Hydrogelated cells and their use as vaccines

Abstract

Described herein are cells that contain a synthetic polymer network which renders the cells incapable of dividing and allows the cells to remain metabolically active. Also described herein are methods for making the metabolically-active cells, and related methods for using the metabolically-active cells to induce an immune response in a subject, such as a vaccine.

Description

PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2HYDROGELATED CELLS AND THEIR USE AS VACCINESCROSS-REFERENCES TO RELATED APPLICATIONS

[0001] The present application claims priority' to U.S. Provisional Application No. 63 / 694,622, filed September 13, 2024, which is incorporated herein by reference in its entirety'.STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under grant no. GM142788 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Synthetic materials can infuse or form inside a living cell to alter the cell’s behavior. It has been shown previously that E. coll cells can be infused with polyethylene glycol diacrylate monomers and that the monomers can polymerize into a cross-linked hydrogel within the cells (Contreras-Llano, L.E., Liu, Y.H., Henson, T., Meyer, C.C., Baghdasaryan, O., Khan. S., Lin. C.L.. Wang, A., Hu. C.M.J. and Tan, C., 2023. Engineering cyborg bacteria through intracellular hydrogelation. Advanced Science, 10(9), p.2204175). The E. coll cells comprising the hydrogel (“hydrogelated E. coli cells”) had lost their ability to replicate but maintained certain cellular processes such as metabolic pathways, cellular respiration, protein synthesis, and motility. The ability to modify a cell through hydrogelation has generated considerable interest in designing hydrogelated cells for broad biomedical applications including vaccines.

[0004] P. aeruginosa (PA) and S. aureus (SA) are two of the most prevalent and opportunistic pathogens in hospital settings, each accounting for -10% of all nosocomial infections (Qin. S. el al. Pseudomonas aeruginosa', pathogenesis, virulence factors, antibiotic resistance, interaction with host, technology advances and emerging therapeutics. Sig Transduct Target Ther 7, 199 (2022)). These bacteria can resist multiple antibiotics, limiting the therapeutic options and resulting in high mortality' rates of >25% (Abbara, S. et al. Antimicrobial Resistance and Mortality in Hospitalized Patients with Bacteremia in the Greater Paris Area from 2016 to 2019. CLEP 14, 1547-1560 (2022)). Moreover, they can1KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2 spread through medical devices and skin surfaces, facilitating their dissemination. Therefore, there is an urgent need for prophylactic vaccines that can elicit protective immunity against these bacteria in hospitalized patients, especially those with chronic conditions, such as cystic fibrosis, diabetes, or pulmonary disease. See, e.g., Pletz, M. W., Uebele, J., Gbtz, K., Hagel, S. & Bekeredjian-Ding, I. Vaccines against major ICU pathogens: where do we stand? Current Opinion in Critical Care 22, 470 (2016); Bekeredjian-Ding, I. Challenges for Clinical Development of Vaccines for Prevention of Hospital- Acquired Bacterial Infections. Frontiers in Immunology 11, (2020); and Osterloh, A. Vaccination against Bacterial Infections: Challenges, Progress, and New Approaches with a Focus on Intracellular Bacteria. Vaccines 10, 751 (2022).

[0005] While several ty pes of experimental vaccines have been developed to target P. aeruginosa and S. aureus, based on different antigens, adjuvants, and delivery systems. See, e.g., Liu, C. et al. Construction of a Protective Vaccine Against Lipopolysaccharide- Heterologous Pseudomonas aeruginosa Strains Based on Expression Profiling of Outer Membrane Proteins During Infection. Frontiers in Immunology 9. (2018); Micoli, F., Bagnoli, F., Rappuoli, R. & Serruto, D. The role of vaccines in combatting antimicrobial resistance. Nat Rev Microbiol 19, 287-302 (2021); Weyant, K. B. et al. A modular vaccine platform enabled by decoration of bacterial outer membrane vesicles with biotinylated antigens. Nat Commun 14, 464 (2023); and Super, M. et al. Biomaterial vaccines capturing pathogen-associated molecular patterns protect against bacterial infections and septic shock. Nat Biomed Eng 6, 8-18 (2022). Some of the promising subunit vaccines (i.e., vaccines that include one or more antigenic components or epitopes from a pathogen instead of a whole entire pathogenic organism) contain bacterial proteins, such as flagellin, pilin, exotoxin A, OprF. protein A, and capsular polysaccharides. These vaccines aim to induce immunity against the surface or secreted molecules of the bacteria. Other types of vaccines include live attenuated or killed whole-bacteria vaccines, which attempt to elicit cellular and humoral immune responses against multiple bacterial antigens (Id.). However, none of these vaccines have reached clinical approval despite showing some efficacy in preclinical tests. The lack of further development or their failure in clinical trials are not fully understood, but may include the following factors: (1) the heterogeneity and evolution of “vaccine-escape” bacteria, and (2) the lack of proper vaccine-immune interactions to robustly activate an adaptive immune response (Wang, S., Gao, J., Li, M., Wang, L. & Wang, Z. A facile approach for development2KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2 of a vaccine made of bacterial double-layered membrane vesicles (DMVs). Biomaterials 187, 28-38 (2018)).

[0006] One main challenge in the development of subunit-based vaccines is to identify the most relevant and conserved antigens that can elicit lasting protective immunity' across different strains and clinical settings. Subunit vaccines must identify and then purify specific bacterial proteins that can be recognized by immune cells, but these proteins may vary in their expression, structure, or function among different bacterial isolates or under different environmental conditions. Moreover, some antigens may not be essential for bacterial survival or pathogenesis, and thus, the bacteria may escape the vaccine-induced immune responses by mutating, deleting, or downregulating them. Therefore, subunit vaccines often need to incorporate multiple antigens to cover antigenic diversity and variability of the bacteria, as well as enhance the immunogenicity and durability of the immune responses (Super, M. et al. Biomaterial vaccines capturing pathogen-associated molecular patterns protect against bacterial infections and septic shock. Nat Biomed Eng 6, 8-18 (2022); and Bianconi, I. et al. Genome-Based Approach Delivers Vaccine Candidates Against Pseudomonas aeruginosa. Front. Immunol. 9, (2019)). Subunit-based vaccines, which are without the bacterial pathogen, also omit native bacteria-host interactions. This omission necessitates additional modifications, e.g., with adjuvants or viral elements, to mimic the natural route and mode of infection to enhance the immune responses to the vaccine.

[0007] Another common vaccine chassis is live-attenuated bacteria that have reduced virulence but retain immunogenicity. Attenuated bacteria can theoretically stimulate both humoral and cellular immunity, as well as mucosal immunity (VanCott, J. L. et al. Regulation of host immune responses by modification of Salmonella virulence genes. Nat Med 4, 1247-1252 (1998); and Grandi, G. Antibacterial vaccine design using genomics and proteomics. Trends in Biotechnology 19.181-188 (2001)). However, the development of attenuated bacterial vaccines faces several scientific challenges, including the safety and stability of the attenuation, which may be reversible or incomplete, leading to reversion to virulence or persistence in the host (Frey, J. Biological safefy concepts of genetically modified live bacterial vaccines. Vaccine 25, 5598-5605 (2007)). Further, significant time required to "attenuate” bacteria. Genetically attenuating new bacteria strains / species requires significant investment in obtaining their genomic, proteomic, or virulence information. Non- genetic methods of attenuating bacteria, using heat, shear forces, or osmotic pressure, often3KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2 result in weak immunity. And finally, classical whole-bacteria-based vaccines can present bystander autoimmunity issues because some cytosolic molecules of bacteria share structural similarities with host molecules. Evidence has emerged showing bacterial DNA from infections could promote autoimmune abnormalities similar to that observed in systemic lupus erythematosus (Pisetsky, D. S. Evolving story of autoantibodies in systemic lupus erythematosus. Journal of Autoimmunity 110, 102356 (2020)). Immune responses against bacterial HSPs can lead to cross-reactive responses against human HSPs, implicated in autoimmune diseases like rheumatoid arthritis (van Eden, W. et al. Heat Shock Proteins Can Be Surrogate Autoantigens for Induction of Antigen Specific Therapeutic Tolerance in Rheumatoid Arthritis. Front. Immunol. 10. (2019)).SUMMARY

[0008] As disclosed herein, the inventors have determined compositions and methods related to hydrogelated cells that overcome the long-standing challenges of classical vaccines, hence resulting in a superior chassis as antibacterial vaccines.

[0009] Disclosed herein are metabolically-active cells comprising cross-linked hydrogel within the cell in sufficient amount to prevent cell replication. In some embodiments, wherein the cell is a bacterial pathogen. In some embodiments, the bacterial pathogen comprises Pseudomonas, Klebsiella, or Staphylococcus. In some embodiments, the bacterial pathogen is Pseudomonas aeruginosa, Klebsiella pneumoniae, or Staphylococcus aureus.

[0010] In some embodiments, the cross-linked hydrogel comprises monosaccharide or polysaccharide monomer subunits, and wherein the cross-linked hydrogel is a homopolymer or co-polymer. In some embodiments, the cross-linked hydrogel comprises substituted or unsubstituted poly(ethylene glycol) or poly(ethylene glycol) diacrylate monomer (PEG-DA) monomer subunits. In some embodiments, the cross-linked hydrogel comprises poly(dimethyl siloxane) (PDMS), poly(ethylene oxide) (PEG), poly(vinyl alcohol) (PVA), polypropylene fumarate) (PPF), alginate, guanosine mono phosphate (GMP), cyclodextrin (CD), fibrin, collagen, polypeptides, decellularized extracellular matrix, or nucleic acids. In some embodiments, the cross-linked hydrogel is substituted.

[0011] In some embodiments, the metabolically-active cell is covalently linked to an adjuvant.

[0012] In some embodiments, the cross-linked hydrogel has a density of 1-2% (w / w) in the cell.4KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2

[0013] In some embodiments, the metabolically-active cell further comprises at least one heterologous nucleic acid. In some embodiments, the heterologous nucleic acid encodes a protein. In some embodiments, the protein is an outer membrane protein derived from a bacterial pathogen.

[0014] In some embodiments, the cell is contacted with a heterologous cryoprotectant.

[0015] Also disclosed herein are vaccines comprising metabolically-active cells of the present disclosure and a pharmaceutically-acceptable excipient. In some embodiments, at least 99% of cells in the vaccine comprise the cross-linked hydrogel.

[0016] Also disclosed herein are methods of inducing an immune response in an animal, the method comprising administering a vaccine of the present disclosure to the animal in a sufficient amount to induce an immune response. In some embodiments, the animal is a bird or mammal. In some embodiments, the animal is a human.

[0017] Also disclosed here are methods of making a vaccine. In some embodiments, the method comprises: (i) providing a plurality of bacterial pathogen cells; (ii) introducing hydrogel monomer subunits into the plurality of metabolically-active bacterial pathogen cells; and (iii) causing the polymerization inducer to initiate formation of cross-linked hydrogel in the plurality’ of bacterial pathogen cells thereby forming metabolically-active bacterial pathogen cells comprising the cross-linked hydrogel, wherein the cross-linked hydrogel is formed from the hydrogel monomer subunits.

[0018] In some embodiments, the method further comprises introducing a polymerization inducer into the plurality of metabolically-active bacterial pathogen cells before, after, or simultaneously with the step of introducing hydrogel monomer subunits.

[0019] In some embodiments, the polymerization inducer is activated by light of a specific wavelength and the step of causing polymerization comprises exposing the plurality of metabolically-active bacterial pathogen cells to light of the specific wavelength.

[0020] In some embodiments, the method further comprises contacting the plurality of metabolically-active bacterial pathogen cells with a replication-specific toxin and / or antibiotics, thereby killing cells in the plurality capable of replicating.5KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2

[0021] In some embodiments, the method further comprises isolating metabolically-active bacterial pathogen cells comprising the cross-linked hydrogel from cells without the crosslinked hydrogel.

[0022] In some embodiments, the introducing step in the method further comprises introducing a marker into the plurality of metabolically-active bacterial pathogen cells, and wherein the isolating comprises isolating the metabolically-active bacterial pathogen comprising the cross-linked hydrogel by presence of the marker.

[0023] In some embodiments, the method further comprises contacting the cell with a heterologous cryoprotectant during the providing, introducing, and / or causing steps.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A shows a schematic workflow for the hydrogelation of cells.

[0025] Figure IB shows flow cytometry and fluorescence microscopy analysis depicting the detection of wild-type and hydrogelated cells in multiple bacterial strains, including Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumonia. The histogram illustrates the fluorescence signal obtained from two distinct markers: PEGDA - Fluorescein Diacrylate, serving as an indicator of intracellular hydrogelation, and Reductive Chromogenic Dye, a marker used to assess metabolic activity. The grey quadrant in the hydrogelated cell plot show's a population of hydrogelated cell that are metabolic active (n= 3 independent experiment).

[0026] Figure 1C shows the percentages of hydrogelation and hydrogelated metabolically active cells in three distinct bacterial strains: S. aureus, P. aeruginosa, and T. pneumoniae while varying the hydrogelation percentages at 5%, 10%, 20%, and 30 %. The left bar (PEGDA - fluorescein diacrylate) of the pair of bars for each percentage represents the proportion of cells exhibition intracellular hydrogelation. The right bar (reductive chromogenic dye) of the pair of bars for each percentage represents the subset of hydrogelated cells that also exhibit metabolic activity. T-test were applied to confirm the statistical significance of the observed difference (n= 3 independent experiment). The error bars represent the standard deviation of the mean.

[0027] Figure ID shows a comparative analysis of the rate of hydrogelation and metabolic activity tracked across six distinct bacterial strains. The chart displays the hydrogelated cells6KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2 and the metabolically active population for each bacterium at four different hydrogelation percentages: 5%, 10%. 20%, and 30%. 10 % hydrogelation exhibited the highest values among the examined percentages (n= 3 independent experiment). The error bars represent the standard deviation of the mean.

[0028] Figure 2A shows a schematic workflow for hydrogelation and purification / isolation of hydrogelated cells using flow cytometry.

[0029] Figure 2B shows colony-forming units (CFU) of hydrogelated S. aureus, P. aeruginosa, and K. pneumoniae. Flow cytometry-sorted hydrogelated S. aureus, P. aeruginosa, and K. pneumoniae cells exhibit non-detectable CFU (below detection limit) after sorting. This observation signifies the successful elimination of non-hydrogelated and dividing cells within the hydrogelated bacterial cell population (n= 3 independent experiment). Statistical significance was determined using a two-tailed t-test with a significance threshold set at p < 0.05. -test.

[0030] Figure 3A shows a schematic of an invasion assay. Macrophages (RAW 264.7) were infected with wild-type bacterial strains, including S. aureus MN8, P. aeruginosa PA14, and K. pneumonia BIDMC 2A at multiplicity' of infection (MOI) of 50. Post-infection, gentamycin treatment was applied to eliminate extracellular bacteria. After gentamycin treatment, the infected cells underwent three rounds of through washing to remove remaining extracellular bacteria. Subsequently, cell lysis was achieved using 0.5 ml of Triton-X, follow ed by enumeration of colony-forming unit (CFU). This assay quantifies surviving intracellular bacteria, shedding light on their ability to persist within macrophages after uptake.

[0031] Figure 3B shows colony-forming units (CFU) for wild-type S. aureus MN8, P. aeruginosa PAI 4, and K. pneumoniae BIDMC 2A. Gentamicin protection assay was performed on wild-type S. aureus, K. pneumoniae, and P. aeruginosa in RAW 264.7 macrophage cell lines. Successful invasion of wild-type pathogens inside the macrophage cells was indicated by this assay. The experiment was performed with three biological replicates (N=3).

[0032] Figures 4A-4G show quantitative analysis of DNA amplification and bacterial uptake. Figures 4A-4C: melt-curve peaks are shown for DNA extracted from S. aureus, P. aeruginosa, and K. pneumoniae. Figures 4D-4F: linear standard curves of Ct (qPCR cycle7KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2Threshold) against log DNA concentration. R2 values exceed 0.995, 0.994, and 0.990 for S. aureus, P. aeruginosa, and K. pneumoniae respectively. Figure 4G: the uptake of wild-type and hydrogelated cells, measured by qPCR of extracted bacterial DNA, followed by conversion to colony forming unit (CFU) using the standard curves.

[0033] Figure 5 shows inflammatory response of macrophages against hydrogelated and wild-type pathogens. Macrophages (RAW 264.7) were subjected to infection with wild-type and hydrogelated bacterial strains, encompassing <S’. aureus, K. pneumonia, and P. aeruginosa, at a multiplicity of infection (MOI) of 50 for 4 hours. Post-infection, supernatants were collected, and the levels of proinfl ammatory cytokines, including TNF- alpha. IL-17, and IFN-gamma, were quantified using ELISA, following the manufacturer's instructions. Figures 5A-5C: standard curves for TNF-alpha, IL-17, and IFN-gamma were develop as per manufacturer instructions. The standard curve was developed in biological replicates (N=2). One sample two-tailed t-test was applied. *P<0.01 for TNF-aipha, APO.01 for IL-17, and APO.01 for IFN-gamma. Figures 5D-5F: TNFa, IL-17, and IFN-gamma from macrophages infected with S. aureus, P. aeruginosa, and K. pneumonia. Assessed via ELISA as per manufactured instruction. The assay was conducted using two biological replicates and two technical replicates. The error bar represents the standard deviation of the mean.

[0034] Figure 6 shows the intracellular level of total ROS in macrophages in response to hydrogelated and wild-type pathogens. The mean fluorescence intensity of H2DCFDA probe in macrophages infected with wild-type and hydrogelated pathogens, including S. aureus, P. aeruginosa, and K. pneumonia. Unstained uninfected and stained uninfected cells are controls. n=2. Error bars = standard errors of the mean. Statistical significance was determined using one-way analysis of variance (ANOVA) with a p-value < 0.0001.

[0035] Figures 7A-7C show7mice (C57BL / 6 LysM-EGFP) vaccination tests using hydrogelated / 5. aeruginosa PA14. Figure 7A shows the immunization and challenge protocol. Figure 7B shows % mouse survival after vaccination. Figure 7C shows % mouse survival after challenge.

[0036] Figure 8 shows results for a co-culture test of hydrogelated PA14 and dendritic cells DC2.4. After co-incubation with or without hydrogelated PA14, DC2.4 was stained with CD86-FITC and analyzed with flow cytometry. N=28KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPC Client Reference No : UC 2025-422-2

[0037] Figure 9A-9C show blood and organ sample test of hydrogelated PA14 vaccinated mice. Figure 9A shows bacterial load (by qPCR) in different harvested organs at the end of mice experiments (Day 60 or time of death). Basal number of bacteria is likely due to dead bacteria. ELISA of for IgG-1 and IgG2 are shown in Figures 9B and 9C, respectively. N=2. Results suggest the activation of adaptive immunity against wild-type PAI 4. IgGl and IgG2 increased significantly in vaccinated mice. However, a slight dip was observed at Day 35 for IgG2, likely due to an experimental error. More replicates and IgG measurements will be performed.

[0038] Figure 10 shows flow cytometry analysis of T cell population from the spleen of treated mice (C57BL / 6 Lysm-eGFP) spleen. The mice were vaccinated and then challenged with PA14. Mice vaccinated with hydrogelated PA14 showed higher levels of live CD3+ T cell population than in the controls. N=2.DETAILED DESCRIPTIONI. Definitions

[0039] Practicing this invention utilizes routine techniques in the field of molecular biology. Basic texts disclosing the general methods of use in this invention include Sambrook and Russell, Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)).

[0040] As used herein, the terms “hydrogelation” and “intracellular hydrogelation” are used interchangeably and refer to a process or method of forming a synthetic polymer within a cell. Typically, a synthetic monomer and a polymerization inducer are first added to a cell in a manner that allows them to enter the cell. Then, an initiation signal is applied to the cell such that the inducer is able to cause the formation of the synthetic polymer, i.e., a crosslinked hydrogel. Hydrogelated cells contain cross-linked hydrogel and are unable to replicate but can perform certain cellular processes, such as metabolic pathways, cellular respiration, protein synthesis, motility, etc.

[0041] As used herein, the term “pathogenic bacteria” refers to bacteria that can cause disease. Pathogenic bacteria come in a variety7of shapes, such as such rods (bacillus), spirals9KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2(twisted), and spheres (coccus). In many cases, pathogenic bacteria can cause bacterial infections and contagious diseases, which can cause serious complications.

[0042] As use herein, the term “vaccine’7(also widely referred to as an “immunogenic compound”) refers to a pharmaceutical preparation formulated to initiate an immune response (e.g., in B cells and / or T cells) that is specific for a pre-determined pathogen. The vaccine can provide one or more epitopes of one or more antigens of the pathogen, which, when administered to a subject, prompts the immune system of the subject to initiate or boost the intended immune response. “Vaccination” or “immunization” is the act of administering such a pharmaceutical composition to a person in need thereof, whether or not they have a particular disease or condition.

[0043] An “immunogenic response” to an antigen or composition is the development in a subject of a humoral and / or a cellular immune response to an antigen present in the composition of interest. For purposes of the present disclosure, a “humoral immune response” refers to an immune response mediated by antibody molecules, while a “cellular immune response” is one mediated by T-lymphocytes and / or other white blood cells. One important aspect of cellular immunity involves an antigen-specific response by cytolytic T-cells (“CTLs”). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. CTLs help induce and promote the destruction of intracellular microbes, or the lysis of cells infected with such microbes. Another aspect of cellular immunity involves an antigen- specific response by helper T-cells. Helper T-cells act to help stimulate the function, and focus the activity of, nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface. A “cellular immune response” also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and / or other white blood cells, including those derived from CD4+ and CD8+ T-cells. Hence, an immunological response may include one or more of the following effects: the production of antibodies by B-cells; and / or the activation of suppressor T-cells and / or yA T-cells directed specifically to an antigen or antigens present in the composition or vaccine of interest. These responses may serve to neutralize infectivity, and / or mediate antibody-complement, or antibody dependent cell cytotoxicity (ADCC) to provide protection to an immunized host. Such responses can be determined using standard immunoassays and neutralization assays, which are well known in the art.10KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2

[0044] As use herein, the term ‘Therapeutically effective amount'’ is an amount of a compound, such as the cells or vaccine of the present disclosure that (i) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein; (ii) prevents or delays progression of the particular disease, condition or disorder; (iii) treats the particular disease, condition, or disorder; (iv) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder; or (v) at least partially reverses damage caused by the condition prior to treatment.

[0045] Successful “treatment” or “vaccination” in a subject according to this disclosure may have any effect that is beneficial to the subject being treated. This includes preventing, delaying, or decreasing the severity’, duration, or progression of a condition disclosed herein, or any’ adverse signs or symptoms resulting therefrom. Treatment or vaccination may also be unsuccessful, resulting in no improvement in typical signs and symptoms of the condition. The subject has still been “treated” if the intention of the managing clinician has been at least in part the improvement or alteration of a condition referred to. A concurrent objective of therapy is to minimize adverse effects on the target tissue or elsewhere in the treated subject.

[0046] For nucleic acids, sizes are given in either kilobases (kb), base pairs (bp), or nucleotides (nt). Sizes of single-stranded DNA and / or RNA can be given in nucleotides. These are estimates derived from agarose or acry lamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or ammo acid residue numbers. Protein sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.

[0047] Oligonucleotides that are not commercially available can be chemically synthesized, e.g., according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers. Tetrahedron Lett. 22: 1859-1862 (1981). using an automated synthesizer, as described in Van Devanter et. al. , Nucleic Acids Res. 12:6159-6168 (1984). Purification of oligonucleotides can be performed using any art-recognized strategy’, e.g., native acrylamide gel electrophoresis or anion-exchange high performance liquid chromatography (HPLC), e.g., as described in Pearson and Reanier. J. Chrom. 255: 137-149 (1983).

[0048] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.11KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2

[0049] The terms “a,” “an,” or “the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherw ise. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth.

[0050] The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and / or deoxyinosine residues (Batzer et al.. Nucleic Acid Res . 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0051] The term “gene” means the segment of DNA involved in producing a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).

[0052] A “promoter” is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. The promoter can be a heterologous promoter. In some embodiments, the promoter is a prokary otic promoter. Typical prokary otic promoters include elements such as short sequences at the -10 and -35 positions upstream from the transcription start site, such as a Pribnow box at the -10 position typically consisting of the six nucleotides TATAAT, and a sequence at the -35 position, e.g., the six nucleotides TTGACA. In some embodiments, the promoter is a eukaryotic promoter.12KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2

[0053] An ‘‘expression cassette’' is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a host cell. An expression cassette may be part of a plasmid, viral genome, or nucleic acid fragment. Typically, an expression cassette includes a polynucleotide to be transcribed, operably linked to a promoter. The promoter can be a heterologous promoter. In the context of promoters operably linked to a polynucleotide, a ■‘heterologous promoter” refers to a promoter that would not be so operably linked to the same polynucleotide as found in a product of nature (e.g., in a wild-type organism).

[0054] As used herein, a first polynucleotide or polypeptide is "heterologous" to an organism or a second polynucleotide or polypeptide sequence if the first polynucleotide or polypeptide originates from a foreign species compared to the organism or second polynucleotide or polypeptide, or, if from the same species, is modified from its original form. For example, when a promoter is said to be operably linked to a heterologous coding sequence, it means that the coding sequence is derived from one species whereas the promoter sequence is derived from another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g.. is a genetically engineered coding sequence).

[0055] “Polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0056] The terms “expression” and “expressed” refer to the production of a transcriptional and / or translational product, e.g.. of an RNA and / or a nucleic acid sequence encoding a protein. Tn some embodiments, the term refers to the production of a transcriptional and / or translational product encoded by a gene (or a portion thereof. The level of expression of a DNA molecule in a cell may be assessed on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell.13KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2II. Introduction

[0057] The inventors have discovered how to generate a synthetic polymer network, e.g.. a hydrogel network, inside cells, which renders the cells incapable of dividing while allowing the cells to retain certain cellular activity, and that when used in pathogenic bacteria, can be used to generate effective vaccines and to generate immune responses in animals. The present disclosure provides cells that comprise the synthetic polymer networks, and these cells are referred to herein as ‘'hydrogel ated cells.’’

[0058] The present disclosure provides methods for making cells that comprise a synthetic polymer network on the inside. Herein, the synthetic polymer network is referred to as an “intracellular hydrogel network” or a “hydrogel network” whereas the cells are referred to as “hydrogelated cells.” In some cases, the hydrogelated cells are useful for inducing an immune response in a subject, for example, as a vaccine. Because the hydrogelated cells are metabolically active and non-replicating, the hydrogelated cells can interact with the host immune system in ways that live bacterial cells would, but without the risk of infection. Hydrogelated cells represent a new class of vaccines with (1) the efficacy of live vaccines (i.e., vaccines made from live or attenuated / weakened virus) that can illicit robust immune responses in patients, and (2) the safety of inactivated / killed vaccines, for example heat-killed vaccines (that have no risk of infection).

[0059] Because the process of hydrogelating cells can preserve the cell’s ability' to produce proteins, the hydrogelated cells can act as vaccines themselves, and / or can be engineered to express heterologous proteins that can interact with the host’s immune system to elicit a stronger or enhanced immune response. For example, the hydrogelated cells can be engineered to express an antigen on the cell surface or an antigen that is secreted into the extracellular environment. In some cases, the hydrogelated cells can also be covalently linked with adjuvants to potentiate an immune response or otherwise administered with an adjuvant. Vaccines comprising hydrogelated cells can elicit a host immune response that has greater breadth and with greater potency compared to subunit or inactivated vaccines. The hydrogelated cells can be generated quickly and efficiently from any bacterial strain without the long production times and high costs associated with developing traditional vaccines. Thus, disclosed herein are significant advancements in vaccine technology, which can provide vaccines with broad-spectrum protection, enhanced safety’, and ease of rapid and low - cost manufacturing.14KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2

[0060] In many cases, the hydrogelated cells can be prokaryotic or eukaryotic cells. The methods disclosed herein can be readily applied to hydrogelate any bacterial cell, including pathogenic bacteria and multi-drug / antibiotic resistant bacteria. The hydrogelated cells can be used as vaccines against bacterial infections, for example infections by pathogenic bacteria, drug / antibiotic-resistant bacteria, or multi-drug / antibiotic resistant bacteria. In some cases, the bacteria possess virulence factors that can enhance therapeutic efficacy through targeted cell invasion, improved persistence, and modulation of host immune responses. For instance, the bacteria can produce toxins or proteins that allow them to invade host cells. Non-limiting examples of bacteria include Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus. Bordetella pertussis. Streptococcus pneumoniae, Mycobacterium tuberculosis, and Neisseria gonorrhoeae.

[0061] Hydrogelated cells can be produced using varying hydrogel densities. In some cases, a population of hydrogelated cells with lower densities correlate with a population of hydrogelated cells having higher metabolic activity7, such as cellular respiration (i.e., the breakdown of glucose to produce ATP). Many methods and materials are known and available to one of ordinary' skill in the art for use in measuring metabolic activity7in cells. In some embodiments, metabolic activity was determined by' staining the cells with 5-Cyno-2,3- ditolyl tetrazolium chloride (CTC) and flow cytometry7and / or microscopy.

[0062] In some embodiments, when the population of hydrogelated cells (i) have a crosslinked hydrogel density of 10%-30% (w / w) or more in the cell, (ii) about 40-70%, about 40- 60%, about 50-70%, about 40%. about 50%. about 60%, about 70%, less than 40%, less than 50%, less than 60%, or less than 70% of the population of hydrogelated cells demonstrate metabolic activity'.

[0063] In some embodiments, when the population of hydrogelated cells (i) have a crosslinked hydrogel density of less than 15% (w / w), about 1%- 15% (w / w), about 5%-l 5% (w / w), 10%-15% (w / w). 5%-10% (w / w), about 1-5% (w / w), about 1-3% (w / w), about 1-2% (w / w), about 1 % (w / w), about 2% (w / w), about 3% (w / w), about 4% (w / w), about 5% (w / w), about 6% (w / w), about 7% (w / w), about 8% (w / w), about 9% (w / w), about 10% (w / w), about 11% (w / w), about 12% (w / w), about 13% (w / w), about 14% (w / w), or about 15% (w / w) in the cell; (ii) about 70-100%, about 80-100%, about 90-100%, about 95-100%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 98%, greater than 99%, or about 100% of the population of hydrogelated cells demonstrate metabolic activity.15KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2

[0064] In some embodiments, the hydrogelated cells were capable of inducing an immune response in a subject. In some embodiments, the hydrogelated cells are engulfed and / or internalized by immune cells. In some embodiments, the hydrogelated cells caused the production of pro-inflammatory cytokines and reactive oxygen species (ROS) in immune cells. In some embodiments, the immune cell is a macrophage or dendritic cell.III. Hydrogelated Cells

[0065] Disclosed herein are hydrogelated cells, which comprise an intracellular synthetic polymer network, i.e.. a cross-linked hydrogel. Hydrogelated cells cannot replicate (e.g., divide) but can continue to perform certain cellular activities for a time period (e.g., a period of days). Typically, a cell type that is intended for hydrogelation is propagated until a sufficient number of cells is attained, and that population of cells is hydrogelated. After the hydrogelation process, the cell can no longer divide.

[0066] Hydrogelated cells are metabolically active and can be used for one or more of their cellular activities without expansion of the cell population. For example, essentially all cellular functions aside from replication will continue to function. Exemplary cellular activity' can include but is not limited to, transcription and translation, enzymatic functions, motility’, REDOX reactions, homeostasis, and active response to stimuli in the environment.

[0067] In some embodiments, the cell is a bacteria, e.g., a bacterial pathogen, including but not limited to those of the genera Escherichia, Proteus, Enterobacter, Klebsiella, Citrobacter, Yersinia, Shigella, Pseudomonas, Staphylococcus, Bordetella, Streptococcus, Mycobacterium, Neisseria, and Salmonella. In some embodiments, the cell is from the genera Pseudomonas, Klebsiella. Bordetella. Streptococcus. Mycobacterium, Neisseria, or Staphylococcus. In some embodiments, the cell is Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Bordetella pertussis, Streptococcus pneumoniae, Mycobacterium tuberculosis, Neisseria gonorrhoeae or E. coli.

[0068] In some embodiments, the hydrogelated cells can be used to induce an immune response in a subject (e.g, as a vaccine). Vaccines comprising hydrogelated cells are discussed below. In some embodiments, the hydrogelated cells will gain additional abilities or functions. In some instances, the cells can gain the ability to survive exposure to stress, such as oxidative stress (e.g., hydrogen peroxide exposure), heat stress, etc.16KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2

[0069] The hydrogel polymeric network (“cross-linked hydrogel’") in the cells can be composed of different hydrogel components as desired. The hydrogel will be formed from monomer subunits that are polymerized once introduced into the cell as discussed in more detail below. The monomer subunits can comprise for example polysaccharides, monosaccharides, and / or proteins. In many embodiments, the hydrogel is a homopolymer or a co-polymer. In some embodiments, the hydrogel can be composed of poly(ethylene glycol) (or “polyethylene glycol”) monomers of any of a variety of lengths. In other embodiments, the hydrogel can comprise, for example, poly(ethylene glycol) diacrylate monomer (PEG- DA; average Mn 700), poly(dimethyl siloxane) (PDMS), polyethylene oxide) (PEO), poly(vinyl alcohol) (PVA). or polypropylene fumarate) (PPF). Polymeric networks in cells can also be formed from components such as, but not limited to, alginate, guanosine mono phosphate (GMP), cyclodextrins (CD), fibrin, collagen, polypeptides, decellularized extracellular matrix (ECM), and nucleic acids. A discussion of hydrogels can be found in, e.g., Ahmed, Journal of Advanced Research. Volume 6, Issue 2. March 2015, Pages 105-121; Drury JL. Mooney DJ. 2003 Biomaterials . 24(24):4337-4351; and Tibbitt MW. Anseth KS. 2009. Biotechnol. Bioeng. 103(4):655-663.

[0070] The hydrogel monomers can be substituted at one or more side chains, e.g., with a side chain moiety or otherwise conjugated to other molecules, providing further functionality' or reactivity of the poly mer in the cell. For example, some or all of the monomers of the polymers can be conjugated to. for example, metals, nucleic acids, nanoparticles, peptides, and drugs. Generally, conjugation to the monomers will occur before the monomers are introduced into the cells but in some embodiments, conjugation can occur after the monomer subunits are introduced into the cells. In some embodiments, the conjugation is carried out using activated monomers that can be conjugated to peptides, drugs, enzymes, etc. An activated monomer is a monomer that has been modified with a reactive (electrophilic) group. For example, PEG or other monomers can be modified with aryl chloride residues, reactive acyl groups, or modified with alky lating reagents or optionally can be purchased commercially. In some cases, the substitution reactions are performed after the hydrogel has formed and the hydrogel is thereby substituted.

[0071] Cells having an internal hydrogel polymer (“cross-linked hydrogel”) can be generated by providing cells, introducing hydrogel monomeric subunits, and, depending on the monomeric subunit, a polymerization inducer (an initiator of polymerization), into the17KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2 cells and then polymerizing the monomeric subunits in the cells to form the polymer network (cross-linked hydrogel) in the cells. The polymerization inducer can be introduced to the cells before, after, or simultaneously with the step of introducing hydrogel monomer subunits to the cells. As noted above, any cell can be used as a starting material. Generally, the cells will be cultured such that the cells are actively dividing. As an example, for bacterial cells, they can be cultured so that the cells are in exponential growth phase. Other cells may not have an exponential growth phase, but can nevertheless be exposed to new culture and nutrients such that the cells are in a phase of cell division.

[0072] The monomer subunits and polymerization inducer can be introduced into the cells as desired. In some embodiments, for example, the cells can be exposed to a freeze / thaw cycle to increase the cells’ ability’ to uptake the monomeric subunits and polymerization inducer. The freezing will be selected to avoid excessive cell death while improving the entry' of the monomer subunits. For example, the cells can be “flash” frozen, e.g, immersed in a liquid below 0°C, e.g., at -80°C, for less than five minutes, e.g., 1-3 minutes. In some embodiments, one or more heterologous cryoprotectants are incubated with the cells during the freeze / thaw cycle to protect the cells from the process. The heterologous cryoprotectant can be introduced to the cells at any method step of hydrogelating cells, e.g., before, during, or after the hydrogel monomer subunits are introduced to the cells; or before, during, or after the polymerization inducer is introduced to the cells; before, during, or after the polymerization steps. In some embodiments where the cell is a gram-positive bacteria, chemical enhancers may be used such as CaCh. In some embodiments where the cell is a gram-positive bacteria, two or more freeze and thaw cycles may be used to infuse hydrogel components inside the gram-positive cells. Exemplary cryoprotectants include, but are not limited to, dimethyl sulfoxide (DMSO) or glycerol. In other embodiments, the monomer subunits can be introduced into the cell by chemical or electncal (e.g.. electroporation)-based methods, or methods including but not limited to, e.g., osmotic shock, sonoporation, electroporation, laser, surfactant-based permeabilization, or shearing. Once the monomers are introduced into the cells, the cells can be washed to remove excess monomers on the exterior of the cells.

[0073] The concentration of monomers introduced into the cells can be selected for optimum results and can vary' depending on the monomer and resulting polymers used. In some embodiments, the concentration of monomers is 1-30% (w / w), e.g., 1-20% (w / w), 1-18KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-210% (w / w). 3-8% (w / w), or 4-6% (w / w) in the cell. In some embodiments, the monomers have a density of about 0.5-5% (w / w), about 0.5-1% (w / w), about 1-1.5% (w / w), about 1.5- 2% (w / w), about 2-2.5% (w / w), about 2.5-3% (w / w), about 3-3.5% (w / w), about 3.5-4% (w / w), about 4-4.5% (w / w), about 4.5-5% (w / w) in the cell.

[0074] In some embodiments, the cross-linked hydrogel has a density' of about 0.5-5% (w / w), about 0.5-1% (w / w), about 1-1.5% (w / w), about 1.5-2% (w / w), about 2-2.5% (w / w), about 2.5-3% (w / w), about 3-3.5% (w / w), about 3.5-4% (w / w), about 4-4.5% (w / w), about 4.5-5% (w / w) in the cell.

[0075] In some embodiments, the monomers are polyethylene glycol (PEG) molecules. As one example, the PEG monomers can be of an average weight of 500-2000 daltons. In some embodiments, the PEG monomers comprise end moieties to assist in polymerization. For example, in some embodiments, the PEG is a diacrylate, meaning both ends comprise acry late moieties.

[0076] In some embodiments, the monomers are sodium alginate, a non-toxic, biocompatible, and biodegradable polysaccharide. The formation of hydrogels from alginate can occur by interactions of the anionic alginates with multivalent inorganic cations through a typical ionotropic gelation method. Exemplary non-limiting divalent cations can include, for example, calcium or magnesium. The monomer and the crosslinker (divalent cation) can be introduced into the cell as desired. In some embodiments, they are introduced together or separately by freezing and thawing, osmotic shock, sonoporation, electroporation, laser, surfactant-based permeabilization, or shearing.

[0077] In some embodiments, the monomers are guanosine. In these embodiments, hydrogelation can occur as self-assembly, e.g., to form a guanosine mono phosphate (GMP) hydrogel. In some embodiments, the hydrogel can be stabilized using hydrazides, aldehydes, or cations such as but not limited to K+, which can also be introduced into the cells. The monomer and the stabilizer can be introduced for example by freezing and thawing, osmotic shock, sonoporation, electroporation, laser, surfactant-based permeabilization, or shearing.

[0078] In some embodiments, the monomers are cyclodextrin (CD), i.e.. a cyclic oligosaccharide of glucopyranoside units linked through a-1,4 glycosidic bonds. Once introduced into cells, a CD polymer can be formed. In some embodiments, the polymer is formed via chemical cross-linking, for example by free-radical polymerization cross-linking-19KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2 based methods; nucleophilic addition / substituti on-based methods; cross-linking methods based on 'click7reactions and / or incorporation of CDs through post-gelation attachment. The CD monomer and any crosslinkers can be introduced, for example, by freezing and thawing, osmotic shock, sonoporation, electroporation, laser, surfactant-based permeabilization, or shearing.

[0079] In some embodiments, the monomers are fibrinogen (e.g., a glycoprotein 45 nm in length). Once introduced into cells, fibrinogen polymerization into fibrin can be initiated by the action of the proteolytic enzyme, thrombin, which can also be introduced into the cells. The monomer and polymerizing enzyme (e.g., thrombin) can be introduced, for example, by freezing and thawing, osmotic shock, sonoporation, electroporation, laser, surfactant-based permeabilization. or shearing.

[0080] In some embodiments, the monomers are collagen. For example, in some embodiments, the collagen is type I collagen subunits (triple-helical protein formed of 67-nm periodic polypeptide chains with a total molecular weight near 300 kDa). Polymerization can be achieved, for example with a mixture of temperature, pH, and ionic strength. In some embodiments, the monomers can be introduced by freezing and thawing, osmotic shock, sonoporation, electroporation, laser, surfactant-based permeabilization, or shearing. In some embodiments, crosslinking factors is controlled, for example, independently of permeation. For example, one can change temperature, ionic strength, and pH of the cell culture medium to induce cross-linking without needing to introduce external factors into the cell.

[0081] In some embodiments, the polymer comprises decellularized components such as decellularized extracellular matrix (ECM) and the monomers variate and their precise composition can depend on the tissue that it is derived from. For example, the composition is a mixture of collagen, glycosaminoglycans, proteoglycans, and ECM proteins. Hydrogel formation can be induced in cells by collagen-based self-assembly, which can also be regulated, for example, by a mixture of temperature. pH, and ionic strength. The ECM components can be introduced by freezing and thawing, osmotic shock, sonoporation, electroporation, laser, surfactant-based permeabilization, or shearing. In some embodiments, crosslinking factors are controlled independently of permeation. For example, one can change temperature, ionic strength, and pH to induce cross-linking without needing to introduce external factors into the cell.20KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2

[0082] In some embodiments, the monomers are nucleic acids or nucleotides. Nucleic acid hydrogels can be formed within the cells. In some embodiments, different classes of nucleic acids (e.g. DNA molecules) with different properties and shapes (e.g. X-shaped, Y-shaped or T-shaped DNA molecules). In some embodiments, the nucleic acids are chemically or enzy matically cross-linked to form a hydrogel in the cells. In some embodiments, the nucleic acids and, if included, crosslinking enzymes (e.g, ligases used to polymerize different shapes of DNA), can be introduced, for example, by freezing and thawing, osmotic shock, sonoporation, electroporation, laser, surfactant-based permeabilization, or shearing.

[0083] In some embodiments, the monomers are polydimethylsiloxane-isocyanatomethyl- 3,5,5-trimethylcyclohexyl isocyanate-2-hydroxyethylmethacrylate (PDMS-IPDI-HEMA) and acry lamide. In some embodiments, these components can be polymerized in the cells, for example, by micellar copolymerization (compartmentalization of PDMS-IPDI-HEMA inside SDS or other detergent micelles). PDMS-IPDI-HEMA micelles and acrylamide can be introduced to cells, for example by freezing and thawing, osmotic shock, sonoporation, electroporation, laser, surfactant-based permeabilization, or shearing.

[0084] In some embodiments, the monomers are poly(ethylene oxide) (PEO). The PEO monomers can be any of a variety of weights as desired. In some embodiments, the lengths of the PEO monomers differ. In some embodiments, the PEO monomers can be of average length of 10000-10 million daltons weights (e.g., 35,000; 900,000; or 5,000,000 Da). In some embodiments, the PEO monomers are of different molecular weights (e.g, 35,000; 900,000; and / or 5,000.000 Da). Polymerization of the monomers, once introduced into the cells, can be achieved for example, by y-irradiation or photo-crosslinking using UV light. PEO monomers can be introduced into cells, for example, by freezing and thawing, osmotic shock, sonoporation. electroporation, laser, surfactant-based permeabilization, or shearing.

[0085] In some embodiments, the monomers are poly(ethylene glycol) diacrylate monomer (PEG-DA; average Mn700). PEG-DA monomers can be introduced into cells, for example, by freezing and thawing, osmotic shock, sonoporation, electroporation, laser, surfactantbased permeabilization, or shearing. Polymerization of the monomers, once introduced into the cells, can be achieved for example, by incubation with a photoinducer, e.g., 2 -hydroxy -4'- (2 -hydroxy ethoxy )-2-methylpropiophenone (Irgacure 2959) or lithium phenyl-2,4,6-tri- methylbenzoylphosphinate, and by activation with irradiation of an appropriate wavelength.21KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2 e.g.. 365 nm for 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959); or 365 nm or 400 nm for lithium phenyl-2,4,6-tri-methylbenzoylphosphinate.

[0086] In some embodiments, the monomers are poly(vinyl alcohol) (PVA). Following introduction into the cells, polymerization can be carried out, for example, by low- temperature cry stallization. PVA can be introduced into cells, for example, by osmotic shock, sonoporation. electroporation, laser, surfactant-based permeabilization, or shearing.

[0087] In some embodiments, the monomers are polypropylene fumarate) (PPF). In some embodiments, the monomers are a mixture of PPF and PEG. For example, in some embodiments, a 1 : 1 w / w mixture of PPF and PEG can be introduced into cells and then formed into a hydrogel with low cytotoxicity'. In some embodiments, a polymerization inducer is also introduced into the cell. Exemplary' polymerization inducer can include, for example, a benzoyl peroxide initiator mixed with a vinyl monomer. N-vinyl pyrrolidinone. The reaction can be further accelerated with, for example, N,N-dimethyl-p-toluidine. These components can be introduced into the cells to initiate polymerization. Monomers and other components described above can be introduced into the cells, optionally in gradual steps, by freezing and thawing, osmotic shock, sonoporation, electroporation, laser, surfactant-based permeabilization, or shearing.

[0088] Depending on the monomer and polymerization inducer employed, the polymerization inducer can be introduced into the cells with the monomers or in a separate step. For efficiency, it can be helpful to introduce both at the same time. However, in circumstances in which mixture of the monomer and inducer causes significant polymerization before the components can be introduced into the cell together, it can be advantageous to introduce each component separately into the cells. The inducer used will depend on the cross-reaction chemistry' involved in linking the monomers in the cell. In some embodiments, the inducer can be selected from, for example, 2-hydroxy-4'-(2- hydroxy ethoxy )-2-methylpropiophenone (Irgacure 2959). Eosin-Y, and lithium phenyl-2,4,6- tri-methylbenzoylphosphinate.

[0089] Once the monomer subunits and polymerization inducer are introduced into the cells, polymerization can be induced. In embodiments in which the inducer is a photoinducer, the appropriate wavelength and intensity of light can be exposed to the cells for sufficient time to polymerize the monomer subunits thereby forming a polymer network within the cell. In other embodiments, the inducer can be activated by' temperature, or other22KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2 environmental stimuli, or in some embodiments, simply by being in proximity' to the monomer subunits.

[0090] In some embodiments, following polymerization, the cells are exposed to a replication-specific toxin and / or antibiotics that target the replication of cells. This will kill cells that remain capable of replication, thereby enriching cells that comprise the hydrogel polymer and that are no longer capable of replication. Exemplary toxins or antibiotics can include, but are not limited to, carbenicillin, which kills replicating bacterial cells. In some embodiments, the antibiotic is gentamicin.

[0091] In some embodiments, following polymerization and / or treatment with an antibiotic, the cells are sorted such that hydrogelated cells are isolated from non-hydrogelated cells. In some embodiments, a marker is introduced to the cells to distinguish hydrogelated cells from non-hydrogelated cells. The marker can be useful for assessing the permeation of the hydrogel components into the bacteria and confirm the success of intracellular hydrogelation. In some embodiments, the marker is introduced to the cells before, after, or in the step that hydrogel monomer units are introduced to the cells. In some embodiments, the marker is a fluorescent dye, for example, without limitations, fluorescein O’O-diacrylate (also referred to as '‘fluorescein diacrylate). In some embodiments, the cells are sorted using fluorescence-activated cell sorting (FACS) using an appropriate channel. For example, as discussed in the Examples below, the incorporation of fluorescein O’O-diacrylate into intracellular hydrogel was detected using the fluorescein (FITC) channel, where the fluorescein is excited at about 498 nm and its emission is detected at about 517 nm. In some cases, the cells comprising fluorescent dye can be analyzed by fluorescence microscopy.

[0092] In some embodiments, in a population of treated cells, before or following antibiotic treatment and / or cell sorting, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% of cells comprise cross-linked hydrogel.

[0093] The resulting cell population are hydrogelated cells that have an internal crosslinked hydrogel polymer that prevents the cells from dividing but that nevertheless retain other biological activities. These cells can then be used in a variety of applications, such as in vaccines.23KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2

[0094] In some embodiments, the cell can be engineered to express one or more heterologous polynucleotides. In some embodiments, the expressed polynucleotide is an RNA that encodes a polypeptide. Thus, in some embodiments, the cells comprise one or more heterologous polynucleotides (e.g., DNA) that is integrated into its genome or provided on a plasmid or other extrachromosomal vector. In some embodiments, the polynucleotide is operably linked to an endogenous or heterologous promoter that controls expression of the polynucleotide. The promoter can, in some embodiments, be constitutive or inducible. When under an inducible promoter, expression of the gene product can be controlled by exposure of the cells to an agent that induces expression from the inducible promoter. In some embodiments, the cells produce or express one or more proteins derived from a bacterial pathogen. In some embodiments, the one or more proteins are capable of inducing an immune response, for example, without limitations, an outer membrane protein or a secreted protein derived from a bacterial pathogen. Non-limiting examples of bacterial pathogen outer membrane proteins include porins, Ton-B transporters, OprF, OprI, FhuA, and FepA. Nonlimiting examples of bacterial pathogen secreted proteins include toxins, invasins, hemolysins, and proteases. In some embodiments, genetic engineering of the cell is performed before the cell is subjected to hydrogelation.

[0095] In some embodiments, the hydrogelated cells are administered with, and optionally covalently or non-covalently linked to, one or more adjuvants and / or nonspecific inflammatory mediators. Adjuvants and / or nonspecific inflammatory mediators are not required, but can be optionally administered with an active ingredient (e.g., a vaccine comprising hydrogelated cells) before, during, or after the priming and / or boosting of the immune response. Adjuvants are substances that are used to specifically or nonspecifically potentiate an antigen-specific immune response, perhaps through activation of antigen presenting cells. The adjuvant can be, e.g.. Montamde-ISA 51 (e.g.. from Seppic Hiltonol (e.g., from Oncovir), anti-CD40 agonistic monoclonal antibodies, polylCLC, Bacillus Calmette-Guerin (BCG) vaccine, an ADP-ribosylating exotoxin (e.g., cholera toxin, diphtheria toxin, E. coli heat-labile enterotoxin, pertussis toxin, P. aeruginosa exotoxin A), a fragment containing the A and / or B subunit of ADP-ribosylating exotoxin, a chemically modified or genetically mutated derivative thereof, or a derivative thereof with reduced toxicity; a chemical conjugate or genetic recombinant containing a bacterial ADP- ribosylating exotoxin or derivative thereof; a chemokine (e.g., defensins, HCC-1, HCC-4, MCP-1 MCP-3, MCP-4, MLP-1 a, M1P-10, MlP-ly. MIP-3a, MIP-2, RANTES); another24KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2 ligand of a chemokine receptor (e.g., CCR1, CCR-2, CCR-5, CCR-6, CXCR-1); a cytokine (e.g., IL-lp. IL -2, IL-6, IL-8. IL-10, IL-12; IFN-y; TNF-a; GM-CSF); another ligand of a cytokine receptor; a salt (e.g., aluminum hydroxide or phosphate, calcium phosphate); lipid A or a derivative thereof (e.g., monophosphoryl or diphosphoryl lipid A, lipid A analogs, AGP, ASO2, ASO4, DC-Choi, Detox, OM-174); a pathogen-associated molecular pattern (PAMP); immunostimulatory CpG motifs in bacterial DNA or an oligonucleotide (see, for example, U.S. Patent 6,218,371); aLeishmania homolog of elF4a or a derivative thereof (see, for example, U.S. Patent 5,876,735); a heat shock protein or derivative thereof; C3d tandem array; a muramyl dipeptide (MDP) or a derivative thereof (e.g., murabutide, threonyl-MDP, muramyl tripeptide); ISCOMS and saponins (e.g., Quil A. QS-21); squalene; superantigens; a ligand of a toll-like receptor, or the like. Adjuvants may be chosen to preferentially induce antibody or cellular effectors, specific antibody isotypes (e.g, IgM, IgD, IgAl, IgA2, secretory IgA, IgE, IgGl, IgG2, IgG3, and / or IgG4), or specific T-cell subsets (e.g., CTL, Thl, Th2 and / or TDTH)- For example, antigen presenting cells may present Class Il-restricted antigen to precursor CD4+ T cells, and the Thl or Th2 pathway may be entered.

[0096] In some embodiments, a biological activity of the cells can be measured, e.g., in response to exposure to one or more agents, expressed gene product, or environmental change. In some embodiments, the cells can be exposed to one or more cell-permeable probes to measure a biological activity. Exemplary' cell-permeable probes include but are not limited to, resazurin-based PrestoBlueTM (ThermoFisher Scientific). In other embodiments, one can measure the ATP / ADP ratio of the cells using a protein biosensor. An exemplary protein biosensor can include but is not limited to, Perceval (see, e.g., Tantama, et al., Nat Commun. 2013; 4: 2550). In yet other embodiments, the cells can be measured for proteomic and metabolomic changes in response to exposure to one or more agents, expressed gene products, or environmental experience.

[0097] The extent of hydrogelation in a population of cells can be assessed. Because hydrogelated cells cannot divide but can continue to perform certain cellular / metabolic activities, cell hydrogelation can be assessed using a colony forming unit (CFU) assays and / or a variety of assays that measure metabolic activity that are known and commercially available to one of ordinary' skill in the art. For example, without limitation. 5-Cyno-2,3- ditolyl tetrazolium chloride (CTC), a reductive chromogenic dye, can be used to assess cell metabolic activity'; the dyed cells can be sorted and / or analyzed using flow cytometry'.25KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2

[0098] The ability of the hydrogelated cells to elicit an immune response can be assessed. Quantitative assessments of bacterial cell count, cytokine levels, and reactive oxygen species (ROS) production in immune cells, e.g, macrophages, RAW 264.7 cells, antigen-presenting cells (APCs), dendritic cells (DCs), DC2.4 cells, etc. can be performed using a variety of assays that are known and commercially available to one of ordinary' skill in the art. In some embodiments, an immune cell’s response to hydrogelated bacteria is evaluated using 2', 7'- dichlorofluorescein (DCF), which forms a fluorescent compound (2', 7'- dichlorodihydrofluorescein diacetate (H2DCFDA)) when reacting with ROS. In some embodiments, cytokine levels are quantified using commercially available assays as an indicator of an immune response elicited by the hydrogelated cells. Non-limiting examples of cytokines include TNF-alpha, IL-17, and IFN-y. In some embodiments, it is useful to detect the presence, activation, and / or differentiation of an immune cell.

[0099] In some embodiments, the immune cell is an APC or a DC and the cell is detected with a label that recognizes an APC surface marker, such as CD86, CD80, and CD11c. In some embodiments, the immune cell is a T cell and the cell is detected with a label that recognizes a T cell marker, such as CD3. In some cases, it is useful to assess the intracellular fate of hydrogelated cells within immune cells, e.g., macrophages and DCs, and comparisons can be made to the intracellular fate of wild-type or unhydrogelated cells in the same immune cells.

[0100] In some embodiments, the impact of hydrogelated cells on antibody production can be assessed. For example, as discussed in the Examples below, a subject administered with hydrogelated cells or a composition of hydrogelated cells of the present disclosure and the levels of IgGl and / or IgG2 antibodies in the serum of the subject are assessed.IV. Compositions

[0101] Disclosed herein are pharmaceutical compositions comprising hydrogelated pathogenic bacterial cells. The hydrogelated cells of the present disclosure can be used to prepare a composition to prevent or treat a bacterial infection by eliciting an immune response, i.e., a vaccine, in an animal subject (e.g., mammal, human, livestock animal, domesticated animal, companion animal, bird, dog, cat, chicken, cow. goat, sheep, horse, mouse, rabbit, rat, etc.). Hydrogelated cells and methods for making and characterizing them are discussed above.26KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2

[0102] In some embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% of cells in the pharmaceutical composition, e.g., vaccine, are nondividing cells.

[0103] In some embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% of cells in the pharmaceutical composition, e.g., vaccine, comprise cross-linked hydrogel.

[0104] Pharmaceutical compositions, e.g., vaccines, comprise hydrogelated cells and a pharmaceutically -acceptable excipient. Vaccines may comprise one or more such excipients and anon-specific immune response enhancer. A non-specific immune response enhancer may be any substance that enhances an immune response to an exogenous antigen. Examples of non-specific immune response enhancers include adjuvants, biodegradable microspheres (e.g, polylactic galactide) and liposomes (into which the compound is incorporated; see, e.g, U.S. Patent No. 4,235,877). Most adjuvants contain a substance designed to protect the hydrogelated cell or the antigen from rapid catabolism or degradation, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins. Suitable adjuvants are commercially available as. for example, Freund’s Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (SmithKline Beecham); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.

[0105] Vaccine preparation is generally described in, for example, Powell and Newman, eds., Vaccine Design (the subunit and adjuvant approach), Plenum Press (NY, 1995). Vaccines may be designed to generate antibody immunity and / or cellular immunity such as that arising from CTL or CD4+ T cells.27KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2

[0106] Pharmaceutical compositions and vaccines within the scope of the present invention may also contain other compounds, which may be biologically active or inactive. For example, one or more immunogenic portions of other antigens may be present, either incorporated into a fusion polypeptide or as a separate compound, within the composition or vaccine. Polypeptides may. but need not. be conjugated to other macromolecules as described, for example, within U.S. Patent Nos. 4,372,945 and 4,474,757. Pharmaceutical compositions and vaccines may generally be used for prophylactic and therapeutic purposes.

[0107] Vaccines and pharmaceutical compositions may be presented in unit-dose or multidose containers, such as sealed ampoules or vials. Such containers are preferably hermetically sealed to preserve sterility of the formulation until use. In general, formulations may be stored as suspensions, solutions or emulsions in oily or aqueous vehicles. Alternatively, a vaccine or pharmaceutical composition may be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier immediately prior to use.

[0108] Pharmaceutically acceptable carriers are determined in part by the particular composition being administered (e.g., hydrogelated bacterial pathogen cells), as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g. , Remington ’s Pharmaceutical Sciences, 17thed., 1989). Administration can be in any convenient manner, e.g., by injection, oral administration, inhalation, transdermal application, or rectal administration.

[0109] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the packaged nucleic acid suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, com starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, e.g. sucrose, as well as pastilles comprising the active ingredient in an inert base, such as gelatin28KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2 and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.

[0110] The compound of choice, alone or in combination with other suitable components, can be made into aerosol formulations (z.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.[OHl] Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally. Parenteral administration and intravenous administration are the preferred methods of administration. The formulations of commends can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.

[0112] Such compositions may also comprise buffers (e.g. neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and / or preservatives. Alternatively, compositions of the present invention may be formulated as a lyophilizate. Compounds may also be encapsulated within liposomes using well known technology.V. Methods of Use

[0113] The hydrogelated cells and compositions, e.g., vaccines, of the present disclosure are useful for inducing an immune response in a subject for the prevention or treatment of a bacterial infection in a subject. In many cases, the subject is an animal subject, for example, a mammal, human, livestock animal, domesticated animal, companion animal, bird, dog. cat, chicken, cow, goat, sheep, horse, mouse, rabbit, rat, etc.29KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2

[0114] Typically, a vaccine is administered via injection, either as a liquid solution or suspension. The injection may be subcutaneous, intramuscular, intravenous, intraperitoneal, intrathecal, intradermal, intraepidermal, or by “gene gun” (Miller, K., Eggenberger, A.L., Lee, K. et al. An improved biolistic delivery7and analysis method for evaluation of DNA and CRISPR-Cas delivery7efficacy in plant tissue. Sci Rep 11, 7695 (2021)). Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Other types of administration comprise electroporation, implantation, suppositories, oral ingestion, enteric application, inhalation, aerosolization or nasal sprays or drops. Solid forms, suitable for dissolving in or suspension in, liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or encapsulated in liposomes for enhancing adjuvant effect.

[0115] The dose administered to a subject, in the context of the present invention can be sufficient to effect a beneficial therapeutic response in the subject over time. The dose can be determined by considering the condition of the subject, as well as the body weight or surface area of the subject to be treated. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of cell type in a particular subject. Dose administration can be accomplished via single or multiple doses.EXAMPLES

[0116] The Examples are meant to exemplify, but not limit, the invention. Examples 1-4 discuss the engineering of hydrogelated cells and their characterization in in vitro experiments. Examples 5-9 discuss the characterization of hydrogelated cell vaccines in a mouse model.Example 1 - Materials and Methods for Example 1-4

[0117] Bacterial strains and cell culture. Methicillin-resistant Staphylococcus aureus MN8 (MRSA HM-162D) and A98010 (MRSA NR-45982), Pseudomonas aeruginosa EmvKY2 (NR-51330) and PA14 (NR-50573), Klebsiella pneumonia BIDMC 2A (NR-41916) and MRSN 28880 (NR-55541), and E. coli Nissle were procured from Biodefense and Emerging Infections Research Resources Repository (BEI Resources), which is managed by American Type Culture Collection (ATCC). All bacterial strains were cultured in sterile LB broth.30KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2

[0118] RAW 264.7 cells (murine macrophage cells) were cultured in 10-cm dishes at 37°C with 5% CO2. The medium used to support cell maintenance and growth was DMEM supplemented with 10 % fetal bovine serum.

[0119] Intracellular hydrogelation of bacterial strains. The hydrogelation of bacterial strains was conducted in accordance with prior studies, with minor modification [1], Brielfy, a 1-ml hydrogelation buffer was prepared by mixing 160 pL of 2-hydroxy-4'-(2- hydroxy ethoxy )-2-methylpropiophenone (1% w / v) and 800 pL of PEG 700-DA (5% w / v). Concurrently, a 1-mL culture of bacterial strain grown overnight in LB medium was subjected to centrifugation. For 5% hydrogelation, 67.5 pL of hydrogel buffer was combined with the cell pellet along with 865 pL of LB media. Likewise, 135 pL for 10%, 270 pL for 20%, and 405 pL for 30% were introduced to the pellet, which resulted in a final volume of 1 mL.

[0120] For fluorescent labeling of the hydrogelated cells, fluorescein O,O'-diacrylate (0.1% w / v) was incorporated into the hydrogelation buffer. Following a 10-minute incubation on a rotating rack at 37°C, the samples were rapidly frozen in supercool methanol at -80°C for 1-2 minutes and subsequently transferred for a 10-minute incubation at -80°C. After the freeze cycle, cell thawing was conducted at 30°C using a dry bath. Cells were then centrifuged at 6.8 g for 5 minutes, with the supernatant discarded and the cells resuspended in 1 mL of LB media. The cells were subjected to 1600 nm irradiation using a UV oven (UVP Crosslinker, CL3000, USA). Following UV crosslinking, samples underwent centrifugation at 6.8 G for 5 minutes. The supernatant was discarded, and the resultant hydrogelated cells were collected and subsequently washed with PBS for subsequent analysis.

[0121] Flow cytometry. In the flow cytometry analysis for the successful generation of hydrogelated bacteria, the hydrogelated bacterial pellet, obtained following the intracellular hydrogelation protocol detailed earlier, underwent a thorough washing process with PBS to eliminate extracellular fluorescein. The percentage of the hydrogelated cells in the treated cell population was determined using the FITC channel to detect fluorescein O’O-diacrylate that was incorporated within the synthetic hydrogel.

[0122] To assess metabolic activity7, hydrogelated bacteria, along with positive (wild-type) and negative control (killed WT bacteria), were stained with 5-Cyno-2,3-ditolyl tetrazolium chloride (CTC), a reductive chromogenic dye. Flow cytometry analysis was performed, and the percentage population of hydrogelated cells exhibiting metabolic activity was determined31KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2 using the PerCp channel of the CytoFLEX Flow cytometry system. This methodology allowed for a comprehensive evaluation of both the successful generation of hydrogelated bacteria and their metabolic activity in comparison to relevant controls.

[0123] Fluorescence activated cell sorting (FACS) and colony-forming unit (CFU) analysis. Hydrogelated bacteria were isolated / purified by detecting fluorescein O’O diacrylate that was integrated into the synthetic hydrogel using the FITC fluorescence channel. The sorting criteria were established by referencing wild-type, non-fluorescent bacteria. Roughly one million cells were sorted based on their fluorescence. Following this, a CFU (colony-forming unit) assay was conducted with the sorted and unsorted groups of one million bacteria each, to assess the non-proliferative ability7of the hydrogelated bacteria.

[0124] Intracellular survival hydrogelated cell in macrophages. In order to determine the ability of hydrogelated cells to invade and survive within RAW 264.7 (macrophages), a gentamicin protection assay was performed as described previously

[0013] , The RAW 264.7 cells were seeded in DMEM media with 10% FBS. The plates were incubated at 37°C in 5% CO2 for 24 h in DMEM media without antibiotic. The RAW 264.7 cells were pulsed with S. aureus MN8, P. aeruginosa PA14. and K. pneumonia BIDMC 2A hydrogelated cells at a multiplicity of infection (MOI) of 50 at 37°C for 45 mins. The RAW 264.7 cells were washed three times with PBS and treated with 100 pg / ml gentamicin for 1.5 h to kill extracellular bacteria. The infected RAW 264.7 cells were washed three times to remove any extracellular bacteria. After washes the cells lysed with 0.5 ml of 0.1% Triton-X in PBS. Total DNA was extracted using Qiagen Total DNA extraction kit. DNA samples were eluted using 100 pL Elution buffer. DNA was kept frozen at -20°C until PCR amplification.

[0125] Real-time qPCR for quantification of hydrogelated cells. The wild-type strains of S. aureus MN8, P. aeruginosa PA14, and K. pneumonia BIDMC 2A were utilized to establish a correlation between amplification threshold cycle (Ct) and colony forming units per volume (CFU / mL). A 12-14 hour-old bacterial culture was inoculated in LB broth for CFU assessment, with its optical density (OD600) adjusted to 0.1 OD. Serial dilutions of 1 ml aliquots were spread-plated for CFU quantification. Concurrently, DNA isolation form a 1 ml aliquot was performed using Qiagen Genomic DNA extraction kit. Tecan microplate reader assessed DNA purity and yield. Purity was gauged via the A260 / 280 absorbance ratio at 260 nm and 280 nm. The study established links between QD0600, CFU / mL and DNA concentration (ng / mL).32KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2

[0126] To construct the standard curve, DNA concentrations were observed from the 0. 1 OD bacterial culture of each strain. Subsequently, 10-fold dilutions were executed to create standard curves for strain-specific genes. The reaction mixture, utilizing PowerUPTM SYBR Green Master Mix (Thermo Fisher Scientific), was prepared in a total of 10 pL volume. The mixture included 0.5 pL of each primer (final concentration 0.5 pM) and 5.0 pL of PowerUPTM SYBR Green Master Mix. For every bacterial strain, six serial DNA dilutions were generated from the 0. 1 OD600 cultures of all three bacteria, serving as the foundation for the standard graph. Real-time PCR runs were conducted in triplicates, with Ct values graphed against the logarithm of DNA concentration. This approach facilitated the determination of the standard curve equation, subsequently applicable for all ensuring calculations.

[0127] Immunofluorescence confocal microscopy. RAW 264.7 cells (3 x 105cells) were seeded on coverslips placed in 4-chamber slide and incubated for 24 h in DMEM media with 10% FBS. RAW 264.7 cells were infected with hydrogelated cells and wild-ty pe cells of the 3 bacterial strains described above at an MOI of 50 at 37°C for 1 h. The infected RAW 264.7 cells were washed three times with PBS and treated with 100 pg / ml gentamicin for 1.5 h to kill extracellular bacteria. The infected RAW 264.7 cells were again washed to remove any extracellular bacteria. Then, the RAW 264.7 cells were washed with PBS and stained with Phalloidin Alexa flour 555.

[0128] Quantification of cytokine levels. The quantification of proin fl ammatoiy cytokine levels, including TNF-alpha. IL-17, and IFN-y, was performed in the supernatants of murine macrophage cell lines (RAW264.7) inoculated with both wild-type and hydrogelated variants of three pathogenic strains. Pro-inflammatory cytokines, TNF-alpha (TNF-alpha Mouse ELISA Kit, catalog no. BMS607; Thermo Fisher Scientific), IFN-y (IFN gamma Mouse ELISA Kit. catalog no. BMS606; Thermo Fisher Scientific), and IL- 17 (IL- 17 Mouse ELISA Kit, catalog no. BMS6001; Thermo Fisher Scientific), were estimated in the cell culture supernatant following the manufacturer's protocols for the respective ELISA kits. Optical absorbance was measured at 450 nm using a microplate reader (Tecan, Infinite Ml 000 PRO), and protein concentrations were calculated using standard curves provided with the ELISA kits.

[0129] Quantification of reactive oxygen species (ROS). Intracellular levels of ROS were assessed using 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester33KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPC Client Reference No : UC 2025-422-2(CM-H2DCFDA; Invitrogen). Macrophages were seeded in DMEM media with 10% FBS. The plates were incubated at 37°C in 5% CO2 in DMEM media without antibiotic.Macrophages were pulsed with S. aureus MN8, P. aeruginosa PA14, and K. pneumonia BIDMC 2A wild-type and hydrogelated cells at a multiplicity of infection (MOI) of 50 at 37°C for 4 hrs. After infection, macrophages were washed to remove exrtacellular bacteria and then incubated with 4 pM CM-H2DCFDA in Hank's balanced salt solution for 30 minutes at 37°C. Following infection, macrophages were washed with HPBS and then trypsinzed , subjected to flow cytometer analysis using the CytoFLEX Flow cytometry.Example 2 - Hydrogelated Pathogens are Non-Replicating But Metabolically-Active Cells

[0130] Hydrogelated versions of six pathogenic strains ofF*. aeruginosa, S. aureus and K pneumonia were produced as previously described with minor modifications [1] (Table 1). The bacterial pathogens were infused with a chemically stable and nondegradable synthetic hydrogel characterized by low biological reactivity. The chosen hydrogel chemistry included poly(ethylene glycol) diacrylate monomer (PEG-DA; average Mn700) and 2-hydroxy-4'-(2- hydroxy ethoxy )-2-methylpropiophenone as the photoinitiator. Additionally, incorporated fluorescein O’O-diacrylate was incorporated in the bacterial pathogens as a fluorescent dye to assess the permeation of the hydrogel components into the bacteria and confirm the success of intracellular hydrogelation (Figure 1 A). Four distinct hydrogel densities were selected, ranging from 5% to 20% wt / vol (g / ml) for intracellular hydrogelation. The successful generation of hydrogelated pathogens was measured using flow cytometry of fluorescein O’O-diacrylate, which is incorporated into the intracellular hydrogel (Figure IB).

[0131] Hydrogelated cells exhibited green fluorescence intensity of the fluorescein diacrylate when compared to wild-type cells. In addition, lower hydrogel densities at 5% and 10% resulted in a bigger population of hydrogelated pathogens (Figures 1C and ID). Across all six bacterial strains, at the optimal gel densities, approximately 60-80% of the total population exhibited positive fluorescein signals (Figure ID), indicating the successful formation of an intracellular hydrogel.34KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2

[0132] Next, the metabolic activity’ of hydrogelated cells was assessed by staining them with 5-Cyno-2,3-ditolyl tetrazolium chloride (CTC), a reductive chromogenic dye widely used to determine the respiratory7activity7of bacteria [3], Flow cytometry results indicated that lower hydrogel densities (5% and 10%) resulted in a higher population of CTC-positive cells. Conversely, an increase in hydrogel density led to a reduction in the metabolic activity of hydrogelated cells (Figure ID). Specifically, for strains N. aureus MN 8 and A980101, CTC-positive cells were halved when the hydrogel density was elevated from 10% to 30%. Similarly, both strains of . aeruginosa exhibited a decline in metabolically active cells, averaging a drop from 60% to 30% when the hydrogel density7was increased from 5% to 30%. Ulis trend, demonstrating a negative impact of increased hydrogel density on the respiratory activity of hydrogelated celts, was consistently replicated in K. pneumoniae strains (Figure ID). These findings show that optimal gelation conditions can create hydrogelated pathogens with optimal metabolic activities while not replicating.

[0133] The non-replicative ability of the hydrogelated bacteria was also investigated using a Colony Forming Unit (CFU) assay (Figure 2A). The hydrogelation protocol resulted in some cells that were not hydrogelated within the hydrogelated bacterial population. To isolate the hydrogelated population from wild-type bacteria, Fluorescence- Activated Cell Sorting (FACS) based on the intensity of the fluorescein diacrylate (Figure 2B) was used. Prior to FACS, the CFU count of the hydrogelated cell population was around three log folds lower than that of the non-hydrogelated controls. Following FACS flow cytometry, the CFU count of hydrogelated cells decreased to a non-detectable level (Figure 2B). The application of FACS flow cytometry7successfully purified the hydrogelated cell population. Despite being non-replicable, the hydrogelated cells remained active and intact after sorting. These results confirm the generation of hydrogelated pathogens that cannot divide due to the intracellular hydrogelation.Example 3 - Hydrogelated Pathogens were Internalized by Macrophages

[0134] Bacterial pathogens often encounter macrophages in vivo and engulfed and / or internalized in the process. Macrophage-pathogen interactions necessitate specific molecular35KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2 recognition and signaling events. Bacterial pathogens must possess pathogen-associated molecular patterns (PAMPs) that can be recognized by pattern recognition receptors (PRRs) on macrophage surfaces, triggering phagocytosis [4], This process, crucial for engulfing and internalizing pathogens, relies on the pathogens being viable or “alive” to elicit a response from macrophages. Leveraging this understanding of macrophage-bacteria interaction, the intracellular fate of hydrogelated bacteria within macrophages was investigated. Gentamicin protection assays were performed using RAW 264.7 macrophages at multiplicities of infection (MOI) 1 :50 for wild-type bacteria S. aureus MN8, P. aeruginosa PA 14. and K. pneumonia BIDMC 2A to investigate the ability7of these pathogens to invade the macrophages (Figure 3A).

[0135] CFU data showed that around 0.96±0. 144, 0.864±0.071. 0.405±0.206 percent of live S. aureus MN8, P. aeruginosa PA 14, and K. pneumonia BIDMC 2A were successfully internalized by the macrophages (Figure 3B). Notably, E. coli BL21, which is an extracellular bacterium, does not show detectable CFU, suggesting a lack of invasion or survival inside the macrophage (Figure 3B). Furthermore, confocal microscopy images demonstrate equal colocalization of both hydrogelated and wild-type bacteria (depicted in Green) within macrophages. Therefore, the intracellular hydrogelation had negligible effects on hydrogelated bacterial uptake by macrophages.

[0136] To better compare the uptake efficacy of hydrogelated bacteria, a qPCR-based method was used instead of the standard CFU assay due to the non-replicative nature of hydrogelated bacteria. The qPCR-based approach was used to measure the number of hydrogelated bacteria (S. aureus MN8, P. aeruginosa PA 14. and K. pneumonia BIDMC 2A) inside macrophages.

[0137] First, correlation curves were established between optical density7(OD@600nm), colony -forming units per milliliter (CFU / mL), and DNA concentration (ng / pL). An OD of 0.1 600 nm corresponded to 2.65 (±1.5) x 107CFU / mL for S. aureus MN8, 6.45 (±1.5) x107CFU / mL for P. aeruginosa PA 14. and 7.41 (±1.5) x 107CFU / mL for K. pneumonia BIDMC 2A. DNA concentrations at an OD of 0.1 were determined as 9.54 ng / pL (S. aureus MN8), 8.87 ng / pL (P. aeruginosa PA 14). and 9.76 ng / pL (K. pneumonia BIDMC 2A).Subsequent 10-fold dilutions of bacterial DNA were used to construct standard curves for amplifying the 16s RNA gene. Melt-curve analysis consistently showed a melting temperature Tmof 84.5°C for S. aureus MN8 (Figure 4A), 84°C for P. aeruginosa PAM36KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2(Figure 4B), and 87°C for A. pneumonia BIDMC 2A (Figure 4C). confirming single-product amplification. The resulting standard curves for Ct values against DNA concentration exhibited high linearity7(R2 > 0.99) for all three bacteria, with PCR efficiency close to 100% (slope: -2.4 for S. aureus, -2.6 for P. aeruginosa, and -2.7 for K. pneumonia) (Figures 4D- 4F).

[0138] After establishing the measurement protocol, the internalization of hydrogelated bacteria into the macrophages was investigated. Minor but insignificant differences was observed in the intra-macrophage count of wild-type and hydrogelated P. aeruginosa PA14 (Figure 4G). Similar to PA, no major difference was observed in intra-macrophage count of WT and hydrogelated S. aureus MN8 and K. pneumonia BIDMC 2A (Figure 4G). These results indicate that intracellular hydrogelation did not affect the invasion or uptake of these bacterial pathogens by the macrophage.Example 4 - Hydrogelated Bacteria Caused the Production of Pro-Inflammatory Cytokines and Reactive Oxygen Species (ROS) in Macrophages

[0139] Previous studies have established that macrophages infected with S', aureus. P. aeruginosa, and K. pneumonia secrete proinflammatory cytokines as part of their response to control infections [5-8], Here, hydrogelated cells were analyzed for the ability7to induce cytokine production; the cytokine levels were compared to cytokine levels observed in their wild-type counterparts. The production of proinflammatory cytokines, specifically TNFa, IL- 17, and IFN-y, were evaluated in murine macrophage cell lines (RAW264.7) infected with both wild-type and hydrogelated cells of all three pathogenic strains using ELISA assays (Figures 5D-5F).

[0140] Upon treating RAW 264.7 cells with P. aeruginosa PAM (MOI 1 : 50) for 4 hours, hydrogelated cell infection resulted in a 1.8-fold higher TNF-alpha secretion compared to wild-type PA14-infected cells (Figure 5D). Conversely, both wild-type and hydrogelated PA14-infected macrophages exhibited similar IL-17 levels (Figure 5E). Similarly, a significant two-fold higher IFN-y level was observed in macrophages infected with hydrogelated PAM compared to those infected with wild-ty pe PAM (Figure 5F). hydrogelated . aureus MN8 also induced higher levels of TNFa than their wild-type counterparts (Figure 5D). IL- 17 and IFN-y levels were similar in both hydrogelated and wildtype SA-infected macrophages (Figure 5E-F). For K. pneumonia, hydrogelated cells induced37KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2 nearly double the level of TNFot compared to wild-ty pe, indicating a heightened immune response (Figure 5D). However, no significant differences were observed in IL- 17 and IFN-y levels between hydrogelated and wild-type-infected macrophages (Figures 5E-5F). These findings underscore the distinctive cytokine profiles induced by hydrogelated cells compared to their wild-type counterparts across different bacterial strains. The heightened TNFa production by macrophages infected with hydrogelated cells, despite similar IL- 17 and IFN-y levels to wild-type-infected macrophages, may result from selective activation of TNFa- related pathways or unique interactions between hydrogelated bacteria and macrophages.

[0141] The capacity of both wild-type and hydrogelated cells to elicit reactive oxygen species (ROS) as part of the immune response by macrophages was also assessed. Prior research has established that immune cells, particularly macrophages, generate ROS to counter infections caused by pathogenic strains such as P. aeruginosa, S. aureus, and K. pneumonia [5, 7, 9], ROS levels produced by macrophages in response to hydrogelated bacteria were evaluated using 2',7'-dichlorofluorescein (DCF), which forms a fluorescent compound (2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA)) when reacting with ROS. H2DCFDA (green) fluorescence intensity increased from the background level (Figure 6B. uninfected cells) when macrophages were infected with either wild-type or hydrogelated cells. No statistically significant difference in ROS levels was observed in macrophages infected with hydrogelated S. aureus and P. aeruginosa (Figure 6), relative to their wild-type counterparts. However, macrophages infected with hydrogelated K. pneumonia exhibited a more pronounced release of ROS than wild-type bacteria (Figure 6). These findings underscore the distinct ROS responses elicited by hydrogelated cells, particularly in the context of K. pneumonia infection.Discussion Related to Examples 1-4

[0142] The research successfully extended the intracellular hydrogelation technique from lab-based E. coli strains to pathogenic bacteria, specifically S. aureus, K. pneumoniae, and P. aeruginosa, demonstrating its potential for biomedical applications. Intracellular hydrogelation involves the infusion and polymerization of gel monomers within bacterial cells, creating hydrogelated cells that retain metabolic activity but are non-replicating. This feature is significant because it allows the pathogens to maintain their invasive capabilities without the risk of proliferation, making them ideal candidates for further development as drug delivery and immunotherapy devices

[0010] ,38KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2

[0143] Flow cytometry analysis revealed significant insights into the generation of hydrogelated pathogens from six bacterial strains. The integration of fluorescein diacrylate within the hydrogel matrix allowed for the quantitative assessment of hydrogelation efficiency, indicated by the fluorescence intensity (Figure IB). This analysis demonstrated vary ing degrees of hydrogel incorporation across the strains, with S', aureus strains (MN8 and A980101) and P. aeruginosa strains (PA14 and EmvKY2) showing anotably higher percentage of hydrogelation compared to the Klebsiella pneumoniae strains (MRSN 28880 and BIDMC 2A) (Figure 1C). This observation may be due to the presence of capsule polysaccharide [2], potentially reducing the uptake of the hydrogel components.

[0144] Moreover, the metabolic activity of these pathogens, as assessed by staining with 5- Cyno-2,3-ditolyl tetrazolium chloride (CTC), a reductive chromogenic dye. varied with hydrogel density. Lower hydrogel densities (5% and 10%) were more conducive to maintaining higher levels of metabolic activity7, particularly in S. aureus and P. aeruginosa strains (Figure ID). This trend was consistent across all strains, with a marked decrease in metabolic activity7observed at higher hydrogel densities (20% and 30%) (Figure ID). These findings highlight the delicate balance between hydrogel density and the preservation of vital cellular functions, suggesting the potential impact of molecular crowding on cellular metabolism [1],

[0145] These engineered pathogens were effectively taken up by macrophages, similar to their wild-type counterparts, suggesting that the intracellular hydrogelation does not impede their recognition and uptake by host immune cells. Importantly, the interaction of hydrogelated pathogens with macrophages did not significantly alter the immune response when compared to the response elicited by wild-ty pe pathogens, indicating that the hydrogelated bacteria maintain crucial pathogenic features that are recognized by the host's immune system. Additionally, the engineered pathogens induced comparable levels of pro- inflammatory cytokines and ROS in macrophages as their wild-type counterparts, reinforcing their potential utility in biomedical applications where controlled immune stimulation is desired (Figures 5A-5F and 6).

[0146] Although hydrogelated pathogens mentioned above are derived from well- characterized bacteria, the specific processes governing the termination of cell division remain unclear. Previous studies propose that cell division is governed by replication activities as well as processes that do not involve replication [11, 12], The presence of the39KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2 hydrogel matrix is thought to inhibit cell division by either interfering with DNA replication, physically constraining cell growth, or potentially a combination of both factors.

[0147] The experimental results validate the versatility of intracellular hydrogelation for creating non-replicative, metabolically active pathogens. The results also open new avenues for developing synthetic bacteria in biomedical applications. The ability to manipulate pathogenic bacteria safely without compromising their native interactions with the host immune system offers a promising strategy for developing novel therapeutic agents.Example 5 - Materials and Methods for Example 6-9

[0148] Hydrogelated cells as vaccines. In certain Examples discussed below, untreated, heat-killed bacteria was used as a benchmark of classical inactivation method. In certain Examples, hydrogelated bacteria (non-replicating but metabolically active) and over- hydrogelated bacteria (as a metabolically-dead control) were also used. In certain Examples, at least 6 replicates were used for each condition.

[0149] Fluorescently labeled hydrogelated bacteria. Hydrogelated bacteria were incubated with the Syto24 fluorescent dye, which can penetrate the bacterial membrane and bind to the nucleic acids inside the bacteria. To monitor the internalization of the hydrogelated bacteria by dendritic cells (DCs; a type of antigen-presenting cell (APC)) were seeded in 24-well plates. The labeled hydrogelated bacteria to the wells at a predetermined multiplicity of infection (MOI, 10:1, 1 : 1, 1: 10 = bacteria:host cells). The co-cultures were incubated for 15 minutes to 24 hours. Next, the extracellular bacteria were removed by doing 3 washes with PBS, followed by gentamicin treatment, and then another 3 washes. At each time point, the cells were stained with a fluorescent antibody that recognizes a surface marker of the APCs, such as CD86, CD80, and CDl lc for DCs. The nuclei of the mammalian cells were also stained with DAPI, a blue fluorescent dye that binds to DNA.

[0150] Mouse model. C57BL / 6 LysM-EGFP mice were used and blood samples and cells were obtained from mice treated with hydrogelated cells. In each experiment, the mice were treated with saline, heat-killed bacteria, hydrogelated bacteria (non-replicating but metabolically active), or over-hydrogelated bacteria (non-replicating and metabolically dead). Where appropriate, heat-killed bacteria served as a control of classical bacteria inactivation method (Figures 7A-7C); and over-hydrogelated bacteria served as a control to examine the need / importance of metabolic activities in hydrogelated cell vaccine.40KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No.: UC 2025-422-2Example 6 - Hydrogelated Cells Provided Protection against P. aeruginosa in Mice

[0151] Hydrogelated P. aeruginosa PAM were tested in mice (C57BL / 6 LysM-EGFP). The immunization and challenge protocol were as shown in Figure 7A. At Day 0, mice were administered hydrogelated PA or heat-killed bacteria (same number of bacteria) intradermally (as “vaccines”). One mouse died due to heat-killed bacteria (Figure 7B). All mice were healthy with hydrogelated PAM. Figure 7C shows mouse survival after challenge with wild-type PAM. Hydrogelated PAM protected >80% of mice (5 out of 6) from PAM infection. In contrast, the control group (i.e., nonvaccinated or vaccinated with heat-killed bacteria) had 100% mortality within 3-days of infection (Figure 7B). Analysis of blood samples and bacterial load in organs suggests that hydrogelated PA generated systemic immunity against the pathogens.Example 7 - Hydrogelated Cells Activate Dendritic Cells

[0152] Hydrogelated bacteria are expected to interact with immune cells like their wild- type / unhydrogelated counterparts to elicit proper immune response. Enhancing the uptake of hydrogelated bacteria by antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages, could improve their immunogenicity and efficacy as vaccines. In this Example, the uptake efficiency of hydrogelated bacteria by dendritic cells DC2.4 was analyzed using fluorescently-labeled hydrogelated bacteria. The amount of surface molecules that are upregulated in DCs, e.g., CD86, was measured. As shown in Figure 8, dendritic cells DC2.4 that were exposed to hydrogelated PAM were activated by hydrogelated PAM, as evidenced by higher levels of CD86.Example 8 - Hydrogelated Cells and Host Immunity in Mice

[0153] This Example relates to characterizing the immune response in mice treated with hydrogelated cells. The dorsal wound was created on mice (n=3 for each condition) using a standardized protocol. At Day 0, One mice group was vaccinated with hydrogelated PAM while non-vaccinated mice were injected with saline. At Day 28, both vaccinated and nonvaccinated mice were challenged with 5xl06of wild-type PA , and then monitored for survival and immune response. Blood samples were collected at 14, 28, 35, 42, and 49 days post- vaccination using the retroorbital method. Levels of IgGl and IgG2 antibodies in the serum by ELISA. As shown in Figures 9B-9C, both antibody subclasses were significantly increased after vaccination compared to the control group across all days. The data suggests41KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPC Client Reference No.: UC 2025-422-2 that hydrogelated bacteria can stimulate both Thl and Th2 responses in mice and induce protective immunity against bacterial infections.|0154| The mice where then sacrificed and the liver, kidney, lungs, and spleen were isolated using sterile necropsy. The organs were then homogenized and qPCR was performed using primers specific to PA 14 to estimate the bacterial load. Positive controls were nonvaccinated mice. The results showed minimal or no bacteria in the organs of vaccinated mice (Figure 9A). In non-vaccinated mice, the bacterial load is high in the lungs and liver, as expected because P. aeruginosa primarily infects the lungs. A basal amount of bacteria is detected, which could be due to the clearance of the bacteria. This preliminary result suggests that hydrogelated bacteria can confer protection against lethal bacterial challenge, and prevent sepsis and subsequent organ damage in mice.Example 9 - Hydrogelated Cells Result in Increased CD3+ T Cells in Mice

[0155] T cells and B cells can develop into memory cells that persist long after the initial infection and protect the host upon re-exposure to the same pathogen. To examine whether these immune cells engage in the protection induced by hydrogelated bacteria vaccines, the spleen of vaccinated mice were analyzed by flow cytometry using specific markers that identify different subsets of immune cells. The mice were vaccinated with hydrogelated P. aeruginosa. Immune cells were isolated from the spleen of vaccinated mice. The cells were stained for the T cell marker CD3 and analyzed by flow cytometry. As shown in Figure 10, hydrogelated P. aeruginosa produced a robust generation of live CD3+ T-cell in the spleen of vaccinated mice, which indicates that hydrogelated cells can induce T cell immunity.References

[0156] The following references relate to the Examples above.1. Contreras-Llano, L.E., Liu, Y.H., Henson, T., Meyer, C.C., Baghdasaryan, O., Khan, S., Lin, C.L., Wang, A., Hu, C.M.J. and Tan, C., 2023. Engineering cyborg bacteria through intracellular hydrogelation. Advanced Science, 10(9), p.2204175.2. Opoku-Temeng, C., Kobayashi, S.D. and DeLeo, F.R., 2019. Klebsiella pneumoniae capsule polysaccharide as a target for therapeutics and vaccines. Computational and structural biotechnology journal, 17, pp.1360-1366.42KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No.: UC 2025-422-23. Creach, V., Baudoux, A.C., Bertru, G. and Le Rouzic, B., 2003. Direct estimate of active bacteria: CTC use and limitations. Journal of microbiological methods, 52(1), pp.19-28.4. Li, D. and Wu, M., 2021. Pattern recognition receptors in health and diseases. Signal transduction and targeted therapy, 6(1), p.291.5. Hickman-Davis, J.M., O’Reilly, P., Davis, I.C., Peti-Peterdi, J., Davis, G., Young, K.R., Devlin, R.B. and Matalon, S., 2002. Killing of Klebsiella pneumoniae by human alveolar macrophages. American Journal of Physiology-Lung Cellular and Molecular Physiology, 282(5), pp.L944-L956.6. Ciszek-Lenda, M., Majka, G., Suski, M., Walczewska, M., Gorska, S., Golihska, E., Fedor, A., Gamian, A., Olszanecki, R., Strus, M. and Marcinkiewicz, J., 2023. Biofilm- forming strains of P. aeruginosa and S. aureus isolated from cystic fibrosis patients differently affect inflammatory phenotype of macrophages. Inflammation Research, 72(6), pp. 1275-1289.7. Pidwill, G.R., Gibson, J.F., Cole, J., Renshaw, S.A. and Foster, S.J., 2021. The role of macrophages in Staphylococcus aureus infection. Frontiers in immunology’, 11, p.620339.8. Liu, D., Chen, Z., Yuan, Y., Jing, H., Zou, J., Zhang, X., Zeng, X., Zhang, W., Zou, Q. and Zhang, J., 2020. Innate immune effectors play essential roles in acute respiratory infection caused by Klebsiella pneumoniae. Journal of Immunology Research, 2020.9. Cifani, N., Pompili, B., Anile, M., Patella, M., Diso, D., Venuta, F., Cimino, G., Quattrucci, S., Di Domenico, E.G., Ascenzioni, F. and Porto, P.D., 2013. Reactive- oxygen-species-mediated P. aeruginosa killing is functional in human cystic fibrosis macrophages. PloS one, 8(8), p.e71717.10. Le Gouellec, A., Chauchet, X., Laurin, D., Aspord, C., Verove, J., Wang, Y., Genestet, C., Trocme, C., Ahmadi, M., Martin, S. and Broisat, A., 2013. A safe bacterial microsyringe for in vivo antigen delivery and immunotherapy. Molecular Therapy, 21 (5), pp. 1076-1086.11. Alpers, K., Vatareck, E., Grobe, L., Miisken, M., Scharfe, M., Haussler, S. and Tomasch, J., 2023. Transcriptome dynamics of Pseudomonas aeruginosa during transition from overlapping to non-overlapping cell cycles. Msystems, 8(2), pp.eO 1130-22.43KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPC Client Reference No.: UC 2025-422-212. Monteiro, J.M., Fernandes, P.B., Vaz, F., Pereira, A.R., Tavares, A.C., Ferreira, M.T., Pereira, P.M., Veiga, H., Kuru, E., VanNieuwenlize, M.S. and Brun, Y.V., 2015. Cell shape dynamics during the staphylococcal cell cycle. Nature communications, 6(1), p.8055. 13. Qin, L., Sun, Y., Zhao,Y., Xu. J., Bi. K., 2017. In vitro model to estimate Edwardsiella tarda -macrophage interactions using RAW264.7 cells. Fish & Shellfish Immunology, 60: 177-184.

[0157] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.44KILPATRICK TOWNSEND 79785391 1

Claims

PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-2WHAT IS CLAIMED IS:

1. A metabolically-active cell comprising a cross-linked hydrogel within the cell in sufficient amount to prevent cell replication, wherein the cell is a bacterial pathogen.

2. The metabolically-active cell of claim 1, wherein the bacterial pathogen comprises Pseudomonas , Klebsiella, or Staphylococcus.

3. The metabolically-active cell of claim 1 or 2. wherein the bacterial pathogen is Pseudomonas aeruginosa, Klebsiella pneumoniae, or Staphylococcus aureus.

4. The metabolically-active cell of any one of claims 1-3, wherein the cross-linked hydrogel comprises monosaccharide or polysaccharide monomer subunits, and wherein the cross-linked hydrogel is a homopolymer or co-polymer.

5. The metabolically-active cell of any one of claims 1-4, wherein the cross-linked hydrogel comprises substituted or unsubstituted polyethylene glycol) monomer subunits.

6. The metabolically-active cell of any one of claims 1-5, wherein the cross-linked hydrogel comprises poly(dimethyl siloxane) (PDMS), poly(ethylene oxide) (PEO), poly(vinyl alcohol) (PVA), poly (propylene fumarate) (PPF), alginate, guanosine mono phosphate (GMP), cyclodextrin (CD), fibrin, collagen, polypeptides, decellularized extracellular matrix, or nucleic acids.

7. The metabolically-active cell of any one of claims 1-6, wherein the cross-linked hydrogel is substituted.

8. The metabolically-active cell of any one of claims 1-7, wherein the metabolically-active cell is covalently linked to an adjuvant.45KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-29. The metabolically-active cell of any one of claims 1-8, wherein the cross-linked hydrogel has a density of 1-2% (w / w) in the cell.

10. The metabolically-active cell of any one of claims 1-9, further comprising at least one heterologous nucleic acid.1 1 . The metabolically-active cell of any one of claims 1 -10, wherein the heterologous nucleic acid encodes a protein.

12. The metabolically-active cell of claim 11, wherein the protein is an outer membrane protein derived from a bacterial pathogen.

13. The metabolically-active cell of any one of claims 1-12, wherein the cell is contacted with a heterologous cryoprotectant.

14. A vaccine comprising the metabolically-active cell of any one of claims 1-13 and a pharmaceutically-acceptable excipient and / or adjuvant.

15. The vaccine of claim 14, wherein at least 99% of cells in the vaccine comprise the cross-linked hydrogel.

16. A method of inducing an immune response in an animal, the method comprising administering the vaccine of claim 14 or 15 to the animal in a sufficient amount to induce an immune response.

17. The method of claim 16, wherein the animal is a bird or mammal.

18. The method of claim 16 or 17, wherein the animal is a human.

19. A method of making the vaccine of 14 or 15. the method comprising,(i) providing a plurality of bacterial pathogen cells;(ii) introducing hydrogel monomer subunits into the plurality of metabolically- active bacterial pathogen cells; and(iii) causing the polymerization inducer to initiate formation of cross-linked hydrogel in the plurality of bacterial pathogen cells thereby forming metabolically-active bacterial pathogen cells comprising the cross-linked hydrogel, wherein the cross-linked hydrogel is formed from the hydrogel monomer subunits.46KILPATRICK TOWNSEND 79785391 1PATENTAttorney Docket No. : 081906- 1513157-25601 OPCClient Reference No : UC 2025-422-220. The method of claim 19, further comprising introducing a polymerization inducer into the plurality of metabolically-active bacterial pathogen cells before, after, or simultaneously with the step of introducing hydrogel monomer subunits.

21. The method of claim 20, wherein the polymerization inducer is activated by light of a specific wavelength and the step of causing polymerization comprises exposing the plurality of metabolically-active bacterial pathogen cells to light of the specific wavelength.

22. The method of any one of claims 19-21, further comprising contacting the plurality of metabolically-active bacterial pathogen cells with a replication-specific toxin and / or antibiotics, thereby killing cells in the plurality capable of replicating.

23. The method of any one of claims 19-22, further comprising isolating metabolically-active bacterial pathogen cells comprising the cross-linked hydrogel from cells without the cross-linked hydrogel.

24. The method of any one of claims 19-23, wherein the introducing step further comprises introducing a marker into the plurality of metabolically-active bacterial pathogen cells, and wherein the isolating comprises isolating the metabolically-active bacterial pathogen comprising the cross-linked hydrogel by presence of the marker.

25. The method of any one of claims 19-24, further comprising contacting the cell with a heterologous cryoprotectant during the providing, introducing, and / or causing steps.47KILPATRICK TOWNSEND 79785391 1

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