Manganese glycerophosphate GELS and methods of use thereof

Abstract

This invention relates to compositions comprising an immunogen and a manganese glycerophosphate (MnGp) gel useful in raising an immune response in a subject, vaccinating a subject, and / or immunotherapy, such as cancer immunotherapy, in a subject. In addition, this invention relates to MnGp gels and methods of synthesizing the same using mechanical shear stress and / or synthesis of nanoparticles.

Description

Attorney Docket No. 5470.985.WOMANGANESE GLYCEROPHOSPHATE GELS AND METHODS OF USE THEREOFSTATEMENT OF PRIORITY

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 714,626, filed October 31, 2024, the entire contents of which are incorporated by reference herein.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant No. 75N93019C00052 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD OF THE INVENTION

[0003] This invention relates to compositions comprising an immunogen and manganese glycerophosphate (MnGp) gel, and methods of use of the same in raising an immune response, vaccinating a subject, and / or providing an immunotherapy to a subject.BACKGROUND OF THE INVENTION

[0004] Vaccination stands as one of the most successful public health measures to date. In the United States alone, extensive childhood vaccination programs have achieved the full eradication of smallpox and polio, as well as significant reductions (>90%) in other previous high morbidity and mortality infectious diseases such as diphtheria, measles, and mumps (Rodrigues and Plotkin, 2020; Roush, 2007). More recently, the SARS-CoV-2 mRNA vaccines demonstrated profound efficacy in preventing severe illness and improving disease outcomes, especially in aged, immunocompromised, and high-risk comorbidity populations (Kelly et al., 2022; Lin et al., 2022; Polack et al., 2020). Nevertheless, complications observed with the vaccine rollout and global distribution - namely, poor stability outside of cold-chain storage (Uddin and Roni, 2021) and carrier-mediated reactogenicity (Sutton et al., 2022) - highlight the continual need to develop novel vaccine formulations.

[0005] Traditional vaccine development is largely focused on the production of live attenuated pathogens, but the capacity for vaccine strains to replicate in and cause illness to the host prompts safety concerns for immunocompromised populations (Minor, 2015; Plotkin, 2014). In contrast, subunit vaccines, which are comprised of purified protein(s), peptide(s), orAttorney Docket No. 5470.985.WO polysaccharide(s) of a pathogen (i.e., antigen) and are therefore non-infectious, represent a promising alternative vaccination strategy that circumvents these safety concerns (Kyriakidis et al., 2021). Subunit antigens are often weakly immunogenic on their own, typically eliciting modest humoral immunity with little to no cellular immunity (Reed et al., 2013). Therefore, subunit vaccines often incorporate adjuvants, or immunostimulatory molecules, to enhance and prolong the immune response. Aluminum-containing adjuvants (i.e., “alum”) are perhaps the most widely used subunit vaccine adjuvants, largely due to their potency, safety profile, and relatively accessible cost (Lindblad, 2004). However, despite proven efficacy in several licensed vaccines, alum primarily elicits a T helper 2 (Th2) cell-biased response (i.e., antibody- mediated, or humoral, immunity) with a marginal T helper 1 (Thl) cell response (i.e., cell- mediated immunity) (Pulendran et al., 2021). Thus, for diseases where a robust Thl response is critical, such as influenza (Aleebrahim-Dehkordi et al., 2022) and certain cancers (Knutson and Disis, 2005), candidate vaccines require more effective adjuvant systems. As such, significant progress has been made in the exploration of adjuvants MF59 (Ko and Kang, 2018), AS04 (Didierlaurent et al., 2009)), toll-like receptor (TLR) agonists (e.g., polyinosinic:polycytidylic acid (poly(I:C)) (Martins et al., 2015), cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs) (Bode et al., 2011)), and cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) agonists (e.g., cGAMP (Li et al., 2013)) to improve the Thl / Th2 balance of vaccine responses.

[0006] Current limitations in conventional adjuvants, like alum described above, require improved design and delivery. Thus, new adjuvants for vaccine and immunotherapy methodologies are desired.SUMMARY OF THE INVENTION

[0007] The present invention is based on the finding that applying mechanical shear stress to a MnGp powder allows the formation of an MnGp gel, and the beneficial functions of said gel as an adjuvant.

[0008] Thus, one aspect of the invention relates to a composition comprising an immunogen and an MnGp gel. In some embodiments, the composition further comprises a cryoprotectant. In some embodiments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0009] Another aspect of the invention relates to a method of raising an immune response in a subject, said method comprising administering to the subject an effective amount of a composition as described herein, thereby raising an immune response in the subject.Attorney Docket No. 5470.985.WO

[0010] Another aspect of the invention relates to a method of vaccinating a subject against a pathogen infection, said method comprising administering to the subject an effective amount of a composition as described herein, thereby vaccinating the subject against the pathogen infection.

[0011] Another aspect of the invention relates to a method of providing an immunotherapy in a subject in need thereof, said method comprising administering to the subject an effective amount of a composition as described herein, thereby providing the immunotherapy to the subject.

[0012] Another aspect of the invention relates to a method of forming an MnGp gel, optionally wherein the method comprises: obtaining MnGp (e.g., powdered MnGp); and applying mechanical shear stress to the MnGp; thereby forming an MnGp gel. In some embodiments, the method optionally comprises synthesizing MnGp nanoparticles and concentrating the nanoparticles in solution, thereby forming an MnGp gel.

[0013] Another aspect of the invention relates to an MnGp gel, optionally wherein the MnGp gel is comprised of MnGp particles. In some embodiments, the MnGp gel further comprises a cryoprotectant.

[0014] These and other aspects of the invention are set forth in more detail in the description of the invention below.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Fig. 1 is a cartoon showing the usages for manganese glycerophosphate (MnGp) gels according to some embodiments of the present invention.

[0016] Fig. 2A is a cartoon showing the synthesis schematic of a manganese glycerophosphate (MnGp) gel according to some embodiments of the present invention. Fig. 2B is a series of images showing a comparison of an MnGp powder in solution (left tube) and a 350 mg / mL MnGp gel formed with 150 mg / mL sucrose (right tube) both in upright containers (left) or with inverted containers (right). Fig. 2C is a scanning electron microscope (SEM) image of a 350 mg / mL MnGp gel formed with 150 mg / mL sucrose according to some embodiments of the present invention. Fig. 2D is a graph showing the ovalbumin (OVA) released by MnGp gels at various MnGp concentrations over 1 week; data are presented as mean ± standard deviation (n=3).

[0017] Fig. 3A is an SEM image of a 350 mg / mL MnGp gel formed with 150 mg / mL sucrose according to some embodiments of the present invention. Fig. 3B is an SEM image of a 350Attorney Docket No. 5470.985.WO mg / mL MnGp gel formed without sucrose according to some embodiments of the present invention.

[0018] Fig. 4A is an SEM image of a 250 mg / mL MnGp gel according to some embodiments of the present invention. Fig. 4B is an SEM image of a 100 mg / mL MnGp gel according to some embodiments of the present invention. Fig. 4C is an SEM image of a 25 mg / mL MnGp gel according to some embodiments of the present invention.

[0019] Figs. 5A-5D are graphs showing bone marrow derived dendritic cells (BMDCs) either untreated (UT) or 12 hours after being treated with a 100 mg / mL MnGp gel or lipopolysaccharide (LPS); the data are presented as mean ± standard deviation (n=3); statistical significance is presented as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 for an ordinary one-way ANOVA with Tukey ’ s multiple comparisons test. Fig. 5A is a graph showing the cell viability normalized to the UT cells. Fig. 5B is a graph showing the frequency of CD80+CD86+cells. Fig. 5C is a graph showing the mean fluorescent intensity (MFI) of the major histocompatibility complex II (MHC-II). Fig. 5D is a graph showing the concentration of interferon beta (IFN-P) in the cell supernatant.

[0020] Figs. 6A and 6B are graphs showing the MFI of the indicated proteins as assessed by flow cytometry. BMDCs were analyzed after either being untreated (UT) or 12 hours after being treated with a 100 mg / mL MnGp gel or lipopolysaccharide (LPS); the data are presented as mean ± standard deviation (n=3); statistical significance is presented as *p<0.05 for an ordinary one-way ANOVA with Tukey ’s multiple comparisons test.

[0021] Figs. 7A-7C are graphs showing the titers of the indicated OVA-specific antibody titers from the serum of five 6-8-week-old C57BL / 6 mice following prime-boost immunization via intramuscular (IM) injection of the indicated composition on days 0, 28, and 140. Data are presented as median ± range. Statistical significance is presented as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 for an ordinary two-way ANOVA with Tukey’s multiple comparisons test.

[0022] Figs. 8A-8C are graphs showing the titers of the indicated OVA-specific antibody titers from the serum of five 6-8-week-old C57BL / 6 mice following prime-boost immunization via IM injection of the indicated composition on days 0 and 28. Data are presented as median ± range. Statistical significance is presented as ***p<0.001 and ****p<0.0001 for an ordinary two-way ANOVA with Tukey’s multiple comparisons test.

[0023] Figs. 9A-9C are graphs showing the titers of the indicated OVA-specific antibody titers from the serum of five 6-8-week-old C57BL / 6 mice following prime-boost immunization viaAttorney Docket No. 5470.985.WOIM injection of the indicated composition on days 0 and 28. Data are presented as median ± range.

[0024] Figs. 10A and 10B are graphs showing the titers of the indicated antibody from three BALB / c mice following prime-boost immunization via IM injection of the indicated composition on days 0 and 21. Data are presented as median ± range. Statistical significance is presented as ****p<0.0001.

[0025] Figs. 11A-11D are graphs showing the antigen recall in splenocytes from five 6-8- week-old C57BL / 6 mice following prime-boost immunization via IM injection of the indicated composition on days 0, 28, and 140. Data are presented as mean ± standard deviation. Statistical significance is presented as **p<0.01 and ****p<0.0001 for ordinary two-way ANOVA with Tukey’s multiple comparisons test. Fig. 11A is a graph showing the number of interferon gamma (IFN-y) spots per 1 million cells. Fig. 1 IB is a graph showing the number of interleukin two (IL-2) spots per 1 million cells. Fig. 11C is a graph showing the concentration of IFN-y in the cell supernatant. Fig. 11D is a graph showing the concentration of IL-2 in the cell supernatant.

[0026] Figs. 12A and 12B are graphs showing the antigen recall in splenocytes from five C57BL / 6 mice following prime-boost immunization via IM injection of the indicated composition on days 0, 28, and 140. Data are presented as mean ± standard deviation. Fig. 12A is a graph showing the concentration of tumor necrosis factor alpha (TNF-a) in the cell supernatant. Fig. 12B is a graph showing the concentration of interleukin six (IL-6) in the cell supernatant.

[0027] Fig. 13 is a series of graphs in a flowchart showing the flow cytometry gating strategy used to characterize T and B cell phenotypes from the spleens and lymph nodes of immunized mice in experiments according to some embodiments of the invention.

[0028] Figs. 14A-14F are a series of graphs showing the flow cytometry counts of the indicated immune cell phenotypes in the spleen from five 6-8-week-old C57BL / 6 mice following primeboost immunization via IM injection of the indicated composition on days 0, 28, and 140. Splenocytes were harvested on day 151. Data are presented as mean ± standard deviation. Statistical significance is presented as *p<0.05, **p<0.01, and ****p<0.0001 for ordinary two- way ANOVA with Tukey’s multiple comparisons test.

[0029] Figs. 15A and 15B are a series of graphs showing the flow cytometry counts of germinal center (GC) or effector B cell phenotypes in the spleen from five 6-8-week-old C57BL / 6 mice following prime-boost immunization via IM injection of the indicatedAttorney Docket No. 5470.985.WO composition on days 0, 28, and 140. Splenocytes were harvested on day 151. Data are presented as mean ± standard deviation.

[0030] Figs. 16A-16E are a series of graphs showing the flow cytometry counts of the indicated T cell phenotypes in the inguinal lymph nodes (iLNs) from five C57BL / 6 mice following prime-boost immunization via IM injection of the indicated composition on days 0, 28, and 140. Splenocytes were harvested on day 151. Data are presented as mean ± standard deviation. Statistical significance is presented as *p<0.05 and **p<0.01 for ordinary two-way ANOVA with Tukey’s multiple comparisons test.

[0031] Figs. 17A and 17B are a series of graphs showing the flow cytometry counts of GC or effector B cell phenotypes in the iLNs from five C57BL / 6 mice following prime-boost immunization via IM injection of the indicated composition on days 0, 28, and 140. Splenocytes were harvested on day 151. Data are presented as mean ± standard deviation. Statistical significance is presented as *p<0.05 for ordinary two-way ANOVA with Tukey’s multiple comparisons test.

[0032] Figs. 18A and 18B are a series of graphs showing the antigen recall in the spleen and draining lymph node (dLN) of five C57BL / 6 18-month-old mice following IM immunization on days 0, 28, and 77 with the indicated composition. Fig. 18A shows the concentration of IL- 4 in the cell supernatant of splenocytes restimulated with antigen for 36 hours. Fig. 18B shows the concentration of IFN-y in the cell supernatant of dLN cells harvested on day 84 and restimulated for 36 hours with antigen. Data is presented as mean ± standard deviation. Statistical significance is presented as *p<0.05, **p<0.01, and ****p<0.0001 for an ordinary two-way ANOVA with Tukey’s multiple comparisons test.

[0033] Figs. 19A-19C are a series of graphs showing the titers of the indicated antibody from 18-month-old C57BL / 6 mice following prime-boost-boost immunization on days 0, 28 and 84 via IM injection of the indicated composition. Blood was drawn every two weeks and serum antibody titers measured through ELISA. Tissues were collected on day 84. Data is presented as mean ± standard deviation. Statistical significance is presented as *p<0.05, **p<0.01 for an ordinary two-way ANOVA with Tukey’s multiple comparisons test.

[0034] Figs. 20A-20B are a series of graphs showing the antigen recall in the spleen and draining lymph node (dLN) of C57BL / 6 18-month-old mice following IM immunization on days 0, 28, and 77 with the indicated composition. Fig. 20A shows the concentration of IFN-y in the cell supernatant of splenocytes or draining lymph node restimulated with antigen for 36 hours. Fig. 20B shows the concentration of IL- 17 in the cell supernatant of splenocytes or dLN cells harvested on day 84 and restimulated for 36 hours with antigen. Data is presented asAttorney Docket No. 5470.985.WO mean ± standard deviation. Statistical significance is presented as *p<0.05, **p<0.01, and ****p<0.0001 for an ordinary two-way ANOVA with Tukey’s multiple comparisons test.

[0035] Figs. 21A-21C are a series of graphs showing the titers of the indicated antibody from 6-8-week-old BALB / c mice (n=10) following IM immunization on days 0 and 28 with the indicated composition and a vaccinia virus challenge on day 56. Fig. 21D shows the percentage of mice from each treatment cohort that survived after the indicated number of days postinfection. Data is presented as mean ± standard deviation. Statistical significance is presented as *p<0.05, **p<0.01, and ****p<0.0001 for an ordinary two-way ANOVA with Tukey’s multiple comparisons test.DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention is explained in greater detail below. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure which do not depart from the instant invention. Hence, the following specification is intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

[0037] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0038] 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. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, and other references mentioned herein areAttorney Docket No. 5470.985.WO incorporated by reference in their entirety. In case of a conflict in terminology, the present specification is controlling.

[0039] Nucleotide sequences are presented herein by single strand only, in the 5’ to 3’ direction, from left to right, unless specifically indicated otherwise. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three- letter code, both in accordance with 37 C.F.R. §1.822 and established usage.

[0040] Except as otherwise indicated, standard methods known to those skilled in the art may be used for production of recombinant and synthetic polypeptides, antibodies or antigenbinding fragments thereof, manipulation of nucleic acid sequences, production of transformed cells, the construction of rAAV constructs, modified capsid proteins, packaging vectors expressing the AAV rep and / or cap sequences, and transiently and stably transfected packaging cells. Such techniques are known to those skilled in the art. See, e.g., M. R. GREEN & J. SAMBROOK, MOLECULAR CLONING: A LABORATORY MANUAL 4th Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012); F. M. AUSUBEL et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, NY).

[0041] All publications, patent applications, patents, nucleotide sequences, amino acid sequences and other references mentioned herein are incorporated by reference in their entirety.

[0042] As used in the description of the invention and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0043] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0044] Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

[0045] Furthermore, the term “about,” as used herein when referring to a measurable value such as an amount of a compound or agent of this invention, dose, time, temperature, and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount.

[0046] As used herein, the transitional phrase “consisting essentially of’ is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basicAttorney Docket No. 5470.985.WO and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of’ as used herein should not be interpreted as equivalent to “comprising.”

[0047] The term “consists essentially of’ (and grammatical variants), as applied to a polynucleotide or polypeptide sequence of this invention, means a polynucleotide or polypeptide that consists of both the recited sequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional nucleotides or amino acids on the 5’ and / or 3’ or N-terminal and / or C-terminal ends of the recited sequence or between the two ends e.g., between domains) such that the function of the polynucleotide or polypeptide is not materially altered. The total of ten or less additional nucleotides or amino acids includes the total number of additional nucleotides or amino acids added together. The term “materially altered,” as applied to polynucleotides of the invention, refers to an increase or decrease in ability to express the encoded polypeptide of at least about 50% or more as compared to the expression level of a polynucleotide consisting of the recited sequence. The term “materially altered,” as applied to polypeptides of the invention, refers to an increase or decrease in biological activity of at least about 50% or more as compared to the activity of a polypeptide consisting of the recited sequence.

[0048] The term “enhance” or “increase” refers to an increase in the specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelvefold, or even fifteen-fold.

[0049] The term “inhibit” or “reduce” or grammatical variations thereof as used herein refers to a decrease or diminishment in the specified level or activity of at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments, the inhibition or reduction results in little or essentially no detectible activity (at most, an insignificant amount, e.g., less than about 10% or even 5%).

[0050] A “therapeutically effective” or “treatment effective” amount as used herein is an amount that provides some improvement or benefit to the subject. Alternatively stated, a “therapeutically effective” or “treatment effective” amount is an amount that will provide some alleviation, mitigation, or decrease in at least one clinical symptom in the subject (e.g., in the case of raising an immune response, activating or increasing the number of immune cells known to produce an immune response). Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

[0051] By the term “treat,” “treating,” or “treatment of’ (or grammatically equivalent terms) is meant to reduce or to at least partially improve or ameliorate the severity of the subject’sAttorney Docket No. 5470.985.WO condition and / or to alleviate, mitigate or decrease in at least one clinical symptom and / or to delay the progression of the condition.

[0052] As used herein, the term “prevent,” “prevents,” or “prevention” (and grammatical equivalents thereof) means to delay or inhibit the onset of a disease. The terms are not meant to require complete abolition of disease, and encompass any type of prophylactic treatment to reduce the incidence of the condition or delay the onset of the condition.

[0053] A “prevention effective” amount as used herein is an amount that is sufficient to prevent and / or delay the onset of a disease, disorder and / or clinical symptoms in a subject and / or to reduce and / or delay the severity of the onset of a disease, disorder and / or clinical symptoms in a subject relative to what would occur in the absence of the methods of the invention. Those skilled in the art will appreciate that the level of prevention need not be complete, as long as some benefit is provided to the subject.

[0054] As used herein, the terms “protein” and “polypeptide” are used interchangeably and encompass both peptides and proteins, unless indicated otherwise.

[0055] The term “fragment,” as applied to a polypeptide, will be understood to mean an amino acid sequence of reduced length relative to a reference polypeptide or amino acid sequence and comprising, consisting essentially of, and / or consisting of an amino acid sequence of contiguous amino acids identical or almost identical (e.g., 90%, 92%, 95%, 98%, 99% identical) to the reference polypeptide or amino acid sequence. Such a polypeptide fragment according to the invention may be, where appropriate, included in a larger polypeptide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially of, and / or consist of peptides having a length of at least about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more consecutive amino acids of a polypeptide or amino acid sequence according to the invention.

[0056] As used herein, an “isolated” polypeptide means a polypeptide that is separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide.

[0057] As used herein, the term “modified,” as applied to a polypeptide sequence, refers to a sequence that differs from a wildtype sequence due to one or more deletions, additions, substitutions, or any combination thereof.

[0058] As used herein “sequence identity” refers to the extent to which two optimally aligned polynucleotide or polypeptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. “Identity” can be readily calculated by knownAttorney Docket No. 5470.985.WO methods including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W ., ed.) Academic Press, New York, NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, Totowa, NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press, New York, NY (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York, NY (1991).

[0059] As used herein, the term “substantially identical” or “corresponding to” means that two nucleic acid or polypeptide sequences have at least 60%, 70%, 80% or 90% sequence identity. In some embodiments, the two nucleic acid or polypeptide sequences can have at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of sequence identity.

[0060] An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence.

[0061] The term “vaccinate” and “immunize” are used interchangeably herein to mean administering a compound and / or composition (e.g., a vaccine) in a prevention effective amount to a subject. Thus, a vaccine may prevent, delay, and / or reduce the severity of a disease, disorder and / or clinical symptoms in a subject after administration to said subject relative to what would occur in the absence of the methods of the invention.

[0062] The term “adjuvant” is used herein to mean a compound and / or substance that is added to a composition to increase the efficacy, speed of action of, and / or longevity in vivo of an immunogen when said composition is administered to a subject. In some embodiments, increasing the efficacy of an immunogen will increase the immune response in said subject. In some embodiments, the adjuvant may increase the efficacy of the immunogen by at least about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000-fold or more. In some embodiments, the adjuvant may reduce the amount of immunogen required to produce an immune response in the subject by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In some embodiments, the adjuvant may prolong the time over which the immunogen produces an immune response in the subject (e.g., increase the longevity in vivo) by at least about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50-fold or more. In some embodiments, the adjuvant may increase the longevity of an immunogen in vivo from about 1 to about 365 days (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30,Attorney Docket No. 5470.985.WO40, 50, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, or 365 days). Those skilled in the art will appreciate that the adjuvant may work through any mechanism to produce the effects listed above, including, but not limited to, stabilizing the immunogen against degradation, increasing the binding affinity of the immunogen to an immune cell, activating other components of the immune system (e.g., cytokine production, interferon production, etc.), altering inflammatory responses, and / or altering the release kinetics of the immunogen (e.g., by sequestering all or a portion of the immunogen within the adjuvant).

[0063] “Pharmaceutically acceptable,” as used herein, means a material that is not biologically or otherwise undesirable, / .< ., the material can be administered to an individual along with the compositions of this invention, without causing substantial deleterious biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. The material would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art (see, e.g., D. B. TROY, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY 21sted. (Lippincott Williams & Wilkins, Philadelphia, PA, 2005). In some embodiments, the pharmaceutically acceptable carrier is a physiological buffer such that it maintains the composition in a physiological pH range (e.g., a pH range between about 7.00 and about 7.50, (e.g., about 7.00, 7.05, 7.10, 7.15, 7.20, 7.25, 7.30, 7.35, 7.40, 7.45, or 7.50)). Exemplary physiological buffers for pharmaceutically acceptable carriers for the compositions of this invention include, but are not limited to, phosphate buffered saline (PBS), HEPES, sterile pyrogen-free water, and other sterile pyrogen-free physiological saline solutions.

[0064] The term “administering” or “administration” of a composition of the present invention to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function (e.g., for raising an immune response in a subject, for vaccinating a subject, and / or for providing an immunotherapy to a subject). In some embodiments, the administration comprises intravenous administration, mucosal administration, intradermal administration, intramuscular administration, vaginal administration, rectal administration, subcutaneous administration, transdermal administration, oral administration, sublingual administration, buccal administration, intranasal administration, topical administration, and / or intratumoral administration

[0065] A “subject” of the invention may include any animal in need thereof. In some embodiments, a subject may be, for example, a livestock animal and / or a domesticated pet. In some embodiments, a subject may be, for example, a mammal, a reptile (e.g., a snake, iguana,Attorney Docket No. 5470.985.WO chameleon, gecko, bearded dragon, monitor lizard, skink, etc.), a bird (e.g., a chicken, turkey, goose, duck, parakeet, parrot, canary, cockatiel, budgerigar etc.), an amphibian (e.g., a frog, toad, salamander, etc.), or a fish (e.g., a koi fish, goldfish, betta fish, etc.). A mammalian subject may include, but is not limited to, a laboratory animal (e.g., a rat, mouse, guinea pig, rabbit, primate, etc.), a farm or commercial animal (e.g., cattle, pig, horse, goat, donkey, sheep, llama, etc.), or a domestic animal (e.g., cat, dog, ferret, gerbil, hamster, etc.). In some embodiments, a mammalian subject may be a primate, or a non-human primate (e.g., a chimpanzee, baboon, macaque (e.g., rhesus macaque, crab-eating macaque, stump-tailed macaque, pig-tailed macaque), monkey (e.g., squirrel monkey, owl monkey, etc.), marmoset, gorilla, etc.). In some embodiments, a mammalian subject may be a human.

[0066] A “subject in need” of the methods of the invention can be any subject known or suspected of having increased risk of developing a pathogen infection (e.g., a viral, a bacterial, a prion, a parasitic, and / or a fungal infection) as described herein to which raising an immune response, vaccinating against said infection, and / or providing an immunotherapy may provide beneficial health effects. In some embodiments, the subject in need of the methods of the invention can be any subject known or suspected of having increased risk of developing a disease and / or a disorder wherein providing an immunotherapy, such as cancer immunotherapy, may provide beneficial health effects. In some embodiments, the subject in need of the methods of the invention can be any subject known to have a disease and / or a disorder wherein providing an immunotherapy, such as cancer immunotherapy, may provide beneficial health effects.

[0067] A “sample”, “biological sample”, and / or “ex vivo sample” of this invention can be any biological material, such as a biological fluid, an extract from a cell, an extracellular matrix isolated from a tissue, a cell (in solution or bound to a solid support), a tissue, a tissue homogenate, and the like as are well known in the art.Compositions

[0068] One aspect of the invention relates to a composition comprising an immunogen and an MnGp gel. In some embodiments, the immunogen is a protein (e.g., a subunit vaccine), a nucleic acid (e.g., an immunostimulatory nucleic acid and / or a nucleic acid encoding an immunogenic protein), and / or a whole-pathogen immunogen (e.g., a live attenuated pathogen, a dead pathogen, and / or an inactivated pathogen). In some embodiments, the MnGp is present in a concentration from about 1 to about 400 mg / mL (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 350, or 400 mg / mL). In some embodiments, theAttorney Docket No. 5470.985.WO immunogen is present in an amount from about 0.01 to about 500 weight percent (e.g., about 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.15, 0.2, 0.25, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or about 500 weight percent) of the composition. In some embodiments, the immunogen is present within the structure of the MnGp gel.

[0069] In some embodiments, a subunit vaccine may be one or more purified components of a pathogen which are known to be immunogenic (i.e., antigenic). In some embodiments, the subunit vaccine comprises antigenic proteins, peptides, and / or polysaccharides.

[0070] In some embodiments, a nucleic acid immunogen may be a nucleic acid that directly produces an immune response when administered to a subject, and / or encodes an antigenic protein that is produced by cells in the subject after administration. In some embodiments, the nucleic acid immunogen is a lipid nanoparticle (LNP) encapsulated mRNA (i.e., an mRNA LNP).

[0071] In some embodiments, a whole-pathogen immunogen may be one or more pathogenic cells which have been killed or inactivated prior to administration to the subject. In some embodiments, the pathogenic cells are killed using physical (e.g., heat), chemical (e.g., formaldehyde), and / or radiation methodologies. In some embodiments, the pathogenic cells are live-attenuated pathogenic cells that have a reduced virulence / pathogenicity, i.e., pathogens capable of replicating and inducing a specific immunological response, but do not induce diseases caused by the corresponding wild type pathogens in a subject. Live-attenuated pathogens can be produced by any method known to those of skill in the art, e.g., by cultivating wild-type pathogens under conditions that disable their virulent properties or using closely- related but less virulent organisms to produce such an immunological response. In some embodiments, the live-attenuated pathogenic cells comprise one or more genetic mutations that reduce the pathogenicity. In some embodiments, the live-attenuated pathogenic cells still produce, or are capable of producing, antigens that are capable of producing an immune response in a subject.

[0072] In some embodiments, the pathogen is a virus, a bacterium, a prion, a parasite, and / or a fungus.

[0073] In some embodiments, the pathogen is a virus and the virus is influenza (e.g., influenza A, influenza B, influenza C, and / or influenza D), coronavirus, norovirus, Ebola virus, herpes virus, human immunodeficiency virus (HIV), hepatitis (e.g., hepatitis A, hepatitis B, hepatitis C), human papillomavirus (HPV), mumps virus, varicella zoster virus, adenovirus, cytomegalovirus (CMV), adeno-associated virus (AAV), Chikungunya virus, Dengue virus,Attorney Docket No. 5470.985.WO enterovirus, Lassa virus, Marburg virus, West Nile virus, Zika virus, poxvirus, encephalitis virus, poliovirus, and the like.

[0074] In some embodiments, the pathogen is a bacterium, and the bacterium is a Bacillus bacterium, a Bordatella bacterium, a Borrelia bacterium, a Brucella bacterium, a Burkholderia bacterium, a Campylobacter bacterium, a Chlamydia bacterium, a Coxiella bacterium, a Ehrlichia bacterium, a Escherichia bacterium, a Francisella bacterium, a Leptospira bacterium, a Listeria bacterium, a Rickettsia bacterium, a Salmonella bacterium, a Shigella bacterium, a Tuberculosis bacterium, and the like.

[0075] In some embodiments, the pathogen is a parasite, and the parasite is a protozoan (e.g., Balamuthia mandrillaris, Cryptosporidium parvum, Cyclospora cayelanensis. Entamoeba histolytica, Giardia lamblia, Naegleria fowleri, Toxoplasma gondii and the like) and / or a helminth (e.g., a Taenia, an Echinococcus, a Schistosoma, a Fasciola, an Ascaris, an Enterobius, a Rhabditis, a Trichuris, a Necator, an Ancylostoma, and the like).

[0076] In some embodiments, the pathogen is a fungus, and the fungus is Coccidioides, Microsporidia, Mucorales, and the like.

[0077] Another aspect of the invention relates to an MnGp gel.

[0078] In some embodiments, the MnGp gel is comprised of MnGp particles. In some embodiments, the MnGp particles have an average particle size (e.g., diameter) from about 25 to 10000 nanometers (e.g., about 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 nanometers). In some embodiments, the MnGp gel has an average pore size from about 0.1 to about 15 pm (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 pm).

[0079] In some embodiments, the composition further comprises a cryoprotectant. In some embodiments, the cryoprotectant is glycerol, propylene glycol, ethylene glycol, galactose, glucose, lactose, mannitol, raffinose, sorbitol, sucrose, trehalose, dimethyl sulfoxide (DMSO), formamide, ethanediol, or propanediol. In some embodiments, the cryoprotectant is sucrose. In some embodiments, the cryoprotectant (e.g., sucrose) is present in a concentration from about 10 to 1000 mg / mL (e.g., about 10, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, 500, 600, 700, 800, 900, or 1000 mg / mL). In some embodiments, the composition is lyophilized. In some embodiments, a lyophilized composition of the present invention has improved stability (e.g., shelf-life) by about 10% to about 1000% (e.g., 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more) compared to a non-lyophilized composition. In some embodiments, the shelf-life of aAttorney Docket No. 5470.985.WO lyophilized composition is from about 1 week to about 52 weeks (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 52 weeks or more).

[0080] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is a physiological buffer such that it maintains the composition in a physiological pH range (e.g., a pH range between about 7.00 and about 7.50, (e.g., about 7.00, 7.05, 7.10, 7.15, 7.20, 7.25, 7.30, 7.35, 7.40, 7.45, or 7.50)). In some embodiments, the pharmaceutically acceptable carrier is PBS, HEPES, water, saline, and the like.

[0081] In some embodiments, the composition further comprises at least one additional adjuvant. In some embodiments, the at least one additional adjuvant is an oil-in-water emulsion, an aluminum salt (e.g., aluminum hydroxide, aluminum phosphate, and / or aluminum potassium sulfate), a STING agonist, cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), a saponin adjuvant (e.g., Quil-A®, QS-21), NOD-2 agonists, a RIG-I agonist, a Mincle agonist, a mast cell agonists, a C-type receptor agonist, ALR agonists, NLR agonist, RLR agonist, and / or a toll-like receptor (TLR) agonist. In some embodiments, the at least one additional adjuvant is cGAMP. In some embodiments, the cGAMP is present in an amount from about 0.001 pg to about 5 pg (e.g., about 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 pg).

[0082] In some embodiments, the at least one additional adjuvant is a TLR agonist. In some embodiments, the TLR agonist is an endosomal TLR agonist, e.g., a TLR3, TLR7, TLR8, and / or TLR9 agonist. In some embodiments, the TLR agonist is monophosphoryl lipid A (MPLA), polykC, flagellin, Pam3CKS4, CpG DNA (e.g., CpG 1018, CpG 1826, CpG 2006, and / or CpG 2395), 2BXy, loxoribine, Motolimod, and / or an imidazoquinoline or imidazoquinoline derivative (e.g., resiquimod, imiquimod, and / or gardiquimod). In some embodiments, the TLR agonist (e.g., imidazoquinoline or imidazoquinoline derivative) is conjugated to dopamine.

[0083] In some embodiments, the oil-in-water emulsion is a squalene-based oil-in-water emulsion. In some embodiments, the oil-in-water emulsion comprises about 1% to about 20% squalene oil by volume of the emulsion (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or about 20% squalene oil). In some embodiments, the aqueous phase of the oil-in-water emulsion is buffered (e.g., the aqueous phase comprises a tris buffer or PBS). In some embodiments, the squalene-based oil-in-water emulsion further comprises one or more vitamin (e.g., vitamin E) and / or surfactant (e.g., polysorbates [e.g., polysorbate 80], sorbitan trioleate, synthetic phosphatidylcholines,Attorney Docket No. 5470.985.WO eumulgin Bl, sucrose fatty acid sulfate esters, and / or pol oxamers). In some embodiments, the squalene-based oil-in-water emulsion is AS03 (e.g., AddaS03), AS04, MF59 (e.g., AddaVax), and / or AF03. In some embodiments, the emulsion is a micro emulsion, a submicron emulsion, or a nanoemulsion. In some embodiments, a composition of the present invention is incorporated into the oil-in-water emulsion by simple mixing. In some embodiments, a composition of the present invention (e.g., non-oil-in-water emulsion adjuvants) remain in the aqueous phase of the emulsion. In some embodiments, a composition of the present invention (e.g., non-oil-in-water emulsion adjuvants) are encapsulated in the oil droplets of the emulsion.Methods of Synthesis

[0084] One aspect of the invention relates to a method of forming an MnGp gel, the method comprising: obtaining MnGp (e.g., powdered MnGp); and applying mechanical shear stress to the MnGp; thereby forming an MnGp gel.

[0085] The mechanical shear stress can be applied using a Top-Down approach through any technique known in the art. In some embodiments, the mechanical shear stress is applied in an aqueous environment. In some embodiments, the mechanical shear stress is applied in a dry environment. In some embodiments, the mechanical shear stress is applied in a vacuum. In some embodiments, the mechanical shear stress is applied using a sonicator, a continuous flow sonicator, a miller (e.g., a roll miller, a hammer miller, an impact miller, a jet mill, a wet mill and / or a ball miller), a homogenizer, and / or a microfluidizer. In some embodiments, the mechanical shear stress is applied using sonication from about 1 minutes to about 60 minutes (e.g., about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes). In some embodiments, applying the mechanical sheer stress reduces the average particle size of the MnGp to between about 50 to 50000 nanometers (e.g., about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, or 50000 nanometers). In some embodiments, the MnGp gel has an average pore size from about 0.1 to about 15 pm (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 pm) after the mechanical shear stress is applied.

[0086] In another aspect of the invention, the MnGp gel can be formed by synthesizing MnGp nanoparticles and concentrating the nanoparticles in solution. In some embodiments, MnGp particles can be formed for the appropriate size to form a gel through a Bottom-Up approach using any technique known in the art. In some embodiments, the particles are formed through an aqueous reaction of manganese chloride and disodium glycerol-l-phosphate. In some embodiments, once MnGp particles are synthesized, the particles are concentrated in solutionAttorney Docket No. 5470.985.WO to form the gel using known techniques in the art. Particles can be formed with various sizes. In some embodiments, the particles are between about 50 to 50000 nanometers (e.g., about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, or 50000 nanometers). In some embodiments, the MnGP gel has an average pore size from about 0.1 to about 15 pm (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 pm).

[0087] In some embodiments, a cryoprotectant is added to the MnGp (e.g., the powdered MnGp) prior to applying the mechanical shear stress. In some embodiments, the cryoprotectant is glycerol, propylene glycol, ethylene glycol, galactose, dextran, mannan, glucose, lactose, mannitol, raffinose, sorbitol, sucrose, trehalose, dimethyl sulfoxide (DMSO), formamide, ethanediol, or propanediol. In some embodiments, the cryoprotectant is sucrose. In some embodiments, the cryoprotectant (e.g., sucrose) is added in a concentration from about 10 to 1000 mg / mL (e.g., about 10, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, 500, 600, 700, 800, 900, or 1000 mg / mL). In some embodiments, the method further comprises lyophilizing the MnGp gel.

[0088] In some embodiment, an immunogen is added to the MnGp gel after the mechanical shear stress has been applied.Methods of Use

[0089] One aspect of the invention relates to a method of raising an immune response in a subject, said method comprising administering an effective amount of a composition as described herein, thereby raising an immune response.

[0090] Another aspect of the invention relates to a method of vaccinating a subject against a pathogen infection, said method comprising administering an effective amount of a composition as described herein, thereby vaccinating the subject. In some embodiments, the pathogen infection is caused by a virus, a bacterium, a prion, a parasite, and / or a fungus.

[0091] Another aspect of the invention relates to a method of providing an immunotherapy in a subject in need thereof, the method comprising administering an effective amount of a composition as described herein, thereby providing the immunotherapy to the subject. In some embodiments, the immunotherapy is cancer immunotherapy. In some embodiments, the composition is administered in combination with other cancer treatments, e.g., radiation therapy and / or chemotherapy.

[0092] In some embodiments, a composition of the present invention is administered to a subject in an amount of about 1 pL to about 1 mL (e.g., about 1 pL, 2 pL, 3 pL, 4 pL, 5 pL,Attorney Docket No. 5470.985.WO10 pL, 15 pL, 20 pL, 25 pL, 30 pL, 35 pL, 40 pL, 45 pL, 50 pL, 100 pL, 150 pL, 200 pL, 250 pL, 300 pL, 350 pL, 400 pL, 450 pL, 500 pL, 600 pL, 700 pL, 800 pL, 900 pL, to about 1 mL).

[0093] In some embodiments, administering a composition of the present invention and / or raising an immune response in a subject comprises activating the cGAS-STING pathway; increasing the frequency of CD80+CD86+ cells in the subject; increasing the level of MHC-II molecules in the subject; activating dendritic cells in the subject; increasing the level of IFN-P in the subject; and / or producing an antibody response in the subject, e.g., a protective antibody response.

[0094] In some embodiments, multiple doses of the composition are administered to the subject to raise an immune response in the subject, vaccinate the subject, and / or provide immunotherapy to the subject. In some embodiments, a second dose of the composition is administered to the subject (e.g., a booster dose). In some embodiments, the second dose is administered from about 7 days to about 365 days (e.g., about 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, or 365 days), or from about 6 months to about 12 months (e.g., about 6, 7, 8, 9, 10, 11, or about 12 months) after the first administration. In some embodiments, at least a third dose is administered to the subject (e.g., a yearly booster dose). In some embodiments, the third dose is administered about 365 days after the second administration.

[0095] In some embodiments, the compositions as described herein are formulated such that, upon administration to a subject, the immunogen is released from the MnGp gel over a predetermined period of time. For example, in some embodiments, the immunogen is released from the MnGp gel from about 1 day to about 365 days (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, or 365 days) after administration to the subject. In some embodiments, the compositions as described herein are formulated such that, upon administration to a subject, a burst of the immunogen is released from the MnGp gel over a shortened pre-determined period of time; e.g., the composition releases about 1% to about 75% (e.g., about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%) of the amount of the immunogen within about 12 hours to about 72 hours after administration (e.g., within about 12, 14, 16, 18, 20, 22, 24, 28, 32, 36, 40, 44, 48, 54, 60, 66, or 72 hours). In some embodiments, the compositions as described herein are formulated such that, after the burst release of the immunogen, the remaining immunogen is released from the MnGp gel from about 1 day to about 365 days (e.g.,Attorney Docket No. 5470.985.WO about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, or 365 days).Subjects, Pharmaceutical Formulations, and Modes of Administration

[0096] The methods of the present invention find use in both veterinary and medical applications. Suitable subjects include avians (e.g., a chicken, turkey, goose, duck, parakeet, parrot, canary, cockatiel, budgerigar), reptiles (e.g., a snake, iguana, chameleon, gecko, bearded dragon, monitor lizard, skink), amphibians (e.g., a frog, toad, salamander), fish (e.g., a koi fish, goldfish, betta fish), and mammals. The term “mammal” as used herein includes, but is not limited to, humans, primates, non-human primates (e.g., monkeys and baboons), cattle, sheep, goats, pigs, horses, llamas, cats, dogs, rabbits, rodents e.g., rats, mice, hamsters, and the like), etc. Human subjects include neonates, infants, juveniles, and adults. Optionally, the subject is “in need of’ the methods of the present invention, e.g., because the subject is believed at risk for a pathogen infection and / or has or is thought to have a disease treatable by immunotherapy, e.g., cancer. As a further option, the subject can be a laboratory animal and / or an animal model of disease. Optionally, the subject is a human.

[0097] In particular embodiments, the present invention provides one or more pharmaceutical compositions comprising an immunogen and an MnGp gel in a pharmaceutically acceptable carrier and, optionally, other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, diluents, etc. For injection, the carrier will typically be a liquid. For other methods of administration, e.g., mucosal administration, the carrier may be either solid or liquid. In some embodiments, the pharmaceutically acceptable carrier is PBS, HEPES, or water. In some embodiments, the pharmaceutical composition of the present invention is suitable for mucosal administration, such as where the composition has high mucoadhesion, optionally by the inclusion of a mucoadhesive polymer such as a hydrogel (e.g., chitosan, alginate, cellulose, poly-lactic-coglycolic acid) and / or a polyethylene glycol (PEG); and / or has physiological pH (e.g., between about 7.0 to about 7.5).

[0098] The compositions of the present invention may be administered to a subject by any suitable method including, but not limited to, intranasal administration (e.g., sprays or gels); oral administration (e.g., lozenges, sprays, or gels), e.g., sublingual sprays or gels; anal administration (e.g., gels or suppositories); ocular administration (e.g., eye drops or gels) and / or vaginal administration (e.g., gels or suppositories). In some embodiments, the administration is mucosal administration.Attorney Docket No. 5470.985.WO

[0099] In some embodiments, the administering comprises intravenous administration (e.g., intravenous injection), mucosal administration, intradermal administration, subdermal administration (e.g., subdermal injection), intramuscular administration (e.g., intramuscular injection), vaginal administration (e.g., gels or suppositories), rectal administration (e.g., gels or suppositories), subcutaneous administration, transdermal administration, oral administration (e.g., lozenges, sprays, or gels), sublingual administration, buccal administration, intranasal administration (e.g., sprays or gels), topical administration (e.g., gels, creams, suppositories) and / or intratumoral administration.

[0100] The amount of the disclosed compositions administered to a subject will vary from subject to subject, depending on the nature of the disclosed compositions and / or formulations, the species, gender, age, weight and general condition of the subject, the mode of administration, and the like. Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the disclosed compositions are those large enough to produce the desired effect (e.g., to raise an immune response, vaccinate a subject, and / or provide an immunotherapy). The dosage should not be so large as to outweigh benefits by causing extensive or severe adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like, although some adverse side effects may be expected. The dosage can be adjusted by the individual clinician in the event of any counterindications.

[0101] In some embodiments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is a physiological buffer (e.g., PBS, HEPES, water, saline, and the like). In some embodiments, the composition consists essentially of the immunogen, the MnGp gel, and the pharmaceutically acceptable carrier.

[0102] Having described the present invention, the same will be explained in greater detail in the following examples, which are included herein for illustration purposes only, and which are not intended to be limiting to the invention.EXAMPLESExample 1

[0103] Manganese (Mn) is an emerging adjuvant that possesses varied effects on the cGAS- STING pathway (Lv et al., 2020; Wang et al., 2018; Zhang et al., 2021; Zhao et al., 2020). Classically, the cGAS-STING pathway orchestrates the immunological responses to doublestranded DNA (dsDNA). Upon binding dsDNA, cGAS catalyzes the synthesis of the secondAttorney Docket No. 5470.985.WO messenger cyclic GMP-AMP (cGAMP), which subsequently binds and activates STING. Activated STING then initiates a number of downstream processes, ultimately resulting in the expression of type I interferons and several interferon-stimulated genes that regulate the antiviral immune response (Hopfner and Hornung, 2020). Interestingly, Mn(II) has been found to augment cGAS-STING through both DNA-dependent and DNA-independent mechanisms. Mn was first discovered to modulate the sensitivity of cGAS-STING to dsDNA, as Mn-deficient WT mice demonstrated increased susceptibility to dsDNA, but not RNA viruses in a STING- dependent manner (Wang et al., 2018). Alternatively, Mn has since been shown to directly activate cGAS to synthesize cGAMP (Zhao et al., 2020), as well as to associate with cGAMP to enhance cGAMP-STING binding affinity (Wang et al., 2018). Given these mechanisms, several groups have already investigated the adjuvanticity of Mn to augment anti-tumor (Lv et al., 2020) and anti-viral (Fan et al., 2022) responses with substantial success.

[0104] Despite the promise of Mn as a novel adjuvant, excessive systemic dosing of Mn results in considerable toxicities in humans (Crossgrove and Zheng, 2004). To mitigate potential adverse off-target effects and enhance delivery to target immune cells, Mn adjuvants have largely been encapsulated in or formulated as nanoparticles (Aikins et al., 2024; Fan et al., 2022; Gao et al., 2023; Hou et al., 2020; Ma et al., 2022; OuYang et al., 2023; Qiao et al., 2023; Sun et al., 2021; Zhang et al., 2022; Zhao et al., 2019). Although improved compared to soluble injections of Mn, many of these formulation strategies require sophisticated fabrication processes that are poorly scalable. Furthermore, nanoparticles often offer limited antigen / adjuvant dosing and are rapidly cleared from the injection site. In contrast, gel -based delivery systems offer several potential advantages that address the limitations of nanoparticlebased formulations. Foremost, given the shear-thinning properties of many gel-like formulations, gels often form a well-defined, long-lasting depot following injection. Considering several adjuvant systems, including nanoparticles, utilize such a phenomenon to prolong the immune response (Awate et al., 2013), developing a gel-based formulation for Mn- adjuvanted vaccines would further enhance the retention and controlled release of antigen and adjuvant at the injection site, potentially resulting in a more robust and sustained immune response. Furthermore, depending on the chemistries, gels can be highly scalable, thermostable, and optimized for a wider range of administration profiles (e.g., mucosal, intradermal) (Li and Mooney, 2016). Therefore, a gel -based formulation for Mn-adjuvanted vaccines represents a promising yet underexplored strategy to improve Mn-adjuvanted formulations.Attorney Docket No. 5470.985.WO

[0105] Herein, we report the fabrication and characterization of a novel gel derived from MnGp. MnGp was selected as the gel precursor as it bypasses the need for adjuvant encapsulation, thereby circumventing a potentially limiting design consideration and instilling the delivery system with inherent adj uvanti city. Following the development of a facile and scalable fabrication method, MnGp gels were first assessed for antigen release kinetics and in vitro activation of relevant immune cell populations at a range of concentrations. Subsequently, we evaluated the potential for MnGp gels to act as vaccine platforms in vivo. Using the model antigen OVA (EndoFit™ OVA protein, Invivogen, San Diego, CA), dosing, immunization schedules, humoral immunity, and cellular immunity were analyzed in comparison to the immunological adjuvant AddaVax™ (InvivoGen, San Diego, CA), an MF59-like depotforming adjuvant, 2,3-cGAMP, a STING agonist, and CpG, a TLR agonist. Overall, the present work seeks to demonstrate the significant promise for the MnGp gel to act as a novel vaccine platform, and the application for this invention is shown in Fig. 1.METHODSMaterials

[0106] Unless otherwise specified, all chemicals were purchased and used unmodified from Sigma Aldrich (St. Louis, MO) and all assays, biologies, and disposables were purchased from Thermo Fisher Scientific (Waltham, MA).Preparation and Characterization of MnGp Gels

[0107] A dry mixture of MnGp (Pfaltz & Bauer, Waterbury, CT) and sucrose was resuspended to 350 mg / mL MnGp and 150 mg / mL sucrose in 0.1M 2-[4-(2-hydroxyethyl)piperazin-l- yl]ethanesulfonic acid (HEPES). The suspension was then placed in an ice bath and probe sonicated using an ultrasonic processor (Q500 Sonicator®, QSonica Sonicators, Newtown, CT) for 30 minutes (1 second on, 1 second off) at maximum amplitude (100%). After sonication, the resultant gel was stored at 4°C until use. Endotoxin level was confirmed to be less than 0.1 EU / dose using a limulus amoebocyte lysate-based assay.

[0108] MnGp gels were imaged using a cold-cathode field emission scanning electron microscopy (SEM) (Hitachi S-4700, Hitachi, Ibaraki, Japan) at the UNC CHANL facility (Chapel Hill, NC). To prepare samples, a droplet of the MnGp gel was spread on an SEM sample mount affixed with adhesive carbon tape. The mount was then submerged in liquid nitrogen for 1 minute and lyophilized for at least 48 hours prior to imaging.Attorney Docket No. 5470.985.WOAssessment of OVA Release from MnGp Gels

[0109] 250, 100, 25, and 5 mg / mL MnGp gels were prepared via dilution of the stock 350 mg / mL MnGp gel in 0.1 M HEPES and subsequently loaded with OVA protein (Fisher Scientific, New Hampton, NH) via mixing. To measure OVA release from the MnGp gels, 50 pL of the OVA-loaded MnGp gels were dispensed into 96-well plates and layered with 200 pL PBS, with three wells prepared for each timepoint. At each timepoint, the plates were centrifuged at 500 x g for 5 minutes, and the supernatant was collected and stored at 4°C prior to analysis. OVA content in the supernatant was quantified using a Bradford’s assay following the manufacturer’s protocol. Percent release was quantified as the amount of OVA present in the supernatant relative to the known OVA content initially loaded in each gel aliquot.Cell Culture and Treatments

[0110] All experiments involving mice were performed with the approval of the University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee (IACUC). BMDCs were differentiated from murine bone marrow as previously described (Jin and Sprent, 2018; Lutz et al., 1999; Roque et al., 2024). In short, C57 / BL6J mice (Jackson Laboratory, Bar Harbor, ME) were humanely euthanized and bone marrow was collected from the femurs and tibias. The bone marrow-derived cells were then cultured in Roswell Park Memorial Institute 1640 medium (RPMI 1640, Corning, Coming, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Coming), 1% penicillin-streptomycin solution (Coming), and 10 ng / mL granulocyte-macrophage colony-stimulating factor (GM-CSF) for 10 days, followed by RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillinstreptomycin solution, 10 ng / mL GM-CSF, and 10 ng / mL interleukin 4 (IL-4) for the next 4 days. After the 14-day culture, cells were immediately used for cell-based assays.[oni] To assess the immunostimulatory properties of the MnGp gel, BMDCs were cultured with the MnGp gel in a transwell. The bottom of a 12-well transwell plate was first pre-coated with the 100 mg / mL MnGp gel, and BMDCs were then plated at 1 x 105cells / well in the upper transwell insert. Additional BMDCs were cultured in base media and base media supplemented with 100 ng / mL LPS as untreated and LPS controls, respectively. To determine changes in cell phenotype, cells were collected after 12 hours and stained with a viability dye (Invitrogen™ eBioscience™ Fixable Viability Dye eFluor™ 506, Fisher Scientific, New Hampton, NH) and the following fluorophore-conjugated antibodies (Biolegend, San Diego, CA): MHC-II (fluorescein isothiocyanate (FITC), clone: M5 / 114.15.2), CD86 (phycoerythrin (PE), clone: GL-1), CD80 (PE / Cyanine 7 (Cy7), clone: 16-10A1), CD40 (allophycocyaninAttorney Docket No. 5470.985.WO(APC) / Cyanine7, clone: 3 / 23), CDl lc (brilliant violet 421 (BV421), clone: N418), and CDl lb (brilliant violet 711 (BV711), clone: MI / 70). To determine cytokine secretion levels, cell supernatants were collected after 24 hours and the concentration levels of IFN-P, IFN-y, TNF- a, IL-2, IL-4, and IL-6 were determined via enzyme linked immunosorbent assay (ELISA) (R&D Systems™ ELISA Kits, R&D Systems, Inc., Minneapolis, MN).In Vivo Immunizations & Sample Collection

[0112] For the initial MnGp gel titration studies, C57 / BL6J mice (n=5) were immunized intramuscularly on day 0 with saline, soluble OVA, OVA + AddaVax, OVA + 5 mg / mL MnGp gel, OVA + 25 mg / mL MnGp gel, OVA + 100 mg / mL MnGp gel, or OVA + 250 mg / mL MnGp gel. All mice were dosed at 25 pL per hind limb (50 pL total) and 25 pg of endotoxin- free OVA (Invivogen; San Diego, CA. AddaVax formulations were prepared via 1 : 1 v / v mixing of AddaVax (Invivogen) with OVA according to the manufacturer’s protocol. MnGp gel formulations were prepared as described for the OVA release studies. Submandibular bleeds were subsequently collected biweekly through week 18 (day 126).

[0113] For subsequent immunization studies, C57 / BL6J mice (n=5) were immunized intramuscularly on days 0, 28 and 140 with saline, soluble OVA, OVA + AddaVax, OVA + 2,3-cGAMP (1 pg), OVA + 250 mg / mL MnGp, or OVA + 250 mg / mL MnGp + 2,3-cGAMP (0.01, 0.1, or 1 pg). Mice were boosted on day 140 to stimulate cellular responses. As before, all mice were dosed at 25 pL per hind limb (50 pL total) and 25 pg of endotoxin-free OVA. AddaVax and MnGp gel formulations were prepared as described above. Submandibular bleeds were collected biweekly through week 18 (day 126), as well as 10 days following the final boost (day 150). On day 151, mice were humanely euthanized to collect spleens and iLNs, which were subsequently processed into single cell suspensions as previously described (Batty et al., 2023).Determination of Antibody Titers

[0114] OVA-specific antibody titers in the collected sera were determined by a previously described indirect ELISA method (Hendy et al., 2023; Roque et al., 2024). Briefly, high- binding 384-well plates (Greiner Bio-One, Kremsmunster, Austria) were coated with 10 pg / mL OVA in PBS and incubated at 4 °C overnight. Plates were then blocked with 3% w / v instant nonfat dry milk in PBS (blocking buffer; Food Lion; Salisbury NC) for 1 hour at room temperature (RT) and incubated with sera samples across a range of dilutions for 2 hours at RT. Plates were subsequently incubated with IgG, IgGl, and IgG2c-specific horseradishAttorney Docket No. 5470.985.WO peroxidase (HRP)-conjugated detection antibodies (SouthernBiotech, Birmingham AL) for 30 minutes at RT. 3,3,5,5-tetramethylbenzidine (TMB) was then added to initiate the detection reaction, and development was quenched with 2 N sulfuric acid. A minimum of three washes with 0.05% v / v Tween 20 in PBS (PBST) were performed between each step. Absorbances at 450 and 570 nm were read on a microplate reader (SpectraMax® M2, Molecular Devices, Sunnyvale, CA), with the 570 nm absorbance subtracted from the 450 nm absorbance to correct for background. Titer was determined using a plot of correct absorbance against serum dilution factor as previously reported (Eckshtain-Levi et al., 2022; Frey et al., 1998).Assessment of Antigen Recall

[0115] Antigen recall was assessed by both ELISA and ELISPOT, as previously described (Batty et al., 2023; Eckshtain-Levi et al., 2022; Hendy et al., 2023; Roque et al., 2024). For ELIS As, splenocytes from immunized mice were plated at 1 x 106cells per well in a 96-well plate and incubated with 10 pg / mL OVA protein for 36 hours. After 36 hours, the cell supernatants were collected and stored at -80 °C until use. IFN-y, IL-2, IL-6, and TNF-a content in the supernatants was measured via ELISA according to the manufacturer’s protocol (Biolegend, San Diego, CA). For ELISPOTs, splenocytes from immunized mice were plated at 1 x 106cells per well in a MultiScreen® 96-well plate (Multi-Screen-IP Filter Plate, 0.45 pm, MilliporeSigma, Burlington, MA) pre-coated with the desired capture antibody. Cells were then incubated with 10 pg / mL OVA protein for 36 hours, and ELISPOTs were developed according to the manufacturer’s protocol (BD Biosciences, Becton, Dickinson and Company, Franklin Lakes, NJ). Spots were counted with an EliSpot reader (AID Classic EliSpot Reader, AID Autoimmun Diagnostika GmbH, StraBberg, Germany).Immune Cell Phenotyping via Flow Cytometry

[0116] Following generation of single cell suspensions from spleens and iLNs, cells were stained to assess immune phenotype and OVA-specificity. To profile immune cell phenotypes, 1 x 106cells were stained with a viability dye (Invitrogen™ eBioscience™ Fixable Viability Dye eFluor™ 506, Fisher Scientific, New Hampton, NH) and the following fluorophore- conjugated antibodies (Biolegend, San Diego, CA): CD3 (Alexa Fluor® 488 (AF488), clone: 17A2), CD4 (APC / Fire™ 750, clone: RM4-5), CD8 (Peridinin-Chlorophyll Protein Complex (PerCP) / Cyanine5.5, clone: 53-6.7), CD44 (BV421, clone: IM7), CD62L (brilliant violet 785 (BV785), clone: MEL-14), CD19 (APC, clone: 6D5), GL7 (PE, clone: GL7), and CD38 (PE / Cy7, clone: 90). Tetramer staining to assess OVA-specificity was performed as a separateAttorney Docket No. 5470.985.WO panel. In short, 1 x 106cells were first incubated with 50 nM desatinib for 30 minutes at 37 °C. Cells were subsequently stained with OVA Class I (Alexa Fluor® 568 (AF568) (Thermo Fisher Scientific, Waltham, MA), clone: H2-Kb / SIINFEKL), OVA Class II (BV421, clone: I- A(b) / AAHAEINEA), or control tetramers (clone: I-A(b) / PVSKMMRMATPLLMQA) acquired from the NIH Tetramer Facility at Emory University (Atlanta, GA). Cells were then stained with the following fluorophore-conjugated antibodies (Biolegend, San Diego, CA): CD3 (Alexa Fluor® 488 (AF488), clone: 17A2), CD4 (APC / Fire™ 750, clone: RM4-5), and CD8 (PerCP / Cyanine5.5, clone: 53-6.7). Following staining, cells were fixed with 1% paraformaldehyde and analyzed with a flow cytometer (Attune NxT Flow Cytometer, Thermo Fisher Scientific, Waltham, MA) at the UNC Flow Core Facility (Chapel Hill, NC). Analysis of flow data was performed with the FlowJo™ vlO.9.0 software (Becton, Dickinson and Company, Franklin Lakes, NJ).Statistical Analysis

[0117] Figures and statistical analyses were prepared with the software GraphPad Prism™ 10 (Dotmatics, Boston, MA). For in vitro adjuvanticity experiments, all data were compared via ordinary one-way ANOVA with Tukey’s multiple comparisons test. For in vivo vaccination studies, all data were compared via ordinary two-way ANOVA with Tukey’s multiple comparisons test.RESULTS & DISCUSSIONSynthesis and Characterization of the MnGp Gel

[0118] Sonication has been previously reported to develop metallogel (Picci et al., 2023) and hydrogel (Wang et al., 2008; Zhang et al., 2011) platforms, with pulsatile forces, local temperature changes, and aeration hypothesized to facilitate cross-linking of the gel-forming reagent(s). Without wishing to be bound by any particular theory, sonication allows the insoluble MnGp crystals to decrease in size. This decrease in size increases the surface area of the MnGp which increases the intermolecular interaction of forces between the individual particles. This decrease in size is what causes the gelling phenomena. In addition, mechanical disruption of the MnGp salts in an aqueous environment forces water molecules between salts and / or salt aggregates, thereby coordinating MnGp molecules via shared hydrogen bonding with intercalating water molecules. Additionally, we opted to incorporate sucrose into some embodiments of the formulation due to its known properties as both a gel stabilizer and cryoprotectant (Lee et al., 2009; Oakenfull and Scott, 1986).Attorney Docket No. 5470.985.WO

[0119] An MnGp gel was successfully synthesized via probe sonication on ice for 30 minutes (Fig 2A). Comparison of the gel mixture before and after sonication demonstrated a significant shift in fluidity and viscosity, as evidenced by the retention of MnGp gel at the top of an inverted scintillation vial (Fig. 2B). SEM of a lyophilized MnGp gel revealed an irregular, microporous architecture, suggesting significant fluid retention in the gel matrix (Fig. 2C). Interestingly, the same architecture was observed for MnGp gels prepared without sucrose, suggesting that interactions between manganese glycerophosphate and water are the chief cross-linking mechanisms (Fig. 3).

[0120] Given the application of many gel-like materials as controlled release formulations, release of the model antigen OVA was measured from MnGp gels under physiological conditions (37°C, pH 7.4). OVA-loaded MnGp gels were prepared at various concentrations post-sonication by dilution of the stock gel in 0.1 M HEPES and simple mixing with OVA. The generated OVA release curves indicated clear concentration-dependent kinetics (Fig. 2D). The 250 mg / mL MnGp gel had the best slowed release, exhibiting minimal burst release and approximately 60% OVA release after 48 hours. The 100 mg / mL MnGp gel demonstrated slightly slowed kinetics, although burst release was nearly 50% and total release leveled off at around 60% OVA release after 24 hours. Both the 25 mg / mL and 5 mg / mL MnGp gels exhibited no control over OVA release. These release kinetics aligned strongly with the SEM of these gels, with increasing porosity and dispersity observed with decreasing concentration of the MnGp gels (Fig. 4). Notably, no gels fully released their cargo, indicating that some OVA might be sequestered within the gel and inaccessible to facile diffusion out of the gel matrix. This release is ideal, since over an extended period of time, the rest of the encapsulated OVA will be fully released.In Vitro Adi uvanti city of the MnGp Gel

[0121] To determine the immunostimulatory properties of the MnGp gel, BMDCs were cultured with the 100 mg / mL MnGp gel and assessed for relevant phenotypic changes and cytokine secretions (Fig. 5). As dendritic cells (DCs) are the primary innate immune cell population involved in T cell education and activation, the response of DCs to the MnGp gel provides an early insight into the potential efficacy of a vaccine platform. After 12 hours, the MnGp gel was well tolerated, with slight reductions in cell viability observed compared to the UT and LPS controls (Fig. 5A). There was a marked increase in co-stimulatory molecule expression: the frequency of CD80+CD86+cells was significantly increased in response to the MnGp gel (Fig. 5B), and the MFI of CD80 (Fig. 6A) and CD86 (Fig. 6B) were more thanAttorney Docket No. 5470.985.WO doubled with MnGp gel-treated cells compared to untreated cells. Furthermore, treatment with the MnGp gel resulted in a considerable increase in MHC-II MFI (Fig. 5C). Given that MHC- II is one of the molecules by which DCs present antigens to T cells, and both CD80 and CD86 are co-stimulatory molecules involved in T cell activation, these data suggest that the MnGp gel aptly poises DCs for presentation of antigen to T cells. Additionally, after 24 hours, there were significantly elevated levels of IFN-P in the cell supernatants of MnGp gel-treated cells compared to the untreated and LPS controls (Fig. 5D), consistent with previous reports of Mn stimulating the cGAS-STING pathway (Zhang et al., 2021). Type I interferons such as IFN-P are also associated with improved T cell activation and differentiation into Thl-like phenotypes (Sinigaglia et al., 1999), further underscoring the potential of the MnGp gel as a vaccine platform in vivo.In Vivo Titration of OVA-Loaded MnGp Gel Vaccine

[0122] Initial titration studies were performed to identify the optimal MnGp gel concentration for use as a vaccine platform. Mice were immunized once with saline, unadjuvanted OVA, OVA + AddaVax (analogous to FDA-approved adjuvant MF59), or OVA-loaded MnGp gels of increasing MnGp concentration, and sera were collected biweekly to measure OVA-specific IgG titers (Fig. 7). Total IgG titers exhibited concentration-dependent responses; wherein higher MnGp gel concentrations elicited superior titers (Fig. 7A). Across all adjuvanted groups, total IgG titers peaked around 70 days post-immunization and subsequently decreased, with the 250 mg / mL MnGp gel inducing the highest and most sustained titers. Without wishing to be bound by any particular theory, it is possible that these results are due in part to concentration of Mn at the injection site, as well as the increased viscosity of the higher concentration MnGp gels. The 250 mg / mL MnGp gel forms a well-defined, long-lasting depot in the intramuscular space, and could, without wishing to be bound by any particular theory, result in continued recruitment and activation of innate immune cells such as dendritic cells. IgGl and IgG2c were additionally measured as markers of Th2 and Thl responses, respectively (Collins, 2016; Nazeri et al., 2020). Both IgGl (Fig. 7B) and IgG2c (Fig. 7C) titers mirrored the trends observed for total IgG, albeit at lower levels. Notably, the 100 mg / mL and 250 mg / mL MnGp gels elicited significantly higher IgG2c titers compared to OVA adjuvanted with AddaVax at peak titers, suggesting that the MnGp gels might offer a more efficacious platform for inducing Thl responses. Based on these data, 250 mg / mL MnGp was identified as the optimal formulation for further studies.Attorney Docket No. 5470.985.WOHumoral Response to Optimal OVA-Loaded MnGp Gel Vaccine

[0123] The initial titration studies examined humoral responses with a single vaccination with the OVA-loaded MnGp gel vaccines. However, many vaccines often utilize sequential immunization, or “boosts”, to bolster immune memory and ensure long-term protection. Therefore, we sought to better characterize the immune response to the MnGp gel in a primeboost regiment. Mice were immunized on days 0 and 28 with saline, unadjuvanted OVA, OVA + AddaVax, OVA + 250 mg / mL MnGp gel, or OVA + 2,3 cGAMP (an established STING agonist). An additional boost was performed on day 140 for subsequent cellular analyses. Additionally, given the ability of Mn to augment the cGAS-STING pathway, the 250 mg / mL MnGp gel was co-loaded with OVA and various doses of cGAMP to assess potential synergy.

[0124] As before, sera were collected biweekly and assessed for OVA-specific IgG titers (Fig. 8). Compared to a single immunization (Fig. 7), boosting 28 days after the initial immunization yielded notably higher overall total IgG (Fig. 8A), IgGl (Fig. 8B), and IgG2c (Fig. 8C) titers at subsequent timepoints. Titer kinetics additionally followed the same trend, with titers initially peaking around day 70 and gradually decreasing thereafter for all IgG subtypes and adjuvanted cohorts. However, following a second boost on day 140, titers rapidly returned to their peak levels (Fig. 8). Consistent with the single immunization titers, the 250 mg / mL MnGp gel outperformed the AddaVax-adjuvanted cohort, with boosting resulting in significantly higher total IgG (Fig. 8A), IgGl (Fig. 8B), and IgG2c (Fig. 8C) titers throughout most of the time course. Similarly, the 250 mg / mL MnGp gel elicited significantly higher titers across all measured subtypes compared to the cGAMP-adjuvanted cohort (Fig. 8). Thus, when compared to both another depot-forming adjuvant system (MF59) and cGAS-STING-targeting adjuvant (cGAMP), the MnGp gel demonstrates significant promise in humoral immunity alone. Furthermore, cGAMP was not shown to benefit the IgG response to the 250 mg / mL MnGp gel across a range of doses in 6-8-week-old C57BL / 6J mice (Fig. 9), suggesting that Mn and cGAMP might be incompatible adjuvants or the 250 mg / mL MnGp gel saturates local cGAS- STING signaling. However, when tested in 6-8-week-old BALB / c mice, MnGp and cGAMP compositions drastically enhanced the immune response (Fig. 10). After only two administrations, the MnGp with cGAMP produced a significant increase in antibody titers of IgGl and IgG2a compared to MnGp alone (Fig. 10). Without wishing to be bound by any particular theory, different strains of mice respond to adjuvants differently and thus the discrepancy in the antibody titers with MnGp alone and MnGp + cGAMP could be due to the different mouse strains used. Consequently, mice immunized with the MnGp-cGAMP combination were not included in subsequent analyses.Attorney Docket No. 5470.985.WOCellular Response to Optimal OVA-Loaded MnGp Gel Vaccine

[0125] In addition to the humoral response, the cellular response to vaccines is critical for durable protection, control of reinfection, and defense against pathogens when antibodies are insufficient. Thus, we sought to profile the specific immune phenotypes associated with sequential immunization with the MnGp gel vaccine formulation. To this end, spleens and dLNs were harvested on day 151 and characterized via ELISPOT, ELISA, and flow cytometry.

[0126] Splenocytes were first cultured with whole OVA for 36 hours to assess antigen-specific immune memory (i.e., “antigen recall”). Following restimulation, a significantly higher number of IFN-y-secreting cells (Fig. 11A) and elevated levels of soluble IFN-y (Fig. 11C) were observed in mice of the 250 mg / mL MnGp gel cohort compared to all other cohorts. While the number of IL-2-secreting cells were not statistically significant (Fig. 11B), the concentration of soluble IL-2 in the cell supernatant was notably higher in splenocytes from mice immunized with the 250 mg / mL MnGp gel (Fig. 11D). IFN-y and IL-2 are integral cytokines in T cell responses; particularly, Th 1 -like responses, driving APC activation, Thl differentiation, and T cell proliferation (Romagnani, 1999). Therefore, the splenocyte restimulation data indicates that the 250 mg / mL MnGp gel is an ideal formulation for eliciting Thl responses compared to more conventional adjuvant systems, consistent with the IgG2c responses (Fig. 7C and Fig. 8C). TNFa (Fig. 12A) and IL-6 (Fig. 12B) were additionally measured from the cell supernatants, but differences were marginal and non-significant.

[0127] Splenocytes and lymphocytes were additionally stained to identify immune cell subsets and effector states (Fig. 13). In the spleen, 6-8-week-old mice immunized with the 250 mg / mL MnGp gel exhibited slightly elevated OVA-specific CD4+(Fig. 14A) and CD8+(Fig. 14B) T cells counts, with the OVA-specific CD4+T cell counts significantly higher than those in mice immunized with AddaVax. There was also significantly higher effector memory CD4+T cells (Fig. 14C, CD4+CD44+CD62L ) counts in the spleens of mice immunized with the 250 mg / mL MnGp gel compared to mice immunized with AddaVax or cGAMP. Without wishing to be bound by any particular theory, these data likely explain the previous IFN-y and IL-2 ELISPOT and ELISA data (Fig. 11), as effector memory CD4+T cells are primarily responsible for rapid mobilization of the immune response upon exposure to their cognate antigen (Kiinzli and Masopust, 2023). Additionally, effector memory CD8+T cells (Fig. 14 D, CD8+CD44+CD62L' ) were also slightly more prevalent in mice immunized with the 250 mg / mL MnGp gel, although these results were comparable to the cGAMP -immunized mice and not statistically different from the AddaVax-immunized mice. Similarly, central memory CD4+T cell countsAttorney Docket No. 5470.985.WO(Fig. 14E, CD4+CD44+CD62L+) were appreciably greater in mice receiving the 250 mg / mL MnGp gel, although the change is not significant relative to mice receiving other adjuvanted formulations. Central memory CD8+T cell counts were also measured but were highest in mice immunized with cGAMP (Fig. 14F, CD8+CD44+CD62L+). In addition to T cells, B cell phenotypes were additionally profiled. Immunization with the 250 mg / mL MnGp gel induced greater counts of both splenic GC (Fig. 15A, CD19+GL7+CD38‘) and activated (Fig. 15B, CD19+GL7'CD38+) B cells compared to AddaVax and cGAMP, although these differences were not significant. These data were consistent with the immune cell counts of the dLNs (Figs. 16A-16E and 17A-17B), which also demonstrated elevated counts of effector CD4+T cells (Fig. 16B), effector CD8+T cells (Fig. 16C), GC B cells (Fig. 17A), and effector B cells (Fig. 17B) Overall, these data align with the understood properties of the 250 mg / mL MnGp gel, known adjuvanticity of Mn, and the previously explored humoral data. Compared to AddaVax and cGAMP, which are bolus injections that are more rapidly cleared, the gel forms a highly immunogenic depot that continually releases antigen and Mn, resulting in continual innate immune stimulation and maintenance of effector-like phenotypes.Conclusion

[0128] The promise of Mn as an adjuvant necessitates improved delivery formulations. As such, the present work described the synthesis, characterization, and administration of a novel Mn-based gel for vaccine applications. As stated previously, without wishing to be bound by any particular theory, sonication allows the insoluble MnGp crystals to decrease in size. This decrease in size increases the surface area of the MnGp which increases the intermolecular interaction of forces between the individual particles. This decrease in size may be causing the gelling phenomena. The MnGp gel was fabricated via a scalable and facile sonication method, and gels of varying MnGp content displayed tunable release of the model antigen OVA over the course of a week. Titration of the MnGp gel in vitro demonstrated significant immunostimulatory efficacy in dendritic cell lines, resulting in elevated type I interferon secretion and increased expression of co-stimulatory markers. Titration of the MnGp gel in vivo as a single-dose vaccine against OVA demonstrated sustained humoral kinetics, with the highest concentration gel significantly outperforming AddaVax, an analog to the FDA- approved adjuvant MF59, by total IgG, IgGl (Th2), and IgG2c (Thl) titers. Subsequently, the MnGp gel vaccine was applied in a prime-boost-boost model and boosting demonstrated significant enhancement of IgG titers across subtypes. Furthermore, in comparison to MF59 and the STING-adjuvant 2,3-cGAMP, the MnGp gel elicited significantly superior Thl-likeAttorney Docket No. 5470.985.WO cellular responses, as well as increased expansion of OVA-specific CD4+ / CD8+T cells, effector memory CD4+T / CD8+cells, and GC B cells in the spleens of immunized mice. In all, our MnGp gel represents a highly efficacious platform for improving and prolonging both the humoral and cellular immune responses to subunit vaccines.Example 2

[0129] Data in Example 1 highlights the generation of cytokines after antigen recall in young (Figs. 9A-9C) and elderly (Figs. 15A-15B) mice. To add to this, we have performed antigen recall assays in the spleen and draining lymph node (dLN) cells of five 18-month-old C57BL / 6 mice. Briefly, the mice were immunized intramuscularly on days 0, 28, and 77 with PBS, unadjuvanted OVA, MnGp (250 mg / mL) + OVA or AddaVax + OVA, and the level of IL-4 and IFN-y production subsequently measured. As can be seen in Figs. 18A-18B, following restimulation with OVA, significantly higher levels of IL-4 production in the spleen and IFN- y production in the dLN cells were observed in the mice of the 250 mg / mL MnGp gel cohort compared to the other cohorts. Thus, the data in Example 1 and the present example together illustrate a balanced immune response stimulated by the MnGp gel adjuvant, further underscoring the potential of the MnGp gel as a vaccine platform.Example 3

[0130] In Example 1 (Figs. 14A-14F & 15A-15B), we presented multiple figures from ‘elderly’ (18mos+) mice showing that the 250 mg / mL MnGp gel stimulates an immune response more efficiently than AddaVax or cGAMP does. In this Example, we further support the data in Example 1 by performing a separate experiment evaluating the MnGp gel-based vaccine in combination with lOpg of CpG in aged (18mos) C57BL / 6 mice. Using a primeboost immunization strategy, we found that IFN-y concentrations were higher in the MnGp gel group than in the CpG group (Fig. 19A) and that IFN- y and IL-17 concentrations were higher in the MnGp gel group compared to the CpG group (Figs. 20A-20B). Remarkably, IFN- y and IL-17 concentrations in the MnGp gel + CpG group were even higher than the levels in the MnGp gel group (Figs. 20A-20B), signifying a synergistic effect between MnGp and CpG. Antibody titers were also significantly higher in the MnGP gel and MnGp gel + CpG groups than in the soluble CpG group (Figs. 19B-19C, 20A). Importantly, the MnGp gel + CpG vaccination also induced notably elevated levels of IgG2c (Fig. 20A) in addition to IgGl (Fig. 19C) - illustrating a balanced immune response in this sensitive population. Since IgG2c is a marker of Thl-type immune responses, this finding suggests that the instant invention can driveAttorney Docket No. 5470.985.WO a Thl-skewed response even in elderly mice. This is particularly noteworthy because aged mice, like elderly humans, typically exhibit diminished immune responses in general, and Thl responses specifically, following vaccination (Fig. 19).Example 4

[0131] Based on the data in Example 1 (Fig. 10), which shows a synergy between the MnGp gel with cGAMP, and the data in the Example 3 (Figs. 19A-19C and 20A-20B), which shows a synergy between the MnGp gel with another PAMP adjuvant, CpG, co-delivery of the MnGp gel with secondary adjuvants appears to drive the generation of correlates of protection. Our preliminary data show that MnGp gels outperform FDA-approved adjuvants, with cGAMP and CpG further amplifying these effects with a balanced immune response.

[0132] In this example, we further support these data by performing a separate experiment evaluating the effect of MnGp in combination with the orthopoxvirus antigen B5R when 6-8- week-old BALB / c mice, 6-8 weeks old, (n=10) are challenged with the vaccinia virus. We found that MnGp-adjuvanted vaccination with the orthopoxvirus antigen B5R elicits stronger humoral and protective responses than current controls, including in aged mice. IgG, IgGl, and IgG2C sera titers from mice immunized with MnGp + B5R were significantly higher than the titers from mice immunized with AddaVax + B5R or CpG + B5R (Figs. 21A-21C). And notably, immunization with MnGP + B5R was the only treatment that was observed to result in full protection (100% survival after 15 days) against the vaccinia virus (Fig. 21D). Unlike the MnGp + B5R group, only 80% of the AddaVax + B5R group survived the vaccinia virus challenge, while the other treatment groups completely succumbed to infection (Fig. 21D). These results, consistently with the results shown in Examples 1-3, show that MnGp gels can potentially be harnessed synergistically with established adjuvants to achieve broad, protective immunity.

[0133] The foregoing is illustrative of the present invention and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.References:Aikins, M.E., Sun, X., Dobson, H., Zhou, X., Xu, Y., Lei, Y.L., Moon, J.J., 2024. STING- activating cyclic dinucleotide-manganese nanoparticles evoke robust immunity against acute myeloid leukemia. J. Controlled Release 368, 768-779. http s : / / doi . org / 10.1016 / j .j conrel .2024.03.022Attorney Docket No. 5470.985.WOAleebrahim-Dehkordi, E., Molavi, B., Mokhtari, M., Deravi, N., Fathi, M., Fazel, T., Mohebalizadeh, M., Koochaki, P., Shobeiri, P., Hasanpour-Dehkordi, A., 2022. T helper type (Thl / Th2) responses to SARS-CoV-2 and influenza A (H1N1) virus: From cytokines produced to immune responses. Transpl. Immunol. 70, 101495. https: / / doi.Org / 10.1016 / j.trim.2021.101495Awate, S., Babiuk, L.A., Mutwiri, G., 2013. Mechanisms of Action of Adjuvants. 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Claims

1. Attorney Docket No. 5470.985.WOWHAT IS CLAIMED IS1. A composition comprising an immunogen and a manganese glycerophosphate (MnGp) gel.

2. The composition of claim 1, wherein the immunogen is a protein (e.g., a subunit vaccine), a nucleic acid (e.g., an immunostimulatory nucleic acid and / or a nucleic acid encoding an immunogenic protein), and / or a whole-pathogen immunogen (e.g., a live attenuated pathogen, a dead pathogen, and / or an inactivated pathogen).

3. The composition of claim 2, wherein the whole-pathogen immunogen is a virus, a bacterium, a prion, a parasite, and / or a fungus.

4. The composition of any preceding claim, wherein the MnGp gel is comprised of MnGp particles.

5. The composition of claim 4, wherein the MnGp particles have an average particle size from about 25 to 10000 nanometers (e.g., about 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 nanometers).

6. The composition of any preceding claim, wherein the MnGp gel has an average pore size from about 0.1 to about 15 pm (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 pm).

7. The composition of any preceding claim, wherein the immunogen is present from about 0.01 to about 500 weight percent of the composition, and the MnGp particles are present in a concentration from about 1 to about 400 mg / mL (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 350, or 400 mg / mL).

8. The composition of any preceding claim, wherein the composition further comprises a cryoprotectant.

9. The composition of claim 8, wherein the cryoprotectant is sucrose.Attorney Docket No. 5470.985.WO10. The composition of claim 9, wherein the sucrose is present in a concentration from about 10 to about 1000 mg / mL (e.g., about 10, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, 500, 600, 700, 800, 900, or 1000 mg / mL).

11. The composition of any one of claims 8-10, wherein the composition is lyophilized.

12. The composition of any preceding claim, wherein the composition further comprises at least one additional adjuvant.

13. The composition of claim 12, wherein the at least one additional adjuvant is an oil-in- water emulsion (e.g., a squalene in water emulsion (e.g., AS03 or MF59)), an aluminum salt (e.g., aluminum hydroxide, aluminum phosphate, and / or aluminum potassium sulfate), cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), a sponin adjuvant (e.g., Quil-A®), QS-21, and / or a toll-like receptor (TLR) agonist (e.g., monophosphoryl lipid A (MPLA), CpG, flagellin, polyinosinic:polycytidylic acid, Pam3CKS4, resiquimod (R848), and / or imiquimod).

14. The composition of claim 13, wherein the at least one additional adjuvant is cGAMP and the cGAMP is present in an amount from about 0.001 pg to about 5 pg (e.g., about 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 pg).

15. The composition of claim 13, wherein the at least one additional adjuvant is a CpG adjuvant, optionally CpG 1018, CpG 1826, CpG 2006, and / or CpG 2395.

16. The composition of any preceding claim, which is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

17. The composition of claim 16, wherein the pharmaceutically acceptable carrier is a physiological buffer (e.g., phosphate buffered saline (PBS), 4-(2 -hydroxy ethyl)- 1- piperazineethanesulfonic acid (HEPES), water, saline, and the like).

18. The composition of any preceding claim, wherein the composition is suitable for intravenous administration, mucosal administration, intradermal administration, intramuscularAttorney Docket No. 5470.985.WO administration, vaginal administration, rectal administration, subcutaneous administration, transdermal administration, oral administration, sublingual administration, buccal administration, intranasal administration, and / or intratumoral administration.

19. The composition of claim 18, wherein the composition persists in vivo from about 1 day to about 365 days (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, or 365 days) after administration.

20. The composition of any preceding claim, wherein the immunogen is present within the structure of the MnGp gel.

21. The composition of claim 20, wherein the immunogen is released from the MnGp gel from about 1 day to about 365 days (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, or 365 days) after administration.

22. The composition of claim 21, wherein about 1% to about 75% (e.g., about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%) of the amount of the immunogen is released within about 12 to about 72 hours after administration.

23. The composition of any one of claims 16-22, wherein the composition consists essentially of the immunogen, the MnGp gel, and the pharmaceutically acceptable carrier.

24. A method of raising an immune response in a subject, said method comprising administering to the subject an effective amount of the composition of any preceding claim, thereby raising an immune response in the subject.

25. The method of claim 243, wherein the administering activates the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, increases the frequency of CD80+CD86+ cells in the subject, increases the level of major histocompatibility complex class II (MHC-II) molecules in the subject, activates dendritic cells in the subject, and / or increases the level of interferon beta (IFN-P) in the subject.Attorney Docket No. 5470.985.WO26. The method of claim 24 or 25, wherein the administering comprises intravenous administration, mucosal administration, intradermal administration, intramuscular administration, vaginal administration, rectal administration, subcutaneous administration, transdermal administration, oral administration, sublingual administration, buccal administration, intranasal administration, and / or intratumoral administration.

27. The method of any one of claims 24-26, wherein the subject is a vertebrate, such as a livestock animal (e.g., a cow, a horse, a pig, a chicken, a turkey, a goose, a duck, a goat, a sheet, a llama, a donkey, and / or a rabbit) or a domestic pet (e.g., a dog, a cat, a mouse, a rat, a hamster, a guinea pig, a koi fish, a goldfish, a betta fish, a parakeet, a parrot, a canary, a cockatiel, a budgerigar, a snake, an iguana, a chameleon, a gecko, a bearded dragon, a monitor lizard, and / or a skink), optionally wherein the subject is a human.

28. The method of any one of claims 24-27, wherein the administering comprises administering from about 1 pL to about 500 pL (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, or 500 pL), of the composition.

29. A method of vaccinating a subj ect against a pathogen infection, said method comprising administering an effective amount of the composition of any one of claims 1-23, thereby vaccinating the subject against the pathogen infection.

30. The method of claim 29, wherein the pathogen infection is caused by a virus, a bacterium, a prion, a parasite, and / or a fungus.

31. The method of claim 29 or 30, wherein the administering activates the cyclic GMP- AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, increases the frequency of CD80+CD86+ cells in the subject, increases the level of major histocompatibility complex class II (MHC-II) molecules in the subject, activates dendritic cells in the subject, and / or increases the level of interferon beta (IFN-P) in the subject.

32. The method of any one of claims 29-31, wherein the administering comprises intravenous administration, mucosal administration, intradermal administration, intramuscular administration, vaginal administration, rectal administration, subcutaneous administration,Attorney Docket No. 5470.985.WO transdermal administration, oral administration, sublingual administration, buccal administration, intranasal administration, and / or intratumoral administration.

33. The method of any one of claims 29-32, wherein the subject is a vertebrate, such as a livestock animal (e.g., a cow, a horse, a pig, a chicken, a turkey, a goose, a duck, a goat, a sheet, a llama, a donkey, and / or a rabbit) or a domestic pet (e.g., a dog, a cat, a mouse, a rat, a hamster, a guinea pig, a koi fish, a goldfish, a betta fish, a parakeet, a parrot, a canary, a cockatiel, a budgerigar, a snake, an iguana, a chameleon, a gecko, a bearded dragon, a monitor lizard, and / or a skink), optionally wherein the subject is a human.

34. The method of any one of claims 29-33, wherein the administering comprises administering from about 1 pL to about 500 pL (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, or 500 pL) of the composition.

35. The method of any one of claims 29-34, wherein at least a second dose of the composition is administered to the subject.

36. The method of claim 35, wherein the second dose is administered from about 7 days to about 365 days (e.g., about 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, or 365 days) after the first administration.

37. The method of claim 35, wherein a yearly booster dose is administered to the subject and the yearly booster dose is administered about 365 days after the preceding administration.

38. A method of providing an immunotherapy in a subject in need thereof, the method comprising administering the composition of any one of claims 1-23, thereby providing the immunotherapy to the subject.

39. The method of claim 38, wherein the immunotherapy is cancer immunotherapy.

40. The method of claim 39, wherein the composition is administered in combination with radiation therapy and / or chemotherapy.Attorney Docket No. 5470.985.WO41. The method of any one of claims 38-40, wherein the administering activates the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, increases the frequency of CD80+CD86+ cells in the subject, increases the level of major histocompatibility complex class II (MHC-II) molecules in the subject, activates dendritic cells in the subject, and / or increases the level of interferon beta (IFN-P) in the subject.

42. The method of any one of claims 38-41, wherein the administering comprises intravenous administration, mucosal administration, intradermal administration, intramuscular administration, vaginal administration, rectal administration, subcutaneous administration, transdermal administration, oral administration, sublingual administration, buccal administration, intranasal administration, and / or intratumoral administration.

43. The method of any one of claims 38-42, wherein the subject is a vertebrate, such as a livestock animal (e.g., a cow, a horse, a pig, a chicken, a turkey, a goose, a duck, a goat, a sheet, a llama, a donkey, and / or a rabbit) or a domestic pet (e.g., a dog, a cat, a mouse, a rat, a hamster, a guinea pig, a koi fish, a goldfish, a betta fish, a parakeet, a parrot, a canary, a cockatiel, a budgerigar, a snake, an iguana, a chameleon, a gecko, a bearded dragon, a monitor lizard, and / or a skink) optionally wherein the subject is a human.

44. The method of any one of claims 38-43, wherein the administering comprises administering from about 1 pL to about 500 pL (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, or 500 pL) of the composition .

45. The method of any one of claims 38-44, wherein at least a second dose of the composition is administered to the subject.

46. The method of claim 45, wherein the second dose is administered from about 7 days to about 365 days (e.g., about 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, or 365 days) after the first administration.

47. The method of claim 45, wherein a yearly booster dose is administered to the subject and the yearly booster dose is administered about 365 days after the preceding administration.

48. A method of forming an MnGp gel, the method comprising:Attorney Docket No. 5470.985.WO obtaining MnGp (e.g., powdered MnGp); and applying mechanical shear stress to the MnGp; thereby forming an MnGp gel.

49. The method of claim 48, wherein the mechanical shear stress is applied in an aqueous environment.

50. The method of claim 48 or 49, wherein a cryoprotectant is added to the MnGp (e.g., the powdered MnGp) prior to applying the mechanical shear stress.

51. The method of claim 50, wherein the cryoprotectant is sucrose.

52. The method of claim 50 or 51, wherein the method further comprises lyophilizing the MnGp gel.

53. The method of any one of claims 48-52, wherein the mechanical shear stress is applied using a sonicator, a continuous flow sonicator, a miller (e.g., a roll miller, a hammer miller, a jet mill, a wet mill, an impact miller, and / or a ball miller), a homogenizer, and / or a microfluidizer.

54. The method of any one of claims 48-53, wherein the mechanical shear stress is applied using sonication from about 1 minute to about 60 minutes (e.g., about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes).

55. The method of any one of claims 48-54, wherein applying the mechanical sheer stress reduces the particle size of the MnGp to between about 50 nanometers to about 50000 nanometers (e.g., about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, or 50000 nanometers).

56. The method of any one of claims 48-55, wherein the MnGp gel has an average pore size from about 0.1 pm to about 15 pm (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 pm).Attorney Docket No. 5470.985.WO57. A method of forming an MnGp gel, comprising synthesizing MnGp nanoparticles and concentrating the nanoparticles in solution, thereby forming an MnGp gel.

58. The method of claim 57, wherein the nanoparticles are synthesized by an aqueous reaction of manganese chloride and disodium glycerol- 1 -phosphate.

59. An MnGp gel prepared by the method of any one of claims 48-58.

60. An MnGp gel.

61. The MnGp gel of claim 60, which is comprised of MnGp particles.

62. The MnGp gel of claim 61, wherein the MnGp particles have an average particle size from about 100 nanometers to about 50000 nanometers (e.g., about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, or 50000 nanometers).

63. The MnGp gel of any one of claims 60-62, wherein the MnGp gel has a pore size from about 0.1 pm to about 15 pm (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 pm).

64. The MnGp gel of any one of claims 60-63, wherein the MnGp gel further comprises a cryoprotectant.

65. The MnGp gel of claim 64, wherein the cryoprotectant is sucrose.

66. The MnGp gel of any one of claims 60-65, wherein the MnGp gel is lyophilized.

67. A pharmaceutical composition comprising the MnGp gel of any one of claims 60-66, and a pharmaceutically acceptable carrier.

68. The pharmaceutical composition of claim 67, wherein the pharmaceutically acceptable carrier is a physiological buffer, (e.g., PBS, HEPES, water, saline, and the like).Attorney Docket No. 5470.985.WO69. The pharmaceutical composition of claim 67 or 68, wherein the composition is suitable for intravenous administration, mucosal administration, intradermal administration, intramuscular administration, vaginal administration, rectal administration, subcutaneous administration, transdermal administration, oral administration, sublingual administration, buccal administration, intranasal administration, and / or intratumoral administration.

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