WO2024010686A2

WO2024010686A2 - Vaccine tissue assays

Info

Publication number: WO2024010686A2
Application number: PCT/US2023/025705
Authority: WO
Inventors: Peter Alan Sims; Donna Lynn FARBER; Alex George; Julia DAVIS-PORADA
Original assignee: Columbia University in the City of New York
Current assignee: Columbia University in the City of New York
Priority date: 2022-06-20
Filing date: 2023-06-20
Publication date: 2024-01-11
Anticipated expiration: 2024-12-20
Also published as: WO2024010686A3; WO2024010686A9

Abstract

Provided herein are methods, compositions, systems and kits for testing immune responses, safety, and efficacy of vaccines. In particular, the methods, compositions, systems and kits of the present invention test immune responses, duration of the immune responses, dose responses and age dependencies in cellular components of lymph nodes, other lymphoid tissues, mucosal tissues, barrier tissues, intestinal tissues, pulmonary tissues, and other solid tissues to vaccines after exposure in cell culture and tissue slices.

Description

VACCINE TISSUE ASSAYS

The present application claims priority to United States Provisional Patent Application Serial Number 63/353,719, filed June 20, 2022, the disclosure of which is herein incorporated by reference in its entirety.

This invention was made with government support under AI128949 and AI106697 awarded by the National Institutes of Health, and W91 INF-23-2-0018 awarded by the Army Research Laboratory - Army Research Office. The U.S. government has certain rights in the invention.

FIELD

BACKGROUND

Although immune responses to vaccines are initiated in tissue sites of administration and the local draining lymph nodes, testing immune responses to vaccines in mammals including humans is largely confined to sampling peripheral blood. Priming of adaptive immune responses to newly encountered antigens occurs in lymph nodes (LNs) by dendritic cells (DC) that present antigens and co-stimulatory signals to naive T cells resulting in activation and differentiation to effector T cells. This priming event also generates T cell-B cell interactions that promote B cell differentiation to antibody-secreting plasma cells. In the case of the SARS-CoV-2 vaccines such as the Moderna mRNA-1273 and other mRNA formulations, the vaccine induces a SARS-CoV-2 Spike (S) protein-specific neutralizing antibody response that is protective (Baden, L.R., et al. Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. N Engl J Med (2020); Thompson, M.G., et al. Prevention and Attenuation of Covid-19 with the BNT162b2 and mRNA-1273 Vaccines. N Engl J Med (2021); Widge, AT, et al. Durability of Responses after SARS-CoV-2 mRNA-1273 Vaccination. N Engl J Med384, 80-82 (2021). Analysis of innate and adaptive immune responses after SARS-CoV-2 mRNA vaccination has been analyzed in peripheral blood. (Arunachalam, P.S., et al. Systems vaccinology of the BNT162b2 mRNA vaccine in humans. Nature (2021). Germinal center B cell responses have also been detected in lymph node aspirates. (Turner, J.S., et al. SARS-CoV-2 mRNA vaccines induce persistent human germinal center responses. Nature (2021). As with many vaccines, the lifespan of the immune response is variable among subjects and is not as long-lasting as desired. New systems and methods are needed to assist in the understanding of and development of long-lasting vaccines and for selecting vaccine administration to subject that will receive maximal benefit.

SUMMARY

The nature of the innate and adaptive immune response that is initiated in tissue sites including lymph nodes and other lymphoid, mucosal, and barrier tissues is unaccounted for using conventional technologies. The cellular composition of human lymphoid sites changes with age from predominantly naive T cells in early life and childhood, to increased frequencies of memory T cells in adults, and predominantly memory T cells in the elderly that affect the ability to respond to newly introduced antigens. Lymphoid sites such as lymph nodes also undergo age- associated changes in morphology, composition and function of myeloid cells.

Accordingly, provided herein are methods, compositions, systems, and kits to test immune responses to vaccines in humans and other mammals that target a diversity of antigens in lymph nodes and a diversity of other lymphoid, mucosal, barrier and solid tissues. In some embodiments, the present invention provides compositions and methods to test a vaccine comprising: exposing tissue or cells from lymph node tissue, other lymphoid tissue, mucosal tissue, barrier tissue, intestinal tissue, and /or pulmonary tissue to a vaccine and evaluating the vaccine. The evaluation may include one or more of: the nature of the immune response generated, the establishment of immune memory, the efficacy of the vaccine for subject that provided the tissue or a category of subject to which the tissue donor belongs (e.g., based on age, health status, drugs or other agents taken (e.g., immunomodulatory drugs), etc.), and the identification of biomarkers that provide an indication of the efficacy of the vaccine and/or the duration of the immune response.

In some embodiments, vaccine evaluation is performed using intact tissue, which preserves the intercellular interactions and structural features of the tissue that are important for immune memory formation.

In some embodiments, the invention analyzes the early cellular and molecular immune responses to vaccination directly in animal (e.g., human) lymph node tissue, or in cultures of total mononuclear cells from animal (e.g., human) lymph nodes. Adaptive immune responses to vaccination are initiated in lymph nodes; T and B cells become activated and interact to promote antibody production, and where the cellular stores of immune memory to vaccines likely reside. Conventional serological measures of vaccine responses simply assess the amount of antibody produced in response to vaccination that is present in peripheral blood, and do not provide any information on the early immune response to vaccination, nor on the cells that produce the antibodies or maintain long-term immune memory. The invention has the advantage of measuring immune responses directly at the tissue site that orchestrates immune memory formation and directly assess the functional responses of lymph node cells which is where vaccine responses are initiated in vivo.

In some embodiments, the present invention provides a method to test a vaccine, comprising: exposing a vaccine to a tissue sample comprising one or more of a lymph node tissue, other lymphoid tissue, mucosal tissue, barrier tissue, intestinal tissue, and/or pulmonary tissue, or cells derived therefrom, and assessing one or more characteristics of the vaccine.

In some embodiments, the present invention provides a method to test a vaccine, comprising: digesting one or more solid tissues to an immune cell suspension; generating one or more cell cultures from the immune cell suspension; and applying the vaccine to the one or more cell cultures.

In some embodiments, the vaccine is an attenuated live vaccine, an inactivated vaccine, a toxoid and/or toxin vaccine, a subunit vaccine, a conjugate vaccine, an outer membrane vesicle vaccine, a heterologous vaccine, a DNA vaccine, an RNA vaccine, a viral vector vaccine, a lipid nanoparticle vaccine, a dendritic cell vaccine, a recombinant vector vaccine, a T-cell receptor vaccine, a killed bacteria vaccine, and existing pathogen vaccine, and emerging pathogen vaccine, a bacterial vector vaccine, a plasmid vector vaccine, or an antigen-presenting cell vaccine. In some embodiments, the vaccine is a mRNA vaccine. In some embodiments, the mRNA vaccine is a SARS-CoV-2 mRNA vaccine.

In some embodiments, the solid tissue is a lymph node tissue, other lymphoid tissue, mucosal tissue, barrier tissue, intestinal tissue, and/or pulmonary tissue. In some embodiments, the solid tissue is a tissue slice sample, a biopsy sample, or a needle aspirate sample.

In some embodiments, the method comprises assessing the cell culture for an immune response. In some embodiments, the assessing comprises supernatant testing for one or more activation marker concentrations, co-stimulatory marker concentrations, cytokine concentrations, chemokine concentrations, secreted factor concentrations, and/or cell free nucleic acid sequences and modifications. In some embodiments, the assessing comprises cell testing for high parameter flow cytometry, T-cell activation and proliferation, B-cell activation, myeloid cell activation, and/or cell-type specific and/or single cell nucleic acid sequences and modifications. In some embodiments, assessing is performed serially at hourly, daily, weekly and/or monthly intervals. In some embodiments, the assessing comprises identification of at least one biomarker of immune response duration. In some embodiments, two or more vaccines with different durations of pathogen protection differ in at least one biomarker of immune response duration.

In some embodiments, the one or more cell cultures comprises two or more cell cultures from two or more solid tissues from an individual or from two or more individuals. In some embodiments, the assessing comprises assessing two or more immune responses from the two or more cell cultures.

In some embodiments, the present invention provides a method to test an immunomodulatory agent, comprising: digesting one or more solid tissues to an immune cell suspension; generating one or more cell cultures from the immune cell suspension; and applying the immunomodulatory agent to the one or more cell cultures. In some embodiments, the immunomodulatory agent is an antibody, a cytokine, an immunomodulating drug, an immunostimulating drug, an immunosuppressive drug, a receptor antagonist, an immunoglobulin, a cytotoxic agent, an antimetabolite, a checkpoint inhibitor, a cytokine, an adjuvant or a small molecule. In some embodiments, the method comprises applying an immunomodulatory agent and a vaccine to the one or more cell cultures.

In some embodiments, the present invention provides a method to test a vaccine or immunomodulatory agent comprising: obtaining a tissue slice from a solid tissue; generating a tissue culture from the tissue slice; applying the vaccine or immunomodulatory agent to the tissue culture; and assessing one or more characteristics of the vaccine or immunomodulatory agent. In some embodiments, the tissue culture is a cultured tissue slice. In some embodiments, the method comprises digesting the tissue culture before the applying. In some embodiments, the solid tissue comprises one or more of a lymph node tissue, other lymphoid tissue, mucosal tissue, barrier tissue, intestinal tissue, and/or pulmonary tissue. In some embodiments, the solid tissue is fresh-frozen tissue and/or formalin-fixed paraffin-embedded (FFPE) tissue. In some embodiments, the assessing comprises one or more of microscopy, histology, spatial profiling, immunohistochemistry, immunofluorescence, RNA-FISH, RNA-ISH, spatial transcriptomics, and highly multiplexed RNA-FISH.

In some embodiments, the present invention provides a method to test a vaccine or immunomodulatory agent comprising: applying the vaccine or immunomodulatory agent to a tissue sample comprising one or more of a lymph node tissue, other lymphoid tissue, mucosal tissue, barrier tissue, intestinal tissue, and/or pulmonary tissue, or cells derived therefrom; digesting the tissue sample to generate an immune cell suspension; generating one or more cell cultures from the immune cell suspension; and assessing one or more characteristics of the vaccine or immunomodulatory agent.

DESCRIPTION OF THE DRAWINGS

A better understanding of the invention and its advantages will be apparent from the detailed description taken in conjunction with the accompanying drawings in which: Figure 1. shows a schematic workflow comprising a comparison of lymph node (LN) suspension and lymph node slice culture. In some embodiments of the present invention, corresponding liquid and slice cultures are generated from an organ donor from a LN. T cells in cultures are stimulated with reagents. scRNA-seq with cell surface proteomics (Cellular Indexing of Transcriptomes and Epitopes or CITE-seq) is performed on liquid and slice culture after dissociation to single cell suspension.

Figure 2. shows a schematic diagram of 2 stimulation reagents used in liquid and slice LN culture. Anti CD3/28 antibody encodes signal 1 and 2 respectively to activate T cells and is independent of antigen presenting cells (APCs). A superantigen mimicking bispecific antibody (Cytostim) brings APCs in contact with T cells by bridging major histocompatibility complex (MHC) Class II and T cell receptor (TCR) thereby providing signal 1), and proximity of APCs to T cells provides costimulatory signals 2 and paracrine signal 3 to facilitate T cell activation.

Figure 3. shows a schematic diagram of slice culture generation workflow. Gross dissection of lymph node is performed to attenuate the collagen rich capsule and subcutaneous fat to facilitate tissue slicing downstream. Dissected and/or cleaned nodes are embedded in low melting point agarose (6% v/w) at 37 degrees C. LNs embedded in agarose are cut into blocks and glued onto a stage of a tissue chopper. Slices are generated (500 pM) and placed in complete culture media (IMDM, 5% FBS, IX penicillin/streptomycin/glutamine - PSQ). 2-3 slices are placed on each transwell insert (in, for example, a 6 well plate well). 1.5 mL of media is placed on the bottom under the transwell insert. 60 uL of complete media or stimulation reagent diluted in complete media is placed on top of slices. Slices are moved to the periphery of a “reagent pool” to prevent slices from floating in the media pool and to establish contact with the transwell on the bottom of the slice, while facilitating gas exchange and contact with complete media pool or treatment at the level of the transwell insert. Slices may be cultured for 18-24 hours or longer. If cultured > 24 hours, 1.5 mL of media under the transwell is replaced every 24 hours.

Figure 4. shows % viability of LN slice culture after dissociation to a single cell suspension in 7 distinct organ donors. Donor 1 was cultured for 96 hours and Donors 2-7 for 21 hours.

Figure 5. shows a schematic diagram of steps to assess lymphocyte activation dynamics in corresponding slice and liquid cultures. Figure 6. shows a heatmap of z-scored average expression values from scRNA-seq of unstimulated LN liquid and slice cultures from 3 organ donors. Expression is evaluated specifically in T cells. Markers provided are associated with cytotoxic CD8 T cells and are found at higher levels in LN slice cultures compared to LN liquid cultures.

Figure 7. shows a heatmap of z-scored average expression values from scRNA-seq of unstimulated and stimulated LN liquid and slice cultures from 3 organ donors. T cells were identified computationally and expression of each marker was computed specifically in T cells. Markers shown are associated with T cell activation. Liquid LN cultures exhibit higher Thl responses (IFNG, IL2), particularly with anti-CD3/anti-CD28 stimulation, whereas CD8 effector memory activating markers including XCL1 and XCL2 are higher in stimulated LN slice cultures.

Figure 8. shows the expression of activation markers as a function of measles, mumps, rubella MMR-II vaccine dose and IFN positive control stimulation in LN liquid cultures as measured by flow cytometry. Dilution values on the x-axis correspond to MMR-II dose.

Figure 9. shows uniform manifold approximation and projection (UMAP) embedding of CITE-seq data for MMR-II vaccine stimulated and unstimulated LN liquid and slice cultures colored by classification of major immune cell types.

Figure 10. shows a heatmap of z-scored average expression values from scRNA-seq of unstimulated and MMR-II- stimulated LN liquid and slice cultures. A) T cells were identified computationally and expression of each marker was computed specifically in T cells. Markers shown are generally associated with resting and activated T cells. Slice cultures exhibit higher induction of CD8 effector memory activation markers XCL1, XCL2, and CCL4 compared to liquid culture. B) B cells were identified computationally and expression of each marker was computed specifically in B cells. Markers shown are associated with resting and activated B cells. Liquid cultures exhibit higher induction of B cell activation markers such as CD80 and CD 86 than slice cultures.

Figure 11. shows representative H&E-stained tissue section from a lymph node slice culture prepared as described in Experimental Example 11.

DEFINITIONS To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below. While the invention will be described in conjunction with certain representative embodiments, it will be understood that the invention is not limited to these illustrative examples. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein may be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

Unless defined otherwise, technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice of the invention, certain methods, devices, and materials are described herein. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description.

As used in this disclosure, including the appended claims, the singular forms "a," "an," and "the" include plural references, unless the content clearly dictates otherwise, and are used interchangeably with "at least one" and "one or more."

As used herein, the term "about" represents an insignificant modification or variation of the numerical value such that the basic function of the item to which the numerical value relates is unchanged.

As used herein, "protein" is used synonymously with "peptide," "polypeptide," or "peptide fragment." A "purified" polypeptide, protein, peptide, or peptide fragment is substantially free of cellular material or other contaminating proteins from the cell, tissue, or cell-free source from which the amino acid sequence is obtained, or substantially free from chemical precursors or other chemicals when chemically synthesized.

As used herein, "modulate" means to alter, either by increasing or decreasing, the activity of a gene or protein. The term “inhibit”, as used herein, means to prevent or reduce the activity of gene or protein.

As used herein, the term "bioactivity" indicates an effect on one or more cellular or extracellular process (e.g., via binding, signaling, etc.) that can impact physiological or pathophysiological processes. Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub -range from 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 (as well as fractions thereof unless the context clearly dictates otherwise). Any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated.

As used herein, the term “cell culture” refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, transformed cell lines, finite cell lines (e.g., non-transformed cells), tissues, and any other cell population maintained in vitro.

As used herein, the term “/// vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell lysate. The term “/// vivo” refers to the natural environment (e.g., an animal or a cell) and to processes or reaction that occur within a natural environment.

DETAILED DESCRIPTION OF THE DISCLOSURE

Immune cells may be referred to as “white blood cells.” But most, including more than 95% of T cells, reside and function in tissues, particularly lymphoid organs including bone marrow, spleen and lymph nodes, and in barrier surfaces including skin, gut and mucous membranes. (Kumar BV, Connors TJ, Farber DL. Human T Cell Development, Localization, and Function throughout Life. Immunity. 2018 Feb 20;48(2):202-213.) Certain immune cells are rarely found in blood. Some fail to enter the circulation. Others change their phenotypes when they do enter the circulation. For example, memory T cells, develop from activated T cells that migrate to tissues following priming in lymph nodes during infection. The tissuehoming T cells take up long-term residence in tissues and develop properties that are distinct in each.

For SARS-CoV-2, examining blood has helped to track responses to infection and vaccines, and to identify correlates of severe disease. However, the bulk of immune responses take place in tissue. The composition of immune cells is distinct in different tissues, with tissuespecific variation in gene expression, metabolic pathways, and functional regulation. (Weisberg SP, Ural BB, Farber DL. Tissue specific immunity for a changing world. Cell. 2021 Mar 18; 184(6): 1517-1529.) RNA transcripts, protein content, DNA methylation, histone and chromatin status, genotype and gene modifications may be pinpointed even for single cells. Combined with computational analyses, these measurements allow' the simultaneous identification of immune-cell composition, lineage, and functional states. In particular, to fully ascertain the response of an organism to a vaccine, and to test a candidate vaccine for efficacy in the tissues of greatest relevance to disease expression, blood samples before and after vaccination are insufficient. Accordingly, in some embodiments, the present invention provides tissue platforms comprising lymph nodes and/or other lymphoid, mucosal, and barrier tissue platforms to assay the efficacy and safety of candidate vaccines, to optimize vaccines, and to select appropriate vaccine for administration to particular subjects or groups of subjects.

In some embodiments, the present invention provides methods, compositions, systems and kits that predict vaccine responses in mammals and humans, including durable immune memory and side effects. Conventional technologies rely on pre-clinical studies in animal models followed by large-scale clinical trials that may take months or years to complete. Prior efforts to identify predictors of vaccine efficacy directly from human immune cells have relied on peripheral blood because of its accessibility. However, the early-stage cellular and molecular events that orchestrate long-term protection by vaccines occur in the lymph nodes (LNs). Similarly, testing immunomodulatory drugs as a general class of therapeutics have relied on peripheral blood rather than LNs. These drugs induce well -recognized immunological side effects including cytokine storm syndrome and autoimmunity arising from interactions with tissues and tissue-associated immune cells. Accordingly, the present invention provides methods and compositions for procuring and culturing human lymph node slices for direct perturbation with immunomodulatory drugs, biologies and vaccines, and for analyzing cell type-specific, molecular responses with single-cell genomics.

In some embodiments, the present invention provides methods, compositions, systems and kits comprising LN assays to test immunomodulatory drugs and vaccines, and for collecting and analyzing single-cell genomic data as a readout for these assays. In some experiments conducted in development of the present invention, T cell responses of liquid mononuclear cell (MNC) cultures derived from human LNs were compared to directly perturbed human LN slice cultures (Figure 1). To elicit T cell responses, 2 mechanistically distinct drugs: 1) an anti- CD3/anti-CD28 tetramer that stimulates T cell receptor (TCR) signaling independent of antigen- presenting cells (APCs); and 2) Cytostim™, a bi-specific TCR/MHC II antibody that stimulates APC-mediated TCR signaling (Figure 2). To generate LN slice cultures, LNs are dissected and embedded in agarose, sectioned to 500-micron slices with a tissue chopper, and cultured on transwell inserts in tissue culture plates (Figure 3). LNs from 7 human organ donors reproducibly yielded high-viability cells after dissociation of LN slice cultures and 21-96 hours of culture (Figure 4). This experimental system was used to analyze immune responses to the 2 drugs described above in human LN slices using Cellular Indexing of Transcriptomes and Epitopes (CITE-seq) as a readout. CITE-seq provides parallel profiling of transcriptome-wide gene expression and surface protein abundance for greater than 130 markers in thousands of single cells. The workflow from tissue procurement to CITE-seq analysis is depicted in Figure 5 with methodological details for slice culture provided in Experimental Example 4 and for slice culture perturbation and single-cell profiling in Example 5.

The experimental results disclosed marked differences in the T cell responses elicited in LN slice cultures compared to liquid cultures. At rest, T cells in LN slice cultures comprise a more cytotoxic phenotype, expressing granzymes, perforin- 1, and other markers at higher levels than in liquid cultures (Figure 6). T cell stimulation, particularly with anti-CD3/anti-CD28, generates strong Thl responses (IFNG, IL2) in liquid culture, whereas a subset of genes associated with CD8 effector memory responses (XCL1, XCL2) are more strongly induced in slice culture (Figure 7). These data reveal that baseline immunophenotypes and T cell responses in liquid and LN slice cultures are distinct, thereby providing newly detected predictive biomarkers of drug and vaccine efficacy wherein the spatial architecture of the LN that is preserved in the slice culture supports cell-cell interactions and kinetics that are not possible in liquid cultures.

In experiments conducted in the development of the present invention, LN liquid and slice cultures were stimulated with measles/mumps/rubella vaccine (Merck MMR-II). To assess immune responses, dose response experiments were conducted in liquid LN culture with MMR- II using cytometry for quantification (Experimental Example 6). Stimulation with IFNb was provided as a positive control. As shown in Figure 8, clear dose-response behavior was observed including for CD25 a marker that decreases in CD3+ T cells as a function of dose, and CD86 and HLA-DR markers that increase in CD45+/CD3-/CD19- cells with dose.

Immune responses to MMR-II in LN liquid and slice cultures were further compared using CITE-seq (Experimental Example 9). Using the CITE-seq surface protein and scRNA-seq data, major immune cell types were observed in the LN including CD4 T cells, CD8 T cells, B cells, plasma cells, and myeloid cells (Figure 9) supporting analysis of cell type-specific responses to MMR-II. Of note, a comparison of liquid and slice LN cultures for MMR-II- stimulated T cells paralleled results for antibody-based T cell stimulation with slice cultures expressing higher levels of CD8 effector memory activation markers including XCL1, XCL2, PRF1, and CCL4 (Figure 10A). Conversely, B cell responses were more prominent in liquid LN cultures stimulated with MMR-II (Figure 10B).

Methods

In some embodiments, the methods of the present invention find use in screening a candidate vaccine for immune responses, safety, and efficacy and for developing and identifying vaccine with desired characteristics. In some embodiments, the method provides a vaccine discovery method. In some embodiments, the method provides a vaccine optimization method for a vaccine function or property comprising, for example, speed of protection onset, duration of protection, concentration range of protection, breadth of protection vs. diverse species and strains, side effects and toxicity of a vaccine, interaction of a vaccine with co-existing solid tissue conditions, interaction of a vaccine with co-administered medications and compounds, combinations of two or more related or unrelated vaccines, and the like. In some embodiments, the method provides a method for precision, personalized vaccination wherein one or more functions or properties of a vaccine differ between individuals. In some embodiments, the method provides a manufacturing method. In some embodiments, the method provides a quality assurance method. In some embodiments, the method finds use in satisfying vaccine regulatory requirements. Methods of the present invention are not limited to a specific pathogen, a specific candidate vaccine, or to a specific assay tissue. In some embodiments, methods of the present invention are performed in parallel with two or more sample sources, for example, a blood sample source and a lymph node tissue sample source, or two or more lymph node sources from, for example, one individual or two or more individuals. In some embodiments, the methods, compositions, reaction mixtures, systems and kits of the present invention find use in the identification of biomarkers of susceptibility, diagnosis, prognosis, response to therapy, and transmissibility of an infection or heritability of a cancer.

In some embodiments, the vaccine is an attenuated vaccine, an inactivated vaccine, a toxoid vaccine, a subunit vaccine, a conjugate vaccine, an outer membrane vesicle vaccine, a heterologous vaccine, a DNA vaccine, an RNA vaccine, a viral vector vaccine, a lipid nanoparticle vaccine, a dendritic cell vaccine, a recombinant vector vaccine, a T-cell receptor vaccine, a bacterial vector vaccine, a plasmid vector vaccine, or an antigen-presenting cell vaccine. In some embodiments, the vaccine comprises an adjuvant, an excipient, and/or a preservative.

In some embodiments, the vaccine is directed to infection by an adenovirus, a papovavirus, a parvovirus, a herpes virus, a poxvirus, a poliovirus, a reovirus, a rotavirus, a pircomavirus, a calicivirus, a togavirus, an arenavirus, a flavivirus, an orthomyxovirus, a paramyxovirus, a bunyavirus, a rhabdovirus, a filovirus, a coronavirus, an astrovirus, a bornavirus, an arterivirus, a hepevirus, a retrovirus, a heptavirus, a hepadnavirus or other viral pathogen. In some embodiments, the vaccine is directed to infection by Corynebacieritim diphtheriae , Clostridium letani, Bordeiella pertussis, Haemophilus influenzae type b, Streptococcus pneumoniae. Neisseria meningitidis, Salmonella typhi, Mycobacterium tuberculosis. Yersinia pestis, Bacillus anfhracis, Vibrio cholerae or other bacterial pathogen. In some embodiments, the vaccine is directed to infection by a fungal pathogen, for example, a Candida fungus, and Cryptococcus fungus, an Aspergillus fungus or other fungal pathogen. In some embodiments, the vaccine is directed to infection by a parasite, for example, a malarial parasite, a leishmaniasis parasite, a schistosomiasis parasite or other parasite infection. In some embodiments, the vaccine is directed to cancer prevention and/or to cancer therapy.

Kits

The present disclosure provides kits comprising components to perform methods of the present invention. For example, in some embodiments, kits of the present invention comprise at least one tissue dissociation reagent, at least one cell culture reagent, and/or at least one detection reagent. In some embodiments, such kits may comprise, for example, two or more of a stabilizer, a buffer, instructions, positive and negative control reagents (for example, positive and negative vaccine control reagents), one or more calibrants, and ligands to identify possible sources of error and contamination. In some embodiments, a positive control vaccine achieves statistical significance indicating that the positive control experiment performs to specification. In some embodiments, a kit of the present invention comprises calibration reagents and ligands including, for example, a detection calibration reagent. In some embodiments, the kit comprises customizable options. In some embodiments, a kit of the present invention comprises frozen (e.g., prepared with glycerol for storing at -20 C degree) components and reagents, and/or desiccated reagents and components.

Compositions and reaction mixtures

The present disclosure provides compositions and reaction mixtures comprising components of the assays described herein. Such compositions and reaction mixtures may comprise, for example, two or more of a stabilizer, a buffer, positive and negative control reagents, and ligands to identify possible sources of error and contamination. In some embodiments, compositions and reaction mixtures of the present invention comprise a cell culture or tissue slice supernatant, and/or a cell culture or tissue slice living cell. EXPERIMENTAL EXAMPLES

The following examples are provided to demonstrate and further illustrate certain embodiments and aspects of the present disclosure and are not to be construed as limiting the scope thereof.

Tissues

Tissues from human organ donors have been acquired through collaboration with organ procurement organizations (OPOs) in the New York Metropolitan area for adults of all ages, and OPOs through the entire US for tissues from infant and pediatric donors aged 0-12 years of age. (Farber, D.L. Tissues, not blood, are where immune cells act. Nature 593, 506-509 (2021) Kumar, B.V., Connors, T.J. & Farber, D.L. Human T Cell Development, Localization, and Function throughout Life. Immunity 48, 202-213 (2018) Multiple LN are obtained, including lung- and gut-associated LN, and peripheral LN, together with spleen, bone marrow, lungs, intestines, and blood to test immune cells and vaccine responses across multiple tissues and ages. (Carpenter, D.J., el al. Human immunology studies using organ donors: Impact of clinical variations on immune parameters in tissues and circulation. Am J Transplant 18, 74-88 (2018); Dogra, P., et al. Tissue Determinants of Human NK Cell Development, Function, and Residence. Cell 180, 749-763 e713 (2020); Gordon, C.L., etal. Tissue reservoirs of antiviral T cell immunity in persistent human CMV infection. J Exp Med 214, 651-667 (2017); Granot, T., et al. Dendritic Cells Display Subset and Tissue-Specific Maturation Dynamics over Human Life. Immunity 46, 504-515 (2017); Thome, J. J., et al. Spatial map of human T cell compartmentalization and maintenance over decades of life. Cell 159, 814-828 (2014). In experiments in the course of development of embodiments of the present invention, these tissues are used to appraise innate and adaptive immune response to SARS-CoV-2 mRNA vaccines, for example the mRNA vaccine encoding SARS-CoV-2 S protein in diverse tissues and tissue ages.

Example 1 - Lymph node vaccine response from donors of different ages

To assess vaccine responses in lymph nodes (LNs) obtained from donors of different ages, total immune cells are isolated from donor LNs after tissue digestion to obtain single cell suspensions. Lymphocytes, DC, and other myeloid cells predominate in LN. Total LN immune cells are cultured with scaled concentrations of a mRNA vaccine formulation for evaluation at serial intervals beginning with 12-24 hours to measure innate immune responses, and 48-96 hours to monitor adaptive immune responses. Supernatants and cells are isolated at each interval. Upregulation of activation markers (e.g., HLA Class II) and co-stimulatory markers (e.g., CD80/86) by DC and myeloid-lineages cells are assessed by high parameter flow cytometry at early serial intervals, while T cell activation and proliferation to S protein are assessed at later serial intervals, with spike (S)-specific B cell activation assessed at 1 week and thereafter in culture. Supernatants are analyzed for 50+plex cytokines and chemokines from both innate and adaptive cells. Lymph nodes acquired from donors aged 2-80 years are used to test whether a candidate mRNA vaccine promotes immune responses in LN obtained from donors that differ by age. The optimal mRNA vaccine dose for each age is identified in vitro by measuring responses to different doses in cultures from LN donors of different ages.

Example 2 - Pulmonary and intestinal vaccine responses

Parallel ex vivo cultures are prepared, and supernatants and cells are tested for immune responses in lung and intestinal tissue after exposure to a vaccine.

Example 3 - Vaccine responses in tissue slice cultures

A candidate vaccine is loaded directly onto a tissue slice, for example, a lymph node tissue slice, followed by incubation, dissociation, cell culture and characterization. (Zhao, W., et al. Deconvolution of cell type-specific drug responses in human tumor tissue with single cell RNA-seq. Genome Med 13, 82 (2021). Immune responses to a candidate vaccine are tested in situ by imaging, flow cytometry, genotype, single cell transcriptome profiling, methylation and/or histone status, chromatin status, immune receptor identity (for example, T-cell receptor identity and/or antibody clonal identity), and/or protein/ surf ace phenotype of cells isolated from tissue slices that have been exposed to the vaccine.

Example 4 - Lymph nodes from organ donors

In some embodiments, the present invention provides methods, compositions, systems, and kits to generate and maintain slice cultures of human lymphoid tissues with high viability (>90%) for up to ~96 hours after sectioning (Figure 3). LNs from organ donors were dissected to attenuate the collagen-rich capsule and remove surrounding fat/connective tissue, embedded in 6% w/v low melting point agarose (Invitrogen, Cat: 16520100) at 37 °C, and sectioned at a thickness of 500 pM (McIlwain Tissue Chopper, Ted Pella, Cat: 10180). Following removal of agarose, 2-3 slice cultures were placed on each cell culture transwell insert (PTFE, 30 mm, 0.4 pM pores, Millipore, Cat: PICM0RG50) in a 6-well plate with 1.5 mL of culture media (IMDM (Thermo Fisher, Cat: 12440053), 5% FBS (Thermo Fisher, Cat: A3160401), IX Penicillin/Streptomycin/Glutamine (Thermo Fisher, Cat: 10378016)) per well. 60 pL of media was added on 2-3 slices per transwell with slices moved to the periphery of the “media/reagent pool” to prevent slices from floating and to establish contact with the transwell on the bottom of the slice while facilitating gas exchange and contact with media/reagent at the level of the transwell insert. Slice cultures were maintained at 5% CO2 37 °C. Growth media was changed without disturbing slice cultures on insert every 24 hours.

Example 5

Slice cultures (pooling all slice cultures per experimental condition) were processed to a single-cell suspension using enzymatic and mechanical dissociation. A corresponding suspension culture as a comparator to slice culture for a specific donor is also generated with the following protocol: LN slices or dissected whole LN were digested with DNAse I (0.1-0.3 mg/mL, Millipore Sigma, Cat: DN25-5G) and Collagenase D (1-3 mg/mL, Millipore Sigma, Cat: 11088882001) for 30 min at 37 °C. The digest was passed through a 100 pM filter (MACS smart strainer, Miltenyi) and mononuclear cells were isolated with density gradient centrifugation (Ficoll-Paque PLUS, Cytiva). Single cell suspensions from slices were quantified for viability and cell counts, incubated with Fc blocking reagents (TruStain FcX, Biolegend, Cat: 422301; FcR blocking reagent human, Miltenyi Biotec, Cat: 130-059-901) for 10 min at 4 °C, incubated with barcoded antibodies for hash-tagging and cell surface proteomics (Total Seq-A human panel, Biolegend) for 30 min at 4 °C, and processed for single cell sequencing using lOx Genomics Chromium 3’ v3.1 chemistry (10X Genomics, Cat: PN-1000121). Libraries were sequenced on a NextSeq 500/550 system (Illumina). Example 6 - Lymph node slice cultures (T Cell Stimulation)

Slice cultures were generated (500 pM thick) together with corresponding single cell (liquid culture) suspensions from the same donor using the procedure in Experimental Example 5. 5xl0⁶ cells in suspension were cultured in complete media (IMDM (Thermo Fisher, Cat: 12440053), 5% FBS (Thermo Fisher, Cat: A3160401), IX Penn/Strep/Glutamine (Thermo Fisher, Cat: 10378016)) as an unstimulated control at a density of IxlO⁶ cells/mL. Liquid culture was stimulated with a-CD3/28 (Immunocult™, Stem Cell Technologies, Cat: 10971) in complete media for 18hrs at 25 pL of reagent per million cells at lxl0⁶ cells/mL (5xl0⁶ cells total). Liquid culture was also stimulated with APC independent superantigen mimicking reagent (Cytostim™, Miltenyi Biotec, Cat: 130-092-172) in complete media for 18hrs as per manufacturer’s protocol (20 pL of reagent per IxlO⁷ cells at IxlO⁷ cells/mL, IxlO⁷ cells total). A subset of slice cultures was set aside as an unstimulated control (for example, 6+ slices w/ 2-3 slices on transwell insert per well in 6 well plate) in complete media with 60 uL of media added on top of slices which were moved to the periphery of the “media/reagent pool” to prevent slices from floating and to establish contact with the transwell on the bottom of the slice while facilitating gas exchange and contact with media/reagent at the level of the transwell insert. Slices were stimulated with aCD3/28 for 18 hours in complete media (60 uL of Immunocult^1M aCD3/28 reagent on top of 2-3 slices per transwell as described in method above). Slices were also stimulated with Cytostim™ reagent for 18 hours prepared in complete media (20 uL of stock Cytostim™ reagent diluted in 40 pL of complete media) with 60 pL of diluted Cytostim™ in media placed on top of slices as described in method above. After 18 hours of stimulated or unstimulated culture, liquid cultures were spun down, supernatants collected for cytokine analysis, and resuspended in cold FBS-DPBS-EDTA buffer (IX DPBS (Thermo Fisher, Cat: 14190144), 5% FBS (Thermo Fisher, Cat: A3160401), 2mM EDTA (Thermo Fisher, Cat: 15575020) until the slice cultures were processed. Slice culture media beneath the transwells was collected for cytokine analysis. Slice cultures were pooled per stimulation/unstimulated condition, digested with DNAse I (0.1-3 mg/mL, Millipore Sigma, Cat: DN25-5G) and Collagenase D (1-3 mg/mL, Millipore Sigma, Cat: 11088882001) for 30 min at 37 °C. The digest was passed through a 100 pM filter (MACS smart strainers, Miltenyi) and mononuclear cells were isolated with density gradient centrifugation (Ficoll-Paque PLUS, Cytiva). Suspension cell cultures on ice and single cell suspension from slices were quantified for viability and cell counts, incubated with Fc blocking reagents (TruStain FcX, Biolegend, Cat: 422301; FcR blocking reagent human, Miltenyi Biotec, Cat: 130-059-901) for 10 min at 4 °C, incubated with barcoded antibodies for hash-tagging and cell surface proteomics (Total Seq-A human panel, Biolegend) for 30 min at 4 °C, and processed for single cell sequencing using lOx Genomics Chromium 3’ v3.1 chemistry (10X Genomics, Cat: PN-1000121). Libraries were sequenced on a NextSeq 500/550 system (Illumina).

Example 7 - Single cell mononuclear suspensions and MMR vaccine

Single cell mononuclear suspensions isolated from human lung-draining lymph nodes as described in Example 4 were thawed as described in Example 8 and plated in a 24-well flat bottom plate (1.5xl0⁷ cells per well) in 1.5mL of complete media (RPMI (Corning, catalog no. 10-040-CM), 10% inactivated human AB serum (Gemini, catalog no. 507533010) and penicillin- streptomycin-glutamate (Thermo Fisher Scientific, catalog no. 10378016)), and incubated overnight at 37°C in 5% CO2. The next day, one dose of Merck Measles, Mumps, and Rubella live virus vaccine (M-M-R II; McKesson catalog no. 304337) was reconstituted in 500uL of sterile diluent. Various doses of MMR vaccine (l OOuL, lOuL, 5uL, or 0.5uL) were added to the cell cultures (two replicates per dose) and incubated for 24 hours. Two replicate wells were unstimulated, and two replicate wells received lOng of IFNb (PeproTech, catalog no. 300- 02BC). After 24 hours, replicate wells were combined and counted. 2xl0⁶ cells were set aside as the “lymphoid fraction.” The remaining cells (~2.5xl0⁷) underwent a CD33 positive selection using the Easy Sep Release Human Biotin Positive Selection Kit (StemCell, catalog no. 17653) and biotinylated human anti-CD33 antibody (Biolegend, catalog no. 366628), and the resulting fraction was set aside as the “myeloid fraction.” The myeloid and lymphoid fraction samples were washed with FACS buffer (PBS (Coming, cat no. 20-030-CV) with 2% heat inactivated FBS (GeminiBio, cat no. 100-106) and ImM EDTA (Coming, cat no. 46-034-CI)) and resuspended in lOuL Human TruStain FcX (Biolegend, cat no. 422302) for 10 minutes at room temperature. Fluorochrome-conjugated antibodies and fixable viability stain eFluor780 in Brilliant Stain buffer (BD Bioscience, catalog no. 566349) were added to cell suspensions for 20 minutes at room temperature protected from light, washed twice, and incubated for 10 minutes with Annexin V BUV395 (BD Bioscience, catalog no. 564871) in the recommended buffer. Cells were washed and resuspended in fixation buffer (Tonbo, catalog no. TNB-0607-KIT) for 30 minutes at room temperature. Flow cytometry data were collected using the five-laser Cytek Aurora flow cytometer and analyzed using FlowJo V10.7.1.

Example 8 - Lymphoid and myeloid cell flow cytometry

Organ donor samples in saline or University of Wisconsin solution were placed on ice, transported to the laboratory, and processed within 2 to 4 hours of procurement. Single cell suspensions were obtained from blood, bone marrow, spleen, lungs, and associated lymph nodes (LN) using mechanical and enzymatic digestion as previously described (Granot et al., ibid. , ' Sathaliyawala et al., Distribution and compartmentalization of human circulating and tissueresident memory T cell subsets, Immunity 38, 187-197 (2013); Thome et al., ibid). Blood and bone marrow samples were diluted with wash buffer (PBS (Coming, cat #20-030-CV) + 2mM EDTA (Corning, cat #46-034-CI) + 5% FBS (GeminiBio, cat #100-106)), and mononuclear cells were isolated by density centrifugation using Ficoll-Paque PLUS (GE Healthcare, catalog no. 17- 1440-03). Lymph nodes were isolated by dissection from the mesentery of the small and large intestine, the tracheobronchial tree of the lung, and inguinal fat. Tissue samples were first mechanically disrupted with scissors. Isolated LNs and lung were then placed into digestion media (IMDM + Collagenase D Img/mL (Millipore Sigma cat# 11088882001) and + DNAse 0. Img/mL (Millipore Sigma cat# DN25-5G)) on a shaker at 37°C for 30 minutes followed by addition of 0.5M EDTA (pH 8.0). Digested LNs and lung as well as mechanically disrupted spleen were then pushed through 100pm filters and washed with buffers. The samples then underwent density centrifugation as above to isolate live leukocytes.

Example 9 - Resuspension of frozen cells

Frozen cryovials were incubated in a water bath at 37C for one minute. ImL of warmed complete media (RMPI, 10% heat inactivated FBS, and penicillin-streptomycin-glutamate) was added dropwise to each vial and the vial contents were carefully decanted into a 50mL conical containing 25mL of complete media. The sample was topped up to 50mL and the cells are pelleted by centrifugation at 400xg for 10 minutes. The pellet was resuspended in lOmL of complete media with lOOuL benzonase (Millipore Sigma, catalog no. E1014-25KU) and incubated in 37C water bath for 20 minutes. The resulting sample is counted and used as required.

Example 10 - Liquid lymph node and slice cultures

Matching LN liquid and slice cultures were prepared for testing Merck MMR-II with CITE-seq as a readout with LN slice cultures generated as described in Experimental Example 4, and culture, perturbation, and CITE-seq as described in Experimental Example 5 with the following modifications. For the liquid culture, stimulation comprised of a 5x dilution of MMR- II (50 pL of MMR-II stock + 200 pL of complete media + 1X10⁶ LLN suspension cells) for 24 hours, 37 °C, 5% CO2 in a 96 well plate. A total of 3xl0⁶ cells were stimulated with vaccine at IxlO⁶ cells per well in 250 pL of MMR and complete media. For slice culture, perturbation comprised of stock MMR (60 pL of MMR stock ‘pool’ on 3 slices per transwell). A total of 9 slices in three transwells were treated with this condition. Unstimulated liquid (3xl0⁶ cells) and slice cultures (9 slices) were prepared and treated as in Experimental Example 5 and CITE-seq was carried out using the procedure described in Experimental Example 5.

Example 11 - Spatial profiling

Human lymph node slice cultures were generated as described in Example 4. To prepare lymph node slice cultures for analysis by microscopy or spatial profiling, formalin-fixed paraffin embedding (FFPE) tissue slices were prepared that were further cut into thin sections. Slice cultures were pooled and fixed in 4% formaldehyde (Thermo Fisher, Cat: 28908) in PBS (Thermo Fisher, Cat: 10010023) for 24 hours. Slices were then transferred and stored in IX PBS for up to two weeks until automated paraffin embedding was performed (Columbia Histology Service, Molecular Pathology Core Facility, New York, NY) to generated FFPE (Formalin-Fixed Paraffin Embedded) tissue, which was further sectioned to 5 microns. Hematoxylin and Eosin (H&E) staining was performed on the 5 micron sections (Columbia Histology Service, Molecular Pathology Core Facility, New York, NY) and images were acquired using an automated slide imaging system (Leica SCN 400). Figure 11 shows a representative image of an H&E-stained tissue section from a lymph node slice culture. Example 12 - Spatial profiling with commercial systems

Human lung lymph node slice cultures (500 microns thick) are generated as described in Example 4. Three slice cultures are set aside as unstimulated controls in complete media (IMDM (Thermo Fisher, Cat: 12440053), 5% FBS (Thermo Fisher, Cat: A3160401), IX Penn/Strep/Glutamine (Thermo Fisher, Cat: 10378016)) with 60 uL of media added on top of the slices. Another three slices are cultured in the same media, but also stimulated for 24 hours with stock MMR-II vaccine (Merck - 60 uL per slice). The two sets of slice cultures are then fixed with formaldehyde, and 5 micron FFPE tissue sections are prepared from each slice culture as described in Experimental Example 11. Following deparaffinization and permeabilization, oligonucleotide probes from the Human Lung Gene Expression Panel (lOx Genomics) are hybridized to target mRNAs at 50C overnight at a concentration of 10 nM followed by probe ligation, primer hybridization, and enzymatic amplification of the resulting padlock probes by rolling circle amplification as described previously (Janesick et al., bioRxiv, Nov. 3, 2022). After extensive washing, tissue sections are loaded into consumables for imaging with sequential fluorescence hybridization using the lOx Genomics Xenium Analyzer platform. The resulting data contain a spatial map of the lymph node slice cultures that localize -300 mRNA species to individual cells. Using unsupervised clustering and differential expression analysis to identify markers of each cluster, each cell is classified and the spatial location of each lymphocyte and myeloid cell subset is determined. For each cell subset, the gene expression profile of the MMR- II-stimulated cells is then compared to the corresponding control to identify gene expression responses to the vaccine. For reach cell, a feature space comprised of the spatially adjacent neighbor cells is further defined and unsupervised clustering in this feature space is performed to further refine the immune cell subsets on the basis of the identities of their neighboring cells (e.g., cell-cell interactions). With this refined classification, differential expression analysis between MMR-II- stimulated cells and the corresponding controls is further performed to identify gene expression-level immune responses.

Similar procedures for preparing FFPE or fresh-frozen tissue sections are applied to measure spatial gene expression patterns and spatially defined immune responses using alternative techniques for multiplexed in situ single-cell profiling of protein and mRNA including the CoxMx platform (Nanostring), MERSCOPE platform (Vizgen), GenePS platform (Spatial Genomics) or sequencing-based spatial profiling including the Visium platform (lOx Genomics), DBiT-seq (AltasXomics), or Slide-seq (Curio Bioscience).

EQUIVALENTS

The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

INCORPORATION BY REFERENCE

All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.

Claims

CLAIMS We claim:

1. A method to test a vaccine, comprising: exposing a vaccine to a tissue sample comprising one or more of a lymph node tissue, other lymphoid tissue, mucosal tissue, barrier tissue, intestinal tissue, and/or pulmonary tissue, or cells derived therefrom, and assessing one or more characteristics of the vaccine.

2. A method to test a vaccine, comprising: a) digesting one or more solid tissues to an immune cell suspension; b) generating one or more cell cultures from said immune cell suspension; and c) applying said vaccine to said one or more cell cultures.

3. The method of claim 1 or 2, wherein said vaccine is an attenuated live vaccine, an inactivated vaccine, a toxoid and/or toxin vaccine, a subunit vaccine, a conjugate vaccine, an outer membrane vesicle vaccine, a heterologous vaccine, a DNA vaccine, an RNA vaccine, a viral vector vaccine, a lipid nanoparticle vaccine, a dendritic cell vaccine, a recombinant vector vaccine, a T-cell receptor vaccine, a killed bacteria vaccine, and existing pathogen vaccine, and emerging pathogen vaccine, a bacterial vector vaccine, a plasmid vector vaccine, or an antigen- presenting cell vaccine.

4. The method of claim 1 or 2, wherein said vaccine is a mRNA vaccine.

5. The method of claim 4, wherein said mRNA vaccine is a SARS-CoV-2 mRNA vaccine.

6. The method of claim 2, wherein said solid tissue is a lymph node tissue, other lymphoid tissue, mucosal tissue, barrier tissue, intestinal tissue, and/or pulmonary tissue.

7. The method of claim 2, wherein said solid tissue is a tissue slice sample, a biopsy sample, or a needle aspirate sample.

8. The method of claim 2, comprising assessing said cell culture for an immune response.

9. The method of claim 1 or 8, wherein said assessing comprises supernatant testing for one or more activation marker concentrations, co-stimulatory marker concentrations, cytokine concentrations, chemokine concentrations, secreted factor concentrations, and/or cell free nucleic acid sequences and modifications.

10. The method of claim 1 or 8, wherein said assessing comprises cell testing for high parameter flow cytometry, T-cell activation and proliferation, B-cell activation, myeloid cell activation, and/or cell-type specific and/or single cell nucleic acid sequences and modifications.

11. The method of claim 1 or 8, wherein said assessing is performed serially at hourly, daily, weekly and/or monthly intervals.

12. The method of claim 1 or 8, wherein said assessing comprises identification of at least one biomarker of immune response duration.

13. The method of claim 12, wherein two or more vaccines with different durations of pathogen protection differ in said at least one biomarker of immune response duration.

14. The method claim 8, wherein said one or more cell cultures comprises two or more cell cultures from two or more solid tissues from an individual or from two or more individuals.

15. The method of claim 14, wherein said assessing comprises assessing two or more immune responses from said two or more cell cultures.

16. A method to test an immunomodulatory agent, comprising: a) digesting one or more solid tissues to an immune cell suspension; b) generating one or more cell cultures from said immune cell suspension; and c) applying said immunomodulatory agent to said one or more cell cultures.

17. The method of claim 16, wherein said immunomodulatory agent is an antibody, a cytokine, an immunomodulating drug, an immunostimulating drug, an immunosuppressive drug, a receptor antagonist, an immunoglobulin, a cytotoxic agent, an antimetabolite, a checkpoint inhibitor, a cytokine, an adjuvant or a small molecule.

18. The method of claim 16, wherein said applying further comprises applying a vaccine to said one or more cell cultures.

19. A method to test a vaccine or immunomodulatory agent, comprising: a) obtaining a tissue slice from a solid tissue; b) generating a tissue culture from said tissue slice; c) applying said vaccine or immunomodulatory agent to said tissue culture; and d) assessing one or more characteristics of said vaccine or immunomodulatory agent.

20. The method of claim 19, wherein said tissue culture is a cultured tissue slice.

21. The method of claim 19, further comprising digesting said tissue culture before said applying.

22. The method of claim 19, wherein said solid tissue comprises one or more of a lymph node tissue, other lymphoid tissue, mucosal tissue, barrier tissue, intestinal tissue, and/or pulmonary tissue.

23. The method of claim 19, wherein said solid tissue is fresh-frozen tissue and/or formalin- fixed paraffin-embedded (FFPE) tissue.

24. The method of claim 19, wherein said assessing comprises one or more of microscopy, histology, spatial profiling, immunohistochemistry, immunofluorescence, RNA-FISH, RNA- ISH, spatial transcriptomics and highly multiplexed RNA-FISH.

25. A method to test a vaccine or immunomodulatory agent, comprising: a) applying said vaccine or immunomodulatory agent to a tissue sample comprising one or more of a lymph node tissue, other lymphoid tissue, mucosal tissue, barrier tissue, intestinal tissue, and/or pulmonary tissue, or cells derived therefrom; b) digesting said tissue sample to generate an immune cell suspension; c) generating one or more cell cultures from said immune cell suspension; and d) assessing one or more characteristics of said vaccine or immunomodulatory agent.