A universal vaccine strategy for conferring protection against diverse pathogens

Abstract

Compositions and methods are provided for immunizing an individual or population of individuals to generate an immune response that protects against pathogen infection in an antigen-agnostic manner. The protective antigen-agnostic immune response is active for a period of time following immunization, from about 7 days, 10 days, 14 days following immunization, and may be active for at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, or more.

Description

A UNIVERSAL VACCINE STRATEGY FOR CONFERRING PROTECTION AGAINST DIVERSE PATHOGENS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Pursuant to 35 U.S.C. § 119 (e), this application claims priority to the filing date of United States Provisional Patent Application Serial No.63 / 533,893 filed August 21, 2023, the disclosure of which application is herein incorporated by reference. BACKGROUND

[0002] The development of a universal vaccine capable of providing broad protection against diverse pathogens would have a transformative impact on pandemic preparedness and global health. The present disclosure addresses this issue. SUMMARY

[0003] Compositions and methods are provided for immunizing an individual, or population of individuals, to generate an immune response that protects against pathogen infection or allergy in an antigen-agnostic manner. The protective antigen-agnostic immune response is active for a period of time following immunization, from about 7 days, 10 days, 14 days following immunization, and may be active for at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or more. A protective response reduces active infection, or reduces the severity of infection, e.g. a reduction of 10%, 25%, 50%, 75%, 90% or more in active infection or severity of infection.

[0004] An antigen-agnostic immunostimulatory agent of the disclosure comprises an effective dose of an adjuvant and an effective dose of an antigen. The antigen may be a protein antigen. Useful protein antigens for this purpose can be of a molecular weight of at least about 25 kD, at least about 30 kD, at least about 40 kD or more. A protein antigen may be a non-pathogen antigen, and is other than a self-antigen. The protein antigen is preferably non-toxic and not biologically active in the host. Exemplary protein antigens include, without limitation, ovalbumin, keyhole limpet hemacyanin (KLH), non-human serum albumins, e.g. bovine serum albumin, and the like.

[0005] An adjuvant of the antigen-agnostic immunostimulatory agent comprises an effective dose of a TLR 4 agonist, and a TLR 7, 8, or 7 / 8 agonist. The adjuvant may be provided in a liposomal, e.g. nanoparticle, composition. The protein antigen may be encapsulated in the liosome, or may be free of the liposome.

[0006] Suitable methods of administration include parenteral, oral, nasal, etc. routes. In some embodiments, where protection against a respiratory pathogen is desired, administration may be via a nasal spray or inoculum. Administration may be performed once or multiple times,e.g. as a priming dose and one or more booster doses. The timing between doses may be from about 3 to about 21 days, e.g. at least 5, at least 7, at least 10, at least 14 days, up to about 3 week, 4 weeks, 5 weeks.

[0007] In some embodiments the immunostimulatory composition of the disclosure is administered prophylactically, prior to a period of time in which an individual or population of individuals will be at increased risk of pathogen exposure, including without limitation epidemic or pandemic conditions, hospital admission, incarceration, travel, communal living, etc. The pathogen may be a virus, e.g. a respiratory virus. It is shown herein that antigen-agnostic immunostimulatory can act as broad immune enhancing agents that engender a broad state of enhanced immune responsiveness. Prophylactic administration may be performed to provide for these periods of increased immune responsiveness during a period of increased risk of pathogen exposure.

[0008] It is also shown that the innumostimulatory compositions of the disclosure are protective in reducing atopic, allergic asthma responses.

[0009] It is shown herein that a liposomal adjuvant comprising TLR 4 and TLR 7 / 8 agonists, coupled with a protein antigen, can confer non-specific viral protection upon administration. Administration may comprise, for example, intranasal delivery in a prime-boost regimen. This approach stimulated heterologous protection by promoting optimal localized T cell responses and innate antiviral immunity, and protected animals against SARS-CoV-2, influenza, omicron coronavirus and Staph. aureus infection in an antigen-agnostic manner even at 3 weeks following the last immunization. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0011] FIGS. 1A-1B. Intranasal liposomal GLA / 3M-052 plus OVA protects against SARS- CoV-2 and influenza. Schematic of experimental design and weight loss of liposomal GLA / 3M- 052 plus OVA immunized and unimmunized control mice following (a) SARS-CoV-2 B.1.351 challenge up till day 3 post-challenge and (b) PR8 challenge up till day 11 post-challenge. Immunization was performed with one prime dose, followed by three booster doses, each given at a one-week interval. Mice were challenged at day 21 following the last boost. Data combined from 2 or 3 independent experiments ((B.1.351 challenge, n=24-26; PR8 challenge, n=24-29, respectively). Statistical analysis was performed with Two-way ANOVA with Sidak multiple comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.0001.

[0012] FIGS, 2A-2C. T cell responses in the lungs to intranasal liposomal GLA / 3M-052 plus OVA immunization at day 21 post-last boost. (a) OVA-specific CD4+ T cell responses in the lungs at day 21 post-last boost (b) Circle plot showing OVA-specific CD4 T cell cytokine secretion profile in the lungs. (c) CD8+ T cell responses in the lungs. (d) T cell responses in the mediastinal lymph nodes. All T cell responses were analyzed following ex vivo stimulation with OVA peptides. Data combined from two independent experiments (n=10-14). Statistical analysis performed by Mann-Whitney test. ****, P < 0.0001.

[0013] FIGS, 3A-3B. Innate immune responses in the lungs to intranasal liposomal GLA / 3M- 052 plus OVA immunization at day 21 post-last boost. (a) CD86 median fluorescence intensity (MFI) on innate cells in the lungs. (b) Frequency of innate cells in the lungs represented as % of live CD45+ cells. Data combined from two independent experiments (n=10-14). Statistical analysis performed by Mann-Whitney test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

[0014] FIGS.4A-4B. PR8 challenge following different challenge timepoint and immunization regimen. (A) Mice given 3 booster doses of intranasal liposomal GLA / 3M-052 plus OVA at one-week interval were protected from weight loss when challenged with PR8 virus at day 7 after the last dose. (B) Mice given only a single dose of intranasal liposomal GLA / 3M-052 plus OVA were not protected from weight loss following PR8 challenge at day 21 post-prime. Data is from one independent experiment (n=6-7). Statistical analysis was performed with Two-way ANOVA with Sidak multiple comparison test. **, P < 0.01.

[0015] FIG.5. T cell responses to intranasal liposomal GLA / 3M-052 plus OVA immunization in the mediastinal lymph nodes at day 21 post-last boost. T cell responses were analyzed following ex vivo stimulation with OVA peptides. Data combined from two independent experiments (n=10-14). Statistical analysis performed by Mann-Whitney test.

[0016] FIGS. 6A-6B. Liposomal GLA / 3M-052 plus OVA protects against SARS-CoV-2 challenge at day 21 post-boost. A. Immunization schematic. B. Viral load in lungs and nasal turbinate.

[0017] FIGS. 7A-7B. Two or three boosts of Liposomal GLA / 3M-052+OVA protects against SARS-CoV-2 challenge at day 21 post-boost. A. Immunization schematic. B Effect of immunization on weight post-challenge.

[0018] FIGS. 8A-8B. Liposomal GLA / 3M-052 plus OVA protects against SARS-CoV-2 challenge at day 42 post-boost. A. Immunization schematic. B. Effect of immunization on viral load in lungs and nasal turbinates.

[0019] FIGS. 9A-9B. Liposomal GLA / 3M-052 plus OVA protects against SARS-CoV-2 challenge 3 months post-boost. A. Immunization schematic. B. Effect of immunization on viral load on lungs and nasal turbinates.

[0020] FIGS. 10A-10B. Liposomal GLA / 3M-052 plus OVA protects against Omicron BA.5 challenge at day 21 post-boost. A. Immunization schematic. B. Effect of immunization on viral load in the lungs and nasal turbinates.

[0021] FIGS. 11A-11B. Liposomal GLA / 3M-052 plus OVA protects against Omicron BA.5 challenge at day 21 post-boost. A. Immunization schematic. B. Effect of immunization on viral load in the lungs and nasal turbinates.

[0022] FIGS.12A-12B. Liposomal GLA / 3M-052 plus OVA protects against PR8 challenge at day 21 post-boost. A. Immunization schematic. B. Effect of immunization on probability of survival post challenge.

[0023] FIGS.13A-13B. Liposomal GLA / 3M-052 plus OVA protects against PR8 challenge at day 42 post-boost. A. Immunization schematic. B. Effect of immunization on probability of survival post-challenge.

[0024] FIGS. 14A-14B. Liposomal GLA / 3M-052 plus OVA protects against S. aureus challenge at day 21 day 42 or 3months post-boost. A. Bacterial load in the lungs. B. Plate counts of bacteria.

[0025] FIGS. 15A-15D. T cell responses in intranasal liposomal GLA / 3M-052 plus OVA immunization. A. Immunization schematic. B. Effect of immunization on cytokines. B. Effect of immunization on T cell populations. D. Ratio of T cells.

[0026] FIGS. 16A-16D. T cell responses in intranasal liposomal GLA / 3M-052 plus OVA immunization, 6 weeks later. A. Immunization schematic. B. Effect of immunization on cytokines. C, D. Effect of immunization on T cell subsets.

[0027] FIGS. 17A-17C. T cell responses in intranasal liposomal GLA / 3M-052 plus OVA immunization, 6 weeks later, in which we are evaluating the capacity of intranasal immunization with liposomes (GLA+3M-052)+OVA to protect against allergic asthma induced by repeated administration of house dust mite (HDM) allergens several weeks later. A. Immunization schematic, animals were challenged with the allergen HDM, house. B. Effect of immunization innate immune cells. C. Effect of immunization on frequencies of cell types in the lungs of mice. Of note, eosinophils, which are key mediators of the allergic response, are greatly diminished in mice that were preimmunized with liposomes (GLA+3M-052)+OVA.

[0028] FIGS. 18. Intranasal liposomal GLA / 3M-052 plus OVA immunization decreased allergic asthma disease. A. Immunization schematic. B, C. shows the frequencies of CD4 T cells that secrete Th1 (IFNg) or Th2 (IL-4, IL-13, IL-5) cytokines. As will be evident, pre immunization with liposomes (GLA+3M-052)+OVA results in a marked suppression of Th2 responses, which are critical mediators of the allergic asthma response.DETAILED DESCRIPTION

[0029] Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0030] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supercedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0032] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.

[0033] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0034] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal being assessed for treatment and / or being treated. In some embodiments, themammal is a human. The terms “subject,” “individual,” and “patient” encompass, without limitation, individuals having a disease. Subjects may be human, but also include other mammals, particularly those mammals useful as laboratory models for human disease, e.g., mice, rats, etc.

[0035] The term “sample” with reference to a patient encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The term also encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations, such as diseased cells. The definition also includes samples that have been enriched for particular types of molecules, e.g., nucleic acids, polypeptides, etc. The term “biological sample” encompasses a clinical sample, and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, and the like.

[0036] The term “diagnosis” is used herein to refer to the identification of a molecular or pathological state, disease or condition in a subject, individual, or patient.

[0037] The term “prognosis” is used herein to refer to the prediction of the likelihood of death or disease progression, including recurrence, spread, and drug resistance, in a subject, individual, or patient. The term “prediction” is used herein to refer to the act of foretelling or estimating, based on observation, experience, or scientific reasoning, the likelihood of a subject, individual, or patient experiencing a particular event or clinical outcome. In one example, a physician may attempt to predict the likelihood that a patient will survive, or the severity of an infection.

[0038] As used herein, the terms “treatment,” “treating,” and the like, refer to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect on or in a subject, individual, or patient. The effect may be prophylactic in terms of completely or partially preventing a disease, for example infection by a pathogen, or symptom thereof and / or may be therapeutic in terms of effecting a partial or complete cure for a disease and / or symptoms of the disease.

[0039] Treating may refer to any indicia of success in the treatment or amelioration or prevention of a disease, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of an examination by a physician. Accordingly, the term "treating" includes the administration of an agent to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with infectiousdisease or other diseases. The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.

[0040] As used herein, a "therapeutically effective amount" refers to that amount of the immunostimulatory composition sufficient to induce an enhanced immune response. A therapeutically effective amount may refer to the amount of immunostimulatory composition sufficient to reduce infection upon pathogen exposure, e.g., to delay or minimize infection. A therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease. Further, a therapeutically effective amount means the amount of immunostimulatory composition alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease.

[0041] As used herein, the term “dosing regimen” refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0042] In some embodiments, for example with clinically approved adjuvants, the unit dose is the same or comparable to the clinically approved dose. For example, a dose for prophylactic purposes disclosed herein may be from about 10% to about 500% of a clinically approved dose of an adjuvant for vaccine administration, and may be from about 25% to about 250%, from about 50% to about 150%, and may be substantially similar in dose.

[0043] "In combination with", "combination therapy" and "combination products" refer, in certain embodiments, to the concurrent administration to a patient of the immunostimulatory compositions described herein in combination with additional therapies, e.g. inclusion of antigenic material, and the like. When administered in combination, each component can beadministered at the same time or sequentially in any order at different points in time. Thus, each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect.

[0044] "Concomitant administration" means administration of one or more components, such as immunostimulatory compositions, known therapeutic agents, etc. at such time that the combination will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of components. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration.

[0045] The use of the term "in combination" does not restrict the order in which prophylactic and / or therapeutic agents are administered to a subject. A first prophylactic or therapeutic agent can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second prophylactic or therapeutic agent to a subject with a disorder.

[0046] The term “isolated” refers to a molecule that is substantially free of its natural environment. For instance, an isolated protein is substantially free of cellular material or other proteins from the cell or tissue source from which it is derived. The term refers to preparations where the isolated protein is sufficiently pure to be administered as a therapeutic composition, or at least 70% to 80% (w / w) pure, more preferably, at least 80%-90% (w / w) pure, even more preferably, 90-95% pure; and, most preferably, at least 95%, 96%, 97%, 98%, 99%, or 100% (w / w) pure. A “separated” compound refers to a compound that is removed from at least 90% of at least one component of a sample from which the compound was obtained. Any compound described herein can be provided as an isolated or separated compound.

[0047] "Antibody" refers to an immunoglobulin molecule that can bind to a specific antigen as the result of an immune response to that antigen. Immunoglobulins are serum proteins composed of "light" and "heavy" polypeptide chains having "constant" and "variable" regions and are divided into classes (e.g., IgA, IgD, IgE, IgG, and IgM) based on the composition of the constant regions.

[0048] "Antigen" or "immunogen" refers to any substance that stimulates an immune response. The term includes killed, inactivated, attenuated, or modified live bacteria, viruses, or parasites. The term antigen also includes polynucleotides, polypeptides, recombinant proteins, synthetic peptides, protein extract, cells (including bacterial cells), tissues,polysaccharides, or lipids, or fragments thereof, individually or in any combination thereof. The term antigen also includes antibodies, such as anti-idiotype antibodies or fragments thereof, and to synthetic peptide mimotopes that can mimic an antigen or antigenic determinant (epitope).

[0049] "Immune response" in a subject refers to the development of an adaptive immune response, e.g. humoral immune response, cellular immune response, or a humoral and a cellular immune response to an antigen. Imune response also refrs to an innate immune response. Immune responses may be determined using standard immunoassays and neutralization assays, which are known in the art.

[0050] "Cellular immune response" or "cell mediated immune response" is one mediated by T-lymphocytes or other white blood cells or both, and includes the production of cytokines, chemokines and similar molecules produced by lymphocyte, leukocytes, or both.

[0051] "Immunogenic" means evoking an immune or antigenic response. Thus an immunogenic composition would be any composition that induces an immune response.

[0052] "Emulsifier" means a substance used to make an emulsion more stable.

[0053] "Emulsion" means a composition of two immiscible liquids in which small droplets of one liquid are suspended in a continuous phase of the other liquid.

[0054] "Pharmaceutically acceptable" refers to substances, which are within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit-to- risk ratio, and effective for their intended use.

[0055] "Reactogenicity" refers to the side effects elicited in a subject in response to the administration of an adjuvant, an immunogen, a vaccine composition, etc. It can occur at the site of administration, and is usually assessed in terms of the development of a number of symptoms. These symptoms can include inflammation, redness, and abscess. It is also assessed in terms of occurrence, duration, and severity. A "low" reaction would, for example, involve swelling that is only detectable by palpitation and not by the eye, or would be of short duration. A more severe reaction would be, for example, one that is visible to the eye or is of longer duration.

[0056] "Immunostimulatory composition" refers to a composition that includes an adjuvant, as defined herein and a non-self, non-pathogen antigen. Administration of the composition to a subject results in an increased antigen-agnostic responsiveness. The amount of a composition that is therapeutically effective may vary depending on the antigen, the adjuvant, and the condition of the subject, and can be determined by one skilled in the art.

[0057] Atopy. Atopy is a predisposition to an immune response against diverse antigens and allergens leading to CD4+Th2 differentiation and overproduction of immunoglobulin E (IgE).The clinical consequence is an increased propensity to hypersensitivity reactions. Allergic bronchial asthma and allergic rhinitis are frequent manifestations of atopy, followed by atopic dermatitis and food allergy. Other diseases described as atopic include allergic conjunctivitis, IgE-mediated drug allergy, urticaria and angioedema, and anaphylactic shock.

[0058] The pathophysiology of atopy characteristically demonstrates by mast cell activation. Antigen binding to IgE cross-links Fc epsilon RI proteins on mast cells. It activates protein tyrosine kinases (Lyn and Syk) that in turn cause activation of a MAP kinase cascade and a phosphatidylinositol-specific phospholipase C, which catalyzes the release of the following molecules: IP3 and DAG from membrane PIP2. Inositol trisphosphate (IP3) causes the release of intracellular calcium from the endoplasmic reticulum. DAG and calcium activate PKC that phosphorylates substrates such as myosin light chain molecule and thus leads to degradation and release of preformed mediators. MAP kinases and calcium react to activate the enzyme cytosolic phospholipase A2, which stimulates the synthesis of lipid mediators including PGD2, LTC4, LTD4, and LTE4. Ras / MAP kinases in the presence of calcium and PKC cause cytokine gene expression, which releases TNF and other cytokines (IL-4, IL-5, IL-6, IL-13 among others). Lipid mediators, cytokines and histamine cause an inflammatory response.

[0059] Atopy presents with a histopathologically characteristic wheal and flare reaction in the skin, which is in response to an allergen-stimulated release of mediators from mast cells, local blood vessels that dilate and become leaky to proteins and fluids, which produces local swelling and redness.

[0060] Allergic asthma is a manifestation of atopy localized in the bronchus. There is a release of critical mediators including histamine, leukotrienes, and cytokines including IL-4, IL-5, IL- 13, TNF and eosinophil chemotactic factor. The aim of symptomatic asthma is controlling the hyperirritable bronchial mucosa using environmental measures, drugs, and other therapies. Atopic individuals have a lifelong tendency for the development of allergic reactions as it is incurable. Nevertheless, the manifestations of atopy often change over some time. Allergic asthma has a prognosis that varies according to the persistence of the causative environmental allergen, the IgE levels in blood or tissues, and the genetic makeup. Compositions

[0061] The term “adjuvant” refers to a composition that increases the humoral or cellular immune response of an individual. The adjuvant portion of the immunostimulatory composition comprises a TLR4 and a TLR 7, 8, or 7 / 8 agonist. The TLR agonists may be present in a liposomal formulation. The formulation may be free of squalene.

[0062] The dose of adjuvant administered may depend on the antigen with which it is used and the antigen dosage to be applied. It is also dependent on the intended species and the desired formulation. Usually the quantity is within the range conventionally used for adjuvants.For example, adjuvants typically comprises from about 1 µg to about 1000 µg, inclusive, of a 1-mL dose.

[0063] TLR4 agonists may be present at a concentration of from about 1 ng / ml, 10 ng / ml, 100 ng / ml, 1 µg / ml, 10 µg / ml include, 100 µg / ml, 1 mg / ml, 10 mg / ml, up to about 100 mg / ml, where the unit dose for a human may range from about 1 µg, 5 µg, 10 µg, 50 µg, 100 µg, 500 µg, 1 mg, 5 mg, 10 mg, 50 mg, up to about 100 mg. Oral and nasal doses may be higher than parenterally delivered doses.

[0064] TLR4 agonists of interest include, without limitation, aminoalkyl glucosaminide phosphates (AGPs), monophosphoryl lipid A (MPLA), MPLA-modified formulation (AS02 and AS04), synthetic glycolipid CCL-34; glucopyranosyl lipid adjuvant; N-cyclohexyl-2-((5-methyl- 4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-b]indol-2-yl)thio)acetamide; 8-(furan-2-yl) substituted pyrimido[5,4-b]indole analog; MPL (3-O-desacyl-4′-monophosphoryl lipid A); phosphorylated hexa-acyl disaccharide (PHAD); RIBI-529 (RC-529), a lipid A mimetic (aminoalkyl glucosaminide 4-phosphate); murine β-defensin 2, etc. In some embodiments the TLR4 agonist is glucopyranosyl lipid adjuvant.

[0065] TLR7, 8 and 7 / 8 agonists include those in Table 1, as well as guanosine analogs; imidazoquinolines such as Aldara ®, R848; Loxoribine, phospholipid conjugated small molecule TLR7 ligand (TLR-L), 1V270, synthetic imidazoquinolinone 3M-052. In some embodiments the TLR7 / 8 agonist is 3M-052. Agonist Targeted Clinical Trial# TLRAgonist Targeted Clinical Trial# TLR

[0066] TLt 1 ng / ml, 10 ng / ml, 100 ng / ml, 1 µg / ml, 10 µg / ml include, 100 µg / ml, 1 mg / ml, 10 mg / ml, up to about 100 mg / ml, where the unit dose for a human may range from about 1 µg, 5 µg, 10 µg, 50 µg, 100 µg, 500 µg, 1 mg, 5 mg, 10 mg, 50 mg, up to about 100 mg. Oral and nasal doses may be higher than parenterally delivered doses.

[0067] The TLR4 and TLR 7, 8, 7 / 8 agonists can be formulated in nanoparticles or other delivery vehicles. In certain embodiments the TLR agonists are encapsulated within a nanoparticle. In particular embodiments, a nanoparticle is a particle having at least one dimension (e.g., a diameter) less than or equal to 1000 nM, less than or equal to 500 nM or less than or equal to 100 nM. In particular embodiments, a nanoparticle comprises a lipid. Lipid nanoparticles include, but are not limited to, liposomes and micelles. Any of a number of lipids may be present, including cationic and / or ionizable lipids, anionic lipids, neutral lipids, amphipathic lipids, PEGylated lipids, and / or structural lipids. Such lipids can be used alone or in combination.

[0068] In some embodiments, the lipid nanoparticle formulations comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) cationic and / or ionizable lipids. Such cationic lipids include, but are not limited to, 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2- (didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25- ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N- dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin- MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane (DLin-KC2-DMA), 2-({8- [(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1- yloxy]propan-1-amine (Octyl-CLinDMA), (2R)-2-({8-[(3 (3)-cholest-5-en-3-yloxy]octyl}oxy)- N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA (2R)), (25)-2-({8-[(3 (3)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca- 9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA (2S)).N,N-dioleyl-N,N- dimethylammonium chloride (“DODAC”); N-(2,3-dioleyloxy)propyl-N,N—N-triethylammonium chloride (“DOTMA”); N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”); N-(2,3- dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”); 1,2-Dioleyloxy-3- trimethylaminopropane chloride salt (“DOTAP.Cl”); 3-β-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”), N-(1-(2,3-dioleyloxy)propyl)-N-2- (sperminecarboxamido)ethyl)-N,N-dimethyl-ammonium trifluoracetate (“DOSPA”), dioctadecylamidoglycyl carboxyspermine (“DOGS”), 1,2-dioleoyl-3-dimethylammonium propane (“DODAP”), N,N-dimethyl-2,3-dioleyloxy)propylamine (“DODMA”), and N-(1,2- dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (“DMRIE”). Additionally, a number of commercial preparations of cationic and / or ionizable lipids can be used, such as, e.g., LIPOFECTIN® (including DOTMA and DOPE, available from GIBCO / BRL), and LIPOFECTAMINE® (including DOSPA and DOPE, available from GIBCO / BRL). KL10, KL22, and KL25 are described, for example, in U.S. Pat. No.8,691,750, which is incorporated herein by reference in its entirety. In particular embodiments, a cationic lipid is one or both of DLin-MC3-DMA and DLin-KC2-DMA.

[0069] Anionic lipids suitable for use in lipid nanoparticles of the disclosure include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl phosphatidylethanoloamine, N-succinyl phosphatidylethanolamine, N- glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, and other anionic modifying groups joined to neutral lipids.

[0070] Neutral lipids suitable for use in lipid nanoparticles of the disclosure include, but are not limited to, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. Lipids having a variety of acyl chain groups of varying chain length and degree of saturation are available or may be isolated or synthesized by well-known techniques. Additionally, lipids having mixtures of saturated and unsaturated fatty acid chains can be used. In some embodiments, the neutral lipids used in the disclosure are DOPE, DSPC, DPPC, POPC, or any related phosphatidylcholine. In some embodiments, the neutral lipid may be composed of sphingomyelin, dihydrosphingomyeline, or phospholipids with other head groups, such as serine and inositol.

[0071] In some embodiments, amphipathic lipids are included in nanoparticles of the disclosure. Exemplary amphipathic lipids suitable for use in nanoparticles of the disclosure include, but are not limited to, sphingolipids, phospholipids, and aminolipids. In some embodiments, a phospholipid is selected from the group consisting of 1,2-dilinoleoyl-sn- glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 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-diundecanoyl-sn-glycero- phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di- O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn- glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoetha nolamine (DOPE), 1,2-diphytanoyl- sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl- sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3- phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin. Other phosphorus-lacking compounds, such as sphingolipids, glycosphingolipid families, diacylglycerols, and β- acyloxyacids, may also be used. Additionally, such amphipathic lipids can be readily mixed with other lipids, such as triglycerides and sterols.

[0072] In some embodiments, the lipid component of a nanoparticle of the disclosure comprises one or more PEGylated lipids. A PEGylated lipid (also known as a PEG lipid or a PEG-modified lipid) is a lipid modified with polyethylene glycol. The lipid component may include one or more PEGylated lipids. A PEGylated lipid may be selected from the non-limiting group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified di alkyl amines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols. For example, a PEGylated lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

[0073] The molecular weight of the PEG conjugated to a lipid includes but is not limited to PEGs having molecular weights between 500Da and 20kDa, and may be between 500Da and 10kDa, between about 500Da and 5kDa, between about 750Da and 2kDa. in some embodiments the PEG has a molecular weight of approximately 750Da, in some embodiments the PEG has a molecular weight of approximately 1kDa, , in some embodiments the PEG has a molecular weight of approximately 2kDa. The PEG may be linear or branched. The processes and chemical reactions necessary to achieve PEGylation of biological compounds is well known in the art.

[0074] A lipid nanoparticle of the disclosure may include one or more structural lipids. Exemplary, non-limiting structural lipids that may be present in the lipid nanoparticles of the disclosure include cholesterol, fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, or alpha-tocopherol).

[0075] An antigen-agnostic immunostimulatory agent of the disclosure comprises an antigen, usually a protein antigen. Useful protein antigens for this purpose can be of a molecular weight of at least about 25 kD, at least about 30 kD, at least about 40 kD or more. The protein antigen may be a non-pathogen antigen, and is other than a self-antigen. The protein antigen is preferably non-toxic and not biologically active in the host. Exemplary protein antigens include, without limitation, gelatin, ovalbumin, keyhole limpet hemacyanin (KLH), non-human serumalbumins, e.g. bovine serum albumin, non-human collagens and other structural proteins, and the like. Preferred proteins are those approved for administration to humans.

[0076] The protein antigen may be present at a concentration of from about 1 ng / ml, 10 ng / ml, 100 ng / ml, 1 µg / ml, 10 µg / ml include, 100 µg / ml, 1 mg / ml, 10 mg / ml, up to about 100 mg / ml, where the unit dose for a human may range from about 1 µg, 5 µg, 10 µg, 50 µg, 100 µg, 500 µg, 1 mg, 5 mg, 10 mg, 50 mg, up to about 100 mg. Oral and nasal doses may be higher than parenterally delivered doses.

[0077] The routes of administration for the adjuvant compositions include parenteral, oral, oronasal, intranasal, intratracheal, topical, etc. In some embodiments administration is nasal. Any suitable device may be used to administer the compositions, including nasal sprays, syringes, droppers, needleless injection devices, patches, and the like. The route and device selected for use will depend on the composition of the adjuvant, the antigen, and the subject, and such are well known to the skilled artisan.

[0078] Some examples of bacteria causing disease for which non-specific immune responsiveness may be obtained include, for example, Aceinetobacter calcoaceticus, Acetobacter paseruianus, Actinobacillus pleuropneumoniae, Aeromonas hydrophila, Alicyclobacillus acidocaldarius, Arhaeglobus fulgidus, Bacillus pumilus, Bacillus stearothermophillus, Bacillus subtilis, Bacillus thermocatenulatus, Bordetella bronchiseptica, Burkholderia cepacia, Burkholderia glumae, Campylobacter coli, Campylobacter fetus, Campylobacter jejuni, Campylobacter hyointestinalis, Chlamydia psittaci, Chlamydia trachomatis, Chlamydophila spp., Chromobacterium viscosum, Erysipelothrix rhusiopathieae, Listeria monocytogenes, Ehrlichia canis, Escherichia coli, Haemophilus influenzae, Haemophilus somnus, Helicobacter suis, Lawsonia intracellularis, Legionella pneumophilia, Moraxellsa sp., Mycobactrium bovis, Mycoplasma hyopneumoniae, Mycoplasma mycoides subsp. mycoides LC, Clostridium perfringens, Odoribacter denticanis, Pasteurella (Mannheimia) haemolytica, Pasteurella multocida, Photorhabdus luminescens, Porphyromonas gulae, Porphyromonas gingivalis, Porphyromonas salivosa, Propionibacterium acnes, Proteus vulgaris, Pseudomnas wisconsinensis, Pseudomonas aeruginosa, Pseudomonas fluorescens C9, Pseudomonas fluorescens SIKW1, Pseudomonas fragi, Pseudomonas luteola, Pseudomonas oleovorans, Pseudomonas sp B11- 1, Alcaliges eutrophus, Psychrobacter immobilis, Rickettsia prowazekii, Rickettsia rickettsia, Salmonella typhimurium, Salmonella bongori, Salmonella enterica, Salmonella dublin, Salmonella typhimurium, Salmonella choleraseuis, Salmonella newport, Serratia marcescens, Spirlina platensis, Staphlyoccocus aureus, Staphyloccoccus epidermidis, Staphylococcus hyicus, Streptomyces albus, Streptomyces cinnamoneus, Streptococcus suis, Streptomycesexfoliates, Streptomyces scabies, Sulfolobus acidocaldarius, Syechocystis sp., Vibrio cholerae, Borrelia burgdorferi, Treponema denticola, Treponema minutum, Treponema phagedenis, Treponema refringens, Treponema vincentii, Treponema palladium, and Leptospira species, such as the known pathogens Leptospira canicola, Leptospira grippotyposa, Leptospira hardjo, Leptospira borgpetersenii hardjo-bovis, Leptospira borgpetersenii hardjo-prajitno, Leptospira interrogans, Leptospira icterohaemorrhagiae, Leptospira pomona, and Leptospira bratislava, and combinations thereof.

[0079] Examples of viruses causing disease for which non-specific immune responsiveness may be obtained include, for example, mpox (monkeypox), SARS-Cov1, SARS-Cov2, and other coronaviruses, Avian herpesviruses, Bovine herpesviruses, Canine herpesviruses, Equine herpesviruses, Feline viral rhinotracheitis virus, Marek's disease virus, Ovine herpesviruses, Porcine herpesviruses, Pseudorabies virus, Avian paramyxoviruses, Bovine respiratory syncytial virus, Canine distemper virus, Canine parainfluenza virus, canine adenovirus, canine parvovirus, Bovine Parainfluenza virus 3, Ovine parainfluenza 3, Rinderpest virus, Border disease virus, Bovine viral diarrhea virus (BVDV), BVDV Type I, BVDV Type II, Classical swine fever virus, Avian Leukosis virus, Bovine immunodeficiency virus, Bovine leukemia virus, Bovine tuberculosis, Equine infectious anemia virus, Feline immunodeficiency virus, Feline leukemia virus (FeLV), Newcastle Disease virus, Ovine progressive pneumonia virus, Ovine pulmonary adenocarcinoma virus, Canine coronavirus (CCV), pantropic CCV, Canine respiratory coronavirus, Bovine coronavirus, Feline Calicivirus, Feline enteric coronavirus, Feline infectious peritonitis, virus, Porcine epidemic diarrhea virus, Porcine hemagglutinating encephalomyletitis virus, Porcine parvovirus, Porcine Circovirus (PCV) Type I, PCV Type II, Porcine Reproductive and Respiratory Syndrome (PRRS) Virus, Transmissible gastroenteritis virus, Turkey coronavirus, Bovine ephemeral fever virus, Rabies, Rotovirus, Vesicular stomatitis virus, lentivirus, Avian influenza, Rhinoviruses, Equine influenza virus, Swine influenza virus, Canine influenza virus, Feline influenza virus, Human influenza virus, Eastern Equine encephalitis virus (EEE), Venezuelan equine encephalitis virus, West Nile virus, Western equine encephalitis virus, human immunodeficiency virus, human papilloma virus, varicella zoster virus, hepatitis B virus, rhinovirus, and measles virus, and combinations thereof.

[0080] Examples of parasites causing disease for which non-specific immune responsiveness may be obtained include, for example, Anaplasma, Fasciola hepatica (liver fluke), Coccidia, Eimeria spp., Neospora caninum, Toxoplasma gondii, Giardia, Dirofilaria (heartworms), Ancylostoma (hookworms), Trypanosoma spp., Leishmania spp., Trichomonas spp., Cryptosporidium parvum, Babesia, Schistosoma, Taenia, Strongyloides, Ascaris, Trichinella, Sarcocystis, Hammondia, and Isopsora, and combinations thereof. Also contemplated are external parasites including, but not limited to, ticks, including Ixodes, Rhipicephalus,Dermacentor, Amblyomma, Boophilus, Hyalomma, and Haemaphysalis species, and combinations thereof.

[0081] Other components of the compositions can include pharmaceutically acceptable excipients, such as carriers, solvents, and diluents, isotonic agents, buffering agents, stabilizers, preservatives, vaso-constrictive agents, antibacterial agents, antifungal agents, and the like. Typical carriers, solvents, and diluents include water, saline, dextrose, ethanol, glycerol, oil, and the like. Representative isotonic agents include sodium chloride, dextrose, mannitol, sorbitol, lactose, and the like. Useful stabilizers include gelatin, albumin, and the like.

[0082] Surfactants are used to assist in the stabilization of the emulsion selected to act as the carrier for the adjuvant and antigen. Surfactants suitable for use in the present inventions include natural biologically compatible surfactants and non-natural synthetic surfactants. Biologically compatible surfactants include phospholipid compounds or a mixture of phospholipids. Preferred phospholipids are phosphatidylcholines (lecithin), such as soy or egg lecithin. Lecithin can be obtained as a mixture of phosphatides and triglycerides by water- washing crude vegetable oils, and separating and drying the resulting hydrated gums. A refined product can be obtained by fractionating the mixture for acetone insoluble phospholipids and glycolipids remaining after removal of the triglycerides and vegetable oil by acetone washing. Alternatively, lecithin can be obtained from various commercial sources. Other suitable phospholipids include phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, phosphatidic acid, cardiolipin, and phosphatidylethanolamine. The phospholipids may be isolated from natural sources or conventionally synthesized.

[0083] Non-natural, synthetic surfactants suitable for use in the present invention include sorbitan-based non-ionic surfactants, e.g. fatty-acid-substituted sorbitan surfactants, fatty acid esters of polyethoxylated sorbitol (TWEENTM), polyethylene glycol esters of fatty acids from sources such as castor oil; polyethoxylated fatty acid, polyethoxylated isooctylphenol / formaldehyde polymer, polyoxyethylene fatty alcohol ethers (BRIJTM); polyoxyethylene nonphenyl ethers (TRITONTM), polyoxyethylene isooctylphenyl ethers (TRITONTMX).

[0084] As used herein, "a pharmaceutically-acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. The carrier(s) must be "acceptable" in the sense of being compatible with the other components of the compositions and not deleterious to the subject. Typically, the carriers will be will be sterile and pyrogen-free, and selected based on the mode of administration to be used. It is well known by those skilled in the art that the preferred formulations for the pharmaceutically acceptable carrier which comprise the compositions are those pharmaceutical carriersapproved in the applicable regulations promulgated by the United States (US) Department of Agriculture or US Food and Drug Administration, or equivalent government agency in a non- US country. Therefore, the pharmaceutically accepted carrier for commercial production of the compositions is a carrier that is already approved or will be approved by the appropriate government agency in the US or foreign country.

[0085] The compositions optionally can include compatible pharmaceutically acceptable (i.e., sterile or non-toxic) liquid, semisolid, or solid diluents that serve as pharmaceutical vehicles, excipients, or media. Diluents can include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin, among others.

[0086] The compositions can also contain antibiotics or preservatives, including, for example, gentamicin, merthiolate, or chlorocresol. The various classes of antibiotics or preservatives from which to select are well known to the skilled artisan.

[0087] In some embodiments, the immunostimulatory composition of the disclosure comprises two synthetic Toll-like receptor (TLR) ligands: Glucopyranosyl lipid adjuvant (GLA) (TLR4) and 3M-052 (TLR7 / 8). In some embodiments the TLR agonists are present in a PEGylated liposomal adjuvant. The adjuvant is combined with an effective dose of a protein antigen. In some embodiments the liposomal component comprises dipalmitoyl phosphatidylcholine (DPPC), PEGylated dipalmitoyl phosphatidylethanolamine or distearoyl (DSPE-PEG) with PEG MW of 500-5000 (DPPE-PEG), and cholesterol.

[0088] In some embodiments, an immunostimulatory composition comprises a protein adjuvant present at a unit dose for human administration of from about 10 µg to about 10 mg, e.g. from about 30 µg to about 3 mg, and may be from about 250 to 750 µg, e.g. around 300 µg.

[0089] In some embodiments, an immunostimulatory composition comprises glucopyranosyl lipid adjuvant present at a unit dose for human administration of from about 10 µg to about 10 mg, e.g. from about 30 µg to about 3 mg, and may be from about 250 to 750 µg, e.g. around 300 µg.

[0090] In some embodiments, an immunostimulatory composition comprises GM-052 present at a unit dose for human administration of from about 10 µg to about 10 mg, e.g. from about 12 µg to about 1.2 mg, and may be from about 75 to 250 µg, e.g. around 120 µg.

[0091] In some embodiments, the liposomal component comprises phospholipid:cholesterol at a ratio of about 10:1, about 5:1, about 2:1, and may be around 12:7 (molar ratio). In some embodiments the phospholipid is DPPC.

[0092] The liposomal component may comprise pegylated lipid at a ratio, relative to phospholipid, of about 20:1, about 15:1, about 10:1, about 5:1 (molar ratio phospholipid topegylated lipid), and may be about 12:1. In some embodiments the pegylated lipid is dipalmitoyl phosphatidylethanolamine (DPPE-PEG) or distearoyl (DSPE-PEG) with a PEG MW of 500-5000 Kd.

[0093] In some embodiments the lipid component comprises phospholipid:PEGylated lipid:cholesterol at a molar ratio of 12.2:1.0:7.1. The unit dose of phospholipid for a human may be from about 50 µg to about 10 mg, e.g. from about 80 µg to about 8 mg, and may be from about 500 to 1000 µg, e.g. around 850 µg. The composition may be formulated for nasal delivery. The volume of a unit dose may be, for example, from around 250 µl to about 1 ml. Methods of Use

[0094] In some embodiments an immunostimulatory composition of the disclosure is administered to an individual to enhance immune responsiveness. The individual may be at risk of exposure to a pathogen, e.g. in a pandemic, in a hospital setting, or other circumstances. The individual may be administered an immunostimulatory composition prior to a period of time in which an individual will be at increased risk of pathogen exposure, including without limitation: hospital admission, incarceration, travel, entering a communal living situation, etc. The pathogen of increased risk may be a bacteria, virus, parasite, etc., e.g. a respiratory virus.

[0095] It is shown herein that the immunostimulants of the disclosure can act as broad immune enhancing agents that engender a broad state of enhanced immune responsiveness for a period of at least about 2 weeks, at least to about 3 weeks, at least to about 4 weeks, and in some instances can be detected after about 2 months or more. Prophylactic administration may be performed to provide for increased immune responsiveness during a period of increased risk of pathogen exposure.

[0096] Administration may be performed once, twice, three or more times as required. Multiple administrations can be spaced apart by about 2, 3, 4, 5, 6, 7, 8 or more weeks initially, and can be further spaced by 2, 3, 4, 5, 6, or more months for subsequent administrations. In some embodiments a dose is administered weekly for at least two, at least 3, at least weeks.

[0097] Although it is not required, individuals selected for treatment with the methods of the disclosure may include those with reduced adaptive immune responses, who particularly benefit from enhanced innate immunity. Such individuals may include without limitation, neonates, elderly, individuals being treated with immunosuppressants, e.g. transplant recipients, autoimmune patients, and the like; cancer patients, e.g. those treated with chemotherapeutic drugs or radiotherapy; and the like. For example, a reduced ability to produce antibodies, or other adaptive immune responses, in response to vaccination or exposure can be an indicator of reduced adaptive immune response.

[0098] Kits may be provided. Kits may further include cells or reagents suitable for isolating and culturing cells in preparation for conversion; reagents suitable for culturing T cells; and reagents useful for determining the epigenomic effect of a vaccine adjuvant. Kits may also include tubes, buffers, etc., and instructions for use. EXPERIMENTAL

[0099] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[0100] Liposomal GLA / 3M-052 plus OVA protects against SARS-CoV-2 and influenza. To evaluate the capacity of liposomal GLA / 3M-052 in inducing persistent antiviral effects, we administered the adjuvant with OVA intranasally into C57BL / 6 mice with booster doses given at day 7, 14, 21 respectively. Mice were then challenged with respiratory SARS-CoV-2 or PR8 viruses at 21 days following the last dose (Fig. 1a and 1b). Interestingly, we found that immunized mice challenged with SARS-CoV-2 barely lost weight as compared to unimmunized control mice (lowest mean % starting weight = ~89.7%) (Fig 1a). In addition, immunized mice challenged with PR8 had significantly reduced weight loss compared to unimmunized control (lowest % starting weight, immunized = ~93.5%; unimmunized control = ~81.7%) (Fig. 1b). Notably, mice were protected from weight loss against PR8 challenge at day 7 post-last dose, whereas there was no difference in weight loss against PR8 challenge when mice were given only one dose of the vaccine (Fig.4).

[0101] T cell responses to intranasal liposomal GLA / 3M-052 plus OVA immunization. We next profiled the T cell responses to intranasal liposomal GLA / 3M-052 plus OVA immunization in the lungs and spleen at 21 days following the last booster dose. Notably, liposomal GLA / 3M- 052 plus OVA induced significantly increased levels of OVA-specific CD4+ T cells that are either single, double, or triple producers of IFN-γ, TNF-α, or IL-17A in the lungs (Fig.2a and Fig 2b). This suggests that antigen-specific T cells stimulated following immunization were predominantly of Th1 and Th17 profile. In addition, enhanced CD8+ T cells that are single producers of IFN-γ or double producers of IFN-γ and TNF-α were observed in the lungs at 21days following immunization (Fig 2c). Notably, an absence of OVA-specific CD4 or CD8 T cells was detected in the draining mediastinal lymph nodes, suggesting that induced T cell responses were mostly confined to the lungs at day 21 post-last boost (Fig.5).

[0102] Innate immune responses to intranasal liposomal GLA / 3M-052 plus OVA immunization. As innate antiviral signatures have been associated with non-specific protection against heterologous viruses, we assessed innate immune responses in the lungs and spleen 21 days following last boost. We found sustained activation across alveolar macrophages, DCs, Ly6C+monocytes, and interstitial macrophages (F4 / 80+) in the lungs at 21 day post-last boost, as represented by elevated CD86 activation marker expression (Fig.2a). In addition, the frequencies of alveolar macrophages and DCs were significantly increased in the lungs, whereas a modest decrease in Ly6C+ monocytes and no change in the frequency of interstitial macrophages were observed (Fig. 2b). These findings show that intranasal liposomal GLA / 3M-052 plus OVA immunization induced persistent innate activation within the lungs present at 21 days after the last boost.

[0103] We hypothesize that the non-specific viral protection observed could be a result of sustained antiviral signatures in lung epithelial cells, which are target cells of the respiratory viruses. Given the enhanced OVA-specific CD4 and CD8 T cell responses and persistent myeloid activation localized within the lungs at day 21 post-last boost, we speculate that persistent innate and epithelial antiviral resistance could be imprinted by a feedback mechanism via IFN-γ signaling from stimulated OVA-specific T cells on the myeloid and epithelial compartment. However, 3M-052 has also been found to elicit sustained innate antiviral or interferon-stimulated gene programs.

[0104] Challenge experiments were performed with SARS COV2 (data shown in FIGS.6-9), omicron BA-5 coronavirus (data shown in FIGS 10-11), influenza virus PR8 (data shown in FIGS 12-13; and Staph aureus (shown in FIG.14).

[0105] In addition, T cells were phenotyped from animals following immunization, shown in FIG.15-16.

[0106] Immunization was also protective against allergic asthma, shown in FIGS.17 and 18, where the animals were challenged with house dust mite. Methods

[0107] Adjuvant. Liposomal GLA / 3M-052 was obtained from Access to Advanced Health Institute (AAHI). For each dose, liposomal GLA / 3M-052 was mixed at 1:1 ratio with Endofit Ovalbumin (Invivogen, Cat #vac-pova) to a final volume of 20 µl containing 25 µg ovalbumin, 4 µg 3M-052 and 10 µg GLA.

[0108] Tissue processing. Lungs and mediastinal lymph nodes (LN) were harvested from mice. Lungs were prepared by gentleMACS Lung_01_01 program, followed by incubation with collagenase IV (1mg / ml) and DNase I (Sigma) in PBS+2%FBS for 30 min. Lung suspension was then processed using gentleMACS Lung_02_01 program and passed through a 70um strainer. Red blood cell lysis was performed with ACK lysis buffer (Quality Biological). Mediastinal LNs were harvested and processed without enzymatic digestion through smashing through a 70um strainer.

[0109] Intracellular cytokine staining assay for OVA-specific T cells. For T cell analysis, lung suspension was further processed with Percoll centrifugation. Single cells were isolated from the interphase of a 70-40% Percoll gradient. Both lung or LN single cell suspension was plated in complete RPMI 1640 medium at 1-2 × 106cells per well in a 96-well U-shaped plate, and restimulated with 10ug / ml class I (SINFEKL) and II (ISQAVHAAHAEINEAGR) OVA peptides (Genscript). Cells were restimulated ex vivo at 37°C, 5% CO2 condition for 1.5-2h. Brefeldin- A (10 μg ml−1) was added and cells were incubated overnight. On day 2, cells were stained with Ghost Dye Violet 510 (Tonbo Biosciences) for 10 min at RT in PBS+1mM EDTA. After washing, cells were blocked with Fc receptor antibody α-CD16 / 32 (clone 2.4G2, BD) for 5 min on ice before staining with fluorochrome-conjugated antibodies in FACS staining buffer (1× PBS, 2% FBS): CD3 (1:50 dilution; clone 145-2C11, BioLegend), CD8α (1:200 dilution; clone 53-6.7, BioLegend), CD4 (1:200 dilution; clone RM4-5, BioLegend), CD44 (1:400 dilution; clone IM7, BioLegend), CD69 (1:200 dilution; clone H1.2F3; BioLegend) and CD45 (1:200 dilution; clone 30-F11, BioLegend). Cells were incubated for 20 min at 4°C and washed 2x with PBS + 2% FBS + 1 mM EDTA. Cells were then permeabilized with BD Fix / Perm for 20 min at room temperature (RT) and stained intracellularly with antibodies in 1x Perm buffer (BD Biosciences): anti-IFN-γ (1:100 dilution; clone XMG1.2, BioLegend), anti-TNF-α (1:100 dilution; clone MP6-XT22, BioLegend), IL-2 (1:100 dilution; clone JES6-5H4, BioLegend) and anti-IL-4 (1:100 dilution; clone 11B11, BioLegend). Cells were then washed twice with 1x Perm buffer and fixed with BD Cytofix for 10 min at room temp. Data was acquired on BD Symphony analyzer and analyzed using FlowJo v10.

[0110] Flow cytometry analysis of innate cells in lungs and mediastinal lymph nodes. For innate analysis, LNs harvested from mice were treated with 1 mg / ml collagenase type IV (Worthington) 20 min at 37 °C. Lung and LN single cells were stained with antibodies: Zombie UVTM(1:200 dilution; BUV496; Biolegend #423107), anti-Ly6C (1:500; BV780; BioLegend #128041), anti-Ly6G (1:400; APC-Cy7; BioLegend #127624), anti-CD19 (1:100; BB700; BD #566411), anti-CD3 (1:100; BB700; BD #742175), anti-MHCII (1:400; AF700; eBioscience #56-5321-82), anti-CD11b (1:300; BV650; BioLegend #101239), anti-CD11c (1:400; BV421; Biolegend #117330), anti-CD86 (1:300; A647; BioLegend #105020), anti-Siglec-F (1:400; PE- CF594; BD #562757), anti-CD45 (1:200; BV610; BioLegend #103140), anti-CD169 (1:200;PE-Cy7; BioLegend #142412), anti- PDCA-1 (1:200; BUV563; BD #749275), anti-CD8a (1:200; BUV805; BD #612898), anti-CD103 (1:100; PE; eBioscience #12-1031-82), anti- NK1.1 (1:200; BV510; BioLegend #108738), anti-F4 / 80 (1:100; BUV737; BD #749283), Ghost DyeTMViolet 510 (1:400; Tonbo Bioscience #13-0870-T100), anti-EpCAM (1:100; Biolegend 118214). Samples were then washed twice with PBS + 2% FBS + 1 mM EDTA and fixed with BD Cytofix (#554655). Samples were then analyzed on a BD FACS Symphony analyzer with BD FACS Diva v.8.0.1. References Yao, Y. et al. Induction of Autonomous Memory Alveolar Macrophages Requires T Cell Help and Is Critical to Trained Immunity. Cell 175, 1634-1650.e17 (2018). Mata, E. et al. Pulmonary BCG induces lung-resident macrophage activation and confers long-term protection against tuberculosis. Sci. Immunol.6, 1–14 (2021). Pulendran, B., S. Arunachalam, P. & O’Hagan, D. T. Emerging concepts in the science of vaccine adjuvants. Nat. Rev. Drug Discov.20, 454–475 (2021). Smirnov, D., Schmidt, J. J., Capecchi, J. T. & Wightman, P. D. Vaccine adjuvant activity of 3m-052: An imidazoquinoline designed for local activity without systemic cytokine induction. Vaccine 29, 5434–5442 (2011). Abhyankar, M. M. et al. Adjuvant composition and delivery route shape immune response quality and protective efficacy of a recombinant vaccine for Entamoeba histolytica. npj Vaccines 3, (2018). Lee, A. et al. A molecular atlas of innate immunity to adjuvanted and live attenuated vaccines, in mice. Nat. Commun.1–13 (2022). doi:10.1038 / s41467-022-28197-9 Arunachalam, P. S. et al. T cell-inducing vaccine durably prevents mucosal SHIV infection even with lower neutralizing antibody titers. Nat. Med.26, 932–940 (2020). Kasturi, S. P. et al. 3M-052, a synthetic TLR-7 / 8 agonist, induces durable HIV-1 envelope-specific plasma cells and humoral immunity in nonhuman primates. Sci. Immunol. 5, (2020).

[0111] The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims.

Claims

THAT WHICH IS CLAIMED IS:

1. A method for generating an antigen-agnostic immune response in an individual, the method comprising: administering to the individual an immunostimulatory composition comprising an effective dose of a TLR4 agonist; a TLR7, 8, or 7 / 8 agonist, and a protein antigen.

2. The method of claim 1, wherein the antigen-agnostic immune response is protective for pathogen infection.

3. The method of claim 1 or claim 2, wherein the antigen-agnostic immune response is protective for a period of time of from about 1 week following immunization to 6 weeks or more following immunization.

4. The method of any of claims 1-3, wherein the protein antigen is a non-self, non- pathogen protein of greater than 25 kD that is non-toxic in the individual.

5. The method of any of claims 1-4, wherein the protein antigen is a non-human albumin.

6. The method of any of claims 1-5, wherein the effective dose of protein antigen is from about 10 µg to about 10 mg.

7. The method of any of claims 1-6, wherein the TLR4 agonist is selected from aminoalkyl glucosaminide phosphates (AGPs), monophosphoryl lipid A (MPLA), MPLA- modified formulation (AS02 and AS04), synthetic glycolipid CCL-34; glucopyranosyl lipid adjuvant; N-cyclohexyl-2-((5-methyl-4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-b]indol-2- yl)thio)acetamide; 8-(furan-2-yl) substituted pyrimido[5,4-b]indole analog; MPL (3-O-desacyl- 4′-monophosphoryl lipid A); phosphorylated hexa-acyl disaccharide (PHAD); RIBI-529 (RC- 529), a lipid A mimetic (aminoalkyl glucosaminide 4-phosphate); and murine β-defensin 2.

8. The method of of any of claims 1-7, wherein the TLR4 agonist is glucopyranosyl lipid adjuvant (GLA).

9. The method of claim 8, wherein the effective dose of GLA is from about 10 µg to about 10 mg.

10. The method of any of claims 1-9, wherein the TLR7, 8 or 7 / 8 agonist is selected from guanosine analogs; imidazoquinoline, R848; Loxoribine, phospholipid conjugated small molecule TLR7 ligand (TLR-L), 1V270, synthetic imidazoquinolinone 3M-052, 852A, BNT411, DN1508052, DSP-0509, Immiquimod, MEDI9197 (3 M-052), resiquimod, TLR agonist PolyICLC, VTX-2337, GS-9620, and GS-9688.

11. The method of claim 10, wherein the the TLR7 / 8 agonist is 3M-052.

12. The method of claim 11, wherein the effective dose of 3M-052 is from about 10 µg to about 10 mg.

13. The method of any of claims 1-12, wherein the adjuvant is encapsulated in a nanoparticle or in other formulations.

14. The method of claim 13, wherein the adjuvant is encapsulated in a liposome comprising phospholipid, pegylated lipid and cholesterol.

15. The method of claim 14, wherein the unit dose of phospholipid is from about 50 µg to about 10 mg.

16. The method of any of claims 13-15, wherein the phospholipid is dipalmitoyl phosphatidylcholine (DPPC).

17. The method of any of claims 13-16, wherein the pegylated lipid is PEGylated dipalmitoyl phosphatidylethanolamine or distearoyl phosphatidylethanolamine.

18. The method of any of claims 1-17, wherein administration is performed prior to a period of time in which the individual will be at increased risk of pathogen exposure.

19. The method of any of claims 1-18, wherein an individual selected for administration has reduced adaptive immune responses.

20. The method of any of claims 1-19, wherein administration is repeated at suitable intervals as the immune responsive state fades.

21. The method of any of claims 1-20, wherein administration is repeated weekly for at least 2 weeks.

22. The method of claim 1, wherein the antigen-agnostic immune response is protective for allergic asthma.

23. A composition for use in the method of any of claims 1-22.

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