Triple vaccine protects against bacterial and fungal pathogens via trained immunity

Abstract

An optimized protein-free three-component vaccine that 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. The present disclosure provides an entirely new vaccine-derived approach based on adjuvants without protein or polypeptide antigens that mediates short- to medium-duration, broad-spectrum protection against lethal HAIs caused by bacteria (including antibiotic-resistant bacteria) and fungi. The approach is based on an entirely new horizontal transmission prevention rather than vertical transmission prevention strategy.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 321,961, filed March 21, 2022, the contents of which are incorporated by reference herein in their entirety.

[0002] Statement of Government Support This invention was made with government support under Grant Nos. AI145759, AI130060, AI106375, and AI139052 awarded by the National Institutes of Health (NIH). The Government has certain rights in this invention. [Background technology]

[0003] background Throughout this application, several technical publications are referenced by Arabic numerals. Full bibliographic citations for each reference are found immediately preceding the claims. The contents of each such referenced publication, and the publications referenced within, are incorporated by reference into this disclosure in order to more fully describe the state of the art to which this invention pertains.

[0004] Approximately 7% of hospitalized patients develop healthcare-associated infections (HAIs), of which approximately half consist of pneumonia, bloodstream infections, or wound infections. This is perhaps the most amenable to immunological intervention (WHOrganization). On any given day, approximately 3% of hospital inpatients have HAIs (SS Magill et al., N Engl J Med 370, 2014). In acute care hospitals in the United States, this results in 722,000 HAIs, more than 75,000 deaths, and an economic burden of over $100 billion annually (A. Marchetti et al., J Med Econ 16, 2013). In the majority of cases, HAIs are caused by antibacterial-resistant bacterial and fungal pathogens, which are associated with worse mortality and morbidity than antimicrobial-susceptible pathogens (LM Weiner et al., Infect Control Hosp Epidemiol 37, 2016). Despite the high incidence of HAIs, there are no FDA-approved vaccines against the most commonly encountered and antibiotic-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Acinetobacter baumannii (CRAB), and carbapenem-resistant Enterobacterales (CRE). Broadly speaking, there are two approaches to prevent infection: horizontal and vertical (RP Wenzel et al., Int J Infect Dis 14 Suppl 4, 2010). Horizontal approaches protect against a broad range of pathogens; examples include disinfecting patient rooms with ultraviolet light or bleach, hand washing, and personal protective equipment (PPE). Horizontal approaches provide protection against a broad range of infectious organisms and offer the best overall value, but implementing and maintaining these approaches is generally challenging. In comparison, vertical approaches provide specific protection against only one organism. Thus, vertical approaches are much narrower in focus; examples include active surveillance and testing for specific pathogen species, decolonization of hospitalized patients with MRSA, and vaccination against certain pathogens (e.g., pneumococcus, influenza). Traditional vaccines only protect against one specific pathogen, whereas providing a powerful way to prevent infection via long-term adaptive immunity. Moreover, vaccine development is costly, time-consuming, and scientifically challenging. The merits of both approaches are often debated, but the horizontal approach has been shown to have a broader impact at less cost (E. Septimus et al., Infect Control Hosp Epidemiol 35, 2014). Nevertheless, strategies that seek to integrate both approaches are most successful in preventing a greater disease burden than either approach alone. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] SS Magill et al., N Engl J Med 370, 2014 [Non-Patent Document 2] A. Marchetti et al., J Med Econ 16, 2013 [Non-Patent Document 3] LM Weiner et al., Infect Control Hosp Epidemiol 37, 2016 Summary of the Invention [Means for solving the problem]

[0006] Disclosure Summary The present disclosure provides a novel vaccine-derived approach based on adjuvants without protein or polypeptide antigens that mediates short- to medium-duration broad-spectrum protection against lethal HAIs caused by bacteria (including antibiotic-resistant bacteria) and fungi. This approach is based on a novel horizontal infection prevention rather than vertical infection prevention strategy. In one aspect, the vaccine or composition does not contain, does not contain, is essentially free of, or is free of substances such as proteins or polypeptides that induce or elicit lymphocyte-mediated (T-cell or B-cell) adaptive immunity. In some embodiments, the antigen can be a virus-, fungus-, or bacteria-derived protein or polypeptide that can induce or elicit T-cell and / or B-cell adaptive immunity.

[0007] A "horizontal" infection prevention strategy refers to an approach that prevents infections caused by many different pathogens at once, which is preferable to approaches that prevent infections by one pathogen at a time. (7) The present disclosure utilized a horizontal infection prevention strategy by immunizing with a broadly active vaccine adjuvant to provide innate immune defense against Gram-positive and Gram-negative bacterial and fungal pathogens.

[0008] The results show that a combination comprising, consisting essentially of, or consisting of an effective amount of each of mannan, monophosphoryl lipid (MPL), and aluminum hydroxide (combined, referred to herein as "MMA or MMMA") induces protection and is associated with epigenetic changes in macrophages. Removal of macrophages (but not B and T lymphocytes) prevented MMA from protecting against infection. Without being bound by theory, this suggests that MMA-induced protection may be induced by macrophage immune memory and not by lymphocyte-mediated adaptive immunity. Without being bound by theory, applicants have shown that immune regulation by macrophages is the main reason why MMA improved survival during lethal infection with a variety of pathogens across taxonomic kingdoms, including gram-positive bacteria, gram-negative bacteria, and fungi.

[0009] Thus, in one aspect, provided herein is a composition comprising, essentially consisting of, or even more consisting of an effective amount of each of MPL monophosphoryl lipid (MPL), mannan, and aluminum hydroxide ("MMA"), provided that said composition does not contain an effective antigen for inducing a B cell or T cell memory immune response against any pathogen (e.g., viral, fungal, or bacterial pathogen). In another aspect, provided herein is a composition consisting essentially of an effective amount of each of MPL monophosphoryl lipid (MPL), mannan, and aluminum hydroxide (collectively "MMA"), provided that said composition does not contain an effective antigen for inducing an immune response against any pathogen (e.g., viral, fungal, or bacterial pathogen). Still further provided is a composition consisting of an effective amount of each of MPL monophosphoryl lipid (MPL), mannan, and aluminum hydroxide (collectively "MMA"), as an active immune-inducing agent. In yet a further aspect, the effective amount of aluminum hydroxide, MPL monophosphoryl lipid (MPL) and mannan 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 not T cell adaptive immunity or B cell adaptive immunity.

[0010] Applicants have unexpectedly found that the triple combination "MMA" provides better protection against S. aureus than a triple adjuvant combination composed of aluminum hydroxide, MPL, and whole glucan particles, and that the triple combination MMA is more effective against A. baumannii blood and lung infections and against mucormycosis than a quadruple regimen including aluminum hydroxide, MPL, whole glucan particles, and mannan. Thus, the unique triple combination "MMA" provides better protection against gram-positive, gram-negative, and fungal infections than triple combinations in which whole glucan particles are used instead of mannan, or even quadruple combinations including both whole glucan particles and mannan.

[0011] For purposes of this disclosure, the aluminum hydroxide is Al(OH)3, typically found in nature as the mineral gibbsite. It is commercially available and can be an aluminum hydroxide gel or a wet suspension, optionally commercially available as Alhydrogel® (Sigma-Aldrich) or Al(OH)3 gel (Accurate Chemical & Scientific Corporation Cat. # A1090S) or Al(OH)3 gel (Croda Cat. # AJV3012). The percentage of Al(OH)3 in the feed can be any percentage, non-limiting examples of such include compositions of 2% or 1% and combinations thereof.

[0012] In one embodiment, the components of the MMA are combined in a liquid suspension containing 0.2-1 mg aluminum hydroxide; about 0.01-1.5 mg MPL; and about 0.1-5 mg mannan in a total volume of 0.5-1 ml.

[0013] As will be apparent to one of skill in the art, the compositions may be combined with pharma- ceutically acceptable carriers, such as phosphate buffered saline (PBS) and DMSO, for ease of storage and for administration. The compositions may further include stabilizers and components that maintain stability, for example, during storage or lyophilization.

[0014] The present disclosure also provides a method of enhancing immunity in a subject against infection caused by a pathogen (e.g., a bacterial or fungal pathogen) by a method comprising, consisting essentially of, or even further consisting of administering to the subject an effective amount of a composition as described herein (e.g., MMA). The present disclosure also provides a composition or medicament for use to enhance immunity in a subject against infection caused by a pathogen (e.g., a bacterial or fungal pathogen), wherein the medicament comprises, consists essentially of, or even further consists of a composition as described herein (e.g., MMA). Boosting immunity contemplates the outcome of protection from disease or symptoms and may be monitored clinically (e.g., fever, cough, or pain) or sub-clinically (e.g., enhanced innate immunity as measured by the presence or increased presence of macrophages and / or monocytes, reduced bacterial or fungal load, lower pro-inflammatory cytokines (e.g., IL-6, IL-12, TNF) and elevated IL-10 / TNF).

[0015] Further provided is a method for treating or preventing bacterial or fungal infection or a disorder caused by bacterial or fungal infection in a subject in need of such treatment or prevention, said method comprising, or alternatively consisting essentially of, or even further consisting of administering an effective amount of the composition (e.g., MMA) of the present disclosure.Also provided is a composition for use or medicine as described herein for treating or preventing bacterial or fungal infection or a disorder caused by bacterial or fungal infection in a subject in need of such treatment or prevention.

[0016] Non-limiting examples of bacteria are selected from S. aureus, A. baumannii, K. pneumoniae, P. aeruginosa, E. coli, Enterobacter spp., Serratia, Stenotrophomonas, and the fungi may be Candida spp or species that cause the disease mucormycosis (e.g., Rhizopus spp).

[0017] Any suitable method of administration may be used. Non-limiting examples include topical, inhalation, intramuscular, subcutaneous, or intravenous administration, as determined by the treating physician. The composition may be administered once, twice, or three times over a period of 1 to 3 months or longer.

[0018] In one aspect, the subject is infected with a bacterial or fungal microorganism or is at risk for a bacterial or fungal infection.

[0019] The treatment methods, uses and medicaments may further be combined with suitable diagnostics to assay the subject for bacterial or fungal infection prior to, during and / or upon administration of the composition.

[0020] The subjects to be treated include mammals, such as rats, mice, dogs, cats, humans, etc. When performed in non-human animals, the methods provide suitable animal models for testing new disease agents or combination therapies. When performed in human subjects, the methods are viable therapeutic modalities.

[0021] Further provided are kits containing the compositions as described herein and instructions for use.

[0022] Still further provided is a method for identifying a compound or agent that provides benefits selected from one or more of: enhancing immunity against bacterial or fungal microorganism infection, or treating bacterial or fungal infection, or disease associated with bacterial or fungal microorganism infection, comprising mixing said compound or agent with a composition as disclosed herein, administering said mixed composition to a non-human subject infected with bacterial or fungal microorganism, and assaying infection or survival rate after administration, wherein said compound or agent enhances the activity of the composition disclosed herein.Methods for determining whether benefits are provided are known in the art, and are provided briefly herein. [Brief description of the drawings]

[0023] [Figure 1-1]Figures 1A-1H show that adjuvant-only vaccines provide short-term protection against lethal bloodstream infection in a dose-dependent manner. All vaccinations were administered as 0.2 ml suspensions in phosphate-buffered saline (PBS). (Figure 1A) Female BALB / c mice (N=8 / group) were immunized subcutaneously (SC) and boosted 3 weeks later with 10 μg monophosphoryl lipid A (MPL), 100 μg whole glucan particles (WGP), 200 μg Al(OH)3, 0.826 μg LPS, and / or recombinant protein antigen (Ag). Three weeks after the boost, mice were infected intravenously (IV) via the tail vein with 4.5×107 CFU S. aureus LAC. (Fig. 1B) Female BALB / c mice (N=8 / group) were immunized SC with 10 μg MPL, 100 μg WGP, and 200 μg Al(OH)3; half of the mice were boosted 3 weeks after the first immunization. Three, seven, or 21 days after the final immunization, mice were infected IV via the tail vein with 4.9×107 CFU S. aureus LAC. (Fig. 1C) Male C3HeB / Fe mice (N=6 / group) were immunized SC with 10 μg MPL, 100 μg WGP, and 200 μg Al(OH)3. Three or seven days after immunization, mice were infected IV via the tail vein with 1.74×107 CFU A. baumannii HUMC1. (Fig. 1D) Male C3HeB / Fe mice (N=6 / group) were immunized SC with 0, 1, 2, or all three doses of adjuvant: 10 μg MPL, 100 μg WGP, and 0.2 mg Al(OH)3. Three days later, mice were infected IV via tail vein with 2.5×107 CFU A. baumannii HUMC1. (Fig. 1E) Male C3HeB / Fe mice (N=8 / group) were immunized SC with 10 μg MPL, 100 μg WGP, and 0.2 mg Al(OH)3. Three days after immunization, mice were infected via oral aspiration (OA) with 1.5×108 CFU A. baumannii HUMC1. (FIG. 1F) Male C3HeB / Fe mice (N=6 / group) were immunized SC with 10 μg MPL, 100 μg WGP, and 0.2 mg Al(OH)3.Three or seven days after immunization, mice were infected IV via the tail vein with 3.8×108 CFU K. pneumoniae KP3. (Figure 1G) Female BALB / c mice (N=10 / group) were immunized SC with phosphate-buffered saline (PBS), MWA (10 μg MPL, 100 μg WGP, and 0.2 mg Al(OH)3), MWA 10% (1 μg MPL, 10 μg WGP, and 0.2 mg Al(OH)3), or MWA 1% (0.1 μg MPL, 1 μg WGP, and 0.2 mg Al(OH)3). Three days after immunization, mice were infected IV via the tail vein with 8.4×107 CFU S. aureus LAC. (Fig. 1H) Male C3HeB / Fe mice (N = 5 / group) were immunized SC with PBS, MWA (10 μg MPL, 100 μg WGP, and 0.2 mg Al(OH)3), MWA 10% (1 μg MPL, 10 μg WGP, and 0.2 mg Al(OH)3), or MWA 30% (3 μg MPL, 30 μg WGP, and 0.2 mg Al(OH)3). Three days after immunization, mice were infected IV via the tail vein with 4.6 × 107 CFU A. baumannii HUMC1. Survival rates were compared by nonparametric log-rank test at α = 0.05. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 vs. PBS group. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above.

[0024] [Figure 2-1]Figures 2A-2E show that replacing WGP with mannan expanded coverage and increased protective efficacy. All vaccinations were administered SC as 0.2 ml suspensions in PBS. MMA (100 μg mannan, 10 μg MPL, and 0.2 mg Al(OH)3); or MMWA (100 μg mannan, 10 μg MPL, 100 μg WGP, and 0.2 mg Al(OH)3). Three days after immunization, (Figure 2A) 6.9 × 107 CFU S. aureus LAC was IV-infected into female BALB / c mice (N = 8 / group) via the tail vein. (Figure 2B) 1.4–2.9 × 107 CFU A. baumannii HUM1 was IV-infected into male C3HeB / Fe mice (N = 5 / group) via the tail vein. (Fig. 2C) 2.4 × 108 CFU K. pneumoniae KPC-KP1 was IV-infected into male C3HeB / Fe mice (N = 5 / group) via the tail vein; (Fig. 2D) 3.0 × 103 CFU Rhizopus delemar 99-880 was IV-infected into female BALB / c mice (N = 5 / group) via the tail vein; and (Fig. 2E) 1.6 × 108 CFU A. baumannii HUMC1 was IV-infected into male C3HeB / Fe mice (N = 5 / group) via oral aspiration (OA). Survival rates were compared by nonparametric log-rank test at α = 0.05. *p ≤ 0.05, **p ≤ 0.01 vs. PBS group. [Figure 2-2] Same as above. [Figure 2-3] Same as above.

[0025] [Figure 3-1]Figures 3A-3H show increased duration of protection with higher adjuvant doses and innate versus adaptive immunity. All vaccinations were administered SC as a 0.2 ml suspension in PBS. MMA 1x (100 μg mannan, 10 μg MPL, and 0.2 mg Al(OH)3); MMA 3x (300 μg mannan, 30 μg MPL, and 0.2 mg Al(OH)3), or MMA 10x (1,000 μg mannan, 100 μg MPL, and 0.2 mg Al(OH)3). Seven, 14, or 21 days after immunization, (Fig. 3A) 9.7 × 107 CFU S. aureus LAC was IV-infected into female BALB / c mice (N = 8 / group) via the tail vein, (Fig. 3B) 2.9 × 107 CFU A. baumannii HUMC1 was IV-infected into male C3HeB / Fe mice (N = 5 / group) via the tail vein, (Fig. 3C) MMA-immunized male C3HeB / Fe and female BALB / c mice (N = 5 / group) did not develop specific antibodies against S. aureus or A. baumannii. (Fig. 3D) 1.2×108 CFU S. aureus LAC was IV-infected into female BALB / c mice (N=10 / group) via the tail vein, (Fig. 3E) 24 h after immunization, 2.9×107 CFU A. baumannii HUMC1 was IV-infected into male C3HeB / Fe mice (N=5 / group) via the tail vein, (Fig. 3F) 3 days after MMA immunization, 1.4×107 CFU A. baumannii HUMC1 was IV-infected into male C57BL / 6 mice (N=4 / group) harboring humanized CD34+ cells via the tail vein. (Figure 3G) 2.4x107 CFU A. baumannii HUMC1 was IV-infected into male C3HeB / Fe mice (N=5 / group) via the tail vein, (Figure 3H) 3x108 CFU S. aureus LAC was IV-infected into female BALB / c mice (N=8 / group) via the tail vein 3 days after immunization with research-grade and GMP-grade MMA. Survival rates were compared by nonparametric log-rank test at α=0.05. *p≦0.05, **p≦0.01 vs. PBS group. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Diagram 3-4] Same as above.

[0026] [Figure 4] Figure 4 (8 panels) shows the percentage of CD45+ immune cell population changes in response to MMA immunization. Male C3HeB / Fe mice (N=5 / group) were immunized SC with MMA (100 μg mannan, 10 μg MPL, and 0.2 mg Al(OH)3) in 0.2 ml suspension in PBS. Splenocytes were harvested before immunization or 1, 3, 7, 14, or 21 days after immunization, and cell populations were determined by multicolor cytometry. Percentages of CD45+ cells that were T cells (TCRβ+), B cells (CD19+), dendritic cells (CD11C+), natural killer (NK) cells (NKp46+CD49b+), and percentages of myeloid cells (CD45+CD11b+) that were neutrophils (Ly6C+Ly6G+), monocytes (Ly6C+Ly6G-), and macrophages (Ly6C-Ly6G-). Cell populations were compared by Kruskal-Wallis test with α=0.05. *p≦0.05, **p≦0.01 vs. day 0.

[0027] [Figure 5-1]Figures 5A-5G show that macrophages are key to induce MMA-mediated protection. (Figure 5A) Male C57BL / 6J wild-type (WT) mice (N=5 / group) and RAG1 knockout (RAG1-KO) mice (N=6 / group) were immunized SC with 0.2 ml suspension of MMA (100 μg mannan, 10 μg MPL, and 0.2 mg Al(OH)3) in PBS and infected IV via the tail vein with 3.9-7.8 × 107 CFU A. baumannii HUMC1. (Figure 5B-5G) Male C3HeB / Fe mice (N=5 / group) and female BALB / c mice (N=8 / group) were depleted of neutrophils with cyclophosphamide, macrophages and monocytes with liposomal clodronate, or natural killer (NK) cells with anti-Asialo-GM1 antibody. Mice were then immunized with MMA (100 μg mannan, 10 μg MPL, and 0.2 mg Al(OH)3) and infected IV via the tail vein with A. baumannii HUMC1 or S. aureus LAC. MMA stimulated primary human macrophages (Figure 5F). [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 5-4] Same as above.

[0028] [Figure 6-1]Figures 6A-6D show the reduction of pro-inflammatory cytokines after infection in MMA-immunized mice. Mice were immunized SC with 0.2 ml suspension in PBS containing MMA (100 μg mannan, 10 μg MPL, and 0.2 mg Al(OH)3) 1, 3, 7, 14, or 21 days before infection. (Figure 6A) 1.5×108 CFU S. aureus LAC was infected IV via the tail vein into female BALB / c mice (N=5 / group). Kidney bacterial burden was assessed 4 hours after infection. (Figure 6B) 1.8×107 CFU A. baumannii HUMC1 was infected IV via the tail vein into male C3HeB / Fe mice (N=5 / group). Blood bacterial burden was assessed 4 hours after infection. (Figures 6C-6D) Plasma from each mouse was then analyzed by Luminex for five cytokines: IL-4, IL-6, IL-10, IL-12 p70, and TNF. [Figure 6-2] Same as above. [Figure 6-3] Same as above.

[0029] [Figure 7] Figure 7 shows that 0.2 ml SC vaccination with PBS containing the MMA triple vaccine (100 μg mannan, 10 μg MPL, and 0.2 mg Al(OH)3) is more effective than other vaccine combinations, including the double vaccine combination, the original MWA triple combination (containing whole glucan particle [WGP] instead of mannan, with aluminum hydroxide and MPL), or even quadruple vaccination with MMA+WGP (mannan, aluminum hydroxide, MPL and WGP) in mice (n=8 mice / group).

[0030] [Figure 8]FIG. 8 shows that 0.2 ml SC vaccination with PBS containing the MMA triple vaccine (1x = 100 μg mannan, 10 μg MPL, and 0.2 mg Al(OH)3; 0.3x = 30 μg mannan, 3 μg MPL, 200 μg Al(OH)3) is at least as effective as the quadruple adjuvant (MMA + WGP) against A. baumannii bloodstream infection (n = 5 mice / group).

[0031] [Figure 9] Figure 9 shows that 0.2 ml SC vaccination with PBS containing MMA triple vaccine (100 μg mannan, 10 μg MPL, and 0.2 mg Al(OH)3) is more effective than the quadruple vaccine (MMA + WGP) against pneumonia caused by A. baumannii (n = 5 mice / group).

[0032] [Figure 10] FIG. 10 shows that 0.2 ml SC vaccination with PBS containing the MMA triple vaccine (100 μg mannan, 10 μg MPL, and 0.2 mg Al(OH) 3 ) protects against PDR K. pneumonia. PDR K pneumonia KPCKP1 (2×10 8 inoculum) in bloodstream infection.

[0033] [Figure 11] FIG. 11 shows that 0.2 ml SC vaccination with PBS containing MMA triple vaccine (100 μg mannan, 10 μg MPL, and 0.2 mg Al(OH)3) protects against Candida albicans. Highly pathogenic C. albicans SC5314 (2×105 inoculum) in bloodstream infection was tested. BALB / c mice were vaccinated and infected. This is an aggressive challenge since the inoculum can be reduced 4-fold from 2×105 and still cause 100% mortality in non-vaccinated mice. N=5 mice / group. P<0.05 vs. phosphate buffered saline.

[0034] [Figure 12] Figures 12A and 12B show that 0.2 ml SC vaccination with PBS containing MMA vaccine (100 μg mannan, 10 μg MPL, and 0.2 mg Al(OH)3) protected against mucormycosis, a fungal disease caused by Rhizopus delamar, in neutropenic mice and was more effective than the quadruple vaccine (MMA + WGP). BALB / c mice were made neutropenic with cyclophosphamide, immunized, and 3 days later, infected IV with the fungus, Rhizopus delamar. (Figure 12A) The quadruple vaccine (MMA + WGP) protected the mice. (Figure 12B) The MMA triple vaccine alone was more effective than the quadruple vaccine (MMA + WGP) even with a larger inoculum. A modified triple vaccine (containing 100 μg WGP + 100 μg mannan + 200 μg aluminum hydroxide) in which MPL was replaced with WGP was not effective. This highlights the non-trivial nature of the MMA triple combination. *p<0.05 vs. PBS.

[0035] [Figure 13] Figure 13 shows that 0.2 ml SC vaccination with PBS containing MMA vaccine protects up to 24 hours even in neutropenic mice. Highly pathogenic A. baumannii HUMC1 was tested in bloodstream infection. C3H mice (half made neutropenic / immunocompromised with cyclophosphamide on day -3) were immunized with 100 μg mannan, 10 μg MPL, and 0.2 mg Al(OH)3 on day -1 and infected with A. baumannii HUMC1 24 hours later (day 0). *p<0.05 vs. PBS.

[0036] [Figure 14]Figures 14A and 14B show that higher doses of the MMA triple vaccine administered SC as a 0.2 ml suspension in PBS protect against A. baumannii for up to 21 days. HUMC1 (2.9x107 inoculum) in bloodstream infection was tested. A. baumannii HUMC1 was infected IV via the tail vein into C3HeB / FeJ mice (n=5 / group) 14 days (Figure 14A) or 21 days (Figure 14B) after 1x (100μg mannan, 10μg MPL, and 0.2mg Al(OH)3), 3x (300μg mannan, 30μg MPL, and 0.2mg Al(OH)3) or 10x dose (1000μg mannan, 100μg MPL, and 0.2mg Al(OH)3) of the MMA triple vaccine regimen.

[0037] [Figure 15] Figures 15A and 15B show that higher doses of the MMA triple vaccine administered SC as a 0.2 ml suspension in PBS protect against S. aureus for up to 21 days. LAC (2.4x107 inoculum) in bloodstream infection was tested. BALB / c mice (n=8 / group) were infected IV via the tail vein with S. aureus LAC 14 days (Figure 15A) or 21 days (Figure 15B) after 1x (100μg mannan, 10μg MPL, and 0.2mg Al(OH)3), 3x (300μg mannan, 30μg MPL, and 0.2mg Al(OH)3) or 10x doses (1000μg mannan, 100μg MPL, and 0.2mg Al(OH)3) of the MAA triple vaccine regimen.

[0038] [Figure 16]Figure 16 shows that humanized mice infected with A. baumannii. HUMC1 in bloodstream infection: Efficacy was tested in CD34+ human stem cell transplants. A. baumannii HUMC1 was IV infected into human immune stem cell transplanted mice (n=5 / group) via tail vein 3 days after 1x dose of MMA triple vaccine administered SC as 0.2ml suspension (100μg mannan, 10μg MPL, and 0.2mg Al(OH)3) in PBS. p<0.05 vs. control.

[0039] [Figure 17-1]Figures 17A-17H. Replacing the entire glucan particle with mannan enhanced protection. Mice were immunized SC with 0.2 ml suspension in PBS containing MMA (100 μg mannan, 10 μg MPL, and 0.2 mg Al(OH)3), MMWA (100 μg mannan, 10 μg MPL, 100 μg WGP, and 0.2 mg Al(OH)3). Three days later, (Figure 17A) 6.9×107 CFU MRSA LAC was IV-infected into female BALB / c mice (N=8). (Figure 17B) 1.4-2.9×107 CFU XDR A. baumannii HUM1 was IV-infected into male C3HeB / Fe mice (N=5). (Fig. 17C) 2.4×108 CFU carbapenem-resistant K. pneumoniae KPC-KP1 was IV-infected into male C57BL / 6 mice (N=5). (Fig. 17D) 3.0×103 CFU R. delemar 99-880 was IV-infected into female BALB / c mice (N=5). (Fig. 17E) 1.7×105 C. albicans was IV-infected into male BALB / c mice (N=5). (Fig. 17F) 1.6×108 CFU XDR A. baumannii HUMC1 was IV-infected into male C3HeB / Fe mice (N=5). (Fig. 17G) 5.7×105 CFU XDR P. aeruginosa PA9019 was IV-infected into male C3HeB / Fe mice (N=3). (FIG. 17H) Female C3HeB / Fe mice (N=6) were infected IV with 1.6-2.8×108 CFU VRE 51299 and blood bacterial load was analyzed 1 hour post-infection. Survival rates were compared by nonparametric log-rank test with α=0.05. Bacterial load was compared by Wilcoxon rank sum test for unpaired comparisons with α=0.05. *p≦0.05 and **p≦0.01 vs. PBS. [Figure 17-2] Same as above.

[0040] [Figure 18-1]FIG. 18A-18G Higher doses help extend the duration of protection. (FIG. 18A)-(FIG. 18B) Female BALB / c mice (N=8) and male C3HeB / Fe mice (N=5) were immunized SC with 0.2 ml suspension of PBS containing MMA 1x (100 μg mannan, 10 μg MPL, and 0.2 mg Al(OH)3); MMA 3x (300 μg mannan, 30 μg MPL, and 0.2 mg Al(OH)3); or MMA 10x (1,000 μg mannan, 100 μg MPL, and 0.2 mg Al(OH)3). After 7, 14, or 21 days, the mice were infected IV with 9.7×107 CFU MRSA LAC and 2.9×107 CFU XDR A. baumannii HUMC1. (FIG. 18C)-(FIG. 18G) Female BALB / c mice (N=8-10), male C3HeB / Fe mice (N=5), and female NSG mice with adoptively transferred human CD34+ hematopoietic stem cells (N=4) were immunized with PBS; research grade or GMP grade MMA (G-MMA) (100 μg mannan, 10 μg MPL, and 0.1% Al(OH)3). One or three days later, mice were infected IV with 1.2-3.0×108 CFU MRSA LAC and 1.4-2.9×107 CFU XDR A. baumannii HUMC1. Survival rates were compared by nonparametric log-rank test at α=0.05. *p≦0.05 and **p≦0.01 vs. PBS. [Figure 18-2] Same as above.

[0041] [Figure 19]Figure 19 (8 panels) CD45+ immune cell population changes in response to MMA immunization. Male C3HeB / Fe mice (N=5) were immunized SC with MMA (100 μg mannan, 10 μg MPL, and 0.2 mg Al(OH)3) in 0.2 ml suspension in PBS. Splenocytes were harvested from naive mice (day 0) and 1, 3, 7, 14, or 21 days prior to immunization of the mice. The percentages of CD45+ cells that were T cells (TCRβ+), B cells (CD19+), dendritic cells (CD11C+), natural killer (NK) cells (NKp46+CD49b+), and the percentages of myeloid cells (CD45+CD11b+) that were neutrophils (Ly6C+Ly6G+), monocytes (Ly6C+Ly6G-), and macrophages (Ly6C-Ly6G-). Cell populations were compared by Kruskal-Wallis test with α = 0.05. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 versus naive mice (day 0 post-immunization).

[0042] [Figure 20]Figures 20A-20G Monocytes and macrophages are key effectors of MMA-mediated protection. (Figure 20A) Male C57BL / 6 wild-type mice (N=5) and RAG1-KO mice (N=6) were immunized SC with a 0.2 ml suspension of MMA (100 μg mannan, 10 μg MPL, and 0.2 mg Al(OH)3) in PBS and infected IV with 3.9-7.8×107 CFU XDR A. baumannii HUMC1. (Figure 20B)-(Figure 20E) Male C3HeB / Fe mice (N=5) and female BALB / c mice (N=8) were depleted of natural killer (NK) cells or macrophages / monocytes. Mice were then immunized SC with a 0.2 ml suspension of MMA (100 μg mannan, 10 μg MPL, and 0.2 mg Al(OH)3) in PBS and infected IV with 2.3-2.7×107 CFU XDR A. baumannii HUMC1 or 1.4-3.0×108 CFU MRSA LAC. (FIG. 20F) RAW 264.7 macrophages and (FIG. 20G) primary human monocytes were stimulated in vitro with MMA (5 μg / mL mannan, 0.5 μg / mL MPL, 10 ng / mL Al(OH)3) or IFNγ and assessed for their ability to take up A. baumannii ATCC17978. Survival rates were compared by nonparametric log-rank test at α=0.05. Macrophage uptake was compared by Wilcoxon rank sum test for unpaired comparisons with α=0.05. *p≦0.05, **p≦0.01, and ***p≦0.001 versus PBS.

[0043] [Figure 21-1]Figures 21A-21D Induction of epigenetic reprogramming resulted in gene expression changes. (Figure 21A) Summary of differential peaks of H3K27ac between naive, non-vaccinated control mice, or samples taken on days 3 or 21 after SC vaccination of mice with 0.2 ml of PBS suspension containing MMA (100 μg mannan, 10 μg MPL, and 0.2 mg Al(OH)3). Differential peaks were identified as adjusted p<0.05, fold change>2, reads / peak>50. (Figure 21B) Summary of transcriptional analysis by RNA-seq. Differentially expressed genes (DEGs) between immunized for 3 days, 21 days, and naive controls. DEGs were identified as those showing p<0.05, FC>2, and RPKM>1. (FIG. 21C) Heatmap of H3K27ac differential peaks and transcriptome DEG analysis selected between 3-day, 21-day immunized and naive controls. Differential peaks and DEGs different between 3-day and naive controls were selected. 3 mice / group. (FIG. 21D) Pathway analysis of H3K27ac differential peaks and DEGs using genes that were more highly expressed in 3-day immunized compared to naive controls. [Figure 21-2] Same as above.

[0044] [Figure 22-1]Figures 22A-22F Reduction of pro-inflammatory cytokines in mice immunized with MMA. Mice were immunized SC with 0.2 ml of PBS suspension containing MMA (100 μg mannan, 10 μg MPL, and 0.2 mg Al(OH)3) 1, 3, 7, 14, or 21 days before infection. (Figure 22A)-(Figure 22B) Gene expression changes of cytokine-related genes. (Figure 22C)-(Figure 22D) 1.5×108 CFU MRSA LAC and 1.8×107 CFU XDR A. baumannii HUMC1 were infected IV via the tail vein into female BALB / c mice (N=5 / group) and male C3HeB / Fe mice (N=5 / group). Bacterial burden was assessed 4 hours after infection. (FIG. 22E)-(FIG. 22F) Plasma from each mouse was then analyzed by Luminex for the cytokines: IL-4, IL-6, IL-10, IL-12 p70, and TNF. Kruskal-Wallis test at α=0.05. *p≦0.05, **p≦0.01, ***p≦0.001 vs. naive mice (day 0 post-immunization). [Figure 22-2] Same as above. [Figure 22-3] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] Detailed Description Before the compositions and methods are described, it is to be understood that the invention is not limited to the particular methodology, 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 invention, and is in no way intended to limit the scope of the invention as set forth in the appended claims.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are described herein. All technical publications and patent publications cited herein are incorporated by reference in their entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0046] The practice of the present invention employs, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which techniques are within the skill of the art. See, e.g., Sambrook and Russell (eds.) (2001) Molecular Cloning: A Laboratory Manual, 3rd Edition; Ausubel et al. (eds.) (2007) Current Protocols in Molecular Biology; Methods in Enzymology (Academic Press, Inc., NY); 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 ed.; Gait, ed. (1984) Oligonucleotide 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, eds. (2003) Gene Transfer and Expression in Mammalian Cells; and Mayer and Walker, eds. (1987). Immunochemical Methods in Cell and Molecular Biology (Academic Press, London). .

[0047] All numerical values, including ranges (e.g., pH, temperature, time, concentration, and molecular weight), are approximate values ​​that vary by (+) or (-) in increments of 0.1. It should be understood, although not always explicitly stated, that all numerical values ​​are preceded by the term "about." It should also 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.

[0048] All numerical values, including ranges (e.g., pH, temperature, time, concentration, and molecular weight), are approximate values ​​that vary by (+) or (-) in increments of 0.1. It should be understood, although not always explicitly stated, that all numerical values ​​are preceded by the term "about." It should also 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.

[0049] definition As used in the specification and claims, the singular forms "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.

[0050] As used herein, the terms "comprising" or "comprises" are intended to mean that the compositions and methods include the recited elements but do not exclude other elements. When used to define compositions and methods, "consisting essentially of" is intended to mean excluding other elements of any essential importance to the combination for its stated purpose. Thus, a composition consisting essentially of the elements as defined herein does not exclude trace contaminants from isolation and purification methods and pharma- ceutically acceptable carriers (e.g., phosphate buffered saline, preservatives, etc.). "Consisting of" is intended to mean excluding trace elements of other ingredients and more than substantial method steps for administering the compositions of the invention or process steps that produce the compositions or achieve the intended results. Embodiments defined by each of these transitional phrases are within the scope of the invention.

[0051] The term "isolated" as used herein with respect to nucleic acids (e.g., DNA or RNA) refers to a molecule that is separated from other DNA or RNA, respectively, present in the natural macromolecular source. The term "isolated peptide fragment" is meant to include peptide fragments that do not naturally occur as fragments and are not 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 that a cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment thereof is separated from components, cells and others with which it is normally associated in nature. For example, an isolated cell is one that is separated from tissues or cells of dissimilar phenotype or genotype. As will be apparent to one of skill in the art, non-naturally occurring polynucleotides, peptides, polypeptides, proteins, antibodies or fragments thereof do not require "isolation" to distinguish them from their naturally occurring counterparts.

[0052] "Composition" is intended to mean a combination of the elements recited in the claims and another compound or composition, an inert substance (e.g., a detectable label) or an active substance (except for the antigen), either alone or, in one embodiment, in combination with a simple carrier such as saline or a carrier that may be pharma- ceutically acceptable or a solid support as defined below.

[0053] A "pharmaceutical composition" is intended to include a combination of the elements recited in the claims with carriers, inert materials, or active materials (except for antigens), making the composition suitable for diagnostic or therapeutic use in vitro, in vivo, or ex vivo.

[0054] As used herein, the term "pharmaceutical acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline solutions, water, and emulsions (e.g., oil / water or water / oil emulsions), DMSO, and various types of wetting agents. The compositions may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see Martin (1975) Remington's Pharm. Sci., 15th Edition. (Mack Publ. Co., Easton).

[0055] "Administration" can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods for determining the most effective means and dosages of administration are known to those skilled in the art and vary with the composition used for treatment, the therapeutic purpose, the infection being treated, and the subject being treated. Single or multiple administrations can be performed with the dosage level and pattern selected by the treating physician. Appropriate dosage formulations and methods for administering the agent are known in the art. Routes of administration can also be determined and methods for determining the most effective route of administration are known to those skilled in the art and vary with the composition used for treatment, the therapeutic purpose, the health or disease state of the subject being treated, and the infecting bacteria or organism. Non-limiting examples of routes of administration include oral administration, intranasal administration, injection, and topical application.

[0056] The term "effective amount" refers to an amount 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 being addressed, as well as the characteristics of the individual subject, such as general health, age, sex, weight, and tolerance to the pharmaceutical composition.

[0057] In the case of in vitro application, in some embodiments, the effective amount depends on the size and nature of the application in question. It also depends on the nature and sensitivity of the in vitro target and the method in use. Those skilled in the art can determine the effective amount based on these and other considerations. The effective amount can include one or more administrations of the composition depending on the embodiment.

[0058] The administration routes applicable to the method of the present invention include intravenous, intranasal, intramuscular, urethral, ​​intratracheal, subcutaneous, intradermal, topical application, rectal, nasal, oral, inhalation, and other oral and parenteral administration routes.The administration routes can be combined or adjusted as desired, depending on the drug and / or the desired effect.The active drug can be administered in a single dose or multiple doses.Embodiments of these methods and routes suitable for delivery include systemic or local routes.

[0059] Parenteral administration routes other than inhalation administration include, but are not limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intrathecal, intrasternal, and intravenous routes, i.e., any administration route other than via the digestive tract.Parenteral administration can be carried out to effect systemic or local delivery of the inhibitor.When systemic delivery is desired, administration typically requires invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations.

[0060] The term "suffering", as it relates 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 said to be "at risk of suffering" from a disease or infection. The patient has not yet developed the characteristic disease symptoms or active infection, but is known to be predisposed to the disease due to family history, having a genetic predisposition to developing the disease, being in an environment that places the subject at substantial risk of acquiring the infection, or because they have been diagnosed with a disease or disorder that predisposes them to developing the disease to be treated.

[0061] Alhydrogel® is a commercially available (Accurate Chemical and Scientific Corporation, Catalogue # A1090S) wet gel colloidal suspension. The InvivoGen catalog (invivogen.com / PDF / Alhydrogel_TDS.pdfd, last accessed October 23, 2017) describes Alhydrogel® adjuvant as an aluminum hydroxide wet gel suspension. Alhydrogel® particles have a net positive charge at pH 5-7. Alhydrogel® Adjuvant 2% is made by Brenntag Biosector, a leader in the global vaccine adjuvant market with a long history of producing high quality products. Alhydrogel® Adjuvant 2% was selected as the international standard preparation for aluminum hydroxide gel. Alhydrogel® Adjuvant 2% is present in many commercially available vaccine formulations.

[0062] Whole Glucan Particles (WGP) refers to a granular formulation of fungal glucan. It is a commercially available (InVivoGen Catalogue # tlrl-wgps) powder that is resuspended in water / saline. It has been used in the past as a vaccine adjuvant.

[0063] By monophosphoryl lipid (MPL) or lipid A is meant the lipid component of endotoxin, which is believed to be responsible for the toxicity of Gram-negative bacteria. It has the following chemical structure: [ka]

[0064] It is commercially available from InVivoGen (Catalog # tlrl-mpls) and Avanti Polar Lipids (catalog # 699800P). It is used as a vaccine adjuvant.

[0065] As used herein, the term "mannan" refers to a polysaccharide found in fungi that is composed of mannose polymers (Sigma-Aldrich, Cat. No. M7504 (last accessed Jan. 3, 2023, or Sigma Cat. #M3460-IG, or listed as "Mannan" by MedicaPharma, or listed as "Mannan Oligosaccharides" by Newgreen Health Industry).

[0066] As used herein, the term "antigen that induces an immune response against a bacterial or fungal pathogen" contemplates, for example, traditional vaccine preparations used prophylactically and for the treatment of infections and diseases associated with these infections, and in one embodiment, only those that induce an immune response mediated by T lymphocytes and / or B lymphocytes against a pathogen (e.g., a viral, bacterial, or fungal infection).

[0067] As used herein, the term "antigen" refers to any substance that causes the body to generate an adaptive immune response by T-lymphocytes and / or B-lymphocytes against that substance. Non-limiting 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 that can cause an immune response against the body. These antigens can also be used as markers in tests to identify those tissues or cells. In one aspect, the term "antigen" as claimed herein refers only to those that induce an immune response mediated by T-lymphocytes and / or B-lymphocytes against a pathogen (e.g., a viral, bacterial, or fungal infection).

[0068] The terms "subject," "individual," or "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, mice, rats, rabbits, monkeys, cows, sheep, pigs, dogs, cats, farm animals, sport animals, pets, horses, and primates, particularly humans.

[0069] composition The present disclosure provides a composition comprising, or alternatively consisting essentially of, or even further consisting of an effective amount of each of aluminum hydroxide, monophosphoryl lipid (MPL), and mannan (a combination referred to herein as "MMA"), with the proviso that said composition does not include an antigen effective to induce an immune response against a pathogen (e.g., a viral, fungal or bacterial pathogen). In one aspect, "antigen" as claimed herein is intended only to induce an immune response mediated by T lymphocytes and / or B lymphocytes against a pathogen (e.g., a viral, bacterial or fungal infection).

[0070] In one aspect, the compositions are combined to achieve a final concentration.For example, the compositions may be in a range of about 0.1mg / ml to about 20mg / ml, or alternatively about 0.5mg / ml to about 20mg / ml, or alternatively about 1.0mg / ml to about 20mg / ml, or alternatively about 0.1mg / ml to about 15mg / ml, or alternatively about 0.1mg / ml to about 12mg / ml, or alternatively about 0.1mg / ml to about 10mg / ml, or alternatively about 0.1mg / ml to about 5mg / ml, or alternatively about 0.1mg / ml to about 3mg / ml, or alternatively 0.1mg / ml to about 2mg / ml, or alternatively about 200μg / ml to about 20mg / ml, or alternatively about 0.1mg / ml to about 15mg / ml, or alternatively about 0.1mg / ml to about 13mg / ml, or alternatively about 0.5mg / ml to about 15mg / ml. or about 13 mg / ml, or alternatively 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.

[0071] MMA may have a range of about 0.1 mg / ml to about 20 mg / ml, or alternatively about 0.5 mg / ml to about 20 mg / ml, or alternatively about 1.0 mg / ml to about 20 mg / ml, or alternatively about 0.1 mg / ml to about 15 mg / ml, or alternatively about 0.1 mg / ml to about 12 mg / ml, or alternatively about 0.1 mg / ml to about 10 mg / ml, or alternatively about 0.1 mg / ml to about 5 mg / ml, or alternatively about 0.1 mg / ml to about 3 mg / ml, or alternatively 0.1 mg / ml to about 2 mg / ml, or alternatively about 200 μg / ml to about 20 mg / ml, or alternatively about 0.1 mg / ml to about 15 mg / ml, or alternatively about 0.1 mg / ml to about 13 mg / ml, or alternatively about 0.5 mg / ml to about 13 mg / ml, or alternatively The compound is suspended in a carrier (e.g., a pharma- ceutical acceptable carrier such as PBS) to a concentration of 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.

[0072] The components of MMA can be combined in any suitable combination, and although the ratio of each component is not intended to be the same, they can be combined in a ratio of 1:1:1 (mannan:MPL:aluminum hydroxide). Alternatively, the MMA can be combined 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.

[0073] In some aspects, the MMA is at 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 / ml, 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 μg / 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 3 mg / ml. or 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.

[0074] The components of MMA may be supplied by commercial sources.

[0075] The composition may 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 from 10 mcg / kg body weight to 50 mcg / kg body weight, or alternatively from 20 mcg / kg body weight to about 50 mcg / kg body weight, or alternatively from 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 mcg / kg body weight to about 200 mcg / kg body weight.

[0076] The composition may be further formulated for storage and distribution, for example, by lyophilization or freeze-drying. Additionally, preservatives and stabilizers may be added to further enhance the shelf life of the composition.

[0077] Treatment The present disclosure also provides a method for enhancing immunity in a subject against infection caused by bacterial or fungal pathogens by administering to the subject an effective amount of a composition as described above. The subject to be treated is any animal or human patient at risk of or has developed an infection from bacteria (gram-positive or gram-negative bacteria) or fungi. Non-limiting examples include sports and livestock animals, pet animals, and human patients. As used herein, the term "enhancing immunity" is intended to increase the innate immune response (including macrophages, neutrophils, dendritic cells, and γδ T cells and / or NK T cells), but does not include B cell-mediated antibody or T cell stimulation, which are characteristic of classical protein antigen-induced adaptive immunity. Methods for determining whether an immune response has been elicited are known in the art and include, for example, taking a suitable sample (blood, saliva, or plasma) from the patient and assaying cytokine levels by ELISA, measuring the counts of various types of white blood cells per ml of blood, and whether antibodies against the pathogen or bacteria are present. Additionally, non-invasive means (eg, reducing the subject's temperature) can be used alone or in combination with clinical methods.

[0078] In one aspect, the bacteria is selected from S. aureus, A. baumannii, K. pneumoniae, P. aeruginosa, E. coli, Enterobacter spp., Serratia, Stenotrophomonas, and the fungus is selected from Candida spp. and fungi that cause the disease mucormycosis (including Rhizopus spp.).

[0079] The composition may be administered in any suitable dose as determined by the treating physician, medical professional or veterinarian. Non-limiting examples of suitable administration methods include intramuscular, subcutaneous, or intravenous administration. The effective amount to be administered is about 5mcg / kg body weight to about 50mcg / kg body weight, or alternatively 10mcg / kg body weight to 50mcg / kg body weight, or alternatively 20mcg / kg body weight to about 50mcg / kg body weight, or alternatively 10mg / kg body weight to about 100mcg / kg body weight, or alternatively about 15mcg / kg body weight to about 150mcg / kg body weight, or alternatively about 20mcg / kg body weight to about 200mcg / kg body weight.

[0080] In one aspect, the doses of MPL and aluminum are 50 μg and 500 μg, respectively, resulting in a weight adjusted dose for a 70 kg adult of about 0.3 μg / kg, or about 0.4 μg / kg, or about 0.7 μg / kg (MPL) and about 3 μg / kg, or about 4 μg / kg, or about 7 μg / kg (aluminum).

[0081] In another aspect, the ranges of components in the MMA are as provided in Table 1 below. [Table 1]

[0082] The dose is repeated as necessary, for example, a second, third, or fourth dose, if necessary. The MMA can be combined with PBS (e.g., 0.5 ml carrier) and administered intramuscularly.

[0083] [Table 2]

[0084] Administration of the MMA vaccine may be combined with pre-administration monitoring. For example, blood may be obtained prior to vaccination and repeated at 1 and 2 weeks. Blood may be tested for standard complete blood counts, blood chemistry, and liver function tests, and may also be stored for immunological testing.

[0085] In one aspect, the composition is administered once, twice or three times over a period of one to three months.

[0086] In another aspect, the method further includes assaying the subject for bacterial or fungal infection using methods known in the art or as described herein prior to administering the composition.

[0087] The compositions may be administered in any suitable dosage, as determined by the treating physician, medical professional, or veterinarian. Non-limiting examples of suitable methods of administration include intramuscular, subcutaneous, or intravenous administration. The effective amount to be administered may be from about 25mcg / kg body weight to about 200mcg / kg body weight, or alternatively from about 50mcg / kg body weight to about 175mcg / kg body weight, or alternatively from about 75mcg / kg body weight to about 200mcg / kg body weight, or from about 25mcg / kg body weight to about 100mcg / kg body weight, or from about 25mcg / kg body weight to about 175mcg / kg body weight, or from about 25mcg / kg body weight to about 150mcg / kg body weight, or from about 75mcg / kg body weight to about 200mcg / kg body weight, or from about 75mcg / kg body weight to about 150mcg / kg body weight, or from about 25mcg / kg body weight to about 150mcg / kg body weight, or from about 50mcg / kg body weight to about 125mcg / kg body weight. body weight, or about 50 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.

[0088] The method can also be used to treat or prevent disorders caused by bacterial or fungal infection in a subject in need thereof, 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 an infection from bacteria (gram-positive or gram-negative bacteria) or fungi. The method of determining whether a subject is being treated comprises, for example, taking an appropriate sample (blood, saliva or plasma) from the patient, and assaying the cytokine levels by ELISA and whether antibodies against the pathogen or bacteria are present. In addition, non-invasive means (e.g., lowering the temperature of the subject) can be used alone or in combination with clinical methods. Furthermore, depending on the disease being treated, a reduction in the clinical symptoms of the disease indicates effective treatment.

[0089] In one aspect, the bacteria is selected from S. aureus, A. baumannii, K. pneumoniae, P. aeruginosa, E. coli, Enterobacter spp., Serratia, Stenotrophomonas, and the fungus is selected from Candida spp. and fungi that cause the disease mucormycosis, including Rhizopus spp.

[0090] The compositions may be administered in any suitable dosage as determined by the treating physician, medical professional, or veterinarian. Non-limiting examples of suitable methods of administration include intramuscular, subcutaneous, or intravenous administration. The effective amount to be administered may be from about 25mcg / kg body weight to about 200mcg / kg body weight, or alternatively from about 50mcg / kg body weight to about 175mcg / kg body weight, or alternatively from about 75mcg / kg body weight to about 200mcg / kg body weight, or from about 25mcg / kg body weight to about 100mcg / kg body weight, or from about 25mcg / kg body weight to about 175mcg / kg body weight, or from about 25mcg / kg body weight to about 150mcg / kg body weight, or from about 75mcg / kg body weight to about 200mcg / kg body weight, or from about 75mcg / kg body weight to about 150mcg / kg body weight, or from about 25mcg / kg body weight to about 150mcg / kg body weight, or from about 50mcg / kg body weight to about 125mcg / kg body weight. body weight, or about 50 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.

[0091] In one aspect, the composition is administered once, twice or three times over a period of 1 to 3 months. In one aspect, the immunization is boosted about 14 to about 28 days after immunization, about 16 to about 26 days after immunization, or about 18 to about 24 days after immunization, or about 20 to about 26 days after immunization, or about 22 to about 23 days after immunization, or about 3 weeks after immunization.

[0092] Doses may be boosted with multiple doses and / or may be administered at about 0.5x, or about 1x, 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 11x, or about 12x, or about 13x, or about 14x, or about 15x or more of the baseline dose as determined by the treating physician. 1x MMA contains 100μg, 10μg MPL and 200μg aluminum hydroxide (2%); 3x MMA contains 300μg mannan, 30μg MPL, and 200μg 2% aluminum hydroxide; and 10x contains 1000μg mannan, 100μg MPL, and 200μg 2% aluminum hydroxide.

[0093] In another aspect, the method further includes assaying the subject for bacterial or fungal infection using methods known in the art or as described herein prior to administering the composition.

[0094] kit Also provided herein are kits comprising a composition or formulation as described herein and instructions for use.

[0095] Drug screening assays Also provided herein is a method for identifying a compound or agent that provides a benefit selected from one or more of enhancing immunity against bacterial or fungal microorganism infection, or treating bacterial or fungal infection or disease associated with bacterial or fungal microorganism infection, comprising mixing the compound or agent with a composition as described herein, administering the mixed composition to a non-human subject infected with bacterial or fungal microorganism, and assaying for infection or survival after administration, wherein a compound or agent that enhances the activity of a composition as described herein is a compound or agent that provides the benefit. Methods for determining whether 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 for cytokine levels by ELISA, and for the presence of antibodies against the pathogen or bacteria. Additionally, non-invasive means (e.g., lowering the temperature of the subject) can be used alone or in combination with clinical methods. The method of determining whether a subject is being treated includes, for example, taking a suitable sample (blood, saliva or plasma) from the patient and assaying whether antibodies against the pathogen or bacteria are present by ELISA.In addition, non-invasive means (e.g., lowering the temperature of the subject) can be used alone or in combination with clinical methods.In addition, depending on the disease being treated, a reduction in the clinical symptoms of the disease indicates effective treatment.

[0096] Data generated from Experiments 1-7 are found in Figures 1-16. Data generated from Experiments 8 and 9 are found in Figures 17-22.

[0097] Preparation of the Composition To prepare the composition, the preferred amount of aluminum hydroxide is 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 / ml, 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 0.1 mg / ml to about 1 mg / ml, or alternatively about 200 μg. / ml to about 8 mg / ml, or alternatively about 0.1 mg / ml to about 5 mg / ml, or alternatively about 0.1 mg / ml to about 3 mg / ml, or alternatively about 0.5 mg / ml to about 3 mg / ml, or alternatively 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 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 It is suspended in saline solution at a concentration of 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.

[0098] In some aspects, the mannans are at 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 / ml, 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 μg / 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

[0043] The aluminum hydroxide may be dissolved in saline prior to combination with the MPL and aluminum hydroxide to provide a concentration in the range of about 0.5 mg / ml, or about 1.0 mg / ml, or about 1.5 mg / ml, 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.

[0099] A preferred amount of MPL is in the range of about 0.1 mg / ml to about 10 mg / ml, or alternatively about 0.5 mg / ml to about 10 mg / ml, or alternatively about 1.0 mg / ml to about 10 mg / ml, or alternatively about 0.1 mg / ml to about 9 mg / ml, or alternatively about 0.1 mg / ml to about 7 mg / ml, or alternatively about 0.1 mg / ml to about 5 mg / ml, or alternatively about 0.1 mg / ml to about 3 mg / ml, or alternatively about 0.1 mg / ml to about 2 mg / ml, or alternatively 0.1 mg / ml to about 1 mg / ml, or alternatively about 200 μg / ml to about 8 mg / ml, or alternatively about 0.1 mg / ml to about 5 mg / ml, or alternatively about 0.1 mg / ml to about 3 mg / ml, or alternatively about 0.5 mg / ml to about 3 mg / ml, Alternatively, is added to the combination of aluminum hydroxide and MPL to a concentration of 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.

[0100] MMA may have a concentration in the range of about 0.1 mg / ml to about 10 mg / ml, or alternatively about 0.5 mg / ml to about 10 mg / ml, or alternatively about 1.0 mg / ml to about 10 mg / ml, or alternatively about 0.1 mg / ml to about 9 mg / ml, or alternatively about 0.1 mg / ml to about 7 mg / ml, or alternatively about 0.1 mg / ml to about 5 mg / ml, or alternatively about 0.1 mg / ml to about 3 mg / ml, or alternatively about 0.1 mg / ml to about 2 mg / ml, or alternatively 0.1 mg / ml to about 1 mg / ml, or alternatively about 200 μg / ml to about 8 mg / ml, or alternatively about 0.1 mg / ml to about 5 mg / ml, or alternatively about 0.1 mg / ml to about 3 mg / ml, or alternatively about 0.5 mg / ml to about 3 mg / ml, or alternatively about 0.5 mg / ml to about 3 mg / ml. The antibody is suspended in a pharma- ceutical acceptable carrier (e.g., PBS and optionally DMSO) to a concentration of about 1.0 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.

[0101] In one aspect, the compositions are combined to achieve a final concentration. For example, the compositions may be in a range of about 0.1 mg / ml to about 20 mg / ml, or alternatively about 0.5 mg / ml to about 20 mg / ml, or alternatively about 1.0 mg / ml to about 20 mg / ml, or alternatively about 0.1 mg / ml to about 15 mg / ml, or alternatively about 0.1 mg / ml to about 12 mg / ml, or alternatively about 0.1 mg / ml to about 10 mg / ml, or alternatively about 0.1 mg / ml to about 5 mg / ml, or alternatively about 0.1 mg / ml to about 3 mg / ml, or alternatively 0.1 mg / ml to about 2 mg / ml, or alternatively about 200 μg / ml to about 20 mg / ml, or alternatively about 0.1 mg / ml to about 15 mg / ml, or alternatively about 0.1 mg / ml to about 13 mg / ml, or alternatively about 0.5 mg / ml to about 15 mg / ml. or about 13 mg / ml, or alternatively 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.

[0102] The compositions may be formulated for in vivo administration (in one or more doses) to administer 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 weight, 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 about 15 mcg / kg body weight to about 150 mcg / kg body weight, or alternatively about 20 mcg / kg body weight to about 200 mcg / kg body weight. Exemplary doses are disclosed herein and are incorporated herein by reference. [Table 3] EXAMPLES

[0103] Experiment 1: Discovery of adjuvant-induced immunity - Materials and methods Adjuvant Immunization Mannan (Sigma Cat. # M3640-1G), monophosphoryl lipid A (MPL) (InvivoGen Cat. # tlrl-mpls), whole glucan particle (WGP) (InvivoGen Cat. # tlrl-wgp), 2% aluminum hydroxide (Al(OH)3) gel (Accurate Chemical & Scientific Corporation Cat. # A1090S) are prepared and stored according to the manufacture's protocol. Mannan (MedicaPharma Cat. # Mannan), PHAD (登録商標) (Avanti Polar Lipids Cat. # 699800P), and 2% (Al(OH)3) gel (Croda Cat. # AJV3012) were used as GMP grade MMA. Mice were immunized subcutaneously (SC) with 200 μL of premixed adjuvant mixture in phosphate buffered saline (PBS) in the scruff of the neck.

[0104] Intravenous (IV) infection Frozen stocks of A. baumannii and K. pneumoniae bacteria grown to mid-log phase were prepared as previously described (15). Inocula were prepared by diluting these concentrated frozen stocks of bacteria in PBS. Inocula were established by plating serial dilutions on tryptic soy agar (TSA) plates and incubating overnight at 37°C. Mice infected with A. baumannii were monitored for 7 days and mice infected with K. pneumoniae were monitored for 14 days. They were then euthanized according to our IACUC protocol.

[0105] S. aureus inocula were prepared from mid-logarithmic 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 at 200 rpm. The overnight cultures were subcultured by diluting 1:100 into sterile TSB and incubated at 37° C. for 3 hours with shaking set at 200 rpm. The subcultures were pelleted in a refrigerated centrifuge at 4,000×g for 5 minutes and rinsed by resuspending the pellet in PBS. After two additional rinses (3 times total), the pellets were resuspended in PBS and the optical density at 600 nm (OD 600 ) to 0.5 using PBS. Applicants used the established correlation coefficient for this optical density to predict the inoculum concentration (OD 600 0.5~2.4×10 8 CFU / mL). Inocula were established by plating serial dilutions onto TSA plates and incubating overnight at 37° C. Mice infected with S. aureus were monitored for 28 days after which they were euthanized according to our IACUC protocol.

[0106] Oral aspiration (OA) infection Mice were infected using the OA model as previously described (9). Briefly, A. baumannii HUMC1 inoculum was prepared from a subculture of an overnight culture as described above, and mice were infected by aspirating 50 μL of bacteria suspended in PBS. Mice infected with A. baumannii via OA were monitored for 7 days, after which they were euthanized according to our IACUC protocol.

[0107] Mouse knockout (KO) strains RAG1 KO (lineage # 034159) and human CD34 + Hematopoietic stem cell engrafted NSG mice were purchased from Jackson Laboratories. Innate immune cell depletion

[0108] Macrophages were depleted by IP injection of 50 μg / g (e.g., a 30-g mouse received 300 μL or 1.5 mg liposomal clodronate) 3 days prior to infection.

[0109] Neutrophils were depleted by IP injection of 230 mg / kg cyclophosphamide (Baxter) 3 days before infection, and neutropenia persisted for 6 days ( 14 , 16 ).

[0110] NK cells were depleted by injecting anti-Asialo-GM1 antibody (Poly21460, Biolegend, 30 μl / mouse) every 3 to 4 days starting on the day of immunization ( 17 ).

[0111] Flow cytometry analysis Mouse spleens were collected, crushed through a 70 μm cell strainer with a 35 mL syringe plunger, and rinsed by volume with Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (DMEM + 10% FBS). The cell suspension was pelleted by centrifugation at 300 × g for 5 min at room temperature, and the supernatant was discarded. After two further rinses (3 total), red blood cells (RBCs) were lysed using RBC lysis buffer (BioLegend Cat. # 420301) according to the manufacturer's protocol. Splenocytes were resuspended in PBS with 5% fetal bovine serum (FACS buffer), counted with a hemocytometer, and incubated with Fc blocker (BD Biosciences Cat. # 553141) on ice for 30 min. Splenocytes were pelleted at 300×g for 5 min in a room temperature centrifuge, resuspended in FACS buffer, and incubated with fluorophore-conjugated antibodies for OMIP-032 (multicolor immunophenotyping of two panels to assess innate and adaptive immune cells as previously described (18)). After incubation, splenocytes were washed twice at 300×g for 5 min in a room temperature centrifuge and resuspended in FACS buffer. Samples were analyzed using a BD FACS Canto II flow cytometer.

[0112] statistics Survival rates were compared by the non-parametric log-rank test at α = 0.05. Mac assay, cfu, cytokines, and chip-seq were compared by the Wilcoxon-Mann-Whitney test at α = 0.05.

[0113] In an effort to develop a vaccine against S. aureus bacteremia, the efficacy of previous combinations was compared. Mice were injected with a panel of protein antigens combined with one or more adjuvants, and mice immunized with adjuvant alone were included as negative controls. All mice were then challenged intravenously (IV) with a clinical blood isolate of S. aureus MRSA USA300 strain LAC. Applicants observed that mice immunized with multiple adjuvants had a survival advantage compared to mice immunized with only one adjuvant, regardless of whether the vaccine contained protein antigens (Figure 1A). Specifically, mice immunized with whole glucan particles (WGP) and lipopolysaccharide (LPS) (WGP + LPS) experienced the same level of survival as mice immunized with the same two adjuvants plus a panel of recombinant S. aureus surface antigens (WGP + LPS + Ag). Similarly, mice further immunized with aluminum hydroxide (Al(OH)3) (Al(OH)3+ WGP + LPS) survived at a similar level to mice further immunized with monophosphoryl lipid A (MPL) and the same panel of recombinant protein antigens (Al(OH)3+ WGP + LPS + MPL + Ag) (Figure 1A).

[0114] To evaluate the impact of immune booster injections on vaccine efficacy, applicants immunized mice with phosphate-buffered saline (PBS) or MPL, WGP, and Al(OH)3 (MWA); half of the mice received the same booster injections 3 weeks after their first immunization. The mice were then challenged IV with S. aureus LAC 3, 7, or 21 days after the last immunization. For single-dose recipients, protection against infection persisted for 3 and 7 days after the dose, but waned by 21 days (Figure 1B). These results demonstrated that the adjuvant-only vaccine provided short-term protection against S. aureus bacteremia with only one immunization. Booster administration restored protection for an additional 3 days, but not for an additional 21 days (Figure 1B).

[0115] Because the innate immune stimulatory components of the tripartite vaccine do not specifically target S. aureus, Applicants investigated the ability of the vaccine to protect against bacteremia caused by the Gram-negative bacterium Acinetobacter baumannii. Mice were immunized with PBS or MWA and challenged with extremely drug-resistant (XDR) clinical lung and blood isolates of A. baumannii HUMC1 3 or 7 days after immunization. Both groups of immunized mice were well protected against an otherwise lethal infection (Figure 1C).

[0116] Experiment 2: Critical contribution of vaccine components Mice immunized with MWA were fully protected against A. baumannii bacteremia, but they were only partially protected against S. aureus bacteremia. Applicants therefore explored how each component of the vaccine contributes to protection against lethal infection. Applicants immunized mice with none, one, two, or all three adjuvants and challenged them 3 days later with a larger inoculum of A. baumannii, allowing differentiation between the survival benefits conferred to each immunization group. Compared to the other groups, MWA provided the greatest protection (Figure 1D).

[0117] With the superiority of MWA established, we next investigated whether the triple-adjuvanted vaccine could protect against another common route of infection in hospitalized patients, aspiration pneumonia, using the oropharyngeal aspiration (OA) pneumonia model we previously described. (9) Despite protecting against bacteremia, MWA did not protect against A. baumannii pneumonia infection (Fig. 1E).

[0118] Applicants also evaluated the ability of the vaccine to protect against lethal infection from Klebsiella pneumoniae, another Gram-negative bacterium in our bacteremia model. Although no mice survived, the immunized group experienced a delayed fate of death compared to the placebo group when infected with K. pneumoniae clinical blood isolate KP3 (Figure 1F). Finally, Applicants explored the effect of reduced dosing and found that reduced doses resulted in a dose-dependent reduction in efficacy (Figures 1G-H).

[0119] Study 3: Optimization of triple regimen with mannan Based on previous experience working with mannosylated proteins (10), applicants sought to enhance the efficacy of the vaccine by incorporating mannan, an oligosaccharide shown to have immunomodulatory properties. Mice were immunized with either mannan in addition to MWA (MMWA) or mannan instead of WGP (MMA). Three days later, mice were challenged with normally lethal inocula of S. aureus, A. baumannii, K. pneumoniae, or neutropenic mice were infected with the fungus Rhizopus delemar (Figure 2A-D). Overall, the vaccine with mannan instead of WGP (MMA) provided superior protection against lethal infection with all four pathogens, including in a neutropenic mouse model of fungal infection (Figure 2A-D). Furthermore, mannan helped confer a dramatic survival advantage in mice infected with A. baumannii pneumonia (Figure 2E).

[0120] Experiment 4: Optimization of vaccine dosage Having established that MMA was superior to MWA or MMWA and that MMA protected mice 3 days after immunization, Applicants next sought to evaluate whether increasing the dose could enhance protection and how long the vaccine would protect following immunization. Applicants immunized with 3x or 10x as much mannan and MPL and challenged with lethal S. aureus or A. baumannii bacteremia 1, 2, or 3 weeks later (Figure 3A-B). Higher doses provided protection for up to 21 days, whereas lower doses terminated protection sooner.

[0121] With regard to A. baumannii bacteremia, all three doses provided complete protection when mice were challenged 1 week after immunization (FIG. 3B). However, only the highest dose (10-fold) provided complete protection when mice were challenged 2 or 3 weeks after immunization (FIG. 3B). To confirm that the protective effect was not due to antibody-mediated immunity, applicants analyzed serum from vaccinated mice and confirmed that immunization did not produce antibodies specific for A. baumannii or S. aureus (FIG. 3C).

[0122] Applicants next sought to clarify the rapidity of onset of protection. Applicants immunized mice with a standard (1x) dose of MMA and challenged them with lethal A. baumannii or S. aureus bacteremia 24 hours after immunization; MMA provided complete protection (Figure 3D) or similar protection as at day -3 (Figure 3E). To assess its translatability to humans, Applicants used CD34 + Human stem cell transplant recipient mice were immunized with MMA 3 days prior to infection. Despite being severely immunocompromised, the vaccine still provided protection against A. baumannii bacteremia (Figure 3F). Applicants also tested several Good Manufacturing Practice (GMP) materials by immunizing mice with GMP grade MMA (labeled G-MMA in the figure) 3 days prior to infection, and G-MMA provided similar protection as MMA (Figures 3G-H).

[0123] Test 5: Cellular sources of defense To begin to elucidate the mechanism of protection, applicants evaluated changes in seven immune cell populations sourced from the spleens of immunized and non-immunized mice. + No significant changes were observed in the proportion of CD45 B cells (Figure 4). + The proportion of dendritic cells was elevated for at least 3 weeks after immunization (Figure 4). Dendritic cell and natural killer (NK) cell populations were CD45 + Although the proportion of cells that are of myeloid lineage rapidly decreased, they returned to pre-immune levels by 2 weeks after immunization (Figure 4). + No trends were observed in the cell percentages (Figure 4). Nevertheless, there were immediate changes in the percentages of neutrophils, monocytes, and macrophages (Figure 4). Specifically, the percentage of neutrophils decreased, whereas the percentages of monocytes and macrophages rapidly increased and remained elevated for at least 3 weeks (Figure 4).

[0124] The lack of antibody-mediated immunity (Figure 3C) and the rapid overnight induction of protective immunity (Figure 3D) suggested that the vaccine mediated protection via the innate immune system. Applicants therefore hypothesized that only the innate immune system was necessary to provide protection. Recombination activating gene 1 (RAG1) is crucial for V(D)J recombination, and mice with non-functional RAG1 lack mature B and T lymphocytes (11). To determine whether lymphocytes play a role in MMA-mediated protection, Applicants immunized wild-type (WT) and RAG1 knockout (RAG1-KO) mice with MMA and challenged them with A. baumannii bacteremia 3 days after immunization. Even in the absence of mature B and T lymphocytes, the MMA vaccine successfully protected mice from an otherwise lethal infection (Figure 5A). These results indicate that the adjuvants provided short-term protection via innate immune cells without lymphocyte involvement, consistent with trained immunity ( 12 , 13 ).

[0125] To identify potentially key innate immune cells providing MMA-mediated protection, applicants selectively depleted innate immune cells from mice, immunized with MMA, and then challenged the mice 3 days later. Specifically, neutrophils were depleted with cyclophosphamide, macrophages / monocytes were depleted with liposomal clodronate, and NK cells were depleted with anti-Asialo-GM1 antibodies. Immunized mice could survive in the absence of neutrophils or NK cells, but mice lacking macrophages died from both S. aureus and A. baumannii infections (Figure 5B-F).

[0126] Since macrophages / monocytes were found to be most important in protecting mice from lethal infection, Applicants then investigated whether MMA could differentiate RAW 264.7 macrophage-like cells and primary human monocytes in vitro by assessing macrophage phagocytosis of A. baumannii after MMA stimulation. Both RAW 264.7 macrophage-like cells incubated with MMA for 1 or 3 days prior to the uptake assay mediated similar phagocytosis as after IFNγ stimulation, a positive control (Figure 5F). Experiment 6: Defense mechanisms through immunomodulation

[0127] After elucidating the importance of macrophages in conferring protection, applicants investigated the immunomodulatory properties of the vaccine by assessing the bacterial load and cytokine profile of immunized mice. Applicants first analyzed the bacterial load in MMA-immunized mice challenged with lethal S. aureus or A. baumannii bacteremia 4 hours after infection (Figure 6A-B). In mice infected with S. aureus, the bacterial load was essentially unchanged regardless of how long the mice were immunized or whether they were immunized (Figure 6A). In mice infected with A. baumannii, the bacterial load was significantly lower in mice infected 1 or 3 days after immunization compared to non-immunized mice (Figure 6B). Despite no statistically significant change in the bacterial load of the majority of immunized mice, the survival outcome of immunized mice was significantly improved compared to non-immunized mice (Figure 2A-B, Figure 3A-B, DE). These data indicate that the reduction in bacterial load was not the only driving factor for adjuvant-based protection.

[0128] Applicants have previously observed that cytokine profiles are better indicators of survival outcome than bacterial burden (14). Thus, Applicants analyzed cytokines in the plasma of the very same immunized mice challenged with lethal S. aureus or A. baumannii bacteremia 4 h after infection. For both pathogens, immunized mice had lower proinflammatory cytokines (IL-6, IL-12, TNF) and elevated IL-10 / TNF ratios that persisted for 3 weeks (Figure 6C-D). This trend was particularly pronounced in mice challenged 1 or 3 days after immunization. Several cytokines were below the limit of detection (IFNγ, IL-1β, IL-13, IL-17A) or unchanged (IL-4).

[0129] Study 7: Comparative MMA vaccine efficacy Applicant's MMA triple vaccine was compared to the prior art MPL triple vaccine, alone or in combination with MPL + mannan vaccine. MPL alone or in combination with mannan is disclosed in WO 2018 / 089475, which is incorporated herein by reference.

[0130] Briefly, the MPL triple vaccine comprises, or alternatively consists essentially of, or even further consists of, an effective amount of each of aluminum hydroxide, monophosphoryl lipid (MPL), and whole glucan particle (WGP); provided that the composition does not include an antigen effective to induce an immune response against a fungal or bacterial pathogen. In a further aspect, the composition further comprises, consists essentially of, or consists of an effective amount of mannan.

[0131] Experiments 8 and 9 extend the studies reported in Experiments 1-7 (above).

[0132] Experiment 8: Discovery and characterization of vaccine components Applicants first explored the contribution of the individual components of the triple-component vaccine to protection against S. aureus bacteremia. Applicants immunized mice with each individual component or with a dual or triple combination of Al(OH)3, MPL, and WGP. All mice were then challenged intravenously (IV) with a USA300 clinical blood isolate of MRSA, LAC. Compared to the individual (Al(OH)3) and dual combinations (Al(OH)3 + MPL or Al(OH)3 + WGP), the mixture of Al(OH)3, MPL, and WGP (MWA) provided the best protection (Figure 1A).

[0133] To evaluate the effect of repeated dosing on vaccine efficacy, applicants immunized mice with phosphate-buffered saline (PBS) or MWA. Three weeks later, half of the mice were revaccinated. Mice were then challenged IV with S. aureus LAC 3, 7, or 21 days after the last immunization. For recipients of a single dose, protection against infection persisted for 3 days (log rank, p=0.02) and 7 days (log rank, p=0.05) after the dose, but gradually waned by 21 days after immunization (Figure 1B). A second dose restored protection for an additional 3 days (log rank, p=0.02), but not for an additional 7 or 21 days (Figure 1B).

[0134] Because the immunostimulatory components of the MMA vaccine were not specific for S. aureus, Applicants investigated the ability of the vaccine to protect against bacteremia caused by the Gram-negative bacterium Acinetobacter baumannii. Applicants immunized mice with PBS or MWA and infected intravenously with an extremely drug-resistant (XDR) clinical lung and blood isolate of A. baumannii, HUMC1, either 3 or 7 days after immunization. The MWA-immunized group was completely protected against otherwise lethal bacteremia (log-rank, p=0.0009) (Figure 1C).

[0135] Similar to S. aureus, applicants tested whether all three components were required to protect against A. baumannii bacteremia. Applicants immunized mice with zero, one, two, or all three components and challenged them 3 days later with a larger inoculum of A. baumannii, allowing differentiation between the survival benefit conferred to each immunization group. Compared to the other groups, MWA provided the best protection (log-rank, p=0.0001) (FIG. 1D).

[0136] Having established the superiority of MWA, applicants next examined whether the three-component vaccine could protect against another common route of infection in hospitalized patients—pneumonia—using the oropharyngeal aspiration (OA) pneumonia model that applicants have previously described (7). Despite protecting against bacteremia, MWA did not protect against A. baumannii pneumonia infection (Figure 1E).

[0137] Applicants also evaluated the vaccine's ability to protect against lethal bloodstream infection from Klebsiella pneumoniae, another Gram-negative bacterium. No mice survived, but the immunized group experienced a significantly delayed fate of death (log-rank, p=0.001) (Figure 1F). Finally, Applicants explored the effect of reducing dosing and found that dose reduction resulted in a dose-dependent decrease in efficacy (Figures 1G-1H).

[0138] Optimization of protein-free vaccines with mannan Based on previous experience working with mannosylated protein vaccines (8), we sought to enhance the efficacy of the vaccine by incorporating mannan, an oligosaccharide shown to have immunomodulatory properties. Mice were immunized with either a quadruple regimen in which mannan was added to the MPL + whole glucan particle + aluminum hydroxide (MWA) combination (creating a new quadruple combination (MMWA)), or a new triple regimen in which mannan was substituted for WGP (consisting of mannan + MPL + aluminum hydroxide (MMA)). Three days later, mice were challenged with a normally lethal inoculum of MRSA, carbapenem-resistant A. baumannii, carbapenem-resistant K. pneumoniae, Rhizopus delemar (in neutropenic mice), or Candida albicans. Overall, the vaccine in which mannan was substituted for WGP (MMA) provided superior protection against lethal infection with four of the above pathogens, including in a neutropenic mouse model of R. delemar mucormycosis (Figures 17A-17E). Furthermore, in contrast to the vaccine with WGP (which did not protect against A. baumannii pneumonia), the inclusion of mannan conferred a dramatic survival advantage (log rank, p=0.003) in mice infected with A. baumannii pneumonia (Figures 17F vs. 17E) and helped protect against carbapenem-resistant P. aeruginosa pneumonia (Figure 17G).

[0139] Finally, applicants have also tested the vaccine against bacteremia caused by vancomycin-resistant Enterococcus (VRE), a highly antibiotic-resistant bacterium. Because VRE tends to be of low virulence, impractically high inocula would be required to achieve a lethal dose in immunocompetent mice (i.e., >5×10 8 CFU). Applicants therefore assessed bacterial blood density rather than survival rate. Mice immunized with MMA had significantly lower blood bacterial loads than control mice (Mann-Whitney, p=0.04) (Figure 17H).

[0140] Optimizing Vaccine Dosage Having established that MMA was superior to previous formulations and that MMA protection lasted for 3 days after immunization, applicants next sought to evaluate the duration of protection and whether increasing the dose could enhance protection. Applicants immunized mice with 3x or 10x the previous doses of mannan and MPL (3x = 300 μg mannan, 30 μg MPL, 200 μg aluminum hydroxide; 10x = 1000 μg mannan, 100 μg MPL, 200 μg aluminum hydroxide) and challenged them with lethal S. aureus or A. baumannii bacteremia 1, 2, or 3 weeks later. At 1 and 2 weeks after immunization, the lowest dose (1x = 100 μg mannan, 10 μg MPL, 200 μg aluminum hydroxide) provided the greatest protection against MRSA (Figure 18A). However, 3 weeks after immunization, the trend was reversed, with the highest dose (10-fold) providing the greatest protection and the lowest dose (1-fold) performing little more than a placebo (Figure 18A).

[0141] With regard to A. baumannii bacteremia, all three doses provided complete protection when mice were challenged one week after immunization (FIG. 18B). However, only the highest dose (10x) provided complete protection when mice were challenged two or three weeks after immunization (FIG. 18B). To confirm that the protective effect was not due to antibody-mediated immunity, applicants analyzed the plasma of vaccinated mice and confirmed that immunization did not produce antibodies specific for A. baumannii or S. aureus (Table 3).

[0142] Applicants next sought to clarify how rapidly the onset of protection occurs. To this end, Applicants immunized mice with a standard (1x) dose of MMA and challenged them with lethal S. aureus or A. baumannii bacteremia 24 hours after immunization. MMA provided either complete protection or protection similar to 3-day immunization (log-rank, p=0.005 S. aureus and p=0.003 A. baumannii) (Figure 17A vs. Figure 18C and Figure 17B vs. Figure 18D).

[0143] To assess its transferability to humans, total body irradiation was followed by CD34 + Mice receiving human stem cell transplants were immunized with MMA 3 days prior to infection (9, 10). Despite being severely immunocompromised, the vaccine still provided significant protection against A. baumannii bacteremia (log-rank, p = 0.008) (Figure 18E). To support potential future clinical use in humans, applicants also tested Good Manufacturing Practice (GMP)-compliant Al(OH)3 and MPL and Good Laboratory Practice (GLP)-compliant mannan by immunizing mice with GMP / GLP-grade MMA (G-MMA) 3 days prior to infection. The GMP / GLP-grade materials provided the same protection as non-GMP / GLP-grade materials (log-rank, p = 0.004 S. aureus and p = 0.003 A. baumannii) (Figures 18F-18G).

[0144] Cellular sources of defense To begin to elucidate the mechanism of protection, applicants evaluated changes in eight immune cell populations sourced from the spleens of immunized and non-immunized mice over a 21-day period following immunization: T cells, B cells, dendritic cells, NK cells, myeloid cells, neutrophils, monocytes, and macrophages. + No significant change was observed in the proportion of CD45 B cells (Figure 19). + The proportion of dendritic cells was elevated by the first day after immunization and persisted for at least three weeks (Figure 19). Dendritic cell and natural killer (NK) cell populations were CD45+ The proportion of CD45 cells decreased proportionally by the first day after immunization and began to increase by 3 to 7 days after immunization (Figure 19). + No trend for change in cell proportions was observed (Figure 4). However, by the first day after immunization, there was a shift in the proportion of myeloid cell types, including neutrophils, monocytes, and macrophages (Figure 19). Specifically, while the proportion of monocytes and macrophages increased, the proportion of neutrophils decreased and remained below baseline for at least 3 weeks (Figure 19).

[0145] The lack of antibody-mediated immunity (Table 3) and the rapid <24 h induction of protective immunity (Figures 18C-18D) suggested that the vaccine mediated protection via the innate immune system, rather than via adaptive immunity. Applicants therefore hypothesized that only the innate immune system was necessary to provide protection. Recombination activating gene 1 (RAG1) is crucial for V(D)J recombination, and mice with non-functional RAG1 lack mature B and T lymphocytes, consistent with severe combined immunodeficiency (SCID) (11-14). To determine whether lymphocytes play a role in protection mediated by MMA, Applicants immunized wild-type and RAG1 knockout (RAG1-KO) mice with MMA and challenged them with A. baumannii bacteremia 3 days after immunization. Even in the absence of B and T lymphocytes in RAG1-KO mice, the MMA vaccine successfully protected the mice from an otherwise lethal infection (log-rank, p=0.0009 RAG 1-KO, PBS vs. RAG 1-KO, MMA) (FIG. 20A). Thus, the vaccine did not induce T- and / or B-lymphocyte-mediated protection as did the older vaccines.

[0146] Applicants therefore hypothesized that innate immune cells are essential in protection mediated by MMA. To identify the innate immune cell types essential for MMA-mediated protection, Applicants immunized mice with MMA and selectively depleted the NK cells, or monocytes / macrophages, from the mice, since studies have shown that NK cells, monocytes, and macrophages are key to induce trained immunity (15-17). Depletion of NK cells partially eliminated the protective efficacy of the vaccine against S. aureus bacteremia, but not against A. baumannii bacteremia (log-rank, p=0.003), but not against S. aureus bacteremia (Figures 20B-20C). Depletion of monocytes and macrophages completely eliminated vaccine protection against both S. aureus and A. baumannii bacteremia (Figures 20D-20E).

[0147] Applicants then investigated whether MMA could activate mouse macrophage-like RAW 264.7 cells in vitro and primary human monocytes ex vivo in a manner similar to interferon gamma (IFNγ) by measuring macrophage uptake of A. baumannii. For both cell types, stimulation with MMA for 1 day resulted in phagocytosis at least as effectively as IFNγ stimulation (Kruskal-Wallis, PBS vs. MMA 1 day p<0.0001 RAW 264.7 and p=0.006 primary human monocytes) (Figures 20F-20G).

[0148] Immunomodulatory defense mechanisms Having established the importance of monocytes and macrophages, applicants investigated their immunomodulatory properties by evaluating cytokine-related gene expression, bacterial load, and cytokine profile in mice immunized with MMA before and after infection with S. aureus or A. baumannii bacteremia. First, applicants attempted to analyze cytokine concentrations in immunized mice. However, cytokines (IFNγ, TNF, IL-1β, IL-4, IL-6, IL-10, IL12 p70, IL-13, IL-17A) were below the detection limit. Applicants then assessed cytokine-related gene expression and found that 3 days after immunization, mice had lower expression of genes encoding the pro-inflammatory cytokines IL-12 p35 (Kruskal-Wallis, p=0.02), IL-17RB (Kruskal-Wallis, p=0.03), and TNFRSF13C (Kruskal-Wallis, p=0.02), and higher expression of the anti-inflammatory cytokine-related genes IL-10 (Kruskal-Wallis, p=0.02) and IL-18BP (Kruskal-Wallis, p=0.03). IL-1α and IL-1R2 (both associated with the pro-inflammatory cytokine IL-1) were also increased in immunized mice, but without statistical significance (Figures 22A-22B).

[0149] Applicants then analyzed bacterial load in immunized mice challenged with lethal S. aureus or A. baumannii bacteremia 4 hours after infection (Figures 22C-22D). In mice infected with S. aureus, the change in bacterial load was small regardless of whether the mice were immunized or how long they were immunized (Figure 21C). In mice infected with A. baumannii, bacterial load was significantly lower when infected 1 day after immunization compared to non-immunized mice (Figure 22D). Despite the fact that bacterial load was not statistically significantly different between most immunized mice compared to naive mice, survival outcomes were significantly improved for immunized mice (Figure 22C vs. Figures 18A and 18C, Figure 22D vs. Figures 18B and 18D). These data indicated that reduction in bacterial load is not the only driving factor for vaccine-mediated protection.

[0150] Applicants have previously observed that cytokine profiles can be better indicators of survival outcomes than changes in bacterial load (18-20). Thus, Applicants analyzed plasma cytokine concentrations in the very same immunized mice challenged with lethal S. aureus or A. baumannii bacteremia 4 h after infection. For both pathogens, immunized mice had lower concentrations of the proinflammatory cytokines IL-6, IL-12, and TNF; higher concentrations of the anti-inflammatory cytokine IL-10; and elevated IL-10 / TNF ratios that persisted even when infected several weeks after immunization (Figures 22E-22F). This trend matched gene expression analysis performed in immunized mice, which was particularly prominent in mice challenged 1 or 3 days after immunization. Several cytokines were below the limit of detection (IFNγ, IL-1β, IL-13, IL-17A) or were unchanged (IL-4).

[0151] MMA vaccination induces epigenetic and gene expression changes To further understand the protective mechanisms mediated by MMA and to evaluate whether the vaccine induces epigenetic biomarkers of trained immunity, applicants tested histone reprogramming and transcriptional changes in immunized mice. Epigenetic reprogramming has been shown to be a key characteristic of trained immunity (16, 21), with histone 3 lysine 27 acetylation (H3K27ac) as a key marker for promoter and enhancer activation (22). To test this, applicants isolated splenic macrophages from mice immunized with MMA 3 or 21 days prior and then performed H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq). Using mice immunized for 3 days, H3K27ac ChIP-seq revealed 1,080 differential peaks compared to naive control mice and 1,039 differential peaks compared to mice immunized for 21 days (Figure 21A). Naive control mice and mice immunized for 21 days showed only a few differential peaks (FIG. 21A).

[0152] Similarly, RNA-seq identified 779 differentially expressed genes (DEGs) between naive and immunized mice 3 days prior, 652 DEGs between immunized mice 3 days prior and immunized mice 21 days prior, but only 170 DEGs between naive and immunized mice 21 days prior (Figure 21B). Most of the highly acetylated H3K27 regions in immunized mice 3 days prior returned to baseline by 21 days after immunization. Most regions with low H3K27 acetylation in immunized mice 3 days prior increased by 21 days after immunization, but not all returned to baseline (Figure 21C). Similar gene expression changes were observed by RNA-seq (Figure 21C), indicating that the changes in H3K27ac caused gene expression in the manner we predicted.

[0153] To understand the biological processes affected by MMA immunization, applicants performed pathway analysis on the DEGs, which found that many genes that were highly upregulated 3 days after immunization were associated with pathways (e.g., lysosome formation and complement cascade) known to be involved in host defense against various infections (e.g., those caused by S. aureus, Bordetella pertussis, Plasmodium spp., and prions) and activation of the innate immune system (Figure 21D).

[0154] Experiment 9: Vaccination Materials and Protocols The following materials and methods relate to and support the data reported in Experiment 8.

[0155] Mannan (Sigma, M3640), monophosphoryl lipid A (MPL) (InvivoGen, tlrl-mpls), whole glucan particles (WGP) (InvivoGen, tlrl-wgp), and 2% aluminum hydroxide (Al(OH)3) (Accurate Chemical & Scientific Corporation, A1090S) were prepared and stored according to the manufacturer's protocol. Mannan (MedicaPharma, Mannan), PHAD® (Avanti Polar Lipids, 699800P), and 2% Al(OH)3 (Croda, AJV3012) were used in GMP / GLP grade MMA. Vaccines were prepared fresh each time. Mice were immunized with 200 μL of premixed vaccine in phosphate buffered saline (PBS) administered subcutaneously (SC) in the scruff of the neck. Unless otherwise specified, each mouse received 0.1% Al(OH)3, 100 μg WGP, and 10 μg MPL, and 100 μg mannan, either alone or in combination. Mice immunized with MWA 30% were immunized with 0.1% Al(OH)3, 3 μg MPL, and 30 μg WGP. Mice immunized with MWA 10% were immunized with 0.1% Al(OH)3, 1 μg MPL, and 10 μg WGP. Mice immunized with MWA 1% were immunized with 0.1% Al(OH)3, 0.1 μg MPL, and 1 μg WGP. Intravenous (IV) infection

[0156] Frozen stocks of A. baumannii and K. pneumoniae bacteria grown to mid-log phase were prepared as previously described (35). Inocula were prepared by diluting these concentrated frozen stocks of bacteria in PBS. Inocula were established by plating serial dilutions on tryptic soy agar (TSA) plates and incubating overnight at 37°C. Mice infected with A. baumannii were monitored for 7 days and mice infected with K. pneumoniae were monitored for 14 days, after which they were euthanized according to IACUC protocols.

[0157] S. aureus and E. faecalis inocula were prepared from mid-logarithmic subcultures of overnight cultures for each infection. Briefly, S. aureus and E. faecalis were inoculated into tryptic soy broth (TSB) and incubated overnight at 37° C. with shaking set at 200 rpm. The overnight cultures were subcultured by diluting 1:100 into sterile TSB and incubated for 3 hours at 37° C. with shaking set at 200 rpm. The subcultures were pelleted in a refrigerated centrifuge at 4,000×g for 5 minutes and rinsed by resuspending the pellet in PBS. After two additional rinses (3 times total), the pellets were resuspended in PBS and the optical density at 600 nm (OD 600 The dilution was adjusted to 0.5 using PBS. The inoculum was established by plating serial dilutions onto TSA plates and incubating overnight at 37°C. Mice infected with S. aureus were monitored for 28 days after which they were euthanized according to IACUC protocol. Mice infected with E. faecalis were monitored for 7 days after which they were euthanized according to IACUC protocol.

[0158] Rhizopus delemar were cultured on plates with potato dextrose agar for 8 days at 37°C and harvested in PBS with 0.05% Tween® 80. The conidial density was determined using a hemocytometer and the viability of the conidial suspension was determined by plating on Sabouraud dextrose agar. Mice infected with R. delemar were monitored for 14 days after which they were euthanized according to IACUC protocols.

[0159] Candida albicans was serially passaged three times in yeast peptone dextrose broth and washed twice with PBS prior to infection. The infectious inoculum was prepared by counting with a hemocytometer. Mice infected with C. albicans were monitored for 7 days after which they were euthanized according to IACUC protocols.

[0160] Oral aspiration (OA) infection Mice were infected using an aspiration pneumonia model as previously described (7). Briefly, A. baumannii and P. aeruginosa inocula were prepared from subcultures of overnight cultures as described above, and mice were infected by aspirating 50 μL of bacteria suspended in PBS. Mice infected via OA were monitored for up to 7 days, after which they were euthanized according to IACUC protocols.

[0161] Innate immune cell depletion Macrophages were depleted by intraperitoneal (IP) injection of 50 mg / kg liposomal clodronate (Foundation Clodronate Liposomes, C-010) 3 days prior to infection (36). Neutrophils were depleted by IP injection of 230 mg / kg cyclophosphamide (Baxter) 3 days prior to infection (18, 37).

[0162] Flow cytometry analysis Mouse spleens were collected, crushed through a 70 μm cell strainer with a 35 mL syringe plunger, and rinsed with Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) by volume (DMEM + 10% FBS). Splenocytes were pelleted by centrifugation at 300 × g for 5 min at room temperature, and the supernatant was discarded. After rinsing three times with PBS, red blood cells (RBCs) were lysed using RBC lysis buffer (BioLegend Cat. # 420301) according to the manufacturer's protocol. Splenocytes were resuspended in PBS with 5% fetal bovine serum (FACS buffer), counted with a hemocytometer, and incubated with Fc blocker (BD Biosciences Cat. # 553141) for 30 min on ice. Splenocytes were pelleted at 300×g for 5 min in a room temperature centrifuge, resuspended in FACS buffer, and incubated with fluorophore-conjugated antibodies for OMIP-032, a two-panel multicolor immunophenotyping assay to assess innate and adaptive immune cell populations as previously described (38). After incubation, splenocytes were pelleted at 300×g for 5 min in a room temperature centrifuge, washed twice with PBS, and resuspended in FACS buffer. Samples were analyzed using a BD FACS Canto II flow cytometer.

[0163] Bacterial binding flow analysis was performed by incubating diluted HUMC1 (1:100) or LAC (1:100,000) with mouse plasma or 10 μg / mL isotype (Fisher, MAB002) or serum from mice collected from 30 min HUMC1 or LAC infection. Bacteria were pelleted in a room temperature centrifuge at 10,000×g for 1 min and rinsed twice with PBS. Samples were then incubated with anti-mouse secondary antibody (Thermo Fisher, A21235) for 30 min, pelleted, rinsed twice with PBS, and resuspended in FACS buffer. Samples were analyzed by BD Accuri TM Analysis was performed using a C6 Plus flow cytometer.

[0164] In vitro macrophage opsonophagocytosis assay Applicants used RAW 264.7 mouse macrophages (ATCC) cultured with DMEM + 10% FBS and stimulated with a mixture of 10 ng / mL Al(OH)3, 0.5 μg / mL MPL, and 5 μg / mL mannan ("MMA") for either 1 or 3 days. RAW 264.7 stimulated overnight with 100 μg / mL IFN-γ (Peprotech, 315-05) and PBS were used as positive and negative controls, as previously described (39). Once the cells were prepared, Applicants performed macrophage opsonophagocytosis assays as follows: Bacteria were prepared from overnight cultures of A. baumannii subcultured to logarithmic growth phase, washed in PBS, and resuspended in Hank's buffered saline solution (HBSS). Cells were rinsed three times with HBSS, and bacteria were added to the wells at a ratio of 20:1 (bacteria to macrophages) in the presence of 10% CD-1 mouse serum (Innovative Research Inc, IMSCD1-COMPL) and centrifuged at 300×g for 5 min. Macrophages were washed three times with HBSS, fixed with 100% methanol, and stained with Hema-3 according to the manufacturer's protocol (Fisher Scientific). To quantify bacteria per macrophage, coverslips were imaged with a Leica DMLS clinical microscope equipped with a Leica ICC50 HD digital camera.

[0165] Ex vivo macrophage opsonophagocytosis assay Applicants used ex vivo macrophages differentiated from human peripheral blood mononuclear cells (PBMCs). Fresh PBMCs were purchased from the UCLA / CFAR Virology Core Laboratory. Monocytes were isolated (StemCell Technologies, 19359) and differentiated into M1 macrophages according to the manufacturer's protocol (StemCell Technologies, 10961). We stimulated with a mixture of 10 ng / mL Al(OH)3, 0.5 μg / mL MPL and 5 μg / mL mannan for either 1 or 2 days. Macrophages stimulated with 50 ng / mL IFN-γ (StemCell Technologies, 78020) and PBS for 3 days were used as positive and negative controls. Macrophage opsonophagocytosis assays were performed and enumerated as described above.

[0166] Chromatin immunoprecipitation sequencing Spleens were harvested from C3HeB / Fe mice and splenocytes were isolated as described above. Splenocytes were rinsed in PBS and resuspended in PBS containing 2% FBS and 1 mM EDTA. f4 / 80+ macrophages were isolated according to the manufacturer's protocol (StemCell Technologies, 100-0659). H3K27ac chromatin immunoprecipitation (ChIP) was performed using anti-H3K27ac polyclonal antibody (Diagenode, C15410196) and iDeal ChIP-seq kit for histones (Diagenode, C01010051). DNA library preparation and Illumina HiSeq 150-bp paired-end sequencing were performed by Azenta Life Sciences. ChIP-sequencing reads were aligned against the mouse genome assembly mm39 (based on NCBI GRCm39) using bwa (PMID: 19451168). BAM files were filtered to remove duplicate reads and poor mapping quality using SAMtools (PMID: 19505943). MACS2 was used to call H3K27ac peaks using default (narrow) settings (PMID: 18798982). The quality of the ChIP-seq data was visualized by creating bigwig files using deepTools (PMID: 27079975) in the UCSC Genome Browser. For differential peak calling, data (H3K27ac reads / peak) were normalized using the R package DESeq2 (PMID: 25516281) and then pairwise comparisons were made between the PBS group and the day 3 and day 21 groups. Differential peaks were identified as adjusted p-values ​​<0.05, fold change >2, reads / peak >50. Peaks were then merged into one file and principal component analysis (PCA) plots were generated.

[0167] RNA sequencing Splenic f4 / 80+ macrophages were harvested from C3HeB / Fe mice as described above. Azenta Life Sciences performed RNA isolation, rRNA removal, library preparation, and Illumina HiSeq 150-bp paired-end sequencing. To infer gene expression levels, RNA-seq reads were aligned to the mm39 mouse transcriptome using Bowtie (PMID: 19261174). Quantification of gene expression was performed using MMSEQ (PMID: 21310039). Statistical analysis was performed using DESeq2, and pairwise comparisons were made between the PBS group, the day 3 group, and the day 21 group. Differentially expressed genes were identified as those exhibiting p-values ​​<0.05, FC>2, and RPKM>1. The differential gene lists from all comparisons were then merged, and the combined list of differential genes was used for plotting.

[0168] statistics Survival rates were compared by the non-parametric log-rank test at α = 0.05. Bacterial burden, cell populations, macrophage assays, and cytokines were compared by Wilcoxon-Mann-Whitney or Kruskal-Wallis tests at α = 0.05.

[0169] Table 3 Immunization did not result in pathogen-specific antibody production. Male C3HeB / Fe and female BALB / c mice immunized with MMA (N=5 / group) did not develop specific antibodies against S. aureus or A. baumannii. [Table 4]

[0170] Consideration Applicant believes that the protein-free three-component vaccine is +We found that the vaccine mediated kingdom-wide protection against high-priority AMR bacterial and fungal bloodstream and lung infections in mice, including humanized mice. Protection began within 24 hours after vaccination and persisted for up to 3 weeks, and protection was mediated by the innate immune system rather than the adaptive immune system. Protection required macrophages, but not lymphocytes or neutrophils. NK cells were required for optimal protection against S. aureus infection, but not against A. baumannii, a gram-negative pathogen. Furthermore, vaccination significantly increased the number of monocytes and macrophages in the blood, and the vaccine directly induced differentiation of monocytes into macrophages in vitro and ex vivo, significantly enhancing phagocytosis. Macrophages were shown to mediate protection in a manner involving cytokine modulation via epigenetic reprogramming. Applicants also showed that the tripartite vaccine induced H3K27ac epigenetic and gene expression changes in splenic macrophages. Taken together, these data confirm that the protective mechanism of the vaccine was the induction of trained immunity (23).

[0171] Traditional vaccines are a “vertical” approach to infection prevention (4), which activates antigen-specific lymphocytes that target one pathogen at a time. This single-pathogen targeting makes it difficult to develop or deploy such vaccines for the prevention of HAIs caused by the myriad of bacterial and fungal pathogens. In contrast, trained immunity offers much broader targeting of pathogens and reflects a “horizontal” approach to infection prevention, rather than being pathogen-specific. The price of horizontal targeting of many pathogens from one vaccine via trained immunity is that protection lasts for a much shorter period of time than adaptive immunity-mediated traditional pathogen-specific vaccines. However, given that the average duration of acute hospitalization is 5 days and that 95% of hospitalizations last less than 21 days (31), the shorter duration of protection offered by trained immunity is not detrimental to vaccine efficacy or deployment.

[0172] While the global crisis of AMR infections continues to grow, the pipeline of new antibiotics to combat the threat has not kept pace (32). This study utilized models of two common routes of infection caused by AMR nosocomial pathogens: central line-associated bloodstream infections and ventilator-associated pneumonia (33). Many such infections are highly drug resistant and have few effective prevention or treatment options. AMR pathogens are most commonly found in hospitals, and new strategies are critically needed to prevent such infections (34). Described herein is a novel trained immunity vaccine that can be used to prevent blood and lung infections caused by the most dangerous and highest priority AMR pathogens that cause HAIs. The breadth and robustness of protection our vaccine provides against these infections suggests substantial promise for reducing antimicrobial use and transforming infection prevention in healthcare.

[0173] Equivalent While the present disclosure has been described in conjunction with the above embodiments, it should be understood that the foregoing description and examples are intended to illustrate, but not limit, the scope of the present disclosure. Other aspects, advantages, and modifications within the scope of the present disclosure will be apparent to those skilled in the art to which the present disclosure pertains.

[0174] The invention illustratively described herein may suitably be practiced in the absence of any element or limitation not specifically disclosed herein. Thus, for example, the terms "comprises," "including," "containing," and the like, are to be understood broadly and without limitation. Moreover, the terms and expressions used herein are used as terms of description and not as terms of limitation, and in the use of such terms and expressions, there is no intention to exclude 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 as described in the claims.

[0175] Thus, while the present invention has been specifically disclosed by preferred embodiments and optional features, it is to be understood that modifications, improvements, and variations of the invention disclosed and embodied therein may occur to those skilled in the art, and that such modifications, improvements, and variations are deemed to be within the scope of the present invention. The materials, methods, and examples provided herein are representative of preferred embodiments, are illustrative, and are not intended as limitations on the scope of the invention.

[0176] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings that fall within the scope of the generic disclosure also form part of the invention. This includes any provisos or negative limitations that remove any subject matter from the genus, along with the generic description of the invention, whether or not the omitted thing is specifically described herein.

[0177] Furthermore, when features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention is also thereby described in terms of any individual members or subgroups of members of that Markush group.

[0178] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each was individually incorporated by reference. In case of conflict, the present specification, including definitions, will control.

[0179] References - Experiments 1-7 [Table 5-1] [Table 5-2]

[0180] References for Experiments 8 and 9:

Table 6-1

Table 6-2

Table 6-3

Table 6-4

Table 6-5

Claims

1. A composition comprising an effective amount of each of MPL monophosphoryl lipid (MPL), mannan, and aluminum hydroxide (collectively MMA), provided that said composition does not contain an antigen effective to induce an immune response against a pathogen, optionally a (parasitic) pathogen 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), mannan, and aluminum hydroxide (MMA), provided that said composition does not contain an antigen effective to induce an immune response against a pathogen, optionally selected from a viral pathogen, a bacterial pathogen, or a fungal pathogen.

3. A composition comprising an effective amount of each of MPL monophosphoryl lipid (MPL), mannan, and aluminum hydroxide (collectively MMA) as active immune-inducing agents.

4. The composition of any one of claims 1 to 3, wherein the antigen does not induce adaptive T cell or B cell immunity.

5. The composition of any one of claims 1 to 4, wherein the effective amounts of aluminum hydroxide, MPL monophosphoryl lipid (MPL), and mannan are jointly effective to induce an immune response when administered to a subject in need thereof.

6. The composition of claim 5 , wherein the immune response is non-specific against the pathogen.

7. The composition according to any one of claims 1 to 6, wherein the antigen is selected from a peptide, a protein or a glycoprotein.

8. A composition according to any one of the preceding claims, wherein the aluminium hydroxide is an aluminium hydroxide wet suspension, optionally Alhydrogel®.

9. The effective amount is a. about 0.1 mg / ml to about 10 mg / ml aluminum hydroxide; b. about 0.1 mg / ml to about 10 mg / ml of MPL; c. about 0.01 mg / ml to about 10 mg / ml of mannan; The composition according to any one of claims 1 to 8, comprising:

10. The composition according to any one of claims 1 to 9, further comprising a pharma- ceutically acceptable carrier, optionally saline or phosphate buffered saline.

11. 11. A method for enhancing immunity in a subject against infection caused by a bacterial or fungal pathogen, the method comprising administering to the subject an effective amount of a composition according to any one of claims 1 to 10.

12. 12. The method of claim 11, wherein the bacteria is selected from S. aureus, A. baumannii, K. pneumoniae, P. aeruginosa, E. coli, Enterobacter spp., Serratia, Stenotrophomonas, and the fungus is selected from Candida spp.

13. 13. The method of claim 11 or 12, wherein the composition is administered by a method comprising inhalation, intramuscular, subcutaneous, or intravenous administration.

14. The method of any one of claims 11 to 13, wherein the composition is administered once, twice, or three times over a period of one to three months.

15. The method of any one of claims 11 to 14, wherein the subject is at risk of a bacterial or fungal infection or the subject is infected with said bacterial or fungal microorganism.

16. The method of any one of claims 11 to 15, wherein the subject is immunocompromised.

17. The method of any one of claims 111 to 16, further comprising assaying the subject for a bacterial or fungal infection prior to administering the composition.

18. 11. A method of treating or preventing 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 a composition according to any one of claims 1 to 10.

19. 19. The method of claim 18, wherein the bacterial or fungal microorganism is selected from S. aureus, A. baumannii, K. pneumoniae, P. aeruginosa, E. coli, Enterobacter spp., Serratia, Stenotrophomonas, Candida spp.

20. 20. The method of claim 18 or 19, wherein the composition is administered by a method comprising parenteral administration, including subcutaneous, intramuscular, or intravenous administration.

21. The method of any one of claims 18 to 20, wherein the composition is administered once, twice or three times.

22. The method of any one of claims 18 to 21, further comprising assaying the subject for a bacterial or fungal infection prior to administering the composition.

23. The composition of any one of claims 18 to 22, wherein the disorder is a blood or lung infection caused by a nosocomial pathogen.

24. A kit comprising the composition of any one of claims 1 to 10 and instructions for use.

25. 11. A method for identifying a compound or agent that provides a benefit selected from one or more of enhancing immunity to bacterial or fungal microbial infection or treating a bacterial or fungal infection or a disease associated with bacterial or fungal microbial infection, comprising the steps of combining the compound or agent with a composition according to any one of claims 1 to 10, administering the combined composition to a non-human subject infected with a bacterial or fungal microbial microorganism, and assaying for infection or survival after administration, wherein the compound or agent that enhances the activity of the composition according to any one of claims 1 to 10 is the agent that provides the benefit.

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