Compositions and methods for the prevention and treatment of pathogens

Abstract

An optimized protein-free tripartite vaccine protects against lethal blood and lung infections caused by a variety of nosocomial pathogens across taxonomic kingdoms, including Gram- positive bacteria, Gram-negative bacteria, and fungi is provided as well as method for using same.

Description

COMPOSITIONS AND METHODS FOR THE PREVENTION AND TREATMENT OF PATHOGENSCROSS-REFERENCE TO RELATED APPLICATION|0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 659,771, filed on June 13, 2024, the contents of which are incorporated herein by reference in their entireties.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant Nos. AI145759, awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.BACKGROUND

[0003] Throughout this application, several technical publications are referenced by an Arabic numeral. The complete bibliographic citation for each reference is found immediately preceding the claims. The contents of each publication so referenced, and the publications referenced within the specification are hereby incorporated into the present disclosure to more fully describe the state of the art to which this invention pertains.

[0004] Approximately 7% of inpatients will develop a healthcare-associated infection (HAI), approximately half of which are comprised of pneumonia, bloodstream infection, or wound infections, which are likely the most amenable to immunological intervention (W.H. Organization). On any given day, approximately 3% of hospital inpatients have an HAI (Magill, S.S. et al. (2014), N Engl J Med 370, 1198-1208)). At acute-care hospitals in the US, this results in 722,000 HAIs, more than 75,000 deaths, and a financial burden exceeding $100 billion, every year (Marchetti, A. et al. (2013), J Med Econ 16, 1399-1404). In most cases, HAIs are caused by antimicrobial-resistant bacterial and fungal pathogens, which are associated with worse mortality and morbidity than antimicrobial-susceptible pathogens (Weiner, L.M. et al. (2016), Infect Control Hosp Epidemiol 37, 1288-1301). Despite the high incidence of HAIs, there are no FDA-approved vaccines against the most commonly encountered and antibiotic-resistant pathogens, like methicillin-resistant Staphylococcus aureus (MRS A), carbapenem-resistant Acinetobacter baumannii (CRAB), and carbapenem-resistant Enterobacterales (CRE).

[0005] Broadly speaking, there are two approaches to preventing infections: horizontal and vertical (Wenzel, R.P. et al. (2010), Int J Infect Dis 14 Suppl 4, S3-5). Horizontal approaches protect against a wide range of pathogens; examples include disinfecting hospital rooms with ultraviolet light or bleach, hand washing, and personal protective equipment (PPE). Horizontal approaches provide protection against a broad spectrum of infectious organisms and offer the best overall value, although the implementation and maintenance of these approaches are generally challenging. By comparison, vertical approaches provide specific protection against only one organism. Vertical approaches are thus much narrower in focus; examples include active surveillance and testing of specific pathogenic species, decolonizing hospitalized patients with MRSA, and vaccinating against certain pathogens (e.g., pneumococcus, influenza). While offering a powerful way to prevent infections through long-term adaptive immunity, traditional vaccines only protect against one specific pathogen. Moreover, the development of vaccines is costly, timeconsuming, and scientifically challenging. Although the merits of both approaches are often debated, horizontal approaches have been shown to have a broader impact at lower costs (Septimus, R.A. et al. (2014), Infect Control Hosp Epidemiol 35, 797-801).Nevertheless, a strategy that seeks to integrate both approaches will be most successful in preventing a greater disease burden than either approach alone.SUMMARY OF THE DISCLOSURE

[0006] This disclosure provides an entirely new vaccine-derived approach, based on adjuvants without a protein or polypeptide antigen, to mediate broad spectrum short-to- intermediate term protection against deadly, HAIs caused by bacteria (including antibioticresistant bacteria) and fungi. This approached is based on entirely new horizontal rather than vertical infection prevention strategy. In one aspect, the vaccine or composition does not contain, comprise, consist essentially of, or consist of a substance such as an antigen, a protein or a polypeptide that induces or raises lymphocyte-mediated (T cell or B cell) immunity, e.g., adaptive immunity. In several embodiments, the antigen can be a protein or polypeptide derived from a virus, a small molecule, fungus or bacteria, that can induce or raise a T cell and / or B cell adaptive immunity. In one aspect, a small molecule is excluded from the term “antigen.”

[0007] Without being bound by theory, this disclosure utilizes a horizontal infection prevention strategy by immunizing with broadly-active adjuvants to provide innate immuneprotection against the Gram-positive and Gram-negative bacterial pathogens and fungal pathogens.

[0008] In one aspect, Applicant discloses herein a protein-free vaccine, composed of, comprising, consisting essentially of, or consisting of: aluminum hydroxide (AHO), monophosphoryl lipid A and D-mannose, to stimulate the innate immune system and confer protection against several common nosocomial pathogens including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis, and Acinetobacter baumannii and fungi such as Candida albicans and mucormycosisin one embodiment, the composition does comprise whole glucan particles (WGP) but does not comprise, consist of, or consist essentially of mannan and / or whole glucan particles (WGP), and does not comprise an antigen effective to induce an immune response against any pathogen, e.g., a virus, a small molecule, a fungal pathogen, or a bacterial pathogen. In one embodiment, the combination does not comprise, consist of, or consist essentially of mannan and / or whole glucan particles (WGP) and does not comprise an antigen effective to induce an immune response against any pathogen, e.g., a virus, a small molecule, a fungal pathogen, or a bacterial pathogen. In one aspect, a small molecule is excluded from the term “antigen.”

[0009] In another embodiment, the combination does not comprise, consist of, or consist essentially of whole glucan particles (WGP) and / or mannan and / or and AHO and does not comprise an antigen effective to induce an immune response against any pathogen, e.g., a virus, a small molecule, a fungal pathogen, or a bacterial pathogen. In one aspect, a small molecule is excluded from the term “antigen.”

[0010] In a further aspect, further provided is a composition comprising, consisting essentially of, or consisting of: aluminum hydroxide (AHO), monophosphoryl lipid A, D- mannose, and whole glucan particles (WGP) to stimulate the innate immune system and confer protection against several common nosocomial pathogens including methicillin- resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis, and Acinetobacter baumannii. In one embodiment, the combination does not comprise, consist of, or consist essentially of an antigen effective to induce an immune response against any pathogen, e.g., a virus, a small molecule, a fungal pathogen, or a bacterial pathogen and / or mannan. In one aspect, a small molecule is excluded from the term “antigen.”

[0011] Without being bound by theory, vaccine efficacy is dependent upon monocytes and macrophages and can be caused epigenetic remodeling in those cells. In another aspect, MPL and D-mannose are provided in IX, 3X or 10X concentration. In one aspect, the concentration is 3X for protection against Staphylococcus and 10X for protection against Acinetobacter .

[0012] Thus, in one aspect, provided herein is a composition comprising, or consisting essentially of, or yet further consisting of an effective amount of each of: MPL monophosphoryl lipid (MPL), D-mannose, and aluminum hydroxide (“AHO”) (“collectively AMMO”), with the proviso that the composition does not comprise an antigen effective to induce a B-cell or T-cell memory immune response against any pathogen, e.g., a virus, a small molecule, a fungal pathogen, or a bacterial pathogen and / or mannan. In one aspect, a small molecule is excluded from the term “antigen.”

[0013] In another aspect, provided herein is a composition consisting essentially of an effective amount of each of: MPL mono-phosphoryl lipid (MPL), D-mannose, and aluminum hydroxide (collectively “AMMO”), with the proviso that the composition does not comprise an antigen effective to induce an immune response against any pathogen, e.g., a virus, a small molecule, a fungal pathogen or a bacterial pathogen. Yet further provided is a composition consisting of as active immune inducing agents, an effective amount of each of: MPL mono-phosphoryl lipid (MPL), D-mannose, and aluminum hydroxide (collectively “AMMO”). In a yet further aspect, the effective amount of the aluminum hydroxide, MPL mono-phosphoryl lipid (MPL), and D-mannose in combination, is effective to induce an immune response when administered to a subject in need thereof, wherein in one embodiment, the immune response is neither a T cell nor B cell adaptive immunity. In a further aspect of each of the embodiments, the combination or composition does not comprise, contain, or consist essentially of mannan and / or whole glucan particles (WGP) and does not comprise an antigen effective to induce an immune response against any pathogen, e.g., a virus, a small molecule, a fungal pathogen, or a bacterial pathogen.

[0014] Thus, in another aspect, provided herein is a composition comprising, or consisting essentially of, or yet further consisting of an effective amount of each of: MPL monophosphoryl lipid (MPL) and D-mannose (collectively “MMD”) with the proviso that the composition does not comprise an antigen effective to induce a B-cell or T-cell memory immune response against any pathogen, e.g., a virus, a small molecule, a fungal pathogen,or a bacterial pathogen and / or AHO and / or mannan and / or WGP. In another aspect, provided herein is a composition consisting essentially of an effective amount of each of: MPL mono-phosphoryl lipid (MPL) and D-mannose, with the proviso that the composition does not comprise an antigen effective to induce an immune response against any pathogen, e.g., a viral, a small molecule, a fungal or a bacterial pathogen and / or an effective amount of AHO and WGP. Yet further provided is a composition consisting of as active immune inducing agents, an effective amount of each of: MPL mono-phosphoryl lipid (MPL) and D- mannose. In a further aspect of each of the embodiments, the combination or composition does not comprise mannan and / or whole glucan particles (WGP) and AHO and / or does not comprise an antigen effective to induce an immune response against any pathogen, e.g., a virus, a small molecule, a fungal pathogen, or a bacterial pathogen.

[0015] Applicant unexpectedly found that the triple combination “MMA” or AMMO provided ease of manufacturing and enhanced composition purity over prior art compositions. As shown in FIG. 1, the AMMO composition resulted in at least equivalent protection against S. aureus bacteremia than a combination of triple adjuvants comprised of aluminum hydroxide, MPL, and mannan.

[0016] For the purpose of this disclosure, the aluminum hydroxide is Al(OH)s typically found in nature as the mineral Gibbsite. It is commercially available and can be an aluminum hydroxide gel or wet suspension, optionally commercially available Alhydrogel® (Sigma-Aldrich) or Al(OH)s gel (Accurate Chemical & Scientific Corporation Cat. # A1090S or Al(OH)s gel, Croda Cat. #AJV3012). The percentage of Al(OH)s in the source material can be any percentage, non-limiting examples of such include a 2% or 1% composition and combinations thereof.

[0017] In one embodiment the components of the AMMO are combined in total volumes of 0.5 to 1 ml liquid suspension, containing 0.2 to 1 mg of aluminum hydroxide (AHO); from about 0.01 to 1.5 mg of MPL; and from about 0.1 to 5 mg of D-mannose.

[0018] In a further aspect of the above compositions, the effective amount comprises from about 0.1 mg / ml to about 10 mg / ml of MPL and from about 0.01 mg / ml to about 10 mg / ml of D-mannose.

[0019] As is apparent to the skilled artisan, the compositions can be combined with a pharmaceutically acceptable carrier, such as phosphate buffered saline (PBS) and DMSO,for ease of storage and for administration. The compositions can further contain stabilizers and components for example, maintaining stability during storage or lyophilization.

[0020] This disclosure also provides a method to enhance immunity in a subject against an infection caused by a pathogen, e.g., a bacterial or fungal pathogen, by a method comprising, or consisting essentially of, or yet further consisting of administering to the subject an effective amount of a composition (e.g., AMMO or MMD, with or without WGP) as described herein. This disclosure also provides compositions or medicaments for use to enhance immunity in a subject against an infection caused by a pathogen, e.g., bacterial or fungal pathogen, the medicament comprising, or consisting essentially of, or yet further consisting of a composition (e.g., AMMO or MMD, with or without WGP) as described herein. To enhance immunity intends a result of protection from disease or symptoms and can be monitored clinically (e.g., fever, cough or pain) or sub-clinically (enhanced innate immunity as measure by, for example, the presence or increased presence of macrophages and / or monocytes, lowered bacterial or fungal burden, lower proinflammatory cytokines, e.g., (IL-6, IL-12, TNF) and elevated IL-10 / TNF).

[0021] Further provided are methods to treat or prevent a bacterial or a fungal infection or a disorder caused by a bacterial or a fungal infection in a subject in need thereof, the methods comprising, or alternatively consisting essentially of, or yet further consisting of, administering an effective amount of the composition (e.g., AMMO or MMD) of this disclosure. Also provided are compositions for use or medicaments as described herein to treat or prevent a bacterial or a fungal infection or a disorder caused by a bacterial or fungal infection in a subject in need thereof.

[0022] Non-limiting examples of bacterium causing infections are selected from S. aureus, A. baumannii, K. Pneumoniae, P. aeruginosa, E. coli, Enterobacter spp., Serratia, Stenotrophomonas, and the fungus can be Candida spp or species that cause the disease mucormycosis (e.g., Rhizopus spp).

[0023] Any appropriate method of administration can be used. Non-limiting examples include a method comprising topical, inhalation, intramuscular, subcutaneous, or intravenous administration as determined by the treating physician. The composition can be administered once, twice, or three times over the period of one to three months or more.

[0024] In one aspect, the subject is infected with the bacterial or fungal microorganism. Alternatively, the subject is at risk of the bacterial or fungal infection.

[0025] The therapeutic methods, uses and medicaments can be further combined with appropriate diagnostics to assaying the subject for a bacterial or fungal infection prior to, during, and / or administration of the composition.

[0026] Subjects to be treated include mammals, e.g., rats, mice, canines, felines, humans and the like. When practiced in non-human animal, the method provides an appropriate animal model for testing against new disease pathogens or combination therapies. When practiced in a human subject, the method is a viable therapeutic and / or vaccine modality.

[0027] Further provided are kits comprising the composition of as described herein and instructions for use.

[0028] Yet further provided are method to identify an compound or agent that provides a benefit selected from one or more of: enhances immunity against a bacterial or fungal microbial infection or treats a bacterial or fungal infection or a disease related to a bacterial or fungal microbial infection, the method comprising admixing the compound or agent with the composition as disclosed herein and administering the admixed composition to a non- human subject infected with a bacterial or fungal microorganism and assaying for postadministration infection or survival, wherein the compound or agent that enhances the activity of the composition of this disclosure. Methods determine if a benefit is provided are known in the art and briefly described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1 : shows that replacing fungal mannan with D-mannose retains protection against S. aureus bacteremia. Balb / c mice (n = 8 mice / group) were vaccinated with aluminum hydroxide + monophosphoryl lipid (MPL) + mannan (“AMMA”) or aluminum hydroxide + MPL + D-mannose (“AMMO” or AM+D-mannose or “MMA”) or PBS control. Mice were infected IV with S. aureus LAC (MRSA) 3 days later. *p < 0.05 vs. PBS. D-mannose was purchased from SIGMA (Cat. No. M6020-25G, https: / / www.sigmaaldrich.com / US / en / product / sigma / m6020, last accessed on June 12, 2024.)

[0030] FIG. 2: AMMO with D-mannose is at least as effective as AMMA with mannan against S. aureus. Balb / C mice were treated at day -3 and infected IV with S. aureus LAC. AM = aluminum hydroxide + MPL. N = 16 mice per group from 2 experiments for PBS and AMMO. N = 8 mice per group for 1 experiment each for D-mannose groups. *p < 0.05 vs. PBS.[0031 J FIGS. 3A-3B: AMMO with D-mannose is at least as effective as AMMA with mannan against Gram negative pathogens. C3H mice were treated at day -3 and infected IV with K. pneumoniaeKPCKPl (FIG. 3A) or infected at day -3 or -1 via oropharyngeal aspiration pneumonia with . baumannii HUMCI (FIG. 3B). AMMA = aluminum hydroxide + MPL + mannan. AM = aluminum hydroxide + MPL. N = 5 mice per group for K. pneumoniae. For A. baumannii, N = 10 mice per group from 2 experiments for PBS, AMMO day -3, AM+100 pg, and 5 mice per group from 1 experiment for AM+30 or 300 pg, and 4 mice for AMMO day -1. *p < 0.05 vs. PBS. Applicant also tested D-mannose against the Gram negative pathogens, pan-drug resistant Klebsiella pneumoniae during IV infection, and A. baumannii during lung infection. D-mannose containing AMMO was again at least as effective as mannan containing AMMA.

[0032] FIG. 4: AMMO with D-mannose is at least as effective as AMMA with mannan against mucormycosis in neutropenic mice. Balb / c mice (n = 5 / group) were made neutropenic with cyclophosphamide, treated with AMMO (D-mannose) or AMMA (mannan), and infected IV 3 days later with Rhizopus delamar. AMMO and AMMA had very similar efficacy, both of which resulted in markedly superior survival compared to negative control. *p < 0.05 vs. PBS. Applicant also made mice neutropenic with cyclophosphamide and treated with AMMA or AMMO and infected with R. oryzae. AMMO was as effective as AMMA against mucormycosis in the neutropenic mice.

[0033] FIG. 5: AMMO stimulated macrophage phagocytosis of killed A aureus from a commercial kit. RAW cell macrophages were stimulated for 24 h with IFNy (positive control), or various concentrations of AMMA (Al(OH)3+MPL+mannan), or AMMO (A1(OH)3+MPL+ D-mannose (mannose)). A1(OH)3 = 5 pg for all conditions; lx = 0.05 pg MPL + 0.5 pg mannan or D-mannose. P > 0.05 vs. PBS.DETAILED DESCRIPTION

[0034] Before the compositions and methods are described, it is to be understood that the invention is not limited to the particular methodologies, protocols, cell lines, assays, and reagents described, as these may vary. It is also to be understood that the terminology used herein is intended to describe particular embodiments of the present invention, and is in no way intended to limit the scope of the present invention as set forth in the appended claims.

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which thisinvention 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, the preferred methods, devices, and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0035] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1 : A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotid Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; and Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London).

[0036] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 0.1. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term “about”. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0037] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 0.1. It is to be understood, although not always explicitly stated that allnumerical designations are preceded by the term “about”. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.Definitions

[0038] As used in the specification and claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.

[0039] As used herein, the term “comprising” or “comprises” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention or process steps to produce a composition or achieve an intended result. Embodiments defined by each of these transition terms are within the scope of this invention.

[0040] The term “isolated” as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule. The term “isolated peptide fragment” is meant to include peptide fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polypeptides and proteins that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. In other embodiments, the term “isolated” means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature. For example, an isolated cell is a cell that is separated form tissue or cells of dissimilar phenotype or genotype. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart.

[0041] A “composition” is intended to mean a combination of the claimed elements and another compound or composition, inert (e.g. a detectable label) or active (with the exclusion of an antigen) alone or in combination with a carrier which can in one embodiment be a simple carrier like saline or pharmaceutically acceptable or a solid support as defined below.

[0042] A “pharmaceutical composition” is intended to include the combination of the claimed elements with a carrier, inert or active (with the exclusion of an antigen), making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.|0043] As used herein, the term “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil / water or water / oil emulsion, DMSO, and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin (1975) Remington’s Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton).

[0044] “Administration” can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the infection being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and the infecting bacteria or organism. Non-limiting examples of route of administration include oral administration, nasal administration, injection, and topical application.

[0045] The term “effective amount” refers to a quantity sufficient to achieve a desired effect. In the context of therapeutic or prophylactic applications, the effective amount will depend on the type and severity of the condition at issue and the characteristics of the individual subject, such as general health, age, sex, body weight, and tolerance to pharmaceutical compositions.

[0046] In the case of an in vitro application, in some embodiments the effective amount will depend on the size and nature of the application in question. It will also depend on the nature and sensitivity of the in vitro target and the methods in use. The skilled artisan will be able to determine the effective amount based on these and other considerations. The effective amount may comprise one or more administrations of a composition depending on the embodiment.

[0047] The routes of administration applicable to the methods of the invention include intravenous, intranasal, intramuscular, urethrally, intratracheal, subcutaneous, intradermal, topical application, rectal, nasal, oral, inhalation, and other enteral and parenteral routes of administration. Routes of administration may be combined, if desired, or adjusted depending upon the agent and / or the desired effect. An active agent can be administered in a single dose or in multiple doses. Embodiments of these methods and routes suitable for delivery, include systemic or localized routes.

[0048] Parenteral routes of administration other than inhalation administration include, but are not limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, and intravenous routes, i.e., any route of administration other than through the alimentary canal. Parenteral administration can be conducted to effect systemic or local delivery of the inhibiting agent. Where systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations.

[0049] The term “suffering” as it related to the term “treatment” refers to a subject or patient or individual who has been diagnosed with or is predisposed to a disease or infection. A patient may also be referred to being “at risk of suffering” from a disease or infection. This patient has not yet developed characteristic disease pathology or an active infection, however are known to be predisposed to the disease due to family history, being genetically predispose to developing the disease, being in an environment that puts the subject at substantial risk of being infected, or diagnosed with a disease or disorder that predisposes them to developing the disease to be treated.

[0050] Aluminum hydroxide is commercially available as Alhydrogel® (Accurate Chemical and Scientific Corporation, Catalogue # A1090S) wet gel colloidal suspension. The InvivoGen catalog (invivogen.com / PDF / Alhydrogel_TDS.pdfd, last accessed on October 23, 2017) describes Alhydrogel ® adjuvant as an aluminium hydroxide wet gelsuspension. Alhydrogel ® particles have a net positive electrical charge at pH 5-7.Alhydrogel ® adjuvant 2% is made by Brenntag Biosector, a leader in the global vaccine adjuvants market with a long history of producing high quality products. Alhydrogel ® adjuvant 2% was elected as the International Standard Preparation for aluminium hydroxide gels. Alhydrogel ® adjuvant 2% is present in multiple commercial vaccine formulations.

[0051] Whole glucan particles (WGP) intend particulate formulations of fungal glucan. It is a commercially available (InVivoGen Catalogue # tlrl-wgps) powder resuspended in water / saline. This has been used in the past as a vaccine adjuvant.

[0052] Mono-phosphoryl lipid (MPL) or lipid A intends a lipid component of an endotoxin held responsible for the toxicity of gram-negative bacteria. It has the chemical structure of:

[0053] It is commercially available from InVivoGen (Catalog # tlrl-mpls) and Avanti Polar Lipids (catalogue # 699800P). It has been used as a vaccine adjuvant.

[0054] As used herein, the term “mannan” intends polysaccharide found in fungi that is comprised of a mannose polymer (Sigma-Aldrich, catalogue number M7504, last accessed on January 3, 2023 or Sigma Cat. #M3460-IG, or from MedicaPharma catalogued as “mannan”, or from Newgreen Health Industry, catalogues as “mannan oligosaccharides”).

[0055] As used herein, the term “D-mannose” intends a sugar monomer of aldohexose series of carbohydrates. It is the C-2 epimer of glucose and has the chemical structure of:

[0056] As used herein, the term “antigen that induces an immune response against a bacterial or fungal pathogen” intends for example, conventional vaccine preparations used prophylactically and for treatment of infections and diseases associated with these infections and in one embodiment, intends only those which induce an immune response mediated by T and / or B lymphocytes against a pathogen, such as a viral, a bacterial or a fungal infection.

[0057] As used herein, the term “antigen” intends any substance that causes the body to make an adaptive immune response by T and / or B lymphocytes against that substance. Nonlimiting examples of such antigens include toxins, chemicals, bacteria, viruses, or other substances that come from outside the body. Body tissues and cells, including cancer cells, also have antigens on them that can cause an immune response. These antigens can also be used as markers in laboratory tests to identify those tissues or cells. In one aspect, the term “antigen” as claimed herein intends only those which induce an immune response mediated by T and / or B lymphocytes against a pathogen, such as a viral, a small molecule, a bacterial or a fungal infection.

[0058] A “subject,” “individual” or “patient” is used interchangeably herein, and refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, rats, rabbits, simians, bovines, ovines, porcines, canines, felines, farm animals, sport animals, pets, equines, and primates, particularly humans.

[0059] To induce an immune response intends to raise acrophage activation of phagocytosis of microbes while switching from a pro-inflammatory cytokine output to a net antiinflammatory output. The compositions and methods provide the benefit of enhanced clearance of microbes without the harm of systemic out of control inflammation / sepsis.

[0060] Acronyms: AM = aluminum hydroxide + MPL; AMMO or MMA or AM+D- mannose = mono-phosphoryl lipid (MPL), D-mannose, and aluminum hydroxide (“AHO”); AMMA = aluminum hydroxide + mono-phosphoryl lipid (MPL) + mannan.

[0061] RAW cells are Mus musculus macrophages isolated from a mouse with Abelson murine leukemia virus-induced tumor. They are commercially available from the American Type Culture Collection under Product No. ATCC TIB-71 (aka RAW 264.7), https: / / www.atcc.org / , last accessed on May 29, 2025.

[0062] Klebsiella pneumoniae KPCKP1 and S. aureus strain LAC are clinical isolates. Similar strains are commercially available from CDC & FDA Antimicrobial Resistance Isolate Bank, K. pneumoniae AR Bank #0112 and CDC & FDA Antimicrobial Resistance Isolate Bank, S. aureus AR Bank #0215. See https: / / wwwn.cdc.gov / ARIsolateBank / , last accessed on May 29, 2025.Modes for Carrying Out the Disclosure

[0063] Quite unexpectedly, Applicant shows a solution to a technical problem solved by switching to D-mannose from mannan as disclosed previously. To the best of Applicant’s knowledge, mannan is not available in GMP format and a protocol to produce GMP mannan does not exist. Applicant unsuccessfully attempted to produce GMP grade mannan but the cost was prohibitively expensive due to technical complexities. Mannan is a highly variable substance prepared via chemical extraction, and the range of molecular weight, carbohydrate branching, and D-mannose fraction of each mannan prep was difficult to reproduce and create analytics around. D-mannose is the monosaccharide that comprises mannan, but prior to this work, it was unknown that D-mannose is the immunologically active component of mannan for this use. Applicant had theorized that the complex structure of mannan would be required to stimulate the proper immune response. However, quite unexpectedly, D-mannose is as effective at mannan and does not have the manufacturing and quality control issues that use of mannan presents.Compositions

[0064] AMMO

[0065] This disclosure provides a composition comprising, or alternatively consisting essentially of, or yet further consisting of, an effective amount of each of: an aluminum hydroxide, a mono-phosphoryl lipid (MPL), and D-mannose (the combination referred toherein as “AMMO” or MMA” or “AM+D), with the proviso that the composition does not comprise an antigen effective to induce an immune response against a pathogen, e.g., a viral, a small molecule, fungal or bacterial pathogen, optionally excluding a small molecule as the antigen. In one aspect, the “antigen” as claimed herein intends only those which induce an immune response mediated by T and / or B lymphocytes against a pathogen, such as a viral, a small molecule, a bacterial or a fungal infection, optionally excluding a small molecule as the antigen. Acronyms: AM = aluminum hydroxide + MPL; AMMO or MMA or AM+D-mannose: mono-phosphoryl lipid (MPL), D-mannose, and aluminum hydroxide (“AHO”)AMMA: aluminum hydroxide + mono-phosphoryl lipid (MPL) + mannan.

[0066] In one aspect, the components are combined to achieve a final concentration. For example, the compositions have an combined concentration in a range from about 0.1 mg / ml to about 20 mg / ml, or alternatively from about 0.5 mg / ml to about 20 mg / ml, or alternatively from about 1.0 mg / ml to about 20 mg / ml, or alternatively from about 0.1 mg / ml to about 15 mg / ml, or alternatively from about 0.1 mg / ml to about 12 mg / ml, or alternatively from about 0.1 mg / ml to about 10 mg / ml, or alternatively from about 0.1 mg / ml to about 5 mg / ml, or alternatively from about 0.1 mg / ml to about 3 mg / ml, or alternatively from 0.1 mg / ml to about 2 mg / ml, or alternatively from about 200 pg / ml to about 20 mg / ml, or alternatively from about 0.1 mg / ml to about 15 mg / ml, or alternatively from about 0.1 mg / ml to about 13 mg / ml, or alternatively from about 0.5 mg / ml to about 13 mg / ml, or alternatively from about 0.5 mg / ml to about 12 mg / ml, or about 0.5 mg / ml, or about 1.0 mg / ml, or about 1.5 mg / ml, or about or about 1.5 mg / ml, or about 2.0 mg / ml, or about 3.5 mg / ml, or about 4.0 mg / ml, or about 8.0 mg / ml, or about 10 mg / ml, or about 12.0 mg / ml, or about 13 mg / ml, or about 15 mg / ml, or about 15.5 mg / ml, or about 6.0 mg / ml, or about 6.5 mg / ml, or about 7.0 mg / ml, or about 7.5 mg / ml, or about 8.0 mg / ml, or about 8.5 mg / ml, or about 9.0 mg / ml, or about 9.5 mg / ml or about 10 mg / ml.

[0067] The AMMO is suspended in a carrier such as a pharmaceutically acceptable carrier such as PBS, to a concentration in a range from about 0.1 mg / ml to about 20 mg / ml, or alternatively from about 0.5 mg / ml to about 20 mg / ml, or alternatively from about 1.0 mg / ml to about 20 mg / ml, or alternatively from about 0.1 mg / ml to about 15 mg / ml, or alternatively from about 0.1 mg / ml to about 12 mg / ml, or alternatively from about 0.1 mg / ml to about 10 mg / ml, or alternatively from about 0.1 mg / ml to about 5 mg / ml, or alternatively from about 0.1 mg / ml to about 3 mg / ml, or alternatively from 0.1 mg / ml to about 2 mg / ml, or alternatively from about 200 pg / ml to about 20 mg / ml, or alternativelyfrom about 0.1 mg / ml to about 15 mg / ml, or alternatively from about 0.1 mg / ml to about 13 mg / ml, or alternatively from about 0.5 mg / ml to about 13 mg / ml, or alternatively from about 0.5 mg / ml to about 12 mg / ml, or about 0.5 mg / ml, or about 1.0 mg / ml, or about 1.5 mg / ml, or about or about 1.5 mg / ml, or about 2.0 mg / ml, or about 3.5 mg / ml, or about 4.0 mg / ml, or about 8.0 mg / ml, or about 10 mg / ml, or about 12.0 mg / ml, or about 13 mg / ml, or about 15 mg / ml, or about 15.5 mg / ml, or about 6.0 mg / ml, or about 6.5 mg / ml, or about 7.0 mg / ml, or about 7.5 mg / ml, or about 8.0 mg / ml, or about 8.5 mg / ml, or about 9.0 mg / ml, or about 9.5 mg / ml or about 10 mg / ml.

[0068] The components of the AMMO are combined in any appropriate combination and it is not intended that the ratios of each component be identical, although they can be combined in a 1 : 1 : 1 (D-mannose:MPL:aluminum hydroxide) ratio. Alternatively, the AMMO in a ratio of about 0.5:0.05: 1; about 1.5:0.15: 1; about 1 :0.1 :1, about 3:0.3: 1, about 5:0.5: 1; about 10:3: 1. Yet further, the components can be combined in the following ratios: about lx : about lx : about lx; or about lx : about 3x : about 3x; or about lx : about lOx : about lOx; or about; lx : about 30x : about 30x; or lx : lx : lx; or lx : 3x : 3x; or lx : lOx : lOx; or lx : 30x : 30x, wherein lx Aluminum = 0.5 mg; and lx MPL = 0.05 mg and lx D- Mannose = 0.5 mg in particular for the treatment or prevention of human subjects.

[0069] In some aspects, the AMMO is combined with a carrier such as a pharmaceutically acceptable carrier such as saline, to provide a concentration from about 0.1 mg / ml to about 10 mg / ml, or alternatively from about 0.5 mg / ml to about 10 mg / ml, or alternatively from about 1.0 mg / ml to about 10 mg / ml, or alternatively from about 0.1 mg / ml to about 9 mg / ml, or alternatively from about 0.1 mg / ml to about 7 mg / ml, or alternatively from about 0.1 mg / ml to about 5 mg / mlO, or alternatively from about 0.1 mg / ml to about 3 mg / ml, or alternatively from about 0.1 mg / ml to about 2 mg / ml, or alternatively from about 0.1 mg / ml to about 1 mg / ml, or alternatively from about 200 pg / ml to about 8 mg / ml, or alternatively from about 0.1 mg / ml to about 5 mg / ml, or alternatively from about 0.1 mg / ml to about 3 mg / ml, or alternatively from about 0.5 mg / ml to about 3 mg / ml, or alternatively from about 0.5 mg / ml to about 2 mg / ml, or about 0.5 mg / ml, or about 1.0 mg / ml, or about 1.5 mg / ml, or about or about 0.5 mg / ml, or about 1.0 mg / ml, or about 1.5 mg / ml, or about 2.0 mg / ml, or about 3.0 mg / ml, or about 3.5 mg / ml, or about 4.0 mg / ml, or about 4.5 mg / ml, or about 5 mg / ml, or about 5.5 mg / ml, or about 6.0 mg / ml, or about 6.5 mg / ml, or about 7.0 mg / ml, or about 7.5 mg / ml, or about 8.0 mg / ml, or about 8.5 mg / ml, or about 9.0 mg / ml, or about 9.5 mg / ml or about 10 mg / ml.

[0070] The components of the AMMO can be sourced from commercial vendors.

[0071] The compositions can be formulated for in vivo administration (in one or more doses) to administer from about 5 mcg / kg body weight to about 50 mcg / kg body weight, or alternatively 10 mcg / kg body weight to 50 mcg / kg body, or alternatively 20 mcg / kg body weight to about 50 mcg / kg body weight, or alternatively 10 mg / kg body weight to about 100 mcg / kg body weight, or alternatively from about 15 mcg / kg body weight to about 150 mcg / kg body weight, or alternatively from about 20 to mcg / kg body weight to about 200 mcg / kg body weight.|0072] The compositions can be further formulated for storage and distribution such as by lyophilization or freeze-drying. In addition, preservative and stabilizing agents can be added to further enhance the shelf-life of the compositions.

[0073] MMD

[0074] This disclosure provides a composition comprising, or alternatively consisting essentially of, or yet further consisting of, an effective amount of each of: a monophosphoryl lipid (MPL or MPLA), and D-mannose (MMD), with the proviso that the composition does not comprise an antigen effective to induce an immune response against a pathogen, e.g., a virus, a small molecule, a fungal or a bacterial pathogen and / or WGP and AHO. In one aspect, the “antigen” as claimed herein intends only those which induce an immune response mediated by T and / or B lymphocytes against a pathogen, such as a virus, a bacterial or a fungal infection.

[0075] The MMD can be combined with a carrier, such as a pharmaceutically acceptable carrier.

[0076] In one aspect, the components are combined to achieve a final concentration. For example, the compositions have an combined concentration in a range from about 0.1 mg / ml to about 20 mg / ml, or alternatively from about 0.5 mg / ml to about 20 mg / ml, or alternatively from about 1.0 mg / ml to about 20 mg / ml, or alternatively from about 0.1 mg / ml to about 15 mg / ml, or alternatively from about 0.1 mg / ml to about 12 mg / ml, or alternatively from about 0.1 mg / ml to about 10 mg / ml, or alternatively from about 0.1 mg / ml to about 5 mg / ml, or alternatively from about 0.1 mg / ml to about 3 mg / ml, or alternatively from 0.1 mg / ml to about 2 mg / ml, or alternatively from about 200 pg / ml to about 20 mg / ml, or alternatively from about 0.1 mg / ml to about 15 mg / ml, or alternatively from about 0.1 mg / ml to about 13 mg / ml, or alternatively from about 0.5 mg / ml to about 13mg / ml, or alternatively from about 0.5 mg / ml to about 12 mg / ml, or about 0.5 mg / ml, or about 1.0 mg / ml, or about 1.5 mg / ml, or about or about 1.5 mg / ml, or about 2.0 mg / ml, or about 3.5 mg / ml, or about 4.0 mg / ml, or about 8.0 mg / ml, or about 10 mg / ml, or about 12.0 mg / ml, or about 13 mg / ml, or about 15 mg / ml, or about 15.5 mg / ml, or about 6.0 mg / ml, or about 6.5 mg / ml, or about 7.0 mg / ml, or about 7.5 mg / ml, or about 8.0 mg / ml, or about 8.5 mg / ml, or about 9.0 mg / ml, or about 9.5 mg / ml or about 10 mg / ml.

[0077] The MMD is suspended in a carrier such as a pharmaceutically acceptable carrier such as PBS, to a concentration in a range from about 0.1 mg / ml to about 20 mg / ml, or alternatively from about 0.5 mg / ml to about 20 mg / ml, or alternatively from about 1.0 mg / ml to about 20 mg / ml, or alternatively from about 0.1 mg / ml to about 15 mg / ml, or alternatively from about 0.1 mg / ml to about 12 mg / ml, or alternatively from about 0.1 mg / ml to about 10 mg / ml, or alternatively from about 0.1 mg / ml to about 5 mg / ml, or alternatively from about 0.1 mg / ml to about 3 mg / ml, or alternatively from 0.1 mg / ml to about 2 mg / ml, or alternatively from about 200 pg / ml to about 20 mg / ml, or alternatively from about 0.1 mg / ml to about 15 mg / ml, or alternatively from about 0.1 mg / ml to about 13 mg / ml, or alternatively from about 0.5 mg / ml to about 13 mg / ml, or alternatively from about 0.5 mg / ml to about 12 mg / ml, or about 0.5 mg / ml, or about 1.0 mg / ml, or about 1.5 mg / ml, or about or about 1.5 mg / ml, or about 2.0 mg / ml, or about 3.5 mg / ml, or about 4.0 mg / ml, or about 8.0 mg / ml, or about 10 mg / ml, or about 12.0 mg / ml, or about 13 mg / ml, or about 15 mg / ml, or about 15.5 mg / ml, or about 6.0 mg / ml, or about 6.5 mg / ml, or about 7.0 mg / ml, or about 7.5 mg / ml, or about 8.0 mg / ml, or about 8.5 mg / ml, or about 9.0 mg / ml, or about 9.5 mg / ml or about 10 mg / ml.

[0078] The components of the MMD are combined in any appropriate combination and it is not intended that the ratios of each component be identical, although they can be combined in a 0.1 : 1 (D-mannose:MPL) ratio. Alternatively, the MA in a ratio of about 0.3:1; about 0.5: 1; about 1 : 1, about 3: 1, about 5: 1; about 10: 1.

[0079] In another aspect, any of the above compositions further comprises a carrier. The carrier can be a solid phase carrier, a gel, an aqueous liquid carrier, a paste, a liposome, a micelle, albumin, polyethylene glycol, a pharmaceutically acceptable polymer, or a pharmaceutically acceptable carrier, such a phosphate buffered saline.

[0080] The compositions of the disclosure can be manufactured by methods well known in the art such as conventional granulating, mixing, dissolving, encapsulating, lyophilizing, oremulsifying processes, among others. Compositions may be produced in various forms, including granules, precipitates, or particulates, powders, including freeze dried, rotary dried or spray dried powders, amorphous powders, injections, emulsions, elixirs, suspensions or solutions. Compositions may optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers and combinations of these.

[0081] Compositions may be prepared as liquid suspensions or solutions using a sterile liquid, such as oil, water, alcohol, and combinations thereof. Pharmaceutically suitable surfactants, suspending agents or emulsifying agents, may be added for oral or parenteral administration. Suspensions may include oils, such as peanut oil, sesame oil, cottonseed oil, corn oil and olive oil. Suspension preparation may also contain esters of fatty acids, such as ethyl oleate, isopropyl myristate, fatty acid glycerides and acetylated fatty acid glycerides. Suspension compositions may include alcohols, such as ethanol, isopropyl alcohol, hexadecyl alcohol, glycerol and propylene glycol. Ethers, such as poly(ethyleneglycol), petroleum hydrocarbons, such as mineral oil and petrolatum, and water may also be used in suspension compositions.

[0082] The compositions of this disclosure are formulated for pharmaceutical administration to a mammal, preferably a human being. Such compositions of the disclosure may be administered in a variety of ways, preferably topically or by injection.

[0083] Sterile injectable forms of the compositions of this disclosure may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long- chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosageforms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation. Compounds may be formulated for parenteral administration by injection such as by bolus injection or continuous infusion. A unit dosage form for injection may be in ampoules or in multi-dose containers.

[0084] In addition to dosage forms described above, pharmaceutically acceptable excipients and carriers and dosage forms are generally known to those skilled in the art and are included in the disclosure. It should be understood that a specific dosage and treatment regimen for any particular subject will depend upon a variety of factors, including the activity of the specific antidote employed, the age, body weight, general health, sex and diet, renal and hepatic function of the subject, and the time of administration, rate of excretion, drug combination, judgment of the treating physician or veterinarian and severity of the particular disease being treated.

[0085] The compositions can be further formulated for storage and distribution such as by lyophilization or freeze-drying. In addition, preservative and stabilizing agents can be added to further enhance the shelf-life of the compositions.

[0086] In some aspects, the AMMO or MMD is combined with a carrier such as a pharmaceutically acceptable carrier such as saline, to provide a concentration from about 0.1 mg / ml to about 10 mg / ml, or alternatively from about 0.5 mg / ml to about 10 mg / ml, or alternatively from about 1.0 mg / ml to about 10 mg / ml, or alternatively from about 0.1 mg / ml to about 9 mg / ml, or alternatively from about 0.1 mg / ml to about 7 mg / ml, or alternatively from about 0.1 mg / ml to about 5 mg / mlO, or alternatively from about 0.1 mg / ml to about 3 mg / ml, or alternatively from about 0.1 mg / ml to about 2 mg / ml, or alternatively from about 0.1 mg / ml to about 1 mg / ml, or alternatively from about 200 pg / ml to about 8 mg / ml, or alternatively from about 0.1 mg / ml to about 5 mg / ml, or alternatively from about 0.1 mg / ml to about 3 mg / ml, or alternatively from about 0.5 mg / ml to about 3 mg / ml, or alternatively from about 0.5 mg / ml to about 2 mg / ml, or about 0.5 mg / ml, or about 1.0 mg / ml, or about 1.5 mg / ml, or about or about 0.5 mg / ml, or about 1.0 mg / ml, or about 1.5 mg / ml, or about 2.0 mg / ml, or about 3.0 mg / ml, or about 3.5 mg / ml, or about 4.0 mg / ml, or about 4.5 mg / ml, or about 5 mg / ml, or about 5.5 mg / ml, or about 6.0 mg / ml, orabout 6.5 mg / ml, or about 7.0 mg / ml, or about 7.5 mg / ml, or about 8.0 mg / ml, or about 8.5 mg / ml, or about 9.0 mg / ml, or about 9.5 mg / ml or about 10 mg / ml.

[0087] The components of the AMMO or MMD can be sourced from commercial vendors.

[0088] The compositions can be formulated for in vivo administration (in one or more doses) to administer from about 5 mcg / kg body weight to about 50 mcg / kg body weight, or alternatively 10 mcg / kg body weight to 50 mcg / kg body, or alternatively 20 mcg / kg body weight to about 50 mcg / kg body weight, or alternatively 10 mg / kg body weight to about 100 mcg / kg body weight, or alternatively from about 15 mcg / kg body weight to about 150 mcg / kg body weight, or alternatively from about 20 to mcg / kg body weight to about 200 mcg / kg body weight.

[0089] Methods of Treatment

[0090] This disclosure also provides methods to enhance immunity in a subject against an infection caused by a bacterial or fungal pathogen by administering to the subject an effective amount of a composition of a composition as described above. The subject to be treated is any animal or human patient at risk of or has developed infection from a bacterial (gram-positive or gram-negative bacteria) or fungus. Non-limiting examples include sport and farm animals, pets and human patients. As used herein, the term “enhance immunity” intends to augment innate immune responses, including macrophage, neutrophil, dendritic cells, and gamma delta T cells and / or NK T cells, but does not include B-cell mediated antibody or T-cell stimulation that are characteristics of traditional, protein antigen-induced adaptive immunity. Methods to determine if an immune response has been elicited are known in the art and include, for example taking a suitable sample (blood, saliva or plasma) from a patient and assaying by ELISA cytokine levels, measuring the counts per ml of blood of various types of white blood cells, and whether antibodies against the pathogen or bacteria are present. In addition, non-invasive means such as a reduction in temperature of the subject can be used alone or in combination with clinical methods.

[0091] In one aspect, the bacterium is selected from S. aureus, A. baumannii, K. Pneumoniae, P. aeruginosa, E. coli, Enter obacter spp., Serratia, Stenotrophomonas, and the fungus is selected from Candida spp and fungi that cause the disease mucormycosis, including Rhizopus spp.

[0092] The composition can be administered in any suitable dose as determined by the treating physician, health care professional or veterinarian. Non-limiting examples ofsuitable methods of administration comprise intramuscular, subcutaneous, or intravenous administration. The effective amount to be administered is from about 5 mcg / kg body weight to about 50 mcg / kg body weight, or alternatively 10 mcg / kg body weight to 50 mcg / kg body, or alternatively 20 mcg / kg body weight to about 50 mcg / kg body weight, or alternatively 10 mg / kg body weight to about 100 mcg / kg body weight, or alternatively from about 15 mcg / kg body weight to about 150 mcg / kg body weight, or alternatively from about 20 to mcg / kg body weight to about 200 mcg / kg body weight .

[0093] In one aspect, the doses of AMMO or MMD are 50 pg and 500 pg, respectively to provide doses at from about 0.3, or about 0.4, or about 0.7 pg / kg (MPL) and from about 3, or about 4, or about 7 pg / kg weight-adjusted for a 70 kg adult.

[0094] In another aspect, the range of components in the AMMO or MMD are as provided below in Table 1.Table 1*Assuming 70 kg adult human and 0.03 kg (30 gram) adult mouseThe doses are repeated as needed, e.g., a second, third or fourth dose as necessary. The AMMO can be combined with PBS (e.g., 0.5 ml carrier), administered intra-muscularly.

[0095] The doses are repeated as needed, e.g., a second, third or fourth dose as necessary. The AMMO can be combined with PBS (e.g., 0.5 ml carrier), administered intramuscularly.

[0096] MMD - Table 2

[0097] Table 3 - Exemplary Dosage Schemes - AMMO

[0098] Administration of the AMMO or MMD vaccine can be combined with preadministration monitoring, e.g., blood can be obtained prior to vaccination, and repeated at 1 and 2 weeks. Blood can be tested for standard complete blood count, chemistries, and liver function tests, and will also be stored for immunological testing.

[0099] In one aspect, the composition is once, twice, or three times over the period of one to three months.

[0100] In another aspect, the method further comprises assaying the subject for a bacterial or fungal infection prior to administration of the composition using methods known in the art or as described herein.

[0101] The composition can be administered in any suitable dose as determined by the treating physician, health care professional or veterinarian. Non-limiting examples of suitable methods of administration comprise intramuscular, subcutaneous, or intravenous administration. The effective amount to be administered is from about 25 to mcg / kg body weight to about 200 mcg / kg body weight, or alternatively from about 50 mcg / kg body weight to about 175 mcg / kg body weight, or alternatively from about 75 to mcg / kg body weight to about 200 mcg / kg body weight, or from about 25 to mcg / kg body weight to about 100 mcg / kg body weight, or from about 25 to mcg / kg body weight to about 175 mcg / kg body weight, or from about 25 to mcg / kg body weight to about 150 mcg / kg body weight, or from about 75 to mcg / kg body weight to about 200 mcg / kg body weight, or from about 75 to mcg / kg body weight to about 150 mcg / kg body weight, or from about 25 to mcg / kg body weight to about 150 mcg / kg body weight, or from about 50 to mcg / kg body weight to about 125 mcg / kg body weight, or from about 50 to mcg / kg body weight to about 100 mcg / kg body weight, or alternatively about 25 mcg / kg body weight, or alternatively about 75 mcg / kg body weight, or alternatively about 100 mcg / kg body weight, or alternatively about 125 mcg / kg body weight, or alternatively about 150 mcg / kg body weight, or alternatively about 175 mcg / kg body weight, or alternatively about 200 mcg / kg body weight.

[0102] The methods also can be used to treat or prevent a disorder caused by a bacterial or fungal infection in a subject in need thereof, the method comprising administering an effective amount of the composition as described above. The subject to be treated is any animal or human patient at risk of or has developed infection from a bacterial (grampositive or gram-negative bacteria) or fungus. Methods to determine if a subject has been treated include, for example taking a suitable sample (blood, saliva or plasma) from a patient and assaying by ELISA cytokine levels, and whether antibodies against the pathogen or bacteria are present. In addition, non-invasive means such as a reduction in temperature of the subject can be used alone or in combination with clinical methods. In addition, depending on the disease being treated, a reduction of clinical or symptoms of the disease is an indication of effective treatment.

[0103] In one aspect, the bacterium is selected from S. aureus, A. baumannii, K. Pneumoniae, P. aeruginosa, E. coli, Enter obacter spp., Serratia, Stenotrophomonas, and the fungus is selected from Candida spp and the fungi that cause the disease mucormycosis, including Rhizopus spp.

[0104] The composition can be administered in any suitable dose as determined by the treating physician, health care professional or veterinarian. Non-limiting examples of suitable methods of administration comprise intramuscular, subcutaneous, or intravenous administration. The effective amount to be administered is from about 25 to mcg / kg body weight to about 200 mcg / kg body weight, or alternatively from about 50 mcg / kg body weight to about 175 mcg / kg body weight, or alternatively from about 75 to mcg / kg body weight to about 200 mcg / kg body weight, or from about 25 to mcg / kg body weight to about 100 mcg / kg body weight, or from about 25 to mcg / kg body weight to about 175 mcg / kg body weight, or from about 25 to mcg / kg body weight to about 150 mcg / kg body weight, or from about 75 to mcg / kg body weight to about 200 mcg / kg body weight, or from about 75 to mcg / kg body weight to about 150 mcg / kg body weight, or from about 25 to mcg / kg body weight to about 150 mcg / kg body weight, or from about 50 to mcg / kg body weight to about 125 mcg / kg body weight, or from about 50 to mcg / kg body weight to about 100 mcg / kg body weight, or alternatively about 25 mcg / kg body weight, or alternatively about 75 mcg / kg body weight, or alternatively about 100 mcg / kg body weight, or alternatively about 125 mcg / kg body weight, or alternatively about 150 mcg / kg body weight, or alternatively about 175 mcg / kg body weight, or alternatively about 200 mcg / kg body weight.

[0105] In one aspect, the composition is once, twice, or three times over the period of one to three months. In one aspect, the immunization is boosted at from about 14 to about 28 days, post-immunization, or from at from about 16 to about 26 days, post-immunization, or at from about 18 to about 24 days, post-immunization, or from about 20 to about 26 days, post-immunization, or from about 22 to about 23 days post-immunization, or from at about 3 weeks post-immunization.

[0106] The administration can be boosted with multiple administrations and / or administered in following ratios of AHO: MPL: D-mannose: about 0.5X, or about IX, or about 2X, or about 3X, or about 4X, or about 5X, or about 6X, or about 7X, or about 8X, or about 9X, or about 10X, or about 1 IX, or about 12X, or about 13, or about 14X, or about 15X or more of the base line dose, as determined by the treating physician. IX AMMO comprises 100 pg, 10 pg MPL and 200 pg aluminum hydroxide (2%); 3X AMMO comprises 300 pg D-mannose, 30 pg MPL, and 200 pg 2% aluminum hydroxide; and 10X comprise 1000 pg D-mannose, 100 pg MPL, and 200 pg 2% aluminum hydroxide.[0107J In another aspect, the method further comprises assaying the subject for a bacterial or fungal infection prior to administration of the composition using methods known in the art or as described herein.

[0108] Kits

[0109] Also provided herein is a kit comprising the compositions or formulations as described herein and instructions for use.

[0110] Drug Screening Assay

[0111] Also provided herein is a method to identify an compound or agent that provides a benefit selected from one or more of: enhances immunity against a bacterial or fungal microbial infection or treats a bacterial or fungal infection or a disease related to a bacterial or fungal microbial infection, the method comprising admixing the compound or agent with the composition as described herein and administering the admixed composition to a nonhuman subject infected with a bacterial or fungal microorganism and assaying for post administration infection or survival, wherein the compound or agent that enhances the activity of the composition as described herein is a compound or an agent that provides the benefit. Methods to determine if an immune response has been elicited are known in the art and include, for example taking a suitable sample (blood, saliva or plasma) from a patient and assaying by ELISA cytokine levels, and whether antibodies against the pathogen or bacteria are present. In addition, non-invasive means such as a reduction in temperature of the subject can be used alone or in combination with clinical methods. Methods to determine if a subject has been treated include, for example taking a suitable sample (blood, saliva or plasma) from a patient and assaying by ELISA whether antibodies against the pathogen or bacteria are present. In addition, non-invasive means such as a reduction in temperature of the subject can be used alone or in combination with clinical methods. In addition, depending on the disease being treated, a reduction of clinical or symptoms of the disease is an indication of effective treatment.

[0112] Preparation of Compositions

[0113] To prepare the compositions, the preferred amount of aluminum hydroxide is suspended in saline solution to a concentration in a range from about 0.1 mg / ml to about 10 mg / ml, or alternatively from about 0.5 mg / ml to about 10 mg / ml, or alternatively from about 1.0 mg / ml to about 10 mg / ml, or alternatively from about 0.1 mg / ml to about 9 mg / ml, or alternatively from about 0.1 mg / ml to about 7 mg / ml, or alternatively from about0.1 mg / ml to about 5 mg / mlO, or alternatively from about 0.1 mg / ml to about 3 mg / ml, or alternatively from about 0.1 mg / ml to about 2 mg / ml, or altemO.l mg / ml to about 1 mg / ml, or alternatively from about 200 pg / ml to about 8 mg / ml, or alternatively from about 0.1 mg / ml to about 5 mg / ml, or alternatively from about 0.1 mg / ml to about 3 mg / ml, or alternatively from about 0.5 mg / ml to about 3 mg / ml, or alternatively from about 0.5 mg / ml to about 2 mg / ml, or about 0.5 mg / ml, or about 1.0 mg / ml, or about 1.5 mg / ml, or about or about 0.5 mg / ml, or about 1.0 mg / ml, or about 1.5 mg / ml, or about 2.0 mg / ml, or about 3.0 mg / ml, or about 3.5 mg / ml, or about 4.0 mg / ml, or about 4.5 mg / ml, or about 5 mg / ml, or about 5.5 mg / ml, or about 6.0 mg / ml, or about 6.5 mg / ml, or about 7.0 mg / ml, or about 7.5 mg / ml, or about 8.0 mg / ml, or about 8.5 mg / ml, or about 9.0 mg / ml, or about 9.5 mg / ml or about 10 mg / ml.

[0114] In some aspect, D-mannose is dissolved in saline to provide a concentration of from about 0.1 mg / ml to about 10 mg / ml, or alternatively from about 0.5 mg / ml to about 10 mg / ml, or alternatively from about 1.0 mg / ml to about 10 mg / ml, or alternatively from about 0.1 mg / ml to about 9 mg / ml, or alternatively from about 0.1 mg / ml to about 7 mg / ml, or alternatively from about 0.1 mg / ml to about 5 mg / mlO, or alternatively from about 0.1 mg / ml to about 3 mg / ml, or alternatively from about 0.1 mg / ml to about 2 mg / ml, or alternatively from about 0.1 mg / ml to about 1 mg / ml, or alternatively from about 200 pg / ml to about 8 mg / ml, or alternatively from about 0.1 mg / ml to about 5 mg / ml, or alternatively from about 0.1 mg / ml to about 3 mg / ml, or alternatively from about 0.5 mg / ml to about 3 mg / ml, or alternatively from about 0.5 mg / ml to about 2 mg / ml, or about 0.5 mg / ml, or about 1.0 mg / ml, or about 1.5 mg / ml, or about or about 0.5 mg / ml, or about 1.0 mg / ml, or about 1.5 mg / ml, or about 2.0 mg / ml, or about 3.0 mg / ml, or about 3.5 mg / ml, or about 4.0 mg / ml, or about 4.5 mg / ml, or about 5 mg / ml, or about 5.5 mg / ml, or about 6.0 mg / ml, or about 6.5 mg / ml, or about 7.0 mg / ml, or about 7.5 mg / ml, or about 8.0 mg / ml, or about 8.5 mg / ml, or about 9.0 mg / ml, or about 9.5 mg / ml or about 10 mg / ml, prior to combination with MPL and aluminum hydroxide.

[0115] The preferred amount of MPL is added to the aluminum hydroxide and MPL combination to a concentration in a range from about 0.1 mg / ml to about 10 mg / ml, or alternatively from about 0.5 mg / ml to about 10 mg / ml, or alternatively from about 1.0 mg / ml to about 10 mg / ml, or alternatively from about 0.1 mg / ml to about 9 mg / ml, or alternatively from about 0.1 mg / ml to about 7 mg / ml, or alternatively from about 0.1 mg / ml to about 5 mg / mlO, or alternatively from about 0.1 mg / ml to about 3 mg / ml, or alternativelyfrom about 0.1 mg / ml to about 2 mg / ml, or altemO.l mg / ml to about 1 mg / ml, or alternatively from about 200 pg / ml to about 8 mg / ml, or alternatively from about 0.1 mg / ml to about 5 mg / ml, or alternatively from about 0.1 mg / ml to about 3 mg / ml, or alternatively from about 0.5 mg / ml to about 3 mg / ml, or alternatively from about 0.5 mg / ml to about 2 mg / ml, or about 0.5 mg / ml, or about 1.0 mg / ml, or about 1.5 mg / ml, or about or about 0.5 mg / ml, or about 1.0 mg / ml, or about 1.5 mg / ml, or about 2.0 mg / ml, or about 3.0 mg / ml, or about 3.5 mg / ml, or about 4.0 mg / ml, or about 4.5 mg / ml, or about 5 mg / ml, or about 5.5 mg / ml, or about 6.0 mg / ml, or about 6.5 mg / ml, or about 7.0 mg / ml, or about 7.5 mg / ml, or about 8.0 mg / ml, or about 8.5 mg / ml, or about 9.0 mg / ml, or about 9.5 mg / ml or about 10 mg / ml.

[0116] Alternatively, the components are mixed to provide the following ratios of AHO: MPL: D-mannose: about lx : about lx : about lx; or about lx; or about 3x : about 3x; or about lx; or about lOx : about lOx; or about lx; or about 30x : about 30x; or lx : lx : lx; or lx; or 3x : 3x; or lx; or lOx : lOx; or lx; or 30x : 30x; wherein lx AHO = 0.5 mg; lx MPL = 0.05 mg; and lx D-Mannose = 0.5 mg. The composition can be combined with a carrier and used in the disclosed methods.

[0117] The AMMO or MMD is suspended in pharmaceutically acceptable carriers such as PBS and optionally DMSO, to a concentration in a range from about 0.1 mg / ml to about 10 mg / ml, or alternatively from about 0.5 mg / ml to about 10 mg / ml, or alternatively from about 1.0 mg / ml to about 10 mg / ml, or alternatively from about 0.1 mg / ml to about 9 mg / ml, or alternatively from about 0.1 mg / ml to about 7 mg / ml, or alternatively from about 0.1 mg / ml to about 5 mg / mlO, or alternatively from about 0.1 mg / ml to about 3 mg / ml, or alternatively from about 0.1 mg / ml to about 2 mg / ml, or altemO.l mg / ml to about 1 mg / ml, or alternatively from about 200 pg / ml to about 8 mg / ml, or alternatively from about 0.1 mg / ml to about 5 mg / ml, or alternatively from about 0.1 mg / ml to about 3 mg / ml, or alternatively from about 0.5 mg / ml to about 3 mg / ml, or alternatively from about 0.5 mg / ml to about 2 mg / ml, or about 0.5 mg / ml, or about 1.0 mg / ml, or about 1.5 mg / ml, or about or about 0.5 mg / ml, or about 1.0 mg / ml, or about 1.5 mg / ml, or about 2.0 mg / ml, or about 3.0 mg / ml, or about 3.5 mg / ml, or about 4.0 mg / ml, or about 4.5 mg / ml, or about 5 mg / ml, or about 5.5 mg / ml, or about 6.0 mg / ml, or about 6.5 mg / ml, or about 7.0 mg / ml, or about 7.5 mg / ml, or about 8.0 mg / ml, or about 8.5 mg / ml, or about 9.0 mg / ml, or about 9.5 mg / ml or about 10 mg / ml.

[0118] In one aspect, the components are combined to achieve a final concentration. For example, the compositions have an combined concentration in a range from about 0.1 mg / ml to about 20 mg / ml, or alternatively from about 0.5 mg / ml to about 20 mg / ml, or alternatively from about 1.0 mg / ml to about 20 mg / ml, or alternatively from about 0.1 mg / ml to about 15 mg / ml, or alternatively from about 0.1 mg / ml to about 12 mg / ml, or alternatively from about 0.1 mg / ml to about 10 mg / ml, or alternatively from about 0.1 mg / ml to about 5 mg / ml, or alternatively from about 0.1 mg / ml to about 3 mg / ml, or alternatively from 0.1 mg / ml to about 2 mg / ml, or alternatively from about 200 pg / ml to about 20 mg / ml, or alternatively from about 0.1 mg / ml to about 15 mg / ml, or alternatively from about 0.1 mg / ml to about 13 mg / ml, or alternatively from about 0.5 mg / ml to about 13 mg / ml, or alternatively from about 0.5 mg / ml to about 12 mg / ml, or about 0.5 mg / ml, or about 1.0 mg / ml, or about 1.5 mg / ml, or about or about 1.5 mg / ml, or about 2.0 mg / ml, or about 3.5 mg / ml, or about 4.0 mg / ml, or about 8.0 mg / ml, or about 10 mg / ml, or about 12.0 mg / ml, or about 13 mg / ml, or about 15 mg / ml, or about 15.5 mg / ml, or about 6.0 mg / ml, or about 6.5 mg / ml, or about 7.0 mg / ml, or about 7.5 mg / ml, or about 8.0 mg / ml, or about 8.5 mg / ml, or about 9.0 mg / ml, or about 9.5 mg / ml or about 10 mg / ml.

[0119] The compositions can be formulated for in vivo administration (in one or more doses) to administer from about 5 mcg / kg body weight to about 50 mcg / kg body weight, or alternatively 10 mcg / kg body weight to 50 mcg / kg body, or alternatively 20 mcg / kg body weight to about 50 mcg / kg body weight, or alternatively 10 mg / kg body weight to about 100 mcg / kg body weight, or alternatively from about 15 mcg / kg body weight to about 150 mcg / kg body weight, or alternatively from about 20 to mcg / kg body weight to about 200 mcg / kg body weight. Exemplary doses are disclosed herein and incorporated herein by reference.Experiment 1: Discovery of Adjuvant-Induced Immunity - Materials and MethodsAdjuvant Immunization

[0120] D-mannose (SIGMA), Monophosphoryl lipid A (MPL) (InvivoGen Cat. # tlrl-mpls), whole glucan particles (WGP) (InvivoGen Cat. # tlrl-wgp), 2% aluminum hydroxide (A1(OH)3) gel (Accurate Chemical & Scientific Corporation Cat. # A1090S) are prepared and stored according to the manufacture protocol. Mice are immunized with 200 pL administered subcutaneously (SC) in the scruff of the neck with pre-mixed adjuvant mixtures in phosphate-buffered saline (PBS).Intravenous (IV) Infection|0121] Frozen stocks of A. baumannii and K. pneumoniae bacteria grown to mid-log phase are prepared as previously described Nielsen, T.B. et al. (2015), BMC Microbiol 15, 252. Inocula are prepared by diluting these concentrated frozen stocks of bacteria in PBS. Inocula are confirmed by plating serial dilutions on tryptic soy agar (TSA) plates and incubating overnight at 37 °C. Mice infected with A. baumannii are monitored for seven days and mice infected with K. pneumoniae are monitored for 14 days, after which they are euthanized according to our IACUC protocol.

[0122] S. aureus inocula are prepared from mid-log phase subcultures of overnight cultures for each infection. Briefly, S. aureus was inoculated into tryptic soy broth (TSB) and incubated overnight at 37 °C with shaking set to 200 rpm. A subculture was set up from a 1 : 100 dilution of the overnight culture into sterile TSB and incubated for 3 h at 37 °C with shaking set to 200 rpm. The subculture was rinsed by pelleting in a refrigerated centrifuge at 4,000*g for 5 min and resuspending the pellet in PBS. After two more rinses (three total), the pellet was resuspended in PBS and adjusted the optical density at 600 nm (ODeoo) to 0.5 using PBS. An established correlation coefficient at this optical density is used to estimate the inoculum concentration (ODeoo 0.5 ~ 2.4* 108CFU / mL). Inocula are confirmed by plating serial dilutions on TSA plates and incubating overnight at 37 °C. Mice infected with S. aureus are monitored for 28 days, after which they are euthanized according to our IACUC protocol.Oral Aspiration (OA) Infection

[0123] Mice are infected using OA model as previously described (Nielsen, T.B. et al. (2018), JoVE). Briefly, A. baumannii HUMCI inocula are prepared from subcultures ofovernight cultures, as stated above, mice are infected by aspirating 50 pL of bacteria suspended in PBS. Mice infected with A. baumannii via OA are monitored for seven days, after which they are euthanized according to Applicant’s IACUC protocol.Mouse Knockout (KO) Strains

[0124] RAG1 KO (strain # 034159) and human CD34+hematopoietic stem-cell -engrafted NSG mice are purchased from Jackson Laboratories.Innate Immune Cell Depletion

[0125] Macrophages are depleted by injecting liposomal clodronate (C-010, Foundation Clodronate Liposomes) IP at 50 pg / g (e.g. 30-g mouse received 300 pL or 1.5 mg liposomal clodronate) three days before infection.

[0126] Neutrophils are depleted by injecting cyclophosphamide (Baxter) IP at 230 mg / kg three days before infection. Neutropenia last 6 days (Nielsen, T.B. et al. (2021), J Infect Dis, Mostafa, H.H. et al. (2016), PLoS Pathog 12, el005875).

[0127] NK cells are depleted by injecting anti-Asialo-GMl antibody (Poly21460, Biolegend, 30pl / mouse) at every three to four days starting on the day of immunization (Bbttcher, J.P. et al. (2018), Cell 172, 1022-1037.el014).Flow Cytometry Analysis

[0128] The spleens of mice are collected, mashed through a 70-pm cell strainer with a 35- mL syringe plunger, and rinsed with Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) by volume (DMEM + 10% FBS). The cell suspension is pelleted in a room-temperature centrifuge at 300*g for 5 min, and the supernatant is discarded. After two more rinses (three total), red blood cells (RBC) are lysed using RBC lysis buffer (BioLegend Cat. # 420301) according to the manufacture’s protocol. Splenocytes are resuspended in PBS with 5% Fetal Bovine Serum (FACS buffer), counted with a hemacytometer, incubated with Fc blocker (BD Biosciences Cat. # 553141) for 30 min on ice. Splenocyte are pelleted in a room -temperature centrifuge at 300*g for 5 min, resuspended in FACS buffer, and incubated with fluorophore-conjugated antibodies for OMIP-032, a two-panel, multi-color immunophenotyping to assess innate and adaptive immune cells as previously described (Unsworth, A. et al. (2016), Cytometry A 89, 527- 530). After incubation, splenocytes are washed twice in a room-temperature centrifuge at300*g for 5 min and resuspended in FACS buffer. Samples are analyzed using a BD FACS Canto II flow cytometer.Statistics

[0129] Survival is compared by the non-parametric Log-Rank test with a = 0.05. Mac assay, cfu, cytokines, chip-seq are compared by Wilcoxon-Mann-Whitney test with a = 0.05.

[0130] Efficacy of prior combinations are compared. Mice are injected with a group of protein antigens combined with one or more adjuvants and incorporating adjuvant-only immunized mice as negative controls. All mice are then challenged intravenously (IV) with a clinical blood isolate of S. aureus MRSA USA300 strain LAC.

[0131] To evaluate the impact of immunizations booster shots on vaccine efficacy, mice are immunized with phosphate-buffered saline (PBS) or MPL, WGP, and Al(0H)3 (MW A); half of the mice are administered an identical booster shot three weeks after their first immunization. Mice are then challenged IV with S. aureus LAC three, seven, or 21 days after the final immunization.

[0132] To investigate the ability of the vaccines to protect against bacteremia caused by the Gram-negative bacterium, Acinetobacter baumannii, mice are immunized with PBS, AMMO or MMD and infected three or seven days post-immunization with an extremely drug-resistant (XDR), clinical lung and blood isolate of baumannii HUMC1.Experiment 2: Defining Contributions of the Vaccine Components

[0133] Mice are immunized mice with zero, one, two, or all three adjuvants and then challenged three days later with a larger inoculum of A. baumannii, enabling differentiation between the survival benefits conferred on each immunization group.

[0134] To determine whether the triple-adjuvant or dual-adjuvant vaccine can protect against another common route of infection in hospitalized patients, aspiration pneumonia, the previously described oropharyngeal aspiration (OA) pneumonia model (Nielsen, T.B. et al. (2018), JoVE) can be used. The ability of the vaccines to protect against lethal infection from Klebsiella pneumoniae, another Gram-negative bacterium can be used in Applicant’s bacteremia model.Experiment 3: Optimization of the Triple Regimen with D-mannose

[0135] Mice are immunized with either D-mannose in addition to MPL (AMMO) or D- mannose, MPL and AHO (AMMO). Three days later, mice are challenged with a normally lethal inoculum of S. aureus, A. baumannii, K. pneumoniae, or neutropenic mice infected with the fungus Rhizopus delemar.Experiment 4: Optimization of Vaccine Dosage

[0136] Applicant also can evaluate whether increased doses could enhance protection and how long the vaccine would protect after immunization. Mice are immunized with three (3x) or ten times (10x) as much D-mannose and MPL, and challenged them one, two, or three weeks later with lethal S. aureus or A. baumannii bacteremia.

[0137] Applicant can define the rapidity of onset of protection. Mice are immunized with the standard (1 x) dose of AMMO or MMD and challenged them with lethal A. baumannii or S. aureus bacteremia 24 h post-immunization. To assess its translatability to humans, Applicant will immunize CD34+human stem-cell transplant-recipient mice with AMMO three days pre-infection. Applicant will also test several good manufacturing practices (GMP) materials by immunizing mice GMP grade AMMO three days pre-infection.Experiment 5: Cellular Source of Protection

[0138] To begin to define the mechanism of protection, Applicant can evaluate changes to seven immune cell populations sourced from the spleen of immunized and non-immunized mice, the proportion of CD45+cells that are B cells are elevated for at least three weeks post-immunization.

[0139] To determine if lymphocytes play a role in AMMO and MMD mediated protection, Applicant will immunize wild-type (WT) and RAG1 -knockout (RAG1-KO) mice with AMMO or MMD and challenge them with A. baumannii bacteremia three days postimmunization.

[0140] To identify the potential key innate immune cells that provide AMMO- or MMD- mediated protection, Applicant will selectively deplete mice of innate immune cells, immunized them with AMMO, and then challenged mice three days later. Specifically, neutrophils are depleted with cyclophosphamide, macrophages / monocytes are depleted with liposomal clodronate, and NK cells are depleted with anti-Asialo-GMl antibody.[0141 J Applicant will investigate whether AMMO or MMD can differentiate RAW 264.7 macrophage-like cells and primary human monocyte in vitro by assessing macrophage phagocytosis of A baumannii after AMMO stimulation. Both RAW 264.7 macrophage-like cells are incubated with AMMO one or three days before uptake assay mediated similar phagocytosis as following the positive control, IFNy stimulation.Experiment 6: Immunomodulatory Mechanism of Protection

[0142] Bacterial burden in AMMO-immunized mice challenged with lethal S. aureus or A. baumannii bacteremia, four hours post-infection is first analyzed.

[0143] Applicant has previously observed that cytokine profiles are a better indicator of survival outcomes than bacterial burden (Nielsen, T.B. et al. (2021), J Infect Dis). As such, cytokines in the plasma of the very same immunized mice challenged with lethal S. aureus or A. baumannii bacteremia are analyzed four hours post-infection.Experiment 7: Comparative AMMO Vaccine Efficacy

[0144] Applicant’s AMMO triple vaccine or MMD vaccine is compared to prior art MPL triple vaccine, alone or in combination with MPL + Mannan vaccine. The MPL alone or in combination with mannan is disclosed in WO 2018 / 089475, incorporated herein by reference.

[0145] Briefly the MPL triple vaccine comprises comprising, or alternatively consisting essentially of, or yet further consisting of, an effective amount of each of: an aluminum hydroxide, a mono-phosphoryl lipid (MPL), and whole glucan particles (WGP), with the proviso that the composition does not comprise an antigen effective to induce an immune response against a fungal, a small molecule or bacterial pathogen.Experiment 8: Discovery and Defining of Vaccine Components

[0146] The individual components of the tripartite vaccine and dual vaccine (MMD or AMMO) are analyzed to determine protection against S. aureus bacteremia. Mice are immunized with each individual component or dual or triple combinations of A1(OH)3, MPL, and WGP. All mice are then challenged intravenously (IV) with a USA300 clinical blood isolate of MRS A, LAC.

[0147] Mice are immunized with phosphate-buffered saline (PBS) or MWA to evaluate the impact of a repeat dose on vaccine efficacy. Three weeks later, half of the mice arevaccinated again. Mice are then challenged IV with S. aureus LAC three, seven, or 21 days after the final immunization.

[0148] The ability of the vaccine to protect against bacteremia caused by the Gram-negative bacterium, Acinetobacter baumannii can be evaluated. Mice are immunized with PBS, AMMO or MMD and infected intravenously either three or seven days post-immunization with an extremely drug -resistant (XDR), clinical lung and blood isolate of A. baumannii, HUMCI.

[0149] Mice are immunized with zero, one, two, or all three components and challenged three days later with a larger inoculum of A. baumannii, enabling differentiation between the survival benefits conferred on each immunization group.

[0150] Applicant can determine if AMMO or MMD can protect against infections from another common route of infection in hospitalized patients — pneumonia — using Applicant’s previously described oropharyngeal aspiration (OA) pneumonia model (Nielsen T.B., et al. (2018), JoVE).

[0151] The ability of the vaccine to protect against lethal bloodstream infection from Klebsiella pneumoniae, another Gram-negative bacterium also is assessed.Optimization of Vaccine Dosage

[0152] Evaluation of the duration of protection and whether increased doses can enhance protection is tested. Mice are immunized with three (3 *) or ten times (10x) the prior doses of D-mannose and MPL (3x = 300 pg D-mannose, 30 pg MPL, 200 pg aluminum hydroxide; lOx = 1000 pg D-mannose, 100 pg MPL, 200 pg aluminum hydroxide), and are challenged one, two, or three weeks later with lethal S. aureus or A. baumannii bacteremia.

[0153] Applicant can define how soon the onset of protection occurs. To this end, mice are immunized mice with the standard (1 x) dose of AMMO and challenged with lethal S. aureus or A. baumannii bacteremia 24 hours post-immunization.

[0154] To assess its translatability to humans, mice are immunized that had undergone whole-body irradiation followed by CD34+human stem-cell transplantation with AMMO three days prior to infection (Ishikawa, F. et al. (2005), Blood 106, 1565-1573, Coughlan A.M. et al. (2016) Stem Cells Dev 25, 530-541). To support potential future clinical use in humans Applicant can also test Good Manufacturing Practice (GMP)-compliant Al(OH)sand MPL and Good Laboratory Practice (GLP)-compliant D-mannose by immunizing mice with GMP / GLP- grade AMMO (G-AMMO) three days prior to infection.Cellular Source of Protection

[0155] Changes over a 21 -day period following immunization in eight immune cell populations sourced from the spleens of immunized and non-immunized mice can be evaluated to define the mechanism of protection: T cells, B cells, dendritic cells, NK cells, myeloid cells, neutrophils, monocytes, and macrophages.

[0156] To identify the innate immune cell type(s) essential in AMMO or MMD medicated protection, mice are immunized them with AMMO, and selectively depleted mice of their NK cells, or monocyte s / macrophages as studies are completed. Whether AMMO or MMD can activate murine macrophage-like RAW 264.7 cells in vitro and primary human monocytes ex vivo can be evaluated in a manner similar to interferon-y (IFNy), by measuring macrophage uptake of A. baumannii.Immunomodulatory Mechanism of Protection

[0157] Immunomodulatory properties are investigated by evaluating cytokine-related gene expression, bacterial burden, and cytokine profiles in mice immunized with AMMO or MMD, before and after being infected with S. aureus or A. baumannii bacteremia. Cytokine-associated gene expression also is evaluated.

[0158] Bacterial burden is analyzed in immunized mice challenged with lethal S. aureus or A. baumannii bacteremia, four hours post-infection.

[0159] Applicant have previously observed that cytokine profiles may be a better indicator of survival outcomes than changes in bacterial burden (Nielsen, T.B. et al. (2021), J Infect Dis, Pirofski, L.A. et al. (2008), Adv Exp Med Biol 635, 135-146, Fidel, Jr., P.L. et al.(2020), J Fungi (Basel) 6). As such, cytokine concentrations in the plasma of the very same immunized mice challenged with lethal S. aureus or A. baumannii bacteremia are analyzed at four hours post-infection.AMMO Vaccination Induce Epigenetic and Gene Expression Changes

[0160] Histone reprogramming and transcription changes in immunized mice is studied to further understand the mechanism of protection mediated by AMMO and evaluate whether the vaccine induces epigenetic biomarkers of trained immunity. Epigenetic reprogramming has been shown to be a key characteristic of trained immunity (Saeed, S. et al. (2014)Science 345, 1251086, de Laval, B. et al. (2020), Cell Stem Cell 26, 657-674.e658), with acetylation of histone 3 lysine 27 (H3K27ac) as a key marker for activation of promoters and enhancers (Rada, A. et al. (2011), Cell Host Microbe 9, 355-361). To test this, splenic macrophages are isolated from mice that are immunized with AMMO othree or 21 days prior, and then performed H3K27ac chromatin immunoprecipitation sequencing (ChlP-seq) and RNA sequencing (RNA-seq). Pathway analyses on DEGs is analyzed to determine if the biological processes are influenced by AMMO immunization.Intravenous (IV) Infection|0161] Frozen stocks of A. baumannii and K. pneumoniae bacteria are grown to mid-log phase and are prepared as previously described (Nielsen, T.B. et al. (2015), BMC Microbiol 15, 252). Inocula are prepared by diluting these concentrated frozen stocks of bacteria in PBS. Inocula are confirmed by plating serial dilutions on tryptic soy agar (TSA) plates and incubating overnight at 37 °C. Mice infected with A. baumannii are monitored for seven days and mice infected with K. pneumoniae are monitored for 14 days, after which they are euthanized according to the IACUC protocol.

[0162] S. aureus and E. faecalis inocula are prepared from mid-log phase subcultures of overnight cultures for each infection. Briefly, S. aureus and E. faecalis inoculated into tryptic soy broth (TSB) and incubated overnight at 37 °C with shaking set to 200 rpm. A subculture is set up from a 1 : 100 dilution of the overnight culture into sterile TSB and incubated for 3 h at 37 °C with shaking set to 200 rpm. The subculture is rinsed by pelleting in a refrigerated centrifuge at 4,000*g for 5 min and resuspending the pellet in PBS. After two more rinses (three total), the pellet is resuspended in PBS and the optical density at 600 nm (ODeoo) is adjusted to 0.5 using PBS. Inocula are confirmed by plating serial dilutions on TSA plates and incubating overnight at 37 °C. Mice infected with S. aureus are monitored for 28 days, after which they are euthanized according to the IACUC protocol. Mice infected with E. faecalis are monitored for seven days, after which they are euthanized according to the IACUC protocol.

[0163] Rhizopus delemar are cultured on plates with Potato dextrose agar for 8 days at 37 °C and harvested with PBS with 0.05% tween 80. The conidial density is determined with a hemocytometer, and the viability of conidial suspensions is determined by plating on Sabouraud’s dextrose agar. Mice infected with R. delemar are monitored for 14 days, after which they are euthanized according to the IACUC protocol.

[0164] Candida albicans is serially passaged three times in yeast peptone dextrose broth and washed twice with PBS prior to infection. The infectious inoculum is prepared by counting in a hemacytometer. Mice infected with C. albicans are monitored for 7 days, after which they are euthanized according to the IACUC protocol.Oral Aspiration (OA) Infection

[0165] Mice are infected using the aspiration pneumonia model as previously described (7). Briefly, inocula of A. baumannii and P. aeruginosa are prepared from subcultures of overnight cultures, as stated above, mice are infected by aspirating 50 pL of bacteria suspended in PBS. Mice infected via OA are monitored for up to seven days, after which they are euthanized according to the IACUC protocol.Innate Immune Cell DepletionMacrophages are depleted by injecting liposomal clodronate (Foundation Clodronate Liposomes, C-010) intraperitoneally (IP) at 50 mg / kg three days before infection (36). Neutrophils are depleted by injecting cyclophosphamide (Baxter) IP at 230 mg / kg three days before infection (Nielsen, T.B. et al. (2021), J Infect Dis, Mostafa, H.H. et al. (2016), PLoS Pathog 12, el 005875) .Flow Cytometry Analysis

[0166] The spleens of mice are collected, mashed through a 70-pm cell strainer with a 35- mL syringe plunger, and rinsed with Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) by volume (DMEM + 10% FBS). Splenocytes are pelleted in a room -temperature centrifuge at 300 / g for 5 min, and the supernatant was discarded. After three rinses with PBS, red blood cells (RBC) are lysed using RBC lysis buffer (BioLegend, 420301) according to the manufacture’s protocol. Splenocytes are resuspended in PBS with 5% Fetal Bovine Serum (FACS buffer), counted with a hemacytometer, and incubated with Fc blocker (BD Biosciences, 553141) for 30 min on ice. Splenocytes are pelleted in a room-temperature centrifuge at 300*g for 5 min, resuspended in FACS buffer, and incubated with fluorophore-conjugated antibodies for OMIP-032, a two-panel, multi-color immunophenotyping assay to assess innate and adaptive immune cell populations as previously described (Unsworth, A. et al. (2016), Cytometry A 89, 527-530). After incubation, splenocytes are pelleted in a roomtemperature centrifuge at 300*g for 5 min, rinsed with PBS twice, and resuspended in FACS buffer. Samples are analyzed using a BD FACS Canto II flow cytometer.

[0167] Bacteria binding flow analysis is performed by incubating diluted HUMCI (1 : 100) or LAC (1 : 100,000) with mouse plasma or lOpg / mL isotype (Fisher, MAB002) or plasma from mouse recovered from HUMCI or LAC infection for 30 min. Bacteria are pelleted in a room-temperature centrifuge at 10,000* for 1 min and rinsed with PBS twice. Then, samples are incubated with anti-mouse secondary antibody (Thermo Fisher, A21235) for 30min, pelleted, rinsed with PBS twice, and resuspended in FACS buffer. Samples are analyzed using a BD Accuri™ C6 Plus flow cytometer.In Vitro Macrophage Opsonophagocytosis Assays

[0168] RAW 264.7 murine macrophages (ATCC TIB-71) cultured with DMEM + 10% FBS stimulated with either mixture of lOng / mL A1(OH)3, 0.5 pg / mL MPL and 5 pg / mL D- mannose (“AMMO”) for one or three days is used. RAW 264.7 stimulated with 100 pg / mL IFN-y (Peprotech, 315-05) and PBS overnight are used as positive and negative controls, as previously described (Baquir, B. et al (2012), Clin Infect Dis 54 Suppl 3, S229-232). Once cells are ready, macrophage opsonophagocytosis assay is performed as follows. Bacteria are prepared from overnight cultures of A. baumannii subcultured to log phase, washed in PBS, and resuspended in Hanks’ balanced salt solution (HBSS). Cells are rinsed three times with HBSS, bacteria are added to wells at a ratio of 20: 1 (bacteria to macrophages) in the presence of 10% CD-I mouse serum (Innovative Research Inc, IMSCD1-COMPL), and centrifuged at 300*g for 5 min. Macrophages are washed three times with HBSS, fixed with 100% methanol, and Hema-3 stained according to the manufacturer's protocol (Fisher Scientific). To quantitate bacteria per macrophage, coverslips are imaged on a Leica DMLS clinical microscope with a Leica ICC50 HD digital camera.Ex Vivo Macrophage Opsonophagocytosis Assay

[0169] Ex vivo macrophages differentiated from human peripheral blood mononuclear cells (PBMC) are used. Fresh PBMC are purchased from UCLA / CFAR virology core laboratory. Monocytes are isolated (StemCell Technologies, 19359) and differentiated to Ml macrophages (StemCell Technologies, 10961) according to the manufacture’s protocol. Either mixture of lOng / mL A1(OH)3, 0.5 pg / mL MPL and 5 pg / mL D-mannose for one or three days is used for stimulation. Macrophages stimulated with 50 ng / mL IFN-y (StemCell Technologies, 78020) and PBS three days are used as positive and negative controls. Macrophage opsonophagocytosis assay is performed and numerated as mentioned above.Chromatin Immunoprecipitation Sequencing

[0170] Spleens are harvested from C3HeB / Fe mice and splenocytes are isolated as above. Splenocytes are rinsed in PBS and resuspended in PBS containing 2% FBS and 1 mM EDTA. Following the manufacturer’s protocol (StemCell Technologies, 100-0659), f4 / 80+ macrophages are isolated. H3K27ac chromatin immunoprecipitation (ChIP) is performed using anti-H3K27ac polyclonal antibody (Diagenode, Cl 5410196) and iDeal ChlP-seq kit for Histones (Diagenode, C01010051). Azenta Life Sciences performed DNA library preparation and Illumina HiSeq 150-bp paired-end sequencing. ChlP-sequencing reads are aligned to mouse genome assembly mm39 (based on NCBI GRCm39) using bwa (PMID: 19451168). BAM files are filtered to remove duplicate reads and those with poor mapping quality using SAMtools (PMID: 19505943). MACS2 is used to call H3K27ac peaks using the default (narrow) setting (PMID: 18798982). The quality of the ChlP-seq data is visualized by making bigwig files using deepTools (PMID: 27079975) in the UCSC Genome browser. For differential peak calling, data (H3K27ac reads / peak) are normalized using the R package DESeq2 (PMID: 25516281), and then pairwise comparisons are performed between the PBS, day 3 and day 21 groups. Differential peaks are identified as adjusted p value <0.05, fold change >2, reads / peak >50. Peaks are then merged into one file to generate principal component analysis (PCA) plots.RNA Sequencing

[0171] Splenic f4 / 80+ macrophages are harvested from C3HeB / Fe mice as mentioned above. Azenta Life Sciences performed RNA isolation, rRNA depletion, library preparation, and Illumina HiSeq 150-bp paired-end sequencing. To infer gene expression levels, RNA-seq reads are aligned to reference mouse genome transcriptome using Bowtie (PMID: 19261174). Quantification of gene expression was performed using MMSEQ (PMID: 21310039). Statistical analysis was performed using DESeq2, with pairwise comparisons performed between the PBS, day 3 and day 21 groups. Differentially expressed genes are identified as those showing p value <0.05, FC >2 and RPKM >1. Differential gene lists from all comparisons are then merged, and the combined list of differential genes was used for plotting.Statistics

[0172] Survival can be compared by the non-parametric log-rank test with a = 0.05. Bacterial burden, cell population, macrophage assay, and cytokine are compared by Wilcoxon-Mann-Whitney test or Kruskal-Wallis test with a = 0.05.Experiment 9: Replacing Mannan with D-Mannose: AMMOAMMO with D-mannose was equally effective as AMMA with mannan against Gram positive and negative pathogens and fungi

[0173] Fungal mannan is prepared experimentally by chemical extraction from yeast cell wall, resulting in a wide distribution of molecular weight fragments, with a tendency to degrade into smaller fragments in solution. Fungal mannan polysaccharide is comprised of D-mannose monomers, and D-mannose is already available commercially in GMP format, and has already been used as an excipient in an FDA approved vaccine.

[0174] Mice were treated with AMMA or AMMO, with the D-mannose at one of three doses (10, 30, or 100 pg). Mice were infected IV 3 days later with S. aureus LAC. All treated groups had significantly improved survival, but the group with the best survival was the highest dose D-mannose group (FIG. 2). Indeed, D-mannose appeared to have a doseresponse effect, and at the highest dose of D-mannose, AMMO trended to superior in efficacy to AMMA.Experiment 10: An In Vitro Phagocytosis Assay Correlates with In Vivo Protection and Can Detect Loss of AMMO PotencyAMMO stimulated macrophage phagocytosis of S. aureus and A. baumannii (FIG. 5)

[0175] AMMO-activated macrophage phagocytized fluorescently-labeled, killed S. aureus from a commercial kit at least as well as AMMA (FIG. 5). Thus, the phagocytosis assay can be used to detect both Gram positive and negative increases in macrophage uptake stimulated by AMMO.Specifically, phagocytosis was greater than control at the 30x and lOOx concentrations, and fell off rapidly below 30x and at the lOOOx concentration. Thus, the assay is capable of detecting loss of potency with 3 -fold changes in the quantity of AMMO components used to stimulate the macrophages, which indicates sensitivity to detect loss of potency in a future manufacturing run.Clauses

[0176] Clause 1. A composition comprising an effective amount of each of: MPL monophosphoryl lipid (MPL), D-mannose, and aluminum hydroxide (AHO) (collectively AMMO), with the proviso that the composition does not comprise an antigen effective to induce an immune response against a pathogen and / or a Whole Glucan Particle (WGP), optionally wherein the pathogen is selected from a viral pathogen, a bacterial pathogen or a fungal pathogen.

[0177] Clause 2. A composition consisting essentially of an effective amount of each of: MPL mono-phosphoryl lipid (MPL), D-mannose, and aluminum hydroxide (AMMO), with the proviso that the composition does not comprise an antigen effective to induce an immune response against a pathogen and / or Whole Glucan Particle (WGP), optionally wherein the pathogen is selected from a viral pathogen, a bacterial pathogen or a fungal pathogen.

[0178] Clause 3. A composition consisting of as active immune inducing agents, an effective amount of each of: MPL mono-phosphoryl lipid (MPL), D-mannose, and aluminum hydroxide (collectively AMMO) with the proviso that the composition does not comprise an antigen effective to induce an immune response against a pathogen and / or Whole Glucan Particle (WGP), optionally wherein the pathogen is selected from a viral pathogen, a bacterial pathogen or a fungal pathogen.

[0179] Clause 4. A composition comprising an effective amount of each of: MPL monophosphoryl lipid (MPL) and D-mannose with the proviso that the composition does not comprise an antigen effective to induce an immune response against a pathogen and / or Whole Glucan Particle (WGP) and AHO, optionally wherein the pathogen is selected from a viral pathogen, a bacterial pathogen or a fungal pathogen.

[0180] Clause 5. A composition consisting essentially of an effective amount of each of: MPL mono-phosphoryl lipid (MPL) and D-mannose, with the proviso that the composition does not comprise an antigen effective to induce an immune response against a pathogen and / or Whole Glucan Particle (WGP) and AHO, optionally wherein the pathogen is selected from a viral pathogen, a bacterial pathogen or a fungal pathogen.

[0181] Clause 6. A composition consisting of as active immune inducing agents, an effective amount of each of: MPL mono-phosphoryl lipid (MPL)and D-mannose with the proviso that the composition does not comprise an antigen effective to induce an immuneresponse against a pathogen and / or Whole Glucan Particle (WGP) and AHO, optionally wherein the pathogen is selected from a viral pathogen, a bacterial pathogen or a fungal pathogen.

[0182] Clause 7. The composition of any one of clauses 1-6, wherein the antigen does not induce a T cell or a B cell adaptive immunity.

[0183] Clause 8. The composition of any one of clauses 1-7, wherein the effective amount of the aluminum hydroxide, MPL mono-phosphoryl lipid (MPL), and D-mannose or MPL and D-mannose, is collectively effective to induce an immune response when administered to a subject in need thereof.

[0184] Clause 9. The composition of any one of clauses 1-8, wherein the composition comprises the following ratios of AHO: MPL: D-mannose: (a) about lx : about lx : about lx; or (b) about lx : about 3x : about 3x; or (c) about lx : about lOx : about lOx; or (d) about lx : about 30x : about 30x, wherein lx AHO = 0.5 mg; lx MPL = 0.05 mg; and lx D- Mannose = 0.5 mg.

[0185] Clause 10. The composition of clause 9, wherein the immune response is nonspecific to the pathogen.

[0186] Clause 11. The composition of any one of clauses 1-10, wherein the antigen is selected from a peptide, a protein or a glycoprotein.

[0187] Clause 12. The composition of any one of clauses 1-11, wherein the aluminum hydroxide (AHO) is an aluminum hydroxide wet suspension, optionally Alhydrogel®.

[0188] Clause 13. The composition of any one of clauses 1-3 or 7-12, wherein the effective amount comprises: (a) from about 0.1 mg / ml to about 10 mg / ml of aluminum hydroxide; (b) from about 0.1 mg / ml to about 10 mg / ml of MPL; and (c) from about 0.01 mg / ml to about 10 mg / ml of D-mannose.

[0189] Clause 14. The composition of any one of clauses 1-13, further comprising a pharmaceutically acceptable carrier, optionally saline or phosphate buffered saline.

[0190] Clause 15. A method to enhance immunity in a subject against an infection caused by a bacterial or fungal pathogen, the method comprising administering to the subject an effective amount of a composition of any one of clauses 1-14.[0191J Clause 16. The method of clause 14, wherein the bacterial pathogen is selected from S. aureus, A. baumannii, K. Pneumoniae, P. aeruginosa, E. coli, Enterobacter spp., Serratia, Stenotrophomonas, and the fungal pathogen is selected from Candida spp.

[0192] Clause 17. The method of clause 15 or 16, wherein the composition is administered by a method comprising inhalation, intramuscular, subcutaneous, or intravenous administration.

[0193] Clause 18. The method of any one of clauses 15-17, wherein the composition is administered once, twice, or three times over the period of one to three months.

[0194] Clause 19. The method of any one of clauses 15-17, wherein the subject is at risk of a bacterial or fungal infection, or wherein the subject is infected with the bacterial or fungal pathogen.

[0195] Clause 20. The method of any one of clauses 15-17, wherein the subject is immunocompromised.

[0196] Clause 21. The method of any one of clauses 15-20, further comprising assaying the subject for a bacterial or fungal infection prior to administration of the composition.

[0197] Clause 22. A method to treat or prevent a bacterial or fungal infection or a disorder caused by a bacterial or fungal infection in a subject in need thereof, the method comprising administering an effective amount of the composition of any one of clauses 1-14.

[0198] Clause 23. The method of clause 22, wherein the bacterial or fungal pathogenis selected from S. aureus, A. baumannii, K. Pneumoniae, P. aeruginosa, E. coli, Enterobacter spp., Serratia, Stenotrophomonas, Candida spp.

[0199] Clause 24. The method of clause 22 or 23, wherein the composition is administered by a method comprising parenteral administration, including subcutaneous, intra-muscular, or intravenous.

[0200] Clause 25. The method of any one of clauses 22-24, wherein the composition is administered once, twice, or three times.

[0201] Clause 26. The method of any one of clauses 22-25, further comprising assaying the subject for a bacterial or fungal infection prior to administration of the composition.

[0202] Clause 27. The method of any one of clauses 22-25, wherein the disorder is a blood or lung infection caused by a nosocomial pathogen.[0203J Clause 28. A kit comprising the composition of any one of clauses 1-14 and instructions for use.

[0204] Clause 29. A method to identify an compound or agent that provides a benefit selected from one or more of: enhances immunity against a bacterial or fungal microbial infection or treats a bacterial or fungal infection or a disease related to a bacterial or fungal microbial infection, the method comprising admixing the compound or agent with the composition of any one of clauses 1-43 and administering the admixed composition to a non-human subject infected with a bacterial or fungal microorganism and assaying for postadministration infection or survival, wherein the compound or agent that enhances the activity of the composition of any one of claims 1-14 is an agent that provides the benefit.Equivalents

[0205] It is to be understood that while the disclosure has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the disclosure. Other aspects, advantages and modifications within the scope of the disclosure will be apparent to those skilled in the art to which the disclosure pertains.

[0206] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” “containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.

[0207] Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this invention. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

[0208] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0209] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0210] All applications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each are incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising an effective amount of each of: MPL mono-phosphoryl lipid (MPL), D-mannose, and aluminum hydroxide (AHO) (collectively AMMO), with the proviso that the composition does not comprise an antigen effective to induce an immune response against a pathogen and / or a Whole Glucan Particle (WGP), optionally wherein the pathogen is selected from a viral pathogen, a bacterial pathogen or a fungal pathogen.

2. A composition consisting essentially of an effective amount of each of: MPL monophosphoryl lipid (MPL), D-mannose, and aluminum hydroxide (AMMO), with the proviso that the composition does not comprise an antigen effective to induce an immune response against a pathogen and / or Whole Glucan Particle (WGP), optionally wherein the pathogen is selected from a viral pathogen, a bacterial pathogen or a fungal pathogen.

3. A composition consisting of as active immune inducing agents, an effective amount of each of: MPL mono-phosphoryl lipid (MPL), D-mannose, and aluminum hydroxide (collectively AMMO) with the proviso that the composition does not comprise an antigen effective to induce an immune response against a pathogen and / or Whole Glucan Particle (WGP), optionally wherein the pathogen is selected from a viral pathogen, a bacterial pathogen or a fungal pathogen.

4. A composition comprising an effective amount of each of: MPL mono-phosphoryl lipid (MPL) and D-mannose with the proviso that the composition does not comprise an antigen effective to induce an immune response against a pathogen and / or Whole Glucan Particle (WGP) and AHO, optionally wherein the pathogen is selected from a viral pathogen, a bacterial pathogen or a fungal pathogen.

5. A composition consisting essentially of an effective amount of each of: MPL monophosphoryl lipid (MPL) and D-mannose, with the proviso that the composition does not comprise an antigen effective to induce an immune response against a pathogen and / or Whole Glucan Particle (WGP) and AHO, optionally wherein the pathogen is selected from a viral pathogen, a bacterial pathogen or a fungal pathogen.

6. A composition consisting of as active immune inducing agents, an effective amount of each of: MPL mono-phosphoryl lipid (MPL)and D-mannose with the proviso that the composition does not comprise an antigen effective to induce an immune response against a pathogen and / or Whole Glucan Particle (WGP) and AHO, optionally wherein the pathogen is selected from a viral pathogen, a bacterial pathogen or a fungal pathogen.

7. The composition of any one of claims 1-6, wherein the antigen does not induce a T cell or a B cell adaptive immunity.

8. The composition of any one of claims 1-7, wherein the effective amount of the aluminum hydroxide, MPL mono-phosphoryl lipid (MPL), and D-mannose or MPL and D- mannose, is collectively effective to induce an immune response when administered to a subject in need thereof.

9. The composition of any one of claims 1-8, wherein the composition comprises the following ratios of AHO: MPL: D-mannose: a) about lx : about lx : about lx; or b) about lx : about 3x : about 3x; or c) about lx : about lOx : about lOx; or d) about lx : about 30x : about 30x, wherein lx AHO = 0.5 mg; lx MPL = 0.05 mg; and lx D-Mannose = 0.5 mg.

10. The composition of claim 9, wherein the immune response is non-specific to the pathogen.

11. The composition of any one of claims 1-10, wherein the antigen is selected from a peptide, a protein or a glycoprotein.

12. The composition of any one of claims 1-11, wherein the aluminum hydroxide (AHO) is an aluminum hydroxide wet suspension, optionally Alhydrogel®.

13. The composition of any one of claims 1-3 or 7-12, wherein the effective amount comprises: a) from about 0.1 mg / ml to about 10 mg / ml of aluminum hydroxide; b) from about 0.1 mg / ml to about 10 mg / ml of MPL; and c) from about 0.01 mg / ml to about 10 mg / ml of D-mannose.

14. The composition of any one of claims 1-13, further comprising a pharmaceutically acceptable carrier, optionally saline or phosphate buffered saline.

15. A method to enhance immunity in a subject against an infection caused by a bacterial or fungal pathogen, the method comprising administering to the subject an effective amount of a composition of any one of claims 1-14.

16. The method of claim 14, wherein the bacterial pathogenis selected from S. aureus,A. baumannii, K. Pneumoniae, P. aeruginosa, E. coli, Enterobacter spp., Serratia, Stenotrophomonas, and the fungal pathogen is selected from Candida spp.

17. The method of claim 15 or 16, wherein the composition is administered by a method comprising inhalation, intramuscular, subcutaneous, or intravenous administration.

18. The method of any one of claims 15-17, wherein the composition is administered once, twice, or three times over the period of one to three months.

19. The method of any one of claims 15-17, wherein the subject is at risk of a bacterial or fungal infection, or wherein the subject is infected with the bacterial or fungal pathogen.

20. The method of any one of claims 15-17, wherein the subject is immunocompromised.

21. The method of any one of claims 15-20, further comprising assaying the subject for a bacterial or fungal infection prior to administration of the composition.

22. A method to treat or prevent a bacterial or fungal infection or a disorder caused by a bacterial or fungal infection in a subject in need thereof, the method comprising administering an effective amount of the composition of any one of claims 1-14.

23. The method of claim 22, wherein the bacterial or fungal pathogenis selected from S. aureus, A. baumannii, K. Pneumoniae, P. aeruginosa, E. coli, Enterobacter spp., Serratia, Stenotrophomonas, Candida spp.

24. The method of claim 22 or 23, wherein the composition is administered by a method comprising parenteral administration, including subcutaneous, intra-muscular, or intravenous.

25. The method of any one of claims 22-24, wherein the composition is administered once, twice, or three times.

26. The method of any one of claims 22-25, further comprising assaying the subject for a bacterial or fungal infection prior to administration of the composition.

27. The method of any one of claims 22-25, wherein the disorder is a blood or lung infection caused by a nosocomial pathogen.

28. A kit comprising the composition of any one of claims 1-14 and instructions for use.-SO-29. A method to identify an compound or agent that provides a benefit selected from one or more of: enhances immunity against a bacterial or fungal microbial infection or treats a bacterial or fungal infection or a disease related to a bacterial or fungal microbial infection, the method comprising admixing the compound or agent with the composition of any one of claims 1-14 and administering the admixed composition to a non-human subject infected with a bacterial or fungal microorganism and assaying for post-administration infection or survival, wherein the compound or agent that enhances the activity of the composition of any one of claims 1-14 is an agent that provides the benefit.

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