reports cite combinations of various TLR agonists that are able to synergistically increase the magnitude of vaccine-mediated immunity and change the type of downstream adaptive immune response generated thereby enhancing the efficacy of these vaccines. An adjuvant combination for influenza virus challenge protection is described herein. Example 3 Influenza Hemagglutinin (HA) as a Vaccine Antigen Strategies to boost broadly neutralizing stalk antibodies include: 1) focus on headless HAs, with the removal of the entire head domain to make the stalk domain more "available" and thus induce antibody responses against the stalk domain, or 2) use chimeric HAs consisting of the stalk domain from HI, H3 or influenza B viruses in combination. It is known that immunization with one antigen blocks robust immune responses to a second, similar antigen (“original antigenic sin"). That is important for infectious diseases where there are repeated infections (influenza), or antigenic evolution (HIV, malaria). For influenza, major neutralizing antibodies made against the head region of the viral hemagglutinin (HA). Different viral strains have different HA head regions, that cross-read weakly with antibodies, but inhibit the response to new epitopes). For HIV, mutated epitopes on the virus do not stimulate antibodies orT cells because of epitope suppression Mechanisms of original antigenic sin in vaccines may be due to epitope exclusion (pre-existing antibodies, especially mucosal IgA, shield the vaccine from all antigen presenting ceils (APCs); dendritic cell access (memory B cells internalize the new vaccine, with reduced DC activation and T cell immunization): and / or T cell competition (memory B cells are activated, consuming cytokines, co-fadors, and trapping T cells that could react with antigen loaded DCs To overcome original antigenic sin in vaccines, dosage may be increased (e.g., a massive vaccine dose (patients over 60 receive 3X dose of influenza vaccine)); encapsulation (put the vaccine in an emulsion or liposome that preferentially delivers the vaccine to dendritic ceils (Shingrix, varicella vaccine for shingles)); and / or dendritic cell activators (TLR agonists may increase the numbers diversity of activated T cells against the vaccine antigens). To study original antigenic sine in mouse models, the following may be used: hapten-protein conjugates (a hapten is a small molecule like Flourescein or DNP that can be coupled to a protein antigen like ovalbumin and KLS); or pre-immunization witn the unconjugated protein antigen inhibits antibody responses to immunization with the hapten-protein conjugate. For influenza in these models, hyper-immumze with one protein, such as influenza HA, for one viral strain, boost with a partially cross-reactive HA from another strain, then analyze B and T cell immune responses to the second HA, including epitopes recognized, clonal diversity by nexgen RNA sequencing, and neutralizing capacity, and then correlate with in vivo protection. Shingrix is recombinant VSV glycoprotein E, nonophosphoryl lipid A from Salmonella, and QS-21 saponin molecule in a liposomal formulation made from dioleoyl phosphatidylcholine and cholesterol in buffered saline, which is reconstituted at time of use. To make an influenza vaccine analogous to Shingrix, the vaccine has a protein antigen, two adjuvants in a liposomal formulation. Exam pie 4 The effectiveness of the annual influenza vaccine is still rated 10 --60 % because of antigenic drift of influenza virus. Synthetic TLR4 and TLR7 agonists (1Z105 and 1V270) enhanced Th2- and Th1-mediated anti-hemagglutinin antibody production, respectively. The combination with 1Z105 and 1V270 promoted the balanced Th1 / Th2 immunity to protect against influenza virus infection. To enhance the adjuvant efficacy, a structure activity relationship study was conducted on 1Z105 and 2B182C was identified; a derivative with higher potency in vitro. In an in vivo vaccination study using the model antigen ovalbumin, 2B182C induced higher serum IgGI levels and additively enhance the release of antigen-specific igG2a induced by 1V270. Furthermore, the liposomal formulation of 2B182C and 1V270 reduced cytotoxicity and reactogenicity and maintained the activity to enhance both Th1- and Th2-mediated antibody production. In an in vivo vaccination study using inactivated A / California / 04 / 2009 (H1N1) (pdm09) as antigen, the liposomal combination adjuvant increased the populations of T follicular helper cells, germinal center B cells and antibody secreting plasma cells. Next generation sequence analyses of B and T lymphocytes in the draining inguinal lymph nodes showed that the combined adjuvants increased B cell clonotypes of immunoglobulin heavy chain (IGH) genes, shared B ceil clones and TCR clonalities. These findings suggested that the combination contributed to enhance antigen specific Th1 immune response. Finally, the vaccine with the combination adjuvants protected against lethal homologous virus challenge with less lung damage. Methods Mouse Female 6-8 week-old BALB / c mice were purchased from Jackson labolatory (Bar Harbor, MA). The animal experiments using ovalbumin, or inactivated influenza virus as antigens which were not required a live virus challenge were performed at University of California San Diego Animal Facility. The influenza challenge study was performed by the Animal Research Center of Utah State University using female 6 week-old BALB / c mice (Charles River Laboratories, Wilmington, MA). All Animal experiments received prior approval by the institutional Animal Care and Use Committee (IACUC) for UC San Diego or Utah State University. Cells and reagents TLR4 / NF-kB reporter cell lines HEK-Blue™ humanTLR4 and HEK-Blue™ murineTLR4 ceiis were purchased from InvivoGen (Catalog numbers, San Diego, CA). Mouse primary BMDCs were prepared from bone marrow cells harvested from femurs ofC57BL / 6 mice. BMDCs were treated with indicated compounds in RPMI supplemented with 10% FBS (Omega, Tarzana, CA) and penicillin / streptomycin (100 unii / mL / 100 ug / mL, Thermo Fisher Scientific, Waitham, MA). Monophospholipid A (MPLA), AddaVaxwere purchased from InvivoGen (Catalog numbers San Diego, CA). Inactivated Influenza A virus [A / California / 04 / 2009 (HI N1) pdm09] (IIAV) were obtained from BEI resources (# NR-49450, Manassas, VA). TLR7 agonist 1V270, TLR4 agonists 1Z105 and it derivatives including 2B182C were synthesized. Liposomal formulation of 1V270 (20 uM), 2B182C (4mM) and 1V270+2B182C (20 pM + 4mM) was performed y Innimune corp. (Missoula, MT). TLR4 / NF-kB Reporter cell assay TLR4 / NF-kB activation was assessed using HEK-Blue™ hTLR4 and HEK-Blue™ mTLR4 (InvivoGen). The cells were treated with 1Z105 and 2B182C (2-fold serial dilution starting from 10 u.M) for 20h.NF-kB inducible secreted embryonic alkaline phosphatase (SEAR) protein in the culture supernatant was measured according to manufacturer's protocol. Evaluation of antibody production in vivo BALB / c mice were intramuscularly (i.m.) immunized with IAV (10 ug / injection) plus indicated adjuvants in gastrocnemius of hind legs on days 0 and 21. Detailed concentrations of adjuvants and the number of animals in each treatment are described in each figure legends. Sera were collected on day 28 and evaluated for antigenspecific antibodies (anti-HA lgG1, anti-NA lgG1, anti-HA lgG2a, anti-NA lgG2a, anti-HA IgM and anti-NA IgM). ELISA for these antibodies were performed as previously described (Ref). For studies with DMSO formulation, 10% DMSO was used as vehicle. In the experiments using the liposomal-formulated adjuvant, 1,2-dioleoyl-sn-glycero-3-phosphochoiine and cholesterol (DOPC / Chol, control liposomes) was used as vehicle. NGS assay for BCR and TCR repertoire Immunization protocol was shown in Figure 28A. Briefly, mice were sacrificed on day 28 and inguinal lymph nodes were harvested. Total RNA was extracted from lymphocytes (bulk) using RNeasy Mini Kit (Qiagen, Hilden, Germany) and the quality of RNA was confirmed by Agilent 4200 Tapestation (Agilent, Santa Ciara, CA). Nextgeneration sequencing was performed with unbiased TCR repertoire analysis technology (Repertoire Genesis Inc., Osaka, Japan). Evaluation for protection from lethal influenza virus challenge BALB / c mice were i.m. vaccinated witn formulated 1V2 / 0 and 2B182C with IIAV (3 ug / injection) on day 0 and intranasally infected with homologous or heterologous influenza A virus, A / California / 04 / 2009 (pdmH1 N1) and A / Victoria / 3 / 75 (H3N2) on day 21, respectively. The immunization dose of IIAV; 3 pg / injection that protect 30-50% of animal from the challenge with homologous virus was determined in the preliminary experiment. For influenza virus challenge, groups of mice were anesthetized by intraperitoneal injection of ketamine / xylazine (50 mg / kg / / 5 mg / kg) prior to intranasal challenge with 1 x 105 (3x LDso) cell culture infectious doses (CCIDso) of influenza A / California / 04 / 2009 (H1N1pdm) virus per mouse; 5 x 102 (3x LDso) CCIDso of influenza A / Victoria / 3 / 75 (H3N2) virus per mouse in a 90-pL suspension. All mice were administered virus challenge on study day 21. influenza virus (H1N1pdm), strain designation 175190, was received from Dr. Elena Govorkova (Department of infectious Diseases, St. Jude Children’s jemResearch Hospital, Memphis TN). The virus was adapted to replication in the lungs of BALB / c mice by 9 sequential passages in mice. Virus was plaque purified in Madin-Darby Canine Kidney (MDCK) ceils and a virus stock was prepared by growth in embryonated chicken eggs and then MDCK cells. Influenza A / Victoria / 3 / 75 (H3N2) virus was obtained from the American Type Culture Collection (Manassas, VA). The virus was not lethal to mice initially, but became lethal after 7 serial passages in the lungs of infected animals. Following mouse-adaptation a virus stock was prepared by growth in MDCK ceils. Determination of lung virus titers and lung inflammation Six days after virus challenge, the bronchioaiveolar lavage (BAL) procedure was performed immediately after blood collection and was completed within 5 to 10 min of each animal's death. A volume of 0,75 mL of phosphate buffered saline (PBS) was slowly delivered into the lung through the tracheal tube. Immediately after delivery the fluid was slowly withdrawn by gentle suction and the samples were stored at -80 °C. The procedure was repeated a total of three times and lavage fluids from each mouse were pooled. To determine lung virus titers, BAL samples were centrifuged at 2000g for 5 minutes. Varying 10-fold dilutions of BAL supernatants were assayed in triplicate for infectious virus in MDCK cells, with virus titers calculated. For determination of lung cytokine levels, a sample (200 pL) from each lung lavage was tested for MCP-1 and IL-6 using a chemiluminescent multiplex ELfSA-based assay according to the manufacturer’s instructions (Quansys Biosciences Q-Plex™ Array, Logan, UT). Hemagglutination inhibition titers For hemagglutination inhibition (HI) titers, sera were pre-treated with receptordestroying enzyme II (RDE; Vibrio cholerae neuraminidase; YCC-340; Accurate Chemical and Scientific, Westbury, NY) to remove non-specific inhibitors by diluting one part serum with three parts enzyme and incubating at 37”C for 18 h. RDE was subsequently inactivated by heating at 56°C for 45 min. Serum samples were diluted in PBS in 96-well round-bottom microtiter plates (Fisher Scientific, Pittsburg, PA). Following dilution of serum, 8 HA unns / well of influenza A / CA / 04 / 2009 (H1N1pdm) or influenza A / Victoria / 3 / 75 (H3N2) viruses plus turkey red biood ceils (Lampire Biological Laboratories, Pipersville, PA) were added (50 pL per well), mixed briefly, and incubated for 60 min at room temperature. The HI titers of serum samples are indicated as the reciprocal of the highest serum dilution at which hemagglutination was completely inhibited. Virus neutralization titers For anti-influenza virus neutralizing antibody assay, MDCK cells were seeded in 96-weii plates at 1x104 cells per well in MEIVI containing 5% FBS (Hyclone, Logan, UT) 24 h prior to use. Serial 2-fold dilutions of serum samples were prepared in serum-free media, containing 10 units / mL trypsin and 1 ,ug / mL EDTA, starting at 1:32 dilution and ending at 1:4096, Each serum dilution was mixed 1:1 (0.1 mL) with serum-free media (containing trypsin and EDTA) containing approximately 100 CCID50 / well H1N1pdm or influenza A / Victoria / 3 / 75 (H3N2) virus. After incubation at room temperature for 1 h, the serum-influenza virus mixture (0.2 mL) was transferred to a well containing MDCK cells and incubated for 3 days. Anti-influenza virus neutralizing antibodies were measured as cytopathic effect (CPE) inhibition, CPE was scored from duplicate samples by examining the MDCK cell monolayers under a light microscope on day 3 post-infection. Statistical analyses Data obtained in in vivo studies are presented as means with standard error of mean (SEM) and in vitro data are indiaced as means with standard deviation (SD). For in vitro data, a two tailed Welch's t test was used to compare two groups. For antigen specific antibodies, flow cytometry analysis for immune ceii populations, BCR-seq, TCR-seq, lung virus titers, HI endpoint titers, and VN endpoint titers, Kruskal-Wallis tests with Dunn's post hoc test were applied. Correlations between lung virus titers and cytokine / chemokine levels were analyzed using a Spearman rank correlation test. For body weight, area under the curve was calculated for each mouse and one-way ANOVA was used for statistical analysis. The log rank (Mantel-Cox) test was used to test for a significant difference between Kaplan-Meier survival curves. Prism 5 software (GraphPad Software, San Diego, CA) was used. A P value less than 0.05 was considered statistically significant. Table 8. Reagents used in ELISA for hlL-8, mlL-12 and mlL-6 Reagents Dilution factor Source Catalog # Capture antibodies Purified mouse anti-human IL-8 250 BD Biosciences 554716 Purified rat anti-mouse IL-12 200 BD Biosciences 551219 Purified rat anti-mouse IL-6 100 BD Biosciences 554400 Detecting antibodies Biotin mouse anti-human IL-8 1000 BD Biosciences 554718 Biotin rat anti-mouse IL-12 1000 BD Biosciences 554476 Biotin rat anti-mouse IL-6 1000 BD Biosciences 554402 Other reagents Streptavidin, HRP woo Thermo Fisher Scientific 43-4323 KPL SureBlue™ TMB Peroxidase Substrate Seracare 5120-0077 Table 9. Reagents used in ELISA for hlL-8, mlL-12 and mlL-6 Antibodies (clone) Dilution factor Source Catalog # Anti-CD86, APC / Cy7 (GL1) 200 BioLegend 105030 Anti-CD40, PE (1C10) 200 eBioscience 12-0401 Anti-CD3, BV510 (145-2C11) 200 BD Biosciences 563024 Anit-CD19. FITC (1D3) 500 BD Biosciences 553785 Anti-CD4, e450 (RM4-5) 1500 eBioscience 48-0042 Anti-CD95, PE / Cy7 (Jo2) 500 BD Biosciences 557653 Anti-CD138, APC (281-2) 200 BD Biosciences 558626 Anti-GL7, Pacific Blue (GL7) 350 BioLegend 144614 Anti-PD-1, APC (J43) 150 BD Biosciences 562671 Anti-CXCR5, Biotin (2G8) 50 BD Biosciences 551960 Anti-CD16 / 32 (FcR) 300 BD Biosciences 553142 Streptavidin PE 500 BD Biosciences 554061 Propidium iodide Staining Solution 400 BD Biosciences 556463 Stain buffer BD Biosciences 554657 Table 10, Reagents used in ELISA for IgGs Reagents Source Catalog # Proteins for coating Concentrations Influenza A H1N1 (A / Califomia / 04 / 2009) Hemagglutinin / HA Protein (His Tag) 100 ng / mL Sino Biological 11055-V08H Influenza A H1N1 (A / Puerto Rico / 8 / 1934) Hemagglutinin / HA Protein (His Tag) 100 ng / mL Sino Biological 11684- V08B Influenza A H3N2 (A / Victoria / 3 / 1975) Hemagglutinin / HAI Protein (His Tag) 100 ng / mL Sino Biological 40396- V08H1 Influenza A H7N7 (A / Netherlands / 219 / 2003) Hemagglutinin / HA Protein (His Tag) 100 ng / mL Sino Biological 11082-V08B Influenza AHI 1N9 (A / mallard / Alberta / 294 / 1977) Hemagglutinin / HA Protein (His Tag) 100 ng / mL Sino Biological 11704-V08H Influenza A H12N5 (A / green-winged teal / ALB / 199 / 1991) Hemagglutinin / HA Protein (His Tag) 100 ng / mL Sino Biological 11718- V08H Influenza A H1N1 (A / Califomia / 04 / 2009) Neuraminidase / NA (Fc Tag) 100 ng / mL Sino Biological 11058-V07B Influenza A H5N1 (A / Thailand / 1 (KAN-1) / 2004) Neuraminidase / NA (His Tag) 100 ng / mL Sino Biological 40064- V07H Influenza A H3N2 (A / Babol / 36 / 2005) Neuraminidase / NA (His Tag) 100 ng / mL Sino Biological 40017- V07H Influenza A H10N8 (A / duck / Guangdong / E1 / 2012) Neuraminidase / NA Protein (His Tag) 100 ng / mL Sino Biological 40352- V07B Influenza A H7N7 (A / Netherlands / 219 / 2003) Neuraminidase / NA Protein (His Tag) 100 ng / mL Sino Biological 40202-VQ7H Antibodies Dilution factor IgGI-AP goat anti-mouse 2000 Southern Biotech 1070-04 lgG2a-AP goat anti-mouse 2000 Southern Biotech 1080-04 IgG-AP goat anti-mouse 2000 Southern Biotech 1030-04 p-Nitrophenyl Phosphate tablets (pNPP) Sigma N2770 Results Structure activity relationship study of 1Z105 yielded 2B182C 5           To improve the potency to the small molecule pyrimidoindole TLR4 ligand, 1Z105, the structure activity relationship analysis was performed (Chemists will fill out). A total of 56 compounds were synthesized, and screened by human and murine HEK TLR4 reporter ceils (HEK-Blue mTLR4 and hTLR4, respectively). Among those SAR compounds, 2B182C was discovered as a derivative with higher TLR4 stimulatory 10 potency in both murine and human reporter cells. The ECso of 2B182C was examined using HEK TLR4 reporter cells and compared to the EC50 of 1Z105 (Figure 21B). EC50 of 2B182C in murine and human TLR4 reporter cells was increased by 5.8 fold and 870-fold, respectively, in comparison with EC50 of 1Z105. These data indicate that SAR study successfully yielded a derivative exhibiting higher TLR4 stimulatory potency, notably human TLR4 potency, TLR4 agonist 2B182c enhanced antigen specific IgGI production TLR4 agonist 1Z105 induced Th2-mediated IgGI production and TLR7 agonist 1V270 enhanced Th1 cellular immunity against influenza virus (Goff et al., J. Virol., 89:3221 (2015); Goff et al., J. Virol., 9l:e01050 (2017)). It was hypothesized that by combining with 1V270, the efficacy of the TLR4 agonist 2B182C as an influenza vaccine adjuvant could be improved. Therefore, it was examined whether 2B182C with 1V270 improved the adjuvanticity in vivo compared to the combo adjuvants with 1Z105 plus 1V270. To develop the effective combined vaccine adjuvants, the potency of 1Z105 and 2B182C, and optimal dose as a single agent, were compared using inactivated Influenza A virus [A / California / 04 / 2009 (H1N1) pdm09] (IIAV) as an antigen. BALB / c mice were immunized on days 0 and 21 with IIAV mixed with the TLR4 agonists, 1Z105 or 2B182C, were bled on day 28 (Figure 22A). Sera were evaluated by ELISA for antibodies (IgM, IgGI and igG2a) against two glycoproteins on the surface of the virus, hemagglutinin (HA) and neuraminidase (NA). 1Z105 and 2B182C were dissolved in DMSO and the final concentration of DMSO was 10%. The results showed that 2B182C with higher dose as 200 nmol / injection significantly increased IgGI antibody against both HA and NA (Figure 22B). Interestingly, 2B182C, but not 1Z105, enhanced anti-NA specific lgG2a (Figure 12C), Anti-HA IgM level was only slightly increased by 2B182C (Figure 24A). Combination with 2B182C and TLR7 agonist 1V270 increased both antigen specific lqG1 and lgG2a Next the co-adjuvant effects of these TLR4 agonists on antibody production was analyzed when combined with TLR7 agonist 1V270 at a dose of 1 nmol / injection, which was reported to induce lgG2a production enhancing Th1 immune responses (Goff et al., 2017). The results indicated that while 1V270 alone induced only anti-HA lgG2a production, when combined with 2B182C, IgGI and igG2a antibodies against both HA and NA were significantly induced. This suggests that these compounds may work in an additive manner (Figures 23A and 23B). On the other hand, 1Z105 failed to enhance lgG2a production induced by 1V270. Animals in 1V270+2B182C-group produced higher amount of both IgGI and lgG2a and the immune balance was inclined toward Th1-mediated igG2a production, suggesting that the treatment contribute to enhance Th1 immune responses (Figure 23C), The combination with 1V270 and 2B182C showed moderate effect on anti-HA IgM production (Figure 24B). Collectively, the combination of 200 nmol / injection 2B182C plus 1 nmol / injection 1V270 induced highest quantity of antigen specific IgGI and lgG2a and Th1-skewing immune responses, which are desirable for protection in the influenza virus infection. Thus, this combination was selected for the next formulation. Liposomal formuiat-on upgraded 2B182C reducing cyioioxicity Given the results above, a 1V270 / 2B182C ratio (TLR4 / TLR7) of 1 / 200 [1 nmol / injection (20 uM) 1V270 and 200 nmoi / injection (4 mM) 2B182C] was used, in order to avoid unwanted cytotoxicity and reactogenicity while maintaining response to vaccine, adjusting formulation of compounds may be important in the development of vaccine adjuvants. Therefore, 1V270 and 2B182c were formulated in liposomes by Inimmune Corp (Missoula, MT). The activity of the formulated compounds was tested in mouse primary BMDCs. These formulated compounds maintained similar levels of IL-12 secretion as DMSO-formulation compounds (Figure 25A). Cytotoxicity induced by DMSO-2B182C or DMSO-1V270+2B182C were significantly improved by liposomal formulation. (Figure 25B). Histological analysis by H&E staining of muscles in the injected sites is shown in Figure 25C. Multiplex cytokine / chemokine analysis of sera after administration of the compounds is shown in Figure 25D. Liposomal 1V270 and 2B182C synergistically enhanced anti-HA and anti-NA lqG1 and lgG2a production The adjuvanticity of the compounds in vivo was evaluated using prime-boost regimen as described in Figure 22A. Sera harvested on day 28 were assessed for antigen specific antibodies by ELISA. The results indicated that lipo-2B182C induced higher level of IgGI, which was consistent with DMSO-2B182C (Figure 26A). Unlike DMSO-1V270, lipo-1 V270 alone did not promote lgG2a production (Figure 26B). Despite these minimal effects on lgG2a by each agonist, when two adjuvants were combined, antigen specific antibody production was synergistically enhanced (Figure 26B). On the other hand, total IgG levels induced by liposomal vehicle, 1V270, 2B182C and 1V270+2B182C, were comparable (Figure 26C). Antigen specific IgM levels were not affected by any treatment (Figure 27). Consistent with the trend observed with DMSO formulation, the liposomal combined adjuvants developed Thl-biased immune balance (Figure 26D). Formulated 1V270 plus 2B182C enhanced antibody secretion responses To investigate whether the formulated adjuvants induces an activation of B cells promoting antigen specific antibody secretion, lymphocytes in inguinal lymph nodes were examined forTfh cells, GC B cells, plasmablasts and plasma cells using flow cytometry. The immunization protocol described above was used and lymphocytes in the inguinal lymph nodes were harvested on day 28 and analyzed by flow cytometry (Figures 28A and 28B). As the results, the percentage of Tfh cells, which were identified as CD3+ CD4+ PD-1+ CXCR5+ cells, was greatly increased by lipo-1V270+2B182C (Figure 28B and Figure 29). Additionally, the combined adjuvants increased the percentage of GC B cells (CD3- CD19+ CD95+ GL7+). Increased plasmablasts and plasma cells were observed in mice vaccinated with lipo-1V270+2B182C. The results suggest that the combined adjuvants enhance GC reaction compared to a single agent. increased BCR d-vers-ry and TCR cionaiity by the combo adiuvantwitn 1V270 plus 2B182C To examine whether the combined adjuvants affect the diversity of BCR, next generation sequencing analysis was performed for IGH genes (by Repertoire Genesis Inc, Osaka, Japan). The prime-boost IIAV model were used and lymphocytes in the inguinal lymph nodes were collected on day 28 (Figure 30A). BCR sequence analyses showed that BCR diversity normalized to total reads indicated by Pieiou’s index was significantly increased by lipo-1V270+2B182C (Figure 30A). Clonotypes of IgG genes were analyzedby similarity analysis, which compare IGH clones between two mice within the group to see if there is a shared clone and calculate Jaccard index; Jaccard index; J (A, B) =(AHB) / (AUB) (Figure 30B). Jaccard indices for IGH, IGHG1 and IGHG2A were significantly increased by hpo-1V270+2B182C, indicating that clones shared between two mice within this group were increased. Furthermore, in the lipo-1V270+2B182C group, 6 clones (0.03%) were shared among three mice. These results suggest that the liposomal combined adjuvant increased BCR diversity in total IGH and IGHG2A. That is consistent to the higher lgG2a level following immunization of combined adjuvant. The common clones detected in the group immunized with the combined adjuvant might recognize dominant epitope(s) of the antigen. TCR sequencing was performed to see whether the formulated adjuvants contribute to increase f TCR cionaiity toward antigens. Expectedly, the combined adjuvants and lipo-2B182C increased clonalities of TCRa and TCRp (Figure 30C). Collectively, animals in iipo-1V270+2B182C showed higher BCR diversity and TCR cionaiity. This may support the data that Th1 response is enhanced by the combined adjuvants. Lipo-2B182C and lipo-1 V270+2B182C protect mice against homologous influenza virus The combined adjuvant induced Th1 biased immune response accompanying diverse BCR and high cionaiity of TCR. To test whether this diversity could be an indication of an immune response against influenza virus, the formulated 1V270 and 2B182c were tested in the homologous and heterologous influenza virus challenge model. Baib / c mice vaccinated with IIAV plus liposomal 1V270, 2B182C or 1V270+2B182C were intranasally challenged with homologous (H1N1) influenza virus on day 21 post vaccination (single dose). Body weight and survival of mouse were monitored through additional 21 days (Figure 31 A). Lipo-2B182C and lipo-1V270+2B182C significantly suppressed body weight loss after viral infection (Figure 31B). Furthermore, lipo-1 V270 showed 90 % protection, and lipo-2B182C and lipo-1V270+2B182C completely protected mice against homologous influenza virus (Figure 31C). To evaluate if the survival of mice is correlated to viral titers in lung, bronchoalveolar lavage were performed for virus titers in lavage fluid. The results indicated that lipo-1 V270+2B182C effectively suppressed virus titers in lungs on day 6 (Figure 31D). in human, there is an upregulation of cytokine and chemokine in airway epithelial ceils (e.g., MCP-1, IL-6, etc.) correlated with lethal lung injury and pneumonia (Gurczynski et al., Mucosal Immun., 12:518 (2019); Zhou et al., Nature, 499:500 (2013)). Therefore, we evaluated pro-inflammatory cytokine (IL-6) and chemokine (MCP-1) level in lung fluids using the Quansys multiplex ELISA. The results showed the combined liposomal adjuvants significantly suppressed both MCP-1 and IL-6 productions (Figure 31E). The levels of pro-inflammatory cytokines were correlated to 5 lung virus titers [MCP-1 (P<0.0001, Spearman r= 0.83), IL-6 (P<0.0001, Spearman r= 0.79) (Figure 31F). This trend was further enhanced in lipo-1V270+2B182C group. These results suggested that the combined adjuvants reduced lung damage by inhibiting virus entry and proliferation after infection. To evaluate if the protection was related to the hemagglutination inhibition titers (HI) and virus neutralization titer (VN), 10 sera were collected on day 21 post immunization and examined for HI and VN (Figure 31 A). The increased HI titers compared to non-immunized group were observed in 19 mice out of 20 mice in the lip-1 V270, lipo-2B182C and lipo-1V270+2B182C (Figure 31G). In addition, lipo-2B182c and lipo-1V270+2B182C induced significantly higher VN compared to liposomal control (Figure 31H). VN titers were negatively correlated with 15 lung virus titers (P<0.01, Spearman r= -0.59, Figure 27I). Protection against heterologous influenza virus A / Victoria3 / 75 (H3N2) was evaluated using the same protocol as homologous challenge experiment (Figure 31 A), There was not significant difference in body weight loss, survival and lung virus titers in comparison to the liposomal control group (Figures 32A-C). Collectively, the formulated combined 20 adjuvants showed significant protection against homologous H1N1 virus without adverse inflammatory effects, although it was insufficient for heterologous protection. Table 11. Number of shared clones of total IgG genes in BCR-seq #clones (%) Vehicle Lipo- 1V270 Lipo-2B182C Lipo- 1V270+2B182C AddaVax # clones (%) Not shared 11418 (99.7) 14387 (100.0) 9157 (99.9) 18037 (99.5) 19019 (99.7) 2 mice 31 (0.27) 4 (0.03) 1Q (0.11) 90 (0.50) 51 (0.27) 3 mice 0(0) 0(0) 1 (0.01) 6 (0.03) 0(0) 4 mice 0(0) 0 (0) 0(0) o (0) 0(0) 5 mice 0 (0) 0(0) 0(0) 0 (0) 0 (0) BALB / c mice were vaccinated on days 0 and 21 with IIAV with formulated adjuvants. Lymphocytes in the inguinal lymph nodes were harvested on day 28 for next generation sequencing for IGH genes. Similarity analysis of IGH clonotype were performed. Number of clones shared in 2, 3, 4 and 5 mice within a group and not shared were shown. Six clones were shared between 3 mice in the combination group. Example 5 Liposomal co-encapsulation of 1V270(TLR7 Hgand) and 2B182C(TLR4 Hgand) broadens antibody epitopes A universal vaccine for influenza virus infections requires the induction of antibodies that recognize broad epitopes of the major antigenic molecules, hemagglutinins (HA), and neuraminidase (NA). Thus, the epitope spreading and crossreactivities of antibodies induced by the combined adjuvant (1V270 and 2B182C) were examined. BALB / c mice (n=5-10) were immunized with inactivated virus mixed with liposomal formulation of 1V270 (Lipo-1V270), 2B182C (Lipo-2B182C), co-encapsulated liposomal 1V270+2B182C [Lipo-(1V270+2B182C)], and add-mixed Lipo-1V270 and Lipo-2B182C in separate liposomes. Blank liposomes were used as a control and immunization was performed on day 0 (prime) and day 21 (boost) and sera were collected on day 28. Epitope spreading was evaluated by HA peptide ELISA. Overlapping HA peptide array (139 peptides) of the Influenza A(H1N1)pdmO9 virus was obtained from BE! Resources. Pooled peptides comprised of 5 consecutive peptides (total of 28 pools) were plated onto the ELISA plates, 1:200 diluted sera were tested for reactivity to each peptide pool by OD405-570. The OD of each serum was plotted on the heatmap (Figure 38A), and the average OD of individual animals were compared. The sera from the mice vaccinated with co-encapsulated liposomal 1V270+2B182C [Lipo-(1V270+2B182C)] showed significantly higher OD compared to the liposomal formulation of single ligands or admix (Figure 38B). These data indicate that Lipo-(1V270+2B182C) induced antibody responses recognizing a wide range of HA epitopes. To test whether the recognition of broad HA epitopes induced by Lipo-(1V270+2B182C) is associated with the cross-protection of different subtypes of influenza virus infection, we tested the cross-reactivity of antibodies against various subtypes of HA and NA by ELISA (Figures 39 and 40). Subtypes HAs and NAs that belong to different phylogenic distances. Geometric mean titer (GMT; of IgG from mice immunized with coencapsulated Lipo-(1V270+2B182C) showed high reactivity not only with HAs from group 1 (H1, H11, H12) but also with HAs in group 2 (H3 and H7) in comparison to liposomal single ligand, or add-mixed two separate liposomes. Broadened reactivities were also observed to different subtypes of NA. in summary, antibodies produced in the animals vaccinated with IIAV plus Lipo-(1 V270+2B182C) were highly cross-reactive to different subtypes of HA and NA. All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.

Claims

1. A method to enhance an immune response in a mammal, comprisingadministering to a mammal in need thereof a composition comprising liposomes comprising an effective amount of a TLR4 agonist and a TLR7 agonist wherein the TLR7 agonist is:andwherein the TLR4 agonist has formula (II):wherein z2 is 1, 2 or 3,wherein z1 is 1 or 2,wherein R5 is an unsubstituted aryl,wherein R6 is an unsubstituted cycloalkyl,wherein R7 is an unsubstituted alkyl, andR8 is an unsubstituted thienyl, furanyl, pyrrolyl, pyrazolyl, thiazolyl, oxazolyl, or imidazolyl, preferably an unsubstituted furanyl or thienyl.

2. The method of claim 1 wherein the TLR4 agonist and a TLR7 agonist areadministered simultaneously.

3. The method of claim 1 or 2 wherein the liposomes comprise PC, DOPC, orDSPC.

4. The method of any one of claims 1 to 3 wherein the liposomes comprisecholesterol.

5. The method of any one of claims 1 to 4 further comprising administering one ormore immunogens.

6. The method of claim 5 wherein the immunogen is a microbial immunogen.2020236254   10 Jun 20267.      The method of claim 6 wherein the microbe is a virus or a bacteria.

8. The method of any one of claims 5 to 7 wherein the liposomes comprise theone or more immunogens.

9. The method of any one of claims 1 to 8 wherein the mammal is a human.

10. The method of any one of claims 1 to 9 wherein the amount of the TLR7 agonistis about 0.01 to 100 nmol, about 0.1 to 10 nmol, or about 100 nmol to about 1000 nmol.

11. The method of any one of claims 1 to 10 wherein the amount of the TLR4agonist is about 2 to 20 pmol, about 20 nmol to 2 pmol, or about 2 pmol to about 100 pmol.

12. The method of any one of claims 1 to 11 wherein the ratio of TLR7 to TLR4agonist is about 1:10, 1:100, 1:200, 5:20, 5:100, or 5:200.

13. The method of any one of claims 1 to 12 wherein the composition is injected,intramuscularly administered, intranasally administered or intravenously administered.

14. The method of any one of claims 1 to 13 wherein the liposomes compriseDOPC and cholesterol.

15. A pharmaceutical formulation comprising liposomes, a TLR4 agonist and aTLR7 agonist, wherein the TLR7 agonist is:andwherein the TLR4 agonist has formula (II):wherein z2 is 1, 2 or 3,wherein z1 is 1 or 2,wherein R5 is an unsubstituted aryl,wherein R6 is an unsubstituted cycloalkyl,wherein R7 is an unsubstituted alkyl, and2020236254   10 Jun 2026wherein R8 is an unsubstituted thienyl, furanyl, pyrrolyl, pyrazolyl, thiazolyl, oxazolyl, or imidazolyl, preferably an unsubstituted furanyl or thienyl.

16. The formulation of claim 15 wherein the liposome comprises 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-[phosphor-L-serine] (DOPS),   1,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dioleoyl-sn-glycero-3- PE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (16:0 PEG-2000 PE), 1-oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl]-sn-glycero-3-phosphocholine (18:1-12:0 NBD PC), 1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl}-sn-glycero-3-phosphocholine (16:0-12:0 NBD PC), and mixtures thereof; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, or a mixture thereof.

17. The formulation of claim 15 or 16 wherein the liposome comprises DOPC,cholesterol or combinations thereof.

18. The formulation of any one of claims 15 to 17 wherein the amount of the TLR7agonist is about 0.01 to 100 nmol, about 0.1 to 10 nmol, or about 100 nmol to about 1000 nmol.

19. The formulation of any one of claims 15 to 18 wherein the amount of the TLR4agonist is about 2 nmol to 20 pmol, about 20 nmol to 2 pmol, or about 2 pmol to about 100 pmol.

20. The formulation of any one of claims 15 to 19 wherein the ratio of TLR7 to TLR4agonist is about 1:10, 1:100, 1:200, 5:20, 5:100, or 5:200.

21. Use of a composition defined by any one of claims 1 to 14, or thepharmaceutical formulation of any one of claims 15 to 20 in the manufacture of a medicament to enhance an immune response in a mammal.

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