CYTOKINE mRNA ADJUVANT COMPOSITIONS

Abstract

Immunostimulatory adjuvant compositions and methods of use are provided. The adjuvants can increase the titer of immune responses, e.g. antigen-specific immunoglobins; and can increase the duration of responses, e.g. the length of time antigen-specific immunoglobulin is present in serum. The adjuvants of the invention comprise a modified mRNA (mmRNA) encoding a cytokine. In one embodiment, the mRNA, e.g.,mmRNA, is administered intravenously encapsulated in a lipid nanoparticle.

Description

CYTOKINE mRNA ADJUVANT COMPOSITIONSCross-Reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No.63 / 779,064, filed March 27, 2025, which application is incorporated herein by reference in its entirety.Incorporation by Reference of Sequence Listing

[0002] A Sequence Listing is provided herewith as a Sequence Listing XML, “STAN-2264WO_SEQLIST” created on March 26, 2026 and having a size of 17,034 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.BACKGROUND

[0003] Adjuvants are often required to launch an efficient immune response against the antigen in a vaccine. The selection and molecular design of antigens may be informed by the molecular characterization of the infectious agents. However, most adjuvants on the market were discovered empirically. For example, QS-21 is used in shingle, RSV, and Novavax COVID-19 vaccines. It is purified from the soapbark tree (Quillaja saponaria). Meanwhile, another adjuvant, MF59, contains squalene derived from shark liver oil.

[0004] Intensive studies in mice have characterized signaling pathways regulating vaccine responses, particularly TLRs, leading to the emergence of molecular adjuvants. However, there are important differences in the antibody induction process between humans and mice. Further, the potency of different molecular candidates to augment vaccine response has not been systematically compared using a uniform test platform. Such knowledge is critical to prioritizing the targets for the rational design of molecular adjuvants.

[0005] The human response to vaccines is highly variable. Each year, approximately 800 million people receive the influenza vaccine globally, with 98% of them receiving the inactivated influenza vaccine / IIV (the remaining 2% receive the live attenuated influenza vaccine, LAIV). The antibody response to II V (and associated viral protection) varies 10 folds or more. The overall protection rates vary between 20% and 60% depending on the season, with most burdens of morbidity and mortality falling on the elderly (>65y) . Understanding the immunological factors shared by strong responders can inform us of the potential mechanisms of vaccine response in humans.

[0006] It was previously found that the influenza vaccine response variability is mostly driven by non-heritable factors. Such factors may include exposure to prior infections, such as antigenic sin or CMV, gut microbiota, which may activate TLR5, etc. It has been hypothesized that certain immune states at baseline or early vaccine responses may correlate, and potentially regulate, the vaccine response. Multiple system vaccinology studies in the pastdecade have characterized the pre-vaccination or early post-vaccination immune transcriptome signatures that correlate with improved antibody responses to the influenza vaccine. However, it is unclear what cytokines are responsible for the signatures and, if so, whether augmenting these cytokines' signaling can indeed augment vaccine response in humans or human tissues.SUMMARY

[0007] Immunostimulatory adjuvant compositions and methods of use are provided. The adjuvants can increase the titer of immune responses, e.g. antigen-specific immunoglobins; and can increase the duration of responses, e.g. the length of time antigen-specific immunoglobulin is present in serum. The adjuvants of the invention comprise an mRNA, e.g. a modified mRNA (mmRNA) encoding an immunostimulatory cytokine. In an embodiment, the mRNA is encapsulated in a lipid nanoparticle. The nanoparticle is optionally free of additional polynucleotides or polypeptides.

[0008] In some embodiments the mRNA encodes a cytokine selected from one or more of human IL-21 , IL-10, IL-12, IFNB1 , IFNA2, IFNW, IL-9, IL-18, IL-5, IL-17A. In some embodiments the mRNA encodes a cytokine selected from one or more of human IL-10, IL- 12, IL-9, IFNB1 , IFNA2, and IFNW. In some specific embodiments the cytokine is human IL- 21 . In some embodiments the cytokine is IL-12. In some specific embodiments the cytokine is atype I human interferon, e.g. IFNB1 , IFNA2, and IFNW. In an embodiment, the mRNA, e.g., mmRNA, comprises a 5' UTR, a codon optimized open reading frame encoding the cytokine, and a 3' tailing region of linked nucleosides. In another embodiment, the mRNA, e.g., mmRNA, comprises a 5' UTR and 3'UTR that are heterologous to the coding region.

[0009] In an embodiment the adjuvant is administered in combination with an antigen of interest, e.g. a pathogen or pathogen antigen, a tumor-specific antigen, etc. The antigen of interest may be provided as a protein, live attenuated pathogen, inactivated pathogen, mRNA, viral vector, etc., as known in the art. For example, the antigen may comprise an influenza vaccine. The antigen may comprise an mmRNA encoding a tumor antigen. The antigen may comprise an attenuated viral pathogen.

[0010] In some embodiments the adjuvant is administered prior to, or following administration of an antigen of interest. In some embodiments the adjuvant is administered in a coformulation with the antigen of interest. In some embodiments the adjuvant and antigen of interest are separately formulated. The adjuvant may be administered once, e.g. with a priming dose, a boost dose, etc. The adjuvant may be administered multiple times, e.g. with the priming dose and subsequent booster doses, which may be administered 1 , 2, 3, 4, 5, or more times as required for the specific immunogen.

[0011] In an embodiment, the modified mRNA, e.g., mmRNA, is fully modified. In other embodiments, the modified mRNA comprises one or more modified nucleobases described further herein. In some embodiments, the mRNA comprises pseudouridine (i ). In some embodiments, the modified nucleobase is 1-methyl-pseudouridine (m1qj), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (41), a-thio-guanosine, or a-thio-adenosine. In some embodiments, an mRNA of the disclosure includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 of the aforementioned modified nucleobases.)

[0012] In an embodiment, the cytokine-encoding mRNA is administered intravenously, encapsulated in a lipid nanoparticle. In one embodiment, the lipid nanoparticle comprises a cationic and / or ionizable lipid. In certain embodiments the lipid nanoparticle is selected to have reduced immunostimulatory activity relative to, for example, nanoparticles in current use for SARS-CoV-2 vaccines.

[0013] In an embodiment a method is providing for the immunization of an individual with an antigen of interest, the method comprising administering to the individual an effective dose of the antigen of interest, which may be provided in a vaccine formulation, and an effective dose of an adjuvant of the disclosure. In some embodiments the adjuvant comprises mmRNA encoding human IL-21. In some embodiments the effective dose is from about 0.5 pig to about 500 pg / dose, and may be at least about 1 pg, at least about 5 pg, at least about 10 pg, at least about 15 pg, at least about 25 pg, at least about 50 pg, at least about 100 pg, at least about 250 pg, and up to about 1 mg, up to about 750 pg, up to about 500 pg. Administration may be 2, 3 or more time to boost the response.

[0014] Individuals selected for treatment with the methods of the disclosure may include those with reduced adaptive immune responses, who particularly benefit from enhanced innate immunity. Such individuals may include without limitation, neonates, elderly, individuals being treated with immunosuppressants, e.g. transplant recipients, autoimmune patients, and the like; cancer patients, e.g. those treated with chemotherapeutic drugs or radiotherapy; and the like. For example, a reduced ability to produce antibodies, or other adaptive immune responses, in response to vaccination or exposure can be an indicator of reduced adaptive immune response.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensionsof the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0016] FIGS. 1A-1C: Age-effects on vaccine responses were only detectable in the subjects with low pre-vaccination flu antibodies, a) The scheme of the human cohort studies investigating the correlation between serum cytokine abundance and antibody response (ratio of HAI flu antibody abundance between Day 28 and Day 0). b) The summary statistics of samples and cohorts included in the study, c) The correlation between Pre-VAX flu hemagglutinin inhibition (HAI) antibody titers and Post- VAX antibody response. The antibody response was defined by the Iog2 ratio of the Day 28 and Day 0 flu HAI antibody titers. All subjects who received a lower dose (15ug / vaccine strain) IIV were included (while the older adults (>65 y) received the high-dose (60ug / vaccine strain) formulation). Subjects were stratified into quartiles based on pre-vaccination (Day 0) HAI titers. Pre-VAX low and high referred to the lower and upper 50% of pre-vaccination HAI titers, respectively; most in the low group had titers <40, a non-protective threshold. We performed Wilcoxon ranking tests between different age groups. “, p<0.01 ; ***, p<0.001 .

[0017] Fig. 2: Meta-analysis identified correlation between Pre-VAX serum abundance of cytokines and Post-VAX antibody response against the flu vaccine (IIV). We examined the correlation for 66 cytokines across the cohorts included in Fig. 1b. The correlations were performed separately in groups with low or high Pre-VAX flu HAI antibody titers, as defined in Fig. 1c. Details of the meta-analysis of the correlation can be found in the supplementary information. Log10 P values of the correlations were provided with the sign denoting the direction of the correlations (positive or negative slopes). False discovery rates were reported for significant or borderline significant hits. Details about the correlation and meta-analysis can be found in the methods.

[0018] FIGS. 3A-3B: Functional screening in human immune organoids identified IIV- adjuvating cytokines, a) We tested the cytokine adjuvant function in 3 tonsil- and 2 spleen-organoids. We added 19 different cytokines together with the vaccine (IIV), and for each cytokine, we tested 3 different concentrations ranging from 1 ng / mL to 100ng / mL, with exception for IL-1 [3 a) (10x lower for each concentration) and IL-18 (10x higher for each concentration). We measured the antibody production (relative to non-cytokine-added IIV only controls) in Day 7. As the results from the tonsil and spleen organoids are similar, they are combined, b) The assay results are presented.

[0019] FIGS. 4A-4E: Type I IFNs drive an immune activation program distinguishing the response induced by live versus inactivated vaccines, a) We stimulated the spleen organoids derived from 3 transplant organ donors with IIV, LAIV, IFN-p, or combinations (see below), b) The heatmap describes the sample-level differential induction of cytokines at Day 3 Post-VAX. The column corresponds to different sample and stimulations, the rows represent cytokinesdifferentially induced by LAIV versus I IV (FDR <0.05 and fold change >2). The color represents the abundance relative to the non-stimulated organoid derived from the same subject, c-e) Differential cytokine profiles between different stimulation conditions as noted in the bottom. The order of the cytokines is the same as in a. f-g) We examined the Type I IFN activities (f) and flu antibody responses (g) of organoids under different stimulation conditions.

[0020] FIGS. 5A-5C: Two distinct cytokine pathways regulating vaccine responses in humans, a) IL-21 abundance measurement 7 days post-stimulation, b) The induction of cytokine adjuvants (capable of boosting vaccine responses, Fig. 3b). The induction profile is based on data on Fig. 4b-d and Fig. 6a. While the NULISA assays for the live vaccine in Figure 4 did not cover the IL-21 measurement, it is known that the live vaccine preferentially activates its main producer (Tfh) in human immune organoids. c)The model describes the different immune activation programs induced by the live and the inactivated influenza vaccines. We performed Wilcoxon ranking tests between different age groups. *, p<0.05; **, p<0.01.

[0021] FIGS. 6A-6E. mRNA-LNP cytokine adjuvants enhance the durability of antibody responses to inactivated influenza vaccination, a) The experimental scheme illustrates testing of the adjuvant functionality of mRNA lipid nanoparticles (mRNA-LNPs) encoding GFP or cytokines. Mice were primed with inactivated influenza vaccine (I IV) on Day 0 and boosted on Day 21 as indicated, with serum collected at the indicated time points through Day 374 for antibody analysis and bone marrow cells harvested at Day 434 for ELISpot analysis; immunization conditions are listed, b) Serum anti-HA IgG binding intensities against influenza hemagglutinins (HAs) from in-vaccine strains were quantified by custom Luminex bead assay and the geometric mean is taken. Vaccination groups are color coded as shown in (d). c) Longitudinal trajectories of anti-HA IgG abundance over extended follow-up are shown for individual mice and expressed as the percentage of peak antibody levels relative to Day 35. d) Anti-HA IgG retention over long-term follow-up is shown as the percentage of antibody levels at Day 374 relative to Day 35. e) Antibody-secreting cell (ASC) responses in the bone marrow are shown by representative ELISpot wells and quantification of influenza-specific IgG ASCs per 10scells measured at Day 434 following immunization with IIV alone or I IV combined with mRNA-LNPs encoding GFP, I FN-p, or IL-21 ; naive mice are shown as a negative control, and each dot represents an individual mouse. All panels show data from N = 5 mice per immunization group. Data are shown as mean ± SEM. Statistical significance was determined using one-way ANOVA with multiple comparisons test. For panels b-d, false discovery rate (FDR)-adjusted P values are shown.

[0022] FIG. 7. Cytokines demonstrated similar IlV-adjuvating effects in spleen and tonsil organoids. This figure is related to Fig. 3. We harvested 3 tonsils and 2 spleens from donors and vaccinated the organoid cultures with IIV. We added 19 different cytokines together with the vaccine (IIV), and for each cytokine, we tested 3 different concentrations ranging from 1ng / ml to 100 ng / ml. We measured the antibody production (relative to non-cytokine-added I IV only controls) on Day 7. The adjuvant effects of 19 cytokines at 3 different concentrations (a total of 57 points) are plotted on the scatter plot, with the x-axis representing the averaged value for tonsil organoids and the y-axis representing the spleen organoids. The Pearson’s correlation coefficient and p values are listed.

[0023] FIG. 8: The cytokine profiles induced by I IV or LAIV in spleen organoids. This figure is related to Fig. 4. We measure the cytokine profiles using supernatants of the spleen organoids (N=3) culture at Day 3 post-VAX. All cytokines significantly altered after vaccination by either vaccine (as compared with the non-stimulated) are shown. Red texts highlight the Type I interferons (IFNs).

[0024] FIG. 9: IFNs induced by IIV or LAIV in spleen organoids. The figure is related to Fig.4. The abundances of Type I / II / III cytokines in the supernatants of spleen organoids stimulated by either IIV or LAIV were reported.

[0025] FIG. 10. The cytokine profiles of spleen organoids under different stimulation conditions. This figure is related to Fig. 4. We measured the cytokine profiles using supernatants of the spleen organoids (N=3) culture at Day 3 post-VAX. Cytokines differentially induced by LAIV versus IIV (FDR <0.05 and fold change >2) are shown.

[0026] FIGS. 11 A-11 C: Pre-VAX I FN-p serum abundances correlate with Post-VAX antibody responses in the older adults (>65 y) receiving low-dose flu vaccines. We divided the dataset by confounders (age, vaccine dose, gender, day 0 flu antibody quantile, cohort, flu season) into 28 sub-populations and performed correlations with each sub-population, a) The left panel described the characteristics of each one of the 28 sub-populations. The slope coefficients panel described the correlation within each sub-population, with summary statistics provided in the bottom, b) We summarized the 28 sub-populations into 4 groups by age and vaccine doses. For each group, summary statistics are provided. Details about the correlation and meta-analysis can be found in the supplementary methods, c) The correlation between I FN-p serum abundance and antibody response in the old receiving a low-dose IIV. The subpopulations are marked accordingly in a. The I FN- serum abundance data are not normalized here, with the batch effects visible.

[0027] FIG. 12. The correlation between pre-VAX IFNp serum abundance and post-VAX flu antibody responses in the older adults (>65 y) receiving a low-dose flu vaccine. This figure is related to Fig. 11 . The correlations (trends highlighted by the lines) of the 9 sub-populations in the “Old, low dose” group (Fig. 11b, lower) are plotted. The title of each panel corresponds to a sub-population listed in Fig. 11a.

[0028] FIGS. 13A-13E: Osteoarthritis incidence modifies the IFN-p abundance and vaccine response. The figure is related to Fig. 11. We examined the correlation between disease incidence, IFN-p serum abundance, and flu vaccine antibody response in a longitudinal cohort(Flu Study 15, one of the 5 cohorts in Fig. 1 a) . This cohort collected medical histories annually. The analysis was conducted among older adults (> 65 years), a) The I FN p serum abundance between the diseased and non-diseased samples. The diseased samples are samples collected within (+ / -) 2 years relative to a diagnosis. The non-diseased samples are from subjects without a medical history of the disease. Wilcoxon ranking tests were performed for the comparisons, b-d) Temporal profile of IFN-p serum abundance relative to the diagnosis of osteoarthritis within the cohorts. The loess smooth line is provided with confidence intervals, c) The outliers (standardized abundance >=3) were removed, and the temporal correlation was retained, d) the temporal profile of individual patients diagnosed with osteoarthritis during the study, e) The flu HAI antibody responses (Day 28 vs. Day 0 ratio) in non-diseased and osteoarthritis subjects. Similar to Figs 2 and 3, the analysis was performed in subjects with a low level of pre-VAX flu antibodies (Fig. 1c). Refer to the supplementary methods.

[0029] FIGS. 14A-14B. Kinetics and antigenic breadth of IgG responses to H5N1 HA following I IV immunization with or without mRNA-LNP adjuvant, a) Heatmap showing serum IgG levels (quantified as IgG binding intensities, Log2 MFI) against a diverse panel of influenza hemagglutinins (HAs), measured by custom Luminex bead assay at Day 21 and Day 35. HA antigens are categorized as Human Flu-In Vaccine, Human Flu-Out of Vaccine, or Zoonotic Flu, as indicated by the top color bar. b) Serum IgG binding intensities (MFI) against recombinant H5N1 HA proteins from A / chicken / Ghana / AVL-763 / 2021 (top) and A / Cambodia / NPH230032 / 2023 (bottom) were quantified over time using a custom Luminex assay. Mice were immunized as shown in Figure 6a. The two vertical dashed lines indicate the timing of the prime and boost immunizations. The horizontal dashed line represents the IgG response (MFI) against in-vaccine influenza strains induced by an unadjuvanted high- dose vaccine (10 ig) at Day 35, whose antigen dose is 10 times that of the adjuvanted condition.

[0030] FIG. 15. Fluorescence imaging of GFP in hind leg muscles and inguinal draining lymph nodes (dLNs) from unimmunized and LNP-GFP immunized mice. The images were taken 24 hours after injection. Data are representative of two independent experiments.DETAILED DESCRIPTION

[0031] Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular method or composition 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 invention will be limited only by the appended claims.

[0032] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, betweenthe upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0033] 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 be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supercedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0034] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.

[0035] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0036] The term “adjuvant” refers to a composition that increases the humoral or cellular immune response of an individual. Adjuvants of interest stimulate the immune system, and as shown herein, alter the epigenomics of innate immune cells to increase responsiveness.

[0037] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal being assessed for treatment and / or being treated. In some embodiments, the mammal is a human. The terms “subject,” “individual,” and “patient” encompass, without limitation, individuals having a disease. Subjects may be human, but also include other mammals, particularly those mammals useful as laboratory models for human disease, e.g., mice, rats, etc.

[0038] The term “sample” with reference to a patient encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The term also encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations, such as diseased cells. The definition also includes samples that have been enriched for particular types of molecules, e.g., nucleic acids, polypeptides, etc. The term “biological sample” encompasses a clinical sample, and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, and the like.

[0039] The term “diagnosis” is used herein to refer to the identification of a molecular or pathological state, disease or condition in a subject, individual, or patient.

[0040] The term “prognosis” is used herein to refer to the prediction of the likelihood of death or disease progression, including recurrence, spread, and drug resistance, in a subject, individual, or patient. The term “prediction” is used herein to refer to the act of foretelling or estimating, based on observation, experience, or scientific reasoning, the likelihood of a subject, individual, or patient experiencing a particular event or clinical outcome. In one example, a physician may attempt to predict the likelihood that a patient will survive, or the severity of an infection.

[0041] As used herein, the terms “treatment,” “treating,” and the like, refer to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect on or in a subject, individual, or patient. The effect may be prophylactic in terms of completely or partially preventing a disease, for example infection by a pathogen, or symptom thereof and / or may be therapeutic in terms of effecting a partial or complete cure for a disease and / or symptoms of the disease.

[0042] Treating may refer to any indicia of success in the treatment or amelioration or prevention of a disease, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of an examination by a physician. Accordingly, the term "treating" includes the administration of an agent to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with infectious disease or other diseases. The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.

[0043] As used herein, a "therapeutically effective amount" refers to that amount of the immunostimulatory composition sufficient to induce an enhanced immune response. A therapeutically effective amount may refer to the amount of immunostimulatory composition sufficient to reduce infection upon pathogen exposure, e.g., to delay or minimize infection. A therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease. Further, a therapeutically effective amount means the amount of immunostimulatory composition alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease.

[0044] As used herein, the term “dosing regimen” refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0045] "In combination with", "combination therapy" and "combination products" refer, in certain embodiments, to the concurrent administration to a patient of the immunostimulatory compositions described herein in combination with additional therapies, e.g. inclusion of antigenic material, and the like. When administered in combination, each component can be administered at the same time or sequentially in any order at different points in time. Thus, each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect.

[0046] "Concomitant administration" means administration of one or more components, such as immunostimulatory compositions, known therapeutic agents, etc. at such time that the combination will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of components. Aperson of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration.

[0047] The use of the term "in combination" does not restrict the order in which prophylactic and / or therapeutic agents are administered to a subject. A first prophylactic or therapeutic agent can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second prophylactic or therapeutic agent to a subject with a disorder.

[0048] The term “isolated” refers to a molecule that is substantially free of its natural environment. For instance, an isolated protein is substantially free of cellular material or other proteins from the cell or tissue source from which it is derived. The term refers to preparations where the isolated protein is sufficiently pure to be administered as a therapeutic composition, or at least 70% to 80% (w / w) pure, more preferably, at least 80%-90% (w / w) pure, even more preferably, 90-95% pure; and, most preferably, at least 95%, 96%, 97%, 98%, 99%, or 100% (w / w) pure. A “separated” compound refers to a compound that is removed from at least 90% of at least one component of a sample from which the compound was obtained. Any compound described herein can be provided as an isolated or separated compound.

[0049] "Antibody" refers to an immunoglobulin molecule that can bind to a specific antigen as the result of an immune response to that antigen. Immunoglobulins are serum proteins composed of "light" and "heavy" polypeptide chains having "constant" and "variable" regions and are divided into classes (e.g., IgA, IgD, IgE, IgG, and IgM) based on the composition of the constant regions.

[0050] "Antigen" or "immunogen" refers to any substance that stimulates an immune response. The term includes killed, inactivated, attenuated, or modified live bacteria, viruses, or parasites. The term antigen also includes polynucleotides, polypeptides, recombinant proteins, synthetic peptides, protein extract, cells (including bacterial cells), tissues, polysaccharides, or lipids, or fragments thereof, individually or in any combination thereof. The term antigen also includes antibodies, such as anti-idiotype antibodies or fragments thereof, and to synthetic peptide mimotopes that can mimic an antigen or antigenic determinant (epitope).

[0051] "Immune response" in a subject refers to the development of an adaptive immune response, e.g. humoral immune response, cellular immune response, or a humoral and a cellular immune response to an antigen. Imune response also refrs to an innate immuneresponse. Immune responses may be determined using standard immunoassays and neutralization assays, which are known in the art.

[0052] “Innate Immunity”. The term “innate immunity” refers to immune responses that rely primarily on cells of the myeloid system, not B or T lymphocytes, and that do not generate a memory response. Innate immune responses are not specific to a particular pathogen in the way that the adaptive immune responses are, but rather utilize conserved features of pathogens of pathogen associated immunostimulants to initiate responses.

[0053] "Cellular immune response" or "cell mediated immune response" is one mediated by T-lymphocytes or other white blood cells or both, and includes the production of cytokines, chemokines and similar molecules produced by lymphocyte, leukocytes, or both.

[0054] "Immunogenic" means evoking an immune or antigenic response. Thus an immunogenic composition would be any composition that induces an immune response.

[0055] "Pharmaceutically acceptable" refers to substances, which are within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit-to- risk ratio, and effective fortheir intended use.

[0056] "Reactogenicity" refers to the side effects elicited in a subject in response to the administration of an adjuvant, an immunogen, a vaccine composition, etc. It can occur at the site of administration, and is usually assessed in terms of the development of a number of symptoms. These symptoms can include inflammation, redness, and abscess. It is also assessed in terms of occurrence, duration, and severity. A "low" reaction would, for example, involve swelling that is only detectable by palpitation and not by the eye, or would be of short duration. A more severe reaction would be, for example, one that is visible to the eye or is of longer duration.

[0057] "Immunostimulatory composition" refers to a composition that includes an adjuvant, as defined herein and may optionally further include an antigen. The amount of a composition that is therapeutically effective may vary depending on the presence of antigen, the adjuvant, and the condition of the subject, and can be determined by one skilled in the art.

[0058] Nanoparticle: As used herein, “nanoparticle” refers to a particle having any one structural feature on a scale of less than about 1000 nm that exhibits novel properties as compared to a bulk sample of the same material. Routinely, nanoparticles have any one structural feature on a scale of less than about 500 nm, less than about 200 nm, or about 100 nm. Also routinely, nanoparticles have any one structural feature on a scale of from about 50 nm to about 500 nm, from about 50 nm to about 200 nm or from about 70 to about 120 nm. In exemplary embodiments, a nanoparticle is a particle having one or more dimensions of the order of about 1 -1000 nm. In other exemplary embodiments, a nanoparticle is a particle havingone or more dimensions of the order of about 10-500 nm. In other exemplary embodiments, a nanoparticle is a particle having one or more dimensions of the order of about 50-200 nm. A spherical nanoparticle would have a diameter, for example, of between about 50-100 or 70- 120 nanometers. A nanoparticle most often behaves as a unit in terms of its transport and properties. It is noted that novel properties that differentiate nanoparticles from the corresponding bulk material typically develop at a size scale of under 1000 nm, or at a size of about 100 nm, but nanoparticles can be of a larger size, for example, for particles that are oblong, tubular, and the like. Although the size of most molecules would fit into the above outline, individual molecules are usually not referred to as nanoparticles.

[0059] IL-21. Interleukin 21 (IL-21) is a cytokine that has potent regulatory effects on cells of the immune system, including natural killer (NK) cells and cytotoxic T cells that can destroy virally infected or cancerous cells. This cytokine induces cell division / proliferation in its target cells. The human IL-21 gene is about 8.43kb, mapped to chromosome 4 and 180kb from IL-2 gene, and the mRNA product is 616 nucleotides long. The reference sequences for the human gene may be found at Genbank, NM_021803, NM_001207006; and the protein reference sequences at NP 001193935; NP 068575. Coding sequences for human IL-21 are provided as SEQ ID NO:1 and SEQ ID NO:2.

[0060] IL-21 is expressed in activated human CD4+ T cells but not in most other tissues. In addition, IL-21 expression is up-regulated in Th2 and Th17 subsets of T helper cells, as well as T follicular cells. IL-21 is also expressed in NK T cells regulating the function of these cells.

[0061] The IL-21 receptor (IL-21 R) is expressed on the surface of T, B and NK cells. IL-21 r is similar in structure to the receptors for other type I cytokines like IL-2R or IL-15 and requires dimerization with the common gamma chain (yc) in order to bind IL-21. When bound to IL-21 , the IL-21 receptor acts through the Jak / STAT pathway, utilizing Jak1 and Jak3 and a STAT3 homodimer to activate its target genes.

[0062] Interleukin-12 (IL-12) is a canonical proinflammatory cytokine and the founding member of a heterodimeric family that includes IL-23, IL-27, and IL-35. Structurally, the bioactive form (p70) is composed of two disulf ide-linked subunits: the p35 a-chain, which is constitutively expressed but restricted by tight translational control, and the p40 p-chain, which is highly inducible in professional antigen-presenting cells such as dendritic cells and macrophages. The assembly of p70 is a critical checkpoint in the innate immune response, typically triggered by the activation of pattern recognition receptors (PRRs) like TLR4 or TLR9, or through CD40-CD40L cognate interactions with T cells.

[0063] At the signaling level, IL-12 exerts its effects by binding to the IL-12 receptor complex (IL-12R i and IL-12Rp2), which is primarily expressed on activated T cells and natural killer(NK) cells. This binding event recruits and activates Janus kinases, specifically Jak2 and Tyk2, leading to the phosphorylation and homodimerization of STAT4. Once translocated to the nucleus, STAT4 drives the transcription of IFNG (Interferon-gamma), effectively polarizing naive CD4+ T cells toward a Th1 phenotype.

[0064] Since human Interleukin-12 (IL-12) is a heterodimeric cytokine, it is encoded by two distinct genes, each with its own RefSeq accession numbers for the mRNA transcript and the protein. IL-12 Subunit Alpha (p35), mRNA RefSeq: NM 000882, Protein RefSeq: NP 000873. IL-12 Subunit Beta (p40), mRNA RefSeq: NM 002187, Protein RefSeq: NP 002178.

[0065] Human Interferon-beta (IFN-p), a cornerstone of the Type I interferon family, is a pleiotropic cytokine primarily encoded by the intronless IFNB1 gene on chromosome 9p21. IFN-p is produced by nearly all nucleated cells, particularly fibroblasts and epithelial cells, in response to the detection of pathogen-associated molecular patterns (PAMPs). The canonical induction pathway involves the activation of pattern recognition receptors (PRRs), such as RIG-l-like receptors (RLRs) or Toll-like receptors (TLR3 / 4), which trigger a kinase cascade involving TBK1 and IKKs. This leads to the C-terminal phosphorylation and nuclear translocation of Interferon Regulatory Factor 3 (IRF3), which cooperatively assembles with NF-KB and c-Jun / ATF-2 onto the IFNB1 enhanceosome to drive high-level transcription.

[0066] Upon secretion, IFN-p exerts its biological effects by binding with high affinity to the heterodimeric IFN-a / p receptor (IFNAR1 and IFNAR2). This ligation activates the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, specifically inducing the phosphorylation of JAK1 and TYK2. These kinases subsequently phosphorylate STAT1 and STAT2, which recruit IRF9 to form the ISGF3 heterotrimeric complex. ISGF3 translocates to the nucleus and binds to Interferon-Stimulated Response Elements (ISREs) within the promoters of hundreds of Interferon-Stimulated Genes (ISGs), such as OAS1 , MX1 , and PKR. This coordinated wave of transcription establishes a robust antiviral state by inhibiting viral translation, degrading viral RNA, and modulating MHO class I expression to enhance CD8+ T cell surveillance.

[0067] Beyond its immediate antiviral role, IFN-p serves as a sophisticated immunomodulator within the cytokine network, often functioning to dampen excessive Th17-mediated inflammation.

[0068] The following RefSeq identifiers represent the curated mRNA and protein sequences for human IFN-p, mRNA refseq is NM 002176, and protein refseq is NP 002167.

[0069] Modified mRNA. In some embodiments, the mRNA comprises pseudouridine (ip). In some embodiments, the mRNA comprises pseudouridine (ip) and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 1-methyl-pseudouridine (rn'ip). In someembodiments, the mRNA comprises 1-methyl-pseudouridine (m1qj) and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 2-thiouridine (s2U). In some embodiments, the mRNA comprises 2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 5-methoxy-uridine (mo5U). In some embodiments, the mRNA comprises 5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 2'-O-methyl uridine. In some embodiments, the mRNA comprises 2'-O-methyl uridine and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises comprises N6-methyl-adenosine (msA). In some embodiments, the mRNA comprises N6-methyl-adenosine (m6A) and 5-methyl-cytidine (m5C). In some embodiments, the modified nucleobase is 1-methyl-pseudouridine (m1i ), 5-methoxy-uridine (mo5U), 5- methyl-cytidine (m5C), pseudouridine (i ), a-thio-guanosine, or a-thio-adenosine. In some embodiments, an mRNA of the disclosure includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 of the aforementioned modified nucleobases.)

[0070] An RNA may be a naturally or non-naturally occurring RNA, e.g., mRNA. An mRNA may include one or more modified nucleobases, nucleosides, or nucleotides, as described below, in which case it may be referred to as a “chemically modified mRNA”, also referred to herein as a “modified mRNA” or “mmRNA.” As described herein “nucleoside” is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). As described herein, “nucleotide” is defined as a nucleoside including a phosphate group.

[0071] An mRNA may include a 5' untranslated region (5'UTR), a 3' untranslated region (3'UTR), and / or a coding region (e.g., an open reading frame). An mRNA may include any suitable number of base pairs, including hundreds (e.g., 200, 300, 400, 500, 600, 700, 800, or 900) or thousands (e.g., 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000) of base pairs. Any number (e.g., all, some, or none) of nucleobases, nucleosides, or nucleotides may be an analog of a canonical species, substituted, modified, or otherwise non-naturally occurring. In certain embodiments, all of a particular nucleotide or nucleobase type may be modified.

[0072] In one embodiment, the mRNA comprises a first flanking region located at the 5' terminus of an open reading frame (coding region) and a second flanking region located at the 3’ terminus of the open reading frame (coding region), wherein the first flanking region comprises a 5' untranslated region (5' UTR) and the second flanking region comprises a 3' untranslated region (3'UTR). The 3’ and 5’ UTRs may comprise SEQ ID NO:3 and SEQ ID NO:4. In one embodiment, the 5'UTR and the 3'UTR of the mRNA are not derived from the same species. In one embodiment, the 5'UTR and / or the 3'UTR of the mRNA are not derivedfrom beta-globin. In one embodiment, the 5' untranslated region is heterologous to the coding region of the mRNA. In another embodiment, the 3' untranslated region is heterologous to the coding region of the mRNA. In yet another embodiment, the 5' untranslated region and the 3' untranslated region are heterologous to the coding region of the mRNA. In yet another embodiment, the mRNA comprises at least two stop codons.

[0073] In some embodiments, an mRNA as described herein may include a 5' cap structure, a chain terminating nucleotide, a Kozak sequence (also known as a Kozak consensus sequence), a stem loop, a polyA sequence, and / or a polyadenylation signal. In other embodiments, the mRNA lacks a poly A sequence and / or a polyadenylation signal but rather contains an alternative structure for stabilizing the mRNA.

[0074] A 5' cap structure or cap species is a compound including two nucleoside moieties joined by a linker and may be selected from a naturally occurring cap, a non-naturally occurring cap or cap analog, or an anti-reverse cap analog (ARCA). A cap species may include one or more modified nucleosides and / or linker moieties. For example, a natural mRNA cap may include a guanine nucleotide and a guanine (G) nucleotide methylated at the 7 position joined by a triphosphate linkage at their 5' positions, e.g., m7G(5')ppp(5')G, commonly written as m7GpppG. A cap species may also be an anti-reverse cap analog. A non-limiting list of possible cap species includes m7GpppG, m7Gpppm7G, m73'dGpppG, m27 O3GpppG, m27 O3GppppG, m27 O2GppppG, m7Gpppm7G, m73'dGpppG, m27 O3GpppG, m27 O3GppppG, and m27O2GppppG. In various embodiments, the mRNA can comprise a 5' terminal cap selected from the group consisting of CapO, Capl, ARCA, inosine, N1-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA- guanosine, and 2-azido-guanosine. In one embodiment, the 5' terminal cap is Capl.

[0075] An mRNA may instead or additionally include a polyA sequence and / or polyadenylation signal. A polyA sequence may be comprised entirely or mostly of adenine nucleotides or analogs or derivatives thereof. A polyA sequence may be a tail located adjacent to a 3' untranslated region of an mRNA. In some embodiments, a polyA sequence may affect the nuclear export, translation, and / or stability of an mRNA.

[0076] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (i ), pyridin-4- one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4- thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5- aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor 5-bromo-uridine), 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5- carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno- uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl- uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl- uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (Tm5U), 1-taurinomethyl- pseudouridine, 5-taurinomethyl-2-thio-uridine (Tm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m1qj, 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ip), 4-thio-1 - methyl-pseudouridine, 3-methyl-pseudouridine (m3qj), 2-thio-1-methyl-pseudouridine, 1- methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio- dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4- methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1 -methyl-pseudouridine, 3-(3- amino-3-carboxypropyl)uridine (acp3U), 1 -methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3w), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio- uridine (inm5s2U), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'- O-methyl-pseudouridine (i m), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl- 2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5- carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1 -thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)]uridine.

[0077] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include 5-aza-cytidine, 6-aza- cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formyl- cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo- cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2 -thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-th io- 1 -methyl-pseudoisocytidine, 4-th io- 1 -methyl-1 -deaza- pseudoisocytidine, 1 -methyl-1 -deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy- 5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1 -methyl-pseudoisocytidine, lysidine (k2C), a-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl-cytidine (m5Cm), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O- methyl-cytidine (f5Cm), N4,N4,2’-O-trimethyl-cytidine (m42Cm), 1 -thio-cytidine, 2'-F-ara- cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

[0078] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include a-thio-adenosine, 2-amino- purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo- purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza- 2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyl-adenosine (m'A), 2-methyl- adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis- hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl- adenosine (ms2g6A), N6,N6-dimethyl-adenosine (ms2A), N6-hydroxynorvalylcarbamoyl- adenosine (hneA), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hnBA), N6- acetyl-adenosine (acsA), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, a-thio- adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O- trimethyl-adenosine (m Arn), 1 ,2'-0-dimethyl-adenosine (m1Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1 -thio-adenosine, 8-azido-adenosine, 2'-F- ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino- pentaoxanonadecyl)-adenosine.

[0079] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include a-thio-guanosine, inosine (I), 1-methyl-inosine (m1l), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OhyW), undermodified hydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano- 7-deaza-guanosine (preQo), 7-aminomethyl-7-deaza-guanosine (preQi), archaeosine (G+), 7- deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza- guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6- methoxy-guanosine, 1-methyl-guanosine (m1G), N2-methyl-guanosine (m2G), N2,N2- dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m27G), N2, N2,7-dimethyl-guanosine (m2’2’7G), (8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2- methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, a-thio-guanosine, 2'-O-methyl- guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl- guanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2’-O-methyl- guanosine (m2Gm), 2'-O-methyl-inosine (Im), 1 ,2'-O-dimethyl-inosine (m1lm), 2'-O- ribosylguanosine (phosphate) (Gr(p)), 1 -thio-guanosine, 06-methyl-guanosine, 2'-F-ara- guanosine, and 2'-F-guanosine.

[0080] In some embodiments, an mmRNA, of the disclosure includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 of the aforementioned modified nucleobases).

[0081] In some embodiments, an mRNA of the disclosure may be modified in a coding region (e.g., an open reading frame encoding a polypeptide). In other embodiments, an mRNA may be modified in regions besides a coding region. For example, in some embodiments, a 5'-UTR and / or a 3'-UTR are provided, wherein either or both may independently contain one or more different nucleoside modifications. In such embodiments, nucleoside modifications may also be present in the coding region.

[0082] The mmRNAs, of the disclosure can include a combination of modifications to the sugar, the nucleobase, and / or the internucleoside linkage. These combinations can include any one or more modifications described herein.Cytokine mRNA

[0083] Adjuvant compositions of the disclosure comprise mRNA encoding one or more cytokines of interest. In an embodiment the mRNA encodes a cytokine selected from one or more of human IL-21 , IL-10, IL-12, IFNB1 , IFNA2, IFNW, IL-9, IL-18, IL-5, IL-17A. In some embodiments the mRNA encodes a cytokine selected from one or more of human IL-10, IL- 12, IL-9, IFNB1 , IFNA2, and IFNW. In some specific embodiments the cytokine is human IL- 21 , e.g. encoded by SEQ ID NO:2. In an embodiment, the mRNA, e.g., mmRNA, comprises a 5' UTR, e.g. SEQ ID NO:3, a codon optimized open reading frame encoding the cytokine, and a 3' tailing region of linked nucleosides, e.g. SEQ ID NO:4. In another embodiment, the mRNA, e.g., mmRNA, comprises a 5' UTR and 3'UTR that are heterologous to the coding region.

[0084] The mRNAs of the present disclosure, or regions thereof, may be codon optimized. Codon optimization methods are known in the art and may be useful for a variety of purposes: matching codon frequencies in host organisms to ensure proper folding, bias GC content to increase mRNA stability or reduce secondary structures, minimize tandem repeat codons or base runs that may impair gene construction or expression, customize transcriptional and translational control regions, insert or remove proteins trafficking sequences, remove / add post translation modification sites in encoded proteins (e.g., glycosylation sites), add, remove or shuffle protein domains, insert or delete restriction sites, modify ribosome binding sites and mRNA degradation sites, adjust translation rates to allow the various domains of the protein to fold properly, or to reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art; nonlimiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park, Calif.) and / or proprietary methods. In one embodiment, the mRNA sequence is optimizedusing optimization algorithms, e.g., to optimize expression in mammalian cells or enhance mRNA stability. In some embodiments the mRNA is encoded by a sequence disclosed herein.

[0085] The mRNAs, of the present disclosure may be produced by means available in the art, including but not limited to in vitro transcription (IVT) and synthetic methods. Enzymatic (IVT), solid-phase, liquid-phase, combined synthetic methods, small region synthesis, and ligation methods may be utilized. In one embodiment, mRNAs, are made using IVT enzymatic synthesis methods. Methods of making polynucleotides by IVT are known in the art. Accordingly, the present disclosure also includes polynucleotides, e.g., DNA, constructs and vectors that may be used to in vitro transcribe an mRNA described herein.

[0086] Non-natural modified nucleobases may be introduced into polynucleotides, e.g., mRNA, during synthesis or post-synthesis. In certain embodiments, modifications may be on internucleoside linkages, purine or pyrimidine bases, or sugar. In particular embodiments, the modification may be introduced at the terminal of a polynucleotide chain or anywhere else in the polynucleotide chain; with chemical synthesis or with a polymerase enzyme.

[0087] Either enzymatic or chemical ligation methods may be used to conjugate polynucleotides or their regions with different functional moieties, such as targeting or delivery agents, fluorescent labels, liquids, nanoparticles, etc. Conjugates of polynucleotides and modified polynucleotides are reviewed in Goodchild, Bioconjugate Chemistry, vol. 1(3), 165- 187 (1990).Lipid Nanoparticles

[0088] The mRNAs are formulated in nanoparticles or other delivery vehicles, e.g., to protect them from degradation when delivered to a subject. In certain embodiments, an RNA, e.g., mRNA, of the disclosure is encapsulated within a nanoparticle. In particular embodiments, a nanoparticle includes a lipid. Lipid nanoparticles include, but are not limited to, liposomes and micelles. Any of a number of lipids may be present, including cationic and / or ionizable lipids, anionic lipids, neutral lipids, amphipathic lipids, PEGylated lipids, and / or structural lipids. Such lipids can be used alone or in combination. In particular embodiments, a lipid nanoparticle comprises one or more RNAs, e.g., mRNAs, described herein, e.g., a mmRNA encoding a cytokine of interest.

[0089] In some embodiments, the lipid nanoparticle formulations of the mRNAs, described herein may include one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, or 8) cationic and / or ionizable lipids. Such cationic lipids include, but are not limited to, 3-(didodecylamino)-N1,N1 ,4-tridodecyl-l- piperazineethanamine (KL10), N1 -[2-(didodecylamino)ethyl]-N1 ,N4,N4-tridodecyl-1 ,4- piperazinediethanamine (KL22), 14,25-ditridecy I- 15,18,21 ,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4- dimethylaminomethyl-[1 ,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)[1 ,3]- dioxolane (DLin-KC2-DMA), 2-({8-[(30)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3- [(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1 -amine (Octyl-CLinDMA), (2R)-2-({8-[(3 (3)- cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1- yloxy]propan-1 -amine (Octyl-CLinDMA (2R)), (25)-2-({8-[(3 (3)-cholest-5-en-3- yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1 -amine (Octyl-CLinDMA (2S)).N,N-dioleyl-N,N-dimethylammonium chloride (“DODAC”); N-(2,3- dioleyloxy)propyl-N,N — N-triethylammonium chloride (“DOTMA”); N,N-distearyl-N,N- dimethylammonium bromide (“DDAB”); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”); 1 ,2-Dioleyloxy-3-trimethylaminopropane chloride salt (“DOTAP.CI”); 3-0- (N — (N',N'-dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”), N-(1 -(2,3- dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethyl-ammonium trifluoracetate (“DOSPA”), dioctadecylamidoglycyl carboxyspermine (“DOGS”), 1 ,2-dioleoyl-3- dimethylammonium propane (“DODAP”), N,N-dimethyl-2,3-dioleyloxy)propylamine (“DODMA”), and N-(1 ,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (“DMRIE”). Additionally, a number of commercial preparations of cationic and / or ionizable lipids can be used, such as, e g., LIPOFECTIN® (including DOTMA and DOPE, available from GIBCO / BRL), and LIPOFECTAMINE® (including DOSPA and DOPE, available from GIBCO / BRL). KL10, KL22, and KL25 are described, for example, in U.S. Pat. No.8,691 ,750, which is incorporated herein by reference in its entirety. In particular embodiments, the lipid is DLin-MC3-DMA or DLin-KC2-DMA.

[0090] Anionic lipids suitable for use in lipid nanoparticles of the disclosure include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl phosphatidylethanoloamine, N-succinyl phosphatidylethanolamine, N- glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, and other anionic modifying groups joined to neutral lipids.

[0091] Neutral lipids suitable for use in lipid nanoparticles of the disclosure include, but are not limited to, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. Lipids having a variety of acyl chain groups of varying chain length and degree of saturation are available or may be isolated or synthesized by well-known techniques. Additionally, lipids having mixtures of saturated and unsaturated fatty acid chains can be used. In some embodiments, the neutral lipids used in the disclosure are DOPE, DSPC, DPPC, POPO, or any related phosphatidylcholine. In some embodiments, the neutral lipid may be composed of sphingomyelin, dihydrosphingomyeline, or phospholipids with other head groups, such as serine and inositol.

[0092] In some embodiments, amphipathic lipids are included in nanoparticles of the disclosure. Exemplary amphipathic lipids suitable for use in nanoparticles of the disclosure include, but are not limited to, sphingolipids, phospholipids, and aminolipids. In some embodiments, a phospholipid is selected from the group consisting of 1 ,2-dilinoleoyl-sn- glycero-3-phosphocholine (DLPC), 1 ,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1 ,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1 ,2-di- O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl-sn- glycero-3-phosphocholine (C16 Lyso PC), 1 ,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1 ,2- diarachidonoyl-sn-glycero-3-phosphocholine, 1 ,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoetha nolamine (DOPE), 1 ,2-diphytanoyl- sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dilinolenoyl- sn-glycero-3-phosphoethanolamine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dioleoyl-sn-glycero-3- phospho-rac-(1 -glycerol) sodium salt (DOPG), and sphingomyelin. Other phosphorus-lacking compounds, such as sphingolipids, glycosphingolipid families, diacylglycerols, and p- acyloxyacids, may also be used. Additionally, such amphipathic lipids can be readily mixed with other lipids, such as triglycerides and sterols.

[0093] In some embodiments, the lipid component of a nanoparticle of the disclosure may include one or more PEGylated lipids. A PEGylated lipid (also known as a PEG lipid or a PEG- modified lipid) is a lipid modified with polyethylene glycol. The lipid component may include one or more PEGylated lipids. A PEGylated lipid may be selected from the non-limiting group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified di alkyl amines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols. For example, a PEGylated lipid may be PEG-c-DOMG, PEG- DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

[0094] A lipid nanoparticle of the disclosure may include one or more structural lipids. Exemplary, non-limiting structural lipids that may be present in the lipid nanoparticles of the disclosure include cholesterol, fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, or alpha-tocopherol).Pharmaceutical Compositions

[0095] The present disclosure includes pharmaceutical compositions comprising an mRNA or a nanoparticle (e.g., a lipid nanoparticle) described herein, in combination with one or morepharmaceutically acceptable excipient, carrier or diluent. In particular embodiments, the mRNA, is present in a nanoparticle, e.g., a lipid nanoparticle. In particular embodiments, the mRNA or nanoparticle is present in a pharmaceutical composition. In various embodiments, the mRNA, present in the pharmaceutical composition is encapsulated in a nanoparticle, e.g., a lipid nanoparticle.

[0096] Pharmaceutical compositions may optionally include one or more additional active substances, for example, therapeutically and / or prophylactically active substances. Pharmaceutical compositions of the present disclosure may be sterile and / or pyrogen-free. General considerations in the formulation and / or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21sted., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

[0097] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, dividing, shaping and / or packaging the product into a desired single- or multi-dose unit.

[0098] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition in accordance with the disclosure will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may include between 0.1% and 100%, e.g., between 0.5% and 70%, between 1% and 30%, between 5% and 80%, or at least 80% (w / w) active ingredient. In some embodiments, the active agent is an mRNA encoding a cytokine of interest.

[0099] Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference in its entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition. Excipients may include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, and waters of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate(dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0100] In some embodiments, the formulations described herein may include at least one pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts that may be included in a formulation of the disclosure include, but are not limited to, acid addition salts, alkali or alkaline earth metal salts, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzene sulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.

[0101] In some embodiments, the formulations described herein may contain at least one type of polynucleotide. As a non-limiting example, the formulations may contain 1 , 2, 3, 4, 5 or more than 5 cytokine mRNAs described herein.

[0102] Liquid dosage forms for e.g., parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to active ingredients, liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions can include adjuvantssuch as wetting agents, emulsifying and / or suspending agents. In certain embodiments for parenteral administration, compositions are mixed with solubilizing agents such as CREMAPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof.

[0103] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and / or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and / or emulsions in nontoxic parenterally acceptable diluents and / or solvents, for example, as a solution in 1 ,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables. Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0104] In some embodiments, pharmaceutical compositions including at least one mRNA described herein are administered to mammals (e.g., humans). Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions that are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to a non-human mammal. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and / or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and / or turkeys.Methods of Use

[0105] In an embodiment a method is providing for the immunization of an individual with an antigen of interest, the method comprising administering to the individual an effective dose of the antigen of interest, which may be provided in a vaccine formulation, and an effective dose of an adjuvant of the disclosure. In some embodiments the adjuvant comprises mmRNAencoding human IL-21. The adjuvant and the antigen may be separately formulated, or may be co-formulated.

[0106] In certain embodiments, compositions of the disclosure may be administered at dosage levels sufficient to deliver from about 0.0001 mg / kg to about 10 mg / kg, from about 0.001 mg / kg to about 10 mg / kg, from about 0.005 mg / kg to about 10 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 10 mg / kg, from about 2 mg / kg to about 10 mg / kg, from about 5 mg / kg to about 10 mg / kg, from about 0.0001 mg / kg to about 5 mg / kg, from about 0.001 mg / kg to about 5 mg / kg, from about 0.005 mg / kg to about 5 mg / kg, from about 0.01 mg / kg to about 5 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 5 mg / kg, from about 2 mg / kg to about 5 mg / kg, from about 0.0001 mg / kg to about 1 mg / kg, from about 0.001 mg / kg to about 1 mg / kg, from about 0.005 mg / kg to about 1 mg / kg, from about 0.01 mg / kg to about 1 mg / kg, or from about 0.1 mg / kg to about 1 mg / kg in a given dose, where a dose of 1 mg / kg provides 1 mg of mRNA or nanoparticle per 1 kg of subject body weight. In particular embodiments, a dose of about 0.005 mg / kg to about 5 mg / kg of mRNA or nanoparticle of the disclosure may be administrated. In particular embodiments, a dose of about 0.002 mg / kg to about 2 mg / kg of mRNA or nanoparticle of the disclosure may be administrated. In particular embodiments, a dose of about 0.02 mg / kg to about 0.2 mg / kg of mRNA or nanoparticle of the disclosure may be administrated.

[0107] In some embodiments the effective dose is from about 0.5 pg to about 500 pg / dose, and may be at least about 1 pg, at least about 5 pg, at least about 10 pg, at least about 15 pg, at least about 25 pg, at least about 50 pg, at least about 100 pg, at least about 250 pg, and up to about 1 mg, up to about 750 pg, up to about 500 pg. Administration may be 2, 3 or more time to boost the response.

[0108] A dose may be administered one or more times per day, in the same or a different amount, to obtain a desired level of mRNA, expression and / or effect (e.g., atherapeutic effect). The desired dosage may be delivered, for example, three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). In some embodiments, a single dose may be administered, for example, prior to or after a surgical procedure or in the instance of an acute disease, disorder, or condition.

[0109] In some embodiments the adjuvant is administered prior to, or following administration of an antigen of interest. In some embodiments the adjuvant is administered in a coformulation with the antigen of interest. In some embodiments the adjuvant and antigen ofinterest are separately formulated. The adjuvant may be administered once, e.g. with a priming dose, a boost dose, etc. The adjuvant may be administered multiple times, e.g. with the priming dose and subsequent booster doses.

[0110] Administration may be performed once, twice, three or more times as required. Multiple administrations can be spaced apart by about 2, 3, 4, 5, 6, 7, 8 or more weeks initially, and can be further spaced by 2, 3, 4, 5, 6, or more months for subsequent administrations.

[0111] Although it is not required, individuals selected for treatment with the methods of the disclosure may include those with reduced adaptive immune responses, who particularly benefit from enhanced innate immunity. Such individuals may include without limitation, neonates, elderly, individuals being treated with immunosuppressants, e.g. transplant recipients, autoimmune patients, and the like; cancer patients, e.g. those treated with chemotherapeutic drugs or radiotherapy; and the like. For example, a reduced ability to produce antibodies, or other adaptive immune responses, in response to vaccination or exposure can be an indicator of reduced adaptive immune response.

[0112] In an embodiment, the cytokine-encoding mRNA is administered intravenously encapsulated in a lipid nanoparticle. In one embodiment, the lipid nanoparticle comprises a cationic and / or ionizable lipid. In certain embodiments the lipid nanoparticle is selected to have reduced immunostimulatory activity relative to, for example, nanoparticles in current use for SARS-CoV-2 vaccines.

[0113] The routes of administration for the adjuvant compositions include parenteral, oral, oronasal, intranasal, intratracheal, topical, etc. Any suitable device may be used to administer the compositions, including syringes, droppers, needleless injection devices, patches, and the like. The route and device selected for use will depend on the composition of the adjuvant, the antigen, and the subject, and such are well known to the skilled artisan.

[0114] Vaccines known and used in the art that may be administered with an adjuvant of the disclosure include, for example, inactivated pathogen vaccines; live-attenuated pathogen vaccines; messenger RNA (mRNA) vaccines; subunit, recombinant, polysaccharide, and conjugate vaccines; toxoid vaccines; and viral vector vaccines. Inactivated vaccines use a killed version of the pathogen that causes a disease, e.g. Hepatitis A, influenza, rabies, etc. Live vaccines use an attenuated form of the pathogen that causes a disease, e.g. measles, mumps, rubella (MMR combined vaccine), rotavirus, smallpox, chickenpox, yellow fever. mRNA vaccines encode pathogen proteins that trigger an immune response, e.g. SRS-CoV2. Subunit, recombinant, polysaccharide, and conjugate vaccines use specific pathogen molecules, e.g. Hib (Haemophilus influenzae type b), Hepatitis B, HPV (Human papillomavirus), Bordetella pertussis, pneumococcal disease, meningococcal disease, Varicella Zoster virus. Toxoid vaccines use a toxin made by the pathogen, e.g. diphtheria, andtetanus. Viral vector vaccines use a modified version of a different virus as a vector to deliver sequences encoding pathogen protein. Several different viruses have been used as vectors, including influenza, vesicular stomatitis virus (VSV), measles virus, and adenovirus. Viral vectors are in use currently for SARS-CoV2.

[0115] Some examples of bacterial pathogens that may be administered as a vaccine include, for example, Aceinetobacter calcoaceticus, Acetobacter paseruianus, Actinobacillus pleuropneumoniae, Aeromonas hydrophila, Alicyclobacillus acidocaldarius, Arhaeglobus fulgidus, Bacillus pumilus, Bacillus stearothermophillus, Bacillus subtilis, Bacillus thermocatenulatus, Bordetella bronchiseptica, Burkholderia cepacia, Burkholderia glumae, Campylobacter coli, Campylobacter fetus, Campylobacter jejuni, Campylobacter hyointestinalis, Chlamydia psittaci, Chlamydia trachomatis, Chlamydophila spp., Chromobacterium viscosum, Erysipelothrix rhusiopathieae, Listeria monocytogenes, Ehrlichia canis, Escherichia coli, Haemophilus influenzae, Haemophilus somnus, Helicobacter suis, Lawsonia intracellularis, Legionella pneumophilia, Moraxellsa sp., Mycobactrium bovis, Mycoplasma hyopneumoniae, Mycoplasma mycoides subsp. mycoides LC, Clostridium perfringens, Odoribacter denticanis, Pasteurella (Mannheimia) haemolytica, Pasteurella multocida, Photorhabdus luminescens, Porphyromonas gulae, Porphyromonas gingivalis, Porphyromonas salivosa, Propionibacterium acnes, Proteus vulgaris, Pseudomnas wisconsinensis, Pseudomonas aeruginosa, Pseudomonas fluorescens C9, Pseudomonas fluorescens SIKW1 , Pseudomonas fragi, Pseudomonas luteola, Pseudomonas oleovorans, Pseudomonas sp B11 -1 , Alcaliges eutrophus, Psychrobacter immobilis, Rickettsia prowazekii, Rickettsia rickettsia, Salmonella typhimurium, Salmonella bongori, Salmonella enterica, Salmonella dublin, Salmonella typhimurium, Salmonella choleraseuis, Salmonella newport, Serratia marcescens, Spirlina platensis, Staphlyoccocus aureus, Staphyloccoccus epidermidis, Staphylococcus hyicus, Streptomyces albus, Streptomyces cinnamoneus, Streptococcus suis, Streptomyces exfoliates, Streptomyces scabies, Sulfolobus acidocaldarius, Syechocystis sp., Vibrio cholerae, Borrelia burgdorferi, Treponema denticola, Treponema minutum, Treponema phagedenis, Treponema refringens, Treponema vincentii, Treponema palladium, and Leptospira species, such as the known pathogens Leptospira canicola, Leptospira grippotyposa, Leptospira hardjo, Leptospira borgpetersenii hardjo-bovis, Leptospira borgpetersenii hardjo-prajitno, Leptospira interrogans, Leptospira icterohaemorrhagiae, Leptospira pomona, and Leptospira bratislava, and combinations thereof.

[0116] Examples of viral pathogens that may be administered as a vaccine include, for example, SARS-Cov1 , SARS-Cov2, and other coronaviruses, Avian herpesviruses, Bovine herpesviruses, Canine herpesviruses, Equine herpesviruses, Feline viral rhinotracheitis virus, Marek's disease virus, Ovine herpesviruses, Porcine herpesviruses, Pseudorabies virus,Avian paramyxoviruses, Bovine respiratory syncytial virus, Canine distemper virus, Canine parainfluenza virus, canine adenovirus, canine parvovirus, Bovine Parainfluenza virus 3, Ovine parainfluenza 3, Rinderpest virus, Border disease virus, Bovine viral diarrhea virus (BVDV), BVDVType I, BVDVType II, Classical swine fever virus, Avian Leukosis virus, Bovine immunodeficiency virus, Bovine leukemia virus, Bovine tuberculosis, Equine infectious anemia virus, Feline immunodeficiency virus, Feline leukemia virus (FeLV), Newcastle Disease virus, Ovine progressive pneumonia virus, Ovine pulmonary adenocarcinoma virus, Canine coronavirus (CCV), pantropic CCV, Canine respiratory coronavirus, Bovine coronavirus, Feline Calicivirus, Feline enteric coronavirus, Feline infectious peritonitis, virus, Porcine epidemic diarrhea virus, Porcine hemagglutinating encephalomyletitis virus, Porcine parvovirus, Porcine Circovirus (PCV) Type I, PCV Type II, Porcine Reproductive and Respiratory Syndrome (PRRS) Virus, Transmissible gastroenteritis virus, Turkey coronavirus, Bovine ephemeral fever virus, Rabies, Rotovirus, Vesicular stomatitis virus, lentivirus, Avian influenza, Rhinoviruses, Equine influenza virus, Swine influenza virus, Canine influenza virus, Feline influenza virus, Human influenza virus, Eastern Equine encephalitis virus (EEE), Venezuelan equine encephalitis virus, West Nile virus, Western equine encephalitis virus, human immunodeficiency virus, human papilloma virus, varicella zoster virus, hepatitis B virus, rhinovirus, and measles virus, and combinations thereof.

[0117] In some embodiments an antigen of interest for vaccination is a tumor-specific antigen. The antigen may be patient specific. For example see Tiwade et al. Mol Pharm. 2025 Mar 25; Kirtane AR, Traverse G. Cancer J. 2025 Mar-Apr 01 ;31 (2):e0764; Wei Z et al. Front Immunol.2025 Mar 4;16:1524317; Zhou et al. Clin Pharmacol Ther. 2025 Mar 14; Zhao et al. Physiology (Bethesda). 2025 Mar 10; and Attia MS et al. Acta Pharm Sin B. 2025 Jan;15(1):52-96, each herein specifically incorporated by reference.

[0118] Tumor-specific antigens (TSAs) are proteins or other molecules that are uniquely expressed on cancer cells and not found in normal healthy tissues. TSAs are selectively expressed in cancer cells. Many TSAs arise from mutations that occur during cancer development or are overexpressed in cancer cells compared to normal cells. A subset of TSAs called neoantigens are newly generated mutations that are unique to the individual tumor. Examples include MAGE-A protein; NY-ESO-1 ; HER2; p53; alpha-fetoprotein, etc.

[0119] Kits may be provided, for example comprising a unit dose of an adjuvant suitable for administration, or for reconstitution before administration. A unit dose may be provided in a pre-filled vial or syringe. Kits may also include tubes, buffers, etc., and instructions for use.EXPERIMENTAL

[0120] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of howto make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.Example 1Rationale design of vaccine adjuvant informed by human cohort and organoid data

[0011] Studies show that human vaccine responses widely vary. Here, we analyzed data measuring 66 cytokines from 4 different inactivated influenza vaccine (IIV) cohorts over 5 seasons (N=581) and identified a significant correlation between baseline / day 0 serum IL-18 and IFN-p concentrations and the vaccine-specific antibody response on day 28, suggesting these factors may have an adjuvant-like effect. To investigate this further, we tested the impact of 19 cytokines on the development of anti-influenza antibody response when administered together with the IIV vaccine in human tonsil and spleen organoids. We found that Type I IFNs (IFN-p and others), IL-21 , IL-12, IL-10, but not IL-18 or IFN-y, enhanced the antibody response. The live attenuated influenza vaccine (LAIV) induced a stronger antibody response than the inactivated one in organoids. Adding a single cytokine, IFN-p, to IIV stimulation recapitulated most of the live vaccine-specific cytokine activation program. It increased the antibody response of the inactivated vaccine to that of the LAIV. Two other antibody-boosting cytokines, IL-12 and IL-21 , were induced by LAIV but not by Type I IFNs. While it has been reported that IL-12 cannot induce IL-21 expression in mouse CD4 T cells1 2, IL-12 strongly induces IL-21 in human spleen organoids, suggesting they function in the same pathway that is Type I IFN-independent. In a mouse model immunized by IIV, we found that the delivery of IL-21 or IFN-p in the form of mRNA lipid nanoparticles substantially enhanced the antibody production at 1 year and promoted formation of long-lived plasma cells. These findings identified human-specific parallel cytokine pathways regulating vaccine responses. Our approach unites high-throughput organoid testing and human cohort studies, establishing a human-centric platform to identify adjuvant candidates.

[0122] Here, we report the identification of multiple cytokines that correlate with elevated antibody responses in human influenza virus vaccination studies. We combined data from four influenza vaccine cohorts in five flu seasons (N=581). We found a robust correlation betweenthe pre-vaccination (pre-VAX) serum abundance of cytokines and post-VAX flu antibody responses in a subpopulation with a lower baseline antibody titer (N=389). Recently, we and others have developed ex vivo human immune cultures. We have shown that human tonsil organoids can model processes underlying the vaccine response, including somatic hypermutation and affinity maturation. To distinguish between cytokines that merely correlate with an enhanced response to a vaccine versus those that could drive the response, we modified a human immune organoid system we initially developed for tonsil organoids to use spleen cells derived from organ donors, using a protocol developed by Kathuria et al. and using a 96-well format. The platform used much less cell input (97% lower). It enabled high- throughput screening and identified several cytokines that can enhance the antibody response elicited by the I IV in humans. Lastly, we wanted to test whether these in vitro results would hold up in an in vivo system and since RNA vaccines are known to be transported to lymph nodes, we created mRNA-LNP constructs encoding some of these cytokines and tested them in a mouse model. While the mRNA-LNP itself had a significant effect, constructs carrying IL- 21 and IFN-p enhanced this even further, showing that some cytokines could be effective adjuvants.

[0123] Baseline antibody titer influences the vaccine response phenotype. Given the central role of cytokines in modulating immune responses, we analyzed specific pre-vaccination (preVAX) serum cytokine profiles and the magnitude of the post-VAX antibody response (Fig. 1 a). To achieve sufficient statistical power, we pooled data from four different flu vaccine cohorts from five different seasons (Fig. 1b). In these studies, inactivated influenza vaccine (IIV) was administered to subjects, and serum was analyzed in pre-VAX (day 0) and post-VAX (day 28) time points. Influenza (flu) hemagglutinin inhibition (HAI) antibodies for the vaccinated strains were determined at both time points, with the ratio defined as the antibody response. Considerable heterogeneity was present among cohorts in age, sex, flu strains, dose included in the vaccine, and cytokine profiling technology in different seasons.

[0124] The correlation between pre-VAX (Day 0) flu antibody titer and post-VAX (Day 28) antibody response also showed great heterogeneity (Fig. 1c). To address this, we divided the population into 4 quartiles based on their pre-VAX flu antibody titers. In line with prior studies, we observed that both the median and dynamic ranges of antibody responses decreased with an increasing abundance of pre-VAX antibodies, potentially due to the antigen clearance and thus lower vaccine “take”. Aging is known to lower vaccine response significantly. Still, interestingly, we found that responses were only lower in older individuals for subjects with low pre-VAX flu antibodies (quantile 1 and 2, lower 50 percentile, Fig. 1c). These results suggested that the correlation between vaccine response and a biological characteristic (such as age) was considerably stronger in people with a low level of pre-VAX flu antibodies (and potentially higher vaccine “take”). Compared with the group with a high level of pre-VAX fluantibodies, the low-level group had a higher risk of flu infection. Among the 389 subjects with a pre-VAX flu HAI antibody titer lower than 40 (deemed not protected), 89% of them were in the group with a low level of pre-VAX flu antibodies. For this reason, our analysis focused on this group.

[0125] Meta-analysis identifies a correlation between pre- VAX cytokine abundance and postVAX antibody response to the flu vaccine. Heterogeneity (due to technical and biological factors) was a major challenge for pooling data from different cohorts. Here, we divided the dataset by confounders (age, vaccine dose, sex, day 0 flu antibody quantile, cohort, flu season) into sub-populations and performed correlations with each sub-population (see an example of the grouping in FIG. 11a). We then summarized the results using meta-analysis, a method capable of identifying robust signals among heterogeneous data.

[0126] We performed a meta-analysis of 66 cytokines measured across the four cohorts. The analysis identified 2 cytokines (IL-18 and IFN-p) and 1 chemokine (GRO-a, also known as CXCL1) whose pre-VAX serum abundance significantly correlated with post- VAX antibody response (False Discovery Rate <5%, Fig. 2). IFN-p and IL-18 activate two different immune signaling pathways. IFN-p is a type I interferon (IFN) that is critical for antiviral immunity, including antibody response. Among the three members of the type I IFNs tested (IFN-a, IFN- p, IFN-co), IFN-p has the highest binding affinity to the Type I IFN receptor (IFNAR1 / 2), which is expressed by many cell types, including B cells. Interleukin-18 (IL-18) is a multifaceted cytokine primarily known for its role in stimulating T cells to produce the Type II interferon (IFN- y). We also identified several cytokines with a borderline significant correlation (FDR <10%), which included T Helper cytokines IL-17 and IL-9 (Fig. 2).

[0127] Functional screen in human spleen organoids identifies cytokine adjuvants, including Type I IFNs but not Type II IFN. Human cohort studies can identify correlations of immune function, but they cannot prove causality without interventional studies. While the functionality of many cytokines has been tested in various mouse vaccination models, the relevance to the human vaccination process has not been examined. Moreover, each mouse model typically tested only 1 or 2 cytokines. When different cytokine adjuvant effects were compared across different mouse studies, there was heterogeneity in cytokine delivery formats (DNA, protein), antigen, and delivery route used (intramuscular, intranasal, intraperitoneal, etc. A functional systems immunology approach that compares different molecules delivered in the same format and assayed against the same antigen in a human culture system can allow quantitative evaluation of adjuvant effects and prioritize the adjuvant candidates. Recently, we developed a human immune organoid system enabling efficient hypothesis testing. The immune organoids are primary culture, different from many other organoids derived from tissue stem cells or induced Pluripotent Stem Cells (iPSCs). The immune organoids retain memory T and B cells from the donors, including those carrying memory against the influenzainfections / vaccines. Meanwhile, the immune organoids recapitulate key antibody response features, including the production of antigen-specific antibodies, somatic hypermutation, and affinity maturation. Building on our prior work, we devised a low-cell-input immune organoid culture system (decreased the cell input number by 97% to 1.6 x 105cells), which allowed us to generate many immune organoid cultures in a 96-well format. We performed a screen and systematically examined cytokines’ adjuvant functionality to the inactivated vaccine using a functional systems immunology approach (Fig. 3a). We harvested 3 tonsils from patients from the clinic. In collaboration with the Donor Network West, an Organ Processing Organization (OPO), that serves Northern California and Northern Nevada, we procured spleens from 2 authorized ventilated deceased donors. Using this higher throughput immune organoid format, we generated 5 sets of immune organoids from 5 different subjects. We administered them with IIV, alone, or in combination with 19 different cytokines. Most of these cytokines have been shown to boost antibody response in one or multiple mouse vaccination models. We combined the data since the cytokines’ adjuvant effects are similar between spleen and tonsil organoids (Fig. 7). For each cytokine, we tested three different concentrations, ranging from I ng / mL to 100 ng / mL. However, for IL1 -p (10 times lower at each concentration), and IL-18 (10 times higher at each concentration), we used different concentrations as their physiological concentrations in human serum differ from those of other cytokines. Antibody production (relative to the IlV-only controls) on day 7 (Fig. 3a) was measured. In total, we assayed 58 different conditions, including the control, across five biological replicates. The screen revealed that all type I IFNs (IFN-p and IFN-w, though IFN-a was less effective) enhanced antibody production induced by the inactivated vaccine (Fig. 3b). Moreover, we discovered potent adjuvant function of several other cytokines. Among them, IL-21 and IL-9, which are secreted by T follicular helper and Th9 cells, respectively. IL-12 induces the formation of Th1 and Tfh cells. IL-10 enhances B cell survival and proliferation. IL-10 can also act as one of the downstream effectors of type I IFN (also see below). For most factors, the effect was detected at concentrations as low as 1 ng / ml, at the picomolar level. Notably, IL-18 or its downstream effector, type II IFN (IFN-y), did not enhance the HV-induced antibody response in human spleen and tonsil organoids (Fig. 3b), which is in contrast to the strong correlation observed between IL-18 abundance and antibody response in human cohorts.

[0128] Type I IFNs drive an immune activation program that mimics a live vaccine. While cytokines can regulate vaccine response, vaccination can also induce cytokines that coordinate the antibody response. We exposed spleen organoids to IIV, and collected the supernatant on Day 3, analyzing the samples using a DNA-barcoded multiplexed cytokine detection technology with femtomolar sensitivity (NULISA) (Fig. 4a). Of the 250 cytokines / chemokines assayed, 40 were significantly induced by IIV treatment (Fig. 8). Type II IFN (IFN-y) was the most induced cytokine. Several other Th2 cytokines (IL-4 / 5 / 13) werealso highly induced (Fig. 8). Notably, none of the Type I IFNs (IFN-a / p / w) were induced by IIV (Fig. 9, with the names of Type I IFNs marked).

[0129] Next, we examined the cytokine-induction profile of another type of influenza vaccine, the live attenuated influenza vaccine (LAIV). Unlike the inactivated format, the live vaccine contains a mutated influenza virus capable of completing a few cell cycles in a human cell. Both IIV and LAIV are FDA-approved (but for different age groups and administration routes). The cytokine-induction profile of the live attenuated vaccine (LAIV) and inactivated vaccine (IIV) shared a strong induction of Th2 cytokines (such as IL-5, IL-13, IL-4), Th9 cytokine (IL- 9), and Type II IFN (Fig. 8, 9). However, the live attenuated vaccine also induced a unique and prominent Type I and III IFN response, with concentrations up to -1 ,000 times (10 Iog2) higher than IIV (Fig. 4b and Fig. 9). Meanwhile, the live attenuated vaccine also induced a broad spectrum of other cytokines, including IL-10, IL-12, CCL-7, CXCL-10, etc. (Fig. 4b and Fig. 10).

[0130] To examine the regulation of other cytokines by Type I IFN, we added IFN-p to the non-vaccinated spleen organoid controls. Strikingly, the cytokine profile induced by IFN-p almost overlapped with the differentially induced cytokines between the live and inactivated vaccine: 14 out of 15 LAIV-II V differentially upregulated cytokines were also induced by IFN- p. In comparison, 4 out of 5 LAIV-IIV differentially downregulated cytokines were also suppressed by IFN-p (Fig. 4c). Similar cytokine-induction profile overlaps also persisted when we added IFN-p into the IlV-vaccinated controls and compared (Fig. 4d). As a result, the cytokine profile of the organoids treated with IFN-p phenocopied the live vaccine (Fig. 10).

[0131] When we added IFN-p to the spleen organoids, it induced the expression of other Type I IFNs (IFN-a, IFN-co, Fig. 4c-d), which bind to the same receptor (IFNAR1 / 2) and induce the same signaling pathway. This observation suggests a positive feedback loop between the induction of Type I IFNs. These data also show that Type I IFN is a key regulator of the live- vaccine-specific cytokine profile, whose abundance differentiates the inactivated versus live vaccine responses (also including the antibody response, see below). Type I IFN triggers positive feedback on its expression and a cascade of other cytokines.

[0132] Next, we examined the functionality of Type I IFN (IFN-p) to adjuvant antibody responses. Due to the existence of multiple Type I IFNs, the concentration of a single protein could not represent the Type I IFN activity. We measured the global Type I IFN activity by taking the geometric mean of the abundance of all cytokines significantly induced by IFN-p. The Type I IFN activity scores showed that the inactivated vaccine was defective in inducing Type I IFN activity, which was rescued by IFN-p addition (Fig. 4e). While the live vaccine efficiently induced Type I IFN activity, adding more IFN-p did not further enhance it, suggesting saturation (Fig. 4e). Importantly, the antibody production in Day 7 post-VAX rose alongside the Type I IFN activity upon antigen challenge (Fig. 4e). Adding type I IFN (IFN-p) reshapedthe downstream global cytokine profile to the live-vaccine-like state. Ultimately, adding IFN-p increased the antibody titer, making it resemble LAIV.

[0133] IFN-p regulates the human vaccine response among older adults (age > 65y) receiving an insufficient dose of antigens. The association studies in human cohorts (Fig. 1-2, and Fig.11a) and functional studies in tonsil and spleen organoids (Fig. 4) support that IFN-p is a natural adjuvant underlying flu vaccine response variability in humans. Interestingly, we found considerable heterogeneity in IFN-p’s correlation with post- VAX antibody response across the subpopulations (Fig. 11a). The heterogeneity was primarily due to age differences, with the correlation only reaching significance in older adults (>65 y). Among older adults, the correlation was more pronounced in those who received low-dose vaccines. When the low dose was found suboptimal for older adults the cohort studies followed the most current knowledge at the time and switched the older adults to a version with high-dose antigen in the 2014-2015 season. Concurrent to this change, the association between baseline IFN-p concentration and antibody response disappeared (Fig. 11 b-c and Fig. 12).

[0134] The age-dependent correlation between pre-VAX IFN-p abundance and post-VAX response prompted the hypothesis that certain aging-associated conditions may underlie this population's IFN-p abundance and vaccine response variation. One of the cohorts in our analysis (the Stanford-Ellison Cohort, Fig. 1b) surveyed medical history annually. The study recorded 648 clinical events from 135 subjects, 628 of which occurred in the older adults (>65 y). Among them, nine types of clinical events accumulated more than 10 incidences in the cohort. We asked whether the serum IFN-p abundance changed within 2 years (before or after) a disease diagnosis. The most significant association identified is from osteoarthritis (Fig. 13a). Osteoarthritis is a highly prevalent condition in older adults, with 20 incidences in the cohort (compared to 3 cases of rheumatoid arthritis). While traditionally deemed a degenerative disease, a significant inflammatory (or infectious) component has been identified in osteoarthritis. We leveraged the longitudinal nature of the study and examined the temporal correlation between serum IFN-p abundance and osteoarthritis incidence. The IFN-p abundance was significantly elevated from the population mean up to 4 years before diagnosis and lasted up to 5 years after (Fig. 13b-d). The temporal correlation was robust, retained significance after outliers were removed (>= 3 standard deviations from the population mean) (Fig. 13c). Within the time frame, we found that the antibody response to the influenza vaccine was significantly elevated in subjects with osteoarthritis (Fig. 13e). Thus, a highly prevalent aging-associated condition, osteoarthritis (including its pre-clinical stage), was one of the modifiers of the IFN-p abundance and vaccine response. Older adults bear a disproportionate burden of influenza-associated morbidity and mortality and are also the population in which vaccine-induced antibody responses are most consistently suboptimal (Fig. 1c). Type I IFN is an immune regulator that selectively influences vaccine responsiveness in this high-risk group.

[0135] IL-12 and IL-21 represent a Type I IFN-independent pathway in live-vaccine-specific response. In the immune organoid screen, we identified numerous cytokine adjuvants capable of boosting antibody response (Fig. 3b). Strikingly, most of the cytokine adjuvants were induced to higher levels by the live vaccine (Fig. 4b), compared to the inactivated vaccine. This highlights the important role of cytokines induced by the live vaccine. Four cytokine adjuvants are either Type I IFN (IFN-a, IFN-p, or I FN-co) or induced by Type I IFN (IL-10) (Fig.4c, d). However, two potent cytokine adjuvants (IL-12 and IL-21, Fig. 3b) are not induced by Type I IFN in human immune organoids (data for IL-12 shown in Fig. 4c, d, and IL-21 shown in Fig. 5a, b), and we identified them as live-vaccine-specific cytokines outside of the Type I IFN pathway. Prior studies have shown that IL-12 is the most potent human cytokine at inducing IL-21 -expressing CD4+ T cells, which are responsible for B cell “help”. Meanwhile, the live attenuated influenza vaccine, but not the inactivated one, is known to activate the Tfh cells preferentially in human tonsil organoids. Indeed, in spleen organoids, IL-12 can induce IL-21 (Fig. 5a). Thus, at least two distinct cytokine pathways (Type I IFN vs. IL-12 / IL-21 ) regulated human vaccine responses (Fig. 5c).

[0136] mRNA LNPs can deliver cytokines to augment the quantity and breadth of antibody responses in mice. The above human studies showed that Type I IFN, IL-12, and downstream cytokines such as IL-21 could enhance antibody responses. Moreover, the screen quantitatively compared 19 cytokines, and we found I FN-|3 and IL-21 demonstrated the most potent adjuvant function at the lower concentration (1ng / ml, Fig. 3b). We next tested the functional relevance of these cytokine pathways in vivo using a murine model. Murine models complement the human model by offering a system where we can track the cross-tissue protein delivery (see below) and examine the antibody response at an organismal level. To this end, we employed an antigen-coding mRNA lipid nanoparticle (mRNA-LNP) platform in mice immunized with the inactivated influenza vaccine. The mRNA component of mRNA- LNPs is known to induce a strong Type I IFN response, while its LNP component promotes the activation of Tfh cells, the primary source of IL-21. This dual functionality enables mRNA- LNPs to mimic critical immunostimulatory features of a live vaccine, activating both Type I IFN and IL-21 pathways in vivo.

[0137] To evaluate this approach, we administered mRNA LNPs encoding GFP in combination with I IV in mice (Fig. 6a). While GFP is a foreign protein to mice, we reasoned that the protein itself would be relatively immunologically inert when delivered alongside micrograms of influenza antigen, and it allowed us to confirm successful antigen expression via fluorescence imaging. Mice were boosted on Day 21 after the primary immunization, and serum samples were collected at multiple timepoints for antibody profiling. The I IV formulation included hemagglutinins (HAs) from human influenza strains (H1 N1 , H3N2, and B) circulating during the 2023-2024 season. Serum antibody responses were analyzed using a customLuminex panel capable of detecting antibodies against both the in-vaccine (on-target) and out- of-vaccine (cross-reactive) hemagglutinins (HAs), including zoonotic strains such as H5N1 and H17N10 (Fig. 14a).

[0138] At day 21 post-primary immunization, 1 pg I IV alone failed to elicit detectable antibody responses, while 10 pg IIV induced moderate titers against the vaccine, including in-vaccine HAs (B / Austria / 1359417 / 2021 , H3 A / Darwin / 9 / 2021 , and H1 A / Victoria / 4897 / 2022, Fig. 14a). In contrast, 1 pg IIV combined with GFP mRNA-LNP markedly enhanced antibody titers against all in-vaccine HAs and exceeded those elicited by 10 pg IIV alone. In addition, this group exhibited cross-reactive antibody responses to out-of-vaccine HAs. By day 35 (14 days post-booster), antibody titers increased further. The 1 pg IlV-alone group showed low but detectable two in-vaccine HAs B / Austria / 1359417 / 2021 and A / Darwin / 9 / 2021 (H3N2), while the 10 pg IIV group produced substantially higher titers to all in-vaccine strains, along with modest cross-reactivity. Notably, the group that received IIV plus GFP mRNA-LNP showed the highest antibody titers overall, including responses to Zoonotic HAs. The cross- reactive antibody titer against the two most recent H5N1 strains in this group was comparable to or higher than the antibody titer against in-vaccine strains induced by the high-dose non- adjuvanted influenza vaccines (10 pg, represented by the dashed lines) (Fig. 14b). Even with an immune-irrelevant encoded protein, mRNA-LNPs boosted the quantity and the breadth of humoral responses to inactivated vaccines.

[0139] To examine the tissue localization of mRNA-LNP activity, we tracked GFP tissue distribution 24 hours post-injection. Fluorescence was detected at both the injection sites, muscles, and draining lymph nodes, with substantially higher intensity in the lymph nodes (Fig.15). This suggested efficient mRNA delivery and protein expression in secondary lymphoid tissues. This supports the possibility that cytokines encoded by mRNA-LNPs could act in lymph nodes locally to modulate vaccine response as an adjuvant. Thus, we next tested whether mRNA-LNPs encoding specific cytokines could enhance vaccine responses beyond the innate adjuvanticity of LNPs themselves (Fig. 6a). Mice were immunized with 1 pg IIV in combination with mRNA-LNPs encoding either mouse IFN-fB or IL-21. At Day 35, inclusion of IL-21 mRNA LNPs significantly outperformed GFP mRNA-LNPs in boosting antibody titers against both in-vaccine HA antigens (Fig. 6b). At the same time, IFN-p mRNA-LNPs showed no advantage at this time point. However, at 12 months post-immunization, both IL-21 and IFN-p mRNA LNP resulted in ~5 times higher antibody titers than the GFP mRNA-LNPs (Fig.6c, median values). Comparison of titers between the 12-month and Day 35 revealed that either IL-21 or IFN-p mRNA-LNPs slowed the waning of antibody response (Fig. 6d). The enhanced antibody durability was accompanied by a much higher rates of antigen-specific long-lived plasma cell formation induced by either cytokine mRNA-LNPs, as shown by bonemarrow B-cell ELISpot assays (Fig. 6e). The data suggest that cytokine mRNAs sustained antibody production by enhancing long-lived plasma cell persistence.6e).

[0140] These results demonstrate that cytokines IFN-p and IL-21 can serve as effective vaccine adjuvants to enhance antibody responses. Using mRNA-LNPs to deliver these cytokines in vivo provides a flexible strategy to boost the immunogenicity of inactivated vaccines. This approach offers a rational path to improve the efficacy of inactivated or subunit vaccines, particularly those with limited innate immunogenicity.

[0141] This study identified several cytokines (IL-18, GROa / CXCL1 , IFN-p) whose prevaccination concentration correlated with the specific antibody response to influenza vaccines. Identifying baseline predictors of an individual's vaccine response is a key goal for systems vaccinology. Prior studies have investigated the correlation between transcriptome signature and influenza vaccine response. Cytokines are likely molecular drivers for many observed transcriptome signatures. In this study, we combined human cohort studies with in vitro analyses using immune organoids to identify cytokines that augmented flu vaccine responses. We stratified subjects by their pre-VAX HAI flu antibody titer and focused only on those with low pre-VAX HAI flu antibody titer. This design was based on our finding that the biological signals (such as aging effects) were concentrated in the population with low pre-VAX HAI flu antibody levels (Fig. 1c). This finding is in line with prior work that showed subjects with low pre-VAX HAI flu antibody launched more robust interferon responses after the vaccination, which may be attributed to less vaccine antigen clearance and thus more vaccine “take We demonstrated that stratification is essential to detect the correlation between pre-VAX cytokine abundance and post- VAX antibody response. The stratification design may be applied to other vaccine research.

[0142] Combining correlative and interventional studies, we found that IFN-p is a natural adjuvant capable of boosting antibody response in the human population (Fig. 1 , 2, and 5). The IFN-related gene expression signature (induced by either Type I or II IFN) at pre- or postvaccination time points correlated with Day 28 antibody responses. A previous study reported a shared transcriptome signature between vaccine high-responders in patients with systemic lupus erythematosus, a disease characterized by chronic activation of Type-I IFN signaling. Meanwhile, higher Type II IFN production (by CD8+T cells) has been reported to correlate with influenza vaccine responses in COVID-19 recoverees. Also, SLE patients who did not respond to the SARS-Cov2 vaccine had almost no interferon-y responses following a booster.

[0143] The relative contribution of Type I or Type II IFNs to vaccine responses has not been tested in a human immune model. We performed a functional screen of cytokine adjuvants in immune organoids. We found that Type I IFNs, but neither Type II IFN nor IL-18 (a potent inducer of Type II IFN), could enhance influenza vaccine responses. Whether the finding isspecific to the influenza vaccine or can be generalized to other vaccines is worth further investigation. Our findings suggest that Type I IFN is a master regulator specific for the live vaccine response, upstream of most other cytokine activation programs. Live vaccines (e.g., measles vaccine, LAIV) tend to generate a more robust human protection than their inactivated counterparts. Comparative studies in mouse models using influenza or rabies vaccines (live versus inactivated) also confirmed the finding. Extended antigen exposure can augment antibody responses. Additionally, the live vaccine may activate a specific immune program to drive an augmented antibody response. In spleen organoids, we performed a comparative study of 250 cytokines using a high-throughput technology with DNA-barcoded antibodies (NULISA). The assay identified 15 live-vaccine-specific cytokines (Fig. 4b). Adding a single cytokine, type I IFN (IFN-p), recapitulates most of the live-vaccine-specific immune activation program (Fig. 4c-d), including other type I IFNs and another cytokine adjuvant, IL- 10 (but not IL-12 or IL-21 , see below). Moreover, adding Type I IFN boosted the inactivated vaccine’s antibody induction quantity to a level similar to the live vaccine (Fig. 4e). Consistent with these findings in humans, mice lacking IFNAR1 or STAT 1 exhibit impaired viral clearance, altered isotype class switching, and reduced lgG2c and IgA production after influenza vaccination.

[0144] In the longitudinal cohort study we identified Type I IFN as an immune regulator that selectively influences vaccine responsiveness in a high-risk group. Older adults bear a disproportionate burden of influenza-associated morbidity and mortality and are also the population in which vaccine-induced antibody responses are most consistently suboptimal (Fig 1c). Baseline IFN-p abundance correlated with antibody response primarily in old adults (>65y) receiving low-dose antigen (Fig. 11a). Moreover, in older adults, osteoarthritis incidence is associated with a higher IFN-p baseline and antibody response to the inactivated vaccine (Fig. 13). We noted that medical history recorded in the cohort relies on self-report during the annual visit, which is a limitation of the study. Also, due to the sample size limitation, we could not examine the correlation between vaccine response and other well-established Type I interferonopathies with a lower incidence rate in the population (such as lupus).

[0145] Our systemic screen also identified a second pathway (IL-12 / IL-21 ) that is Type I IFN- independent (Fig. 5b-c). Human type I interferon deficiency- due to inborn errors (e.g., IFNAR1 / 2, IRF7 mutations) or neutralizing auto-antibodies- impairs antiviral immunity and underlies severe infectious diseases (influenza, COVID-19, herpes simplex) or severe adverse reactions to live vaccines (measles and yellow fever). Despite profound defects in the innate antiviral defense, individuals with type I IFN deficiency can still mount humoral responses. For example, YFV-neutralizing antibodies in I FNAR1 -deficient patients can develop following vaccination and exposure, indicating that B cell priming and antibody production can proceed independently of intact type I IFN signaling. We found that in humanspleen organoids IL-12 and IL-21 were outside the Type I IFN pathway (not induced by adding Type I IFN) (Figs 4c, d and 5). We also demonstrated that IL-21 can be induced by IL-12 (Fig.5). Meanwhile, in a recent report, IL-12 can also regulate B cell function directly. Consistent with this, the live vaccine augments antibody responses by activating numerous cytokines (at least six distinct adjuvants) (Figs 5b and 6b) through orthogonal pathways (Type I IFN vs. IL- 12 / IL-21 ). Importantly, IL-12 and IL-21 are outside the Type I IFN pathway (not induced by exogenous IFN-p, (Figs 4c, d and 5a, b). We further demonstrated that IL-12 can induce IL-21 (Fig. 5a), likely through Tfh activation. RNA replication of the influenza virus (occurred for a few cycles for the live attenuated vaccine) results in 5'-triphosphate dsRNA, which can activate pattern recognition receptors (RIG-I). Further investigation of the innate immunity sensing process can reveal the upstream mechanisms for the differential cytokine activation program between the live and inactivated vaccines.

[0146] We then tested the adjuvant effect of the above cytokines delivered in mRNA-LNP format in mice. Numerous cytokines have been delivered in protein form into mouse models and have demonstrated adjuvant effects. For example, IL-21 -IgFc protein enhances influenza vaccine responses in aged macaques with suppressed SIV infection. However, mRNA-LNP has several advantages over protein to deliver cytokines, including lower cost and targeted trafficking to the lymph nodes (Fig. 15). In line with our findings in human cohorts and organoids, we found that mRNA LNP, a potent inducer of both Type I IFN and IL-21 , substantially enhanced the quantity and breadth of the antibody response to the inactivated influenza vaccine. Non-antigen coding mRNA LNP was so potent that the H5N1 HA antibody levels against the two most recent H5N1 strains induced by the adjuvanted vaccine (1 pig IIV + 3 pg GFP mRNA LNP, Fig. 14a) were comparable to the human influenza HA antibody levels induced by the high-dose non-adjuvanted vaccine. Our results aligned with the prior finding that lipid nanoparticles exert a potent adjuvant effect by augmenting Tfh expansion. Importantly, including IL-21 mRNA or IFN-p further augmented the durability of the antibody response, which was already potently adjuvanted by mRNA-LNP. Intriguingly, IFN-p mRNA LNP did so without further increasing the peak antibody titer (compared to GFP mRNA LNP at 14 days post-vaccination). This finding is in line with recent work showing that there is a distinct regulatory mechanism governing antibody response durability beyond short-term quantity. While mRNA-LNP has been widely used to encode antigens (such as in COVID-19 vaccines), its potential for delivering cytokine adjuvants has not been fully explored. Both IL- 21 and IL-12 showed a potent adjuvant effect in our organoid screen of 19 cytokines (Fig. 3b). However, the lipid components of mRNA-LNP vaccines, particularly the ionizable and PEGylated lipids, can induce potent and broad inflammatory effects, including the activation of complement, and the release of multiple cytokines, including IL-1 a, IL-1 p, IL-6, IL-8, TNF-a,and IFN-y. Cytokine mRNA adjuvants may be encapsulated with immunologically inert lipids to improve precision in adjuvant engineering.

[0147] Our study focused on antibody responses. Most cohorts included only assayed antibody quantity / titers, which correlated with protection for influenza infections. Using a Luminex platform capable of detecting both in-vaccine and cross-reactive antibodies, we also measured the impact of mRNA-LNP and cytokines on breadth of antibody response (Fig. 6). Future studies can investigate the cytokine regulation of cellular immunity in human vaccine responses.

[0148] Our study has also identified differences between human and mouse vaccine responses. For example, studies in mice show that IL-12 does not induce mouse CD4+T cells to express IL-21. But here, we found that IL-12 strongly induces IL-21 expression in human spleen organoids, while both are regulated in a Type I IFN-independent manner (Fig 6). This is consistent with prior studies in human CD4+ T cells, which showed that IL-12 can induce IL-21 expression. Meanwhile, numerous cytokines, including IFN-y, IL-18, IL-1 a / p, IL-2, and IL-4, have been reported to enhance antibody responses to influenza vaccination in diverse murine models. However, in our human spleen organoid system, these cytokines did not have this effect in the organoid system (Fig. 3b). It is possible that the discrepancy is due to recall (in humans) vs. primary responses (in mice). However, it is also possible that some of them are due to species differences.

[0149] Overall, our study identified a cytokine, IFN-p that acts as a natural adjuvant which contributes significantly to vaccine response variability in human populations with low baseline Ab titers and is also a key regulator of the live-vaccine-specific immune activation program. Moreover, we used a functional systems immunology approach to screen the adjuvanticity of numerous cytokines in immune organoids. The screen identified six cytokine adjuvants involved in the live-vaccine-specific antibody response, in at least two different pathways. This knowledge can inform the design of adjuvants that mimic the live-vaccine-specific effect without the risk of viral replication. While animal testing remains the cornerstone for drug development (including adjuvants), it has limited predictive value for efficacy in humans. Recently, the U.S. FDA announced a transformative plan to adopt “human-relevant methods” for drug evaluation. Our approach unites high-throughput organoid testing and human cohort studies, establishing a human-centric platform to identify adjuvant candidates.References

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[0160] Kotliarov, Y. et al. Broad immune activation underlies shared set point signatures for vaccine responsiveness in healthy individuals and disease activity in patients with lupus. Nat Med 26, 618-629 (2020).

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[0162] Mule, M. P. et al. Integrating population and single-cell variations in vaccine responses identifies a naturally adjuvanted human immune setpoint. Immunity 57, 1160-1176.e1167 (2024).

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[0174] Zhang, Q. et al. Inborn errors of type I IFN immunity in patients with life-threatening COVID-19. Science 370, eabd4570 (2020).

[0175] Hadjadj, J. et al. Impaired type I interferon activity and inflammatory responses in severe COVID-19 patients. Science 369, 718-724 (2020).

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[0177] Nguyen, K. H. et al. COVID-19 Vaccination Intent, Perceptions, and Reasons for Not Vaccinating Among Groups Prioritized for Early Vaccination - United States, September and December 2020. MMWR Morb Mortal Wkly Rep 70, 217-222 (2021).Example 2RNA Vaccine Synthesis Protocol

[0178] DNA sequences for vaccines were codon optimized using an in-house algorithm and were synthesized by Synbio Technologies (Monmouth Junction, NJ). 3’ and 5’ UTRs are taken from the mRNA sequence of commercial Pfizer SARS-CoV-2 vaccine, (see WHO document 11889, proposing the International Nonproprietary Name (INN) for a messenger RNA (mRNA) encoding the full-length SARS-CoV-2 spike glycoprotein).

[0179] Gene sequences were PGR amplified and cloned into a backbone containing 3’ and 5’ UTRs (NEBuilder, NEB). Then, constructs were amplified, linearized with a T7 promoter 5’ overhang, and polyadenylated via a 2-step PGR. PGR Steps were performed using 0.5uM each primer, 1-2ng template per 50uL reaction, and Platinum SuperFi II polymerase (Thermo Fisher). PCRs were cleaned up via standard protocol (Qiagen).

[0180] In-vitro transcription of purified PGR products was performed with the T7 mScript Standard mRNA production kit (Cellscript) while using N1 -methylpseudouridine (Trilink Biotech) in place of uracil. Then, RNA was capped using the ScriptCap Cap 1 Capping System (Cellscript). RNA was purified via Monarch RNA Cleanup kit. IVT RNA was stored at -80C.

[0181] The following lipid solution is prepared to make the LNPs. The lipids are the same as those used in the Pfizer SARS-CoV-2 vaccine.| Reagent | Working | Working Cone. | Stock Cone. | Volume per 1 mL |[ Cone. i (rnM) [ Stock (uL) j [ (mg / mL) | [ ALC-0315 i 5.41 7.06 150mg / mL 1082 [ 100% Ethanol [ N / A | N / A | N / A [ 150.9 [ ALC-0159 10.629 0.26 10mg / mL 162.9 [ Cholesterol [ 2.52 6.52 10mg / mL [ 252 [ DSPC h .13 1 1.43 [ 5mg / mL 226 [[ 50% Ethanol [ N / A j N / A | N / A [i 200ALC-0315 ([(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate)) is a synthetic lipid. ALC-0159 is a PEG / lipid conjugate (i.e. PEGylated lipid), specifically, it is the N,N-dimyristylamide of 2-hydroxyacetic acid, O-pegylated to a PEG chain mass of about 2 kilodaltons (corresponding to about 45-46 ethylene oxide units per molecule of N,N-dimyristyl hydroxyacetamide). It is a non-ionic surfactant by its nature. DSPC is distearoylphosphatidylcholine.

[0182] RNA is thawed and diluted to final concentrations of 141ng / uL RNA, 25mM Acetate, 200mM NaCI, pH 4. Then, RNA solution and lipid solution undergoes microfluidic mixing at a ratio of 3 volumes RNA : 1 volume lipid. Solutions are loaded into 1mL syringes, as larger plastic syringes flex under pressure and are not suitable for mixing. It is essential that the syringes contain nearly no air pockets, or mixing will not occur at a reliable rate. For small batches, syringes can be pre-filled with buffer and a small air pocket can be used to separate RNA from RNA buffer along the length of the tubing. For large batches, the 1 mL syringes are filled completely. Solutions flow from the syringe to the microfluidic mixer via Cole ParmerMasterflex Microbore Transfer Tubing (Tygon® ND-100-80, 0.020" ID x 0.060" OD), and into the device via custom tubing adapters (0.025” OD, 0.013” ID, 0.5” length type 304 stainless steel, New England Small Tube). The overall flow rate is 500uL per minute through the device, controlled via syringe pump (New Era Pump Systems Inc. NE-4000).

[0183] The output solution is dialyzed against PBS to remove the ethanol (Pierce microdialysis devices, 0.3mL capacity, 3.5kd MWCO), typically diluting the final vaccine to 30 pg RNA I 400 pL. If the LNPs are to be frozen, the vaccine is further diluted by adding 1 / 2 volume sterile-filtered 60% sucrose in PBS (final sucrose is 20% w / v). LNPs are then flash frozen in liquid nitrogen and placed in LN2 storage.

[0184] The microfluidic mixer was fabricated using standard photolithography techniques at the Stanford microfluidics foundry, and the photomasks used for production are attached. Photomasks were ordered from Artnet Pro Inc. (San Jose, CA). The flow channel layer was fabricated to a height of roughly 63 microns, and the herringbone layer was fabricated to a height of roughly 21 microns. The photomask was drafted in-house, based on Chen et al. (2012).Methods

[0185] Cohort Design and Data Retrieval. \Ne retrieved human serum cytokine and antibody data from previous influenza vaccine studies conducted by the Stanford Human Immune Monitoring Center (HIMC). Only individuals who received non-adjuvanted, inactivated influenza vaccines (Fluzone®, standard or high dose) were included; subjects who received FluMist® (live attenuated) or Fluad® (MF59-adjuvanted) vaccines were excluded. Various laboratories measured hemagglutination inhibition (HAI) antibody titers between 2007 and 2015. To ensure data quality across years, we analyzed a longitudinal cohort (Flu Vaccine Study No. 15) to assess year-to-year consistency. The 2015 measurements (conducted by a CDC-accredited lab) were treated as the gold standard, and pre-vaccination HAI titers from other years were compared against 2015 values for the same individuals. Years with poor correlation (Pearson’s r < 0.5; e.g., 2007, 2008, 2011 , 2012) were excluded from downstream analyses. The cohort size is provided in Fig. 1b.

[0186] Cytokine Measurement. Cytokine levels were measured using Luminex-based multiplex liquid array assays at the Stanford HIMC. All samples were run in duplicate, and control beads (Radix Biosolutions, Georgetwon, TX) were included in every well. Depending on the year and platform version, the following kits were used:

[0187] EMD Millipore Human 80-plex kits (these included 3 panels: Panel 1 was Milliplex HCYTA-60K-PX48; Panel 2 was Milliplex HCP2MAG-62K-PX23; Panel 3 includes the Milliplex HSP1MAG-63K-06 and HADCYMAG-61K-03 (Resistin, Leptin and HGF) to generate a 9 plex): Samples were diluted 3x for Panels 1 & 2 or 10x for Panel 3, with 25 pL of sample used per well. After overnight incubation with antibody-linked magnetic beads at4°C with shaking, plates were washed twice with wash buffer in a BioTek ELx405 washer (BioTek Instruments, Winooski, VT). Cytokines were detected with biotinylated secondary antibodies and streptavidin-phycoerythrin (PE) (incubations of 1 h and 30 min, respectively, at room temperature with shaking). Plates were washed again as above. The readout was performed on a Luminex FlexMap3D instrument, with a minimum bead count threshold of 50 per cytokine.

[0188] EMD Millipore Magnetic Bead kits (62- or 63-plex): Similar protocol as above, with overnight sample incubation and FlexMap3D readout. Wells yielding bead counts <50 for a given analyte were flagged for quality control.

[0189] Affymetrix / eBioscience Polystyrene Bead kits (37-, 50-, or 51-plex): Samples were incubated on filter-bottom plates (room temperature followed by 4 °C), then washed and detected as per kit instructions. Plates were read on a Luminex 200 instrument, requiring at least 100 beads per cytokine for data inclusion.

[0190] Antibody Response Quantification. HAI titers were measured from serum collected on Day 0 (pre-vaccination) and Day 28 (post-vaccination, as previously described. Serially diluted sera (25 pL in PBS) were mixed with 25 pL of virus containing 4 HA units in V-bottom 96-well plates. After 15 minutes at room temperature, 50 pL of 0.5% chicken red blood cells (cRBCs) were added, followed by 1-hour incubation. The HAI titer was defined as the reciprocal of the highest serum dilution that inhibited hemagglutination, indicated by a compact cRBC pellet. For each individual, the antibody response was defined as the geometric mean fold-rise in titer across vaccine strains, calculated as the ratio of the Day 28 titer to the Day 0 titer. Fold-rise values were log2-transformed to normalize distributions. To allow comparison across study years, titers were further adjusted using quantile normalization. Subjects with baseline titers >40 were optionally excluded in some analyses to minimize the effects of pre-existing immunity.

[0191] Multi-cohort Correlation and Meta-analysis. Subjects were stratified into subpopulations based on age group, gender, vaccine dose (standard vs. high), baseline HAI titer quantile, study cohort, and influenza season. Each subpopulation (n > 5) was analyzed using linear regression to determine the relationship between the standardized prevaccination cytokine level and the log2antibody response. The resulting regression slope (with its standard error) was taken as the subgroup's cytokine-response effect size. We then performed a meta-analysis to synthesize these effects across all subgroups, using the rmeta package in R to fit a random-effects model. This approach provided an overall estimate of the correlation between pre-vaccine cytokine levels and antibody response while accounting for between-group heterogeneity.

[0192] Standardization of Cytokine Concentrations. \Ne standardized cytokine abundances using z-score normalization, allowing us to plot them across different years. Specifically, wecomputed a z-score for each cytokine using the mean and standard deviation derived from a reference subset of relatively young subjects (<40 years old) across all years. We selected this younger group, assuming they have more stable immune profiles over time, providing a consistent baseline. This approach is analogous to previously published methods for longitudinal immune monitoring.

[0193] Disease Correlation Analysis. We investigated associations between baseline cytokine levels and subsequent disease diagnoses in older adults. Seniors (>65 years) from Flu Vaccine Study No. 15 reported new disease diagnoses in annual medical surveys. For each disease of interest, we compared the pre-vaccination IFN-p levels in samples collected within 2 years before diagnosis (cases) with those from disease-free, age-matched subjects (controls). Only cytokine data from subjects >65 years were used in this analysis. Group differences in IFN-p were evaluated to identify any pre-diagnostic cytokine elevations.

[0194] Spleen Organoid Culture and Vaccination. Spleen tissues were retrieved from the Donor Network West (DNW, an authorized organ processing organization). The procedure is approved by Stanford IRB (Exemption as donors are deceased). Spleen and tonsil organoid cultures were established using previously described methods. Tissue was dissected into roughly 3-5mm and manually disrupted into a single-cell suspension by processing through a 100-pm strainer with a 5mL syringe plunger. Enzymatic dissociation was unnecessary and did not improve the response to LAIV from cryopreserved cells. Tissue debris was reduced by Ficoll density gradient separation, although this step was not required for tonsil organoid development. After washing with complete medium (RPMI with glutamax (ThermoFisher # 61870127), 10% FBS, 1 x nonessential amino acids (ThermoFisher #11140050), 1 x sodium pyruvate (ThermoFisher # 11360070), 1 x penicillin-streptomycin (ThermoFisher # 15240062), 1 x Normocin (InvivoGen), and 1 x insulin / selenium / transferrin cocktail ( ThermoFisher # 41400045) (Gibco), cells were enumerated and frozen into aliquots in FBS+ 10% DMSO. Frozen cells were stored at -140 °C until use.

[0195] Aliquots were thawed into complete medium for culture of cryopreserved cells, enumerated, and resuspended to 6 x 107celuls per ml for larger cultures or 2 x 107cells per ml for smaller cultures. Cells were plated, 100 pL per well, into permeable (0.4-pm pore size) membranes (24-well size PTFE or polycarbonate membranes in standard 12-well plates or 96-well polycarbonate membrane plates with single-well receiver trays; Corning or Millipore), with the lower chamber consisting of complete medium (1 mL for 12-well plates, 200 pL for 96- well plates) supplemented with 1 pg / mL of recombinant human B cell-activating factor (BAFF; BioLegend # 559608). Adding a small amount of BAFF improved total B cell survival (and thus increased overall cell recovery) but was not a requirement for plasmablast differentiation or antibody secretion. Vaccination was performed using Fluzone (II V, diluted 1 :10,000) or FluMist (LAIV, diluted 1 :2,000), dose-optimized for organoid stimulation.

[0196] Cytokine and Antibody Detection in Organoids. Cytokine Quantification - Cytokine abundance in organoid culture supernatants was measured using a DNA-barcoded, nanoparticle-based ultrasensitive immunoassay (NULISA, Alamar Biosciences), capable of femtomolar-level detection. Supernatants were collected at Day 3 post-stimulation for cytokine profiling. Each sample (25 pL) was analyzed on the NULISA Inflammation Panel targeting approximately 250 cytokines and chemokines, using the automated ARGO HT platform at the Stanford Human Immune Monitoring Center (HIMC). For stimulation studies comparing IIV, LAIV, IFN-p, and combinations thereof, culture conditions were standardized, and cytokine abundance was reported relative to unstimulated controls matched by donor.

[0197] Influenza-A-specific IgG Detection - To evaluate antigen-specific antibody responses, influenza hemagglutinin (HA)-specific IgG was quantified in organoid supernatants at Day 7 post-vaccination using a Luminex-based multiplex immunoassay. For the cytokine adjuvant screen, 19 recombinant human cytokines were co-administered with inactivated influenza vaccine (IIV) at three concentrations (1 , 10, or 100 ng / mL, with adjusted ranges for IL-1 p and IL-18), and influenza-specific IgG was measured relative to IlV-only controls. Organoids were generated from dissociated human spleen and tonsil tissue and maintained in a 96-well format for high-throughput screening. All measurements were performed in biological replicates (n = 5 donors), and fluorescence intensities were normalized and log-transformed to assess fold changes over background stimulation levels.

[0198] Low-Cell-Input Organoid Culture. Organoids were established using low-input cell seeding to scale up culture throughput. Cells were seeded in ultra-low attachment 96-well plates at 1 .6 x 105cells / well in 200 pL of culture media. Media was partially exchanged (30%) every two days. On Day 7, culture supernatants were harvested for antibody quantification as described above.

[0199] NULISAseq Inflammation Panel. The NULISAseq Inflammation Panel (Alamar Biosciences, Fremont, California), performed at the Stanford University HIMC, is a multiplexed proximity ligation assay targeting 250 inflammation-associated proteins. The assay was processed automatically in the ARGO HT system (Alamar BioSciences). 25 pL of each sample was loaded on the sample plate, along with three sample controls (SC), four negative controls (NC), and three inter-plate controls (IPC). After completion of the automated run, nextgeneration sequencing (Illumina, Foster City, California) was performed on the pooled library. Data were generated using ACC (Alamar Command Center) and NAS (NULISA Analysis Software) via normalization to Internal Controls (IC) and Inter-Plate Controls (IPC). Raw counts were normalized to internal and inter-plate controls and then log2-transformed to yield normalized protein quantity (NPQ) values.

[0200] Viral Antibody Assay. As previously described, a custom Luminex assay was created to detect antibody responses to SARS-CoV-2 and other viral antigens. Antigens of interestwere coupled to barcoded beads (Luminex Corporation, Austin, Texas) according to the manufacturer’s instructions. Supernatant samples were run undiluted, with 25 pL of sample added to the assay plate containing the antigen-coupled bead mixture and incubated for 2 hours at room temperature or overnight at 4°C, shaking on an orbital shaker. Samples were then washed, and 25 pL of secondary Goat-anti Human IgG (Fc fragment) coupled to PE (Phycoerythrin) (Anti IgG-Cat# NC9822979, Jackson ImmunoResearch, West Grove, PA) was added. After incubation with shaking for 30 minutes at room temperature, a second wash was performed before adding 130 pL Reading Buffer (Luminex). Samples were read on a Luminex Flex 3D instrument with a lower bound of 50 beads per target antigen. This assay was performed by the Stanford University HIMC and is presented as MFI (Median Fluorescence Intensity).

[0201] RNA Vaccine Synthesis Protocol. DNA sequences for vaccines were codon optimized using an in-house algorithm and were synthesized by Synbio Technologies (Monmouth Junction, NJ). 3’ and 5’ UTRs are taken from the commercial Pfizer vaccine. Gene sequences were PGR amplified and cloned into a backbone containing 3’ and 5’ UTRs (NEBuilder, NEB). Then, constructs were amplified, linearized with a T7 promoter 5’ overhang, and polyadenylated via a 2-step PCR. PCR Steps were performed using 0.5 pm each primer, 1-2 ng template per 50 pL reaction, and Platinum SuperFi II polymerase (Thermo Fisher). PCRs were cleaned up via standard protocol (Qiagen). In vitro transcription of purified PCR products was performed with the T7 mScript Standard mRNA production kit (Cellscript) while using N1- methylpseudouridine (Trilink Biotech) instead of uracil. Then, RNA was capped using the ScriptCap Cap 1 Capping System (Cellscript). RNA was purified via the Monarch RNA Cleanup kit. IVT RNA was stored at -80 °C. The following lipid solution is prepared to make the LNPs. The lipids are the same as those used in the commercial Pfizer vaccine.Table 1Reagent Working Cone. Working Cone. Stock Cone. Volume per 1 mL (mg / mL) (mM) Stock (pL) ALC-0315 5.41 7.06 50mg / mL 108.2100% Ethanol N / A N / A N / A 150.9ALC-0159 0.629 0.26 10mg / mL 62.9 Cholesterol 2.52 6 52 10mg / mL 252DSPC 1.13 1.43 5mg / mL 22650% Ethanol N / A N / A N / A 200

[0202] RNA is thawed and diluted to final concentrations of 141 ng / pL RNA, 25mM Acetate, 200mM NaCI, pH 4. Then, RNA and lipid solutions undergo microfluidic mixing at a 3 volume ratio of RNA: 1 volume lipid. Solutions are loaded into 1 mL syringes, as larger plastic syringes flex under pressure and are unsuitable for mixing. It is essential that the syringes contain nearly no air pockets, or mixing will not occur at a reliable rate. For small batches, syringes can be pre-filled with buffer, and a small air pocket can be used to separate RNA from RNA buffer along the length of the tubing. For large batches, the 1 mL syringes are filled. Solutions flow from the syringe to the microfluidic mixer via Cole Parmer Masterflex Microbore Transfer Tubing (Tygon® ND-100-80, 0.020" ID x 0.060" OD), and into the device via custom tubing adapters (0.025” OD, 0.013” ID, 0.5” length type 304 stainless steel, New England Small Tube). The overall flow rate is 500 pL per minute through the device, controlled via syringe pump (New Era Pump Systems Inc. NE-4000).

[0203] The output solution is dialyzed against PBS to remove the ethanol (Pierce microdialysis devices, 0.3mL capacity, 3.5 kDa MWCO), typically diluting the final vaccine to 30ug RNA I 400uL. If the LNPs are to be frozen, the vaccine is further diluted by adding 1 / 2 volume sterile-filtered 60% sucrose in PBS (final sucrose is 20% w / v). LNPs are then flash frozen in liquid nitrogen and placed in LN2 storage. The microfluidic mixer used for LNP synthesis was fabricated using standard photolithography; the corresponding photomask design is available upon request. Photomasks were ordered from Artnet Pro Inc. (San Jose, CA). The flow channel layer was fabricated to a height of roughly 63 microns, and the herringbone layer was manufactured to a height of approximately 21 microns. The photomask was drafted in-house, but the basis for this mixer was previously published.

[0204] Mice and Immunization. Male C57BL / 6J mice (6-10 weeks old) were obtained from the Jackson Laboratory and used for all experiments described in this study. Mouse were housed in the Stanford Animal Facility under specific pathogen-free (SPF) conditions, maintained on a 12-hour light / 12-hour dark cycle at a temperature of -18-23 °C and 40-60% humidity. All animal procedures were reviewed and approved by the University Administrative Panel on Laboratory Animal Care (APLAC; protocol No. 34513).

[0205] For immunization, each mouse received a total of 120pL of intramuscular injections, administered as 60pL into each hind leg (left and right caudal thigh muscles). The injection mixture contained 2023 / 24 inactivated influenza vaccine (I IV; Fluzone® Quadrivalent) at a dose of 1 or 10pg per mouse, combined with lipid nanoparticle (LNP)-encapsulated mRNA encoding mouse cytokine adjuvants at 3pg per mouse.

[0206] ELISpot analysis. Bone marrow ELISpoi assays were performed at day 434 after first immunization as previously described. Briefly, multiscreen 96-well plates with Immobilon-P membrane (Millipore, # MAIPS4510) were coated overnight at 4°C with 50 pL per well of Fluzone® Quadrivalent 2023 / 24 seasonal influenza virus vaccine at a final concentration of 2pg / mL in PBS. Plates were washed with PBS and blocked with RPM 11640 / 10%FBS medium at 37°C for 2 h. Single-cell suspensions were prepared from bone marrow of femurs and tibias, followed by red blood cell lysis using ACK lysis buffer. Cells were then plated onto antigen- coated wells and incubated for 15 h at 37°C. After washing, an HRP-conjugated anti-mouse IgG antibody (SouthernBiotech, #1031-05, 1 :2,500) was added, and antigen-specific responses were visualized using an AEG substrate detection kit (BD Biosciences, #551951). ELISpot plates were scanned using ImmunoSplot S6 Universal M2 analyzer and analyzed using an automated ELISpot counting system (Cellular Technology). The number of spots per million cells was calculated.

[0207] Fluorescence imaging. To assess the distribution of LNP-encapsulated mRNA transcripts, mice were intramuscularly immunized with PBS or LNP-encapsulated mRNA encoding GFP (3pg per mouse) as described. Draining lymph nodes and hind limb muscles were harvested at 24 hours post-immunization. Whole-tissue fluorescence was then measured using a Largo spectral imaging system with an excitation wavelength of 465 nm and an emission wavelength of 510 nm. The data were analyzed using Aura imaging software (Spectral Instruments Imaging) and values represent the integrated fluorescence intensity.References

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[0238] World Health Organization International Nonproprietary Names document 11889. (2020).

[0239] Chen, D. et al. Rapid Discovery of Potent siRNA-Containing Lipid Nanoparticles Enabled by Controlled Microfluidic Formulation. J. Am. Chem. Soc. 134, 6948-6951 (2012).DNA Sequences, written (5'->3')IL21 Human IVT Template, SEQ ID NOR TAATACGACTCACTATAGAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGA GAACCCGCCACCATGAGATCATCCCCCGGCAACATGGAGAGGATCGTGATCTGCCTGA TGGTGATCTTCTTAGGCACCCTGGTGCACAAGAGTAGCAGCCAGGGGCAGGATCGGC ATATGATCCGCATGCGCCAGCTGATCGACATCGTCGATCAGCTGAAAAATTATGTGAAT GATTTGGTGCCGGAATTCCTTCCGGCACCGGAGGACGTGGAGACCAATTGCGAGTGGT CAGCGTTCTCCTGTTTCCAGAAGGCGCAGCTGAAGAGCGCCAATACTGGAAACAACGA GCGCATCATCAACGTTTCTATCAAGAAACTGAAGCGCAAGCCTCCCAGCACCAATGCTG GGAGGCGGCAGAAGCACCGGCTGACCTGCCCTTCTTGTGACAGTTACGAGAAGAAGC CTCCCAAGGAGTTCCTTGAGAGGTTTAAGAGTCTGCTGCAGAAGATGATTCATCAGCAT CTCAGTTCTCGGACCCACGGGTCCGAGGACTCATGACTCGAGCTGGTACTGCATGCAC GCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGT CCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACAC CTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAG GGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGCAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAIL21 Human SEQ ID N0:2 ATGAGATCATCCCCCGGCAACATGGAGAGGATCGTGATCTGCCTGATGGTGATCTTCTT AGGCACCCTGGTGCACAAGAGTAGCAGCCAGGGGCAGGATCGGCATATGATCCGCAT GCGCCAGCTGATCGACATCGTCGATCAGCTGAAAAATTATGTGAATGATTTGGTGCCG GAATTCCTTCCGGCACCGGAGGACGTGGAGACCAATTGCGAGTGGTCAGCGTTCTCCT GTTTCCAGAAGGCGCAGCTGAAGAGCGCCAATACTGGAAACAACGAGCGCATCATCAA CGTTTCTATCAAGAAACTGAAGCGCAAGCCTCCCAGCACCAATGCTGGGAGGCGGCAG AAGCACCGGCTGACCTGCCCTTCTTGTGACAGTTACGAGAAGAAGCCTCCCAAGGAGT TCCTTGAGAGGTTTAAGAGTCTGCTGCAGAAGATGATTCATCAGCATCTCAGTTCTCGG ACCCACGGGTCCGAGGACTCATGA5' UTR SEQ ID N0:3 GAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC3'UTR SEQ ID N0:4 CTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCC CGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCAC CACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTA GCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTT TAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCT AGCPoly A SEQ ID NO:5 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAIL21 Mouse SEQ ID NO:6 ATGGAGAGGACCCTGGTGTGTCTGGTGGTCATCTTCCTGGGGACTGTGGCTCACAAGT CCAGTCCCCAGGGGCCTGATCGGCTGCTGATCAGGCTGCGGCATCTGATCGACATCG TGGAGCAGCTGAAGATCTACGAGAACGATCTGGACCCCGAGCTCCTCAGTGCCCCTCA GGACGTGAAGGGGCACTGTGAGCACGCCGCCTTCGCCTGTTTCCAGAAGGCTAAGCT GAAGCCTTCAAACCCGGGCAACAACAAAACCTTCATCATCGACCTGGTTGCCCAGTTGCGAAGGCGGCTGCCCGCCCGCAGGGGGGGCAAGAAGCAAAAGCACATTGCTAAGTGC CCCTCCTGCGACTCTTACGAGAAGAGGACTCCTAAGGAGTTTCTGGAGCGGCTGAAGT GGCTGCTCCAGAAGATGATCCACCAGCACCTGAGCTGAInsert for vaccine cloning vector with 5' and 3' UTRs SEQ ID NO:7 ggggtaccgaagccgctagcgctG AG AAT AAACT AGT ATT CTT CTGGTCCCC AC AG ACT GAG AG A GAACCCGCCACCaccggtagatctcgagctcCTCGAGCTGGTACTGCATGCACGCAATGCTAGC TGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGC TCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCAC GCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGA TTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAAT TTCGTGCCAGCCACACCCTGGAGCTAGCgatgagtttggacaaaccacaacIL21 Human Cloning Primer Fwd SEQ ID NO:8 ACAGACTCAGAGAGAACCCGCCACCATGAGATCATCCCCCGGC IL21 Human Cloning Primer Rev SEQ ID NO:9 TGCGTGCATGCAGTACCAGCTCGAGTCATGAGTCCTCGGACCC IL21 Mouse Cloning Primer Fwd SEQ ID NQ:10 ACAGACTCAGAGAGAACCCGCCACCATGGAGAGGACCCTGGTG IL21 Mouse Cloning Primer Rev SEQ ID NOH 1 TGCGTGCATGCAGTACCAGCTCGAGTCAGCTCAGGTGCTGGTGPCR I Fwd SEQ ID NO:12T AAT ACGACTCACTATAGAGAAT AAACT AGTATTCPCR I Rev SEQ ID NO:13 TTTTTTTTTTTTTTTTTTAGTCATATGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCTAGC TCCAGGGTGTGGPCR II Fwd SEQ ID NO:14T AAT ACGACTCACTATAGAGAAT AAACT AGPCR II Rev SEQ ID NO:15 TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTAGTCATATGC

[0240] The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims.

Claims

T HAT WHICH IS CLAIMED IS:

1. An adjuvant composition, comprising an effective dose of an mRNA encoding an immunostimulatory cytokine encapsulated in a lipid nanoparticle.

2. The composition of claim 1 , wherein the cytokine comprises one or more of human IL-21, IL-10, IL-12, IFNB1, IFNA2, IFNW, IL-9, IL-18, IL-5, and IL-17A.

3. The composition of claim 1 or claim 2, wherein the cytokine is selected from one or more of human IL-10, IL-12, IL-9, IFNB1, IFNA2, and IFNW.

4. The composition of any of the preceding claims, wherein the cytokine is human IL-21.

5. The composition of any of the preceding claims, wherein the cytokine is a type I interferon.

6. The composition of any of the previous claims, wherein the mRNA comprises a 5' UTR, a codon optimized open reading frame encoding the cytokine, and a 3' UTR.

7. The composition of claim 5, wherein the mRNA comprises a 5' UTR and 3'UTR that are heterologous to the coding region.

8. The composition of any of the previous claims, wherein the mRNA is a modified mRNA (mmRNA).

9. The composition of claim 8, wherein the mmRNA comprises pseudouridine (i ).

10. The composition of any of the preceding claims, wherein the lipid nanoparticle comprises a cationic and / or ionizable lipid.

11. The composition of any of the preceding claims, wherein the adjuvant is coformulated with an antigen of interest.

12. The composition of claim 11 , wherein the antigen of interest comprises a vaccine.

13. The composition of claim 12, wherein the vaccine is selected from inactivated pathogen vaccines; live-attenuated pathogen vaccines; messenger RNA (mRNA) vaccines;subunit, recombinant, polysaccharide, and conjugate vaccines; toxoid vaccines; and viral vector vaccines.

14. The composition of any of claims 11-13, wherein the antigen of interest is a pathogen antigen.

15. The composition of any of claims 11-13, wherein the antigen of interest is a tumorspecific antigen.

16. The composition of any of the preceding claims, wherein the lipid nanoparticle comprises ACL-0315, Cholesterol, ACL-0519, and DSPC.

17. A method of increasing an immune response of an individual to an antigen of interest, the method comprising:administering an effective dose of a composition according to any of claim 1-11 or 16, in combination with an effective dose of an antigen of interest, wherein one or both of the strength and duration of immune response is increased.

18. The method of claim 17, wherein the antigen of interest is co-formulated with the adjuvant composition.

19. The method of claim 17, wherein the antigen of interest is separately administered with the adjuvant composition.

20. The method of any of claims 17-19, wherein the antigen of interest comprises a vaccine.21 . The method of claim 20, wherein the vaccine is selected from inactivated pathogen vaccines; live-attenuated pathogen vaccines; messenger RNA (mRNA) vaccines; subunit, recombinant, polysaccharide, and conjugate vaccines; toxoid vaccines; and viral vector vaccines.

22. The method of any of claims 16-20, wherein the antigen of interest is a pathogen antigen.

23. The method of any of claims 16-20, wherein the antigen of interest is a tumorspecific antigen.

24. The method of any of claims 16-22, wherein the adjuvant composition is administered multiple times.

25. The method of any of claims 16-23, wherein the effective dose of adjuvant composition is from about 0.5 ug to about 1 mg.

26. A kit comprising an adjuvant composition according to any of claims 1-15, and instructions for use.

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