EP4680272A1 - Compositions and methods for predicting immune responses to subunit vaccines using synthetic immune organoids - Google Patents

Abstract

The present disclosure relates to an ex vivo method of identifying highly immunogenic glycoconjugates. This method involves providing a plurality of murine B cell organoids; contacting each of the plurality of murine B cell organoids with a plurality of distinct glycoconjugate candidates, where each of the distinct glycoconjugate candidates comprises a distinct polysaccharide of interest and a carrier protein, and where the contacting is carried out to form a plurality of contacted B cell organoids; culturing the plurality of contacted B cell organoids under conditions effective to support B cell maturation, whereby said culturing produces a plurality of cultured B cell organoids; detecting presence or absence of one or more B cell biomarker(s) in each of the plurality of cultured B cell organoids, where the detecting presence of one or more B cell biomarker(s) indicates immunogenicity of the glycoconjugate candidate; and identifying highly immunogenic glycoconjugate candidates.

Description

COMPOSITIONS AND METHODS FOR PREDICTING IMMUNE RESPONSES TO SUBUNIT VACCINES USING SYNTHETIC IMMUNE ORGANOIDS

[0001] This application claims the priority benefit of U.S. Provisional Patent Application Serial No. 63/490,869, filed March 17, 2023, which is hereby incorporated by reference in its entirety.

[0002] This invention was made with government support under HDTRA1-20- 10004 awarded by Defense Threat Reduction Agency, R01GM137314, R01GM127578, 5R01AH32738-06, 1R21AH60136-01, and 5R01CA238745-03 awarded by National Institutes of Health and CBET-1605242 and CBET-1936823 awarded by National Science Foundation. The government has certain rights in this invention.

[0003] The Sequence Listing is being submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on March 18, 2024, is named 147402.009241. xml and is 19,677 bytes in size. No new matter is being introduced.

FIELD

[0004] The present disclosure relates to methods of identifying highly immunogenic glycoconjugates, identified highly immunogenic glycoconjugates, methods of identifying glycoconjugate vaccine candidates, methods of identifying scFvs specific for a polysaccharide of interest, murine B cell organoids, and methods of their making.

BACKGROUND

[0005] For decades, vaccines have been important pillars in preventative medicine, protecting against a wide array of disease-causing pathogens by inducing humoral and/or cellular immunity. Of the many possible candidate antigens for subunit vaccine development, carbohydrates are particularly appealing because of their ubiquitous presence on the surface of diverse pathogens such as bacteria, viruses, parasites, and even human cancers. In the case of pathogenic bacteria, high-molecular-weight polysaccharides in the form of capsular polysaccharides (CPS) and lipopolysaccharides (LPS) decorate the microbial exterior.

Unfortunately, when free CPS or LPS antigens are administered as vaccines, they typically stimulate T-cell -independent humoral immune responses, but such responses are relatively weak (Avci and Kasper, “How Bacterial Carbohydrates Influence the Adaptive Immune System,” Annu. Rev. Immunol. 28: 107-130 (2010)). These responses are characterized by a lack of IgM- to-IgG class switching in B cells, failure to induce a secondary antibody response after recall immunization, and no sustained T-cell memory (Guttormsen et al., “Cognate Stimulatory B-cell- T-cell Interactions are Critical for T-Cell Help Recruited by Glycoconjugate Vaccines,” Infect. Immun. 67(12):6375-6384 (1999)). T-cell-independent polysaccharide antigens can be readily converted into more potent immunogens by covalent conjugation to a CD4⁺ T-cell-dependent antigen such as an immunostimulatory protein carrier (Astronomo and Burton, “Carbohydrate Vaccines: Developing Sweet Solutions to Sticky Situations?,” Nat. Rev. DrugDiscov. 9(4):308- 324 (2010); Weintraub, A., “Immunology of Bacterial Polysaccharide Antigens,” Carbohydr. Res. 338(23):2539-2547 (2003); Lockhart S., “Conjugate Vaccines,” Expert Rev. Vaccines 2(5): 633-648 (2003); and Rappuoli R., “Glycoconjugate Vaccines: Principles and Mechanisms,” Sci. Transl. Med. 10(456):eaat4615 (2018)). Indeed, conjugate vaccines composed of CPS or LPS- based antigens chemically bound to the Clostridium tetani tetanus toxin (TT) or the Corynebacterium diphtheriae diphtheria toxin (DT) induce polysaccharide-specific IgM-to- IgG switching, memory B cell development, and long-lived T-cell memory (Rappuoli R., “Glycoconjugate Vaccines: Principles and Mechanisms,” Sci. Transl. Med. 10(456):eaat4615 (2018); Schneerson et al., “Preparation, Characterization, and Immunogenicity of Haemophilus influenzae Type b Polysaccharide-Protein Conjugates,” J. Exp Med. 152(2):361—376 (1980); and Beuvery et al., “Comparison of the Induction of Immunoglobulin M and G antibodies in Mice with Purified Pneumococcal Type 3 and Meningococcal Group C Polysaccharides and their Protein Conjugates,” Infect. Immun. 37(1): 15-22 (1982)). Such conjugates have proven to be a highly efficacious and safe strategy for protecting against virulent pathogens, including Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumonia. This effective glycoconjugate vaccine format is used in currently approved pneumococcal, meningococcal, and Haemophilus influenzae type B vaccines, and several recent reviews discuss the mechanism of such glycoconjugate vaccines in detail (Rappuoli R., “Glycoconjugate Vaccines: Principles and Mechanisms,” Sci. Transl. Med. 10(456):eaat4615 (2018) and Rappuoli et al., “On the Mechanisms of Conjugate Vaccines,” Proc. Natl. Acad. Sci. U. S. A. 116(1): 14-16 (2019)). The robustness of B cell activation induced by a polysaccharide-carrier conjugate depends on a variety of factors including carrier immunogenicity, location of glycan attachment, and glycan composition and size, all of which are known to modulate the immune response as characterized by serum titer strength and elicitation of protective antibodies. Such antibodies are produced when B cells bind the antigen and initiate the formation of tightly regulated transient germinal center (GC) structures in cooperation with other immune and stromal cells, followed by immunoglobulin isotype class-switching (Victora and Nussenzweig, “Germinal Centers,” Annu. Rev. Immunol. 30:429-457 (2012); Mesin et al., “Germinal Center B Cell Dynamics,” Immunity 45(3):471-482 (2016); Cyster and Allen, “B Cell Responses: Cell Interaction Dynamics and Decisions,” Cell 177 (3): 524-540 (2019); and De Silva and Klein, “Dynamics of B Cells in Germinal Centres,” Nat. Rev. Immunol. 15(3): 137-148 (2015)). Within the GC, B cell programming includes somatic hypermutation (SHM) and multiple rounds of selection to rapidly increase binding affinity and promote differentiation into antibody-secreting cells and memory cells. Eliciting GCs and their ensuing high-affinity antibodies, as well as understanding GC immunobiology and the interactions of conjugate vaccine components with naive B cells as they undergo maturation, is central to the design of better polysaccharide antigens and carriers. However, due to the complexity of GC formation with its myriad of cellular, chemical, and physical signals, animal models are the standard means of eliciting antibody responses to empirically evaluate vaccine immunogenicity and efficacy. While insightful, animal immunization is cost-prohibitive, time-consuming, and has a relatively low throughput. Moreover, these issues are at odds with newer preparation methods based on glycoengineered bacteria or their cell-free extracts that enable facile biosynthesis of conjugate vaccine candidates (Dow et al., “Improving Protein Glycan Coupling Technology (PGCT) for Glycoconjugate Vaccine Production,” Expert Rev. Vaccines 19(6):507-527 (2020); Kay et al., Recent Advances in the Production of Recombinant Glycoconjugate Vaccines,” NPJ. Vaccines 1 :4: 16 (2019); Jaroentomeechai et al., “Cell-Free Synthetic Glycobiology: Designing and Engineering Glycomolecules Outside of Living Cells,” Front Chem. 29:8:645 (2020); and Kightlinger et al., “Synthetic Glycobiology: Parts, Systems, and Applications,” ACS Synth. Biol. 9(7): 1534-1562 (2020)) with greater control over critical design parameters and the potential for generating large libraries (Li et al., “Shotgun Scanning Glycomutagenesis: A Simple and Efficient Strategy for Constructing and Characterizing Neoglycoproteins,” Proc. Natl. Acad. Sci. U. S. A.

118(39):e2107440118 (2021)). Consequently, there is a need for new tools that allow rapid screening of potentially large libraries of different glycoconjugate configurations to identify immunologically superior designs that can be down-selected for resource-intensive animal studies.

[0006] The present disclosure is directed to overcoming these and other deficiencies in the art.

SUMMARY

[0007] One aspect of the present disclosure is directed to a murine B cell organoid comprising: murine B cells; a CD40 ligand selected from the group consisting of soluble CD40 ligand (sCD40L), cells expressing CD40 ligand, scaffolds presenting CD40 ligand, beads presenting CD40 ligand, and combinations thereof; and a hydrogel, where the murine B cells and the CD40 ligand are encapsulated in the hydrogel and where the hydrogel comprises cross-linked multi-arm 9olyethylene glycol (PEG) macromers.

[0008] Another aspect of the present disclosure relates to a method of making a murine B cell organoid. This method involves: providing a population of B cells; providing a CD40 ligand selected from the group consisting of soluble CD40 ligand (sCD40L), cells expressing CD40 ligand, scaffolds presenting CD40 ligand, beads presenting CD40 ligand, and combinations thereof; providing a crosslinker solution comprising a degradable and/or a non-degradable crosslinker; suspending the population of B cells and the CD40 ligand in the crosslinker solution to form a cell suspension; providing a functionalized macromer solution comprising a functionalized polyethylene glycol macromer; combining the cell suspension and the functionalized macromer solution to form a hydrogel solution; and curing the hydrogel solution to form a murine B cell organoid comprising encapsulated murine B cells.

[0009] Another aspect of the present disclosure relates to an ex vivo method of identifying highly immunogenic glycoconjugates. This method involves providing a first plurality of murine B cell organoids; contacting each of the first plurality of murine B cell organoids with a plurality of distinct glycoconjugate candidates, where each of the distinct glycoconjugate candidates comprises a distinct polysaccharide of interest and a carrier protein, and where the contacting is carried out to form a first plurality of contacted B cell organoids; culturing the first plurality of contacted B cell organoids under conditions effective to support B cell maturation, whereby said culturing produces a first plurality of cultured B cell organoids; detecting presence or absence of one or more B cell biomarker(s) in each of the first plurality of cultured B cell organoids, where the detecting presence of one or more B cell biomarker(s) indicates immunogenicity of the glycoconjugate candidate; and identifying highly immunogenic glycoconjugate candidates based on a greater presence of B cell biomarker(s) in one or more of said cultured B cell organoids relative to others of said cultured B cell organoids.

[0010] Also disclosed is a highly immunogenic glycoconjugate candidate identified by the ex vivo method of identifying highly immunogenic glycoconjugates disclosed herein.

[0011] Also disclosed is a plurality of distinct highly immunogenic glycoconjugate candidates identified by the ex vivo method of identifying highly immunogenic glycoconjugates disclosed herein.

[0012] Another aspect of the disclosure is directed to a method of identifying glycoconjugate vaccine candidates. This method involves providing a plurality of mice; immunizing each of the plurality of mice with distinct highly immunogenic glycoconjugate candidates of the plurality of distinct highly immunogenic glycoconjugate candidates of the present disclosure; evaluating an immune response of each of the plurality of mice to each of the highly immunogenic glycoconjugate candidates; and identifying glycoconjugate vaccine candidates based on a greater presence of antibodies specific for a polysaccharide of interest in the sera of one or more immunized mice relative to others of said immunized mice.

[0013] Another aspect of the present disclosure relates to a method of identifying scFvs specific for a polysaccharide of interest. This method involves providing a second plurality of murine B cell organoids; contacting each of the second plurality of murine B cell organoids with the plurality of distinct highly immunogenic glycoconjugate candidates according to the present disclosure, where the contacting is carried out to form a second plurality of contacted B cell organoids; culturing the second plurality of contacted B cell organoids under conditions effective to support B cell maturation, whereby said culturing produces a second plurality of cultured B cell organoids; enzymatically digesting the second plurality of cultured B cell organoids; isolating the cultured B cells to produce a plurality of isolated B cells; sequencing the plurality of isolated B cells to identify nucleic acid sequences encoding heavy chain variable region (VH) and light chain variable region (VL) genes; randomly joining VH and VL genes with a nucleic acid sequence encoding a glycine-serine linker to generate a library of candidate single chain variable fragments (scFvs); cloning each of the library of candidate scFvs into a yeast cell surface expression vector; transforming a plurality of yeast cells with the library of yeast cell surface expression vectors to produce a plurality of transformed yeast cells; expressing the library of yeast expression vectors in the plurality of transformed yeast cells to produce a library of scFv- expressing yeast cells, whereby the candidate scFvs are displayed on the surface of the yeast cells; detecting binding of a fluorescently labelled glycoconjugate to each of the library of scFv- expressing yeast cells, where the fluorescently labelled glycoconjugate comprises the polysaccharide of interest and a carrier protein that is different than the carrier protein of the plurality of distinct highly immunogenic glycoconjugate candidates; and identifying scFvs specific for the polysaccharide of interest based on said detecting.

[0014] The Examples of the present disclosure demonstrate the engineering and characterization of murine B cell follicle organoids to systematically understand the impact of glycoconjugate vaccine candidates on B cell maturation and signaling. Since polysaccharide antigens and carriers exhibit differing degrees of immunogenicity in vivo as measured by the magnitude and protective efficacy of the elicited antibodies, it was hypothesized that organoids potentiated by different antigens would also exhibit differences in measurable outputs. To test this hypothesis, an experimental framework was developed, in which glycoconjugate vaccine candidates derived from glycoengineered Escherichia coli cells were immunologically evaluated in vivo using mice and ex vivo using recently reported hydrogel-based immune organoids (Beguelin et al., “EZH2 Enables Germinal Centre Formation through Epigenetic Silencing of CDKN1 A and an Rb-E2F1 Feedback Loop,” Nat. Commun. 8(1): 877 (2017) and Purwada et al., “Ex vivo Synthetic Immune Tissues with T Cell Signals for Differentiating Antigen-Specific, High Affinity Germinal Center B cells,” Biomaterials 198:27-36 (2019), which are hereby incorporated by reference in their entirety). Importantly, the ex vivo GC responses were observed to (i) depend on the relative immunogenicity of the carrier protein, (ii) increase further by the addition of an O-polysaccharide (O-PS) antigen, and (iii) correlate with the humoral responses in vivo. In a more application-oriented goal, the occurrence of these mechanisms in immune organoids was exploited for identifying antigen-specific antibodies. Specifically, antibody repertoires of GC-like B cells derived from antigen-exposed organoids were screened using yeast surface display (YSD) and high-affinity antibodies against both carrier and O-PS antigens were identified. By sidestepping animal immunization, this immune organoid-based approach enabled antibody discovery on a time scale that was significantly shorter than for conventional immunization-based workflows.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIGs. 1 A-1B show the experimental framework for profiling GC-like B cell responses to glycoconjugate vaccine candidates. FIG. 1 A is schematic of strategies for preparing designer glycoconjugate vaccine candidates (left) and evaluating their immunogenicity in mice and in synthetic immune organoids (right). Glycoengineered bacteria enable the assembly of a single O-PS repeat unit (a tetrasaccharide structure in the case of F. tularensis Schu S4) on a undecaprenol lipid carrier in the cytoplasmic membrane, which is subsequently flipped into the periplasm and polymerized to form variable-length O-PS antigens by the endogenous Wzx flippase and Wzy O-antigen polymerase Wzy, respectively. In the presence of C. jejuni PglB (C/PglB), the lipid-linked O-PS is site-specifically transferred to asparagine residues in recombinant carrier proteins bearing a DQNAT motif. Schematic created with BioRender.com. FIG. IB is an image showing immunoblot analysis of purified carrier proteins derived from E. coli CLM24 cells carrying a plasmid encoding either MBP^4xDQNAT or CRMi97^4xDQNAT along with plasmid pGAB2 encoding the FtO-PS biosynthetic pathway and with (+) or without (-) plasmid pMAFlO encoding C/PglB as indicated. Blots were probed with anti -FLAG antibody to detect acceptor proteins (green signal) and FBI 1 antibody to detect FtO-PS antigens (red signal).

Images depict an overlay of anti -FLAG and FBI 1 blots. Arrows denote aglycosylated (agly) and multiply glycosylated (gly) forms of MBP^4xDQNAT and CRMi97^4xDQNAT Molecular weight (Mw) markers are indicated on the left. Results are representative of three biological replicates.

[0016] FIGs. 2A-2C demonstrate that glycoconjugates differentially boost antigenspecific IgG titers in vivo. FIG. 2A is a schematic of the prime-boost immunization schedule. Mice received an initial injection on day 0 (DO) and identically formulated booster injections on days 21 and 42. Blood was drawn on days 49 and 63. FIG. 2B is a graph showing FtLPS- specific IgG titers in day 63 serum of individual mice (black dots) and mean titers of each group (red lines) as determined by ELISA with FtLPS as immobilized antigen. Groups of three BALB/c mice were immunized s.c. with 100 pL of PBS alone or PBS containing 10 pg of glycoconjugate (MBP-F/O-PS or CR.M197-F/O-PS) adjuvanted with IF A or 10 pg of aglycosylated carrier protein (MBP or CRM197) adjuvanted with IFA. Mice were boosted on days 21 and 42 with the same doses. ForFtLPS-specific IgG titers at day 49, see FIGs. 8A-8C . FIG. 2C shows bar graphs of carrier protein-specific serum IgG titers in day 63 serum of individual mice (black dots) determined as in FIG. 2A but with MBP (left panel) and CRM197 (right panel) as immobilized antigens. For F/LPS-specific IgG titers at day 49, see FIGs. 8A- 8C. Significant differences were determined via one-way ANOVA with Tukey’s posthoc test (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, not significant).

[0017] FIGs. 3 A-3E demonstrate that glycoconjugates shape the GC-like B cell subpopulations in synthetic organoids. FIG. 3 A is a schematic of ex vivo lymphoid immune organoids. B cells are isolated from spleens of C57BL/6 mice and encapsulated with CD40L- presenting fibroblasts in protease-degradable PEG-4MAL hydrogels functionalized with bioadhesive REDV peptide. Schematic created with BioRender.com. FIGs. 3B-3E are graphs showing quantitative flow cytometric analysis of CD19⁺ GL7⁺ GC-like B cells (FIG. 3B), CD86⁺ GC-like B cells (FIG. 3C), ratio of CXCR4^loCD86^hi (LZ) and CXCR4^hiCD86¹⁰ (DZ) GC-like B cells (FIG. 3D), and CD138⁺ plasmablasts (FIG. 3E). Organoids were exposed to either PBS or PBS containing 10 pg of glycoconjugate (MBP-F/O-PS or CR.M197-F/O-PS) or 10 pg of aglycosylated carrier protein (MBP or CRM197). Data represent the mean ± standard error of the mean (SEM). See FIG. 9 and FIG. 10 for all flow cytometric gating strategies used to identify GC-like B cell subpopulations. Significant differences were determined via one-way ANOVA with Tukey’s posthoc test (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, not significant; n = 24 in FIG. 3B; n = 6 in FIG. 3C and FIG. 3D; n = 8 in FIG. 3E).

[0018] FIGs. 4A-4D demonstrate that glycoconjugates shape BCR clustering and signaling of GC-like B cells in synthetic organoids. FIG. 4A is a graph showing quantitative flow cytometric analysis of CD19⁺ GL7⁺ GC-like B cells for expression of IgM after 4 days of in vitro culture. FIG. 4B shows single-cell images of CD19⁺ B cells with IgM BCR puncta. Staining of nuclei was performed with 4',6-diamidino-2-phenylindole (DAPI). Scale bars are 5 pm. Shown at right is the number of IgM BCR puncta per cell quantified from single cell images. FIGs. 4C-4D are graphs showing CD19⁺ GL7⁺ GC-like B cells for expression of phosphorylated BTK (pBTK) and phosphorylated NF-KB (pNF-xB) (FIG. 4C) and BLIMP-1, EZH2, and IRF4 (FIG. 4D) after 4 days of ex vivo culture. Synthetic immune organoids were exposed to either PBS or PBS containing the following: 10 pg of glycoconjugate (MBP-FtO-PS or CRMi97-FtO-PS) or 10 pg of aglycosylated carrier protein (MBP or CRM197). Data represent the mean ± standard error of the mean (SEM). Values are reported as geometric mean fluorescence intensity (MFI). Significant differences were determined via one-way ANOVA with Tukey’s posthoc test (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, not significant; n = 8). See FIG. 10 for flow cytometric gating strategies used to identify GC-like B cell responses.

[0019] FIGs. 5A-5B show BCR clustering on immunogen-exposed GC-like B cells and representative images of B cells in immune organoids exposed to PBS, PBS containing CRM197, or PBS containing CRMi97-FtO-PS after 4 days of culture. FIG. 5 A shows cell clusters in organoids. Scale bars are 10 pm. FIG. 5B shows single cell images of B cells showing the presence or absence of CRM197 and FtO-PS. Staining of nuclei was performed with 4', 6- diamidino-2-phenylindole (DAPI). Scale bars are 2 pm. Merge represents IgM, CRM197, FtO- PS, and DAPI.

[0020] FIGs. 6A-6D shows analysis of GC-like organoid B cell immunoglobulin repertoire. FIG. 6A is a table showing sequence convergence of VH repertoires as measured by Morisita’s overlap index. Libraries of VH genes were prepared from RNA of naive B cells from day 0 prior to organoid culture (naive) and after 4 days of organoid culture treated with PBS, CRM197 carrier protein, or CRMi97-FtO-PS glycoconjugate. FIG. 6B is a graph showing SHM analysis performed by sequencing the Sp immunoglobulin variable locus from organoid GC-like B cells. Data are the mean of three biological replicates. Statistical significance was determined by Welch’s two-sided t-test (*p < 0.05, **p < 0.01). FIG. 6C is a schematic of YSD strategy for antibody repertoire analysis of GC-like organoid B cells. Libraries of VH and VL genes from organoid B cells were assembled as single-chain Fv (scFv) antibodies in which VL genes were randomly paired to VH genes via a flexible GlySe linker. The resulting scFv libraries were cloned into plasmid pCT-CON that enables display of HA/c-Myc tagged scFv antibodies on the yeast cell surface. FIG. 6D shows flow cytometric analysis of antigen-specific scFv expression from organoid-derived YSD libraries constructed using the same RNA described in FIG. 6 A for day 0 naive B cells and day 4 B cells treated with either CRM197 carrier protein or CR.M197-F/O- PS glycoconjugate as indicated. For CRM197 binding (top row), yeast cells were preincubated with a nonspecific, unlabeled carrier protein, protein D (PD) from Haemophilus influenzae, and analyzed by flow cytometry after staining with fluorescently labeled CRM197 (y-axis) and anti-c- Myc-antibody (x-axis). For CR.M197-F/O-PS binding (bottom row), yeast cells were preincubated with unlabeled, aglycosylated CRM197, MBP, and PD carrier proteins and analyzed by flow cytometry after staining with fluorescently labeled MBP-F/O-PS (y-axis) and anti-c- Myc-antibody (x-axis). All proteins were incubated at a concentration of 300 nM, and each data point corresponds to a single yeast cell. Values in each quadrant are the percentage of cells in that quadrant; red dashed-line box denotes the quadrant of interest in which immunogen binding and full-length scFv expression are both high. Results are representative of three biological replicates.

[0021] FIGs. 7A-7C demonstrate immune organoid-enabled discovery of antigenspecific, monoclonal antibodies. FIG. 7A is a schematic of YSD strategy for the isolation of antigen-specific, monoclonal antibodies from GC-like organoid B cell antibody repertoires. Libraries of VH and VL genes from organoid B cells were assembled as scFv antibodies in the YSD plasmid pCT-CON, after which individual yeast cells expressing antigen-specific antibody clones were isolated by FACS. FIG. 7B shows graphs demonstrating antigen-binding activity and specificity for the top 3 clones, N36.1, N36.61, and N36.94, isolated from YSD library corresponding to GC-like B cells treated with CR i97-FtO-PS glycoconjugate as determined by quantitative ELISA using FtLPS (gray circles) or aglycosylated CRM197 (white circles) as immobilized antigen. Data are the average of three biological replicates and error bars reported as standard deviation. Inset boxes show the equilibrium dissociation constant, FD, and the coefficient of determination, R², determined for each clone using Prism 9 software. FIG. 7C shows alignment of VL and VH domains of anti-F/O-PS antibodies along with their putative germline sequences. The putative germline amino acid structure is shown at the top with row(s) below representing mutations from the germline antibody (dash = no mutation).

Complementarity determining region (CDR) 1, 2, and 3 in the variable light and heavy chains are colored blue, green, and orange, respectively, as designated by IMGT analyses.

[0022] FIG. 8A-8C demonstrate that glycoconjugates differentially boost antigenspecific IgG antibody titers in vivo. FIG. 8A is a schematic of the prime-boost immunization schedule. Mice received an initial injection on day 0 (DO) and identically formulated booster injections on days 21 and 42. Blood was drawn on days 49 and 63. FIG. 8B is a graph showing that F/LPS-specific IgG titers in day 49 serum of individual mice (black dots) and median titers of each group (red lines) measured by ELISA with FtLPS as immobilized antigen. Groups of three BALB/c mice were immunized s.c. with 100 pL of PBS alone or PBS containing 10 pg of glycoconjugate (MBP-FtO-PS or CRM197-FtO-PS) adjuvanted with IF A or 10 pg of aglycosylated carrier protein (MBP or CRM197) adjuvanted with IFA. Mice were boosted on days 21 and 42 with the same doses. FIG. 8C are graphs showing carrier protein-specific serum IgG titers in day 49 serum of individual mice (black dots) determined as in FIG. 8A but with MBP (left panel) and CRM197 (right panel) as immobilized antigens. Significant differences were determined via one-way ANOVA with Tukey’s post-hoc test (*p < 0.05, **p < 0.01; ns, not significant).

[0023] FIG. 9 shows the flow cytometry gating strategy to define cell populations. The gating strategy is outlined beginning with size selection of lymphocytes (top left) followed by singlets (top middle) and then live cells (top right). Within the live cell population, CD19⁺ (middle left) and CD138⁺ (middle right) populations were defined. From the CD19⁺ cell population, a GC-like cell population (GL7⁺) was identified, and from the GC-like cells, LZ-like cells (CD86⁺) were found.

[0024] FIG. 10 shows flow cytometric analysis of GC-like B cell responses in synthetic organoids. Flow cytometry histograms for expression specified markers (IgM, pBTK, pNF-xB, BLIMP-1, EZH2, and IRF4) within GL7⁺ cells (GC-like) with respect to PBS, MBP, MBP-FtO- PS, CRM197, and CRM197-FZO-PS. Unstained controls included in light gray.

[0025] FIG. 11 is a table showing GC-like organoid B cell immunoglobulin repertoire analysis. Sequence convergence of VH repertoires as measured by Morisita’s overlap index. Libraries of VH genes were prepared from RNA of naive B cells from day 0 prior to organoid culture (naive) and after four days of organoid culture treated with PBS, MBP carrier protein, or MBP-FtO-PS glycoconjugate.

[0026] FIGs. 12A- 2B show binding analysis of organoid-derived scFv antibodies. FIG. 12A are graphs showing large-scale antigen-binding analysis for 96 putative binders isolated from N35 library for binding to aglycosylated CRM197 (top panel) or N36 library for binding to CRM197-FtO-PS (bottom panel). ELISA with different immobilized antigens was performed to determine binding activity and specificity, with binding ratios determined by normalizing the binding values measured with immobilized CRM197 or FtLPS to those measured with immobilized PD or CRM197. Ratio data are representative of three biological replicates with positive hits having ratios >1 (dashed red line). Red bars denote clones that were arbitrarily chosen for quantitative ELISA. FIG. 12B shows quantitative ELISA for arbitrarily selected clones in FIG. 12 A. ELISA for N35 hits (top row) was performed using aglycosylated CRM197 (gray circles) or BSA (white circles) as immobilized antigen. ELISA for N36 hits (bottom two rows) was performed with FtLPS (gray circles) or aglycosylated CRM197 (white circles) as immobilized antigen. Data are average of three biological replicates and error bars are standard deviation.

[0027] FIGs. 13A-13B demonstrate immune organoid-enabled discovery of antigenspecific, monoclonal antibodies. FIG. 13A shows antigen-binding activity and specificity for the top 3 clones, N35.27, N35.33, and N35.52, isolated from YSD library corresponding to GC-like B cells treated with aglycosylated CRM197 carrier protein. Quantitative ELISA was performed using aglycosylated CRM197 (gray circles) or BSA (white circles) as immobilized antigen. Data are the average of three biological replicates and error bars are standard deviation. Inset boxes show the equilibrium dissociation constant, KD, and the coefficient of determination, R², determined for each clone using Prism 9 software. FIG. 13B shows the alignment of VL and VH domains of anti-CRMi97 antibodies along with their putative germline sequences. The putative germline amino acid structure is shown at the top with rows below representing mutations from the germline antibody (dash = no mutation). Complementarity determining region (CDR) 1, 2 and 3 in the variable light and heavy chains are colored blue, green, and orange, respectively, as designated by IMGT analyses.

[0028] FIG. 14 shows the flow cytometry gating strategy for YSD library screening. Forward scatter (FSC-A) and side scatter (SSC-A) plots were used to select EBY100 yeast cells. The gated cells were sequentially narrowed to single cells using FSC-A/FSC-H and SSC-A/SSC- H plots. Cells incubated with streptavidin Alexa Fluor 488 (AF488) and anti-rabbit Alexa Fluor 647 (AF647) were used to determine the unstained population (grey). Similarly, cells labeled with anti-c-Myc AF647 (red) or streptavidin AF488 (green) were used to determine the singlelabeled populations. The sorting gate was set along the diagonal for expression normalization and without any overlap with the single-labeled or unstained populations.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0029] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present application herein described for which they are suitable as would be understood by a person skilled in the art. [0030] Singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure.

[0031] The term “about” or “approximately” includes being within a statistically meaningful range of a value. Such a range can be within an order of magnitude, such as within 50%, or within 20%, or within 10%, or within 5% (or any amount or range within 5-50%) of a given value or range. The allowable variation encompassed by the term “about” or “approximately” may depend on the context.

[0032] The term “and/or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of’ or “one or more” of the listed items is used or present.

[0033] As will be understood by a person of ordinary skill in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof, as well as any value within a range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, and so on. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, and so on. As will also be understood by a person of ordinary skill in the art all language such as “up to,” “at least,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges or specific values therein as discussed above. Finally, as will be understood by a person of ordinary skill in the art, and as discussed above, a range includes each individual value.

[0034] In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “involving”, “having”, and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The term “consisting essentially of’, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and/or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and/or steps. In embodiments or claims where the term comprising (or the like) is used as the transition phrase, such embodiments can also be envisioned with replacement of the term “comprising” with the terms “consisting of’ or “consisting essentially of.” The methods, kits, systems, and/or compositions of the present disclosure can comprise, consist essentially of, or consist of, the components disclosed.

[0035] In embodiments comprising an “additional” or “second” component, the second component as used herein is different from the other components or first component. A “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.

[0036] The terms “nucleic acid” and “nucleotide” encompass both DNA and RNA unless specified otherwise.

[0037] The term “polypeptide,” “peptide”, or “protein” are used interchangeably and to refer to a polymer of amino acid residues. The terms encompass all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins and modified proteins, including without limitation, glycoproteins, as well as all other types of modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP- ribosylation, pegylation, biotinylation, etc.).

[0038] The terms “express” and “expression” mean allowing or causing the information in a DNA sequence to become produced, for example producing an RNA by activating the cellular functions involved in transcription of a DNA sequence.

[0039] As used herein, the “DNA constructs” of the disclosure are nucleic acid molecules containing a combination of two or more genetic elements not naturally occurring together.

Each DNA construct comprises a non-naturally occurring nucleotide sequence that can be in the form of linear DNA or circular DNA, i.e., placed within a vector.

[0040] As used herein, the term “glycan” refers to a complex carbohydrate molecule comprising sugar molecules linked together in a branched or linear form. The term “glycan” is inclusive of both oligosaccharides and polysaccharides, and includes both branched and unbranched polymers.

[0041] Certain terms employed in the specification, examples, and claims are collected herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, some embodiments of the methods and materials are now described.

[0042] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Murine B Cell Organoids

[0043] The rise of biomaterials in tissue engineering has afforded intriguing new opportunities to explore B cell activation and maturation. Moving beyond traditional stimulation and differentiation of B cells in vitro, immune tissues create a 3D cell culture architecture that more closely mimics native conditions by encapsulating B cells, costimulatory signals, and cytokines in a hydrogel matrix (Shah and Singh, “Cellular Self-Assembly and Biomaterials- Based Organoid Models of Development and Diseases,” Acta Biomater. 53:29-45 (2017) and Kim et al., “Multiscale Engineering of Immune Cells and Lymphoid Organs,” Nat. Rev. Mater. 4(6):355-378 (2019), which are hereby incorporated by reference in their entirety). Recent work using a bottom-up organoid tissue approach was able to faithfully recapitulate key phenotypes of GC B cells, including GC-relevant RNA and gene expression profiles that closely resembled in vivo GC B cells (Beguelin et al., “EZH2 Enables Germinal Centre Formation through Epigenetic Silencing of CDKN1A and an Rb-E2F1 Feedback Loop,” Nat. Commun. 8(1):877 (2017), which is hereby incorporated by reference in its entirety). As occurs during SHM in vivo, significant increases in expression of aicda, which encodes activation-induced cytidine deaminase (AID), and immunoglobulin variable loci mutation rate were observed in B cells after organoid incubation. Building upon this finding, Purwada et al. characterized the response of immune organoids after addition of the small molecule hapten 4-hydroxy-3 -nitrophenyl acetyl (NP) conjugated to ovalbumin (Purwada et al., “Ex vivo Synthetic Immune Tissues with T Cell Signals for Differentiating Antigen-Specific, High Affinity Germinal Center B cells,” Biomaterials 198:27-36 (2019), which is hereby incorporated by reference in its entirety). NP- specific B cells were identified from organoids prepared with B cells derived from wild-type mice. Due to the GC-like phenomena of engineered immune organoids, they are a compelling platform to investigate intricacies of the humoral immune response to an infection or vaccination in a timely and scalable manner (Wagar et al., “Modeling Human Adaptive Immune Responses with Tonsil Organoids,” Nat. Med. 27 y. 25- 35 (2021), which is hereby incorporated by reference in its entirety). Immune organoid culture systems not only provide an accurate immune tissue microenvironment to model GC-like behavior, but are also high-throughput. A single mouse spleen is sufficient to prepare >800 organoids, which can be arrayed in 96-well plates for high-throughput experimentation and analyzed after 4 days of incubation as compared to the weeks or months normally needed for animal immunization work.

[0044] Accordingly, one aspect of the present disclosure is directed to a murine B cell organoid comprising: murine B cells; a CD40 ligand selected from the group consisting of soluble CD40 ligand (sCD40L), cells expressing CD40 ligand, scaffolds presenting CD40 ligand, beads presenting CD40 ligand, and combinations thereof; and a hydrogel, where the murine B cells and the CD40 ligand are encapsulated in the hydrogel and where the hydrogel comprises cross-linked multi -arm 9olyethylene glycol (PEG) macromers.

[0045] Murine B cell organoids may comprise primary B cells from a mouse, e.g., a laboratory-bred strain of mouse. Suitable laboratory-bred strains of mice include, without limitation, BALB/c mice, C57BL/6 mice, C57B1/10 mice, and Swiss Webster (SW) mice. Primary B cells may be derived from any murine lymphoid organ. In some embodiments, the primary B cells are spleen-derived B cells.

[0046] In some embodiments, the murine B cells are naive murine B cells.

[0047] The murine B cells may be primary murine B cells. In some embodiments, the murine B cells are primary naive murine B cells.

[0048] According to the present disclosure, the murine B cell organoid may comprise murine B cells within a range having a lower limit selected from about 500 cells, about 600 cells, about 700 cells, about 800 cells, about 900 cells, about 1,000 cells, about 2,000 cells, about 3,000 cells, about 4,000 cells, about 5,000 cells, about 6,000 cells, about 7,000 cells, about 8,000 cells, about 9,000 cells, about 10,000 cells, about 20,000 cells, about 30,000 cells, about 40,000 cells, about 50,000 cells, about 60,000 cells, about 70,000 cells, about 80,000 cells, about 90,000 cells, about 100,000 cells, about 200,000 cells, about 300,000 cells, about 400,000 cells, about 500,000 cells, about 600,000 cells, about 700,000 cells, about 800,000 cells, about 900,000 cells, and about 950,000 cells; and an upper limit selected from about 600 cells, about 700 cells, about 800 cells, about 900 cells, about 1,000 cells, about 2,000 cells, about 3,000 cells, about 4,000 cells, about 5,000 cells, about 6,000 cells, about 7,000 cells, about 8,000 cells, about 9,000 cells, about 10,000 cells, about 20,000 cells, about 30,000 cells, about 40,000 cells, about 50,000 cells, about 60,000 cells, about 70,000 cells, about 80,000 cells, about 90,000 cells, about 100,000 cells, about 200,000 cells, about 300,000 cells, about 400,000 cells, about 500,000 cells, about 600,000 cells, about 700,000 cells, about 800,000 cells, about 900,000 cells, about 950,000 cells, about 1,000,000 cells. In accordance with such embodiments, the murine B cell organoids may comprise about 500-1,000,000 murine B cells. For example, the murine B cell organoids may comprise about 40,000-80,000 murine B cells. In some embodiments, the murine B cell organoids comprise about 40,000 cells, e.g., primary naive murine B cells.

[0049] CD40 is a 45-50 kDa type I transmembrane protein which belongs to the TNF receptor superfamily because of its homology with the TNF receptor (Smith et al., The TNF Receptor Superfamily of Cellular and Viral Proteins: Activation, Costimulation, and Death,” Cell 76:956-62 (1994), which is hereby incorporated by reference in its entirety). It is ubiquitously expressed on the surface of immune cells, including B cells, monocytes, macrophages, and dendritic cells (DCs), as well as non-immune cells such as epithelial, endothelial, and mesenchymal (fibroblasts, myofibroblasts, synoviocytes, stellate cells, etc) cells, and platelets (Schonbeck and Libby, “The CD40/CD154 Receptor/Ligand Dyad,” Cell Mol Life Sci. 58:4-43 (2001), which is hereby incorporated by reference in its entirety). CD40 signaling of B cells promotes germinal center (GC) formation, immunoglobulin (Ig) isotype switching, somatic hypermutation (SHM) of the Ig to enhance affinity for antigen, and the formation of long-lived plasma cells and memory B cells (Danese et al., “The CD40/CD40L Costimulatory Pathway in Inflammatory Bowel Disease,” Gut. 53(7): 1035-1043 (2004), which is hereby incorporated by reference in its entirety).

[0050] CD40L is a 39 kDa type II transmembrane protein member of the TNF gene superfamily (which includes TNF-a, lymphotoxin a and lymphotoxin P, FasL, etc), and is expressed preferentially by activated CD4⁺ T cells and activated platelets, although it can also be variably expressed by monocytic cells, natural killer cells, B cells, CD8⁺ T cells, mast cells, and basophils. After its expression, CD40L is cleaved and shed from the cell’s surface and circulates systemically in a biologically active soluble form (sCD40L) (Pietravalle et al., “Human Native Soluble CD40L is a Biologically Active Trimer, Processed Inside Microsomes,” J. Biol. Chem. 271 :5965-5967 (1996), which is hereby incorporated by reference in its entirety). CD40 forms a trimer that binds to CD40L, triggering a complex signaling cascade eventually leading to activation of CD40 bearing cells which produce multiple bioactive molecules whose ultimate effects depend on the differentiation state of the cells involved, the expression level of receptor and ligand, and the tissue microenvironment where the binding occurs (Grammer and Lipsky, “CD40-Mediated Regulation of Immune Responses by TRAF -Dependent and TRAF- Independent Signaling Mechanisms,” Adv. Immunol. 76:61-178 (2001), which is hereby incorporated by reference in its entirety). [0051] In some embodiments, the murine B cell organoids comprise cells that express or present CD40 ligand. The cells that express or present CD40 ligand may be present within a range having a lower limit selected from about 500 cells, about 600 cells, about 700 cells, about 800 cells, about 900 cells, about 1,000 cells, about 2,000 cells, about 3,000 cells, about 4,000 cells, about 5,000 cells, about 6,000 cells, about 7,000 cells, about 8,000 cells, about 9,000 cells, about 10,000 cells, about 20,000 cells, about 30,000 cells, about 40,000 cells, about 50,000 cells, about 60,000 cells, about 70,000 cells, about 80,000 cells, about 90,000 cells, about 100,000 cells, about 200,000 cells, about 300,000 cells, about 400,000 cells, about 500,000 cells, about 600,000 cells, about 700,000 cells, about 800,000 cells, about 900,000 cells, and about 950,000 cells; and an upper limit selected from about 600 cells, about 700 cells, about 800 cells, about 900 cells, about 1,000 cells, about 2,000 cells, about 3,000 cells, about 4,000 cells, about 5,000 cells, about 6,000 cells, about 7,000 cells, about 8,000 cells, about 9,000 cells, about 10,000 cells, about 20,000 cells, about 30,000 cells, about 40,000 cells, about 50,000 cells, about 60,000 cells, about 70,000 cells, about 80,000 cells, about 90,000 cells, about 100,000 cells, about 200,000 cells, about 300,000 cells, about 400,000 cells, about 500,000 cells, about 600,000 cells, about 700,000 cells, about 800,000 cells, about 900,000 cells, about 950,000 cells, about 1,000,000 cells. In accordance with such embodiments, the murine B cell organoids may comprise about 500-1,000,000 cells that express or present CD40 ligand. For example, the murine B cell organoids may comprise about 40,000-80,000 cells expressing or presenting CD40 ligand. In some embodiments, the B cell organoids comprise about 40,000 cells that express or present CD40 ligand.

[0052] Suitable exemplary cells expressing or presenting CD40 ligand include immune cells such as T cells and platelets. In some embodiments, the cells expressing or presenting CD40 ligand are T cells. In other embodiments, the cells expressing or presenting CD40 ligand are not T cells.

[0053] In some embodiments, the murine B cell organoids do not comprise T cells. [0054] Suitable exemplary non-immune cells that may express or present CD40 ligand include, without limitation, non-immune cells that have been transfected with a nucleic acid molecule encoding CD40 ligand, e.g., non-immune cells transfected with a nucleic acid molecule encoding murine CD40 ligand. In some embodiments, the non-immune cells are fibroblast cells (e.g., mouse fibroblast cells).

[0055] In some embodiments, the cells that express or present CD40 ligand are primary cells. In some embodiments, the cells that express or present CD40 ligand are cell line cells. In some embodiments, the cells that express CD40 ligand are stromal cells, e.g., stromal cells transfected with a nucleic acid molecule encoding a CD40 ligand gene.

[0056] B-cell activating factor (BAFF) is a major cytokine that regulates B-cell survival, maturation and differentiation through its binding with its receptors: BAFF receptor (BAFF-R), transmembrane activator and cyclophilin ligand interactor (TACI) and B-cell maturation antigen (BCMA). BAFF signaling is implicated in the maintenance of germinal centers (Carrillo- Ballesteros, “B-Cell Activating Factor Receptor Expression is Associated with Germinal Center B-Cell Maintenance,” Exp. Ther. Med. 17(3): 2053-2060 (2019), which is hereby incorporated by reference in its entirety). In some embodiments, the cells that express CD40 ligand do not express B cell activating factor (BAFF).

[0057] In some embodiments, the murine B cell organoids comprise scaffolds and/or beads presenting CD40 ligand. In some embodiments, the murine B cell organoids comprises 10,000 to 200,000 scaffolds presenting CD40 ligand or beads presenting CD40 ligand. Thus, the scaffolds presenting CD40 ligand or beads presenting CD40 ligand may be present within a range having a lower limit selected from about 10,000, about 20,000, about 30,000, about 40,000, about 50,000, about 60,000, about 70,000, about 80,000, about 90,000, about 100,000, about 110,000, about 120,000, about 130,000, about 140,000, about 150,000, about 160,000, about 170,000, about 180,000, and about 190,000 cells; and an upper limit selected from about 20,000, about 30,000, about 40,000, about 50,000, about 60,000, about 70,000, about 80,000, about 90,000, about 100,000, about 110,000, about 120,000, about 130,000, about 140,000, about 150,000, about 160,000, about 170,000 cells, about 180,000, about 190,000, and about 200,000. In accordance with such embodiments, the murine B cell organoids may comprise about 20,000-100,000 cells that express or present CD40 ligand. For example, the murine B cell organoids may comprise about 40,000-80,000 scaffolds presenting CD40 ligand or beads presenting CD40 ligand. In some embodiments, the B cell organoids comprise about 40,000 scaffolds presenting CD40 ligand or beads presenting CD40 ligand.

[0058] In some embodiments, the murine B cell organoids comprises about 1 ng/ml - 1000 pg/ml soluble CD40 (sCD40) ligand. Thus, the soluble CD40 ligand may be present within a range having a lower limit selected from about 100 ng/ml sCD40, about 200 ng/ml sCD40, about 300 ng/ml sCD40, about 400 ng/ml sCD40, about 500 ng/ml sCD40, about 600 ng/ml sCD40, about 700 ng/ml sCD40, about 800 ng/ml sCD40, about 900 ng/ml sCD40; and an upper limit selected from about 2 pg/ml sCD40, about 3 pg/ml sCD40, about 4 pg/ml sCD40, about 5 pg/ml sCD40, about 6 pg/ml sCD40, about 7 pg/ml sCD40, about 8 pg/ml sCD40, about 9 pg/ml sCD40, about 10 pg/ml sCD40, about 20 pg/ml sCD40, about 30 pg/ml sCD40, about 40 pg/ml sCD40, about 50 pg/ml sCD40, about 60 pg/ml sCD40, about 70 pg/ml sCD40, about 80 pg/ml sCD40, about 90 pg/ml sCD40, about 100 pg/ml sCD40, about 200 pg/ml sCD40, about 300 pg/ml sCD40, about 400 pg/ml sCD40, about 500 pg/ml sCD40, about 600 pg/ml sCD40, about 700 pg/ml sCD40, about 800 pg/ml sCD40, about 900 pg/ml sCD40, and about 1,000 pg/ml sCD40. For example, the murine B cell organoids may comprise about 50-150 ng/ml soluble CD40. In some embodiments, the murine B cell organoids may comprise about 100 ng/ml soluble CD40 ligand.

[0059] In some embodiments, the murine B cell organoids may further comprise CD40 ligand expressing cells from murine lymphoid tissues, other mammalian cells, or polymeric beads.

[0060] As used herein, the term “polyethylene glycol” (also polyethylene glycol); polyethylene oxide) is the name given to molecules with the general structure of H-(O-CH2- CH₂)_n-OH. The term “PEG” is the abbreviated form of “polyethylene glycol”. The multi-arm PEG macromers may comprise four-arm PEG macromers and/or eight-arm PEG macromers. In some embodiments, the multi-arm PEG macromers are four-arm PEG macromers. In other embodiments, the multi-arm PEG macromers are 8-arm PEG macromers.

[0061] In some embodiments, the multi-arm PEG macromers are PEG-maleimide (PEG- MAL) macromers, vinyl sulfonate-PEG (PEG- VS) macromers, acrylate-PEG (PEG- Ac) macromers, or combinations thereof. For example, the multi-arm PEG macromers may be selected from the group consisting of four-arm PEG-maleimide (PEG-4MAL) macromers, four- arm vinyl sulfonate-PEG (PEG-4 VS) macromers, four-arm acrylate-PEG (PEG-4 Ac) macromers, or combinations thereof. In another example, the multi -arm PEG macromers may be selected from the group consisting of eight-arm PEG-maleimide (PEG-8MAL) macromers, eight-arm vinyl sulfonate-PEG (PEG-8 VS) macromers, eight-arm acrylate-PEG (PEG-8 Ac) macromers, or combinations thereof.

[0062] In some embodiments, the multi -arm PEG macromers are PEG-maleimide macromers. Maleimide selectively reacts with free thiol, SH, sulfhydryl, or mercapto group via Michael addition to form a stable thioether bond. In some embodiments, the multi-arm PEG macromers are four-arm PEG-maleimide (PEG-4MAL) macromers. In accordance with such embodiments, the PEG-4MAL macromers comprise a four-armed PEG derivative with maleimide at each terminal of its four arms connected to a core. In some embodiments, the core is a pentaerythritol core. For example, the PEG-4MAL macromers may have the following structure:

, wherein n is independently 1 to

2,500. Accordingly, n may be independently selected from 1 to 2,500; from 1 to 2,000; from 1 to 1,750; from 1 to 1,500; from 1 to 1,250; from 1 to 1,000; from 1 to 750; from 1 to 500; from 1 to 250; from 1 to 200; or from 1 to 150. In some embodiments, the PEG-4MAL macromers have a MW of 2kDa, 5kDa, lOkDa, or 20kDa. In some embodiments, the PEG-4MAL macromers have a MW of 20kDa.

[0063] In some embodiments, the multi-arm PEG macromers are eight-arm PEG maleimide (PEG-8MAL) macromers. In accordance with such embodiments, the PEG-4MAL macromers comprise an eight-armed PEG derivative with maleimide at each terminal of its eight arms connected to a core. In some embodiments, the core is a hexaglycerol core. For example, the PEG-8MAL macromers may have the following structure: independently 1 to 2,500. Accordingly, n may be independently selected from 1 to 2,500; from 1 to 2,000; from 1 to 1,750; from 1 to 1,500; from 1 to 1,250; from 1 to 1,000; from 1 to 750; from 1 to 500; from 1 to 250; from 1 to 200; or from 1 to 150. In some embodiments, the PEG-8MAL macromers have a MW of lOkDa, 20kDa, or 40kDa.

[0064] In some embodiments, the multi-arm PEG macromers are crosslinked with one or more crosslinkers. The one or more crosslinkers may comprise an enzymatically degradable crosslinker (Purwada et al., “Ex Vivo Synthetic Immune Tissues with T Cell Signals for Differentiating Antigen-Specific, High Affinity Germinal Center B Cells,” Biomaterials 198: 27- 36 (2019), which is hereby incorporated by reference in its entirety). For example, the enzymatically degradable crosslinker may be a protease degradable cross-linker such as a peptide. In some embodiments, the protease-degradable cross-linker is a cleavable bacterial peptide.

[0065] In some embodiments, the one or more cross-linkers comprise a multi-arm or dithiol protease-cleavable peptide crosslinker.

[0066] In some embodiments, the one or more crosslinkers is a protease cleavable peptide such as matrix metalloproteinases (MMP)-9 degradable VPM peptide (GCRDVPMSMRGGDRCG; SEQ ID NO: 1). Thus, in some embodiments, the protease cleavable peptide is VPM peptide (GCRDVPMSMRGGDRCG; SEQ ID NO: 1).

[0067] In some embodiments, the non-degradable crosslinker is selected from the group consisting of dithiothreitol (DTT), scrambled peptide crosslinkers (e.g., VMP), thiol polymers (e.g., 4-ARM PEG-SH).

[0068] In some embodiments, the scrambled peptide crosslinker is VMP.

[0069] In some embodiments, the thiol polymers comprise 2-arm PEG-SH, 4-arm PEG-

SH, 8-arm PEG-SH, and combinations thereof.

[0070] In some embodiments, the organoid comprises both a degradable crosslinker and a non-degradable crosslinker. The degradable crosslinker and a non-degradable crosslinker are in a molar ratio of 1 : 1.

[0071] According to the present disclosure the murine B cell organoid has a pH within a range having a lower limit selected from about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, and an upper limit selected from about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, and about 8.0. Thus, in some embodiments, the murine B cell organoid is at a pH of about 6.0 to about 8.0, about 7.2 to about 7.6, or about 7.3 to about 7.5. In some embodiments, the organoid is at a pH of about 7.4.

[0072] In some embodiments, culturing is carried out at a temperature of about 37°C.

[0073] In some embodiments, the murine B cell organoid has a volume of about 1 pl to 2 mL. Thus, the B cell organoid may have a volume within a range having a lower limit selected from about 1 pl, about 2 pl, about 3 pl, about 4 pl, about 5 pl, about 6 pl, about 7 pl, about 8 pl, about 9 pl, about 10 pl, about 15 pl, about 20 pl, about 25 pl, about 30 pl, about 35 pl, about 40 pl, about 45 pl, about 50 pl, about 55 pl, about 60 pl, about 65 pl, about 70 pl, about 75 pl, about 80 pl, about 95 pl, about 90 pl, about 95 pl, about 100 pl, about 150 pl, about 200 pl, about 250 pl, about 300 pl, about 350 pl, about 400 pl, about 450 pl, about 500 pl, about 550 pl, about 600 pl, about 650 pl, about 700 pl, about 750 pl, about 800 pl, about 850 pl, about 900 pl, about 950 pl, about 1 mL, about 1.1 mL, about 1.2 mL, about 1.3 mL, about 1.4 mL, about 1.5 mL, about 1.6 mL, about 1.7 mL, about 1.8 mL, and about 1.9 mL; and an upper limit selected from about 2 pl, about 3 pl, about 4 pl, about 5 pl, about 6 pl, about 7 pl, about 8 pl, about 9 pl, about 10 pl, about 15 pl, about 20 pl, about 25 pl, about 30 pl, about 35 pl, about 40 pl, about 45 pl, about 50 pl, about 55 pl, about 60 pl, about 65 pl, about 70 pl, about 75 pl, about 80 pl, about 95 pl, about 90 pl, about 95 pl, about 100 pl, about 150 pl, about 200 pl, about 250 pl, about 300 pl, about 350 pl, about 400 pl, about 450 pl, about 500 pl, about 550 pl, about 600 pl, about 650 pl, about 700 pl, about 750 pl, about 800 pl, about 850 pl, about 900 pl, about 950 pl, about 1 mL, about 1.1 mL, about 1.2 mL, about 1.3 mL, about 1.4 mL, about 1.5 mL, about 1.6 mL, about 1.7 mL, about 1.8 mL, about 1.9 mL, and about 2.0 mL. In some embodiments, the murine B cell organoid has a volume of about 30 pl to about 80 pl. In some embodiments, the organoid has a volume of about 50 pl.

Methods of Making Murine B Cell Organoids

[0074] Another aspect of the present disclosure relates to a method of making a murine B cell organoid. This method involves: providing a population of B cells; providing a CD40 ligand selected from the group consisting of soluble CD40 ligand (sCD40L), cells expressing CD40 ligand, scaffolds presenting CD40 ligand, beads presenting CD40 ligand, and combinations thereof; providing a crosslinker solution comprising a degradable and/or a non-degradable crosslinker; suspending the population of B cells and the CD40 ligand in the crosslinker solution to form a cell suspension; providing a functionalized macromer solution comprising a functionalized polyethylene glycol macromer; combining the cell suspension and the functionalized macromer solution to form a hydrogel solution; and curing the hydrogel solution to form a murine B cell organoid comprising encapsulated murine B cells.

[0075] Suitable murine B cells are described in detail supra. In some embodiments, the murine B cells are primary murine B cells. In some embodiments, the murine B cells are naive murine B cells. In some embodiments, the murine B cells are primary naive splenic B cells. [0076] Suitable CD40 ligands are described in detail supra. In some embodiments, the CD40 ligand comprises cells expressing CD40 ligand. In some embodiments, the cells expressing CD40 ligand are primary cells.

[0077] Suitable crosslinkers are described in detail supra. In some embodiments, the one or more crosslinkers comprise degradable crosslinkers and non-degradable crosslinkers.

[0078] In some embodiments, the functionalized polyethylene glycol macromer comprises a multi-arm PEG macromer. Suitable multi-arm PEG macromers are described in detail supra. In some embodiments, the multi-arm PEG macromers comprise four-arm PEG macromers and/or eight-arm PEG macromers.

[0079] The multi-arm PEG macromers may be selected from the group consisting of PEG-mal eimide (PEG-MAL) macromers, vinyl sulfonate-PEG (PEG- VS) macromers, acrylate- PEG (PEG- Ac) macromers, or combinations thereof (see, e.g., Day et al., “The Impact of Functional Groups of Poly(ethylene glycol) Macromers on the Physical Properties of Photo- Polymerized Hydrogels and the Local Inflammatory Response in the Host,” Acta Biomater. 67:42-52 (2018), which is hereby incorporated by reference in its entirety). For example, the multi-arm PEG macromers may be selected from the group consisting of four-arm PEG- mal eimide (PEG-4MAL) macromers, four-arm vinyl sulfonate-PEG (PEG-4 VS) macromers, four-arm acrylate-PEG (PEG-4Ac) macromers, or combinations thereof. In another example, the multi-arm PEG macromers may be selected from the group consisting of eight-arm PEG- mal eimide (PEG-8MAL) macromers, eight-arm vinyl sulfonate-PEG (PEG-8 VS) macromers, eight-arm acrylate-PEG (PEG-8Ac) macromers, or combinations thereof.

[0080] In some embodiments, the multi -arm PEG macromers are four-arm PEG- maleimide (PEG-4MAL) macromers. In other embodiments, the muti-arm PEG macromers are eight-arm PEG maleimide (PEG-8MAL) macromers.

[0081] In some embodiments, the multi-arm PEG macromer (e.g., PEG-4MAL or PEG- 8MAL) is functionalized with a peptide selected from the group consisting of integrin avb3- binding RGD peptide (GRGDSPC; SEQ ID NO: 8), integrin a4pi-binding REDV peptide (GREDVGC; SEQ ID NO: 2), GFOGER peptide (GYGGGPGPPG PPGPPGPPGP PGFXGERGPP GPPGPPGPPG PPGPC, where P at position 24 is 4-hydroxyproline; SEQ ID NO: 14), and GFOGER (GFXGER, where X at position 3 is hydroxyproline; SEQ ID NO: 15) (see, e.g., Purwada et al., “Ex Vivo Synthetic Immune Tissues with T Cell Signals for Differentiating Antigen-Specific, High Affinity Germinal Center B Cells,” Biomaterials 198: 27- 36 (2019); Graney et al., “Organoid Polymer Functionality and Mode of Klebsiella Pneumoniae Membrane Antigen Presentation Regulates Ex Vivo Germinal Center Epigenetics in Young and Aged B Cells,” Adv. Funct. Matter. 30(48): 2001232 (2020); and Sipila et al., “Proline Hydroxylation in Collagen Supports Integrin Binding by Two Distinct Mechanisms,” J. Biol. Chem. 293(20):2018, which are hereby incorporated by reference in their entirety). In some embodiments, the multi-arm PEG macromer (e.g., PEG-4MAL or PEG-8MAL) is functionalized with a peptide having the sequence of GREDVGC (SEQ ID NO: 2).

[0082] In some embodiments, the multi -arm PEG macromer (e.g., PEG-4MAL or PEG- 8MAL) is functionalized with a thiolated adhesive peptide having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 8, and/or SEQ ID NO: 14.

[0083] In some embodiments, curing the hydrogel solution is carried out for at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, at least about 60 minutes, or more.

[0084] In some embodiments, curing the hydrogen solution is carried out at a temperature of about 37°C.

[0085] In some embodiments, the method further involves introducing medium supplemented with one or more growth factors or cytokines. Suitable growth factors and cytokines are described infra. In some embodiments, the one or more growth factors or cytokines includes, without limitation, IL-4, IL-21, B cell activating factor (BAFF), or a combination thereof (see, e.g., Robinson et al., “BAFF, IL-4 and IL-21 Separably Program Germinal Center-Like Phenotype Acquisition, BCL6 Expression, Proliferation and Survival of CD40L-Activated B Cells in Vitro,” Immunol. Cell. Biol. 97(9): 826-839 (2019), which is hereby incorporated by reference in its entirety).

[0086] In some embodiments, the method further involves introducing a glycoconjugate comprising a polysaccharide of interest and a carrier protein. Suitable glycoconjugates are described infra. In some embodiments, the glycoconjugate is present at a concentration of 1 pM to 5 pM. Suitable glycoconjugate concentrations are described in more detail infra.

[0087] In some embodiments, the method further involves culturing said organoid comprising the glycoconjugate. Suitable culturing conditions are described in more detail infra. Methods of Identifying Immunogenic Glycoconjugate Vaccine Candidates

[0088] As used herein, the term “glycoconjugate” refers to a compound comprising a glycan bound to another molecule, e.g., a carrier protein. Glycoconjugates can include carbohydrate components of peptides, proteins, glycoproteins, polysaccharides, complex carbohydrates, sugars, gangliosides, lipids, and phospholipids; portions thereof and combinations thereof. Suitable glycoconjugates are those derived from, for example, pathogenic mammalian, human, fungal, or protozoan organisms.

[0089] Another aspect of the present disclosure relates to an ex vivo method of identifying highly immunogenic glycoconjugates. This method involves providing a first plurality of murine B cell organoids; contacting each of the first plurality of murine B cell organoids with a plurality of distinct glycoconjugate candidates, where each of the distinct glycoconjugate candidates comprises a distinct polysaccharide of interest and a carrier protein, and where the contacting is carried out to form a first plurality of contacted B cell organoids; culturing the first plurality of contacted B cell organoids under conditions effective to support B cell maturation, whereby said culturing produces a first plurality of cultured B cell organoids; detecting presence or absence of one or more B cell biomarker(s) in each of the first plurality of cultured B cell organoids, where the detecting presence of one or more B cell biomarker(s) indicates immunogenicity of the glycoconjugate candidate; and identifying highly immunogenic glycoconjugate candidates based on a greater presence of B cell biomarker(s) in one or more of said cultured B cell organoids relative to others of said cultured B cell organoids.

[0090] Suitable exemplary murine B cell organoids are described in detail supra. In some embodiments, the murine B cells are naive murine B cells, primary murine B cells, or primary naive murine B cells. The murine B cells may be splenic murine B cells. In some embodiments, the murine B cells are CD19⁺ B cells.

[0091] The first plurality of murine B cell organoids may be configured so as to be amenable for high throughput screening. Thus, in some embodiments, the first plurality of murine B cell organoids is organized in an array. Conventional multi-well plates, dishes, tissue culture plates, or glass coverslips can be used to prepare the array.

[0092] In some embodiments, the array is a 6-well array, a 12-well array, a 48-well array, a 96-well array, a 384-well array, or any other configuration of an array. In accordance with such embodiments, the first plurality of B cell organoids may be seeded in a 6-well plate, a 12- well plate, a 48-well plate, a 96-well plate, a 384-well plate, or larger culture platform.

[0093] In some embodiments, the plurality of murine B cell organoids is organized in one or more arrays. In some embodiments, the plurality of murine B cells organoids is organized in at least 1 array, at least 2 arrays, at least 3 arrays, at least 4 arrays, at least 5 arrays, at least 6 arrays, at least 7 arrays, at least 8 arrays, at least 9 arrays, at least 10 arrays, or more.

[0094] Methods of generating glycoconjugate candidates are well known in the art. See, e.g., Williams et al., “A Low-Cost Recombinant Glycoconjugate Vaccine Confers Immunogenicity and Protection against Enterotoxigenic Escherichia coli Infections in Mice,” Front. Mol. Biosci. 10: 1085887 (2023), Kay et al., “Recent Advances in the Production of Recombinant Glycoconjugate Vaccines,” NPJ Vaccines 4: 16 (2019), U.S. Patent No.

11,542,538 to Jewett et al., U.S. Patent Application Publication No. 2022/0267821 to Jewett et al., U.S. Patent No. 11,530,432 to Jewett et. al., U.S. Patent No. 9,512,434 to DeLisa et al., each of which is hereby incorporated by reference in its entirety. In some embodiments, glycoconjugate vaccine candidates are produced by covalently linking a bacterial polysaccharide to a carrier protein (e.g., by metabolic engineering of bacteria (Kay et al., “Recent Advances in the Production of Recombinant Glycoconjugate Vaccines,” NPJ Vaccines 4: 16 (2019), which is hereby incorporated by reference in its entirety)).

[0095] It will be appreciated by one of skill in the art that while numerous glycan structures are known, many more exist, since only a small fraction of the antigenic or immunogenic glycans have been identified thus far. Examples of the many glycan antigens discovered thus far can be found in, e.g., Moeller et al., “Profiling Germinal Center-like B Cell Responses to Conjugate Vaccines Using Synthetic Immune Organoids,” ACS Cent. Sci.

9(4):787-804 (2023); Zimmerman and Lepenies, “Glycans as Vaccine Antigens and Adjuvants: Immunological Considerations,” Methods Mol. Biol. 1331 : 11-26 (2015); Moreau, M. “A New Vaccine Concept: Polysaccharide Conjugate Vaccines,” Ann. Phar. Fr. 57(3):223-231 (1999); Goyette-Desjardins et al., “Protection against Streptococcus suis Serotype 2 Infection Using a Capsular Polysaccharide Glycoconjugate Vaccine,” Infect. Immun. 84(7):2059-2075 (2016); Nyame et al., “Antigenic Glycans in Parasitic Infections: Implications for Vaccines and Diagnostics,” Arch. Biochem. Biophys. 426(2): 182-200 (2004); Fox et al., Carbohydrates and Glycoproteins of Bacillus anthracis and Related Bacilli: Targets for Biodetection,” J. Microbiol. Meth. 54(2): 143-152 (2003); Hakomori, “Cancer-Associated Glycosphingolipid Antigens: Their Structure, Organization, and Function,” ActaAnat. 161(l-4):79-90 (1998); Hakomori et al., “Glycosphingolipid Antigens and Cancer Therapy,” Chem. Biol. 4(2):97-104 (1997); Jones, “Vaccines Based on the Cell Surface Carbohydrates of Pathogenic Bacteria,” Anais da Academia Brasileira de Ciencias 77(2):293-324 (2005); Pier, “Application of Vaccine Technology to Prevention of Pseudomonas aeruginosa Infections,” Expert Rev. Vaccines 4(5): 645-656 (2005); Goldblatt, “Recent Developments in Bacterial Conjugate Vaccines,” J. Med. Microbiol. 47(7):563-567 (1998); Ada et al., “Carbohydrate-Protein Conjugate Vaccines,” Clin. Microbiol. Inf. 9(2):79-85 (2003); Vliegenthart, “Carbohydrate Based Vaccines,” FEBS Let. 580(12):2945- 2950 (2006); Mendonca-Previato et al., “Protozoan Parasite- Specific Carbohydrate Structures,” Curr. Opin. Struct. Biol. 15(5):499-505 (2005); Croce and Segal -Eiras, “The Use of Carbohydrate Antigens for the Preparation of Vaccines for Therapy in Breast Cancer,” Drugs of Today 38(11): 759-768 (2002), which are hereby incorporated by reference in their entirety. Accordingly, distinct polysaccharides for use in the methods of the present disclosure may be derived from any one of the following: a bacterium, a virus, a parasite, or a cancer.

[0096] In some embodiments, each distinct polysaccharide is derived from a bacterium. In some embodiments, the bacterium is a pathogenic bacterium.

[0097] In some embodiments, the bacterium is a gram-positive bacterium. The grampositive bacterium may be selected from the group consisting of Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Erysipelothrix rhusiopathiae, Gardnerella vaginalis, Group A Streptococcus, Group B Streptococcus, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Nocardia asteroids, Propionibacterium acnes, Rhodococcus equi, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus mutans, Streptococcus pneumoniae, and Streptococcus pyogenes.

[0098] In some embodiments, the bacterium is a gram-negative bacterium. The gramnegative-bacterium may be selected from the group consisting of Acinetobacter baumannii, Bordetella pertussis, Brucella abortus, Burkholderia pseudomallei, Burkholderia mallei, Burkholderia pseudomallei, Campylobacter fetus, Campylobacter jejuni, Coxiella burnetii, Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumoniae, Legionella pneumophila, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia prowazekii, Salmonella enterica, Salmonella typhi, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Vibrio cholerae, Yersinia enter ocolitica, Yersinia pestis, and Yersinia pseudotuberculosis.

[0099] In some embodiments, the bacterium is Francisella tularensis.

[0100] In some embodiments, the bacterium is Escherichia coli. Exemplary pathogenic

E. coli strains include, without limitation, enterotoxigenic Escherichia coli (ETEC), enterohemorrhagic Escherichia coli (EHEC), extraintestinal pathogenic Escherichia coli (ExPEC).

[0101] Suitable polysaccharides derived from a bacterium include, without limitation, O- antigen (e.g., Francisella tularensis O-antigen ( O) or pathogenic Escherichia coli O-antigen), O-PSII (e.g., Burkholderia pseudomallei O-PSII), and capsule polysaccharides (e.g., Streptococcus pneumoniae capsule polysaccharides, Shigella dysenteriae capsule polysaccharides, and Shigella flexneri capsule pholysaccharides). See, e.g., Kay et al., “Recent Advances in the Production of Recombinant Glycoconjugate Vaccines,” NPJ Vaccines 4: 16 (2019), which is hereby incorporated by reference in its entirety.

[0102] In some embodiments, each distinct polysaccharide is derived from a virus. In some embodiments, the virus is selected from the group consisting of Adenovirus, Andes virus, Chikungunya virus, Coconut Creek virus, Coxsackievirus, Crimean-Congo Hemorrhagic Fever virus, Cytomegalovirus, Dengue virus, Eastern Equine Encephalitis virus, Ebola virus, Epstein- Barr virus, Hantavirus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Herpes Simplex Virus 1 (HSV-1), Herpes Simplex Virus 2 (HSV-2), Human Immunodeficiency Virus 1 (HIV-1), Human Immunodeficiency Virus 2 (HIV-2), Human Papillomavirus (HPV), Influenza A virus, Influenza B virus, Japanese Encephalitis virus, Junin virus, La Crosse virus, Lassa fever virus, Marburg virus, Measles virus, MERS-CoV, Mumps virus, Nipah virus, Norovirus, Parvovirus Bl 9, Poliovirus, Rabies virus, Respiratory Syncytial Virus (RSV), Rhinovirus, Ross River virus, Rotavirus, Rubella virus, SARS-CoV, SARS-CoV-2, Severe Fever with Thrombocytopenia Syndrome virus, Varicella-Zoster Virus (VZV), Venezuelan Equine Encephalitis virus, West Nile virus, Yellow Fever virus, and Zika virus.

[0103] In some embodiments, each distinct polysaccharide is derived from a parasite. In some embodiments, the parasite is a protozoan parasite. Suitable protozoan parasites include, without limitation, Cryptosporidium spp., Cyclospora cayetanenensis, Entamoeba histolytica, Giardia intestinalis, Plasmodium falciparum, Plasmodium malar iae, Toxoplasma gondii, and Trypanosoma cruzi (see, e.g., Hague, R., “Human Intestinal Parasites,” J. Health PopuL Nutr. 25(4): 384-391 (2007), which is hereby incorporated by reference in its entirety).

[0104] In some embodiments, the parasite is a helminth. Suitable helminth parasites include, without limitation, Ancylostoma duodenale, Ascaris lumbricoides, Necator americanus, Trichuris trichiura (see, e.g., Geiger et al., “Necator americanus and Helminth Co-Infections: Further Down-Modulation of Hookworm-Specific Type 1 Immune Responses,” PLoS NegL Trop. Dis. 5(9): el280 (2011), which is hereby incorporated by reference in its entirety). [0105] In some embodiments, each distinct polysaccharide is derived from a cancer. In some embodiments, the cancer is selected from the group consisting of adrenal cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, gallbladder cancer, head and neck cancer, hodgkin lymphoma, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, multiple myeloma, non-hodgkin lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, uterine cancer, vaginal cancer, and vulvar cancer. [0106] Each of the distinct glycoconjugate candidates comprises a distinct polysaccharide of interest and a carrier protein. The term “carrier” refers to a protein to which the polysaccharide is coupled or attached or conjugated, typically for the purpose of enhancing or facilitating detection of an antigen by the immune system. The term “carrier protein” is intended to cover both small peptides and large polypeptides (>10 kDa).

[0107] In some embodiments, useful carrier proteins include bacterial toxins or toxoids, such as diphtheria toxoid or tetanus toxoid. Fragments of toxins or toxoids can also be used. For example, fragment C of tetanus toxoid, which is commercially available. The cross-reacting material (CRM) 197 mutant of diphtheria toxin may be particularly useful. Other suitable carrier proteins include, for example and without limitation, the Neisseria meningitidis outer membrane protein, synthetic peptides, heat shock proteins, pertussis proteins, cytokines, lymphokines, hormones, growth factors, human serum albumin (preferably recombinant) in particular for diagnostic aspects, universal CD4⁺ cell epitopes, in particular artificial proteins comprising multiple human CD4⁺ T cell epitopes from various pathogen- derived antigens such as N19 or tetanus toxoid (Cancer Immunol Immunother. 65(3) :315-25 (2016), which is hereby incorporated by reference in its entirety), protein D from Haemophilus influenzae, pneumococcal surface protein PspA, pneumolysin, iron-uptake proteins, toxin A or B from Clostridium difficile, recombinant P. aeruginosa exoprotein A (rEPA), a GBS protein, and the like, as for example described in Micoli et al., Molecules 23(6): 1451 (2018), which is hereby incorporated by reference in its entirety.

[0108] Particularly suitable carrier proteins may include, for example, CRM 197, tetanus toxoid (TT), tetanus toxoid fragment C, protein D, non-toxic mutants of tetanus toxin and diphtheria toxoid (DT). Other suitable carrier proteins may include protein antigens GBS80, GBS67 and GBS59 from Streptococcus agalactiae and fusion proteins, for example, GBS59(6xD3) disclosed in WO 2011/121576, which is hereby incorporated by reference in its entirety, and GBS59(6xD3)-1523 disclosed in EP14179945.2, which is hereby incorporated by reference in its entirety. The use of other suitable carrier proteins of proteins antigens that are common to several Shigella serotypes such as IpaD, IpaB, MxiH and all their possible combinations may also be useful. Another suitable carrier could be genetically modified OMVs (GMMA), for example those developed by the pharmaceutical industry. Synthetic peptides bearing immunodominant T-helper cell epitopes can also act as carriers in polysaccharide and oligosaccharide conjugates. The peptide carriers include polypeptides containing multiple T- helper epitopes addressing the extensive polymorphism of HLA molecules (Pediatrics 92:827- 832 (1993), which is hereby incorporated by reference in its entirety), and universal T-helper epitopes compatible with human use. Exemplary T-helper epitopes include, but are not limited to, natural epitopes characterized from tetanus toxoid (J. Immunol. 149:717-721 (1992), which is hereby incorporated by reference in its entirety), and non-natural epitopes or engineered epitopes such as the pan HLA DR-binding epitope PADRE (Immunity 1 :751-761 (1994) and Vaccine 22(19):2362-7 (2004), which are hereby incorporated by reference in their entirety. [0109] Carriers may also include lipopeptides, for example Pam(3)CAG

(Vaccine 27(39):5419-26 (2009), which is hereby incorporated by reference in its entirety, as an adjuvant. Carriers may also include zwitterionic polysaccharides, as for example described in Chem Sci 11(48): 13052-13059 (2020), which is hereby incorporated by reference in its entirety.

[0110] In some embodiments, the carrier protein is selected from the group consisting of wherein the carrier protein is selected from the group consisting of bovine serum albumin (BSA), cross-reacting material 197 (CRM197), egg ovalbumin (OVA), keyhole limpet hemocyanin (KLH), maltose binding protein (MBP), tetanus toxoid (TT), meningococcal outer membrane protein (OMPC), diphtheria toxoid (DT), H. influenzae protein D (PD).

[OlH] In the ex vivo method of identifying highly immunoconjugate glycoconjugates described herein, the step of contacting each of the first plurality of murine B cell organoids with a plurality of distinct glycoconjugate candidates may be carried out a single time, meaning in some embodiments each organoid is contacted with a distinct glycoconjugate only once. In some embodiments, contacting each of the first plurality of murine B cell organoids with a plurality of distinct glycoconjugate candidates is carried out more than once, such as twice, three times, four times, five times, six times, or more. In some embodiments, said contacting is carried out twice.

[0112] Organoids may be contacted with the distinct glycoconjugates in varying amounts or doses depending on the specific reagents or design of the method and/or array used in carrying out the method. In some embodiments, the contacting is carried out at a dose ranging from 0.5 pM/organoid to 5.0 pM/organoid, or any range or specific amount therein. In some embodiments, the contacting is carried out at a dose of at least 0.5 gM/organoid, at least 0.6 gM/organoid, at least 0.7 gM/organoid, at least 0.8 gM/organoid, at least 0.9 gM/organoid, or at least 1.0 gM/organoid. In some embodiments, the contacting is carried out at a dose ranging from less than 5.0 gM/organoid, or less than 4.9 gM/organoid, or less than 4.8 gM/organoid, or less than 4.7 gM/organoid, or less than 4.6 gM/organoid, or less than to 4.5 gM/organoid. In some embodiments, contacting is carried out at a dose of about 1.75 gM/organoid.

[0113] In culturing the first plurality of contacted B cell organoids under conditions effective to support B cell maturation, certain conditions may be varied to obtain desirable results. For example, one variable may be the time or length of culturing, and other factors include temperature, pH conditions, etc. In some embodiments, culturing is carried out for at least an hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours, or more. In some embodiments, culturing is carried out for at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, or more. In some embodiments, culturing is carried out for 4 days.

[0114] Culturing the first plurality of contacted B cell organoids may be carried out in the presence of one or more growth factors or cytokines. Examples of cytokines include, but are not limited to, interleukins (such as, e.g., IL-la, IL-ip, IL-lra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36a, IL-36p, IL-36y, IL-36ra, IL-37, IL-38, IFNa, IFN , IFNy, IFNK, IFNco, GM-CSF, oncostatin M, leukemia inhibitory factor, ciliary neurotrophic factor, cardiotrophin-1), chemokines (such as, e.g., chemokine C-C motif ligand (CCL) 1, CCL2/MCP1, CCL3/MIPla, CCL4/MIP1P, CCL5/RANTES, CCL6, CCL7, CCL8/MCP2, CCL9, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18/PARC/DCCK1/AMAC1/MIP4, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, chemokine C-X-C motif ligand (CXCL) 1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8/IL-8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL 17, fractalkine, chemokine C motif ligand (XCL) 1 and XCL2), and tumor necrosis factors (such as, e.g., tumor necrosis factor (TNF) a, lymphotoxin, OX40L, CD40LG, Fas ligand, CD70, CD153, 4-1BB ligand, TNF-related apoptosis-inducing ligand (TRAIL), receptor activator of nuclear factor K-B ligand (RANKL), a proliferation-inducing ligand (APRIL), B-cell activating factor (BAFF) and ectodysplasin A (EDA)). [0115] Examples of growth factors include, but are not limited to fibroblast growth factor (FGF) 1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF 14, FGF 16, FGF 17, FGF 18, FGF 19, FGF20, FGF21, FGF23, transforming growth factor (TGF) a, epidermal growth factor (EGF), heparin-binding EGF-like growth factor (HB- EGF), transforming growth factor (TGF) P, insulin-like growth factor (IGF) 1, IGF2, Platelet- derived growth factor (PDGF) subunit A (PDGFA), PDGF subunit B (PDGFB), PDGF subunit C (PDGFC), PDGF subunit D (PDGFD), vascular endothelial growth factor (VEGF)-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor (PGF), nerve growth factor (NGF) and hepatocyte growth factor (HGF).

[0116] In some embodiments, suitable cytokines and growth factors include, without limitation, IL-4, IL-21, B cell activating factor (BAFF), or a combination thereof (see, e.g., Robinson et al., “BAFF, IL-4 and IL-21 Separably Program Germinal Center-Like Phenotype Acquisition, BCL6 Expression, Proliferation and Survival of CD40L-Activated B Cells in Vitro,” Immunol. Cell. Biol. 97(9): 826-839 (2019), which is hereby incorporated by reference in its entirety).

[0117] In some embodiments of the ex vivo method of identifying highly immunogenic glycoconjugates, the one or more B cell biomarkers is selected from the group consisting of IgM, phosphorylated Brunton’s tyrosine kinase (pBTK), phosphorylated nuclear factor-xB (pNFKB), EZH2, IRF4, and/or BLIMP- 1.

[0118] In some embodiments of the ex vivo method of identifying highly immunogenic glycoconjugates, the one or more biomarkers consists of two biomarkers, three biomarkers, four biomarkers, five biomarkers, or six biomarkers.

[0119] As described infra, glycoconjugates shape B cell receptor (BCR) clustering and signaling of germinal center (GC)-like B cells in synthetic organoids. In some embodiments of the ex vivo method of identifying highly immunogenic glycoconjugates, the one or more B cell biomarkers can comprise or consist of any B cell biomarker (e.g., signaling molecule, enzyme, receptor, transcription factor, kinase) that defines the activation state of murine B cells.

[0120] In some embodiments of the ex vivo method of identifying highly immunogenic glycoconjugates, the one or more B cell biomarkers comprise surface proteins and/or intracellular proteins.

[0121] For example, in some embodiments of the ex vivo method of identifying highly immunogenic glycoconjugates, the one or more B cell biomarkers comprises IgM. Upon binding of an antigen (e.g., a glycoconjugate according to the present disclosure), B cell activation is regulated by B cell receptor (BCR) signaling and the nanoscale organization of the BCR on the cell surface. These nanoscale organizations lead to the formation of individual puncta on the surface of B cells. As shown in FIGs. 4A-4B, the Examples of the present disclosure demonstrate that organoids contacted with glycoconjugates comprising CRM197 as the carrier protein demonstrated increased expression of IgM and IgM BCR puncta than organoids contacted with carrier protein alone (aglycosylated carrier proteins). FIG. 4B further demonstrate that the glycoconjugate comprising CRM197 as the carrier protein induced the formation of a significantly higher number of BCR clusters per single B cell than the aglycosylated CRM197 carrier, indicating that the glycoconjugate functions by enhanced BCR expression and clustering.

[0122] Bruton’s tyrosine kinase (BTK) is an important proximal component of B cell receptor (BCR) signaling pathways. Upon BCR engagement, the BTK PH domain binds to PIP3, a signaling intermediate generated by PI3 kinase (PI3K), thus localizing BTK to the plasma membrane, which facilitates its phosphorylation and activation by Src kinases and promotes access to its substrates (Satterthwaite, A., “Bruton’s Tyrosine Kinase, a Component of B Cell Signaling Pathways, Has Multiple Roles in the Pathogenesis of Lupus,” Front. Immunol. 8: 1986 (2017), which is hereby incorporated by reference in its entirety). Phosphorylation of PLCy2 by BTK leads to increased Ca⁺⁺ flux and activation of NF-KB, which is a downstream transcription factor responsible for important B cell maturation processes including classswitching, in the GC-like B cell population.

[0123] FIG. 4C demonstrates that glycoconjugates comprising either CR 197 or MBP as carrier proteins stimulated a statistically significant increase in pBTK and pNF-xB levels relative to their cognate aglycosylated carrier proteins, suggesting a role for the glycoconjugate itself in further activation of BTK and NF-KB, consistent with stronger immunogenicity of such conjugates when evaluated in vivo. Thus, in some embodiments, the one or more B cell biomarkers comprises or consists of phosphorylated Brunton’s tyrosine kinase (pBTK) and/or phosphorylated nuclear factor-xB (pNFKB).

[0124] FIG. 4D examines the expression levels of factors associated with the GC response, including BLIMP-1, EZH2, and IRF4. Both EZH2 and IRF4 are upstream of multiple GC programming factors and have been shown to be required for GC formation. During the late stages of the GC response, IRF4 levels are again elevated and BLIMP-1 is also expressed, with both working together and required for GC B cell differentiation into plasma cells. For all three of these GC factors, glycoconjugates comprising CRM197 as the carrier protein induced significantly greater expression relative to all other treatment groups including both the aglycosylated CRM197 carrier and the glycosylated MBP conjugate. Thus, in some embodiments, the one or more B cell biomarkers comprises or consists of EZH2, IRF4, and/or BLIMP- 1.

[0125] FIG. 4D demonstrates that glycoconjugates comprising MBP as the carrier protein led to statistically higher expression of IRF4 relative to aglycosylated MBP. Thus, in some embodiments, the one or more B cell biomarkers IRF4. For example, the one or more B cell biomarkers may comprise or consist of phosphorylated Brunton’s tyrosine kinase (pBTK), phosphorylated nuclear factor-xB (pNFKB), and/or IRF4.

[0126] Taken together, these results provide further support that the polysaccharide component of the glycoconjugate promotes activation of GC-like responses, which were most pronounced in the context of the glycoconjugates comprising CRM197 as the carrier protein. [0127] In some embodiments of the ex vivo method of identifying highly immunogenic glycoconjugates, detecting presence or absence of one or more B cell biomarker(s) in each of the first plurality of cultured B cell organoids is carried out by fluorescence-based microscopy and/or fluorescence activated cell sorting (FACS). Standard fluorescence-based microscopy and/or fluorescence activated cell sorting (FACS) assays well known in the art.

[0128] In some embodiments, detecting presence or absence of one or more B cell biomarker(s) in each of the first plurality of cultured B cell organoids is carried out by fluorescence-based microscopy. For example, detecting presence or absence of one or more B cell biomarker(s) in each of the first plurality of cultured B cell organoids may be carried out by confocal microscopy.

[0129] In some embodiments, detecting presence or absence of one or more B cell biomarker(s) in each of the first plurality of cultured B cell organoids is carried out by fluorescence activated cell sorting (FACS) or imaging flow cytometry. In accordance with such embodiments, the first plurality of cultured B cell organoids is enzymatically digested prior to said detecting. In some embodiments, the first plurality of cultured B cell organoids is enzymatically digested with a collagenase.

[0130] For example, enzymatically digesting the first plurality of cultured B cell organoids may be carried out by contacting each of the first plurality of cultured B cell organoids with a solution comprising an enzyme (e.g., a collagenase such as collagenase type 1) at a concentration of 1 to 200 U/mL. In some embodiments, the enzyme is at a concentration of about 125 U/mL. In accordance with such embodiments, enzymatically digesting the first plurality of cultured B cell organoids may be carried out for at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, or more. In some embodiments, enzymatically digesting is carried out for at least about 60 minutes. In some embodiments, enzymatically digesting produces hydrogel debris.

[0131] In some embodiments, identifying highly immunogenic glycoconjugate candidates based on a greater presence of B cell biomarker(s) in one or more of said cultured B cell organoids relative to others of said cultured B cell organoids.

[0132] In some embodiments, the highly immunogenic glycoconjugate candidates comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% of the plurality of distinct glycoconjugate candidates. For example, the highly immunogenic glycoconjugate candidates may comprise about 1% of the plurality of distinct glycoconjugate candidates.

[0133] In some embodiments, identifying highly immunogenic glycoconjugate candidates is carried out based on a greater presence of B cell biomarker(s) in one or more of said cultured B cell organoids relative to a control cultured B cell organoid contacted with the carrier protein, wherein the carrier protein is aglycosylated. In accordance with such embodiments, the greater presence is determined by providing a biomarker level.

[0134] The provided biomarker level depends on the assay used to carry out the detecting step. In some embodiments, the detecting step is carried out using biochemical assays, gene expression assays, spatial multinomics assays, and/or proteomic assays.

[0135] In some embodiments, when the detecting step is carried out by fluorescence- based microscopy, the biomarker level may be, without limitation, counts of IgM clustering, observation of the presence of one or more B cell biomarker(s), observation of the absence of the one or more B cell biomarker(s), and/or observation of the overlap between one or more B cell biomarker(s).

[0136] In some embodiments, when the detecting step is carried out by fluorescence activated cell sorting (FACS), the biomarker level may be, without limitation, intensity measurements such as mean fluorescence intensity (MFI), absolute counts, and/or frequency measurements. In some embodiments, the biomarker level is mean fluorescence intensity (MFI). [0137] In some embodiments, the highly immunogenic glycoconjugate candidates comprise at least a 1.2 fold higher level of the one or more B cell biomarker(s) as compared to the control. For example, the one or more B cell biomarkers may have a biomarker level that is at least 1.2 fold higher, at least 1.3 fold higher, at least 1.4 fold higher, at least 1.5 fold higher, at least 1.6 fold higher, at least 1.7 fold higher, at least 1.8 fold higher, at least 1.9 fold higher, at least 2.0 fold higher, 3.0 fold higher, 4.0 fold higher, 5.0 fold higher, 6.0 fold higher, 7.0 fold higher, 8.0 fold higher, 9.0 fold higher, 10.0 fold higher, 20.0 fold higher, 30.0 fold higher, 40.0 fold higher, 50.0 fold higher, 60.0 fold higher, 70.0 fold higher, 80.0 fold higher, 90.0 fold higher, 100.0 fold higher, 200.0 fold higher, 300.0 fold higher, 400.0 fold higher, 500.0 fold higher, 600.0 fold higher, 700.0 fold higher, 800.0 fold higher, 900.0 fold higher, 1,000.0 fold higher, 1,100.0 fold higher, or 1,200.0 fold higher than the control one or more B cell biomarker level.

[0138] Another aspect of the present disclosure pertains to highly immunogenic glycoconjugate candidates identified by the ex vivo method of identifying highly immunogenic glycoconjugates disclosed herein.

[0139] A further aspect of the present disclosure pertains to a plurality of distinct highly immunogenic glycoconjugate candidates identified by the ex vivo method of identifying highly immunogenic glycoconjugates disclosed herein.

Methods of Identifying Immunogenic Glycoconjugate Vaccine Candidates

[0140] Another aspect of the disclosure is directed to a method of identifying glycoconjugate vaccine candidates. This method involves providing a plurality of mice; immunizing each of the plurality of mice with distinct highly immunogenic glycoconjugate candidates of the plurality of distinct highly immunogenic glycoconjugate candidates of the present disclosure; evaluating an immune response of each of the plurality of mice to each of the highly immunogenic glycoconjugate candidates; and identifying glycoconjugate vaccine candidates based on a greater presence of antibodies specific for a polysaccharide of interest in the sera of one or more immunized mice relative to others of said immunized mice.

[0141] In some embodiments of the method of identifying glycoconjugate vaccine candidates described herein, the mice are laboratory-bred strains of mice. Suitable laboratory- bred strains of mice include, without limitation, BALB/c mice, C57BL/6 mice, C57B1/10 mice, and Swiss Webster (SW) mice. In some embodiments, the mice are BALB/c mice. In some embodiments, the mice are C57BL/6 mice.

[0142] In some embodiments, the plurality of mice is arranged in groups of mice. In some embodiments, the groups comprise 2 mice, 3 mice, 4 mice, 5 mice, 6 mice, 7 mice, 8 mice, 9 mice, or more. In some embodiments, the plurality of mice is arranged in groups of three mice.

[0143] Suitable highly immunogenic glycoconjugate candidates and method of identifying such highly immunogenic glycoconjugates are described in detail supra. [0144] In the method of identifying glycoconjugate vaccine candidates described herein, the step of immunizing each of the plurality of mice (or each of the mice within a group of the plurality of mice) with distinct highly immunogenic glycoconjugate candidates of the plurality of distinct highly immunogenic glycoconjugate candidates of the present disclosure may be carried out a single time, meaning in some embodiments each mouse or group of mice is immunized with a distinct highly immunogenic glycoconjugate candidate only once. In some embodiments, immunizing each of the plurality of mice (or each of the mice within a group of the plurality of mice) with distinct highly immunogenic glycoconjugate candidates of the plurality of distinct highly immunogenic glycoconjugate candidates of the present disclosure is carried out more than once, such as twice, three times, four time, five time, six times, or more. In some embodiments, immunizing each of the plurality of mice (or each of the mice within a group of the plurality of mice) with distinct highly immunogenic glycoconjugate candidates of the plurality of distinct highly immunogenic glycoconjugate candidates of the present disclosure is carried out at least twice. In some embodiments, immunizing each of the plurality of mice (or each of the mice within a group of the plurality of mice) with distinct highly immunogenic glycoconjugate candidates of the plurality of distinct highly immunogenic glycoconjugate candidates of the present disclosure is carried out at least three times. Thus, the immunizing step may be carried out once, twice, three times, or more.

[0145] In some embodiments, the step of immunizing each of the plurality of mice (or each of the mice within a group of the plurality of mice) with distinct highly immunogenic glycoconjugate candidates of the plurality of distinct highly immunogenic glycoconjugate candidates of the present disclosure is carried out at a dose within a range having a lower limit selected from about 1 pg, 2 pg, 3 pg, 4 pg, 5 pg, 6 pg, 7 pg, 8 pg, 9 pg, 10 pg, 11 pg, 12 pg, 13 pg, 14 pg, 15 pg, 16 pg, 17 pg, 18 pg, 19 pg, 20 pg, 21 pg, 22 pg, 23 pg, 24 pg, 25 pg, 26 pg,

27 pg, 28 pg, 29 pg, 30 pg, 31 pg, 32 pg, 33 pg, 34 pg, 35 pg, 36 pg, 37 pg, 38 pg, 39 pg, 40 pg, 41 pg, 42 pg, 43 pg, 44 pg, 45 pg, 46 pg, 47 pg, 48 pg, 49 pg, 50 pg, 51 pg, 52pg, 53 pg,

54 pg, 55 pg, 56 pg, 57 pg, 58 pg, 59 pg, 60 pg, 61 pg, 62 pg, 63 pg, 64 pg, 65 pg, 66 pg, 67 pg, 68 pg, 69 pg, 70 pg, 71 pg, 72 pg, 73 pg, 74 pg, 75 pg, 76 pg, 77 pg, 78 pg, 79 pg, 80 pg,

81 pg, 82 pg, 83 pg, 84 pg, 85 pg, 86 pg, 87 pg, 88 pg, 89 pg, 90 pg, 91 pg, 92 pg, 93 pg, 94 pg, 95 pg, 96 pg, 97 pg, 98 pg, and 99 pg; and an upper limit selected from about 2 pg, 3 pg, 4 pg, 5 pg, 6 pg, 7 pg, 8 pg, 9 pg, 10 pg, 11 pg, 12 pg, 13 pg, 14 pg, 15 pg, 16 pg, 17 pg, 18 pg, 19 pg, 20 pg, 21 pg, 22 pg, 23 pg, 24 pg, 25 pg, 26 pg, 27 pg, 28 pg, 29 pg, 30 pg, 31 pg, 32 pg, 33 pg, 34 pg, 35 pg, 36 pg, 37 pg, 38 pg, 39 pg, 40 pg, 41 pg, 42 pg, 43 pg, 44 pg, 45 pg,

46 pg, 47 pg, 48 pg, 49 pg, 50 pg, 51 pg, 52pg, 53 pg, 54 pg, 55 pg, 56 pg, 57 pg, 58 pg, 59 Hg, 60 pg, 61 pg, 62 pg, 63 pg, 64 pg, 65 pg, 66 pg, 67 pg, 68 pg, 69 pg, 70 pg, 71 pg, 72 pg,

73 pg, 74 pg, 75 pg, 76 pg, 77 pg, 78 pg, 79 pg, 80 pg, 81 pg, 82 pg, 83 pg, 84 pg, 85 pg, 86 pg, 87 pg, 88 pg, 89 pg, 90 pg, 91 pg, 92 pg, 93 pg, 94 pg, 95 pg, 96 pg, 97 pg, 98 pg, 99 pg, and 100 pg.

[0146] In some embodiments, the step of immunizing each of the plurality of mice (or each of the mice within a group of the plurality of mice) with distinct highly immunogenic glycoconjugate candidates of the plurality of distinct highly immunogenic glycoconjugate candidates of the present disclosure is carried out at a dose of about 1 pg - about 100 pg of the highly immunogenic glycoconjugate candidate. In accordance with such embodiments, the immunizing may be carried out at a dose of about 10 pg of the highly immunogenic glycoconjugate candidate.

[0147] The term “booster” refers to an additional dose of the highly immunogenic glycoconjugate candidate following the initial dose. As noted above, the step of immunizing may be carried out one or more times. Thus, the step of immunizing may be carried out with an initial dose of a highly immunogenic glycoconjugate candidate and one or more subsequent doses of the same highly immunogenic glycoconjugate candidate. In accordance with such embodiments, the step of immunizing involves a first dose and one or more booster doses.

[0148] In some embodiments, when the immunizing step is carried out more than once, the time between each immunizing step may be, but is not limited to, 1 day, 2 days, 3 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, or any amount of time there between.

[0149] In some embodiments, when the immunizing step is carried out twice, the time between the initial dose and the second (or booster dose) is 21 days. In some embodiments, when the immunizing step is carried out three times, the time between the initial dose and the second (or first booster dose) is 21 days and the time between the second dose (or first booster dose) and the third dose (or second booster dose) is 21 days. This, in some embodiments, the immunizing step is carried out on day 0, day 21, and day 42.

[0150] In some embodiments of the method of identifying glycoconjugate vaccine candidates described herein, immunizing each of the plurality of mice with distinct highly immunogenic glycoconjugate candidates of the plurality of distinct highly immunogenic glycoconjugate candidates of the present disclosure may be carried out using methods known in the art including parenteral, topical, intravenous, oral, subcutaneous, intraperitoneal, intranasal, or intramuscular means. In some embodiments, the plurality of distinct highly immunogenic glycoconjugate candidates of the present disclosure are formulated for injection. In accordance with such embodiments, the plurality of distinct highly immunogenic glycoconjugate candidates of the present disclosure may be formulated for intramuscular injection, intraperitoneal injection, intraarterial injection, intracranial injection, or intradermal injection.

[0151] In some embodiments, the plurality of distinct highly immunogenic glycoconjugate candidates of the present disclosure are formulated for intramuscular injection. In accordance with such embodiments, immunizing each of the plurality of mice with distinct highly immunogenic glycoconjugate candidates of the plurality of distinct highly immunogenic glycoconjugate candidates of the present disclosure may is carried out by intramuscular injection. In some embodiments, this type of injection is performed in the arm or leg muscles. [0152] As used herein, the term “immune response” refers to the development in a subject of a humoral and/or a cellular immune response to, e.g., a glycoconjugate according to 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.

[0153] An immune response may include one or more of the following effects: the production of antibodies by B-cells and/or the activation of suppressor, cytotoxic, or helper T- cells and/or 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 antibodycomplement, or antibody-dependent cell cytotoxicity (ADCC) to provide protection to an immunized host. Such responses can be determined using standard immunoassays and neutralization assays well known in the art.

[0154] Thus, the step of immunizing each of the plurality of mice with distinct highly immunogenic glycoconjugate candidates of the plurality of distinct highly immunogenic glycoconjugate candidates of the present disclosure, may be effective to induce a humoral immune response and/or a cellular immune response.

[0155] In some embodiments of the method of identifying glycoconjugate vaccine candidates described herein, the immune response is a humoral immune response.

[0156] The presence of a humoral immune response can be determined and monitored by testing a biological sample (e.g., blood, plasma, serum, urine, saliva feces, CSF or lymph fluid) from an immunized mouse for the presence of antibodies directed to a component of the glycoconjugate candidate used to immunize the immunization mouse or the presence of antibodies directed to, e.g., a polysaccharide of interest. Methods for detecting antibodies in a biological sample are well known in the art, e.g, ELISA, Dot blots, SDS-PAGE gels or ELISPOT. The presence of a cell-mediated immunological response can be determined by proliferation assays (CD4⁺ T cells) or CTL (cytotoxic T lymphocyte) assays which are known in the art.

[0157] In some embodiments of the method of identifying glycoconjugate vaccine candidates described herein, the step of evaluating an immune response of each of the plurality of mice to each of the highly immunogenic glycoconjugate candidates is carried out by determining mouse serum antibody titers against a polysaccharide of interest.

[0158] As used herein, the term “immunoassay” refers to a biochemical test that measures the presence or concentration of a substance in a sample, such as a biological sample. It is common to use the reaction of an antibody to its cognate antigen, for example the specific binding of an antibody to a protein. Both the presence of antigen and the amount of antigen present can be measured. The presence and amount (z.e., abundance) of the protein can determined or measured. Measuring the quantity of antigen (such as a biomarker) can be achieved by a variety of methods. A common method is to label either the antigen or antibody with a detectable label (e.g., a fluorescent tag, enzymatic linkage or radioactive isotope).

[0159] As used herein, the term “enzyme-linked immunosorbent assay” or “ELISA” refers to an assay that uses a solid-phase type of enzyme immunoassay (EIA). In ELISA assays, one of the reaction components is nonspecifically adsorbed or covalently bound to the surface of a solid phase, such as a microtiter well, a magnetic particle, or a plastic bead. This attachment facilitates the separation of bound and free-labeled reactants.

[0160] Specific antibodies in a sample can be quantified using an ELISA procedure in which an antigen (e.g., a glycoconjugate, a polysaccharide of interest, or a carrier protein according to the present disclosure) is bound to a solid phase. A sample may then be contacted with the solid phase to allow binding to the antigen-specific antibodies, if present, to bind to the antigen. Any unbound antibodies may be removed by one or more washing steps and bound antibodies may be linked to an enzyme. In the final step, a substance containing the enzyme’s substrate is added. If there the sample contained an antigen-specific antibody, the subsequent reaction between the enzyme and substrate produces a detectable signal, typically a color change. There are variations of ELISA tests based on how the analytes and antibodies are bonded and used which include Direct ELISA, Sandwich ELISA, Competitive ELISA and Reverse ELISA. [0161] In some embodiments of the method of identifying glycoconjugate vaccine candidates described herein, serum antibody titers are determined by ELISA. In accordance with such embodiments, serum antibody titers are determined by measuring the highest dilution that results in a signal to a polysaccharide of interest which is three standard deviations above a control (e.g., a no-serum background control).

[0162] In some embodiments, the serum antibody titers are IgM serum antibody titers, IgM serum antibody titers, IgG serum antibody titers, IgA serum antibody titers, IgD serum antibody titers, and/or IgE serum antibody titers.

[0163] The first antibodies to be produced in a humoral immune response are IgM antibodies, because IgM can be expressed without isotype switching. These early IgM antibodies are produced before B cells have undergone somatic hypermutation and therefore tend to be of low affinity. In some embodiments, the antibody titers are IgM antibody titers.

[0164] Mice typically encode for IgGl, IgG2b, and IgG3 and, depending on their strain, will express either IgG2a or IgG2c (see, e.g., Collins, A., “IgG Subclass Co-Expression brings Harmony to the Quartet Model of Murine IgG Function,” Immunology & Cell Biology 94(10): 949-954 (2016), which is hereby incorporated by reference in its entirety). IgG2a, IgG2b, and IgG2c subclasses have similar functions (see, e.g., Collins, A., “IgG Subclass Co-Expression brings Harmony to the Quartet Model of Murine IgG Function,” Immunology & Cell Biology 94(10): 949-954 (2016), which is hereby incorporated by reference in its entirety). In some embodiments, the serum antibody titers are IgGl serum antibody titers selected from the group consisting of IgG2a serum antibody titers, IgG2b serum antibody titers, IgG2c serum antibody titers, and/or IgG3 serum antibody titers.

[0165] Inbred mouse strains, such as BALB/c and Swiss Webster mice, possess the Ighl- a allele, which results in the expression of IgG2a, the gene for IgG2c being absent. Thus, in some embodiments, the antibody titers are IgGl and/or IgG2a serum antibody titers.

[0166] In mouse strains such as C57B1/6 and C57B1/10, the IgG2a gene is deleted. The possession of an Ighl-b allele results in the expression of IgG2c instead of the IgG2a subclass (Martin et al., “The Need for IgG2c Specific Antiserum when Isotyping Antibodies from C57BL/6 and NOD Mice,” J. Immunol. Methods 212(2): 187-192 (1998), which is hereby incorporated by reference in its entirety). Thus, in some embodiments, the antibody titers are IgGl and/or IgG2c serum antibody titers.

[0167] In some embodiments, the polysaccharide of interest is derived from any one of the following: a bacterium, a virus, a parasite, or a cancer. In some embodiments, the polysaccharide of interest is derived from a bacterium. The bacterium may be a pathogenic bacterium.

[0168] In some embodiments, each distinct polysaccharide is derived from a bacterium. In some embodiments, the bacterium is a pathogenic bacterium. [0169] In some embodiments, the bacterium is a gram-positive bacterium. The grampositive bacterium may be selected from the group consisting of Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Erysipelothrix rhusiopathiae, Gardnerella vaginalis, Group A Streptococcus, Group B Streptococcus, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Nocardia asteroids, Propionibacterium acnes, Rhodococcus equi, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus mutans, Streptococcus pneumoniae, and Streptococcus pyogenes.

[0170] In some embodiments, the bacterium is a gram-negative bacterium. The gramnegative-bacterium may be selected from the group consisting of Acinetobacter baumannii, Bordetella pertussis, Brucella abortus, Burkholderia pseudomallei, Burkholderia mallei, Burkholderia pseudomallei, Campylobacter fetus, Campylobacter jejuni, Coxiella burnetii, Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumoniae, Legionella pneumophila, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia prowazekii, Salmonella enterica, Salmonella typhi, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Vibrio cholerae, Yersinia enter ocolitica, Yersinia pestis, and Yersinia pseudotuberculosis.

[0171] In some embodiments, the bacterium is Francisella tularensis.

[0172] In some embodiments, the bacterium is Escherichia coli. Exemplary pathogenic

E. coli strains include, without limitation, enterotoxigenic Escherichia coli (ETEC), enterohemorrhagic Escherichia coli (EHEC), extraintestinal pathogenic Escherichia coli (ExPEC).

[0173] In some embodiments, when the bacterium is Francisella tularensis, the polysaccharide of interest may be Francisella tularensis lipopolysaccharides (FtLPS).

[0174] Also disclosed are the glycoconjugate vaccine candidates identified by the methods of identifying glycoconjugate vaccine candidates according to the present disclosure.

Methods of Identifying scFvs Specific for a Polysaccharide of Interest

[0175] An “antibody” or “Ab” is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. [0176] As used herein, the term “antibody” encompasses not only intact polyclonal or monoclonal antibodies, but also any antigen binding portion (e.g., “antigen-binding fragment”) thereof of an intact antibody that retains the ability to specifically bind to a given antigen (e.g., a carbohydrate antigen) or single chain thereof, fusion proteins comprising an antibody, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site, for example without limitation, Fab; Fab'; F(ab')2; an Fd fragment; an Fv fragment; a single domain antibody (dAb) fragment; an isolated complementarity determining region (CDR); single chain (scFv) and single domain antibodies (e.g., shark and camelid antibodies), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23(9): 1126-1136 (2005), which is hereby incorporated by reference in its entirety).

[0177] An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or subclass thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, and IgG3. The heavy chain (HC) constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0178] A single immunoglobulin molecule is comprised of two identical light chains (LCs) and two identical heavy chains (HCs). Light chains are composed of one constant domain (CL) and one variable domain (VL) while heavy chains are consist of three constant domains (CHi, CH2 and CH3) and one variable domain (VH).

[0179] Each heavy chain variable region (VH) and each light chain variable region (VL) comprises relatively invariant stretches called framework regions (FRs) separated by shorter regions of extreme variability called “hypervariable regions” or “complementary determining regions (CDRs)”, arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0180] Together, the VH and VL domains compose the antigen-binding portion of the molecule known as the Fv. The Fc portion is glycosylated at a conserved Asn297 residue. Attachment of N-glycan at this position results in an “open” conformation that is essential for effector interaction. [0181] A “single-chain variable fragment (scFv)” is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with an amino acid linker. The scFv retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker.

[0182] Another aspect of the present disclosure relates to a method of identifying scFvs specific for a polysaccharide of interest. This method involves providing a second plurality of murine B cell organoids; contacting each of the second plurality of murine B cell organoids with the plurality of distinct highly immunogenic glycoconjugate candidates according to the present disclosure, where the contacting is carried out to form a second plurality of contacted B cell organoids; culturing the second plurality of contacted B cell organoids under conditions effective to support B cell maturation, whereby said culturing produces a second plurality of cultured B cell organoids; enzymatically digesting the second plurality of cultured B cell organoids; isolating the cultured B cells to produce a plurality of isolated B cells; sequencing the plurality of isolated B cells to identify nucleic acid sequences encoding heavy chain variable region (VH) and light chain variable region (VL) genes; randomly joining VH and VL genes with a nucleic acid sequence encoding a glycine-serine linker to generate a library of candidate single chain variable fragments (scFvs); cloning each of the library of candidate scFvs into a yeast cell surface expression vector; transforming a plurality of yeast cells with the library of yeast cell surface expression vectors to produce a plurality of transformed yeast cells; expressing the library of yeast expression vectors in the plurality of transformed yeast cells to produce a library of scFv- expressing yeast cells, whereby the candidate scFvs are displayed on the surface of the yeast cells; detecting binding of a fluorescently labelled glycoconjugate to each of the library of scFv- expressing yeast cells, where the fluorescently labelled glycoconjugate comprises the polysaccharide of interest and a carrier protein that is different than the carrier protein of the plurality of distinct highly immunogenic glycoconjugate candidates; and identifying scFvs specific for the polysaccharide of interest based on said detecting.

[0183] Suitable murine B cell organoids are described in detail supra. In some embodiments, the murine B cells are naive murine B cells, primary murine B cells, or primary naive murine B cells. The murine B cells may be splenic murine B cells. In some embodiments, the murine B cells are CD19⁺ B cells.

[0184] The second plurality of murine B cell organoids may be configured so as to be amenable for high throughput screening. Thus, in some embodiments, the second plurality of murine B cell organoids are organized in an array. Conventional multi-well plates, dishes, tissue culture plates, or glass coverslips can be used to prepare the array. [0185] In some embodiments, the array is a 6-well array, a 12-well array, a 48-well array, a 96-well array, a 384-well array, or any other configuration of an array. In some embodiments, the array is larger than a 384-well array. In accordance with such embodiments, the second plurality of B cell organoids may be seeded in a 6-well plate, a 12-well plate, a 48-well plate, a 96-well plate, a 384-well plate, or larger culture platform.

[0186] In some embodiments, the second plurality of murine B cell organoids are organized in one or more arrays.

[0187] In some embodiments, contacting is carried out at a dose within a range having a lower limit selected from about 0.1 pM/organoid, 0.2 pM/organoid, 0.3 pM/organoid, 0.4 pM/organoid, 0.5 pM/organoid, 0.6 pM/organoid, 0.7 pM/organoid, 0.8 pM/organoid, 0.9 pM/organoid, 1.0 pM/organoid, 1.1 pM/organoid, 1.2 pM/organoid, 1.3 pM/organoid, 1.4 pM/organoid, 1.5 pM/organoid, 1.6 pM/organoid, 1.7 pM/organoid, 1.8 pM/organoid, 1.9 pM/organoid, 2.0 pM/organoid, 2.1 pM/organoid, 2.2 pM/organoid, 2.3 pM/organoid, 2.4 pM/organoid, 2.5 pM/organoid, 2.6 pM/organoid, 2.7 pM/organoid, 2.8 pM/organoid, 2.9 pM/organoid, 3.0 pM/organoid, 3.1 pM/organoid, 3.2 pM/organoid, 3.3 pM/organoid, 3.4 pM/organoid, 3.5 pM/organoid, 3.6 pM/organoid, 3.7 pM/organoid, 3.8 pM/organoid, 3.9 pM/organoid, 4.0 pM/organoid, 4.1 pM/organoid, 4.2 pM/organoid, 4.3 pM/organoid, 4.4 pM/organoid, 4.5 pM/organoid, 4.6 pM/organoid, 4.7 pM/organoid, 4.8 pM/organoid, 4.9 pM/organoid, 5.0 pM/organoid, 6.1 pM/organoid, 6.2 pM/organoid, 6.3 pM/organoid, 6.4 pM/organoid, 6.5 pM/organoid, 6.6 pM/organoid, 6.7 pM/organoid, 6.8 pM/organoid, 6.9 pM/organoid, 7.0 pM/organoid, 7.1 pM/organoid, 7.2 pM/organoid, 7.3 pM/organoid, 7.4 pM/organoid, 7.5 pM/organoid, 7.6 pM/organoid, 7.7 pM/organoid, 7.8 pM/organoid, 7.9 pM/organoid, 8.0 pM/organoid, 8.1 pM/organoid, 8.2 pM/organoid, 8.3 pM/organoid, 8.4 pM/organoid, 8.5 pM/organoid, 8.6 pM/organoid, 8.7 pM/organoid, 8.8 pM/organoid, 8.9 pM/organoid, 9.0 pM/organoid, 9.1 pM/organoid, 9.2 pM/organoid, 9.3 pM/organoid, 9.4 pM/organoid, 9.5 pM/organoid, 9.6 pM/organoid, 9.7 pM/organoid, 9.8 pM/organoid, and 9.9 pM/organoid; and an upper limit selected from about 0.2 pM/organoid, 0.3 pM/organoid, 0.4 pM/organoid, 0.5 pM/organoid, 0.6 pM/organoid, 0.7 pM/organoid, 0.8 pM/organoid, 0.9 pM/organoid, 1.0 pM/organoid, 1.1 pM/organoid, 1.2 pM/organoid, 1.3 pM/organoid, 1.4 pM/organoid, 1.5 pM/organoid, 1.6 pM/organoid, 1.7 pM/organoid, 1.8 pM/organoid, 1.9 pM/organoid, 2.0 pM/organoid, 2.1 pM/organoid, 2.2 pM/organoid, 2.3 pM/organoid, 2.4 pM/organoid, 2.5 pM/organoid, 2.6 pM/organoid, 2.7 pM/organoid, 2.8 pM/organoid, 2.9 pM/organoid, 3.0 pM/organoid, 3.1 pM/organoid, 3.2 pM/organoid, 3.3 pM/organoid, 3.4 pM/organoid, 3.5 pM/organoid, 3.6 pM/organoid, 3.7 pM/organoid, 3.8 pM/organoid, 3.9 gM/organoid, 4.0 gM/organoid, 4.1 gM/organoid, 4.2 gM/organoid, 4.3 gM/organoid, 4.4 gM/organoid, 4.5 gM/organoid, 4.6 gM/organoid, 4.7 gM/organoid, 4.8 gM/organoid, 4.9 gM/organoid, 5.0 gM/organoid, 6.1 gM/organoid, 6.2 gM/organoid, 6.3 gM/organoid, 6.4 gM/organoid, 6.5 gM/organoid, 6.6 gM/organoid, 6.7 gM/organoid, 6.8 gM/organoid, 6.9 gM/organoid, 7.0 gM/organoid, 7.1 gM/organoid, 7.2 gM/organoid, 7.3 gM/organoid, 7.4 gM/organoid, 7.5 gM/organoid, 7.6 gM/organoid, 7.7 gM/organoid, 7.8 gM/organoid, 7.9 gM/organoid, 8.0 gM/organoid, 8.1 gM/organoid, 8.2 gM/organoid, 8.3 gM/organoid, 8.4 gM/organoid, 8.5 gM/organoid, 8.6 gM/organoid, 8.7 gM/organoid, 8.8 gM/organoid, 8.9 gM/organoid, 9.0 gM/organoid, 9.1 gM/organoid, 9.2 gM/organoid, 9.3 gM/organoid, 9.4 gM/organoid, 9.5 gM/organoid, 9.6 gM/organoid, 9.7 gM/organoid, 9.8 gM/organoid, 9.9 gM/organoid, and 10 gM/organoid. In some embodiments, contacting is carried out at a dose of about 0.1 gM/organoid - about 10 gM/organoid. In some embodiments, contacting is carried out at a dose of about 0.5 gM/organoid - about 5 gM/organoid. In some embodiments, contacting is carried out at a dose of 0.5 gM/organoid - 5 gM/organoid. In some embodiments, said contacting is carried out at a dose of about 1.75 gM/organoid.

[0188] In some embodiments, culturing is carried out for at least 2 days, at least 3 days, at least 4 days, at least 5 days, or more. In some embodiments, culturing is carried out for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or more. In some embodiments, culturing is carried out for 4 days.

[0189] Suitable growth factors are described in detail supra. In some embodiments, culturing is carried out in the presence of one or more growth factors or cytokines. In some embodiments, the one or more growth factors or cytokines is IL-4, IL-21, B cell activating factor (BAFF), or a combination thereof.

[0190] In some embodiments, enzymatically digesting the second plurality of cultured B cell organoids was carried out by contacting each of the second plurality of cultured B cell organoids with a solution comprising an enzyme. In some embodiments, the enzyme is at a concentration of 1 to 200 U/mL. In some embodiments, the solution has a concentration of enzyme within a range having a lower limit selected from about 1 U/mL, 2 U/mL, 3 U/mL, 4 U/mL, 5 U/mL, 6 U/mL, 7 U/mL, 8 U/mL, 9 U/mL, 10 U/mL, 20 U/mL, 30 U/mL, 40 U/mL, 50 U/mL, 60 U/mL, 70 U/mL, 80 U/mL, 90 U/mL, 100 U/mL, 120 U/mL, 130 U/mL, 140 U/mL, 150 U/mL, 160 U/mL, 170 U/mL, 180 U/mL, and 190 U/mL; and an upper limit selected from about 2 U/mL, 3 U/mL, 4 U/mL, 5 U/mL, 6 U/mL, 7 U/mL, 8 U/mL, 9 U/mL, 10 U/mL, 20 U/mL, 30 U/mL, 40 U/mL, 50 U/mL, 60 U/mL, 70 U/mL, 80 U/mL, 90 U/mL, 100 U/mL, 120 U/mL, 130 U/mL, 140 U/mL, 150 U/mL, 160 U/mL, 170 U/mL, 180 U/mL, 190 U/mL, and 200 U/mL. In some embodiments, the enzyme is at a concentration of about 50 to about 150 U/mL. the enzyme is at a concentration of about 100 U/mL to 150 U/mL. In some embodiments, the enzyme is at a concentration of about 125 U/mL.

[0191] In some embodiments, the enzyme is collagenase type 1. In accordance with such embodiments, enzymatically digesting is carried out by contacting each of the second plurality of cultured B cell organoids with a solution comprising about 50 to about 150 U/mL collagenase type 1, about 100 U/mL to 150 U/mL collagenase type 1, or about 125 U/mL collagenase type 1. [0192] In some embodiments, enzymatically digesting is carried out for at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, or more. In some embodiments, enzymatically digesting is carried out for at least about 60 minutes. In some embodiments, enzymatically digesting produces hydrogel debris.

[0193] Methods of isolated cultured cells are well known in the art. In some embodiments, cultured B cells can be isolated from, e.g., hydrogel debris, by washing cells and/or filtering cells (e.g., with a mesh filter as described in the Examples of the present disclosure).

[0194] In some embodiments, sequencing is carried out by extracting RNA from the isolated B cells and performing RT-PCT on the extracted RNA to generate cDNA libraries. In some embodiments, sequence further involves amplifying heavy chain variable region (VH) and light chain variable region (VL) genes from the cDNA.

[0195] As used herein, the term “linker” refers to a molecule joining two or more amino acids, or two or more peptides together. The length and composition is generally selected taking into consideration the intended function of the linker.

[0196] In some embodiments, randomly joining VH and VL genes involves connecting the N-terminus of the VH with the C-terminus of the VL through the nucleic acid sequence encoding a linker. In some embodiments, randomly joining VH and VL genes involves connecting the N-terminus of the VL with the C-terminus of the VH through the nucleic acid sequence encoding a linker.

[0197] The length of and composition of the linker is generally selected taking into consideration the intended function of the linker. For example, the linker can be peptide which includes one or more amino acids, such as from 1 to about 50 amino acid residues. Accordingly, the linker may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid residues. [0198] In accordance with this aspect of the invention, the nucleic acid sequence encodes a linker comprising at least 10 amino acid residues, at least 11 amino acid residues, at least 12 amino acid residues, at least 13 amino acid residues, at least 14 amino acid residues, at least 15 amino acid residues, at least 16 amino acid residues, at least 17 amino acid residues, at least 18 amino acid residues, at least 19 amino acid residues, at least 20 amino acid residues, at least 21 amino acid residues, at least 22 amino acid residues, at least 23 amino acid residues, at least 24 amino acid residues, at least 25 amino acid residues, at least 26 amino acid residues, at least 27 amino acid residue s, at least 28 amino acid residues, at least 29 amino acid residues, at least 30 amino acid residues, at least 31 amino acid residues, at least 32 amino acid residues, at least 33 amino acid residues, at least 34 amino acid residues, at least 35 amino acid residues, at least 36 amino acid residues, at least 37 amino acid residues, at least 38 amino acid residues, at least 39 amino acid residues, at least 40 amino acid residues, at least 41 amino acid residues, at least 42 amino acid residues, at least 43 amino acid residues, at least 44 amino acid residues, at least 45 amino acid residues, at least 46 amino acid residues, at least 47 amino acid residues, at least 48 amino acid residues, at least 49 amino acid vs, or at least 50 amino acid residues.

[0199] Suitable linkers may include one or more of the following amino acid residues in any combination: Gly, Ser, Ala, or Thr. In some embodiments, the link er is from 1 to 30 amino acids in length, 1 to 20 amino acids in length, 1 to 10 amino acids in length, or 1 to 5 amino acids in length. Exemplary peptide linkers include glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers.

[0200] Exemplary glycine-serine linkers include (GS)n, (GGS)n, (GGGS; SEQ ID NO: 13)n, (GGSG; SEQ ID NO: 9)n (GGSGG, SEQ ID NO: 10)n, (GSGGS, SEQ ID NO: 1 l)n, and (GGGGS, SEQ ID NO: 12)n, wherein n is an integer of at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In one exemplary embodiment, the linker is GGGS (SEQ ID NO: 13). In another exemplary embodiment, the linker is GGGGS (SEQ ID NO: 12). In some embodiments, the linker is a (Gly4Ser)4 linker having the sequence of SEQ ID NO: 4.

[0201] In some embodiments, the vector is a pCT-CON vector.

[0202] In some embodiments, the yest cells are Saccharomyces cerevisiae cells. In some embodiments, the yeast cells are Saccharomyces cerevisiae strain EBY100 cells.

[0203] In some embodiments, the polysaccharide of interest is derived from any one of the following: a bacterium, a virus, a parasite, or a cancer.

[0204] In some embodiments, the polysaccharide of interest is derived from a bacterium. The bacterium may be a gram-positive or a gram-negative bacterium. Suitable gram-positive and gram-negative bacteria are described in detail supra. [0205] In some embodiments, the bacterium is Francisella tularensis.

[0206] In some embodiments, the bacterium is Escherichia coli. Exemplary pathogenic

E. coli strains are described in detail supra.

[0207] In some embodiments, the polysaccharide of interest is conjugated to a carrier protein. Suitable carrier proteins are described in detail infra. In some embodiments, the polysaccharide of interest is conjugated to a carrier protein and the carrier protein is maltose binding protein (MBP).

[0208] The above disclosure generally describes the present disclosure. A more specific description is provided below in the following examples. The examples are described solely for the purpose of illustration and are not intended to limit the scope of the present disclosure. Changes in form and substitution of equivalents are contemplated as circumstances suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

EXAMPLES

[0209] The examples below are intended to exemplify the practice of embodiments of the disclosure but are by no means intended to limit the scope thereof.

Materials and Methods

Strains and Plasmids

[0210] E. coli strain CLM24 was used for all protein expression work. Plasmids used in this study included pGAB2 encoding the F. tularensis O-PS antigen biosynthesis pathway (Cuccui et al., “Exploitation of Bacterial N-Linked Glycosylation to Develop a Novel Recombinant Glycoconjugate Vaccine Against Francisella tularensis,” Open Biol. 3(5): 130002 (2013), which is hereby incorporated by reference in its entirety); pMAFlO encoding an hemagglutinin (HA)-tagged version of C/PglB in plasmid pMLBAD (Boder and Wittrup, “Yeast Surface Display for Screening Combinatorial Polypeptide Libraries,” Nat. Biotechnol. 15(6): 553-557 (1997), which is hereby incorporated by reference in its entirety); pTrc-spDsbA-MBP- GT encoding the MBP carrier protein modified at its N-terminus with the E. coli DsbA signal peptide and at its C-terminus with four tandem repeats of the DQNAT glycosylation motif and a polyhistidine (6xHis) tag in plasmid pTrc99A (Fisher et al., “Production of Secretory and Extracellular N-Linked Glycoproteins in Escherichia coli,” AppL Environ. Microbiol. 77(3): 871- 881 (2011), which is hereby incorporated by reference in its entirety); and pTrc-spDspA- CRM197-GT encoding an identical construct except with CRM197 cloned in place of MBP. S. cerevisiae strain EBY100 was used for cell surface display of scFv libraries (Chao et al., “Isolating and Engineering Human Antibodies Using Yeast Surface Display,” Nat. Protoc.

1 (2):755— 768 (2006), which is hereby incorporated by reference in its entirety), and S. cerevisiae strain YVH10 (Sterner et al., “Therapeutic Antibodies to Ganglioside GD2 Evolved from Highly Selective Germline Antibodies,” Cell Rep 20(7): 1681-1691 (2017), which is hereby incorporated by reference in its entirety) was used for extracellular secretion of scFv hits isolated by FACS. Plasmid pCT-CON was used for both cell-surface and extracellular expression of scFv clones (Reddy et al., “Monoclonal Antibodies Isolated Without Screening by Analyzing the Variable-Gene Repertoire of Plasma Cells,” Nat. Biotechnol. 28(9):965-969 (2010), which is hereby incorporated by reference in its entirety).

Glycoconjugate Expression and Purification

[0211] To prepare glycoconjugates, CLM24 cells transformed with pGAB2, pMAFlO, and either pTrc-spDspA-MBP-GT or pTrc-spDspA-CRMi97-GT were cultured in Luria-Bertani (LB) broth at 30°C until the optical density at 600 nm (ODeoo) reached ~0.8. At this point, C/PglB expression was induced with 0.2% arabinose (w/v) for 16 hours at 30°C, and then expression of the spDspA-MBP-GT or spDspA-CRM -GT carrier proteins was induced with 1 mM isopropyl P-D-l -thiogalactopyranoside (IPTG) for an additional 8 hours. To prepare aglycosylated carrier proteins, a similar protocol was followed but using CLM24 cells that were transformed with only the pTrc-spDsbA-MBP-GT or pTrc-spDsbA-CRMi97-GT plasmid and induced with 1 mM IPTG for 16 hours at 30°C. Following protein expression, cultures were spun down and stored at -20°C. Frozen cell pellets were resuspended in 10 mL of equilibration buffer (20 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole) per 50 mL culture volume. After cells were fully resuspended in solution, cells were lysed via homogenization (Avestin EmulsiFlex) through three cycles at 15000 psi. The lysate was spun down at 15000 rpm for 30 minutes at 4°C. Ni-NTA resin (1 mL per 100 mL cell culture) was washed with equilibration buffer three times, added to lysate supernatant, and rotated 1 hour at 4 °C. Lysate resin was applied to a 5 mL column (Pierce) followed by three column volumes (CV) of equilibration buffer and one CV of wash buffer (20 mM phosphate, 300 mM NaCl, 25 mM imidazole).

Fractions were collected by addition of 1 mL elution buffer (20 mM sodium phosphate, 300 mM NaCl, 250 mM imidazole) to column. Elution fractions containing protein were pooled and buffer exchanged into phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KC1, 8 mM Na2HPO4, and 2 mM KH2PO4, pH 7.4) with 10K molecular weight cutoff (MWCO) protein concentrators (Pierce). Protein Analysis and Imnuinobloting

[0212] Purified protein samples were loaded onto 10% SDS-PAGE gels (Bio-Rad) and separated at 200 V. Detection of proteins was performed by staining gels with Coomassie blue (Bio-Rad) and by immunoblot analysis. For the latter, proteins were transferred to nitrocellulose membranes (Bio-Rad), blocked with 5% (w/v) bovine serum albumin (BSA)-TBST at room temperature for at least 1 hour, washed three times with TBST (TBS, 0.05% (v/v) Tween 20) for 10 min, and probed with anti-His antibody clone ADI.1.10 (Bio-Rad Cat # MCA1396GA;

1 :5000 dilution), anti-F tularensis LPS antibody clone FBI 1 (Invitrogen; Cat # MAI -21690; 1 : 10000 dilution), goat antimouse DyLight 800 antibody (Bio-Rad Cat # STAR117D800GA; 1 :2500 dilution), and goat antirabbit StarBright Blue 700 antibody (Bio-Rad Cat # 12004161; 1 :2500 dilution). Blots were detected by fluorescence using a ChemiDoc MP imager (Bio-Rad).

Immune Organoid Fabrication

[0213] Wildtype B cells were purified from spleens harvested from 10-to-18-week-old female C57BL/6 mice (Jackson Laboratory). After red blood cell lysis, naive B cells were isolated from splenocyte mixtures with EasySep Mouse B Cell Isolation Kit (Stem Cell Technologies). 40LB cells, which are NH4/3T3 fibroblasts genetically engineered to express CD40L and BAFF, were obtained from Dr. Daisuke Kitamura and generated as previously described (Purwada et al., “Modular Immune Organoids with Integrin Ligand Specificity Differentially Regulate Ex Vivo B Cell Activation,” ACS Biomater Sci. Eng. 3(2):214-225 (2017), which is hereby incorporated by reference in its entirety). 40LB cells were cultured with high glucose Dulbecco’s Modified Eagle Medium (DMEM) medium containing 10% (v/v) FBS and 1% (w/v) penicillin streptomycin (P/S) with all components obtained from ThermoFisher Scientific. Prior to encapsulation in organoids, 40LB cells were mitotically inhibited via incubation in cell culture complete medium containing 0.01 mg/mL mitomycin C (Sigma- Aldrich) at 37°C for 45 minutes. 40LB cells were then rinsed twice with 10 mL of PBS, detached with trypsin, and counted before encapsulation.

[0214] Immune organoids containing 7.5% (w/v) PEG-4MAL were fabricated using four-arm PEG-4MAL macromer with 20-kDa molecular weight with >90% purity (Laysan Bio), adhesive thiolated peptides, and dithiolated crosslinkers. PEG-4MAL macromers were initially functionalized with thiolated adhesive integrin a4bi-binding REDV peptide (GREDVGC (SEQ ID NO: 2), >90% purity; AAPPTec) with a 4: 1 MAL-to peptide molar ratio for 30 minutes at 37°C. Matrix metalloproteinase (MMP)-9 degradable VPM peptide (GCRDVPMSMRGGDRCG (SEQ ID NO: 1), >90% purity; AAPPTec) and nondegradable dithiothreitol (DTT) crosslinkers were combined at a 50:50 VPM-to-DTT molar ratio. All components were diluted using PBS supplemented with calcium and magnesium (PBS⁺⁺, pH 7.4) with 1% (v/v) 4-(2-hydroxyethyl)-l-piperazineethanesulfonicacid (HEPES). 40000 naive B cells and 80000 40LB stromal cells per organoid were suspended in the cross-linker solution prior to cell encapsulation. After 5 pL of PEG-4MAL macromer solution was placed in the middle of a well of a nontreated 96-well plate, 5 pL of cell-containing crosslinker solution was injected into the droplet and mixed by pipetting 5 times. Hydrogel droplets were prepared and cured for 15 minutes at 37°C for complete cross-linking. Fresh Roswell Park Memorial Institute (RPMI 1640) medium supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (w/v) P/S, and 10 ng/mL IL-4 (Peprotech) was then added to each immune organoid. Purified carrier proteins, with or without conjugated FtO-PS antigens, in PBS were filter-sterilized using 0.2 pm syringe filters (Nalgene) and administered to organoids at a final concentration of 1.75 pM.

Flow Cytometry Analysis

[0215] After a specified culture time, typically 4 days, cells were harvested from organoids with 1 hour enzymatic degradation accomplished using a solution of 125 U/mL of collagenase type 1 (Worthington Biochemical) dissolved in serum-free RPMI medium. Gel debris was removed from cell suspensions using Multi Screen-Mesh filter plates with 96-well receiver plate (EMD Millipore). Cells were washed with PBS and then FACS buffer, which was composed of PBS⁺⁺ containing 2% (v/v) FBS and 5 mM ethylenediaminetetraacetic acid (EDTA). Cells were resuspended in FACS buffer containing antibodies, incubated on ice in the dark for 45 minutes, and then resuspended in FACS buffer. Intracellular marker staining was performed with a one-step protocol for intracellular proteins using a Foxp3/Transcription Factor Staining Buffer Set (eBioscience). Phosphorylation signaling protein staining was performed with a two-step protocol using an Intracellular Fixation and Permeabilization Buffer Set (eBioscience). Flow cytometry data were acquired using an Accuri C6 Flow Cytometer (BD Biosciences), FACSymphony (BD Biosciences), or LSRFortessa (BD Biosciences) and analyzed with FlowJo software. Antimouse antibodies used for organoid flow cytometry included the following: anti -CD 19 clone 1D3 (BD Biosciences Cat # 565965); anti-CD19 clone 1D3 phycoerythrin-Cyanine7 (PE-Cy7) conjugate (ThermoFisher Cat # 25-0193-82); anti-CD19 clone 1D3 BUV395 conjugate (BD Biosciences Cat # 563557); anti-GL7 PE conjugate (ThermoFisher Cat # 12-5902-82); anti-GL7 Alexa Fluor 488 conjugate (ThermoFisher Cat # 53-5902-82); anti-GL7 Alexa Fluor 647 conjugate (BD Biosciences Cat # 561529); anti-EZH2 clone AC22 eFluor 660 conjugate (ThermoFisher Cat # 50-9867-82); anti-IRF4 clone 3E4 PE conjugate (ThermoFisher Cat # 12-9858-82); anti-IRF4 clone 3E4 PE-Cy7 conjugate (ThermoFisher Cat # 25-9858-82); anti-BLIMP-1 clone 5E7 (BD Biosciences Cat # 564270); anti-BLIMP-1 clone 6D3 PE-CF594 conjugate (BD Biosciences Cat # 565274); antiphospho- BTK (Tyr551, Tyr511) clone M4G3LN PE conjugate (ThermoFisher Cat # 12-9015-42); antiphospho-NF-xB p65 (Ser529) clone B33B4WP PerCP-eFluor 710 conjugate (ThermoFisher Cat # 46-9863-42); anti-CD138 clone 281-2 Brilliant Violet (BV) 711 conjugate (BioLegend Cat # 142519); anti-IgM clone eB 121-15F9 FITC conjugate (ThermoFisher Cat # 11-5890-85); IgGl clone M1-14D12 FITC conjugate (ThermoFisher Cat # 11-4015-82); LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (ThermoFisher Cat # L34976); and LIVE/DEAD Fixable Blue Dead Cell Stain Kit (ThermoFisher Cat # L23105).

Confocal Microscopy Imaging

[0216] Immune organoids were prepared as described above. After 4 days the organoids were washed with PBS and incubated in 200 pL fixing solution (4% (v/v) paraformaldehyde in PBS) for 15 minutes at room temperature in the dark. Samples were then washed twice with PBS⁺⁺ and permeabilized with addition of 200 pL 0.5% (v/v) Triton X-100 in PBS for 30 minutes. After two more washes with PBS⁺⁺, the organoids were blocked with 200 pL blocking buffer (20% (v/v) goat serum in PBS⁺⁺ or 20% (v/v) donkey serum in PBS⁺⁺) for 30 minutes. For samples using a mouse IgG primary antibody, samples were blocked with Mouse-on-Mouse IgG Blocking Solution (ThermoFisher). Samples were then stained with relevant primary antibody (1 : 100 dilution in 100 pL blocking buffer) overnight at 4°C. The next day, secondary antibodies (1 : 100 dilution in 100 pL blocking buffer) and DAPI stain were added for 4 hours on ice in the dark. Prepared immune tissues were washed twice more, stored in PBS⁺⁺, and imaged using an LSM 710 confocal microscope (Zeiss) or LSM 900 confocal microscope (Zeiss). For IgM BCR quantification, four organoids of each antigen condition were imaged, with 12 single cell images per organoid, for a total of 48 cells per antigen condition. Images were analyzed in Zen (Zeiss), and IgM BCR puncta were quantified manually on single-cell images. Antimouse antibodies used for confocal imaging included the following: anti-CD19 polyclonal (ThermoFisher Cat # PA5-27442); anti-IgM IE41 clone (ThermoFisher Cat # 14-5790-82); antidiphtheria toxin polyclonal (Abeam Cat # ab 151222); and -Francisella tularensis LPS FBI 1 clone (ThermoFisher Cat # MAI -21690). Secondary antibodies used for confocal imaging included the following: goat antirabbit IgG (H+L) Alexa Fluor Plus 488 conjugate (ThermoFisher Cat # A32231); goat antirat IgG (H+L) Alexa Fluor Plus 647 conjugate (Thermo Fisher Cat # A-21247); goat anti-mouse IgG (H+L) Alexa Fluor Plus 555 conjugate (Thermo Fisher Cat # A32727),- donkey antirabbit IgG (H+L) Alexa Fluor Plus 488 conjugate (ThermoFisher Cat # A32790); and donkey antirat IgG (H+L) Alexa Fluor Plus 647 conjugate (ThermoFisher Cat # A48272). Mouse Immunization

[0217] Six-week-old BALB/c mice (Harlan Sprague-Dawley; Jackson Laboratory) were injected s.c. with 100 pL PBS (pH 7.4) alone or with purified aglycosylated or glycosylated carrier proteins. Each group was composed of three mice, and 10 pg antigen on a total protein basis was used for immunization of all groups. Protein concentration was determined by Bradford assay. Purified proteins resuspended in PBS were mixed with an equal volume of IFA (Sigma-Aldrich) before injection. After initial immunizations, boosts of identical doses were given 21 and 42 days later. Blood was obtained at study termination on day 63 via cardiac puncture. Mice were observed at 24 and 48 hours after each injection for change in behavior and physical health. No abnormal responses were observed. This work was carried out under Protocol 2012-0132 approved by the Cornell University Institutional Animal Care and Use Committee (IACUC).

Serum Antibody Titering

[0218] To determine F. tularensis LPS-specific antibody titers, sera from immunized mice were subjected to ELISA. Whole blood was centrifuged at 5000g for 10 minutes, and fractionated sera was stored at -20°C. F. tularensis LPS (BEI resources) prepared at a concentration of 5 pg/mL in PBS was incubated overnight at 4°C in 96-well MaxiSorp plates (Nunc Nalgene). Plates were washed three times with PBST (PBS, 0.05% (v/v) Tween-20, 0.3% (w/v) BSA) and blocked overnight at 4°C with 5% (w/v) nonfat dry milk (Carnation) in PBS. Sera samples were serially diluted by a factor of 2 in triplicate between 1 : 100 and 1 : 12,800,000 in blocking buffer. Plates were incubated with sera for 2 hours at 37°C. Plates were washed three times and incubated for 1 hour at 37°C in the presence of one of the following HRP- conjugated antibodies: goat anti-mouse IgG (Abeam Cat # ab6789; 1 :25000); anti -mouse IgGl (Abeam Cat # ab97240; 1 :25000), and anti-mouse IgG2a (Abeam Cat # ab97245; 1 :25000). After three additional washes with PBST, 1-Step Ultra TMB (3,3',5,5'-tetramethylbenxidine)- ELISA substrate solution (Thermo-Fisher) was added, the plate was incubated at room temperature for 30 minutes, the reaction was halted with 2 M H2SO4, and absorbance was quantified via microplate spectrophotometer (Molecular Devices) at a wavelength of 450 nm. Serum antibody titers were determined by measuring the lowest dilution that resulted in signal 3 standard deviations (SDs) above no serum background controls.

Next-Generation Sequencing and SHM Analysis

[0219] After degradation of organoids on day 4 as previously described, cells were washed in PBS and processed using a RNeasy Mini Kit (Qiagen) to extract RNA. RT-PCR was performed using a SuperScript III kit (ThermoFisher) with 500 ng RNA and oligo(dT) primers according to manufacturer’s instructions. IgH and IgZ. regions were PCR amplified using the resulting cDNA along with primers that anneal to the framework region of the most abundant families of Ig rearrangements, as described previously (Dobin et al., “STAR: Ultrafast Universal RNA-Seq Aligner,” Bioinformatics 29(1): 15-21 (2013), which is hereby incorporated by reference in its entirety). PCR products were cleaned-up using a PCR purification kit (Qiagen) and subsequently purified from DNA gels using a gel extraction kit (QIAGEN). After DNA quantification by Qubit fluorometer (ThermoFisher), Nextera adaptors were attached by PCR reaction, and the amplicon libraries were purified 1 : 1 (v/v) using magnetic AMPure XP beads (Beckman Coulter). Quality control of each library was performed by the Genomics Facility of the Cornell Biotechnology Resource Center with an AATI Fragment Analyzer (Agilent). Libraries were diluted to 6 nM concentration, pooled, and sequenced using 2 x 250 bp MiSeq (Illumina). Bioinformatic analysis was performed using the analyze command of MiXCR (Koboldt et al., “VarScan: Variant Detection in Massively Parallel Sequencing of Individual and Pooled Samples,” Bioinformatics 2009, 25(17):2283-2285 (2009), which is hereby incorporated by reference in its entirety). Paired-end sequence reads were mapped against the Mus musculus primary assembly GRCm38 using aligner Star 2.4.0 (Benatuil et al., “An Improved Yeast Transformation Method for the Generation of Very Large Human Antibody Libraries,” Protein Eng. Des. Sei. 23(4): 155-159 (2010), which is hereby incorporated by reference in its entirety). A pileup of the resulting sorted bam files was made in samtools for each targeted region was made filtering by quality score >20. A list of all single nucleotide polymorphisms was made using VarScan 2.3.4 (Chen et al., “Engineering Fibronectin-Based Binding Proteins by Yeast Surface Display,” Methods EnzymoL 523:303-326 (2013), which is hereby incorporated by reference in its entirety) on each base with minimum read depth of 10 reads and tabulated per targeted region into bed-files. These bed-files were then used to create the mutation rate per kilobase for the Sp gene by dividing the average missense per base by the average coverage of sequenced read and multiplying by 1000 to obtain the missense rate per kilobase. Primers used for immunoglobulin mutation analysis are described elsewhere (Beguelin et al., “EZH2 Enables Germinal Centre Formation Through Epigenetic Silencing of CDKN1A and an Rb-E2F1 Feedback Loop,” Nat. Commun. 8(1):877 (2017), which is hereby incorporated by reference in its entirety).

Immunoglobulin Library Construction

[0220] Immunoglobulin VH and VL regions were amplified from the cDNA preparations described above and randomly paired in the scFv format via a flexible (Gly4Ser)4 linker according to previously described PCR primers and protocols (Robinson et al., “Protein Disulfide Isomerase Overexpression Increases Secretion of Foreign Proteins in Saccharomyces cerevisiae,” Biotechnology (N Y) 12(4):381-384 (1994), which is hereby incorporated by reference in is entirety). The primers were modified to insert additional nucleotides on either end of the scFv to permit homologous recombination with yeast surface display vector pCT- CON with the following designs: 5'- CGACGATTGA AGGTAGATAC CCATACGACG TTCCAGACTA CGCTCTGCAG (SEQ ID NO: 3) - V_L - (GGGGSGGGGS GGGGSGGGGS; SEQ ID NO: 4) linker - V_H - GGATCCGAAC AAAAGCTTA TTTCTGAAGA GGACTTGTAA TAGCTCGAGAT (SEQ ID NO: 5) -3'. Paired V_L/V_H DNA libraries were purified by the QIAquick PCR Purification Kit (Qiagen), quantified by spectrophotometer (NanoDrop), and used to transform yeast as described previously (Benatuil et al., “An Improved Yeast Transformation Method for the Generation of Very Large Human Antibody Libraries,” Protein Eng. Des SeL 23(4): 155-159 (2010), which is hereby incorporated by reference in its entirety). Briefly, 4 pg of plasmid pCT-CON, which was linearized using the above overlapping nucleotide sequences, together with 12 pg of scFv library DNA was gently mixed with 400 pL electrocompetent EBY100 yeast cells, chilled on ice for at least 5 minutes, and electroporated at 2.5 kV and 25 pF in a 0.2 cm gap cuvette. Cells were immediately rescued in 8 mL 1 : 1 1 M sorbitol and 0.5 mM CaCHYPD and incubated at 250 rpm for 1 hour at 30°C. Following centrifugation at 3000g for 5 minutes, cells were resuspended in 200 mL SD-CAA media (20 g/L D-glucose, 6.7 g/L yeast nitrogen base, 5 g/L casamino acids, 5.4 g/L Na2HPO4, and 8.6 g/L NaH2PO4'H2O) and incubated overnight with shaking at 250 rpm at 30°C.

Yeast Surface Display

[0221] The surface display procedure was adapted from work described by Chen and colleagues (Chen et al., “Engineering Fibronectin-Based Binding Proteins by Yeast Surface Display,” Methods EnzymoL 523 :303-326 (2013), which is hereby incorporated by reference in its entirety). Here, cells were pelleted by centrifugation at 3000g for 5 minutes, resuspended in 200 mL SG-CAA media (18 g/L galactose, 2 g/L D-glucose, 6.7 g/L yeast nitrogen base, 5 g/L casamino acids, 5.4 g/L Na2HPO4, and 8.6 g/L NaH2PO4 H2O), and induced overnight at 250 rpm at 20°C. The following day, cells were pelleted, washed with 1 mL PBSA (0.1% (w/v) BSA in PBS), and resuspended in 1 mL PBSA. A series of negative selections was performed by MACS to deplete the libraries of scFv clones that bound to undesired targets. Briefly, yeast cells were incubated with 4 x 10⁶ unconjugated Dynabeads Biotin Binder (ThermoFisher) under gentle rotation for 2 hours at 4°C, and nonbinding cells were collected in the flow-through after magnetic separation. This process was repeated using BSA-conjugated beads, which were prepared by incubation of 4 * 10⁶ Dynabeads and 33 pmol of purified BSA that was biotinylated using EZ-Link Sulfo-NHS-LC-Biotinylation kit (ThermoFisher) according to manufacturer’s instructions, in 100 pL PBSA for 2 h at 4°C. Additional rounds of negative selection were performed using Dynabeads conjugated to the following biotinylated proteins: aglycosylated MBP carrier, aglycosylated H. influenzae PD carrier, and, for identification of FtO-PS-specific binders, aglycosylated CRM197. Afterward, cells were pelleted, washed, resuspended in 5 mL SD-CAA, and incubated at 30°C at 250 rpm for 16 hours.

[0222] Candidate scFvs with affinity for either CRM197 or F/O-PS were isolated by FACS. On the day of sorting, cells were incubated at room temperature for 1 hour with 300 nM of biotinylated antigens, aglycosylated CRM197 or MBP-F/O-PS conjugate, to detect positive binders and 20 nM of rabbit monoclonal anti-c-Myc (EQKLISEEDL; SEQ ID NO: 6) phospho S62 antibody (Abeam Cat # ab51156) to detect cell-surface expression of full-length scFv clones. The total volume of the reaction was calculated so that the antigen of interest was in excess by 1 order of magnitude by assuming a surface concentration of 10⁵ scFv proteins per yeast cell. After washing three times by centrifugation at 3000g for 3 minutes in 1 mL PBSA, cells were incubated with 20 nM streptavidin Alexa Fluor 488 conjugate (ThermoFisher Cat # S32354) and 20 nM of goat antirabbit Alexa Fluor 647 (Invitrogen Cat # A-21244) in the dark and under rotation for 1 hour. Cells were washed four more times by centrifugation in 1 mL PBSA before sorting. FACS was performed using a FACSMelody Cell Sorter (BD Biosciences). The 1% of cells displaying the highest signals for antigen binding and scFv expression were collected, incubated in 5 mL SD-CAA media at 250 rpm at 30°C 250 rpm, pelleted, resuspended in 5 mL SG-CAA media, and incubated overnight at 250 rpm at 20°C. An additional round of FACS was performed as described above using 100 nM of the same biotinylated antigens, aglycosylated CRM197 or MBP-F/O-PS conjugate, and the top 0.1% binders were isolated. For labeling controls, induced cells were incubated with (i) biotinylated antigens but in the absence of anti-c-Myc antibody; (ii) anti-c-Myc antibody and goat antirabbit Alexa Fluor 647 in the absence of biotinylated antigens, and (iii) goat antirabbit Alexa Fluor 647 only but in absence of biotinylated antigens and anti-c-Myc antibody. Sorting gates were set such that there was no overlap between the double positives and the control groups (see FIG. 14). FACS analysis was performed using FlowJo software.

Extracellular Expression of scFv Candidates

[0223] The yeast libraries were sequentially sorted as described above until reaching a final size of ~10³ cells. Plasmid DNA from each positively selected sublibrary was extracted using the Zymoprep Yeast Plasmid Miniprep II kit (Zymo Research) using ~10⁷ cells as the initial material. To enable soluble expression and secretion of pCT-CON-encoded scFv clones into the culture supernatant, sublibrary plasmids were incorporated into S. cerevisiae strain YVH10 (Sterner et al., “Therapeutic Antibodies to Ganglioside GD2 Evolved from Highly Selective Germline Antibodies,” Cell Rep. 20(7): 1681-1691 (2017), which is hereby incorporated by reference in its entirety) by electroporation and plated in SD-CAA agar plates (l x SD-CAA, 20 g/L agar, and 182 g/L sorbitol). Single colonies were grown overnight in 5 mL of SD-CAA at 30°C and with shaking at 250 rpm. The cells were induced by changing the media after 24 hours of growth to 10 mL of SG-CAA and 0.1% (w/v) BSA, and by incubation for 48 hours at 20°C with shaking at 250 rpm. Cells were fed 1 mL of 10x nutrient stock (67 g/L yeast nitrogen base, 50 g/L casamino acids) and incubated overnight at 20°C and with shaking at 250 rpm. Media supernatant was collected by centrifuging the cells at 3000g for 5 minutes. Individual scFv clones were purified using Pierce anti-c-Myc agarose (ThermoFisher) following manufacturer’s instructions. Following purification, scFvs were desalted and stored in PBS at -20°C. Purity of the samples was confirmed via SDS-PAGE and Western blot analysis, while final scFv concentrations were determined using the QuantiPro BCA protein assay kit (Sigma- Aldrich).

Analysis of scFv Candidates

[0224] Antigen was diluted to 5 pg/mL in 0.05 M NaCCL buffer, pH 9.6, and refrigerated overnight at 4°C in 96-well high-binding plates (Corning). The plates were washed three times with 200 pL PBST (PBS + 0.1% (v/v) Tween 20) per well and blocked overnight using 200 pL of 5% (w/v) nonfat dry milk in PBS per well at 4°C. The plates were washed three more times with PBST and incubated for 2 hours with 1 :4 serial dilutions of 10 mg/mL purified scFvs in 100 pL of PBS per well in triplicate, with slow mixing at room temperature. The plates were washed three times to remove unbound protein and were further incubated for 1 hour at room temperature with 100 pL of anti-Myc antibody conjugated to horseradish peroxidase (HRP) (Abeam Cat # abl326) diluted 1 :25,000 in PBS. The plates were washed three times with PBST followed by the addition of 100 pL per well of 1-Step Ultra TMB-ELISA substrate solution (ThermoFisher). The reaction was allowed to develop for a maximum of 30 minutes with incubation in the dark followed by quenching with 100 pL of 2 M H2SO4. The absorbance of each well was recorded at 450 nm in a standard plate reader (Molecular Devices).

[0225] Plasmid DNA of positive clones was extracted using the Zymoprep Yeast Plasmid Miniprep II kit (Zymo Research) and cleaned using a PCR cleanup kit (QIAgen). Plasmid DNA was used to transform electrocompetent E. coli strain DH5a. Single bacterial colonies were grown overnight at 37°C and with shaking in 5 mL of LB media supplemented with 100 pg/mL of carbenicillin. Plasmid DNA from single clones was extracted using the QIAprep Spin Miniprep Kit (QIAgen, USA) and subjected to Sanger sequencing at the Genomics Facility of the Cornell Biotechnology Resource Center.

Statistical Analysis

[0226] Statistical significance between groups was determined by one-way ANOVA with Tukey’s posthoc test or Welch’s two-sided /-test (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, not significant) using GraphPad Prism 9 for MacOS software (version 9.4.1). Statistical parameters including the definitions and values of n, p values, and SDs are reported in the figures and corresponding figure legends.

Example 1 - Biosynthesis of Designer Glycoconjugate Vaccine Candidates

[0227] Candidate glycoconjugates were generated using bacterial glycoprotein expression technology (Dow et al., “Improving Protein Glycan Coupling Technology (PGCT) for Glycoconjugate Vaccine Production,” Expert Rev. Vaccines 19(6): 507-527 (2020); and Kay et al., Recent Advances in the Production of Recombinant Glycoconjugate Vaccines,” NPJ Vaccines 1 :4: 16 (2019), which are hereby incorporated by reference in their entirety), which enabled covalent conjugation of pathogen-specific polysaccharides to specific sites in a carrier protein. Specifically, engineered E. coli cells capable of assembling heterologous O-PS antigens on the cytoplasmic membrane lipid undecaprenol and subsequently transferring lipid-linked O- PS onto specific asparagine residues in recombinantly expressed carrier proteins via the activity of the oligosaccharyltransferase (OST) enzyme PglB from Campylobacter jejuni were leveraged (FIG. 1 A). The O-PS antigen of Francisella tularensis (type A) strain Schu S4 (/ /O-PS), a highly virulent pathogen that causes tularemia and for which there is no currently approved vaccine (Oyston et al., “Tularaemia: Bioterrorism Defence Renews Interest in Francisella tularensis,” Nat. Rev. Microbiol. 2(12):967-978 (2004), which is hereby incorporated by reference in its entirety), was used as the pathogen-specific polysaccharide component. The choice of / /O-PS was motivated by the fact that this glycan has previously been produced as a protein-linked conjugate using engineered E. coli or its cell-free extracts (Stark et al., “On- Demand Biomanufacturing of Protective Conjugate Vaccines,” Sci. Adv. 7(6):eabe9444 (2021); Cuccui et al., “Exploitation of Bacterial N-Linked Glycosylation to Develop a Novel Recombinant Glycoconjugate Vaccine Against Francisella tularensis,” Open Biol. 3(5): 130002 (2013); Marshall et al., “An O-Antigen Glycoconjugate Vaccine Produced Using Protein Glycan Coupling Technology is Protective in an Inhalational Rat Model of Tularemia,” J. Immunol Res. 2018:8087916 (2018), which are hereby incorporated by reference in their entirety). When evaluated in vivo, the resulting conjugates elicited strong /OO-PS-specific IgG antibody titers that were protective against lethal doses of F. tularensis (Stark et al., “On-Demand Biomanufacturing of Protective Conjugate Vaccines,” Sci. Adv. 7(6):eabe9444 (2021); Cuccui et al., “Exploitation of Bacterial N-Linked Glycosylation to Develop a Novel Recombinant Glycoconjugate Vaccine Against Francisella tularensis,” Open Biol. 3(5): 130002 (2013); Marshall et al., “An O-Antigen Glycoconjugate Vaccine Produced Using Protein Glycan Coupling Technology is Protective in an Inhalational Rat Model of Tularemia,” J. Immunol Res. 2018:8087916 (2018), which are hereby incorporated by reference in their entirety).

[0228] For the carrier protein component, two different designs were investigated. The first involved the most widely used and highly effective carrier protein cross-reactive material 197 (CRM197), a mutant version of the diphtheria toxin carrying a single amino acid substitution (G52E) that renders the protein nontoxic (Giannini et al., “The Amino- Acid Sequence of Two Non-Toxic Mutants of Diphtheria Toxin: CRM45 and CRM197,” Nucleic Acids Res. 12(10): 4063-4069 (1984), which is hereby incorporated by reference in its entirety). CRM197 is used in several licensed conjugate vaccines including HibTITER, Prevnar, and Menveo. The second involved E. coli maltose-binding protein (MBP), which has demonstrated compatibility with bacterial protein glycosylation technologies (Stark et al., “On-Demand Biomanufacturing of Protective Conjugate Vaccines,” Sci. Adv. 7(6):eabe9444 (2021); Fisher et al., “Production of Secretory and Extracellular N-Linked Glycoproteins in Escherichia coli,” AppL Environ. Microbiol. 77(3): 871—881 (2011); and Ma et al., “Glycoconjugate Vaccine Containing Escherichia coli O157:H7 O-Antigen Linked with Maltose-Binding Protein Elicits Humoral and Cellular Responses,” PloS One 9(8):el05215 (2014), which is hereby incorporated by reference in its entirety). While not a prototypic carrier, MBP might enhance immune responses to vaccine fusion proteins or conjugated polysaccharides on account of its ability to induce dendritic cell activation and production of proinflammatory cytokines (Fernandez et al., “Potential Role for Toll-Like Receptor 4 in Mediating Escherichia coli Maltose-Binding Protein Activation of Dendritic Cells,” Infect. Immun. 75(3): 1359-1363 (2007), which is hereby incorporated by reference in its entirety). Indeed, linked to O-PS, MBP was found to elicit polysaccharidespecific humoral and cellular immune responses in mice (Stark et al., “On-Demand Biomanufacturing of Protective Conjugate Vaccines,” Sci. Adv. 7(6):eabe9444 (2021); and Ma et al., “Glycoconjugate Vaccine Containing Escherichia coli O157:H7 O-Antigen Linked with Maltose-Binding Protein Elicits Humoral and Cellular Responses,” PloS One 9(8):el05215 (2014), which is hereby incorporated by reference in its entirety) including against FtO-PS, with the latter protecting against pathogen challenge (Stark et al., “On-Demand Biomanufacturing of Protective Conjugate Vaccines,” Sci. Adv. 7(6):eabe9444 (2021), which is hereby incorporated by reference in its entirety). However, little is known about the interactions of these carrier proteins and conjugated antigens with B cells undergoing GC differentiation.

[0229] To enable conjugation with F/O-PS, both CRM197 and MBP carriers were engineered with a C-terminal tag containing four tandem repeats of an optimized bacterial glycosylation acceptor motif, DQNAT, that is preferentially glycosylated by C. jejuni PglB (Chen et al., “From Peptide to Protein: Comparative Analysis of the Substrate Specificity of N- Linked Glycosylation in C. jejuni,” Biochemistry 46(18): 5579— 5585 (2007), which is hereby incorporated by reference in its entirety). Additional C-terminal polyhistidine and FLAG (DYKDDDDK; SEQ ID NO: 7) epitope tags were also introduced to enable purification and immunoblot detection, respectively. Following expression in /v coli strain CLM24 that carried plasmids encoding the / /O-PS biosynthetic pathway and C. jejuni PglB, both carrier proteins were purified and found to be efficiently glycosylated with heterologous F/-O-PS as evidenced by the appearance of a ladder-like banding pattern in immunoblots probed with either anti-His antibody or an antibody, anti-F/LPS, against pathogen-derived F tularensis lipopolysaccharide (FIG. IB). This ladder was characteristic of / /O-PS attachment and reflected O-PS chain length variability through the action of the O-antigen polymerase, Wzy, responsible for adding O-PS repeat units to lipid-A core (Stark et al., “On-Demand Biomanufacturing of Protective Conjugate Vaccines,” Sci. Adv. 7(6):eabe9444 (2021); Cuccui et al., “Exploitation of Bacterial N-Linked Glycosylation to Develop a Novel Recombinant Glycoconjugate Vaccine Against Francisella tularensis,” Open Biol. 3(5): 130002 (2013); and Chen et al., “Outer Membrane Vesicles Displaying Engineered Glycotopes Elicit Protective Antibodies,” Proc. Natl. Acad. Sci. U. S. A. 113(26):E3609-3618 (2016), which are hereby incorporated by reference in their entirety). In contrast, when C. jejuni PglB was absent from cells, no / /O-PS glycan-specific signal from immunoblots was detected (FIG. IB), confirming the lack of protein glycosylation.

Example 2 - Glycoconjugates Differentially Elicit Antigen-Specific Antibodies in Vivo

[0230] To benchmark the immunogenicity of the two different glycoconjugate formulations, conventional animal immunizations were performed. Specifically, BALB/c mice were immunized subcutaneously with 10 pg (on a total protein basis) of purified conjugates or aglycosylated carrier protein controls lacking / /O-PS antigens, all of which were adjuvanted with Incomplete Freund’s Adjuvant (IF A). Following the initial injections, identically prepared booster doses were given subcutaneously at 21 and 42 days, and blood was drawn at day 49 and 63 (FIG. 2A). Serum IgG titers specific for F/LPS, which includes the O-PS component, were significantly increased in mice receiving the CRM197 conjugate after the first booster with relatively little change in titers following the second booster (FIG. 2B and FIGs. 8A-8C), consistent with titers measured previously for /OO-PS-based glycoconjugates (Stark et al., “On- Demand Biomanufacturing of Protective Conjugate Vaccines,” Sci. Adv. 7(6):eabe9444 (2021), which is hereby incorporated by reference in its entirety). The FtLPS-specific IgG titers elicited by the MBP conjugate were significantly lower compared to the CRM197 conjugate, which may have been due to the more extensive polysaccharide decoration observed on CRM197 relative to its MBP counterpart (FIG. IB). Another possible explanation that was considered is that the CRM197 carrier might be more immunogenic than the MBP carrier. Nevertheless, strong serum IgG titers specific to the MBP and CRM197 carrier proteins were elicited by the respective glycoconjugates; with each boosting, carrier protein-specific IgG titers were approximately 3 logs above the background titers measured for mice receiving PBS (FIG. 2C and FIGs. 8A-8C). Importantly, the distinct humoral responses observed for the MBP and CRM197 conjugates suggested that these would be a useful set of reagents for interrogating the ability of synthetic immune organoids to discriminate conjugate vaccine immunogenicity.

Example 3 - Glycoconjugate-Exposed Organoids Exhibit Enhanced GC-like Responses

[0231] It was hypothesized that the MBP and CRM197 glycoconjugates elicited these different humoral immune responses in vivo by distinctly modulating the phenotypic and transcriptional regulatory characteristics of primary GC B cells. To test this hypothesis, a recently developed designer 3D murine B cell immune organoid culture system that induces GC reactions ex vivo in a controlled manner was leveraged (Kim et al., “Multiscale Engineering of Immune Cells and Lymphoid Organs,” Nat. Rev. Mater. 4(6) :355— 378 (2019); Beguelin et al., “EZH2 Enables Germinal Centre Formation through Epigenetic Silencing of CDKN1A and an Rb-E2F1 Feedback Loop,” Nat. Commun. 8(1): 877 (2017); Purwada et al., “Ex Vivo Synthetic Immune Tissues with T Cell Signals for Differentiating Antigen-Specific, High Affinity Germinal Center B Cells,” Biomaterials 98:27-36 (2019); Graney et al., “Engineering Early Memory B-Cell-Like Phenotype in Hydrogel -Based Immune Organoids,” J. Biomed Mater. Res. A 110(8): 1435-1447 (2022); Purwada and Singh, “Immuno-Engineered Organoids for Regulating the Kinetics of B cell Development and Antibody Production,” Nat.

Protoc. 12(1): 168-182 (2017); Graney et al., “Organoid Polymer Functionality and Mode of Klebsiella Pneumoniae Membrane Antigen Presentation Regulates Ex Vivo Germinal Center Epigenetics in Young and Aged B Cells,” Adv. Funct. Mater. 30(48):2001232 (2020); Purwada et al., “Ex Vivo Engineered Immune Organoids for Controlled Germinal Center Reactions,” Biomaterials 63:24-34 (2015); and Nojima et al., “In-Vitro Derived Germinal Centre B Cells Differentially Generate Memory B or Plasma Cells In Vivo,” Nat. Commun. 6:2:465 (2011), which are hereby incorporated by reference in their entirety). Here, primary B cells from the spleens of nonimmunized wild-type mice were isolated and combined with stromal 40LB cells (Nojima et al., “In-Vitro Derived Germinal Centre B Cells Differentially Generate Memory B or Plasma Cells In Vivo,” Nat. Commun. 6:2:465 (2011); and Purwada et al., “Modular Immune Organoids with Integrin Ligand Specificity Differentially Regulate Ex Vivo B Cell Activation,” ACS Biomater Sci. Eng. 3(2):214-225 (2017), which are hereby incorporated by reference in their entirety), which are murine 3T3 fibroblasts genetically engineered to express T cell signal CD40L and follicular dendritic cell signal B cell activation factor (BAFF) (FIG. 3 A), prior to coencapsulation in maleimide-functionalized polyethylene glycol (PEG-4MAL) hydrogels. This was achieved by first functionalizing PEG-4MAL hydrogels with thiolated integrin-binding peptide REDV, which mimics the extracellular matrix fibronectin domain and vascular cell adhesion molecule (VCAM-1) on follicular dendritic cell networks (Graney et al., “Organoid Polymer Functionality and Mode of Klebsiella Pneumoniae Membrane Antigen Presentation Regulates Ex Vivo Germinal Center Epigenetics in Young and Aged B Cells,” Adv. Funct Mater. 30(48):2001232 (2020), which is hereby incorporated by reference in its entirety) found within B cell follicles where GCs form. After functionalization, PEG-4MAL droplets were plated on a 96-well plate, mixed with an equal volume of cell-containing crosslinker solution, and cured at 37°C to form hydrogels. Organoids were designed for a 96-well format to facilitate high-throughput and scalable studies by simplifying cell culture, addition of antigen, imaging, and processing for downstream analysis. This approach was previously shown to induce a robust early GC-like phenotype (CD19⁺ GL7⁺) in a ligand concentration-dependent manner (Kwak et al., “B Cell Signaling in Context,” Nature Immunol. 20(8): 963-969 (2019), which is hereby incorporated by reference in its entirety) as well as several hallmark epigenetic and transcriptional regulators of the GC process (Graney et al., “Organoid Polymer Functionality and Mode of Klebsiella Pneumoniae Membrane Antigen Presentation Regulates Ex Vivo Germinal Center Epigenetics in Young and Aged B Cells,” Adv. Funct Mater. 30(48):2001232 (2020), which is hereby incorporated by reference in its entirety). Recombinant murine IL4, alongside the immunogen of interest, was added to the media where the hydrogel capsules were embedded. After 4 days, the hydrogels were enzymatically digested, and all cells were analyzed using flow cytometry. B cells displaying a GC-like (CD19⁺ GL7⁺) phenotype were isolated from the organoid hydrogel. Approximately similar amounts of GC-like B cells (~80% of the total B cell population) were recovered from organoids regardless of the immunogen treatment (FIG. 3B, FIG. 9, and FIG. 10), consistent with previous work (Purwada et al., “Ex Vivo Synthetic Immune Tissues with T Cell Signals for Differentiating Antigen-Specific, High Affinity Germinal Center B Cells,” Biomaterials 98:27-36 (2019); and Kwak et al., “B Cell Signaling in Context,” Nature Immunol. 20(8): 963-969 (2019), each of which are hereby incorporated by reference in their entirety. Because CD40L is provided by 40LB cells in PBS control groups, high levels of GC- like B cells were expected; however, previous work demonstrates that these are not antigenspecific cells (Purwada et al., “Ex Vivo Synthetic Immune Tissues with T Cell Signals for Differentiating Antigen-Specific, High Affinity Germinal Center B Cells,” Biomaterials 98:27- 36 (2019), which is hereby incorporated by reference in its entirety). Next, the abundance of B cell populations known to be relevant for achieving robust humoral immunity was characterized. B cells within the GC are compartmentalized into two anatomically distinct compartments: the dark zone (DZ), representing the site of intense B cell proliferation and SHM; and the light zone (LZ), where B cells bind antigen and undergo selection aided by the presence of follicular helper T cells and antigen-presenting follicular dendritic cells (Victora and Nussenzweig, “Germinal Centers,” Annu. Rev. Immunol. 30:429-457 (2012); Mesin et al., “Germinal Center B Cell Dynamics,” Immunity 45(3):471-482 (2016); Cyster and Allen, “B Cell Responses: Cell Interaction Dynamics and Decisions,” Cell Y17( y. 524-540 (2019); and De Silva and Klein, “Dynamics of B Cells in Germinal Centres,” Nat. Rev. Immunol. 15(3): 137-148 (2015), each of which are hereby incorporated by reference in their entirety). B cells that do not bind antigens and do not receive survival signals from these auxiliary cells undergo apoptosis. When B cells are primed with antigens and CD40L in vivo, GC B cells segregate into centroblasts (CXCR4^hl CD86¹⁰) in the DZ and centrocytes (CXCR4¹⁰ CD86^hl) in the LZ, prior to selection and exit of the GC response. Indeed, flow cytometric analysis on GC-like B cells indicated significant upregulation of CD86, which is a marker of B cell activation, in CRMi97-FtO-PS conjugate as compared to MBP-FtO-PS conjugate or either of the aglycosylated carrier proteins (FIG. 3C and FIG. 9). Interestingly, the CRM197 carrier protein induced a significantly higher fraction of CD86⁺ B cells than MBP alone, demonstrating a distinct carrier effect on B cell maturation. Lastly, MBP or CR 197 glycoconjugates differentially modulated the centrocyte phenotype, where the CRMi97-FtO-PS conjugate induced a significantly higher percentage of LZ-like (CXCR4¹⁰ CD86^hl) B cells than MBP-FtO-PS conjugate or aglycosylated CR 197 carrier protein (FIG. 3D). In contrast, no significant differences between glycosylated and aglycosylated MBP or between the two aglycosylated carrier proteins was observed. [0232] The downstream effect of B cell maturation is the generation of CD138⁺ plasmablasts. Similar to the centrocyte phenotype, it was observed that the CRMi97-FtO-PS conjugate induced a significantly higher percentage of CD138⁺ plasmablasts compared to the MBP-F/O-PS conjugate or aglycosylated carrier proteins (FIG. 3E and FIG. 9). However, unlike centrocytes, the MBP-FtO-PS conjugate induced a significantly higher percentage of CD138⁺ cells than aglycosylated MBP. The carrier effect on B cell terminal differentiation was again observed, with aglycosylated CRM197 inducing a significantly higher fraction of CD138⁺ cells than aglycosylated MBP. Taken together, these results indicate that while most organoid B cells were GC-like B cells for all groups, those from organoids treated with the CRM197 glycoconjugate exhibited the highest amounts of proteins representative of both GC- associated activation as well as differentiated LZ-like B cells, indicating a shift toward CD138⁺ plasmablast populations. These results were significant when compared to organoids treated with CRM197 carrier protein alone or with the less immunogenic MBP glycoconjugate.

Example 4 - Glycoconjugates Shape BCR Clustering and Signaling in GC-like B Cells

[0233] Another key facet of the humoral immune response is antigen binding to BCRs, which initiate intracellular signaling that promotes a GC response (Pierce and Liu, “The Tipping Points in the Initiation of B cell Signalling: How Small Changes Make Big Differences,” Nature Rev. Immunol. 10(11): 767 -777 (2010); and Beguelin et al., “EZH2 is Required for Germinal Center Formation and Somatic EZH2 Mutations Promote Lymphoid Transformation,” Cancer Cell 23(5):677-692 (2013), which are hereby incorporated by reference in their entirety). Upon binding of antigen, B cell activation is regulated by BCR signaling and the nanoscale organization of the BCR on the cell surface. These nanoscale organizations lead to the formation of individual puncta on the surface of B cells. It was hypothesized that organoids treated with the CRM197 glycoconjugate would exhibit higher BCR clusters than other comparative groups. To test this hypothesis, IgM expression on GC-like B cells was quantified and BCR puncta was imaged on B-cell surfaces. The CR.M197-/ /O-PS conjugate significantly upregulated IgM expression on activated B cells as compared to MBP-F/O-PS conjugate or either of the aglycosylated carrier proteins as determined using mean fluorescence intensity (FIG. 4A). In contrast, these were not a significant increase in IgM expression for the MBP-F/O-PS conjugate or MBP carrier relative to the PBS group. The IgM expression was further impacted by the carrier effect, whereby aglycosylated CRM197 carrier induced significantly higher IgM fluorescence than aglycosylated MBP. Spatially, the CRMi97-FtO-PS conjugate induced the formation of a significantly higher number of BCR clusters per single B cell than the aglycosylated CRM197 carrier (FIG. 4B), indicating that the glycoconjugate functions by enhanced BCR expression and clustering. [0234] It was hypothesized that the different immunostimulatory effects of the two glycoconjugates on B cells undergoing maturation in ex vivo immune organoids would involve distinct activation of signaling in B cells. Specifically, using flow cytometry, the levels of activated Bruton’s tyrosine kinase (BTK), an intermediary kinase, and nuclear factor-xB (NF- KB), a downstream transcription factor responsible for important B cell maturation processes including class-switching, was measured in the GC-like B cell population. GC-like B cells derived from organoids exposed to CRM197, with and without conjugation to FtO-PS, exhibited increased staining of phosphorylated BTK (pBTK) and phosphorylated NF-KB (pNF-xB) relative to organoids exposed to their respective MBP counterparts (FIG. 4C and FIG. 10). Importantly, the observation that glycosylated CRM197 stimulated greater activation of BTK and NF-KB was consistent with its stronger immunogenicity in vivo. Surprisingly, both the CRM197 and MBP glycoconjugates stimulated a statistically significant increase in pBTK and pNF-xB levels relative to their cognate aglycosylated carrier proteins, suggesting a role for the conjugated FtQ- PS antigen itself in further activation of BTK and NF-KB.

[0235] Next, the expression levels of factors associated with the GC response, including BLIMP- 1, EZH2, and IRF4, were examined. Both EZH2 and IRF4 are upstream of multiple GC programming factors and have been shown to be required for GC formation (Beguelin et al., “EZH2 Enables Germinal Centre Formation Through Epigenetic Silencing of CDKN1A and an Rb-E2F1 Feedback Loop,” Nat. Commun. 8(1):877 (2017); Ochiai et al., “Transcriptional Regulation of Germinal Center B and Plasma Cell Fates by Dynamical Control of IRF4,” Immunity 38(5):918— 929 (2013); Jacob, “Intraclonal Generation of Antibody Mutants in Germinal Centres,” Nature 354(6352):389-392 (1991); and Berek et al., “Maturation of the Immune Response in Germinal Centers,” Cell 67(6): 1121-1129 (1991), each of which are hereby incorporated by reference in their entirety). During the late stages of the GC response, IRF4 levels are again elevated and BLIMP-1 is also expressed, with both working together and required for GC B cell differentiation into plasma cells (Jacob, “Intraclonal Generation of Antibody Mutants in Germinal Centres,” Nature 354(6352):389-392 (1991); and Berek et al., “Maturation of the Immune Response in Germinal Centers,” Cell 67(6): 1121-1129 (1991), each of which are hereby incorporated by reference in their entirety). For all three of these GC factors, glycosylated CRM197 induced significantly greater expression relative to all other treatment groups including both the aglycosylated CRM197 carrier and the glycosylated MBP conjugate (FIG. 4D and FIG. 10). In contrast, treatment with glycosylated and aglycosylated MBP did not lead to any statistically meaningful differences in BLIMP-1 and EZH2 expression levels. Only in the case of IRF4 did glycosylated MBP trigger higher expression relative to aglycosylated MBP. Interestingly, both the glycosylated and aglycosylated

CRM197 immunogens stimulated greater expression of BLIMP- 1 and IRF4 relative to their respective MBP counterparts. Taken together, these results provide further support that the FtO- PS component of the conjugate promotes activation of GC-like responses, which were most pronounced in the context of the CRM197 carrier protein.

Example 5 - Glycoconjugate-Exposed Organoids Exhibit Increased BCR Engagement by Immunogens

[0236] It was hypothesized that differences observed above in GC-like B cell outputs, in particular the levels of pBTK and pNF-xB that are known to be activated via BCR signal transduction, could reflect varying degrees of BCR engagement by the different immunogens. To test this hypothesis, CRM -based immunogen binding to BCRs on the surface of organoid B cells was visualized. These immunogens were chosen because of their superior GC-like responses compared to the respective MBP immunogens. Following 4 days of ex vivo culture, GC-like organoid B cells were stained with antibodies specific for BCR, CRM197, and F. tularensis LPS and subsequently imaged by confocal microscopy. Imaging analysis revealed that all three signals were present on the surface of CRM197 glycoconjugate-exposed organoid B cells (FIG. 5A). Higher magnification at the single cell level revealed a clear overlap of the BCR, CRM197, and /70-PS signals indicative of BCR and glycoconjugate colocalization (FIG. 5B). Interestingly, B cells associated with PBS-treated organoids had a diffuse BCR signal throughout the membrane, while B cells from organoids exposed to CRMwbased immunogens had distinct BCR puncta, consistent with the well-known phenomenon of antigen-induced BCR clustering that potentiates intracellular signaling through phosphorylation of immunoreceptor tyrosine-based activation motifs present in the cytoplasmic tail of BCR-associated proteins (Pierce and Liu, “The Tipping Points in the Initiation of B cell Signalling: How Small Changes Make Big Differences,” Nature Rev. Immunol. 10(11): 767 -777 (2010), which is hereby incorporated by reference in its entirety).

Example 6 - Antigen-Exposed Organoids Exhibit Diversification of Immunoglobulin Expression

[0237] The GC is the main structure where antigen-activated B cells diversify their immunoglobulin repertoires via antibody gene mutation (De Silva and Klein, “Dynamics of B Cells in Germinal Centres,” Nat. Rev. Immunol. 15(3): 137-148 (2015), which is hereby incorporated by reference in its entirety). Thus, to further assess the extent to which synthetic immune organoids mimic complex GC immunobiology, whether organoid-derived GC B cells manifested altered immunoglobulin gene expression in response to the different immunogens was investigated. To this end, clonal diversity among the different treatment groups was evaluated by sequencing their expressed immunoglobulin repertoires. Specifically, RNA was isolated from GC-like organoid B cells after 4 days of ex vivo culture and subjected to RT-PCR to generate cDNA libraries encoding variable heavy (VH) and variable light (VL) chain domain sequences that were subsequently used to generate next-generation sequencing (NGS) libraries. Intriguingly, sequencing of the resulting NGS libraries indicated that the expressed repertoires corresponding to B cells derived from organoids exposed to either of the CRM 197-based immunogens shared a high level of sequence convergence and were more similar to each other than to the sequenced repertoires of the initial (day 0) naive B cell population used in preparing organoid tissues or the B cells derived from PBS-treated (day 4) organoids (FIG. 6A). Nearly identical convergence results were observed with the MBP-based immunogens (FIG. 11). The observation that exposure to a common immunogen triggered the emergence of overlapping B cell repertoires that were distinctly different from control groups suggested that antigen-driven repertoire diversification was active in organoid tissues. GC B cells are well-known to diversify their immunoglobulin variable region genes by SHM, resulting in the generation of mutant clones that have a broad range of affinities for the immunizing antigen (Krebber et al., “Reliable Cloning of Functional Antibody Variable Domains from Hybridomas and Spleen Cell Repertoires Employing a Reengineered Phage Display System,” J. Immunol Methods 201(l):35— 55 (1997), which is hereby incorporated by reference in its entirety). Therefore, it was next evaluated whether the immunoglobulin gene variable regions of antigen-exposed GC organoid B cells manifested evidence of SHM. Specifically, the Sp immunoglobulin variable locus was PCR-amplified in genomic DNA harvested from the day 0 naive B cell population and day 4 organoid B cells and subjected the resulting amplicon libraries to NGS. Analysis of sequencing data revealed that GC organoid B cells exposed to CRM 197-based immunogens showed a significant increase in missense mutations compared to the day 0 naive B cell group (FIG. 6B). Collectively, these results indicate that the GC organoid system was capable of reproducing select core aspects of the GC B cell phenotype, in particular diversification of immunoglobulin gene expression, consistent with previous work (Beguelin et al., “EZH2 Enables Germinal Centre Formation through Epigenetic Silencing of CDKN1A and an Rb-E2F1 Feedback Loop,” Nat. Commun. 8(1):877 (2017), which is hereby incorporated by reference in its entirety). While the above data demonstrate significant differences across treatments that suggest immunogenspecific SHM, at present the reason behind the higher missense rate in MBP-treated organoids compared to CRM -treated organoids is not fully understood. A possible explanation is that B cells undergo iterative rounds of somatic hypermutation and selection in GCs in vivo, however, in organoids, recycling or repeated rounds of SHM was not investigated. It is possible, therefore, that the final mutation rate could differ with iterative rounds and would require a more advanced immune organoid system that can induce recycling.

[0238] Given the observed diversification of immunoglobulin repertoires, the antigenspecificity of the B cell antibody populations was next investigated. From the RNA-derived cDNA libraries, VH and VL genes were PCR amplified and randomly joined by a

(Gly4Ser)4 linker to generate single-chain Fv (scFv) antibodies as described (Robinson et al., “Protein Disulfide Isomerase Overexpression Increases Secretion of Foreign Proteins in Saccharomyces cerevisiae,” Biotechnology (N Y) 12(4):381- 384 (1994), which is hereby incorporated by reference in its entirety). Three antibody libraries were constructed: one corresponding to the day 0 naive B cell population that was used to prepare immune organoids and one each corresponding to the day 4 B cells that were exposed to either CRM197 or the CRMi97-based conjugate. Each of the resulting scFv gene libraries was cloned into the YSD plasmid pCT-CON, and the resulting plasmid libraries were used to transform Saccharomyces cerevisiae strain EBY100. The pCT-CON plasmid introduced a C-terminal c-Myc epitope tag to each scFv clone (FIG. 6C) such that recipient yeast cells could be probed simultaneously for full- length scFv expression using an anti-c-Myc antibody and antigen-binding activity using a fluorescently labeled antigen. Accordingly, double-positive yeast cells in scatter plots were indicative of clones displaying full-length scFvs on their surface that bind the antigen of interest. [0239] Yeast cells displaying the three different libraries were preincubated with nonlabeled protein(s) to prevent nonspecific scFvs from binding to the antigen of interest and to reduce false positive results. These cells were then interrogated for binding to fluorescently labeled versions of either the aglycosylated CRM197 carrier protein or the CRMi97-FtO-PS glycoconjugate by flow cytometry. Following incubation with aglycosylated CRM197, the library corresponding to the CRMi97-exposed B cells was observed to contain a significant population of antigen-positive clones as evidenced by the high percentage (60.7%) of double-positive yeast cells in the library (FIG. 6D). In contrast, the library corresponding to the naive B cell population was nearly devoid of CRMwbinding clones with just 2.82% of the library appearing as double positive cells. A low level of CRMw-positive clones (21.4%) was detected in the library corresponding to the glycoconjugate-treated B cells following incubation with the same antigen, consistent with the fact that these immune organoid-derived, GC-like B cells were exposed to CRM197 in the context of the FtO-PS antigen. Likewise, the library corresponding to CRMi97-FtO-PS glycoconjugate-treated B cells, but not day 0 naive B cells, contained a high percentage of double-positive cells (79.4% vs 6.38%, respectively) when incubated with MBP- F/O-PS glycoconjugate. Note that the mismatched MBP-based glycoconjugate was used for staining to enable detection of FtO-PS-specific clones given that these organoid B cells never experienced the MBP carrier. Importantly, the library corresponding to the CRM -exposed B cells, which were strongly double positive for the carrier protein alone, contained only 12.4% double positives following incubation with the MBP-F/O-PS glycoconjugate, indicating a strong bias of the clones in this library for the aglycosylated CRM197 carrier protein. Moreover, the substantial difference in glycoconjugate binders detected in the library derived from glycoconjugate-exposed B cells compared to carrier protein-exposed B cells (79.4% vs 12.4%) suggests that the clones from the former library are specific for the FtO-PS antigen.

Example 7 - Discovery of High- Affinity Monoclonal Antibodies from Antigen-Exposed Organoids

[0240] Encouraged by these population-level observations, it was investigated whether individual antibody clones with high affinity and specificity for CRM197 or the F/O-PS antigen could be discovered from immune organoids that had been immunized with the carrier protein or glycoconjugate, respectively (FIG. 7A). Organoid-derived YSD libraries, corresponding to organoids treated with either CRM197 or the CRM197 glycoconjugate, were subjected to several rounds of negative selection using magnetic-activated cell sorting (MACS) to deplete the libraries of binders to undesired targets (z.e., nonspecific proteins such as MBP or streptavidin) or to the carrier proteins in the case of libraries generated from glycoconjugate-treated organoids. Then, an additional round of MACS was performed to positively select binders, with the YSD library corresponding to the CRMi97-exposed organoids being selected against CRM197 and the YSD library corresponding to the CRM197 glycoconjugate-exposed organoids being selected against MBP bearing the FtO-PS glycan. Lastly, two rounds of fluorescence-activated cell (FACS) sorting were performed with the same CRM197 and MBP-F/O-PS antigens at a concentration of 300 nM and then 100 nM.

[0241] During the final round of FACS sorting, individual yeast cells representing putative antigen-positive clones were sorted into 96-well plates. Clonal plasmid DNA from these selected clones was used to transform the S. cerevisiae strain YVH10, which permits soluble expression and secretion of pCT-CON-encoded scFvs into the media supernatant (Sterner et al., “Therapeutic Antibodies to Ganglioside GD2 Evolved from Highly Selective Germline Antibodies,” Cell Rep. 20(7): 1681-1691 (2017), which is hereby incorporated by reference in its entirety). Screening of supernatants for antigen-specific binders uncovered a total of 46/96 and 62/96 clones with affinity and specificity for CRM197 and the F/O-PS antigen, respectively, as determined by a qualitative enzyme-linked immunosorbent assay (ELISA) of YVH10 culture supernatants (FIGs. 12A-12B ). Three of the strongest scFv binders from each library were expressed in YVH10 cells and purified from extracellular supernatants, and then subjected to quantitative ELISA. All six clones exhibited high affinity to their respective antigen with equilibrium binding constants in the low nanomolar to subnanomolar range (FIG. 7B and FIG. 13 A). Importantly, the clones were also observed to be specific for their antigen with little to no cross-reactivity to either a nonspecific protein in the case of the three CRMw-specific scFvs or to the aglycosylated CRM197 carrier protein in the case of the three FtO-PS-specific scFvs. Sequencing of the six clones enabled the assignment of putative germline genes and subsequent alignment of organoid-derived sequences with the putative germline sequences. It was observed that all isolated clones exhibited 63-95% similarity with the respective germline VH and VL sequences (FIG. 7C and FIG. 13B). Moreover, each clone contained more than 10 germline mutations, with mutations occurring both in the frameworks and complementarity determining regions (CDRs) for each. The high degree of homology and number of germline mutations observed were on par with previous studies that have explored the immunological evolution from germline to affinity -mature antibody structures, including those against carbohydrate antigens (Wang et al., “Escherichia coli Maltose-Binding Protein Induces Ml Polarity of RAW264.7 Macrophage Cells via a TLR2- and TLR4-Dependent Manner,” Int. J. Mol. Sci. 16(5): 9896-9909 (2015), which is hereby incorporated by reference in its entirety).

Discussion of Examples 1-7

[0242] Here, using an engineered B cell immune organoid platform, the impact of designer glycoconjugate vaccine candidates on B cell maturation and signaling was investigated. In general, CR.M197-F/O-PS, a conjugate shown to elicit robust antipolysaccharide antibody titers in vivo, demonstrated the highest levels of BCR clustering and activation of intracellular signaling pathways in organoid B cells relative to all other immunogens tested. Downstream of these important activation signals, the CRMi97-FtO-PS-exposed B cells showed elevated levels of (i) genes important for early- and late-stage GC development including EZH2, IRF4, and BLIMP- 1; (ii) cell populations generated during and post-GC formation, and (iii) immunoglobulin mutation rates reminiscent of activated B cells undergoing SHM. Importantly, the stronger polysaccharide-specific IgG response measured in vivo for the CRM197 glycoconjugate relative to the MBP glycoconjugate was recapitulated in the totality of the outputs measured from organoids. These differences could be due to CRM197 having more numerous immunogenic epitopes that are recognized by BCRs, thereby permitting binding by larger pools of B cells in the starting naive populations. Indeed, the CRM197 carrier alone was significantly more immunoreactive in the organoid system than MBP alone, with the former stimulating BCR expression and activating BTK and pNF-xB to a much greater extent. However, these differences in immunoreactivity were not as clearly observed in vivo, with both carrier protein immunogens triggering comparable carrier-specific IgG titers at day 63. One reason for this discrepancy might be the fact that the mice received three doses (prime plus two boosters), whereas the organoids only received two doses of immunogen. It is also worth pointing out that the IgG responses at day 49 exhibited a trend that more closely matched the organoid data. Moreover, it should be noted that IgG titers are not a complete reflection of B cell activation in vivo, since an increase in pBTK and pNF-kB could still occur without a concomitant increase in IgG production. Likewise, it is possible that the cytokines released by the innate response as a consequence of both the adjuvant and the interactions with the protein carrier might enhance the B cell activation in vivo following immunization with MBP (Fernandez et al., “Potential Role for Toll-Like Receptor 4 in Mediating Escherichia coli Maltose-Binding Protein Activation of Dendritic Cells,” Infect. Immun. 75(3): 1359-1363 (2007) and Richard et al., “In vivo Neutralization of a-Cobratoxin with High- Affinity llama Single-Domain Antibodies (VHHs) and a VHH-Fc antibody,” PloS One 8(7):e69495 (2013), which are hereby incorporated reference in their entirety).

[0243] One unexpected observation was that both the CRMi97-FtO-PS and MBP-/ /O-PS glycoconjugates generally triggered stronger organoid responses, especially activation of BTK and NF-KB, compared to their respective carrier proteins alone. At present, the reasons for why glycosylation boosts the strength of B cell activation by the glycoconjugates relative to the carrier protein remain unknown and are seemingly at odds with the observation that, in vivo, the conjugates and carriers stimulated roughly equal carrier-specific IgG responses. One possible explanation for this discrepancy is the ability of glycoconjugates to activate a pool of polysaccharide-responsive B cells in addition to the protein-responsive B cells in the initial naive B cell populations. This population of B cells, which are responsible for the polysaccharidespecific IgG antibodies, could account for stronger B cell activation in terms of pBTK and pNF- KB without a corresponding increase in IgG titers to the carrier protein. Another possible explanation stems from the lack of T cells in the disclosed organoid system. Within organoids, T cell activation cues to B cells are present for all B cells instead of being selective for B cells displaying the appropriate antigen epitopes on their cell surface for cognate T cell recognition and binding. What this means is that the strength of B cell activation in the organoids greatly depends on the antigen’s ability to promote BCR clustering, as seen previously in the context of integrin ligand specificity (Kwak et al., “B Cell Signaling in Context,” Nature Immunol. 20(8):963-969 (2019), which is hereby incorporated by reference in its entirety). Hence, in the organoid system of the present disclosure, where the T cell signal is binary, a protein might be at a disadvantage compared to a repetitive polysaccharide that can promote more efficient BCR clustering as observed in microscopy analysis of immunogen-exposed GC-like B cells (FIGs. 4A-4D and FIGs.5A-5B). In contrast, the enhanced immunoreactivity of the glycoconjugate relative to the carrier was not observed in vivo where presumably the processing and presentation of antigen by B cells and the modulatory effect these events have on T cell help promote strong CRMi97-specific responses regardless of whether a polysaccharide is attached or not. It is also worth noting that the measurement of IgG titers in vivo does not always correlate with the amount of activated B cells, since there are B cells that may react to the glycoconjugate without class-switching, making it possible to have a stronger B cell activation to the glycoconjugate in terms of pBTK and pNF-xB without a contemporaneous change in the levels of antigen-specific IgG in circulation.

[0244] The immunoglobulin repertoires from organoids were found to be responsive to treatment with the glycoconjugates, as supported by the following observations: (i) variable region mutation rates were increased, indicative of SHM; and (ii) repertoires from organoids exposed to related antigens (e.g., CRM197 and CRMi97-FtO-PS) were more similar than the initial naive B cell population. Furthermore, these repertoires were shown to be enriched for antigen-specific binders, yielding immune libraries suitable for downstream antibody discovery applications. YSD was used in conjunction with these organoid-derived libraries to successfully identify sequences targeting protein (CRM197) and carbohydrate (F/O-PS) motifs. This is especially relevant given the increasing importance in recent years of display-based approaches for antibody discovery. It has previously been shown that immune-focused libraries can be used to identify higher affinity antibodies compared to the use of naive libraries (Lim et al., “Principles and Application of Antibody Libraries for Infectious Diseases,” BiotechnoL Lett. 36(12):2381-2392 (2014), which is hereby incorporated by reference in its entirety). However, immunized libraries are inherently limited to the discovery of antibodies against the antigen or infection generating the response (Jaroentomeechai et al., “Single-Pot Glycoprotein Biosynthesis Using a Cell-Free Transcription-Translation System Enriched with Glycosylation Machinery,” Nat. Commun. 9(1):2686 (2018), which is hereby incorporated by reference in its entirety). Organoids have the potential to provide the benefit of an enriched, immunized library increasing the likelihood of mining clinically relevant sequences without the need to immunize living organisms. Furthermore, the organoids of the present disclosure may provide the capability to sequence repertoires against large variant libraries, which could prove useful for understanding humoral response decision-making and immunodominance. How an immune response becomes biased toward one or several of the most frequent binders is an area of particular interest that is being investigated.

[0245] The “T cell-free” organoid system according to the present disclosure holds great promise for learning more about B cell activation without having to account for differences in T cell activation and thus could be advantageous for antigen-specific antibody discovery, especially in the context of self-antigens. B cells that recognize a self-antigen but receive no simultaneous T cell activation signal enter a state of anergy and become nonresponsive. With CD40L T-cell signals “on”, organoids may generate stronger B cell maturation for weakly immunogenic antigens such as tumor-associated glycans. The current organoid system could also be readily used to provide a rapid, preliminary assessment of large vaccine candidate libraries. Due to the difficulty in conjugate production, including the expression, purification, and attachment of carbohydrate motifs, a small subset of vaccine candidates is normally generated to characterize the immunological response in lieu of screening large libraries of candidates that thoroughly cover every possible design configuration. However, recent advances in glycoengineering, including the use of engineered E. coli and their cell-free extracts for polysaccharide synthesis and conjugation (Dow et al., “Improving Protein Glycan Coupling Technology (PGCT) for Glycoconjugate Vaccine Production,” Expert Rev. Vaccines 19(6): 507— 527 (2020); Kay et al., Recent Advances in the Production of Recombinant Glycoconjugate Vaccines,” NPJ. Vaccines 1 :4: 16 (2019); Jaroentomeechai et al., “Cell-Free Synthetic Glycobiology: Designing and Engineering Glycomolecules Outside of Living Cells,” Front Chem. 29:8:645 (2020); Kightlinger et al., “Synthetic Glycobiology: Parts, Systems, and Applications,” ACS Synth. Biol. 9(7): 1534-1562 (2020); Stark et al., “On-Demand Biomanufacturing of Protective Conjugate Vaccines,” Sci. Adv. 7(6):eabe9444 (2021); and Stefanetti et al., “Glycoconjugate Vaccine Using a Genetically Modified O Antigen Induces Protective Antibodies to Francisella tularensis,” Proc. Natl. Acad. Sci. U. S. A. 116(14)7062- 7070 (2019), which are hereby incorporated by reference in their entirety), have made such coverage possible. A newly developed shotgun-scanning glycomutagenesis (SSGM) method leverages bacterial glycosylation to generate neoglycoprotein libraries that differ in the site of glycan attachment and spans every amino acid position (Li et al., “Shotgun Scanning Glycomutagenesis: A Simple and Efficient Strategy for Constructing and Characterizing Neoglycoproteins,” Proc. Natl. Acad. Sci. U. S. A. 118(39):e2107440118 (2021), which is hereby incorporated by reference in its entirety). A complete SSGM library using CRM197 as the carrier protein for a single polysaccharide structure results in over 500 distinct conjugates, making it infeasible to systematically test all glycosylation site variants using traditional animal immunization pipelines. The number of conjugate library members can expand even further by varying other important design variables. For example, the length of the polysaccharide chain can be controllably altered by heterologous expression of different chain-length regulator genes, generating structures of varying immunogenicity (Joseph et al., “Total Synthesis of Polysaccharides by Automated Glycan Assembly,” J. Am. Chem. Soc. 142(19): 8561—8564 (2020), which is hereby incorporated by reference in its entirety). Likewise, the density of the polysaccharide epitope can be controlled by introducing additional glycan attachment sites, leading to more heavily glycosylated conjugates that elicit varied immune responses (Marshall et al., “An O-Antigen Glycoconjugate Vaccine Produced Using Protein Glycan Coupling Technology is Protective in an Inhalational Rat Model of Tularemia,” J. Immunol Res.

2018:8087916 (2018), which is hereby incorporated by reference in its entirety). Other advanced glycoengineering tools such as automated glycan assembly could eventually be leveraged for producing defined, pure glycans with control over composition and length (Feldman et al., “Engineering N-Linked Protein Glycosylation with Diverse O Antigen Lipopolysaccharide Structures in Escherichia coli.,” Proc. Natl. Acad. Sci. U. S. A. 102(8): 3016-3021 (2005), which is hereby incorporated by reference in its entirety). Altogether, it is envisioned that a significant reduction in the number of animals, costs, and time can be achieved by employing organoidbased prescreening of such large conjugate libraries to identify candidates of interest for further characterization. It is also imagined that future organoid designs involving cocultures of B and T cells, as recently demonstrated in a top-down approach (Wagar et al., “Modeling Human Adaptive Immune Responses with Tonsil Organoids,” Nat. Med. 27(1): 125— 135 (2021), which is hereby incorporated by reference in its entirety), which would open the door to in vitro investigations of glycoconjugate processing and presentation by B cells and the modulatory effect that these events have on T cell help.

[0246] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Claims

WHAT IS CLAIMED IS:

1. An ex vivo method of identifying highly immunogenic glycoconjugates, said method comprising: providing a first plurality of murine B cell organoids; contacting each of the first plurality of murine B cell organoids with a plurality of distinct glycoconjugate candidates, wherein each of the distinct glycoconjugate candidates comprises a distinct polysaccharide of interest and a carrier protein, and wherein said contacting is carried out to form a first plurality of contacted B cell organoids; culturing the first plurality of contacted B cell organoids under conditions effective to support B cell maturation, whereby said culturing produces a first plurality of cultured B cell organoids; detecting presence or absence of one or more B cell biomarker(s) in each of the first plurality of cultured B cell organoids, wherein said detecting presence of one or more B cell biomarker(s) indicates immunogenicity of the glycoconjugate candidate; and identifying highly immunogenic glycoconjugate candidates based on a greater presence of B cell biomarker(s) in one or more of said cultured B cell organoids relative to others of said cultured B cell organoids or relative to a control cultured B cell organoid contacted with the carrier protein, wherein the carrier protein is aglycosylated.

2. The method of claim 1, wherein the first plurality of murine B cell organoids are organized in an array.

3. The method of claim 1 or claim 2, wherein the first plurality of B cell organoids are seeded in a 6-well plate, a 12-well plate, a 48-well plate, a 96-well plate, a 384- well plate, or larger culture platform.

4. The method of any one of the preceding claims, wherein each distinct polysaccharide is derived from any one of the following: a bacterium, a virus, a parasite, or a cancer.

5. The method of any one of the preceding claims, wherein each distinct polysaccharide is derived from a bacterium.

6. The method of claim 5, wherein the bacterium is a pathogenic bacterium.

7. The method of claim 5 or claim 6, wherein the bacterium is a grampositive bacterium.

8. The method of claim 7, wherein the bacterium is selected from the group consisting of Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Erysipelothrix rhusiopathiae, Gardnerella vaginalis, Group A Streptococcus, Group B Streptococcus, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Nocardia asteroids, Propionibacterium acnes, Rhodococcus equi, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus mutans, Streptococcus pneumoniae, and Streptococcus pyogenes.

9. The method of claim 5 or claim 6, wherein the bacterium is a gramnegative bacterium.

10. The method of claim 9, wherein the gram -negative bacterium is selected from the group consisting of Acinetobacter baumannii, Bordetella pertussis, Brucella abortus, Burkholderia pseudomallei, Burkholderia mallei, Burkholderia pseudomallei, Campylobacter fetus, Campylobacter jejuni, Coxiella burnetii, Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumoniae, Legionella pneumophila, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia prowazekii, Salmonella enterica, Salmonella typhi, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Vibrio cholerae, Yersinia enter ocolitica, Yersinia pestis, and Yersinia pseudotuberculosis.

11. The method of claim 10, wherein the bacterium is Francisella tularensis,

12. The method of any one of the preceding claims, wherein the carrier protein is selected from the group consisting of bovine serum albumin (BSA), cross-reacting material 197 (CRM197), egg ovalbumin (OVA), keyhole limpet hemocyanin (KLH), maltose binding protein (MBP), tetanus toxoid (TT), meningococcal outer membrane protein (OMPC), diphtheria toxoid (DT), H. influenzae protein D (PD).

13. The method of any one of the preceding claims, wherein the carrier protein is CRM197.

14. The method of any one of the preceding claims, wherein said contacting is carried out more than once.

15. The method of any one of the preceding claims, wherein said contacting is carried out twice, three times, four times, five times, six times, or more.

16. The method of any one of the preceding claims, wherein said contacting is carried out at a dose of about 0.5 pM/organoid to about 5 pM/organoid.

17. The method of claim 16, wherein said contacting is carried out at a dose of about 1.75 pM/organoid.

18. The method of any one of the preceding claims, wherein said culturing is carried out for at least about 2 days, at about least 3 days, at least about 4 days, at least about 5 days, or more.

19. The method of claim 18, wherein said culturing is carried out for 4 days.

20. The method of any one of the preceding claims, wherein said culturing is carried out in the presence of one or more growth factors or cytokines.

21. The method of claim 20, wherein the one or more growth factors or cytokines is IL-4, IL-21, B cell activating factor (BAFF), or a combination thereof.

22. The method of any of the preceding claims, wherein the one or more B cell biomarkers comprises surface proteins and/or intracellular proteins.

23. The method of any of the preceding claims, wherein the one or more B cell biomarkers comprises any B cell biomarker that defines the activation state of murine B cells.

24. The method of any one of the preceding claims, wherein the one or more B cell biomarkers is selected from the group consisting of IgM, phosphorylated Brunton’s tyrosine kinase (pBTK), phosphorylated nuclear factor-xB (pNFKB), EZH2, IRF4, and/or BLIMP- 1.

25. The method of any one of the preceding claims, wherein the one or more biomarkers consists of two biomarkers, three biomarkers, four biomarkers, five biomarkers, or six biomarkers.

26. The method of any one of the preceding claims, wherein the one or more B cell biomarkers are phosphorylated Brunton’s tyrosine kinase (pBTK) and phosphorylated nuclear factor-xB (pNFKB).

27. The method of any one of the preceding claims, wherein the one or more B cell biomarkers are EZH2, IRF4, and BLIMP- 1.

28. The method of any one of the preceding claims, wherein the one or more B cell biomarkers are phosphorylated Brunton’s tyrosine kinase (pBTK), phosphorylated nuclear factor-xB (pNFKB), and IRF4.

29. The method of any one of the preceding claims, wherein the one or more B cell biomarkers are phosphorylated Brunton’s tyrosine kinase (pBTK), phosphorylated nuclear factor-xB (pNFKB), EZH2, IRF4, and BLIMP-1.

30. The method of any one of the preceding claims, wherein the one or more B cell biomarkers are IgM, phosphorylated Brunton’s tyrosine kinase (pBTK), phosphorylated nuclear factor-xB (pNFKB), EZH2, IRF4, and BLIMP- 1.

31. The method of any one of the preceding claims, wherein the detecting is carried out by fluorescence-based microscopy and/or fluorescence activated cell sorting (FACS).

32. The method of claim 31, wherein said detecting is carried out by fluorescence microscopy.

33. The method of claim 32, wherein said fluorescence microscopy is confocal microscopy.

34. The method of claim 31, wherein said detecting is carried out by fluorescence activated cell sorting (FACS) or imaging flow cytometry.

35. The method of claim 34, wherein each of the first plurality of cultured B cell organoids is enzymatically digested prior to said detecting.

36. The method of claim 35, wherein each of the first plurality of cultured B cell organoids is enzymatically digested with a collagenase.

37. The method of any of the preceding claims, wherein said identifying highly immunogenic glycoconjugate candidates is based on a greater presence of one or more B cell biomarker(s) in one or more of said cultured B cell organoids relative to others of said cultured B cell organoids.

38. The method of claim 37, wherein said highly immunogenic glycoconjugate candidates comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% of the plurality of distinct glycoconjugate candidates.

39. The method of claim 38, wherein said highly immunogenic glycoconjugate candidates comprise about 1% of the plurality of distinct glycoconjugate candidates.

40. The method of any of the preceding claims, wherein said identifying highly immunogenic glycoconjugate candidates is based on a greater presence of one or more B cell biomarker(s) relative to a control cultured B cell organoid contacted with the carrier protein, wherein the carrier protein is aglycosylated.

41. The method of claim 40, wherein said greater presence is determined by providing a biomarker level.

42. The method of claim 41, wherein the biomarker level is mean fluorescence intensity.

43. The method of any one of claim 41 or claim 42, wherein the highly immunogenic glycoconjugate candidates comprise at least a 1.2 fold higher level of the one or more B cell biomarker(s) as compared to the control.

44. The method of any one of the preceding claims, wherein each of the first plurality of murine B cell organoids comprise: murine B cells; a CD40 ligand selected from the group consisting of soluble CD40 ligand (sCD40L), cells expressing CD40 ligand, scaffolds presenting CD40 ligand, beads presenting CD40 ligand, and combinations thereof; and a hydrogel, wherein the murine B cells and the CD40 ligand are encapsulated in the hydrogel and wherein the hydrogel comprises cross-linked multi-arm Polyethylene glycol (PEG) macromers.

45. The method of claim 44, wherein the multi -arm PEG macromers comprise four-arm PEG macromers and/or eight-arm PEG macromers.

46. The method of claim 44 or claim 45, wherein the multi-arm PEG macromers are four-arm PEG macromers.

47. The method of any one of claims 44 to 46, wherein the multi-arm PEG macromers are PEG-mal eimide (PEG-MAL) macromers, vinyl sulfonate-PEG (PEG-VS) macromers, acrylate-PEG (PEG-Ac) macromers, or combinations thereof.

48. The method of claim 47, wherein the multi-arm PEG macromers are four- arm PEG-maleimide (PEG-4MAL) macromers.

49. The method of any one of claims 44 to 48, wherein each of the first plurality of murine B cell organoids further comprises CD40 ligand expressing cells from murine lymphoid tissues, other mammalian cells, or polymeric beads.

50. The method of any one of claims 44 to 49, wherein the murine B cells are naive murine B cells.

51. The method of any one of claims 44 to 50, wherein the murine B cells are primary murine B cells.

52. The method of any one of claims 44 to 51, wherein each of the first plurality of murine B cell organoids comprises murine cells that express or present CD40 ligand.

53. The method of claim 52, wherein the murine cells expressing CD40 ligand are primary cells.

54. The method of any one of the preceding claims, wherein each of the first plurality of B cell organoids does not comprise T cells.

55. The method of any one of the preceding claims, wherein each of the first plurality of B cell organoids further comprises T cells.

56. The method of any one of claims 44 to 55, wherein each of the first plurality of B cell organoids comprises 500 - l x 1,000,000 B cells.

57. The method of claim 56, wherein each of the first plurality of B cell organoids comprises 40,000 - 80,000 cells.

58. The method of any one of the claims 44 to 57, wherein each of the first plurality of B cell organoids comprises 10,000 to 200,000 soluble CD40 ligand (sCD40L), cells expressing CD40 ligand, scaffolds presenting CD40 ligand, or beads presenting CD40 ligand.

59. The method of claim 58, wherein each of the first plurality of B cell organoids comprises about 500 -1,000,000 cells expressing CD40 ligand.

60. The method of claim 59, wherein each of the first plurality of B cell organoids comprises about 40,000 - 80,000 cells expressing CD40 ligand.

61. The method of any one of claims 58 to 60, wherein each of the first plurality of B cell organoids comprises about 10 ng/ml - 10 pg/ml soluble CD40 ligand, scaffolds presenting CD40 ligand, and/or beads presenting CD40 ligand.

62. The method of claim 61, wherein each of the first plurality of B cell organoids comprises about 100 ng/ml soluble CD40 ligand, scaffolds presenting CD40 ligand, and/or beads presenting CD40 ligand.

63. The method of any one of claims 44 to 62, wherein the multi-arm PEG- macromers are crosslinked with one or more crosslinkers.

64. The method of claim 63, wherein the one or more crosslinkers comprise an enzymatically degradable crosslinker.

65. The method of claim 64, wherein the enzymatically degradable crosslinker is a protease degradable crosslinker.

66. The method of claim 65, wherein the protease-degradable crosslinker is a peptide.

67. The method of claim 66, wherein the protease-degradable crosslinker is a cleavable bacterial peptide.

68. The method of any one of claims 63 to 67, wherein the one or more crosslinkers comprise a multi-arm or dithiol protease-cleavable peptide crosslinker.

69. The method of any one of claims 63 to 68, wherein the one or more crosslinkers is a protease cleavable peptide.

70. The method of claim 69, wherein the protease cleavable peptide is VPM peptide (GCRDVPMSMRGGDRCG; SEQ ID NO: 1).

71. The method of any one of claims 63 to 70, wherein the non-degradable crosslinker is selected from the group consisting of dithiothreitol (DTT), scrambled peptide crosslinkers, and thiol polymers.

72. The method of claim 70, wherein the scrambled peptide crosslinker is VMP.

73. The method of claim 70, wherein the thiol polymers comprise 2-arm PEG- SH, 4-arm PEG-SH, 8-arm PEG-SH, and combinations thereof.

74. The method of any one of claims 63 to 73, wherein the organoid comprises both a degradable crosslinker and a non-degradable crosslinker.

75. The method of any one of the preceding claims, wherein the organoid is at a pH of about 6.0 to about 8.0.

76. The method of any one of the preceding claims, wherein the organoid is at a pH of about 7.4.

77. The method of any one of the preceding claims, wherein said culturing is carried out at a temperature of 37°C.

78. The method of any one of the preceding claims, wherein the organoid has a volume of about 1 pl to 2 mL.

79. The method of any one of the preceding claims, wherein the organoid has a volume of about 50 pl.

80. The method of any one of the preceding claims, wherein said identifying highly immunogenic glycoconjugate candidates further comprises: identifying highly immunogenic glycoconjugate candidates based on a greater presence of B cell biomarker(s) in one or more of said cultured B cell organoids relative to when the plurality of B cell organoids are contacted with an aglycosylated antigen comprising the carrier protein.

81. The method of claim 80, wherein the carrier protein is CRM197.

82. A highly immunogenic glycoconjugate candidate identified by the method of any one of claims 1 to 81.

83. A plurality of distinct highly immunogenic glycoconjugate candidates identified by the method of any one of claims 1 to 81.

84. A method of identifying glycoconjugate vaccine candidates, said method comprising: providing a plurality of mice; immunizing each of the plurality of mice with distinct highly immunogenic glycoconjugate candidates of the plurality of distinct highly immunogenic glycoconjugate candidates of claim 83; evaluating an immune response of each of the plurality of mice to each of the highly immunogenic glycoconjugate candidates; and identifying glycoconjugate vaccine candidates based on a greater presence of antibodies specific for a polypeptide of interest in the sera of one or more immunized mice relative to others of said immunized mice.

85. The method of claim 84, wherein the mice are selected from the group consisting of Balb/c mice and C57BL/6 mice.

86. The method of claim 85, wherein the mice are Balb/c mice.

87. The method of any one of claims 84 to 86, wherein said immunizing is carried out once, twice, three times, or more.

88. The method of any one of claims 84 to 86, wherein said immunizing is carried out on Day 0, Day 21, and Day 42.

89. The method of any one of claims 84 to 88, wherein said immunizing is carried out at a dose of 1-100 pg of the highly immunogenic glycoconjugate candidate.

90. The method of claim 89, wherein said immunizing is carried out at a dose of about 10 pg of the highly immunogenic glycoconjugate candidate.

91. The method of any one of claims 84 to 90, wherein the immune response is a humoral immune response.

92. The method of claim 91, wherein said evaluating is carried out by determining mouse serum antibody titers against a polysaccharide of interest.

93. The method of claim 92, wherein said serum antibody titers are IgM serum antibody titers, IgG serum antibody titers, IgA serum antibody titers, IgD serum antibody titers, and/or IgE serum antibody titers.

94. The method of claim 93, wherein said serum antibody titers are IgGl serum antibody titers selected from the group consisting of IgG2a serum antibody titers, IgG2b serum antibody titers, IgG2c serum antibody titers, and/or IgG3 serum antibody titers.

95. The method of any one of claims 84 to 94, wherein said polysaccharide of interest is derived from any one of the following: a bacterium, a virus, a parasite, or a cancer.

96. The method of claim 95, wherein the polysaccharide of interest is derived from a bacterium.

97. The method of claim 96, wherein the bacterium is a gram-positive bacterium.

98. The method of claim 97, wherein the bacterium is selected from the group consisting of Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Erysipelothrix rhusiopathiae, Gardnerella vaginalis, Group A Streptococcus, Group B Streptococcus, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Nocardia asteroids, Propionibacterium acnes, Rhodococcus equi, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus mutans, Streptococcus pneumoniae, and Streptococcus pyogenes.

99. The method of claim 95 or claim 96, wherein the bacterium is a gramnegative bacterium.

100. The method of claim 99, wherein the gram -negative bacterium is selected from the group consisting of Acinetobacter baumannii, Bordetella pertussis, Brucella abortus, Burkholderia pseudomallei, Burkholderia mallei, Burkholderia pseudomallei, Campylobacter fetus, Campylobacter jejuni, Coxiella burnetii, Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumoniae, Legionella pneumophila, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia prowazekii, Salmonella enterica, Salmonella typhi, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Vibrio cholerae, Yersinia enter ocolitica, Yersinia pestis, and Yersinia pseudotuberculosis.

101. The method of claim 100, wherein the bacterium is Francisella tularensis.

102. The method of claim 100, wherein the polysaccharide of interest is Francisella tularensis lipopolysaccharides (FtLPS).

103. A method of identifying scFvs specific for a polysaccharide of interest, said method comprising: providing a second plurality of murine B cell organoids; contacting each of the second plurality of murine B cell organoids with the plurality of distinct highly immunogenic glycoconjugate candidates of claim 83, wherein said contacting is carried out to form a second plurality of contacted B cell organoids; culturing the second plurality of contacted B cell organoids under conditions effective to support B cell maturation, whereby said culturing produces a second plurality of cultured B cell organoids; enzymatically digesting the second plurality of cultured B cell organoids; isolating the cultured B cells to produce a plurality of isolated B cells; sequencing the plurality of isolated B cells to identify nucleic acid sequences encoding heavy chain variable region (VH) and light chain variable region (VL) genes; randomly joining VH and VL genes with a nucleic acid sequence encoding a linker to generate a library of candidate single chain variable fragments (scFvs); cloning each of the library of candidate scFvs into a yeast cell surface expression vector; transforming a plurality of yeast cells with the library of yeast cell surface expression vectors to produce a plurality of transformed yeast cells; expressing the library of yeast expression vectors in the plurality of transformed yeast cells to produce a library of scFv-expressing yeast cells, whereby the candidate scFvs are displayed on the surface of the yeast cells; detecting binding of a fluorescently labelled glycoconjugate to each of the library of scFv-expressing yeast cells, wherein the fluorescently labelled glycoconjugate comprises the polysaccharide of interest and a carrier protein that is different than the carrier protein of the plurality of distinct highly immunogenic glycoconjugate candidates; and identifying scFvs specific for the polysaccharide of interest based on said detecting.

104. The method of claim 103, wherein the second plurality of murine B cell organoids are organized in an array.

105. The method of claim 103 or claim 104, wherein the second plurality of murine B cell organoids are seeded in a 6-well plate, a 12-well plate, a 48-well plate, a 96-well plate, a 384-well plate, or larger culture platform.

106. The method of any one of claims 103 to 105, wherein said contacting is carried out at a dose of 0.5 pM/organoid to 5 pM/organoid.

107. The method of claim 106, wherein said contacting is carried out at a dose of about 1.75 pM/organoid.

108. The method of any one of claims 103 to 107, wherein said culturing is carried out for at least 2 days, at least 3 days, at least 4 days, at least 5 days, or more.

109. The method of claim 108, wherein said culturing is carried out for 4 days.

110. The method of any one of claims 103 to 109, wherein said culturing is carried out in the presence of one or more growth factors or cytokines.

111. The method of claim 110, wherein the one or more growth factors or cytokines is IL-4, IL-21, B cell activating factor (BAFF), or a combination thereof.

112. The method of any one of claims 103 to 111, wherein said sequencing is carried out by isolating RNA from the isolated B cells and subjecting the RNA to RT-PCT to generate cDNA libraries.

113. The method of claim 112, wherein said cDNA libraries encode heavy chain variable region (VH) and light chain variable region (VL) genes.

114. The method of any one of claims 103 to 113, wherein the linker is a serine-glycine linker.

115. The method of claim 114, wherein the serine-glycine linker is a (Gly4Ser)4 linker.

116. The method of any one of claims 103 to 115, wherein the vector is a pCT- CON vector.

117. The method of any one of claims 103 to 116, wherein the yeast cells are Saccharomyces cerevisiae cells.

118. The method of claim 117, wherein the yeast cells are Saccharomyces cerevisiae strain EBY100 cells.

119. The method of any one of claims 103 to 118, wherein the polysaccharide of interest is derived from any one of the following: a bacterium, a virus, a parasite, or a cancer.

120. The method of claim 119, wherein the polysaccharide of interest is derived from a bacterium.

121. The method of claim 120, wherein the bacterium is a gram-positive bacterium.

122. The method of claim 121, wherein the bacterium is selected from the group consisting of Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Erysipelothrix rhusiopathiae, Gardnerella vaginalis, Group A Streptococcus, Group B Streptococcus, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Nocardia asteroids, Propionibacterium acnes, Rhodococcus equi, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus mutans, Streptococcus pneumoniae, and Streptococcus pyogenes.

123. The method of claim 119 or claim 120, wherein the bacterium is a gramnegative bacterium.

124. The method of claim 122, wherein the gram-negative bacterium is selected from the group consisting of Acinetobacter baumannii, Bordetella pertussis, Brucella abortus, Burkholderia pseudomallei, Burkholderia mallei, Burkholderia pseudomallei, Campylobacter fetus, Campylobacter jejuni, Coxiella burnetii, Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumoniae, Legionella pneumophila, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia prowazekii, Salmonella enterica, Salmonella typhi, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Vibrio cholerae, Yersinia enter ocolitica, Yersinia pestis, and Yersinia pseudotuberculosis.

125. The method of claim 124, wherein the bacterium is Francisella tularensis.

126. The method of any one of claims 103 to 125, wherein the polysaccharide of interest is conjugated to a carrier protein and wherein the carrier protein is MBP.

127. A murine B cell organoid comprising: murine B cells; a CD40 ligand selected from the group consisting of soluble CD40 ligand (sCD40L), cells expressing CD40 ligand, scaffolds presenting CD40 ligand, beads presenting CD40 ligand, and combinations thereof; and a hydrogel, wherein the murine B cells and the CD40 ligand are encapsulated in the hydrogel and wherein the hydrogel comprises cross-linked multi-arm polyethylene glycol (PEG) macromers.

128. The murine B cell organoid of claim 127, wherein the multi-arm PEG macromers comprise four-arm PEG macromers and/or eight-arm PEG macromers.

129. The murine B cell organoid of claim 127 or claim 128, wherein the multiarm PEG macromers are four-arm PEG macromers.

130. The murine B cell organoid of any one of claims 127 to 129, wherein the multi-arm PEG macromers are PEG-maleimide (PEG-MAL) macromers, vinyl sulfonate-PEG (PEG-VS) macromers, acrylate-PEG (PEG-Ac) macromers, or combinations thereof.

131. The murine B cell organoid of claim 130, wherein the multi-arm PEG macromers are four-arm PEG-maleimide (PEG-4MAL) macromers.

132. A method of making a murine B cell organoid, said method comprising: providing a population of B cells; providing a CD40 ligand selected from the group consisting of soluble CD40 ligand (sCD40L), cells expressing CD40 ligand, scaffolds presenting CD40 ligand, beads presenting CD40 ligand, and combinations thereof; providing a crosslinker solution comprising one or more crosslinkers; suspending the population of B cells and the CD40 ligand in the crosslinker solution to form a cell suspension; providing a functionalized macromer solution comprising a functionalized polyethylene glycol macromer; combining the cell suspension and the functionalized macromer solution to form a hydrogel solution; and curing the hydrogel solution to form a murine B cell organoid comprising encapsulated murine B cells.

133. The murine B cell organoid of any one of claims 127-131 or the method of claim 132, wherein the murine B cells are naive murine B cells.

134. The murine B cell organoid of any one of claims 127-131 or the method of any one of claims 132 to 133, wherein the murine B cells are primary murine B cells.

135. The murine B cell organoid of any one of claims 104-108 or the method of any one of claims 132 to 133, wherein the CD40 ligand is a cell expressing CD40 ligand.

136. The murine B cell organoid of any one of claims 104-108 or 110-112 or the method of any one of claims 132-135, wherein the cells expressing CD40 ligand are primary cells.

137. The method of any one of claims 132 to 136 further comprising introducing medium supplemented with one or more growth factors or cytokines.

138. The method of any one of claims 132 to 137 further comprising introducing a glycoconjugate comprising a polysaccharide of interest and a carrier protein.

139. The method of any one of claims 132 to 138 further comprising culturing said organoid comprising the glycoconjugate.