Polysaccharide adjuvants for viral vaccines

Abstract

Provided herein are adjuvant systems comprising fungal polysaccharides for use in betacoronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) and influenza (influenza A and B) vaccines, as well as immunogenic compositions comprising the adjuvant systems and betacoronavirus or influenza virus antigens.

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 USC § 119(e) of U.S. Provisional Application No. 63 / 160,667, entitled "POLYSACCHARIDE ADJUVANTS FOR SEVERE ACUTE RESPIRATORY SYNDROME-RELATED CORONAVIRUS (SARS-COV) VACCINES," filed on March 12, 2021, and U.S. Provisional Application No. 63 / 257,076, entitled "POLYSACCHARIDE ADJUVANTS FOR VIRUS VACCINES," filed on October 18, 2021; the entire contents of each are incorporated herein by reference.

[0002] Reference to sequence listing submitted as a text file via EFS-WEB This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web, which is incorporated herein by reference in its entirety. The said ASCII copy, created on Mar. 11, 2022, is entitled C123370205WO00-SEQ-ZJG, and is 76,363 bytes in length.

[0003] Federally Sponsored Research This invention was made with Government support under Grant Nos. R01AI121066 and 75N93019C00044 awarded by the National Institutes of Health (NIH). The Government has certain rights in the invention. [Background technology]

[0004] background Epidemic and pandemic infectious diseases are often caused by viruses that are easily transmitted from birds and other mammals to humans. Such viruses include several families of viruses, such as flaviviruses, coronaviruses, orthomyxoviruses, paramyxoviruses, rhabdoviruses, and filoviruses. Of greatest concern are betacoronaviruses and influenza viruses, both of which are highly transmissible to and between humans and are often virulent. The pandemic potential of these viruses is clear. Severe acute respiratory syndrome coronavirus (SARS-CoV or SARS-CoV-1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are betacoronaviruses that caused the 2002–2004 severe acute respiratory syndrome (SARS) pandemic and the ongoing 2019–2021 coronavirus disease 2019 (COVID-19) pandemic, respectively. Influenza A and B viruses are responsible for causing multiple influenza pandemics as well as seasonal influenza epidemics in the last century. In addition to their high transmissibility and virulence, these viruses have a high mutation rate that complicates efforts to prevent their spread. SARS-CoV-1 and SARS-CoV-2 have each mutated to generate hundreds of strains with increasingly more transmissible concerns. Influenza A viruses continually undergo genetic reassortment to generate multiple viral subtypes (e.g., H1N1, H3N2) that co-circulate within human and other natural host populations. DISCLOSURE OF THEINVENTION

[0005] overview The discovery, development, and deployment of safe and effective vaccines remains critical not only to address the SARS-CoV-2 pandemic, but also to prepare for future potential epidemics, such as those caused by influenza viruses. Immunization of apparently vulnerable populations, such as the elderly and immunocompromised, may result in suboptimal responses, require multiple booster doses, and may be limited by waning immunity. Adjuvantation is an important approach to enhance vaccine-induced immunity. Adjuvants can enhance, prolong, and modulate immune responses to vaccine antigens to maximize protective immunity and may allow for more effective immunization of vulnerable populations (e.g., in the very young and elderly, or for diseases for which an effective vaccine is lacking). In addition, the risk of SARS-CoV-2 vaccine-induced antibody-induced disease enhancement (ADE) must be addressed. Some aspects of the disclosure provide a method of inducing an immune response against a virus in a subject in need thereof, the method comprising administering to the subject a viral antigen and an adjuvant system comprising a fungal polysaccharide.

[0006] In some embodiments, the fungal polysaccharide comprises a soluble polysaccharide. In some embodiments, the fungal polysaccharide comprises a mannan. In some embodiments, the fungal polysaccharide is isolated from Candida albicans. In some embodiments, the adjuvant system further comprises alum. In some embodiments, the fungal polysaccharide is adsorbed to alum. In some embodiments, the fungal polysaccharide is conjugated to alum. In some embodiments, the virus is a betacoronavirus selected from Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome (SARS)-associated coronavirus (SARS-CoV)-1, and SARS-CoV-2. In some embodiments, the viral antigen comprises a betacoronavirus protein or polypeptide. In some embodiments, the viral antigen comprises a nucleic acid encoding a betacoronavirus protein or polypeptide. In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the RNA is messenger RNA (mRNA). In some embodiments, the betacoronavirus protein or polypeptide comprises a betacoronavirus spike protein or spike protein receptor binding domain. In some embodiments, the betacoronavirus spike protein is a MERS-CoV spike protein or spike protein receptor binding domain, a SARS-CoV-1 spike protein or spike protein receptor binding domain, or a SARS-CoV-2 spike protein or spike protein receptor binding domain. In some embodiments, the viral antigen comprises a MERS-CoV, SARS-CoV-1, or SARS-CoV-2 virus particle. In some embodiments, the viral antigen comprises a killed or inactivated MERS-CoV, SARS-CoV-1, or SARS-CoV-2. In some embodiments, the viral antigen comprises killed or live attenuated MERS-CoV, SARS-CoV-1, or SARS-CoV-2.

[0007] In some embodiments, the virus is an influenza A virus or an influenza B virus. In some embodiments, the viral antigen comprises an influenza A virus or influenza B virus protein or polypeptide. In some embodiments, the viral antigen comprises a nucleic acid encoding an influenza A virus or influenza B virus protein or polypeptide. In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the RNA is messenger RNA (mRNA). In some embodiments, the influenza A virus or influenza B virus protein or polypeptide comprises a hemagglutinin (HA) protein, a neuraminidase (NA) protein, or a polypeptide thereof. In some embodiments, the viral antigen comprises an influenza A virus or influenza B virus virus particle. In some embodiments, the viral antigen comprises a killed or inactivated influenza A virus or influenza B virus. In some embodiments, the viral antigen comprises a killed or attenuated live influenza A virus or influenza B virus.

[0008] In some embodiments, the subject is a human.In some embodiments, the subject is a human neonate, a human infant, an adult, or an elderly person.In some embodiments, the subject is a companion animal or a research animal.In some embodiments, the subject is immunocompromised and has chronic lung disease, asthma, cardiovascular disease, cancer, obesity, diabetes, chronic kidney disease, and / or liver disease. In some embodiments, the viral antigen and the adjuvant system are administered simultaneously. In some embodiments, the viral antigen and the adjuvant system are administered separately. In some embodiments, administration is intramuscular, intradermal, oral, intravenous, topical, intranasal, or sublingual. In some embodiments, administration is prophylactic.

[0009] In some embodiments, the adjuvant system induces a type 1 immune response in the subject. In some embodiments, the adjuvant system promotes activation of dendritic cell-associated C-type lectin 2 (Dectin 2) in the subject. In some embodiments, the adjuvant system triggers an innate immune response in the subject. In some embodiments, the adjuvant system enhances B cell immunity. In some embodiments, the adjuvant system enhances the production of antigen-specific antibodies compared to administering the viral antigen alone. In some embodiments, the adjuvant system enhances the production of antigen-specific IgG2c antibodies. In some embodiments, the adjuvant system enhances the cytokine production of antigen-specific T cells compared to administering the viral antigen alone. In some embodiments, the adjuvant system enhances the production of IFNγ. In some embodiments, the adjuvant system polarizes the innate immune response toward follicular helper T (Tfh) cell immunity. In some embodiments, the adjuvant system polarizes the innate immune response toward T helper 1 (Th1) cell immunity. In some embodiments, the adjuvant system extends the protective effect against the viral antigen in the subject compared to administering the viral antigen alone. In some embodiments, the adjuvant system increases the speed of immune response compared to administering the viral antigen alone. In some embodiments, the viral antigen generates the same level of immune response against the antigen at a lower dose in the presence of the adjuvant system compared to administering the viral antigen alone. In some embodiments, the possibility of antibody-induced disease enhancement (ADE) is reduced in the subject compared to administering the viral antigen alone.

[0010] Another aspect of the present disclosure provides an adjuvant system comprising a fungal polysaccharide for use in inducing an immune response against a virus (e.g., a betacoronavirus such as MERS-CoV, SARS-CoV-1, or SARS-CoV-2, or an influenza virus, e.g., influenza A virus or influenza B virus) in a subject in need thereof. In some aspects, the present disclosure provides an adjuvant system comprising a fungal polysaccharide and alum for use in inducing an immune response against a virus (e.g., a betacoronavirus such as MERS-CoV, SARS-CoV-1, or SARS-CoV-2, or an influenza virus, e.g., influenza A virus or influenza B virus) in a subject in need thereof. Also provided herein is an immunogenic composition comprising a viral antigen and an adjuvant system comprising a fungal polysaccharide. In some embodiments, the fungal polysaccharide comprises mannan. In some embodiments, the fungal polysaccharide comprises a fungal polysaccharide isolated from Candida albicans. In some embodiments, the adjuvant system further comprises alum. In some embodiments, the fungal polysaccharide is adsorbed to the alum. In some embodiments, the fungal polysaccharide is conjugated to the alum.

[0011] In some embodiments, the virus is a betacoronavirus selected from Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome (SARS)-associated coronavirus (SARS-CoV)-1, and SARS-CoV-2. In some embodiments, the viral antigen comprises a betacoronavirus protein or polypeptide. In some embodiments, the viral antigen comprises a nucleic acid encoding a betacoronavirus protein or polypeptide. In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the RNA is messenger RNA (mRNA). In some embodiments, the betacoronavirus protein or polypeptide comprises a betacoronavirus spike protein or spike protein receptor binding domain. In some embodiments, the betacoronavirus spike protein is a MERS-CoV spike protein or spike protein receptor binding domain, a SARS-CoV-1 spike protein or spike protein receptor binding domain, or a SARS-CoV-2 spike protein or spike protein receptor binding domain. In some embodiments, the viral antigen comprises a MERS-CoV, SARS-CoV-1, or SARS-CoV-2 virus particle. In some embodiments, the viral antigens comprise killed or inactivated MERS-CoV, SARS-CoV-1, or SARS-CoV-2. In some embodiments, the viral antigens comprise killed or live attenuated MERS-CoV, SARS-CoV-1, or SARS-CoV-2.

[0012] In some embodiments, the virus is an influenza virus selected from influenza A virus and influenza B virus. In some embodiments, the viral antigen comprises an influenza A virus or influenza B virus protein or polypeptide. In some embodiments, the viral antigen comprises a nucleic acid encoding an influenza A virus or influenza B virus protein or polypeptide. In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the RNA is messenger RNA (mRNA). In some embodiments, the influenza A virus or influenza B virus protein or polypeptide comprises a hemagglutinin (HA) protein, a neuraminidase (NA) protein, or a polypeptide thereof. In some embodiments, the viral antigen comprises an influenza A virus or influenza B virus virus particle. In some embodiments, the viral antigen comprises a killed or inactivated influenza A virus or influenza B virus. In some embodiments, the viral antigen comprises a killed or attenuated live influenza A virus or influenza B virus. The above summary is intended to describe in a non-limiting manner some of the aspects, advantages, features, and applications of the technology disclosed herein. Other aspects, advantages, features, and applications of the technology disclosed herein will become apparent from the detailed description, drawings, examples, and claims. [Brief description of the drawings]

[0013] The accompanying drawings are not intended to be drawn to scale. In the drawings, identical or nearly identical components that are shown in various figures are respectively represented by like numerals. For clarity, not every component is labeled in every drawing. In the drawings:

[0014] [Figure 1A-1B]Mannan induces an IFN signature restricted to lymph nodes, which drives lymph node expansion. Figure 1A: Mice were injected intradermally with saline (Sal.), mannan (Mann.), or β-glucan (β-gluc.). 24 hours later, injection sites were assessed for the presence of abscesses and the presence or absence of skin lesions. Graphs show the percentage of mice in each indicated category. Representative photographs of the skin appearance at the saline, mannan, and β-glucan injection sites are also shown. N=5 mice per group. Figure 1B: Transcriptomic analysis of skin samples collected 6 hours after saline (Sal.), β-glucan (β-gluc.), or mannan (Mann.) injection. Heatmap of abundance (log2-normalized counts with Z-score) of genes induced by β-glucan and / or mannan compared to saline control, ranked by difference in abundance between β-glucan and mannan. Genes are split by gaps into two clusters: one highly upregulated by β-glucan and one highly upregulated by mannan. N=3 mice per group. [Fig. 1C-1D] Figure 1C: Mice were injected intradermally with saline, mannan (Mann.) or β-glucan (β-gluc.). 6 or 24 h later, dLNs were collected and analyzed for weight as well as absolute numbers of CD45+, B, and T cells. Results are expressed as fold relative to the contralateral saline-injected LNs. N=5–9 mice per group. Figure 1D: Mice were injected intravenously with a blocking anti-CD62L antibody (αCD62L) or an equal dose of isotype control (Iso CTRL) 1 day prior to intradermal injection of saline or mannan. 24 h later, dLNs were collected and their weight was measured. Results are expressed as fold relative to the contralateral saline-injected LNs. N=4 mice per group. [Fig. 1E-1G]Fig. 1E: WT and Ccr7- / - mice were injected intradermally with saline (Sal.) or fluorescently labeled mannan (Mann.). dLNs were collected 1, 6, and 24 h later, homogenized, and total fluorescence was measured. Results are expressed as arbitrary units of fluorescence (AU) and show mean + SEM. N = 3 mice per time point. Fig. 1F: Ccr7- / - mice were injected intradermally with saline or mannan. 24 h later, dLNs were collected and analyzed as shown in Fig. 1C. N = 6 mice. Fig. 1G: Transcriptome analysis of dLNs collected 6 and 24 h after intradermal injection of saline (Sal.), β-glucan (β-gluc.), or mannan (Mann.). Heatmap of abundance (log2-normalized counts with Z-score) of genes induced by β-glucan and / or mannan compared to saline control, ranked by the difference in abundance between β-glucan and mannan. Genes are split by gaps into two clusters: one highly upregulated by β-glucan and one highly upregulated by mannan. N=4-5 mice per group.

[0015] [Fig. 1H-1I] Figure 1H: Pathway enrichment analysis of genes belonging to the clusters upregulated by mannan as shown in Figure 1G. Figure 1I: Heatmap representation of the average expression levels of the top 50 genes upregulated in mannan-treated dLNs 24 hours after injection compared to saline controls. N=4-5 mice per group. [Fig. 1J-1K]Figure 1J: WT and Ifnar- / - mice were injected intravenously with anti-IFNγ blocking antibody (aIFNγ) or the same dose of isotype control (Iso CTRL) on days -1 and 0. On day 0, mice were also injected intradermally with saline (Sal.) or mannan (Mann.). 24 h later, dLNs were collected, weighed, and RNA extracted for gene expression analysis. Results are expressed as fold over contralateral saline-injected LNs (weight) or relative expression compared to Gapdh. N=4 mice per group. Figure 1K: WT and Ifnar- / -Ifngr- / - mice were injected intradermally with saline (Sal.) or Lipo-CpG. Samples were collected and analyzed as in Figure 1J. N=5 mice per group. # and ## indicate p≦0.05 and 0.01, respectively, when comparing each group with a value of 1 (representing the contralateral control sample, expressed as a multiple). * and ** indicate p≦0.05 and 0.01, respectively, when comparing between different experimental groups.

[0016] [Figure 2A-2B] Mannan-induced lymph node innate responses require CD169+ sinus macrophages expressing Dectin-2. Figure 2A: WT, Clec4n- / -, and Fcer1g- / - mice were injected intradermally with saline or mannan. 24 h later, dLNs were collected, weighed, and RNA was extracted for gene expression analysis. Results are expressed as fold increase relative to contralateral saline-injected LNs. N=3–5 mice per genotype. Figure 2B: WT mice were injected intradermally with fluorescently labeled mannan (Mann.-AF488). 6 h later, dLNs were collected and absolute numbers of mannan-containing (Mann.+) CD3 / CD19 / NK1.1- cells were quantified by flow cytometry. N=6 mice. [Fig. 2C-2D]Figure 2C: Mice were treated as in Figure 2B. Imaging cytometry analysis and quantification of mannan internalization was performed on CD3 / CD19 / NK1.1-deficient CD45+ mannan-containing (Mann.+) cells. N=4 mice. Figure 2D: WT mice were injected intradermally with fluorescently labeled mannan (Mann.). One hour later dLNs were collected for confocal microscopy analysis using antibodies against B220 and phospho-Syk (pSyk). DAPI was used for nuclear counterstain. One representative image is shown. [Fig. 2E-2F] Figure 2E: WT and Fcer1g- / - mice were injected with saline or fluorescently labeled mannan. 6 hours later dLNs were collected and CD86 expression levels were assessed by flow cytometry for CD3 / CD19 / NK1.1- CD45+mannan-containing (Mann.+) cells, CD45+ cells without mannan capture (Mann.-) and CD45+ cells from saline-injected dLNs (Sal.). N=6 mice per genotype. Figure 2F: WT mice were injected intradermally with fluorescently labeled mannan. 6 hours later dLNs were collected and the phenotype of CD3 / CD19 / NK1.1- CD45+mannan-containing (Mann.+) cells was assessed by flow cytometry. N=6 mice. [Fig. 2G-2I] Figures 2G-2I: DT-treated CD11c-DTR, Ccr2- / -, and isotype control (IsoCTRL)- or anti-Ly6G (αLy6G)-treated mice were treated and analyzed as in Figure 2A. N=4 mice per group. [Fig. 2J-2L] Figure 2J, 2K: LNs were isolated from untreated WT mice and expression of Dectin 2 (D2) was assessed by flow cytometry as the percentage of expression in the indicated CD3 / CD19 / NK1.1- CD45+ cell subsets. N=6 for Figure 2J or N=3 for Figure 2K. Figure 2L: Confocal microscopy analysis of untreated LNs stained with antibodies against Dectin 2 (D2), B220, and CD169. DAPI was used for nuclear counterstain. One representative image is shown. [Figure 2M]Figure 2M: DT-treated CD169-DTR mice were treated and analyzed as in Figure 2A. N=4 mice per group. # and ## indicate p≦0.05 and 0.01, respectively, when comparing each group with a value of 1 (representing the contralateral control sample, by fold expression) or saline control. * and ** indicate p≦0.05 and 0.01, respectively, when comparing between different experimental groups.

[0017] [Figure 3A] Activation of the non-canonical NF-κB subunit RelB controls mannan-induced lymph node innate responses. Figure 3A: WT and Card9- / - mice were treated and analyzed as in Figure 2A. N = 9 (for LN weight) or 4 (for gene expression analysis) mice per genotype. [Figure 3B-3D] Figure 3B: CD3- CD19- NK1.1- Ter119- CD45+ AF488-mannan+ Ly6G (CD11b+ Ly6C+) CD11b+ CD11c+ cells were sorted from dLN of WT, Fcer1g- / -, and Card9- / - mice 6 hours after AF488-mannan injection and transcriptional profiles were assessed by targeted transcriptome sequencing. Results are shown as heatmaps of genes with F-test FDR < 0.05 and log2 fold change (FC) > 1 (or < -1) between mutants and WT controls. Figure 3C, 3D: Relbfl / fl and Cd11ccre Relbfl / fl mice were treated with saline, mannan or Lipo-CpG and analyzed as in Figure 2A. N = 4–13 mice per genotype. # and ## indicate p≦0.05 and 0.01, respectively, when comparing each group with a value of 1 (representing the contralateral control sample, by fold expression) or with saline control. * and ** indicate p≦0.05 and 0.01, respectively, when comparing between different experimental groups.

[0018] [Figure 4A-4D]Molecular pathways required for mannan-induced lymph node innate responses modulate the magnitude of mannan adjuvant activity. CFSE-labeled OT-II (Figure 4A-4D) or OT-I (Figure 4E-4H) CD4+ T cells were injected intravenously into WT mice on day -1. On day 0, mice were injected intradermally with saline, ovalbumin (OVA), or OVA combined with mannan (Mann). Three days later, dLNs were isolated and the absolute number of CFSElo cells (i.e., cells that had undergone at least one cycle of cell division) (Figure 4A, 4E) or the percentage of cells at each division peak (Figure 4B, 4F) were quantified by flow cytometry. N=4 mice per group. ## indicates p ≤ 0.01 when comparing each group to saline control (Figure 4A, 4E). * and ** indicate p ≤ 0.05 and p ≤ 0.01 when comparing OVA to OVA+Mann, respectively. (Fig. 4A, 4B, 4E, 4F). Fig. 4C, 4D, 4G, 4H: WT, Fcer1g- / -, and Card9- / - mice were treated and analyzed as in Fig. 4A, 4B, 4E, and 4F, except that all mice received OVA combined with mannan. N=4 mice per genotype. # and ## indicate p≦0.05 and p≦0.01 for WT vs. Fcer1g- / - (black) or Card9- / - vs. Fcer1g- / - (blue), respectively. * and ** indicate p≦0.05 and 0.01 for WT vs. Card9- / -, respectively. Results in Fig. 4B and 4F and 4D and 4H are presented as mean + SD. [Fig. 4E-4H]Molecular pathways required for mannan-induced lymph node innate responses modulate the magnitude of mannan adjuvant activity. CFSE-labeled OT-II (Figure 4A-4D) or OT-I (Figure 4E-4H) CD4+ T cells were injected intravenously into WT mice on day -1. On day 0, mice were injected intradermally with saline, ovalbumin (OVA), or OVA combined with mannan (Mann). Three days later, dLNs were isolated and the absolute number of CFSElo cells (i.e., cells that had undergone at least one cycle of cell division) (Figure 4A, 4E) or the percentage of cells at each division peak (Figure 4B, 4F) were quantified by flow cytometry. N=4 mice per group. ## indicates p ≤ 0.01 when comparing each group to saline control (Figure 4A, 4E). * and ** indicate p ≤ 0.05 and p ≤ 0.01 when comparing OVA to OVA+Mann, respectively. (Fig. 4A, 4B, 4E, 4F). Fig. 4C, 4D, 4G, 4H: WT, Fcer1g- / -, and Card9- / - mice were treated and analyzed as in Fig. 4A, 4B, 4E, and 4F, except that all mice received OVA combined with mannan. N=4 mice per genotype. # and ## indicate p≦0.05 and p≦0.01 for WT vs. Fcer1g- / - (black) or Card9- / - vs. Fcer1g- / - (blue), respectively. * and ** indicate p≦0.05 and 0.01 for WT vs. Card9- / -, respectively. Results in Fig. 4B and 4F and 4D and 4H are presented as mean + SD.

[0019] [Figure 5A-5C]Mannan formulated with aluminum hydroxide acquires novel physical properties predictive of immunological function. Figure 5A: GM-CSF-differentiated bone marrow-derived phagocytes were generated from WT and stimulated with LPS, curdlan, β-glucan (β-gluc.), mannan (Mann.), and aluminum hydroxide / mannan (AlumOH / mann.). After 18–21 h, supernatants were collected and TNF and IL-2 protein concentrations were measured by ELISA, while cells were harvested and expression levels of CD86 and OX40L were measured by flow cytometry. N=5 independent experiments. Figure 5B: Mice were injected intradermally with saline (sal.), aluminum hydroxide (AH), β-glucan (β-gluc), mannan (Mann.), or aluminum hydroxide / mannan (AH / mann). After 24 h, skin samples were collected and RNA was extracted for gene expression analysis. Results are expressed as the fold of contralateral saline-injected skin samples. N=4–5 mice per group. Figure 5C: Mice were injected intradermally with saline (Sal.), fluorescently labeled β-glucan (β-gluc), fluorescently labeled mannan (Mann) or its formulation with aluminum hydroxide (AH / mann). 24 hours later, dLNs were collected, homogenized and total fluorescence was measured. Results are expressed as arbitrary units (AU) of fluorescence and shown as individual data points (horizontal bars represent the mean). N=3 mice. [Fig. 5D-5G](Fig. 5D) Mice were treated as in Fig. 5A. After 1, 7, and 14 days, dLNs were collected, weighed, and expressed as fold over contralateral saline-injected LNs. Results are expressed as mean + SEM (left panel) or area under the curve (AUC, right panel). (Fig. 5E) Mice were treated as in Fig. 5A. After 24 h, dLNs were collected and RNA was extracted for gene expression analysis. Results are expressed as fold over contralateral saline-injected LNs. N = 5 per group. (Fig. 5F) Ifnar- / - and WT mice were treated with blocking anti-IFNγ antibody (αIFNγ) or the same dose of isotype control (Iso CTRL) on days -1 and 0, respectively, and on day 0, mice were injected intradermally with saline (sal.), β-glucan (β-gluc), or AH / mannan (AH / mann). After 24 h, dLNs were collected and weighed. Results are expressed as fold over contralateral saline-injected LNs. N=5 mice per group. (FIG. 5G) WT mice were treated with blocking anti-IFNAR and anti-IFNγ (αIFNAR / IFNγ) antibodies or the same dose of isotype control (Iso CTRL) on days -1 and 0, and on day 0, mice were injected intradermally with saline (sal.) or AH / mannan (AH / mann). 24 hours later, dLNs were collected and their weights were measured. Results are expressed as fold over contralateral saline-injected LNs. N=5 mice per group.

[0020] [Fig. 5H-5J](Fig. 5H, 5I) Mice were treated with soluble (Sol) or particulate (Part) fractions of AH / mannan. After 24 h, skin samples (Fig. 5H) and dLNs (Fig. 5I) were collected, dLN weights were measured, and RNA was extracted for gene expression analysis. Results are expressed as fold over contralateral saline-injected skin samples or LNs. N=5 mice per group. (Fig. 5J) Mice of the indicated backgrounds were injected with saline (sal.) or AH / mannan (AH / mann). After 24 h, dLNs were collected, their weights were measured, and RNA was extracted for gene expression analysis. Results are expressed as fold over contralateral saline-injected samples or as relative expression compared to Gapdh. [Figure 5K-5L] (Fig. 5K, 5L) WT mice were injected with the same amount of PBS or anti-asialo GM1 depletion antibody (αAsGM1) on days -1 and 0 (Fig. 5K), or WT and Batf3- / - mice (Fig. 5L) were injected intradermally with saline (Sal) or AH / mannan (AH / mann) on day 0. dLNs were collected 24 h later and RNA was extracted for gene expression analysis. Results are reported as relative expression compared to Gapdh. N=5 mice per group. # and ## indicate p ≤ 0.05 and 0.01, respectively, when comparing each group with its untreated control (CTRL) or value 1 (representing the contralateral control sample, by fold expression). * and ** indicate p ≤ 0.05 and 0.01, respectively, when comparing between different experimental groups.

[0021] [Figure 6A]Immunization with SARS-CoV-2 spike protein and aluminum hydroxide / mannan generates anti-spike type 1 immunity and neutralizing antibodies. Figures 6A-6E: Mice were intradermally injected with saline (Sal), prefusion stabilized SARS-CoV-2 trimer alone (S) or in combination with AH (S / AH), β-glucan (S / β-gluc.), mannan (S / mann) or AH / mannan (S / AH / mann) on days 0 (prime) and 14 (boost). Serum samples were collected on day 28 to assess anti-spike (Figure 6A) and anti-RBD (Figure 6B) antibody levels, SARS-CoV-2 surrogate virus neutralization test (Figure 6D) and neutralization titers (Figure 6E). In selected experiments (Figure 6C), mice were sacrificed on day 35, spleens were collected and splenocytes were isolated for in vitro restimulation with spike peptide. After 96 h, supernatants were collected and IFNg protein levels were measured by ELISA. N = 16–18 (Figures 6A, 6B), 10 (Figure 6C), 8–10 (Figure 6D), or 13–15 (Figure 6E) mice per group. [Figure 6B-6E]Immunization with SARS-CoV-2 spike protein and aluminum hydroxide / mannan generates anti-spike type 1 immunity and neutralizing antibodies. Figures 6A-6E: Mice were intradermally injected with saline (Sal), prefusion stabilized SARS-CoV-2 trimer alone (S) or in combination with AH (S / AH), β-glucan (S / β-gluc.), mannan (S / mann) or AH / mannan (S / AH / mann) on days 0 (prime) and 14 (boost). Serum samples were collected on day 28 to assess anti-spike (Figure 6A) and anti-RBD (Figure 6B) antibody levels, SARS-CoV-2 surrogate virus neutralization test (Figure 6D) and neutralization titers (Figure 6E). In selected experiments (Figure 6C), mice were sacrificed on day 35, spleens were collected and splenocytes were isolated for in vitro restimulation with spike peptide. After 96 h, supernatants were collected and IFNg protein levels were measured by ELISA. N = 16–18 (Figures 6A, 6B), 10 (Figure 6C), 8–10 (Figure 6D), or 13–15 (Figure 6E) mice per group. [Fig. 6F-6H] Figure 6F-6H: Mice were injected intradermally with saline (Sal), prefusion stabilized SARS-CoV-2 trimer alone (S), or this combined with AH (S / AH). Mannan (Mann) was injected separately on the same side as the S / AH injection at the proximal site, either on the same day (S / AH+Mann (D0)) or the day before (S / AH+Mann (D-1)). As a control, SARS-CoV-2 trimer combined with AH and mannan (S / AH / Mann) was also injected. The formulations were injected on day 0 (prime) and day 14 (boost). Serum samples were collected on day 28 to assess anti-spike antibody levels (Figure 6F) and SARS-CoV-2 neutralization titers (Figure 6G). In selected experiments (Figure 6H), mice were sacrificed on day 35, spleens were collected, and splenocytes were isolated for in vitro restimulation as in C. N=6-8 mice per group. #, * and ##, ** indicate p≦0.05 and 0.01, respectively, for comparisons between different experimental groups. Comparisons are indicated by shading.

[0022] [Figure 7-1] Immunization with SARS-CoV-2 spike protein and aluminum hydroxide / mannan generates cross-reactive anti-spike antibodies with broad epitope specificity. Mice were immunized as in Figures 6A-6E. VirScan analysis was performed on serum samples collected on day 28. Each column represents a single serum sample collected from an individual mouse, and each row represents a peptide tile. Tiles are labeled by amino acid start and end positions. The intensity of the shading represents the enrichment (Z-score) of each peptide. Shaded lines indicate the approximate amino acid positions (AA positions) of the RBD, fusion peptide, and heptad repeat 2 of each virus. N=6 mice per group. [Figure 7-2] Immunization with SARS-CoV-2 spike protein and aluminum hydroxide / mannan generates cross-reactive anti-spike antibodies with broad epitope specificity. Mice were immunized as in Figures 6A-6E. VirScan analysis was performed on serum samples collected on day 28. Each column represents a single serum sample collected from an individual mouse, and each row represents a peptide tile. Tiles are labeled by amino acid start and end positions. The intensity of the shading represents the enrichment (Z-score) of each peptide. Shaded lines indicate the approximate amino acid positions (AA positions) of the RBD, fusion peptide, and heptad repeat 2 of each virus. N=6 mice per group. [Figure 7-3] Immunization with SARS-CoV-2 spike protein and aluminum hydroxide / mannan generates cross-reactive anti-spike antibodies with broad epitope specificity. Mice were immunized as in Figures 6A-6E. VirScan analysis was performed on serum samples collected on day 28. Each column represents a single serum sample collected from an individual mouse, and each row represents a peptide tile. Tiles are labeled by amino acid start and end positions. The intensity of the shading represents the enrichment (Z-score) of each peptide. Shaded lines indicate the approximate amino acid positions (AA positions) of the RBD, fusion peptide, and heptad repeat 2 of each virus. N=6 mice per group.

[0023] [Figure 8A-8C] AH / mannan adjuvant formulations confer protection against viral infection in the lungs. Figure 8A, 8B: Mice were injected intradermally on days 0 (prime) and 14 (boost) with saline (Sal), prefusion stabilized SARS-CoV-2 trimer alone (S) or in combination with AH (S / AH), AH / mannan (S / AH / mann), AddaS03 (S / AddaS03), or AH / PHAD (S / AH / PHAD). Serum samples were collected on day 28 to assess anti-spike and anti-RBD antibody levels (Figure 8A). Mice were infected intranasally with SARS-CoV-2 MA10 on day 35 and the number of plaque forming units (PFU) in the lungs was quantified 2 days later (Figure 8B). N=4–5 mice per group. Figure 8C-8F: Mice were injected intradermally on days 0 (prime) and 14 (boost) with saline (Sal), Flublok alone (rHA), or in combination with AH (rHA / AH), AH / mannan (rHA / AH / mann), AddaVax (rHA / AddaVax), or AH / PHAD (rHA / AH / PHAD). Serum samples were collected on day 28 to assess antibodies against rHA (anti-rHA, C) or IAV A / PR / 8 / 1934 recombinant hemagglutinin (anti-rPR8, Figure 8E). Mice were infected intranasally with IAV A / PR / 8 / 1934 on day 35 and body weights were recorded for 7 days (Figure 8D). N=5 (Figure 8C, 8E) or 8 (Figure 8D) mice per group. Mice were sacrificed 7 days post-infection and lungs were collected for histological analysis (hematoxylin-eosin staining, Figure 8F). One representative image per group is shown. #, * and ##, ** indicate p ≤ 0.05 and 0.01, respectively. Comparisons are indicated by shading. [Fig. 8D-8F]AH / mannan adjuvant formulations confer protection against viral infection in the lungs. Figure 8A, 8B: Mice were injected intradermally on days 0 (prime) and 14 (boost) with saline (Sal), prefusion stabilized SARS-CoV-2 trimer alone (S) or in combination with AH (S / AH), AH / mannan (S / AH / mann), AddaS03 (S / AddaS03), or AH / PHAD (S / AH / PHAD). Serum samples were collected on day 28 to assess anti-spike and anti-RBD antibody levels (Figure 8A). Mice were infected intranasally with SARS-CoV-2 MA10 on day 35 and the number of plaque forming units (PFU) in the lungs was quantified 2 days later (Figure 8B). N=4–5 mice per group. Figure 8C-8F: Mice were injected intradermally on days 0 (prime) and 14 (boost) with saline (Sal), Flublok alone (rHA), or in combination with AH (rHA / AH), AH / mannan (rHA / AH / mann), AddaVax (rHA / AddaVax), or AH / PHAD (rHA / AH / PHAD). Serum samples were collected on day 28 to assess antibodies against rHA (anti-rHA, C) or IAV A / PR / 8 / 1934 recombinant hemagglutinin (anti-rPR8, Figure 8E). Mice were infected intranasally with IAV A / PR / 8 / 1934 on day 35 and body weights were recorded for 7 days (Figure 8D). N=5 (Figure 8C, 8E) or 8 (Figure 8D) mice per group. Mice were sacrificed 7 days post-infection and lungs were collected for histological analysis (hematoxylin-eosin staining, Figure 8F). One representative image per group is shown. #, * and ##, ** indicate p ≤ 0.05 and 0.01, respectively. Comparisons are indicated by shading.

[0024] [Figure 9] Mannan and β-glucan show different diameters. The hydrodynamic diameters of mannan (Mann.) and β-glucan (β-gluc.) preparations were measured by dynamic light scattering. Results of one representative experiment are shown. [Figure 10A]Soluble mannan is inactive in vitro. GM-CSF-differentiated bone marrow-derived phagocytes were generated from WT (Fig. 10A, 10B), Clec7a- / -, Clec4n- / - and Fcer1g- / - mice (Fig. 10B) and stimulated with LPS, curdlan, β-glucan (β-gluc), soluble mannan (Mann) (Fig. 10A, 10B), uncoated microbeads (B only) and microbeads covalently bound to mannan (B:Mann) (Fig. 10B). After 18–21 h, supernatants were collected and TNF and IL-2 concentrations were measured by ELISA, while cells were harvested and CD86 and OX40L expression levels were measured by flow cytometry. N=4–6 independent experiments. ## indicates p≦0.01 when comparing each group with its untreated control (CTRL). Results are presented as mean + SEM. [Figure 10B] Soluble mannan is inactive in vitro. GM-CSF-differentiated bone marrow-derived phagocytes were generated from WT (Fig. 10A, 10B), Clec7a- / -, Clec4n- / - and Fcer1g- / - mice (Fig. 10B) and stimulated with LPS, curdlan, β-glucan (β-gluc), soluble mannan (Mann) (Fig. 10A, 10B), uncoated microbeads (B only) and microbeads covalently bound to mannan (B:Mann) (Fig. 10B). After 18–21 h, supernatants were collected and TNF and IL-2 concentrations were measured by ELISA, while cells were harvested and CD86 and OX40L expression levels were measured by flow cytometry. N=4–6 independent experiments. ## indicates p≦0.01 when comparing each group with its untreated control (CTRL). Results are presented as mean + SEM. [Figure 11] FIG. 11 shows pathway enrichment analysis of genes belonging to the cluster upregulated by β-glucan as shown in FIG. 1B.

[0025] [Figure 12A-12B]Mannan and β-glucan induce unique patterns of immune cell recruitment and activation in draining LNs. Figures 12A-12H: Mice were injected intradermally with saline (black plots), mannan (Mann, red plots) or β-glucan (β-gluc, red plots). 6 or 24 hours later, dLNs were collected and analyzed by flow cytometry for absolute numbers of the indicated populations (Figures 12A-12G) or CD86 expression (Figure 12H). N=5-6 mice per group. [Fig. 12C-12H] Mannan and β-glucan induce unique patterns of immune cell recruitment and activation in draining LNs. Figures 12A-12H: Mice were injected intradermally with saline (black plots), mannan (Mann, red plots) or β-glucan (β-gluc, red plots). 6 or 24 hours later, dLNs were collected and analyzed by flow cytometry for absolute numbers of the indicated populations (Figures 12A-12G) or CD86 expression (Figure 12H). N=5-6 mice per group. [Fig. 12I-12M]Figures 12I-12K: Mice were intradermally injected with saline (Sal) or mannan (Mann). 24 hours later, dLNs were collected and analyzed for absolute numbers of the indicated cell populations in total IFNγ+ cells (Figure 12I) or CD45+ cells (Figure 12J). IFNγ production levels in CD8+ T cells or NK cells were assessed by measuring the MFI of IFNγ in IFNγ-expressing cells (Figure 12K). Figures 12L-12N: WT mice (Figures 12L, 12N), or WT and Batf3- / - mice (Figure 12M), treated with the same amount of PBS or anti-asialo GM1 depletion antibody (aAsGM1) on days -1 and 0, were intradermally injected with saline or mannan on day 0. After 24 hours, dLNs were collected and RNA was extracted for gene expression analysis (Figure 12L, 12M) or dLNs were weighed and absolute numbers of the indicated populations were analyzed by flow cytometry (Figure 12N). Results are expressed as fold relative to contralateral saline-injected LNs. N=4-5 mice per group. # and ## indicate p≦0.05 and 0.01, respectively, when comparing between treatments (Mann vs. β-gluc, Figures 12A-12H) or when comparing each group to a value of 1 (representing the contralateral control sample, Figures 12L, 12M, by fold expression). * and ** indicate p≦0.05 and 0.01, respectively, when comparing between treatments (Mann, β-gluc) and the respective saline control (Figures 12A-12H) or between the indicated experimental groups (Figures 12K, 12L). [Figure 12N]Figures 12I-12K: Mice were intradermally injected with saline (Sal) or mannan (Mann). 24 hours later, dLNs were collected and analyzed for absolute numbers of the indicated cell populations in total IFNγ+ cells (Figure 12I) or CD45+ cells (Figure 12J). IFNγ production levels in CD8+ T cells or NK cells were assessed by measuring the MFI of IFNγ in IFNγ-expressing cells (Figure 12K). Figures 12L-12N: WT mice (Figures 12L, 12N), or WT and Batf3- / - mice (Figure 12M), treated with the same amount of PBS or anti-asialo GM1 depletion antibody (aAsGM1) on days -1 and 0, were intradermally injected with saline or mannan on day 0. After 24 hours, dLNs were collected and RNA was extracted for gene expression analysis (Figure 12L, 12M) or dLNs were weighed and absolute numbers of the indicated populations were analyzed by flow cytometry (Figure 12N). Results are expressed as fold relative to contralateral saline-injected LNs. N=4-5 mice per group. # and ## indicate p≦0.05 and 0.01, respectively, when comparing between treatments (Mann vs. β-gluc, Figures 12A-12H) or when comparing each group to a value of 1 (representing the contralateral control sample, Figures 12L, 12M, by fold expression). * and ** indicate p≦0.05 and 0.01, respectively, when comparing between treatments (Mann, β-gluc) and the respective saline control (Figures 12A-12H) or between the indicated experimental groups (Figures 12K, 12L).

[0026] [Figure 13A] Soluble whole glucan particles induce LN expansion and ISG expression. Mice were injected intradermally with saline (Sal.), dispersible (WGP-D) or soluble (WGP-S) whole glucan particles. After 24 hours, the injection sites were evaluated as shown in Figure 1A. Figure 13A: Skin samples and dLNs were collected, LN weights were measured, and RNA was extracted for gene expression analysis. [Figure 13B]Figure 13B: Results are expressed as fold relative to the median value of saline-injected skin samples or LNs. N=4 mice per group. # and ## indicate p≦0.05 and 0.01, respectively, when comparing each group with a value of 1 (representing the saline control sample, by fold expression). * and ** indicate p≦0.05 and 0.01, respectively, when comparing between different experimental groups. [Figure 14] Mannan induces a CARD-9-independent response in dLNs. Pathway analysis of genes significantly induced in WT compared to Fcer1g− / − mice, as shown in Figure 3B.

[0027] [Figures 15A-15C] Mannans formulated with aluminum hydroxide acquire novel physical properties predictive of immunological function. Figure 15A: Soluble mannan (mann), AH, and blends of mannan and AH (AH / mann) were incubated at room temperature for 30 min, then spun down and the supernatants collected for 1H nuclear magnetic resonance quantification of unbound mannan. Reactions contained excess mannan. Results show that the mannan absorption capacity of AH is approximately twice its mass with this formulation strategy. Results are expressed as a percentage of soluble mannan and shown as mean + SD. Figure 15B-15I: GM-CSF-differentiated bone marrow-derived phagocytes were generated from WT or the indicated knockout mice and stimulated with AH, mannan (Mann), and AH / mannan (AH / mann). After 18-21 h, supernatants were collected and TNF and IL-2 protein concentrations were measured by ELISA (Figure 15B, 15C). Cells were harvested and expression levels of CD86 and OX40L were measured by flow cytometry (Figure 15D, 15E). Alternatively, cells were stimulated for 6 h and RNA was extracted for gene expression analysis. Results are reported as relative expression compared to Rpl13a (Figure 15F-15I). N=3 (Figure 15A) or 4 (Figure 15B, 15I) independent experiments. # and ## indicate p≦0.05 and 0.01, respectively, when comparing the same genotypes between treatments. * and ** indicate p≦0.05 and 0.01, respectively, when comparing between genotypes of the same treatment. Comparisons are indicated by shading. [Fig. 15D-15G] Mannans formulated with aluminum hydroxide acquire novel physical properties predictive of immunological function. Figure 15A: Soluble mannan (mann), AH, and blends of mannan and AH (AH / mann) were incubated at room temperature for 30 min, then spun down and the supernatants collected for 1H nuclear magnetic resonance quantification of unbound mannan. Reactions contained excess mannan. Results show that the mannan absorption capacity of AH is approximately twice its mass with this formulation strategy. Results are expressed as a percentage of soluble mannan and shown as mean + SD. Figure 15B-15I: GM-CSF-differentiated bone marrow-derived phagocytes were generated from WT or the indicated knockout mice and stimulated with AH, mannan (Mann), and AH / mannan (AH / mann). After 18-21 h, supernatants were collected and TNF and IL-2 protein concentrations were measured by ELISA (Figure 15B, 15C). Cells were harvested and expression levels of CD86 and OX40L were measured by flow cytometry (Figure 15D, 15E). Alternatively, cells were stimulated for 6 h and RNA was extracted for gene expression analysis. Results are reported as relative expression compared to Rpl13a (Figure 15F-15I). N=3 (Figure 15A) or 4 (Figure 15B, 15I) independent experiments. # and ## indicate p≦0.05 and 0.01, respectively, when comparing the same genotypes between treatments. * and ** indicate p≦0.05 and 0.01, respectively, when comparing between genotypes of the same treatment. Comparisons are indicated by shading. [Fig. 15H-15I]Mannans formulated with aluminum hydroxide acquire novel physical properties predictive of immunological function. Figure 15A: Soluble mannan (mann), AH, and blends of mannan and AH (AH / mann) were incubated at room temperature for 30 min, then spun down and the supernatants collected for 1H nuclear magnetic resonance quantification of unbound mannan. Reactions contained excess mannan. Results show that the mannan absorption capacity of AH is approximately twice its mass with this formulation strategy. Results are expressed as a percentage of soluble mannan and shown as mean + SD. Figure 15B-15I: GM-CSF-differentiated bone marrow-derived phagocytes were generated from WT or the indicated knockout mice and stimulated with AH, mannan (Mann), and AH / mannan (AH / mann). After 18-21 h, supernatants were collected and TNF and IL-2 protein concentrations were measured by ELISA (Figure 15B, 15C). Cells were harvested and expression levels of CD86 and OX40L were measured by flow cytometry (Figure 15D, 15E). Alternatively, cells were stimulated for 6 h and RNA was extracted for gene expression analysis. Results are reported as relative expression compared to Rpl13a (Figure 15F-15I). N=3 (Figure 15A) or 4 (Figure 15B, 15I) independent experiments. # and ## indicate p≦0.05 and 0.01, respectively, when comparing the same genotypes between treatments. * and ** indicate p≦0.05 and 0.01, respectively, when comparing between genotypes of the same treatment. Comparisons are indicated by shading.

[0028] [Figure 16A-16B]B and T cell responses activated by immunization with SARS-CoV-2 spike in AH / mannan formulation have the same cellular and molecular requirements as AH / mannan-induced LIR. WT, transgenic or WT mice were treated with blocking anti-IFNAR and anti-IFNγ (αIFNAR / IFNγ) antibodies or the same dose of isotype control (Iso CTRL) on days -1, 0, 13 and 14, and these mice were injected intradermally with prefusion stabilized SARS-CoV-2 trimers in combination with AH (S / AH) or AH / mannan (S / AH / mann) on days 0 (prime) and 14 (boost). Serum samples were collected on days 28 (Figures 16B-16G) or 98 (Figure 16A, WT mice) to assess anti-spike antibody levels. In selected experiments (Figures 16B, 16C), mice were sacrificed on day 35, spleens were collected, and splenocytes were isolated for in vitro restimulation with spike peptide. After 96 hours, supernatants were collected and IFNγ protein levels were measured by ELISA. N=4-7 mice per group. # and ## indicate p≦0.05 and 0.01, respectively, when comparing S / AH and S / AH / mann vs. Sal (Figure 16A) or S / AH / mann vs. S / AH (Figures 16B-16G). * and ** indicate p≦0.05 and 0.01, respectively, when comparing S / AH / mann vs. S / AH (Figure 16A) or S / AH / mann across treatments or genotypes (Figures 16B-16G). Comparisons are indicated by shading. [Fig. 16C-16G]B and T cell responses activated by immunization with SARS-CoV-2 spike in AH / mannan formulation have the same cellular and molecular requirements as AH / mannan-induced LIR. WT, transgenic or WT mice were treated with blocking anti-IFNAR and anti-IFNγ (αIFNAR / IFNγ) antibodies or the same dose of isotype control (Iso CTRL) on days -1, 0, 13 and 14, and these mice were injected intradermally with prefusion stabilized SARS-CoV-2 trimers in combination with AH (S / AH) or AH / mannan (S / AH / mann) on days 0 (prime) and 14 (boost). Serum samples were collected on days 28 (Figures 16B-16G) or 98 (Figure 16A, WT mice) to assess anti-spike antibody levels. In selected experiments (Figures 16B, 16C), mice were sacrificed on day 35, spleens were collected, and splenocytes were isolated for in vitro restimulation with spike peptide. After 96 hours, supernatants were collected and IFNγ protein levels were measured by ELISA. N=4-7 mice per group. # and ## indicate p≦0.05 and 0.01, respectively, when comparing S / AH and S / AH / mann vs. Sal (Figure 16A) or S / AH / mann vs. S / AH (Figures 16B-16G). * and ** indicate p≦0.05 and 0.01, respectively, when comparing S / AH / mann vs. S / AH (Figure 16A) or S / AH / mann across treatments or genotypes (Figures 16B-16G). Comparisons are indicated by shading.

[0029] [Figures 17A-17D]AH / mann adjuvant formulations confer protection against pulmonary viral infection. Figures 17A, 17B: Mice were injected intradermally on days 0 (prime) and 14 (boost) with saline (Sal), prefusion stabilized SARS-CoV-2 trimer alone (S) or in combination with AH (S / AH), AH / mannan (S / AH / mann), AddaS03 (S / AddaS03), or AH / PHAD (S / AH / PHAD). Serum samples were collected on day 28 to assess SARS-CoV-2 surrogate virus neutralization tests (Figure 17A) and neutralization titers (Figure 17B). N=4-5 mice per group. Figure 17C, 17D: Mice were intradermally injected with saline (Sal), Flublok alone (rHA), or in combination with AH (rHA / AH), AH / mannan (rHA / AH / mann), AddaVax (rHA / AddaVax), or AH / PHAD (rHA / AH / PHAD) on days 0 (prime) and 14 (boost). On day 35, mice were intranasally infected with IAV A / PR / 8 / 1934. Serum samples were collected on day 42 to assess anti-IAV A / PR / 8 / 1934 recombinant hemagglutinin (anti-rPR8) levels (Figure 17C). Histological scores of lung images were collected as in Figure 8F (Figure 17D). N=5 mice per group. # and ## indicate p≦0.05 and 0.01, respectively. Comparison is indicated by shading.

[0030] Detailed Description of Specific Embodiments Human immunity is crucial for both health and disease, playing a key role in multiple major diseases, including infectious diseases, allergies, and cancer. Infectious diseases are the leading cause of morbidity and mortality at the extreme ends of life. SARS coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, first emerged in China in late 2019. Over 115 million people have been infected worldwide, with over 2.5 million deaths occurring, especially among the elderly. However, even during the SARS-CoV-2 pandemic, many other viruses, including influenza viruses, still continue to pose great concern to human health. The discovery, development, and deployment of safe and effective vaccines will be key to addressing both the current SARS-CoV-2 pandemic, as well as other epidemics that may emerge in the future. Immunization of obviously vulnerable populations, such as the elderly and immunocompromised, may result in suboptimal responses, require multiple booster doses, and may be limited by waning immunity. Adjuvanting is an important approach to enhance vaccine-induced immunity. Adjuvants can enhance, prolong, and modulate immune responses to vaccine antigens to maximize protective immunity and may allow more effective immunization of vulnerable populations (e.g., in the very young and elderly, or for diseases lacking an effective vaccine). In addition, the risk of SARS-CoV-2 vaccine-induced antibody-induced disease enhancement (ADE) must be addressed.

[0031] Some aspects of the disclosure provide immunogenic compositions (e.g., vaccine compositions) comprising a viral antigen and an adjuvant system comprising a polysaccharide from a fungal species. In some embodiments, the adjuvant system further comprises alum (e.g., alum is combined with the fungal polysaccharide). In some embodiments, the fungal polysaccharide (e.g., mannan) is adsorbed into the alum. In some embodiments, the fungal polysaccharide (e.g., mannan) is covalently attached to the alum. The immunogenic compositions (e.g., vaccine compositions) provided herein can be used in a method of inducing an immune response to an antigen in a subject in need thereof, the method comprising administering to the subject an effective amount of a viral antigen and an effective amount of an adjuvant system (e.g., comprising only a fungal polysaccharide or comprising a fungal polysaccharide and alum). In some embodiments, the immunogenic compositions (e.g., vaccine compositions) described herein can be used to induce an immune response in a subject that is a neonate, an adult, or an elderly subject (e.g., 65 years of age or older).

[0032] "Betacoronavirus" is one of the four genera of coronaviruses (alpha, beta, gamma, delta). Betacoronaviruses belong to the subfamily Orthocoronavirinae in the family Coronaviridae of the order Nidovirales. They are enveloped, positive-sense, single-stranded RNA viruses of zoonotic origin. The most clinically important betacoronaviruses in humans include SARS-CoV-1 (the cause of SARS), SARS-CoV-2 (the cause of COVID-19), and MERS-CoV (the cause of MERS). "Influenza virus" refers to one of four types of influenza viruses: influenza A virus (IAV), influenza B virus (IBV), influenza C virus, and influenza D virus. Influenza viruses belong to the order Articulavirales and the family Orthomyxoviridae. All viruses except influenza D virus are known to cause disease in humans, but influenza A virus and influenza B virus have the highest risk of epidemics and are therefore the most clinically important. Influenza viruses are often classified according to subtype. Influenza A virus in particular is classified according to which variants of the hemagglutinin protein (HA) and neuraminidase protein (NA) it encodes. There are 18 different hemagglutinin variants (i.e. H1-H18) and 11 different neuraminidase variants (i.e. N1-N11), theoretically giving a total of 198 possible influenza A subtypes. Influenza A and influenza strains may be further classified according to clades and subclades. Influenza A subtypes of particular clinical importance include A / H1N1 (1918 "Spanish flu" and 2009 swine flu), A / H5N1 (2008 avian flu), and A / H7N9 (2013 avian flu). Influenza B subtypes of particular clinical importance include B / Victoria and B / Yamagata.

[0033] A "fungal polysaccharide" is a polymer of carbohydrates (e.g., sugars) synthesized by any cell species belonging to the kingdom Fungi. Fungal polysaccharides may be produced and secreted by fungal cells or may be present as a component of the fungal cell (e.g., as a structural component of the fungal cell wall). Fungal polysaccharides may be covalently linked to another chemical moiety, such as, but not limited to, a protein (e.g., a glycoprotein expressed on the surface of a fungal cell). Fungal polysaccharides may be composed of one or more types of carbohydrate monomers covalently linked in such a way to form a polysaccharide. Fungal polysaccharides may be soluble, partially soluble, or insoluble in solution, particularly aqueous solutions. Fungal polysaccharides can be measured according to their length (diameter) and can have a diameter of 1 nm or more, 5 nm or more, 10 nm or more, 15 nm or more, 20 nm or more, 25 nm or more, 50 nm or more, 75 nm or more, 100 nm or more, 200 nm or more, 300 nm or more, 400 nm or more, 500 nm or more, 600 nm or more, 700 nm or more, 800 nm or more, 900 nm or more, or 1000 nm or more. Fungal polysaccharides may be uniform or heterogeneous in length, e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the polysaccharides have approximately the same diameter. Fungal polysaccharides may be fungal oligosaccharides. Fungal polysaccharides may be derivative fungal polysaccharides, such as shortened (i.e., low molecular weight) or elongated (i.e., high molecular weight) forms of polysaccharides originally isolated from fungal cells. Fungal polysaccharides may have antigenic properties (i.e., activate an immune response in an animal or human subject). Fungal polysaccharides may be ligands for antigen-specific receptors on cells of an animal or human subject.

[0034] In some embodiments, the fungal polysaccharides for use in the immunogenic compositions (e.g., vaccine compositions) and methods described herein are mannans (i.e., mannose polymers). In some embodiments, the fungal polysaccharides for use in the immunogenic compositions (e.g., vaccine compositions) and methods described herein are β-glucans. In some embodiments, the fungal polysaccharides for use in the immunogenic compositions (e.g., vaccine compositions) and methods described herein elicit an immune response in a subject. In some embodiments, the fungal polysaccharides for use in the immunogenic compositions (e.g., vaccine compositions) and methods described herein are isolated from pathogenic fungi that elicit an immune response, such as Candida albicans. The efficacy of fungal polysaccharides (e.g., mannans, β-glucans, etc.) as vaccine adjuvants in immunization against betacoronaviruses, particularly SARS-CoV-1 and SARS-CoV-2, or influenza viruses has not been previously investigated or demonstrated.

[0035] "Adjuvant system" refers to a composition that includes one or more adjuvants. "Adjuvant" refers to a pharmacological or immunological agent that modifies the effect of other agents, such as antigens in vaccines. Adjuvants are typically included in vaccines to enhance the immune response of recipient subjects to antigens. The use of adjuvants can induce a stronger immune response in subjects with the same dose of antigen, or induce a similar level of immune response by injecting a lower dose of antigen. Adjuvants are believed to function in several ways, including by increasing the surface area of ​​antigens, increasing the retention of antigens in the body and thus allowing time for the lymphatic system to access the antigens, delaying release of antigens, targeting antigens to macrophages, activating macrophages, activating white blood cells such as antigen-presenting cells (e.g., monocytes, macrophages, and / or dendritic cells), or inducing broad activation of cells of the immune system, see, e.g., HS Warren et al, Annu. Rev. Immunol., 4:369 (1986); incorporated herein by reference. Thus, the ability of an adjuvant to induce and increase a particular type of immune response, and identification of that ability, is an important factor in selecting a particular adjuvant for vaccine use against a particular pathogen. Adjuvants known to those of skill in the art include, but are not limited to, aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, etc., collectively referred to herein as "alum"), liposomes, lipopolysaccharide (LPS) or its derivatives, such as monophosphoryl lipid A (MPLA) and glycopyranosyl lipid A (GLA), molecular cages of antigens, components of bacterial cell walls, endocytosed nucleic acids, such as double-stranded RNA (dsRNA), single-stranded DNA (ssDNA), and unmethylated CpG dinucleotide-containing DNA, etc. Exemplary adjuvants include water and oil emulsions, such as Freund's adjuvant and MF59, and compounds such as alum.Currently, vaccines licensed in the United States contain only a limited number of adjuvants, including alum, which promotes the production of T helper type 2 (Th2) cells, and MPLA, which activates innate immunity via Toll-like receptor 4 (TLR4). Many of the most effective adjuvants include bacteria or their products, such as attenuated strains of Mycobacterium bovis, Bacille Calmette-Guerin (BCG); microbial components, such as alum-precipitated diphtheria toxoid, bacterial lipopolysaccharides ("endotoxins"), and their derivatives, such as MPLA and GLA.

[0036] In some embodiments, the adjuvant system of the present disclosure comprises a fungal polysaccharide (e.g., mannan). In some embodiments, the adjuvant system of the present disclosure comprises a fungal polysaccharide (e.g., mannan) and an aluminum salt (referred to herein as "alum"). In some embodiments, the alum is Alhydrogel® (InvivoGen, USA). In some embodiments, in an adjuvant system comprising a fungal polysaccharide (e.g., mannan) and alum, the fungal polysaccharide (e.g., mannan) is adsorbed to the alum (e.g., as described in Jones et al., Journal of Biological Chemistry 280, 13406-13414, 2005; which is incorporated herein by reference). In some embodiments, in adjuvant systems comprising a fungal polysaccharide (e.g., mannan), the fungal polysaccharide (e.g., mannan) is covalently attached to an adjuvant such as alum, liposomes, or lipopolysaccharides (LPS) or derivatives thereof, such as monophosphoryl lipid A (MPLA) and glycopyranosyl lipid A (GLA).

[0037] Adjuvants or adjuvant systems are used in immunogenic compositions, e.g., viral immunogenic compositions (e.g., vaccine compositions) described herein. The terms "vaccine composition" and "vaccine" are used interchangeably herein. An "immunogenic composition" is a composition that activates or enhances a subject's immune response to an antigen after the vaccine is administered to the subject. A vaccine composition is a type of immunogenic composition. In some embodiments, the immunogenic composition stimulates the subject's immune system to recognize the antigen (e.g., betacoronavirus antigen, influenza virus antigen) as foreign and enhances the subject's immune response when the subject is subsequently exposed to the pathogen (e.g., betacoronavirus, influenza virus), whether attenuated, inactivated, or killed. A vaccine can be prophylactic, e.g., to prevent or ameliorate the deleterious effects of future exposure to a pathogen (e.g., betacoronavirus, influenza virus), or therapeutic, e.g., to activate a subject's immune response to a pathogen (e.g., betacoronavirus, influenza virus) after the subject is infected with the pathogen. In some embodiments, the immunogenic composition (e.g., a vaccine composition) is used to protect or treat an organism against a disease (e.g., MERS, SARS, COVID-19, and / or influenza).

[0038] In some embodiments, the vaccine is a subunit vaccine (e.g., a recombinant subunit betacoronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) or influenza virus (e.g., influenza A virus or influenza B virus) vaccine), an attenuated vaccine (e.g., one that contains the viral genome of an attenuated betacoronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) or an attenuated influenza virus (e.g., influenza A virus or influenza B virus)), a live vaccine (e.g., one that contains a live attenuated betacoronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) or a live attenuated influenza virus (e.g., influenza A virus or influenza B virus)), or a conjugated vaccine. vaccine) (e.g., a vaccine containing an antigen that is weakly immunogenic and covalently linked to a relatively highly immunogenic antigen (e.g., a betacoronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) antigen, influenza virus (e.g., influenza A virus or influenza B virus) antigen). One non-limiting example of a conjugate vaccine includes LPS conjugated to a potent protein antigen. In some embodiments, the vaccine includes a killed / inactivated betacoronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) or a killed / inactivated influenza virus (e.g., influenza A virus or influenza B virus). In some embodiments, the vaccine includes virus particles of a betacoronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) or influenza virus (e.g., influenza A virus or influenza B virus).

[0039] "Antigen" refers to an entity that an antibody or receptor binds to, or that induces the production of an antibody. In some embodiments, an antigen increases the production of an antibody that specifically binds to the antigen. In some embodiments, an antigen comprises a protein or a polypeptide. Such proteins or peptides are referred to herein as "immunogenic polypeptides." In some embodiments, the term "antigen" encompasses a nucleic acid (e.g., a DNA or RNA molecule) that encodes an immunogenic polypeptide. In some embodiments, an antigen is derived from a microbial pathogen. For example, antigens can include parts of bacteria, viruses, fungi, and other microorganisms (e.g., coats, capsules, cell walls, flagella, pili, and toxins). As used herein, the term "viral antigen" can refer to an antigen derived from a virus, or, in the case of protein / polypeptide and nucleic acid antigens, an antigen that has a sequence identical or substantially similar (homologous) to an endogenous viral protein / polypeptide or nucleic acid. For purposes of this disclosure, an antigen may include a portion of a betacoronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) or an influenza virus (e.g., influenza A virus or influenza B virus).

[0040] In some embodiments, the protein or polypeptide antigen is a wild-type (i.e., "natural") protein or polypeptide. In some embodiments, the protein or polypeptide antigen is a polypeptide variant relative to the wild-type protein or polypeptide. The term "polypeptide variant" refers to a molecule whose amino acid sequence differs from a native or reference sequence. The amino acid sequence variant may have substitutions, deletions, and / or insertions at specific positions within the amino acid sequence compared to the native or reference sequence. In some embodiments, the polypeptide variant has at least 50% identity to the native or reference sequence. In some embodiments, the variant shares at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity with the native or reference sequence. In some embodiments, polypeptide variants include substitutions, insertions, and / or deletions. In some embodiments, polypeptide variants include covalent variants and derivatives. The term "derivative" is used interchangeably with the term "variant," but generally refers to a molecule that is modified and / or altered in some way relative to a reference or starting molecule.

[0041] In some embodiments, sequence tags or amino acids such as one or more lysines can be added to the peptide sequence (e.g., at the N-terminus or C-terminus). Sequence tags can be used for peptide detection, purification, or localization. Lysines can be used to increase peptide solubility or allow biotinylation. Alternatively, amino acid residues located in the carboxy- and amino-terminal regions of the amino acid sequence of a peptide or protein can be optionally deleted to provide a truncated sequence. Alternatively, specific amino acids (e.g., C-terminus or N-terminus residues) can be deleted depending on the use of the sequence, such as, for example, expression of the sequence as part of a larger sequence linked to a soluble or solid support. In some embodiments, polypeptide variants comprise at least one amino acid residue in native or starting sequence that is removed and a different amino acid is inserted in the same position instead.Substitutions can be single, with only one amino acid being substituted in the molecule, or multiple, with two or more amino acids being substituted in the same molecule.In some embodiments, antigen is a polypeptide that comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more substitutions compared to reference protein.

[0042] In some embodiments, the substitution is a conservative amino acid substitution. The term "conservative amino acid substitution" refers to the substitution of an amino acid that is normally present in a sequence with a different amino acid that has a similar size, charge, or polarity. Examples of conservative substitution include the substitution of a non-polar (hydrophobic) residue, such as isoleucine, valine, and leucine, with another non-polar residue. Similarly, examples of conservative substitution include the substitution of one polar (hydrophilic) residue with another, such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Furthermore, the substitution of a basic residue, such as lysine, arginine, or histidine, with another basic residue, or the substitution of an acidic residue, such as aspartic acid or glutamic acid, with another acidic residue, are additional examples of conservative substitution. Examples of non-conservative substitutions include replacing a non-polar (hydrophobic) amino acid residue, such as isoleucine, valine, leucine, alanine, methionine, with a polar (hydrophilic) residue, such as cysteine, glutamine, glutamic acid, or lysine, or replacing a non-polar residue with a polar residue.

[0043] In some embodiments, protein fragments, functional protein domains, and homologous proteins are used as antigens according to the present disclosure.For example, antigens can include any protein fragments (meaning polypeptide sequences that are at least one amino acid residue shorter than the reference polypeptide sequence, but otherwise identical) of a reference protein that is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 amino acids in length.In another example, any protein that includes a stretch of 20, 30, 40, 50, or 100 amino acids that is 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% identical to a reference protein herein (e.g., a protein from a microbial pathogen) can be utilized according to the present disclosure. In some embodiments, the antigen comprises two or more immunogenic proteins or polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). In some embodiments, the two or more immunogenic proteins or polypeptides are derived from one protein (e.g., different fragments or one protein). In some embodiments, the two or more immunogenic proteins or polypeptides are derived from multiple proteins (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more proteins).

[0044] In some embodiments, the antigen comprises a nucleic acid encoding an immunogenic protein or polypeptide. In some embodiments, the antigen comprises an immunogenic protein or polypeptide and a nucleic acid encoding an immunogenic protein or polypeptide. The term "nucleic acid" or "polynucleotide" in its broadest sense includes any compound and / or substance that comprises a polymer of nucleotides. A nucleic acid encoding an immunogenic protein or polypeptide typically includes an open reading frame (ORF) and one or more control sequences. A nucleic acid (also called a polynucleotide) may be or may include, for example, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, which includes: LNA with a β-D-ribo configuration, α-LNA (a diastereomer of LNA) with an α-L-ribo configuration, 2'-amino-LNA with a 2'-amino functionalization, and 2'-amino-α-LNA with a 2'-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), or chimeras or combinations thereof.

[0045] In some embodiments, the nucleic acid encoding the immunogenic polypeptide is DNA (e.g., an expression vector for an immunogenic protein or polypeptide). In some embodiments, the nucleic acid encoding the immunogenic polypeptide is RNA (e.g., messenger RNA). "Messenger RNA" (mRNA) refers to any polynucleotide that encodes (at least one) polypeptide (a naturally occurring, non-naturally occurring, or modified, polymer of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ, or ex vivo. The basic components of an mRNA molecule typically include at least a coding region, a 5' untranslated region (UTR), a 3'UTR, a 5' cap, and a polyA tail.

[0046] In some embodiments, the coding region of the nucleic acid (e.g., DNA or RNA) encoding the immunogenic polypeptide is codon-optimized. Codon optimization methods are known in the art and can be used as provided herein. In some embodiments, codon optimization can be used to match the codon frequency of the target organism and the host organism to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structure; minimize tandem repeat codons or base runs that may impair gene assembly or expression; customize transcriptional and translational control regions; insert or remove protein transport sequences; remove / add post-translational modification sites (e.g., glycosylation sites) of the encoded protein; add, remove, or shuffle protein domains; insert or remove restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translation rates to ensure that various domains of the protein fold properly; or reduce or remove problematic secondary structures within the polynucleotide. Codon optimization tools, algorithms, and services are known in the art, non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA), and / or proprietary methods. In some embodiments, open reading frame (ORF) sequences are optimized using an optimization algorithm.

[0047] In some embodiments, the codon-optimized sequence shares less than 95% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding an immunogenic protein or polypeptide). In some embodiments, the codon-optimized sequence shares less than 90% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding an immunogenic protein or polypeptide). In some embodiments, the codon-optimized sequence shares less than 85% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding an immunogenic protein or polypeptide). In some embodiments, the codon-optimized sequence shares less than 80% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding an immunogenic protein or polypeptide). In some embodiments, the codon-optimized sequence shares less than 75% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding an immunogenic protein or polypeptide).

[0048] In some embodiments, a nucleic acid encoding an immunogenic protein or polypeptide comprises one or more chemical modifications. The terms "chemical modification" and "chemically modified" refer to a modification in at least one of the following positions, patterns, percentages, or populations of adenosine (A), guanosine (G), uridine (U), thymidine (T), or cytidine (C) ribonucleosides or deoxyribonucleosides. In some embodiments, the nucleic acid (e.g., DNA or RNA) contains a variety of (two or more) different modifications. In some embodiments, a particular region of the nucleic acid (e.g., DNA or RNA) contains one, two or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, the modified nucleic acid (e.g., DNA or RNA) introduced into a cell or organism exhibits reduced degradation in the cell or organism, respectively, compared to the unmodified nucleic acid. In some embodiments, the modified nucleic acid (e.g., DNA or RNA) introduced into a cell or organism may exhibit reduced immunogenicity (e.g., reduced natural response) in the cell or organism, respectively.

[0049] Modified nucleic acids (e.g., DNA or RNA) can include naturally occurring modifications, non-naturally occurring modifications, or polynucleotides can include a combination of naturally occurring and non-naturally occurring modifications. Polynucleotides can include any useful modification, for example, of the sugar, nucleobase, or internucleoside linkage (e.g., to the phosphate linkage, to the phosphodiester linkage, or to the phosphodiester backbone). In some embodiments, modified nucleic acids (e.g., DNA or RNA) include non-naturally modified nucleotides that are introduced during or after the synthesis of the polynucleotide to achieve a desired function or property. Modifications can be present in the internucleotide linkage, the purine or pyrimidine base, or the sugar. Modifications can be introduced at the end of the chain or elsewhere in the chain by chemical synthesis or by polymerase enzymes. Any region of the nucleic acid can be chemically modified.

[0050] In some embodiments, chemically modified nucleic acids contain one or more modified nucleosides. "Nucleoside" refers to a compound containing a sugar molecule (e.g., pentose or ribose) or a derivative thereof in combination with an organic base (e.g., purine or pyrimidine) or a derivative thereof (also referred to herein as "nucleobase"). "Nucleotide" refers to a nucleoside containing a phosphate group. Modified nucleotides can be synthesized by any useful method, e.g., chemical, enzymatic, or recombinant, to include one or more modified or non-natural nucleosides. A polynucleotide may contain a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages may be standard phosphodiester linkages, in which case the polynucleotide will contain a region of nucleotides. In some embodiments, the modified nucleobase is modified uridine.Exemplary nucleobases and nucleosides having modified cytosine include N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and 2-thio-5-methyl-cytidine.

[0051] In some embodiments, the modified nucleobase is a modified uridine. Exemplary nucleobases and in some embodiments, the modified nucleobase is a modified cytosine. Nucleosides having modified uridine include 5-cyanouridine and 4'-thiouridine. In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having modified adenines include 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), and N6-methyl-adenosine (m6A). In some embodiments, modified nucleobase is modified guanine.Exemplary nucleobases and nucleosides with modified guanine include inosine (I), 1-methyl-inosine (m1I), viosine (imG), methyl viosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine. In some embodiments, the antigen comprises a viral protein and / or a nucleic acid encoding a viral protein (e.g., a viral structural or nonstructural protein). In some embodiments, the antigen comprises a nucleic acid encoding a viral genome. In some embodiments, the viral genome is modified to generate an attenuated modified virus.

[0052] A polypeptide or polynucleotide molecule of the present disclosure may share a degree of sequence similarity or identity with a reference molecule (e.g., a reference polypeptide or polynucleotide), such as a wild-type molecule. The term "identity" as known in the art refers to the relationship between two or more polypeptide or polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between them, as determined by the number of matches between strings of two or more amino acid or nucleic acid residues. Identity measures the percentage of identical matches between the smaller of two or more sequences, with gap alignment (if any) addressed by a particular mathematical model or computer program (e.g., an "algorithm"). The identity of related peptides can be easily calculated by known methods. "% identity" as applied to a polypeptide or polynucleotide sequence is defined as the percentage of residues (amino acid or nucleic acid residues) in a candidate amino acid or nucleic acid sequence that are identical to the residues in the amino acid or nucleic acid sequence of a second sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for alignment are well known in the art. Identity is dependent on the calculation of identity percentage, but it should be understood that the value may vary due to gaps and penalties introduced in the calculation. In general, a variant of a particular polynucleotide or polypeptide has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, but less than 100% sequence identity with its particular reference polynucleotide or polypeptide, as determined by sequence alignment programs and parameters described herein and known to those skilled in the art.Such alignment tools include the BLAST suite of tools (Stephen F. Altschul, et al (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402). Another common local alignment technique is based on the Smith-Waterman algorithm (Smith, TF & Waterman, MS (1981) "Identification of common molecular subsequences." J. Mol. Biol. 147:195-197). A common global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, SB & Wunsch, CD (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins." J. Mol. Biol. 48:443-453). Recently, the Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed, which is said to generate global alignments of nucleotide and protein sequences faster than other optimal global alignment methods such as the Needleman-Wunsch algorithm. Other tools are described herein, particularly in the definition of "identity" below.

[0053] The term "homology" as used herein refers to the overall relatedness between polymer molecules, for example between nucleic acid molecules (e.g., DNA and / or RNA molecules) and / or polypeptide molecules. Polymer molecules (e.g., nucleic acid molecules (e.g., DNA and / or RNA molecules) and / or polypeptide molecules) that share a threshold level of similarity or identity determined by alignment of matching residues are said to be homologous. Homology is a qualitative term that describes the relationship between molecules and can be based on quantitative similarity or identity. Similarity or identity is a quantitative term that describes the degree of sequence match between two sequences being compared. In some embodiments, polymer molecules are considered to be "homologous" to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar. The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). Two polynucleotide sequences are considered to be homologous if the polypeptides they encode are at least 50%, 60%, 70%, 80%, 90%, 95%, or even 99% identical or similar for at least one stretch of at least 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by their ability to encode a stretch of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by their ability to encode a stretch of at least 4-5 uniquely specified amino acids. Two protein sequences are considered to be homologous if the proteins are at least 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least 20 amino acids.

[0054] Homology means that the compared sequences have diverged from a common origin during evolution. The term "homolog" refers to a first amino acid or nucleic acid sequence (e.g., a gene (DNA or RNA) or a protein sequence) that is related to a second amino acid or nucleic acid sequence by descent from a common ancestral sequence. The term "homolog" can apply to the relationship between genes and / or proteins separated by the event of speciation, or the relationship between genes and / or proteins separated by the event of gene duplication. An "ortholog" is a gene (or protein) from different species that has evolved from a common ancestral gene (or protein) by speciation. Typically, orthologs retain the same function during evolution. A "paralog" is a gene (or protein) that is related by duplication within a genome. Orthologs retain the same function during evolution, while paralogs evolve novel functions, even if they are related to the original function.

[0055] The term "identity" refers to the overall relatedness between polymer molecules, e.g., between polynucleotide molecules (e.g., DNA and / or RNA molecules) and / or between polypeptide molecules. Calculation of percent identity of two polynucleic acid sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., for optimal alignment, gaps can be introduced in one or both of the first and second nucleic acid sequences, and non-identical sequences can be ignored for comparison purposes). In some embodiments, the length of the aligned sequences for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. If a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.For example, the percent identity between two nucleic acid sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., AcADEmic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., AcADEmic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; each of which is incorporated herein by reference. For example, the percent identity between two nucleic acid sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which is incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percent identity between two nucleic acid sequences can be determined using the GAP program in the GCG software package, using the NWSgapdna.CMP matrix. Commonly used methods for determining percent identity between sequences include, but are not limited to, those described in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); which is incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs.Exemplary computer software for determining homology between two sequences include, but are not limited to, the GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984), BLASTP, BLASTN, and FASTA, Altschul, SF et al., J. Molec. Biol., 215, 403 (1990).

[0056] In some embodiments, the immunogenic compositions (e.g., vaccine compositions) described herein induce an immune response against a betacoronavirus antigen (e.g., an antigen from any betacoronavirus, such as MERS-CoV, SARS-CoV-1, or SARS-CoV-2) or against a betacoronavirus (any betacoronavirus species, such as MERS-CoV, SARS-CoV-1, or SARS-CoV-2). In some embodiments, the betacoronavirus antigen used in the immunogenic compositions described herein comprises an antigen (e.g., a protein or nucleic acid) from MERS-CoV. In some embodiments, the betacoronavirus antigen used in the immunogenic compositions described herein comprises an antigen (e.g., a protein or nucleic acid) from SARS-CoV-1. In some embodiments, the betacoronavirus antigen used in the immunogenic compositions described herein comprises an antigen (e.g., a protein or nucleic acid) from SARS-CoV-2. In some embodiments, the immunogenic compositions (e.g., vaccine compositions) induce an immune response against MERS-CoV, SARS-CoV-1, and / or SARS-CoV-2. Heterologous immunity is contemplated herein. Heterologous immunity refers to the phenomenon in which an antigen-specific response generated against one pathogen is reactivated in response to a second pathogen. For example, an immunogenic composition (e.g., a vaccine composition) may contain a SARS-CoV-1 antigen and induce an immune response against both SARS-CoV-1 and SARS-CoV-2. Similarly, an immunogenic composition (e.g., a vaccine composition) may contain a SARS-CoV-2 antigen and induce an immune response against both SARS-CoV-1 and SARS-CoV-2.

[0057] In some embodiments, the betacoronavirus antigen used in the immunogenic compositions (e.g., vaccine compositions) described herein comprises a betacoronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) protein or polypeptide, or an immunogenic fragment or variant thereof. In some embodiments, the betacoronavirus antigen used in the immunogenic compositions (e.g., vaccine compositions) described herein comprises a nucleic acid (e.g., DNA or RNA such as mRNA) encoding a betacoronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) protein or polypeptide, or an immunogenic fragment or variant thereof. In some embodiments, the betacoronavirus antigen in the immunogenic compositions (e.g., vaccine compositions) described herein comprises a MERS-CoV spike protein, a MERS-CoV envelope protein, a MERS-CoV membrane protein, a MERS-CoV nucleocapsid protein, or an immunogenic fragment thereof (e.g., the receptor binding domain (RBD) of the spike protein). In some embodiments, the betacoronavirus antigen in the immunogenic compositions (e.g., vaccine compositions) described herein comprises a nucleic acid (e.g., DNA or RNA such as mRNA) encoding a MERS-CoV spike protein, a MERS-CoV envelope protein, a MERS-CoV membrane protein, a MERS-CoV nucleocapsid protein, or an immunogenic fragment thereof (e.g., the receptor binding domain (RBD) of the spike protein).

[0058] In some embodiments, the betacoronavirus antigen in the immunogenic compositions (e.g., vaccine compositions) described herein comprises a SARS-CoV-1 spike protein, a SARS-CoV-1 envelope protein, a SARS-CoV-1 membrane protein, a SARS-CoV-1 nucleocapsid protein, or an immunogenic fragment thereof (e.g., the receptor binding domain (RBD) of the spike protein). In some embodiments, the betacoronavirus antigen in the immunogenic compositions (e.g., vaccine compositions) described herein comprises a nucleic acid (e.g., DNA or RNA such as mRNA) encoding a SARS-CoV-1 spike protein, a SARS-CoV-1 envelope protein, a SARS-CoV-1 membrane protein, a SARS-CoV-1 nucleocapsid protein, or an immunogenic fragment thereof (e.g., the receptor binding domain (RBD) of the spike protein). In some embodiments, the betacoronavirus antigen in the immunogenic compositions (e.g., vaccine compositions) described herein comprises a SARS-CoV-2 spike protein, a SARS-CoV-2 envelope protein, a SARS-CoV-2 membrane protein, a SARS-CoV-2 nucleocapsid protein, or an immunogenic fragment thereof (e.g., the receptor binding domain (RBD) of the spike protein). In some embodiments, the betacoronavirus antigen in the immunogenic compositions (e.g., vaccine compositions) described herein comprises a nucleic acid (e.g., DNA or RNA such as mRNA) encoding a SARS-CoV-2 spike protein, a SARS-CoV-2 envelope protein, a SARS-CoV-2 membrane protein, a SARS-CoV-2 nucleocapsid protein, or an immunogenic fragment thereof (e.g., the receptor binding domain (RBD) of the spike protein). The amino acid sequences of exemplary betacoronavirus antigens included in the immunogenic compositions (e.g., vaccine compositions) described herein are provided in Table 1.

[0059] Table 1. Betacoronavirus protein antigens [Table 1-1] [Table 1-2] [Table 1-3]

[0060] In some embodiments, the betacoronavirus antigen in the immunogenic compositions (e.g., vaccine compositions) described herein comprises a protein having an amino acid sequence that is at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identical to any one of SEQ ID NOs: 1-15. In some embodiments, the betacoronavirus antigen in the immunogenic compositions (e.g., vaccine compositions) described herein comprises a protein having an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 1-15. In some embodiments, the betacoronavirus antigen in the immunogenic compositions (e.g., vaccine compositions) described herein comprises a protein comprising an amino acid sequence of any one of SEQ ID NOs: 1-15.

[0061] In some embodiments, the betacoronavirus antigen in the immunogenic compositions (e.g., vaccine compositions) described herein comprises a nucleic acid (e.g., DNA or RNA such as mRNA) encoding a protein having an amino acid sequence that is at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identical to any one of SEQ ID NOs: 1-15. In some embodiments, the betacoronavirus antigen in the immunogenic compositions (e.g., vaccine compositions) described herein comprises a nucleic acid (e.g., DNA or RNA such as mRNA) encoding a protein having an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 1-15. In some embodiments, the betacoronavirus antigen in the immunogenic compositions (e.g., vaccine compositions) described herein comprises a nucleic acid (e.g., DNA or RNA such as mRNA) encoding a protein comprising an amino acid sequence of any one of SEQ ID NOs: 1-15.

[0062] In some embodiments, the immunogenic compositions (e.g., vaccine compositions) described herein induce an immune response against influenza virus antigens (e.g., antigens from any influenza virus, such as antigens from influenza A virus or influenza B virus), or against influenza A virus or influenza B virus (i.e., any strain of influenza A virus or influenza B virus). In some embodiments, the influenza virus antigens used in the immunogenic compositions described herein comprise antigens (e.g., proteins or nucleic acids) from influenza A virus. In some embodiments, the influenza virus antigens used in the immunogenic compositions described herein comprise antigens (e.g., proteins or nucleic acids) from influenza B virus. In some embodiments, the immunogenic compositions (e.g., vaccine compositions) induce an immune response against influenza A virus or influenza B virus. Heterologous immunity is contemplated herein. Heterologous immunity refers to the phenomenon in which an antigen-specific response generated against one pathogen is reactivated in response to a second pathogen. For example, an immunogenic composition (e.g., a vaccine composition) may include an influenza A virus antigen and induce an immune response against both influenza A virus and influenza B virus. Similarly, an immunogenic composition (e.g., a vaccine composition) may include an influenza B virus antigen and induce an immune response against both influenza B virus and influenza A virus. Similarly, an immunogenic composition (e.g., a vaccine composition) may induce an immune response against multiple subtypes (strains) of influenza A virus or multiple subtypes (strains) of influenza B virus.

[0063] In some embodiments, an influenza virus antigen used in an immunogenic composition (e.g., vaccine composition) described herein comprises an influenza virus (e.g., influenza A virus, influenza B virus) protein or polypeptide, or an immunogenic fragment or variant thereof. In some embodiments, an influenza virus antigen used in an immunogenic composition (e.g., vaccine composition) described herein comprises a nucleic acid (e.g., DNA or RNA such as mRNA) encoding an influenza virus (e.g., influenza A virus, influenza B virus) protein or polypeptide, or an immunogenic fragment or variant thereof. In some embodiments, the influenza virus antigen in the immunogenic compositions (e.g., vaccine compositions) described herein comprises an influenza A virus hemagglutinin (HA) protein, an influenza A virus neuraminidase (NA) protein, or an immunogenic fragment thereof. In some embodiments, the influenza A virus antigen in the immunogenic compositions (e.g., vaccine compositions) described herein comprises an influenza A virus hemagglutinin (HA) protein, a nucleic acid (e.g., DNA or RNA such as mRNA) encoding an influenza A virus neuraminidase (NA) protein, or an immunogenic fragment thereof.

[0064] In some embodiments, an influenza virus antigen in an immunogenic composition (e.g., vaccine composition) described herein comprises an influenza B virus hemagglutinin (HA) protein, an influenza B virus neuraminidase (NA) protein, or an immunogenic fragment thereof. In some embodiments, an influenza A virus antigen in an immunogenic composition (e.g., vaccine composition) described herein comprises an influenza B virus hemagglutinin (HA) protein, a nucleic acid (e.g., DNA or RNA such as mRNA) encoding an influenza B virus neuraminidase (NA) protein, or an immunogenic fragment thereof. The amino acid sequences of exemplary influenza virus antigens included in the immunogenic compositions (eg, vaccine compositions) described herein are provided in Table 2.

[0065] Table 2. Influenza virus protein antigens [Table 2]

[0066] In some embodiments, an influenza virus antigen in an immunogenic composition (e.g., a vaccine composition) described herein comprises a protein having an amino acid sequence at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identical to any one of SEQ ID NOs: 16-19, or to the amino acid sequence of another influenza virus antigen (e.g., another subtype of influenza virus antigen) known in the art. In some embodiments, an influenza virus antigen in an immunogenic composition (e.g., a vaccine composition) described herein comprises a protein having an amino acid sequence 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 16-19, or to the amino acid sequence of another influenza virus antigen (e.g., another subtype of influenza virus antigen) known in the art. In some embodiments, the influenza virus antigen in the immunogenic compositions (e.g., vaccine compositions) described herein comprises a protein that includes the amino acid sequence of any one of SEQ ID NOs: 16-19, or the amino acid sequence of another influenza virus antigen (e.g., another subtype of influenza virus antigen) known in the art.

[0067] In some embodiments, the influenza virus antigen in the immunogenic compositions (e.g., vaccine compositions) described herein comprises a nucleic acid (e.g., DNA or RNA such as mRNA) encoding a protein having an amino acid sequence at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identical to any one of SEQ ID NOs: 16-19, or to the amino acid sequence of another influenza virus antigen (e.g., another subtype of influenza virus antigen) known in the art. In some embodiments, the influenza virus antigen in the immunogenic compositions (e.g., vaccine compositions) described herein comprises a nucleic acid (e.g., DNA or RNA such as mRNA) encoding a protein having an amino acid sequence 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 16-19, or to the amino acid sequence of another influenza virus antigen (e.g., another subtype of influenza virus antigen) known in the art. In some embodiments, the influenza virus antigen in the immunogenic compositions (e.g., vaccine compositions) described herein comprises a nucleic acid (e.g., DNA or RNA such as mRNA) encoding a protein that includes the amino acid sequence of any one of SEQ ID NOs: 16-19, or the amino acid sequence of another influenza virus antigen (e.g., another subtype of influenza virus antigen) known in the art.

[0068] In some embodiments, the immunogenic compositions (e.g., vaccine compositions) described herein are formulated for administration to a subject. In some embodiments, the immunogenic compositions (e.g., vaccine compositions) are formulated or administered in combination with one or more pharma- ceutically acceptable excipients. In some embodiments, the immunogenic compositions (e.g., vaccine compositions) include at least one additional active agent, such as, for example, a therapeutic active agent, a prophylactic active agent, or a combination of both. The immunogenic compositions (e.g., vaccine compositions) can be sterile, pyrogen-free, or both sterile and pyrogen-free. General considerations in the formulation and / or manufacture of pharmaceutical agents, such as immunogenic compositions (e.g., vaccine compositions), can be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005, which is incorporated herein by reference in its entirety. Formulations of the immunogenic compositions (e.g., vaccine compositions) described herein can be prepared by any method known or hereafter developed in the art of pharmacology. Generally, such methods involve bringing into association the antigen and / or adjuvant (e.g., fungal polysaccharide, or fungal polysaccharide and alum) with an excipient and / or one or more other accessory ingredients, and then dividing, shaping and / or packaging the product into the desired single-dose or multi-dose unit as necessary and / or desired.

[0069] The relative amounts of antigen, adjuvant, pharma- ceutically acceptable excipient, and / or any additional components in a pharmaceutical composition according to the present disclosure will vary depending on the identity, size, and / or condition of the subject being treated, as well as the route by which the composition is administered. By way of example, the composition may contain 0.1%-100%, e.g., 0.5-50%, 1-30%, 5-80%, at least 80% (w / w) active ingredient. In some embodiments, the immunogenic compositions (e.g., vaccine compositions) described herein are formulated with one or more excipients to (1) increase stability; (2) increase transfection of cells; (3) allow sustained or delayed release (e.g., from a depot formulation); (4) modify biodistribution (e.g., target specific tissues or cell types); (5) increase translation of the encoded protein in vivo; and / or (6) modify the release profile of the encoded protein (antigen) in vivo. In addition to conventional excipients such as any solvent, dispersion medium, diluent, or other liquid vehicle, dispersion or suspension aid, surfactant, isotonicity agent, thickening or emulsifying agent, preservative, excipients can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, cells transfected with peptides, proteins, DNA or RNA vaccines (e.g., for implantation into a subject), hyaluronidase, nanoparticle mimics, and combinations thereof.

[0070] In some embodiments, the immunogenic composition (e.g., vaccine composition) is formulated in an aqueous solution. In some embodiments, the immunogenic composition (e.g., vaccine composition) is formulated in nanoparticles. In some embodiments, the immunogenic composition (e.g., vaccine composition) is formulated in lipid nanoparticles. In some embodiments, the immunogenic composition (e.g., vaccine composition) is formulated in lipid-polycation complexes called lipid nanoparticles. The formation of lipid nanoparticles can be accomplished by methods known in the art and / or methods described in U.S. Patent No. 20120178702; which is incorporated herein by reference. As a non-limiting example, the polycation can include cationic peptides or polypeptides, such as, without limitation, polylysine, polyornithine and / or polyarginine, and cationic peptides described in U.S. Patent Publication WO2012013326 or U.S. Patent US20130142818; each of which is incorporated herein by reference. In some embodiments, the immunogenic composition (e.g., vaccine composition) is formulated in lipid nanoparticles comprising a non-cationic lipid, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0071] In some embodiments, the vaccine formulations described herein are nanoparticles comprising at least one lipid (referred to as "lipid nanoparticles" or "LNPs"). The lipids may be selected from, but are not limited to: DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids, and aminoalcohol lipids. In some embodiments, the lipids may be cationic lipids, such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, and aminoalcohol lipids. The aminoalcohol cationic lipids may be lipids described in U.S. Patent Publication US20130150625, which is incorporated herein by reference, and / or lipids made by the methods described therein. As non-limiting examples, the cationic lipid may be: 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol (compound 2 in US20130150625); 2 -amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol (compound 3 of US20130150625); and 2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (compound 4 of US20130150625); or any pharma- ceutically acceptable salt or stereoisomer thereof.

[0072] Non-limiting examples of lipid nanoparticle compositions and methods for their manufacture are described, for example, in Semple et al. (2010) Nat. Biotechnol. 28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51: 8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578, the contents of each of which are incorporated herein by reference in their entirety. In some embodiments, the immunogenic compositions (e.g., vaccine compositions) described herein have a diameter of about 10 to about 100 nm, for example, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about The lipid nanoparticles may be formulated into lipid nanoparticles having a diameter of about 30 to about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50 to about 70 nm, about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm, and / or about 90 to about 100 nm.

[0073] In some embodiments, the lipid nanoparticles may have a diameter of about 10-500 nm. In some embodiments, the lipid nanoparticles have a diameter of more than 100 nm, more than 150 nm, more than 200 nm, more than 250 nm, more than 300 nm, more than 350 nm, more than 400 nm, more than 450 nm, more than 500 nm, more than 550 nm, more than 600 nm, more than 650 nm, more than 700 nm, more than 750 nm, more than 800 nm, more than 850 nm, more than 900 nm, more than 950 nm, or more than 1000 nm. In some embodiments, the immunogenic composition (e.g., vaccine composition) is formulated in liposomes. Liposomes are artificially prepared vesicles, composed primarily of lipid bilayers, that can be used as delivery vehicles for administering nutrients and pharmaceutical formulations. Liposomes can be of different sizes, including but not limited to multilamellar vesicles (MLVs), which can be hundreds of nanometers in diameter and contain a series of concentric bilayers separated by narrow aqueous compartments, small unilamellar vesicles (SUVs), which are less than 50 nm in diameter, and large unilamellar vesicles (LUVs), which are between 50-500 nm in diameter. Liposome designs can include, but are not limited to, opsonins or ligands to improve attachment of liposomes to unhealthy tissues or to activate events such as, but not limited to, endocytosis. Liposomes may contain low or high pH to improve delivery of pharmaceutical formulations.

[0074] The formation of liposomes may depend on physicochemical properties such as, but not limited to, the encapsulated pharmaceutical agent and liposomal components, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the encapsulated substance and its potential toxicity, additional processes involved during application and / or delivery of the vesicles, optimized size, polydispersity and shelf life of the vesicles for the intended application, and the ability to produce large quantities of the liposomal product with batch-to-batch reproducibility and safety and efficiency. As non-limiting examples, liposomes, such as synthetic membrane vesicles, may be prepared by the methods, apparatus, and devices described in U.S. Patent Publications US20130177638, US20130177637, US20130177636, US20130177635, US20130177634, US20130177633, US20130183375, US20130183373, and US20130183372; each of which is incorporated herein by reference.

[0075] In some embodiments, the immunogenic compositions (e.g., vaccine compositions) described herein can include liposomes formed from, but are not limited to, 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, WA), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLIN-KC2-DMA), and MC3 (US20100324120; incorporated herein by reference), and liposomes that can deliver small molecule agents, such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, PA). In some embodiments, the antigen and / or adjuvant system may be formulated in a water-in-oil emulsion comprising a continuous hydrophobic phase in which a hydrophilic phase is dispersed. As a non-limiting example, the emulsion may be prepared by the method described in International Publication WO201087791, the contents of which are incorporated herein by reference. Antigen, adjuvant system, and / or optional second adjuvant can be formulated using any of the methods described herein or known in the art, either individually or together.For example, antigen and adjuvant system can be formulated in one lipid nanoparticle or two separate lipid nanoparticles.In some embodiments, antigen, adjuvant system are formulated in the same aqueous solution or two separate aqueous solutions.

[0076] Another aspect of the present disclosure provides a method of inducing an immune response to a virus or viral antigen in a subject in need thereof, the method comprising administering to the subject an effective amount of the virus or viral antigen and an effective amount of an adjuvant system comprising a fungal polysaccharide (e.g., mannan). In some embodiments, the present disclosure provides a method of inducing an immune response to a betacoronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) or influenza virus (e.g., influenza A or B) or to an antigen of a betacoronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) or influenza virus (e.g., influenza A or B) in a subject in need thereof, the method comprising administering to the subject an effective amount of an antigen of a betacoronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) or influenza virus (e.g., influenza A or B) and an effective amount of an adjuvant system comprising a fungal polysaccharide (e.g., mannan). In some embodiments, the adjuvant system further comprises alum. In some embodiments, the fungal polysaccharide (e.g., mannan) is adsorbed to the alum.

[0077] In some embodiments, the adjuvant system (e.g., fungal polysaccharides such as mannan alone or fungal polysaccharides and alum) is administered separately from the viral antigen. In some embodiments, the adjuvant system (e.g., fungal polysaccharides such as mannan alone or fungal polysaccharides and alum) is administered prior to administration of the viral antigen. The adjuvant system (e.g., fungal polysaccharides such as mannan alone or fungal polysaccharides and alum) is administered after administration of the viral antigen. In some embodiments, the adjuvant system (e.g., fungal polysaccharides such as mannan alone or fungal polysaccharides and alum) and the viral antigen are administered simultaneously. In some embodiments, the adjuvant system (e.g., fungal polysaccharides such as mannan alone or fungal polysaccharides and alum) and the viral antigen are administered as a mixture.

[0078] A "subject" to which administration is contemplated refers to a human (i.e., male or female of any age, such as a pediatric subject (e.g., an infant, child, or adolescent) or an adult subject (e.g., a young adult, a middle-aged adult, or an elderly adult)), or a non-human animal. In some embodiments, the non-human animal is a mammal (e.g., a primate (e.g., a cynomolgus or rhesus monkey), a commercially relevant mammal (e.g., a cow, a pig, a horse, a sheep, a goat, a cat, or a dog), or a bird (e.g., a commercially relevant bird such as a chicken, a duck, a goose, or a turkey)). In some embodiments, the non-human animal is a fish, a reptile, or an amphibian. The non-human animal may be male or female and at any stage of development. The non-human animal may be a transgenic or genetically engineered animal. A "subject in need thereof" refers to a subject (e.g., a human subject or a non-human mammal) in need of treatment for infection with a virus, such as a betacoronavirus (e.g., a subject having MERS, SARS, or COVID-19) or an influenza virus (e.g., a subject having influenza), or a subject in need of reducing the risk of developing an infection with a virus, such as a betacoronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) or an influenza virus (e.g., influenza A virus or influenza B virus). In some embodiments, administering a viral antigen, such as an antigen of a betacoronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) or influenza virus (e.g., influenza A virus or influenza B virus), and an adjuvant system described herein (e.g., comprising a fungal polysaccharide, such as mannan, alone or with alum) to a subject having an infection caused by a virus, such as a betacoronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) or influenza virus (e.g., influenza A virus or influenza B virus), treats (i.e., has therapeutic use against) the disease (MERS, SARS, COVID-19, or influenza).In some embodiments, administering an antigen and adjuvant system described herein (e.g., comprising a fungal polysaccharide such as mannan alone or comprising a fungal polysaccharide and alum) to a subject at risk of developing an infection with a virus, such as a betacoronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) or an influenza virus (e.g., influenza A virus or influenza B virus) reduces the likelihood of the subject developing an infection (e.g., by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more) (prophylactic use).

[0079] In some embodiments, the subject is a human subject, e.g., a human neonate, infant, child, adult, or elderly. In some embodiments, the fungal polysaccharide used in the adjuvant system for enhancing the immune response to a virus, such as a betacoronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) or influenza virus (e.g., influenza A virus or influenza B virus) in a human subject is a mannan (e.g., mannan alone or in combination with alum). In some embodiments, the fungal polysaccharide used in the adjuvant system for enhancing the immune response to a virus, such as a betacoronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) or influenza virus (e.g., influenza A virus or influenza B virus) in a human subject is a β-glucan (e.g., β-glucan alone or in combination with alum). In some embodiments, the fungal polysaccharide used in the adjuvant system for enhancing an immune response to a virus, such as a betacoronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) or an influenza virus (e.g., influenza A virus or influenza B virus) in a human subject is a fungal polysaccharide isolated from Candida albicans (e.g., alone or in combination with alum).

[0080] In some embodiments, the human subject to which the adjuvant system designed to enhance the immune response to a virus (e.g., a betacoronavirus or an influenza virus) is administered is a newborn, or 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year old, 2 years old, 3 years old, 4 years old, 5 years old, 10 years old, 11 years old, 12 years old, 13 years old, 14 years old, 15 years old, 16 years old, 17 years old, 18 years old, 19 years old, 20 years old, 25 years old, 30 years old, 35 years old, 40 years old, 45 years old, 50 years old, 55 years old, 60 years old, or older than 65 years old. In some embodiments, the human subject is an adult (e.g., older than 18 years old). In some embodiments, the human subject is an elderly person (e.g., older than 60 years old). In some embodiments, the human subject is older than 65 years old. In some embodiments, a human subject receives (ie, is administered) one or more doses of a vaccine described herein.

[0081] In some embodiments, the human subject has an underdeveloped immune system (e.g., an infant or newborn), a weak immune system (elderly), or a compromised immune system. Immunocompromised subjects include subjects with primary or acquired immune deficiencies, such as subjects suffering from sepsis, HIV infection, and cancer, including, but not limited to, subjects undergoing chemotherapy and / or radiation therapy. In some embodiments, the human subject has an underlying disease that makes them more susceptible to infection caused by viruses, such as betacoronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) and / or influenza virus (e.g., influenza A virus or influenza B virus) infection. In some embodiments, the human subject is immunocompromised and has chronic lung disease, asthma, cardiovascular disease, cancer, obesity, diabetes, chronic kidney disease, and / or liver disease. In some embodiments, the subject is a companion animal (i.e., a pet or a service animal). The use of the immunogenic compositions (e.g., vaccine compositions) described herein in veterinary vaccines is also within the scope of this disclosure. As used herein, "companion animal" refers to pets and other livestock. Non-limiting examples of companion animals include dogs and cats; livestock such as horses, cows, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters. In some embodiments, the subject is a research animal. Non-limiting examples of research animals include rodents (e.g., ferrets, pigs, rats, mice, guinea pigs, and hamsters), rabbits, or non-human primates.

[0082] The immunogenic compositions (e.g., vaccine compositions) described herein, when administered, induce an immune response in a subject. In some embodiments, the immune response is a type 1 immune response, characterized by production and secretion of IgE antibodies from B cells, vasodilation, and extravasation of leukocytes. In some embodiments, the immune response is an innate immune response. In some embodiments, the immune response is an adaptive immune response, specific for the antigen in the composition or vaccine. In some embodiments, the immunogenic compositions (e.g., vaccine compositions) described herein activate B cell immunity. In some embodiments, the immunogenic compositions (e.g., vaccine compositions) induce the production of antibodies (immunoglobulins, e.g., IgE, IgG, IgA, IgM, or subtypes thereof, e.g., IgG1, IgG2, IgG3, and IgG4) against the antigen. In some embodiments, the immunogenic compositions (e.g., vaccine compositions) activate cytotoxic T cells specific for the antigen. In some embodiments, the immunogenic composition (eg, a vaccine composition) induces the production of cytokines (eg, chemokines, interferons, interleukins, etc.) by antigen-specific T cells.

[0083] In some embodiments, fungal polysaccharides used in adjuvant systems to enhance immune responses to viruses, such as betacoronaviruses (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) or influenza viruses (e.g., influenza A or B viruses) in human subjects, induce an innate immune response by activating pattern recognition receptors (PRRs) on the subject's immune cells. PRRs are important for innate immunity and act by detecting the presence of specific microbial antigens and signaling the production and secretion of inflammatory cytokines in response. In some embodiments, the PRR is a lectin (i.e., a receptor that binds to specific carbohydrates), such as a C-type lectin receptor (CLR). In some embodiments, the PRR is dendritic cell-associated C-type lectin 1 (Dectin 1). In some embodiments, the PRR is dendritic cell-associated C-type lectin 2 (Dectin 2).

[0084] In some embodiments, the adjuvant systems described herein (e.g., fungal polysaccharides alone or in combination with alum), when administered alone or in admixture with a viral antigen, result in the production of antibodies (immunoglobulins, e.g., IgE, IgG, IgA, IgM, or subtypes thereof (e.g., IgG1, IgG2, IgG3, and IgG4)) that target a broader (i.e., more extensive) range of epitopes of the antigen compared to the absence of the adjuvant system or the administration of the viral antigen alone. An "epitope" is defined as the amino acid sequence of an antigen that is specifically targeted by an antibody compared to other amino acid sequences of the antigen. In some embodiments, for example, the range of epitopes against which antigen-specific antibodies are produced may be increased by 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold in the presence of an adjuvant system compared to the absence of the adjuvant system or when the viral antigen is administered alone.

[0085] In some embodiments, the adjuvant systems described herein (e.g., fungal polysaccharides alone or in combination with alum) reduce pro-inflammatory cytokines (e.g., IFNγ) in a subject. + In some embodiments, the production of pro-inflammatory cytokines (e.g., IFNγ) is enhanced. + ) is increased by at least 20% in the presence of the adjuvant system compared to the absence of the adjuvant system or administration of the viral antigen alone. For example, the levels of pro-inflammatory cytokines (e.g., IFNγ + ) may be increased by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 5-fold, at least 10-fold, at least 100-fold, at least 1000-fold or more in the presence of the adjuvant system compared to the absence of the adjuvant system or administration of the viral antigen alone. In some embodiments, the levels of pro-inflammatory cytokines (e.g., IFNγ + ) is increased by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 1000-fold or more in the presence of the adjuvant system compared to the absence of the adjuvant system or when the viral antigen is administered alone.

[0086] In some embodiments, the adjuvant systems described herein (e.g., fungal polysaccharides alone or in combination with alum) enhance the innate immune response when administered alone or in a mixture with a viral antigen, as compared to the absence of the adjuvant system or administration of the viral antigen alone. In some embodiments, the adjuvant systems described herein (e.g., fungal polysaccharides alone or in combination with alum) activate peripheral blood mononuclear cells (PBMCs) from neonates or elderly subjects. In some embodiments, the number of activated PBMCs is increased by at least 20% in the presence of the adjuvant systems described herein (e.g., fungal polysaccharides alone or in combination with alum) as compared to the absence of the adjuvant system or administration of the viral antigen alone. For example, the number of activated PBMCs may be increased by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 5-fold, at least 10-fold, at least 1000-fold or more in the presence of an adjuvant system compared to the absence of the adjuvant system or administration of the viral antigen alone. In some embodiments, the number of activated PBMCs is increased by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold or more in the presence of an adjuvant system compared to the absence of the adjuvant system or administration of the viral antigen alone.

[0087] In some embodiments, the adjuvant system enhances innate immune memory (also called trained immunity). "Innate immune memory" confers heterologous immunity, providing broad protection against a variety of pathogens. In some embodiments, innate immune memory is increased by at least 20% in the presence of the adjuvant system compared to the absence of the adjuvant system or administration of the viral antigen alone. For example, innate immune memory is increased by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% at least 100%, at least 2-fold, at least 5-fold, at least 10-fold, at least 100-fold, at least 1000-fold or more in the presence of the adjuvant system compared to the absence of the adjuvant system or administration of the viral antigen alone. In some embodiments, the innate immune memory is increased by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 1000-fold or more in the presence of the adjuvant system compared to the absence of the adjuvant system or when the viral antigen is administered alone.

[0088] In some embodiments, the adjuvant system, when administered in a mixture with a viral antigen, enhances an anti-specific immune response to a viral antigen, such as an antigen of a betacoronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) or influenza virus (e.g., influenza A virus or influenza B virus), or an anti-specific immune response to a virus, such as a betacoronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) or influenza virus (e.g., influenza A virus or influenza B virus), compared to the absence of the adjuvant system or the administration of the viral antigen alone. In some embodiments, the adjuvant system enhances (e.g., by at least 20%) the generation of antigen-specific antibody titers in a subject compared to the absence of the adjuvant system or the administration of the viral antigen alone. For example, the adjuvant system may enhance the production of antigen-specific antibody titers in a subject by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 5-fold, at least 10-fold, at least 1000-fold or more compared to the absence of the adjuvant system or administration of the viral antigen alone. In some embodiments, the adjuvant system enhances the production of antigen-specific antibody titers by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 100-fold or more compared to the absence of the adjuvant system or administration of the viral antigen alone. Those skilled in the art are familiar with methods for assessing the level of antibody titers, for example, by ELISA.

[0089] In some embodiments, the adjuvant system polarizes the innate and adaptive immune response by shaping the pattern of cytokine and / or chemokine responses toward T helper 1 (Th1) immunity, which is important for host defense against intracellular pathogens, hi some embodiments, the adjuvant system polarizes the innate immune response toward T follicular helper (Tfh) cell immunity. In some embodiments, the adjuvant system extends the efficacy of the vaccine in a subject (e.g., by at least 20%) compared to the absence of the adjuvant system or administration of the viral antigen alone. For example, the adjuvant system may extend the efficacy of the vaccine in a subject by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 5-fold, at least 10-fold, at least 1000-fold or more compared to the absence of the adjuvant system or administration of the viral antigen alone. In some embodiments, the adjuvant system extends the effectiveness of the vaccine by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 1000-fold or more in the presence of the adjuvant system compared to the absence of the adjuvant system or when the viral antigen is administered alone.

[0090] In some embodiments, the adjuvant system increases (accelerates) the rate of immune response compared to the absence of the adjuvant system or the administration of the viral antigen alone. For example, the adjuvant system can increase the rate of immune response in a subject by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 5-fold, at least 10-fold, at least 1000-fold or more compared to the absence of the adjuvant system or the administration of the viral antigen alone. In some embodiments, the adjuvant system increases the rate of immune response by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold or more in the presence of the adjuvant system compared to the absence of the adjuvant system or the administration of the viral antigen alone. "Increasing the rate of immune response" means reducing the time required for a subject's immune system to respond to an invading virus, such as a betacoronavirus (e.g., MERS-CoV, SARS-CoV-1, SARS-CoV-2) or influenza virus (e.g., influenza A virus or influenza B virus), or a viral antigen, such as a betacoronavirus antigen or an influenza virus antigen.

[0091] In some embodiments, the antigen generates the same level of immune response to a viral antigen, such as an antigen of a betacoronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) or influenza virus (e.g., influenza A virus or influenza B virus), at a lower dose in the presence of the adjuvant system compared to the absence of the adjuvant system or when the viral antigen is administered alone. In some embodiments, the amount of viral antigen required to generate the same level of immune response is reduced by at least 20% in the presence of the adjuvant system compared to the absence of the adjuvant system or when the viral antigen is administered alone. For example, the amount of antigen required to generate the same level of immune response can be reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more in the presence of the adjuvant system compared to the absence of the adjuvant system or when the viral antigen is administered alone. In some embodiments, the amount of antigen required to generate the same level of immune response is reduced by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more in the presence of the adjuvant system compared to the absence of the adjuvant system or when the viral antigen is administered alone.

[0092] Prophylactic or therapeutic uses of the adjuvant systems or immunogenic compositions (e.g., vaccine compositions) described herein are also within the scope of the present disclosure. In some embodiments, the compositions or immunogenic compositions (e.g., vaccine compositions) described herein are used in a method of vaccinating a subject by prophylactically administering to the subject an effective amount of the compositions or immunogenic compositions (e.g., vaccine compositions) described herein. "Vaccinating a subject" refers to the process of administering an immunogen, typically an antigen formulated in a vaccine, to a subject in an amount effective to increase or activate an immune response against a viral antigen, such as a betacoronavirus antigen (e.g., MERS-COV, SARS-COV-1, SARS-COV-2) or influenza virus (e.g., influenza A virus or influenza B virus) antigen, and thus against a virus (e.g., betacoronavirus (e.g., MERS-COV, SARS-COV-1, SARS-COV-2) or influenza virus (e.g., influenza A virus or influenza B virus)). In some embodiments, the term "vaccinating a subject" does not require the creation of complete immunity against the virus. In some embodiments, the term "vaccinating a subject" encompasses a clinically favorable enhancement of an immune response against a viral antigen or pathogen. Methods of immunization, including formulation of an immunogenic composition (e.g., a vaccine composition) and selection of a dose, route of administration, and administration schedule (e.g., an initial dose and one or more booster doses), are well known in the art. In some embodiments, vaccinating a subject reduces the risk of developing a viral infection, such as a betacoronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) or influenza virus (e.g., influenza A virus or influenza B virus) infection, and a disease resulting from a viral infection, such as a disease caused by a betacoronavirus infection (e.g., MERS, SARS, and / or COVID-19) or influenza virus infection (e.g., influenza).

[0093] In some embodiments, the immunogenic compositions (e.g., vaccine compositions) described herein are formulated for administration to a subject. In some embodiments, the composition or immunogenic composition (e.g., vaccine composition) further comprises a pharma- ceutically acceptable carrier. The phrase "pharma-ceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, and are commensurate with a reasonable benefit / risk ratio. The phrase "pharma-ceutically acceptable carrier" refers to a pharma-ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that is involved in carrying or transporting a subject agent from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other components of the formulation and non-invasive to the patient's tissues (e.g., physiological compatibility, sterility, physiological pH, etc.). The term "carrier" refers to an organic or inorganic component, natural or synthetic, with which an active ingredient is combined to facilitate application. The components of the compositions or immunogenic compositions (e.g., vaccine compositions) described herein also can be mixed with the molecules of the present disclosure, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.Some examples of substances which may function as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol. (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, colorants, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulation.

[0094] The immunogenic compositions (e.g., vaccine compositions) described herein may be conveniently provided in unit dosage form and prepared by any of the methods well known in the art of pharmacy. The term "unit dose," as used with respect to the compositions or immunogenic compositions (e.g., vaccine compositions) described herein of the present disclosure, refers to physically discrete units suitable as unitary dosages for subjects, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent, i.e., vehicle carrier or vehicle. The formulation of the compositions or immunogenic compositions (e.g., vaccine compositions) described herein may depend on the route of administration. Injectable preparations suitable for parenteral administration or intratumoral, peritumoral, intralesional or perilesional administration include, for example, sterile injectable aqueous or oily suspensions, which can be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may also be sterile injectable solutions, suspensions or emulsions in non-toxic parenterally acceptable diluents or solvents, such as, for example, solutions in 1,3 propanediol or 1,3 butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, USP and isotonic sodium chloride solution. Additionally, sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, any non-irritating fixed oil can be used, including synthetic mono- or diglycerides. Additionally, fatty acids such as oleic acid are used in the preparation of injectables. Sterilization of the injectable preparations can be accomplished, for example, by filtration through a bacterial-retaining filter, 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.

[0095] For topical administration, the compositions or immunogenic compositions (e.g., vaccine compositions) described herein can be formulated into ointments, salves, gels, or creams, as is generally known in the art. Topical administration can utilize transdermal delivery systems, which are well known in the art. One example is a skin patch. Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, etc., each containing a predetermined amount of the anti-inflammatory agent. Other compositions include suspensions in aqueous liquids or non-aqueous liquids, such as a syrup, elixir or emulsion.

[0096] Other delivery systems may include time-release, delayed release, or sustained release delivery systems. Such systems may avoid repeated administration of anti-inflammatory agents, and may increase the convenience of the subject and the physician. Many types of release delivery systems are available and known to those skilled in the art. These include polymer-based systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the aforementioned polymers containing drugs are described, for example, in U.S. Pat. No. 5,075,109. Delivery systems also include non-polymeric systems such as: lipids, including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats, such as monoglycerides, diglycerides, and triglycerides; hydrogel release systems; silastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants, and the like. Specific examples include, but are not limited to, (a) erosive systems in which the anti-inflammatory agent is contained in a matrix, such as those described in U.S. Patent Nos. 4,452,775, 4,667,014, 4,748,034 and 5,239,660, and (b) diffusion systems in which the active ingredient permeates through a polymer at a controlled rate, such as those described in U.S. Patent Nos. 3,832,253 and 3,854,480. Additionally, pump-based hardware delivery systems can also be used, some of which are suitable for implantation.

[0097] The use of long-term sustained release implants may be particularly suitable for treating chronic conditions.Long-term release as used herein means that the implant is constructed and arranged to deliver therapeutic levels of active ingredient for at least 30 days, preferably 60 days.Long-term sustained release implants are well known to those skilled in the art and include some of the release systems described above. In some embodiments, the immunogenic compositions (e.g., vaccine compositions) described herein used for therapeutic administration must be sterile. Sterility is readily achieved by filtration through a sterile filtration membrane (e.g., a 0.2 micron membrane). Alternatively, a preservative can be used to prevent the growth or action of microorganisms. A variety of preservatives are well known, including, for example, phenol and ascorbic acid. The cyclic Psap peptides and / or compositions or immunogenic compositions (e.g., vaccine compositions) described herein will typically be stored in lyophilized form, where they are highly stable against thermal and oxidative denaturation, or as an aqueous solution. The pH of the preparation is typically about 6-8, although higher or lower pH values ​​may be appropriate in some cases. The chimeric constructs of the present disclosure can be used as vaccines by conjugation to a soluble immunogenic carrier molecule. Suitable carrier molecules include proteins, including keyhole limpet hemocyanin, which is a preferred carrier protein. The chimeric constructs can be conjugated to the carrier molecule using standard methods. (Hancock et al., “Synthesis of Peptides for Use as Immunogens,” in Methods in Molecular Biology: Immunochemical Protocols, Manson (ed.), pages 23-32 (Humana Press 1992)).

[0098] In some embodiments, the present disclosure contemplates an immunogenic composition (e.g., a vaccine composition) that includes a pharma- ceutically acceptable injectable vehicle. The vaccines of the present disclosure may be administered in the form of an injectable solution or suspension in a conventional vehicle, with or without other standard carriers. Additional carriers may be selected from agents that enhance the overall immune response during the immunization procedure. Liposomes have been proposed as suitable carriers. Insoluble salts of aluminum, namely aluminum phosphate or aluminum hydroxide, have been utilized as carriers in routine clinical applications in humans. Water-soluble carriers such as polynucleotides and polyelectrolytes, and muramyl dipeptide have been used. The preparation of the injectable vaccine of the present disclosure includes mixing the immunogenic composition (e.g., vaccine composition) with muramyl dipeptide or other carrier. The resulting mixture can be emulsified in mannide monooleate / squalene or squalane vehicle. Four parts by volume of squalene and / or squalane are used per part by volume of mannide monooleate. Methods of formulating immunogenic compositions (e.g., vaccine compositions) are well known to those skilled in the art. (Rola, Immunizing Agents and Diagnostic Skin Antigens. In: Remington's Pharmaceutical Sciences,18th Edition, Gennaro (ed.), (Mack Publishing Company 1990) pages 1389-1404).

[0099] Additional pharmaceutical carriers can be used to control the duration of action of the vaccine in therapeutic applications. Controlled release formulations can be prepared by complexing or adsorbing the chimeric construct with a polymer. For example, biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of polyanhydride copolymers of stearic acid dimer and sebacic acid. (Sherwood et al. (1992) Bio / Technology 10: 1446). The release rate of the chimeric construct from such matrices depends on the molecular weight of the construct, the amount of the construct within the matrix, and the size of the dispersed particles. (Saltzman et al. (1989) Biophys. J. 55: 163; Sherwood et al, supra; Ansel et al. Pharmaceutical Dosage Forms and Drug Delivery Systems, 5th Edition (Lea & Febiger 1990); and Gennaro (ed.), Remington's Pharmaceutical Sciences, 18th Edition (Mack Publishing Company 1990). Chimeric constructs can also be conjugated to polyethylene glycol (PEG) to improve stability and extend bioavailability time (e.g., Katre et al.; U.S. Pat. No. 4,766,106).

[0100] The terms "treatment", "treat" and "treating" refer to reversing, alleviating, delaying onset or inhibiting progression of the diseases described herein. In some embodiments, treatment can be administered after one or more signs or symptoms of the disease have developed or been observed. In other embodiments, treatment can be administered in the absence of signs or symptoms of the disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms and / or exposure to a pathogen). Treatment can continue after symptoms have resolved, e.g., to delay or prevent recurrence. Prophylactic treatment refers to treatment of a subject who does not have or has never had the disease, but is at risk of developing the disease, or a subject who has had the disease but does not now have it, but is at risk of relapsing. In some embodiments, the subject is at higher risk of developing the disease or at higher risk of relapsing the disease than the average healthy member of the population.

[0101] An "effective amount" of a composition described herein refers to an amount sufficient to induce a desired biological response. The effective amount of a composition described herein may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the condition to be treated, the mode of administration, the age and health of the subject. In some embodiments, the effective amount is a therapeutically effective amount. In some embodiments, the effective amount is a prophylactic treatment. In some embodiments, the effective amount is the amount in a single dose of a compound described herein. In some embodiments, the effective amount is the total amount in multiple doses of a compound described herein. When referring to an effective amount of a composition herein, it means that the amount is prophylactically and / or therapeutically effective depending on the subject and / or the disease to be treated. The determination of an effective amount or dosage is within the capabilities of one of ordinary skill in the art.

[0102] The terms "administer", "administering" or "administration" refer to implanting, absorbing, ingesting, injecting, inhaling or otherwise introducing a compound or composition thereof described herein into or on a subject. The compositions of the immunogenic compositions (e.g., vaccine compositions) described herein can be administered systemically (e.g., via intravenous injection) or locally (e.g., via local injection). In some embodiments, the compositions of the immunogenic compositions (e.g., vaccine compositions) described herein are administered orally, intravenously, topically, intranasally, or sublingually. Parenteral administration is also contemplated. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. In some embodiments, the compositions are administered prophylactically.

[0103] In some embodiments, the composition or immunogenic composition (e.g., vaccine composition) is administered one or more times (e.g., 2, 3, 4, 5 or more times). If administered multiple times, the administrations may occur over a period of time (e.g., 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5 years, 10 years, or more than 10 years). In some embodiments, the composition or immunogenic composition (e.g., a vaccine composition) is administered twice (e.g., on days 0 and 7, 0 and 14, 0 and 21, 0 and 28, 0 and 60, 0 and 90, 0 and 120, 0 and 150, 0 and 180, 0 and 3 months later, 0 and 6 months later, 0 and 9 months later, 0 and 12 months later, 0 and 18 months later, 0 and 2 years later, 0 and 5 years later, or 0 and 10 years later). EXAMPLES

[0104] Introduction The dialogue between the innate and adaptive immune systems is a central paradigm in modern immunology and is important for defense against infections, as well as in the pathogenesis of autoimmune, allergic and inflammatory diseases (Banchereau and Steinman, 1998;Iwasaki and Medzhitov, 2004;Janeway and Medzhitov, 2002;Matzinger, 1994). According to current models, infection and / or damage of peripheral tissues triggers the activation and migration of innate immune phagocytes to draining lymph nodes (dLNs), where phagocytes initiate antigen-dependent adaptive immune responses. Alternatively, innate stimuli, or microbes with specific physical properties (e.g., diameters in the nanometer range), can directly enter dLNs and activate LN-resident innate and adaptive immune cells (Bachmann and Jennings, 2010;Irvine et al., 2020). dLNs have been thoroughly scrutinized for their ability to host adaptive immune responses, with recent reports highlighting that antigen-dependent adaptive immune responses precede and are supported by antigen-independent lymph node innate responses (LIR) (Acton et al., 2014;Coccia et al., 2017;De Giovanni et al., 2020;Didierlaurent et al., 2014;Kastenmuller et al., 2012;Lian et al., 2020;Lynn et al., 2015;Martin-Fontecha et al., 2004;Soderberg et al., 2005;Wong et al., 2019;Wong et al., 2018;Xu et al., 2015b)). Although incompletely characterized, LIR is composed of at least two components that are critical for the development of an effective adaptive immune response: the expansion of antigen-independent LNs and the establishment of a pro-inflammatory environment ( Acton and Reis e Sousa, 2016 ; Grant et al., 2020 ).It remains enigmatic whether and how LIR differs when it is driven by migration of phagocytes from the periphery or when it is governed by direct targeting of LN-resident innate immune cells.

[0105] Innate immune cells recognize pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs) (Janeway and Medzhitov, 2002). Several classes of PRRs have been identified to date, namely, Toll-like receptors (TLRs), RIG-I-like receptors, cytosolic DNA sensors, NOD-like receptors, and C-type lectin receptors (CLRs) (Brubaker et al., 2015). Activation of PRRs within innate immune cells is critical for the induction of inflammation and the subsequent development of adaptive immune responses. For this reason, PRR targeting has been exploited in vaccine development (O'Hagan et al., 2020). Adjuvant formulations containing TLR4 and TLR9 ligands (monophosphoryl lipid A (MPL) and CpG, respectively) promote potent and long-lasting adaptive immune responses and have become adjuvant components of FDA-approved vaccines. Several additional PRR ligands have been tested in preclinical models of immunization and are currently under evaluation in clinical trials ( O'Hagan et al., 2020 ). Among PRRs, the biology of CLRs and their potential as vaccine adjuvant targets is less explored.

[0106] The CLR superfamily is composed of hundreds of proteins that share carbohydrate-binding domains and play key roles in the development of innate and adaptive immune responses against fungal infections through the recognition of cell wall polysaccharides (Borriello et al., 2020;Brown et al., 2018). Preclinical and clinical data support the concept of targeting the CLR Mincle to induce potent cellular and humoral immunity upon vaccination (Pedersen et al., 2018). Furthermore, there is evidence that activation of the CLRs Dectin 1 (Clec7a) and Dectin 2 (Clec4n) by their respective polysaccharide ligands β-glucan and mannan promotes antigen-specific adaptive immunity in experimental models of immunization (Petrovsky and Cooper, 2011). However, the mechanism of action of dectins remains largely ignored. Fungal polysaccharides such as β-glucan and mannan differ by their chemical structure and physical form (e.g., dimensions and solubility), further complicating mechanistic analysis. The physical properties of PRR ligands not only influence their localization (peripheral retention vs. LN egress), but also their recognition and activation by innate immune cells. Dectin-1 and Dectin-2 bind to fungal polysaccharides in soluble and insoluble forms, but only the latter induce efficient receptor clustering and activation (Goodridge et al., 2011;Zhu et al., 2013). As a result, it is widely believed that only particulate polysaccharides are capable of immune stimulation.

[0107] In this study, we revisited this paradigm and found that soluble mannan is largely inactive at the site of injection in vitro and in vivo, but its diameter allows it to be transported to lymph nodes where it induces a robust LIR. Notably, the observed response circumvents the need for dendritic cell migration from the periphery to the dLN. We also demonstrated that further modulation of the physical properties of mannan results in novel immunological properties, such as full activation of innate immune cells in vitro and simultaneous targeting of the periphery and LNs. When tested as an adjuvant system formulated with viral glycoprotein antigens (influenza A virus (IAV) hemagglutinin or SARS-CoV-2 spike), mannan enabled the induction of neutralizing antibodies with broad epitope specificity and protected against infection by IAV or SARS-CoV-2, respectively. Overall, the data demonstrate that the quality of LN innate and adaptive immune responses can be tuned by modulating the physical properties of mannan, supporting important implications for adjuvant design and vaccine development.

[0108] result Mannan induces an LN-restricted IFN signature that promotes LN expansion Dectins 1 and 2 bind to the fungal cell wall polysaccharides β-glucan and mannan, both of which activate the kinase Syk, either directly (Dectin 1) or via the FcRγ chain (Dectin 2) (Borriello et al., 2020;Lionakis et al., 2017;Netea et al., 2008). Using preparations of β-glucan and mannan isolated from Candida albicans; each exhibits a distinct physical morphology, insoluble and approximately 500 nm in diameter, and soluble and approximately 20 nm in diameter (Figure 9). In line with current models of dectin activation (Goodridge et al., 2011;Zhu et al., 2013), particulate β-glucan, but not soluble mannan, was able to induce cytokine production and expression of costimulatory molecules by GM-CSF-differentiated bone marrow-derived phagocytes (Figure 10A). As expected, β-glucan signaling required Dectin-1 (Figure 10B). In contrast to soluble mannan, immobilization of mannan on microbeads resulted in Dectin-2 and FcRγ-dependent phagocyte activation (Figure 10B; lipopolysaccharide (LPS) and curdlan were used as controls). Thus, β-glucan induced the formation of skin abscesses and lesions upon intradermal injection in vivo, whereas mannan did not induce signs of skin inflammation (Figure 1A). These results were confirmed by transcriptomic analysis of saline-, β-glucan- and mannan-injected skin samples (Figure 1B). Pathway analysis of clusters of differentially expressed genes (DEGs) upregulated by β-glucan revealed enrichment of pro-inflammatory and type II interferon (IFN) pathways, consistent with the innate immune response elicited by C. albicans skin infection (Figure 11). (Santus et al., 2017). Although soluble mannan did not induce skin inflammation, these fungal ligands induced an expansion of dLNs and an increase in lymphocytes as early as 6 h postinjection (hpi) that persisted up to 24 hpi (Figures 1C and 12A-12C) and was dependent on the recruitment of circulating leukocytes (Figure 1D).

[0109] β-glucan also induced LN expansion, but only at 24 h.pi (Figs. 1C, 12A-12C). Both mannan and β-glucan increased myeloid cell numbers in dLNs (Figs. 12D-12G), with β-glucan preferentially increasing neutrophil numbers (Fig. 12D), likely due to neutrophil egress from the skin injection site. However, only mannan was able to induce myeloid cell activation, as measured by increased CD86 expression (Fig. 12H). Thus, β-glucan and mannan differ in their ability to induce local and dLN inflammatory responses, with the latter inducing LIR at a faster rate than the former. Given the kinetics of the response to mannan and its diameter (compatible with lymphatic drainage), it was reasoned that mannan may activate LN-intrinsic circuits that ultimately lead to dLN expansion. Consistent with this hypothesis, mannan rapidly accumulated in the dLN (within 6 h.pi) (Figure 1E), and migration of immune cells from the periphery (i.e., skin) to the dLN was abolished in Ccr7 - / - Even in mice, mannan was able to induce dLN expansion (Figure 1F) (Ohl et al., 2004). Taken together, these data demonstrate that mannan activates a potent LIR in the absence of peripheral (skin) inflammation and / or migration of phagocytes to dLN. Transcriptome analysis of dLN isolated from mice injected with saline, β-glucan, and mannan was performed to further characterize the molecular events associated with LN expansion. A completely opposite profile was found when compared to skin, with mannan eliciting an earlier and more pronounced transcriptional response compared to β-glucan (Figure 1G). Pathway analysis revealed that the cluster of DEGs upregulated by mannan was enriched in type I and type II IFN pathways (Figure 1H); this was confirmed by the expression levels of the top 50 genes upregulated in mannan-treated dLN compared to controls (Figure 1I).

[0110] The presence of type II IFN (IFNγ)-producing cells in mannan-treated dLN was confirmed by flow cytometry (Figure 12I). The majority of IFNγ-producing cells were CD8 +T cells and NK cells (Figure 12J), with NK cells found to express higher levels of IFNγ (Figure 12K). To assess whether NK cells are the main source of IFNγ in mannan-treated dLN, we removed NK cells and observed impaired mannan-induced Ifng expression (Figure 12L). cDC1s produce cytokines that induce NK cell activation and IFNγ production (Cancel et al., 2019). Thus, they may contribute to mannan-induced LIR. To evaluate this hypothesis, we investigated the effect of cDC1 lacking Batf3 - / - We used mice and did not observe any impairment of mannan-induced Ifng expression (Fig. S12M). Of note, depletion of NK cells only partially affected LN expansion and cell development (Fig. S12N). Consistent with this, blockade of IFNγ only partially reduced LN expansion after mannan injection (Fig. S1J). Furthermore, simultaneous blockade of type I and type II IFN (Ifnar) reduced LN expansion after mannan injection (Fig. S1J). - / - Using mice and an anti-IFNγ blocking antibody, mannan-induced LN expansion and ISG induction were prevented (Figure 1J). This mechanism was not limited to mannan, as LN expansion and ISG induction induced by Lipo-CpG, a well-characterized LN-targeting TLR9 ligand (Liu et al., 2014), was also inhibited by Ifnar - / - Ifngr - / -The results thus demonstrate that soluble mannan isolated from C. albicans induces a LIR distinct from that induced by β-glucan, i.e., in the induction of ISGs. To further establish whether these differences are due to the different physical forms of fungal polysaccharides, responses to whole glucan particles (WGP) were assessed in dispersed (D) or soluble (S) forms, which have been characterized as dectin-1 agonists and antagonists, respectively (Goodridge et al., 2011). Consistent with the pattern observed with mannan, WGP-S did not induce skin inflammation but induced LN expansion and ISG expression (Figures 13A-13B). Taken together, these results support a model in which the physical form of fungal polysaccharides drives an LIR characterized by dLN expansion and ISG induction.

[0111] Mannan-induced LIR requires CD169+ sinus macrophages expressing Dectin-2 Dectin-2 is the main receptor for mannan (Borriello et al., 2020; Lionakis et al., 2017; Netea et al., 2008), and we found that Dectin-2 and FcRγ were also required for mannan-induced LIR, consistent with our in vitro data (Figure 2A). Dectin-2 is mainly expressed by myeloid cells (Taylor et al., 2005). Therefore, we used fluorescently labeled mannan to identify non-lymphoid (CD3 / CD19 / NK1.1) cells in dLNs. - ) compartment to identify the cellular targets of mannan. The majority of mannan-containing cells express CD45 +Mannan-containing cells were the most abundant cells (Figure 2B). Imaging cytometry analysis confirmed that these cells internalized mannan (Figure 2C). Furthermore, confocal microscopy analysis of mannan-containing dLNs at 1 h.pi showed colocalization of phospho-Syk and mannan (Figure 2D), indicative of Dectin-2 / FcRγ-mediated activation (Borriello et al., 2020). Accordingly, mannan-containing cells showed the highest levels of expression of CD86, a marker of innate immune cell activation, in an FcRγ-dependent manner (Figure 2E). Further characterization of the phenotype of mannan-containing cells revealed that more than 50% were Ly6G - (CD11b + Ly6C + ) - CD11c + cells, whereas the less abundant cell subset is neutrophils (CD11b + Ly6G + ) and monocytes / monocyte-derived cells (MoCsLy6G - CD11b + Ly6C + ) (Figure 2F). Interestingly, diphtheria toxin (DT)-mediated CD11c expression in CD11c-DT receptor (DTR) mice was + Using cell depletion, mannan-induced LIR was abolished (Figure 2G). On the other hand, mannan-induced LIR was not abolished by Ccr2 - / - In mice, where monocyte egress from the bone marrow is impaired (Fig. 2H) or antibody-mediated neutrophil depletion (Fig. 2I), there was no effect. + The cells could be further differentiated based on their expression of CD11b (Figure 2F). Dectin-2 is important for mannan-induced LIR, so we compared its expression with that of steady-state CD11b - CD11c + Cells and CD11b + CD11c + Dectin-2 was mainly expressed on CD11b cells. + CD11c + cells (Figure 2J), and CD11b + CD11c + Dectin 2 +The majority of cells expressed the subcapsular and medullary sinus macrophage marker CD169 (Fig. 2K). Confocal microscopy analysis confirmed the colocalization of CD169 and Dectin-2 on cells lining the LN subcapsular sinus (Fig. 2L). Furthermore, DT-mediated CD169 upregulation in CD169-DTR mice was not observed in the control mice. + Depletion of CD11c-DTR cells phenocopied the results obtained in CD11c-DTR mice and completely abolished mannan-induced LIR (Figure 2M). These results support the role of CD169 as a sentinel for lymphoid material. + demonstrating the role of sinus macrophages ( Moran et al., 2019 ).

[0112] Activation of the non-canonical NF-κB subunit RelB regulates mannan-induced LIR Evidence of Syk phosphorylation as early as 1 h.pi in LN-resident mannan-containing cells points to activation of signaling pathways downstream of Dectin-2. A key step in the Dectin receptor-Syk pathway is activation of CARD9, which regulates the activity of several signaling molecules and transcription factors, including MAPK and canonical NF-κB (Borriello et al., 2020). Thus, Card9 - / - We used mice to characterize the molecular events associated with mannan-induced LIR. Surprisingly, mannan-induced LN expansion was consistent with wild-type (WT) mice and Card9 mice. - / - Consistent with the relevance of the IFN signature in driving LIR, ISG induction was comparable between WT and Clec4n mice (Figure 3A). - / - and Fcer1g - / - In mice, the expression was completely abolished (Fig. 2A). - / - In mice, most of the genes were maintained (Fig. 3A). In particular, type I IFN-dependent genes, such as Card9, - / -CARD9-dependent and -independent activation of mannan-containing cells was not altered in WT, whereas type II IFN-dependent ISGs were significantly reduced compared to WT mice but were still partially induced (Figure 3A). - / - , and Card9 - / - Mannan-containing CD11b isolated from mice + CD11c + Targeted transcriptome analysis of cells was performed. Several genes were detected in WT mice or Card9 mice. - / - Mouse and Fcer1g - / - Differentially expressed between mice, but not between WT mice and Card9 - / - Cells isolated from WT and Fcer1g mice displayed strikingly similar transcriptomes (Figure 3B). Pathway enrichment analysis revealed that WT and Fcer1g - / - It was revealed that DEGs between cells isolated from CD11c are displayed in the TNF / NF-κB, type I and type II IFN pathways (Figure 14). To explain this phenotype, the model inferred that the Dectin2-Syk axis may activate a CARD9-independent pathway. Notably, Syk activates the kinase NIK, which in turn leads to CARD9-independent activation of the non-canonical NF-κB transcription factor RelB (Gringhuis et al., 2009;Xu et al., 2018). Thus, CD11c + Mice with a conditional deletion of RelB in the compartment (Cd11c cre Relb fl / fl In particular, mannan-induced LN expansion and expression of both type I and type II IFN-dependent ISGs were significantly higher than in the control (Relb fl / fl To test whether RelB requirement is mannan-specific, we used Lipo-CpG and found that Lipo-CpG-induced LN expansion and type I IFN-dependent ISG expression were significantly reduced in Cd11c mice compared with Cd11c mice (Figure 3C). cre Relb fl / flWe found that RelB was impaired in mice (Figure 3D). These results support a model in which RelB regulates LN expansion and type I IFN-dependent ISG expression induced by LN-targeted stimuli and further cooperates with a CARD9-dependent pathway to regulate mannan-induced type II IFN-dependent ISG expression.

[0113] Molecular pathways required for mannan-induced LIR regulate the magnitude of mannan adjuvant activity Evidence exists suggesting that LIRs are essential for controlling the development of adaptive immune responses (Coccia et al., 2017;De Giovanni et al., 2020;Lynn et al., 2015;Martin-Fontecha et al., 2004;Soderberg et al., 2005). It was reasoned that mannan-induced lymphocyte expansion and IFN signature may facilitate T cells meeting their cognate antigen and its efficient presentation by the innate immune compartment, thereby improving adaptive immune responses. CFSE-labeled OVA-specific OT-II CD4 + A model of adoptive transfer of T cells was used to evaluate the regulation of T cell proliferation (Figures 4A-4H). Combining mannan with OVA significantly increased the number of OT-II and OT-I cells in the dLN compared to mice injected with saline or OVA alone (Figures 4A and 4E). This increase was consistent with the uptake of OT-II CD4 + This may be due to increased T cell recruitment and more efficient antigen presentation / co-stimulation by LN-resident innate immune cells. Indeed, a lower percentage of non-proliferating T cells and a higher percentage of T cells that had undergone 6 or 7 divisions were detected in mice treated with OVA and mannan compared to OVA alone (Figures 4B and 4F). Notably, the effect of mannan on T cell proliferation was downregulated by Fcer1g. - / - It disappeared in mice, but Card9 - / -The effect of mannan on LIR was only attenuated in mice (Figures 4C, 4D, 4G, and 4H). These results indicate that the pathways required for mannan-induced LIR are also important for the adjuvant activity of mannan on antigen-specific adaptive immune responses.

[0114] Mannan formulated with aluminum hydroxide acquires novel physical properties predictive of immunological function To further modulate the physical properties of mannan, it was reasoned that the presence of phosphate groups in mannan preparations may facilitate their adsorption to aluminum hydroxide (AH), as has been shown for other molecules (Morefield et al., 2005;Moyer et al., 2020). To characterize the mannan-AH blends, 1 H-NMR analysis was performed on the supernatant collected after adsorption of mannan to AH. It was found that approximately 40% of the mannan was adsorbed to AH, while the remaining portion remained soluble (Figure 15A). It was also quantified that in the formulations used in these experiments, AH was bound to approximately twice its mass of mannan.

[0115] In vitro experiments with GM-CSF-differentiated bone marrow-derived phagocytes revealed that formulation of mannan with AH (AH / mann) completely altered the pro-inflammatory activity compared to soluble mannan. Mannan formulated with AH (alumOH / mann) induced significant costimulatory molecule expression and cytokine production (Figure 5A). AH / mann, but not AH or mannan alone, induced significant TNF and IL-2 cytokine production in a dectin-2- and CARD9-dependent manner (Figures 15B and 15C). AH / mann also induced costimulatory molecule expression in a dectin-2-dependent but CARD9-independent manner (Figures 15D and 15E). Finally, both mannan and AH / mann, but not AH alone, induced ISG expression in a dectin-2-dependent but CARD9-independent manner, except for CXCL1, which was significantly upregulated only in response to AH / mann in WT cells (Figures 15F-15I).

[0116] AH / mann formulations, when injected into mice, resulted in a phenotype similar to the combined action of glucan and mannan. Notably, AH / mann induced skin inflammation (a phenotype observed with glucan but not soluble mannan) (Figure 5B), but was also excreted in LNs in a CCR7-independent manner (Figure 5C). Assessment of LN weight over time revealed that AH / mann induced higher cumulative LN expansion compared to AH, mannan or β-glucan (Figure 5D). Furthermore, mannan and AH / mann induced comparable ISG expression in dLNs at 24 h.pi. (Figure 5E). As expected, β-glucan induced LN expansion but was largely excluded from LNs and did not induce ISG expression (Figures 5C-5E). Furthermore, Ifnar treated with anti-IFNγ blocking antibody reduced LN expansion, but was largely excluded from LNs and did not induce ISG expression (Figures 5C-5E). - / - In mice, LN expansion induced by AH / mann was impaired, but not by β-glucan (Figure 5F). Similar results were obtained when IFN production was temporarily blocked by treating wild-type mice with anti-IFNAR and anti-IFNγ blocking antibodies (Figure 5G). Overall, these results are compatible with a model in which the particulate fraction (mannan adsorbed to AH) promotes skin inflammation, while the soluble fraction (unbound mannan) is excreted into LNs and induces the ISG expression observed with AH / mann formulations. We validated this model by injecting mice side-by-side with either the particulate or soluble fraction of AH / mann. As expected, the particulate and soluble fractions induced skin inflammation (Figure 5H) and strong LN expression of ISGs (Figure 5I), respectively. Finally, we assessed whether AH / mann-induced LIR requires identical cellular and molecular mechanisms as mannan-induced LIR or whether the previously described immunomodulatory functions of AH (Eisenbarth et al., 2008) completely rewire these requirements. AH / mann-induced LN expansion and ISG induction were significantly enhanced in mice lacking the Dectin-2 receptor complex (i.e., WT, Clec4n - / - and Fcer1g - / -) was found to be impaired in mice lacking cDC1s (Figure 5J). Furthermore, AH / mann-induced Ifng expression in dLNs was reduced in mice treated with NK cell-depleting anti-asialoGM1 antibody (Figure 5K), as observed in mannan-induced LIR, and Batf3 expression in cDC1s lacking cDC1s was also impaired in mice lacking cDC1s (Figure 5J). - / - This effect was maintained in mice (Figure 5L). Overall, the incorporation of AH confers on mannan enhanced immunological functions that are predictable based on physical properties (i.e., particulate vs. soluble) and reflect the induction of a mannan-dependent dectin-2 activation pathway.

[0117] Immunization with SARS-CoV-2 spike protein and aluminum hydroxide / mannan generates anti-spike type 1 immunity and cross-reactive neutralizing antibodies with broad epitope specificity The novel immunological properties observed by formulating mannan with AH prompted us to investigate its adjuvant activity in a translationally relevant immunization model. To this end, we used a prefusion stabilized SARS-CoV-2 spike trimer; which has been validated as an antigenic protein to prevent COVID-19 (Corbett et al., 2020; Jackson et al., 2020; Keech et al., 2020; Mercado et al., 2020; Walls et al., 2020; Walsh et al., 2020; Wrapp et al., 2020). Mice were immunized with spike alone or spike mixed with AH, β-glucan, mannan or AH / mannan in a prime (day 0)-boost (day 14) schedule. β-glucan was used to control dectin activation restricted to the skin (Figures 1A, 1B and 5B-5D). At day 14 post-prime, the mannan formulations elicited anti-spike antibodies (Figure 6A). At day 28 (14 days post-boost), AH / mannan induced the highest levels of anti-spike antibodies (Figure 6A). The same was true for antibodies against the receptor-binding domain (RBD) of spike (Figure 6B), which is involved in binding to human ACE2 and SARS-CoV-2 cell entry. Notably, both soluble mannan and AH / mannan promoted anti-spike type 1 immunity by inducing anti-spike IgG2c (an antibody subclass induced by IFNγ) and antigen-specific T cells biased towards IFNγ production (Figure 6A-6C), thus correlating with LN expression of ISGs at early time points (Figure 5D). This evidence has clinical relevance, since antigen-specific type 1 immunity is associated with a reduced risk of vaccine-associated enhanced respiratory disease upon viral infection (Graham, 2020). Notably, the elevated levels of anti-spike IgG1 and IgG2c were maintained up to 98 days after immunization (FIG. 16A).To further evaluate the protective potential of anti-spike antibodies elicited in the immunization model, we performed surrogate virus neutralization assays as well as actual SARS-CoV-2 neutralization assays; here, the neutralizing activity of serum samples is measured by the degree of inhibition of recombinant RBD binding to human ACE2 or SARS-CoV-2 cell infection in vitro, respectively. In both assays, mice immunized with spike and AH / mannan showed the highest neutralization levels (Figures 6D and 6E).

[0118] We also tested whether the adjuvant effect of the AH / mann formulation had spatial and temporal constraints by injecting mice with spikes formulated with AH / mann or spikes formulated with AH and mannan at adjacent injection sites on the same or previous day. Interestingly, enhanced anti-spike neutralizing antibodies were observed only when spikes were formulated with AH / mann (Figures 6F and 6G). In contrast to B cell responses, IFNγ production by antigen-specific T cells was observed in all conditions in which mice were immunized with AH and mannan, regardless of when and where mannan was injected (Figure 6H).

[0119] We next investigated the cellular and molecular requirements for the adjuvant effect of AH / mann. AH / mann-induced anti-spike IgG responses and IFNγ production by antigen-specific T cells were significantly suppressed in WT, Clec4n - / - In mice, Card9 was suppressed (Fig. 16B). - / - In mice, Cd11c was only partially impaired (Figure 16C), consistent with the observations of mannan- and AH / mann-induced LIR. Consistent with a critical role for the transcription factor RelB in driving mannan-dependent responses, Cd11c cre Relb fl / fl Immunization of mice also showed impaired anti-spike IgG responses (Figure 16D). We next tested whether the AH / mann adjuvant effect required the type I / II IFN pathway. - / - Ifngr - / -Either AH / mann mice or WT mice in which IFN signaling was transiently suppressed were used during the immunization phase by administration of anti-IFNAR and anti-IFNγ antibodies. It was observed that both constitutive and acute inhibition of IFN signaling abolished the effect of the AH / alum formulation (Figures 16E and 16F). Finally, the AH / mann adjuvant effect was associated with the upregulation of Batf3. - / - cDC1 were required, as assessed by impaired IgG production in mice (FIG. 16G).

[0120] Due to the superior adjuvant activity of the AH / mann formulation, we tested its ability to induce a broader range of anti-spike antibodies. To address this point, we performed VirScan analysis to investigate the spike epitopes targeted by antibodies elicited by the various adjuvant formulations (Shrock et al., 2020; Xu et al., 2015a). Interestingly, all formulations induced antibodies against the region corresponding to SARS-CoV-2 heptad repeat 2, while only AH / mannan induced antibodies against the SARS-CoV-2 fusion peptide and RBD (Figure 7). This epitope targeting profile was comparable between SARS-CoV-2 and SARS-CoV spike proteins, whereas fewer epitopes targeted the MERS spike (Figure 7), consistent with the spike protein sequence homology of these three coronaviruses (Hicks et al., 2020). Taken together, the results indicate that mannan formulations enhance anti-spike antibody levels and promote anti-spike type 1 immunity, with AH / mannan being particularly effective at inducing anti-spike neutralizing antibodies with broad epitope specificity.

[0121] The AH / mann combination provides protection against viral infections of the lungs. The ability of AH / mann to induce neutralizing antibodies with broad epitope specificity prompted a comparison of the AH / mann formulation with other adjuvants that are part of current FDA-approved vaccines (O'Hagane et al., 2020). AH / mann was benchmarked against squalene-based oil-in-water nanoemulsions (AS03-like AddaS03 or MF59-like AddaVax) and an AS04-like formulation, AH / PHAD, prepared by simple mixing of AH with PHAD, a synthetic structural analogue of monophosphoryl lipid A. Immunization of mice with spike formulated with AH / mann, AddaS03, or AH / PHAD elicited comparable levels of anti-spike and anti-RBD antibody IgG (Figure 8A). Consistent with previous results, the increase in anti-spike and anti-RBD antibodies correlated with a significant increase in neutralization capacity compared to AH alone (Figures 17A and 17B). To prove the functional relevance of these antibody responses, immunized mice were infected with the mouse-adapted SARS-CoV-2 MA10 strain (Leist et al., 2020), and a significant reduction in viral lung titers was found in mice immunized with AH / mann, AddaS03, or AH / PHAD compared to saline-treated or AH-immunized mice (Figure 8B).

[0122] These results indicate that AH / mann enhances both the magnitude and breadth of antigen-specific antibody responses. These attributes may be related to additional viral glycoproteins that are characterized by high antigenic variation, such as influenza A virus (IAV) hemagglutinin (HA) and neuraminidase (NA). Based on antigenic diversity and phylogenetic analysis, 18 HA and 11 NA proteins have been identified to date, which combine to form multiple IAV subtypes (e.g., H1N1). Many viral strains have been identified within each subtype, generating high subtype diversity (Sangesland and Lingwood, 2021). Therefore, it was reasoned that AH / mann may not only promote strong antibody responses against target antigens but also promote heterosubtypic immunity during influenza vaccine immunization. To this end, we used the clinically relevant recombinant HA (rHA) vaccine Flublok (2020-2021 season, composed of HA from IAV A / Guangdong-Maonan / SWL1536 / 2019 [H1N1], IAV A / HongKong / 2671 / 2019 [H3N2], influenza B virus B / Washington / 02 / 2019, and influenza B virus B / Phuket / 3073 / 2013). Mice were immunized with Flublok alone or Flublok combined with AH, AH / mann, Addavax, or AH / PHAD. Anti-rHA antibodies were significantly increased in mice immunized with rHA and AH / mann, AddaVax, or AH / PHAD (Figure 8C). Mice were then challenged intranasally with IAV strain A / PR / 8 / 1934, which belongs to the H1N1 subtype but whose HA is not part of the Flublok vaccine. Surprisingly, only mice pre-immunized with Flublok formulated with AH / mann were significantly protected, as measured by reduced weight loss (Figure 8D) and cellular infiltrates in the lungs (Figures 8F and 17D).These results correlated with high IgG levels against recombinant HA derived from A / PR / 8 / 1934 (rPR8) in mice immunized with Flublok and AH / mann, but not with other adjuvant formulations (Fig. 8E). Overall, these data demonstrate that the AH / mann adjuvant formulation enhances both the magnitude and breadth of antibody responses to multiple viral glycoproteins in a clinically relevant immunization model.

[0123] Consideration Activation of innate immune cells by PRR ligands is considered a critical step for the initiation of adaptive immune responses (Banchereau and Steinman, 1998;Iwasaki and Medzhitov, 2004;Janeway and Medzhitov, 2002;Matzinger, 1994). The cellular and molecular events triggered by PRR-mediated activation of innate immune cells have become an area of ​​intensive research, identifying the signaling organelles, metabolic pathways, and gene expression profiles that shape the innate immune response (Brubaker et al., 2015). However, the cell-specific characteristics of PRR activation and signaling are not sufficient to explain the complexity of in vivo inflammatory responses triggered by innate stimuli. Indeed, their localization at the cellular and organismal levels plays a key role in determining the activation state of innate immune cells (Evavold and Kagan, 2019). Recent evidence indicates that tuning the physical properties (e.g., solubility and diameter) of PRR ligands, especially TLR agonists, has a significant impact on tissue targeting and modulation of innate immune activation profiles. Conjugation of TLR agonists to polymers forming submicron-sized particles, compared to soluble ligands, allows more effective transport to lymph nodes and activation of innate immune cells. This translates to stronger B- and T-cell responses in immunization models (Lynn et al., 2015). These results demonstrate the importance of defining how modulation of the physical properties of PRR ligands impacts the location and magnitude of innate immune cell activation. In this regard, the study of CLRs, especially Dectin 1 and 2, is of particular interest. These receptors bind to the fungal cell wall polysaccharides β-glucan (Dectin 1) and mannan (Dectin 2) and are particularly sensitive to their physical properties, as only the particulate forms of fungal polysaccharides induce efficient receptor clustering and activation (Goodridge et al., 2011; Zhu et al., 2013).Soluble mannan was shown to stimulate a potent LIR characterized by LN expansion and expression of type I and type II IFN transcriptional responses. Soluble mannan upregulates CD169 in dLN. + The LIR induced by these fungal ligands was specific for this anatomical location, as these fungal components targeted sinus macrophages and did not induce an inflammatory response in vitro and in vivo at the injection site (skin). Further modulation of the physical and immunological properties of mannan was made by adsorbing mannan to aluminum hydroxide (AH), generating a formulation in which mannan was present in approximately equal proportions in both soluble and particulate (i.e., bound to AH) forms. As predicted by its physical properties, this formulation induces a proinflammatory response in vitro and in vivo in both the periphery and dLN. When tested in an experimental model of immunization with SARS-CoV-2 spike protein, both soluble mannan and mannan formulated with AH promote anti-spike type 1 immunity. However, only the latter elicits anti-spike neutralizing antibodies with broad epitope specificity that cross-react with the SARS-CoV spike and protect against SARS-CoV-2 MA10 challenge to the same extent as the adjuvant formulation that is part of the FDA-approved vaccine. Notably, immunization of mice with the recombinant hemagglutinin (rHA)-based Flublok vaccine formulated with AH and mannan elicited heterosubtypic immunity as assessed by induction of IgG directed against the rHA of an influenza A H1N1 strain (A / PR / 8 / 1934) not included in the Flublok vaccine, and protective efficacy against A / PR / 8 / 1934 challenge. Overall, this study sheds light on the molecular pathways activated by the mannan formulation that trigger lymph node innate and adaptive responses and result in enhanced magnitude and scope of antibody responses against viral glycoproteins.

[0124] Dectins 1 and 2 are primarily involved in the development of innate and adaptive immune responses against fungal infections (Borriello et al., 2020;Brown et al., 2018;Lionakis et al., 2017;Netea et al., 2008). There is some evidence that their ligands (e.g., β-glucan and mannan) can be used as vaccine adjuvants. However, a detailed understanding of their mechanism of action is lacking (Petrovsky and Cooper, 2011). As previously reported, β-glucan, but not mannan, was found to activate innate immune cells at the injection site in vitro and in vivo. Unexpectedly, both β-glucan and mannan were found to induce LN expansion, with the latter also inducing a unique type I and type II IFN signature restricted to LNs. These results can be explained by the physical form of these polysaccharides rather than differences in signaling pathways, since both dectin-1 and dectin-2 activate the same pathway, but mannan is soluble and small in diameter, allowing it to be transported to LNs, whereas β-glucan is large and insoluble, allowing it to remain in the skin. To further confirm this point, studies have demonstrated that a preparation of soluble glucan can induce LN expansion and ISG expression even in the absence of skin inflammation. These results are intriguing for several reasons: 1) they establish a link between the physical form of fungal polysaccharides and LN innate immune responses; 2) they show the feasibility of targeting dectin receptors to LNs, an effective strategy to induce adaptive immunity (Lian et al., 2020; Liu et al., 2014; Lynn et al., 2015; Scales et al., 2018; Woodruff et al., 2014); 3) early adjuvant-induced type II IFNs in LNs are conserved across mammalian species and promote vaccine immunogenicity (Coccia et al., 2017).

[0125] Both type I and type II IFNs are required to maintain mannan-induced lymphocytosis and LN expansion. It remains unclear whether type I and type II IFNs act on the same or different cell subsets in this model, possibly due to the ubiquitous distribution of their receptors. IFNs act on both myeloid and lymph node stromal cells, modulating a range of functions including chemokine expression and vascular permeability (Barrat et al., 2019; Ivashkiv, 2018). This result therefore raises the possibility that a mannan-induced IFN signature may affect LN-resident myeloid and stromal compartments, ultimately leading to lymphocyte recruitment.

[0126] A detailed mechanistic analysis of mannan-induced LIR is provided herein, which is involved in the induction of dectin-2-expressing CD169 +Our results show that sinus macrophages, but not cDC1s, are required for the induction of mannan-induced LIR. An unexpected finding is the partial requirement for CARD9, a key signaling molecule downstream of dectin receptors and required for activation of canonical NFkB subunits (Borriello et al., 2020). However, CARD9-independent pathways have also been reported, including NIK-dependent activation of the non-canonical NFkB subunits p52 and RelB (Gringhuis et al., 2009;Xu et al., 2018). Mice with selective deletion of RelB in CD11c-expressing cells were used because mice lacking NIK, p52, or RelB have severe defects in secondary lymphoid organ development (Sun, 2017); these cells are required for mannan-induced LIR and preserve secondary lymphoid organ development. The results indicate that RelB regulates mannan-induced expression of type I ISGs and cooperates with CARD9 to regulate type II ISGs. Contrasting evidence on the role of RelB in regulating IFN responses and ISG expression has been presented so far (Le Bon et al., 2006; Saha et al., 2020). Receptor- and cell-specific differences likely play an important role and will need to be taken into account to fully understand the role of RelB in mannan-induced ISG expression.

[0127] A notable feature of this study is that mannan formulated with AH acquires a novel immunological property, namely the induction of a proinflammatory response both at the injection site and in dLN in vitro and in vivo. Although synergy between AH and mannan cannot be excluded, these results are more easily explained by the simultaneous presence of unbound and AH-adsorbed mannan in the AH / mannan formulation. The former is responsible for ISG expression in dLN, while the latter mediates skin inflammation. Evaluation of AH / mann in an immunization model with prefusion-stabilized SARS-CoV-2 spike trimers supports this model. When AH and mannan were injected separately but in close proximity, the direct synergy between the inflammatory activity of AH and mannan was eliminated and their ability to boost anti-spike IgG was lost. Interestingly, this is not the case for the T cell-mediated response, which was promoted by the presence of mannan independent of coformulation with AH. Analysis of soluble mannan and AH / mannan in a model of immunization with prefusion-stabilized SARS-CoV-2 spike trimers revealed that both formulations induced humoral and cellular type 1 immunity, suggesting that early induction of type I and type II IFN signatures in dLNs is sufficient to explain the polarization of adaptive immune responses. IFNγ can promote both IgG2c antibody switching and Th1 polarization (Finkelman et al., 1988;Martin-Fontecha et al., 2004). Notably, transient inhibition of type I / II IFN signaling by administration of blocking antibodies significantly reduced anti-spike IgG, suggesting that early IFN signatures in dLNs lead to long-term enhancement of immune responses.

[0128] A unique property of the AH / mannan formulation is the induction of neutralizing anti-spike antibodies in mice immunized with the SARS-CoV-2 spike that have broad epitope specificity and cross-react with the SARS-CoV spike and, to a lesser extent, the MERS spike. The production of these antibodies has the same molecular and cellular requirements as the LIR induced by AH / mann, except for cDC1s, which play a key role in driving the IgG response but not the LIR. Neutralizing antibodies play a key role in protection against SARS-CoV-2 infection in experimental animal models (Cao et al., 2020; Hassan et al., 2020; Lv et al., 2020; McMahan et al., 2020; Rogers et al., 2020; Schafer et al., 2021; Shi et al., 2020; Tortorici et al., 2020; Zheng et al., 2020; Zost et al., 2020). Accordingly, mice immunized with spike and AH / mann show undetectable lung viral titers following SARS-CoV-2 MA10 infection, as do mice immunized with clinically relevant adjuvant formulations such as AddaS03 and AH / PHAD. Moreover, the epitope specificity profile observed in mice immunized with AH / mannan was comparable to that observed in COVID-19 patients, highlighting the translational relevance of the results (Shrock et al., 2020). To further extend these findings, AH / mann was tested in an influenza immunization model using a clinically relevant Flublok vaccine, composed of recombinant HA (rHA) from two influenza A and two influenza B strains, and compared with MF59-like Adda Vax and AS04-like AH / PHAD adjuvant formulations. While all adjuvants enhanced antibody responses to Flublok, only AH / mann induced heterosubtypic immunity, i.e., protection against challenge with an H1N1 influenza A strain (A / PR / 8 / 1934) whose HA was not included in the Flublok vaccine.These results were similar to those shown by the detection of antibodies against A / PR / 8 / 1934 HA only in mice immunized with AH / mann. There are at least two possible explanations for this phenomenon, which are not mutually exclusive: 1) AH / mannan formulations induce a high degree of innate immune activation by simultaneously targeting the periphery and dLNs, thereby enhancing adaptive immunity; 2) AH generates depots that slowly release antigens to the dLNs, and / or AH can promote the formation of antigen multimers and promote germinal center responses, together with the LIR induced by unbound mannan ((Moyer et al., 2020; Pedersen et al., 2020). In the future, it will be important to elucidate how AH / mannan enhances antigen-specific adaptive immunity and regulates germinal center dynamics and B cell repertoire selection. To that end, the in vivo experiments described herein utilized an effective formulation of AH / mannan at a molar ratio of 1:5, while subsequent experiments demonstrated that a 2:5 AH / mannan formulation enhanced the innate immune activation of bone marrow-derived in vivo antigens. These results suggest that AH may further enhance cytokine expression in vitro, thereby highlighting the importance of AH for enhancing the immunogenicity of mannan. From a translational perspective, the increased epitope specificity suggests that AH / mann formulations in combination with appropriate antigens may be promising candidates for developmental vaccines targeting multiple coronaviruses or influenza A strains.

[0129] Overall, this study provides a mechanistic understanding of the different modes of Dectin-2 targeting enabled by modulation of the physical form of mannan and their translational impact for the development of more effective vaccines, further extending our knowledge of the relationship between the physical form of innate stimuli and the innate and adaptive immune responses in LNs.

[0130] method Mouse: C57BL / 6J (Jax 00664) (wild type), CB6F1 (Jax 100007), B6.129P2(C)-Ccr7 tm1Rfor / J(Ccr7 - / -, Jax 006621), B6.129S2-Ifnar1 tm1Agt / Mmjax(Ifnar - / - , Jax 32045-JAX), B6.Cg-Ifngr1 tm1Agt Ifnar1 tm1.2Ees / J(Ifnar - / - Ifngr - / - , Jax 029098), B6.FVB-1700016L21Rik Tg(Itgax-DTR / EGFP)57Lan / J(CD11c-DTR, Jax 004509), B6.Cg-Tg(Itgax-cre)1-1Reiz / J(Cd11c cre , Jax 008068), B6.Cg-Relbt m1Ukl / J(Relb fl / fl , Jax 028719), B6.129S4-Ccr2 tm1Ifc / J(Ccr2 - / - , Jax 004999), B6.129-Card9 tm1Xlin / J(Card9 - / - , Jax 028652) and B6.129S6-Clec7a tm1Gdb / J(Clec7a - / - , Jax 012337) was purchased from Jackson Labs. tm1Rav N12(Fcer1g - / - The Clec4n (Model 583) was purchased from Taconic. - / -Mice were kindly provided by Dr. Nora A. Barrett and Dr. Yoichiro Iwakura. B6;129-Siglec1(CD169-DTR) mice were kindly provided by Dr. F. Pucci from the Riken Institute (No. RBRC04395) and were deposited by Dr. Kenji Kohno and Dr. Masato Tanaka (Miyake et al., 2007; Saito et al., 2001). B6.Cg-Tg(TcraTcrb)425Cbn / J (OT-II, Jax 004194) were kindly provided by Juan Manuel Leyva-Castillo. Female mice were used in all experiments. Mice were housed under specific pathogen-free conditions at Boston Children's Hospital, and all procedures were approved by the Institutional Animal Care and Use Committee (IACUC) and performed under the supervision of the department of Animal Resources (ARCH) at Children's Hospital.

[0131] Reagents and antibodies: For flow cytometry, imaging cytometry, fluorescence-activated cell sorting (FACS), and confocal microscopy experiments, the following reagents and antibodies were used: anti-CD45 BV510 (30-F11), anti-CD45 Alexa Fluor 700 (30-F11), anti-CD45 APC (30-F11), anti-CD3 PE / Dazzle 594 (17A2), anti-CD3 BV510 (17A2), anti-CD19 PE / Dazzle 594 (6D5), anti-CD19 BV650 (6D5), anti-NK1.1 PE / Dazzle 594 (PK136), anti-Ter119 PE / Dazzle 594 (TER-119), anti-IA / IE PE / Cy7 (M5 / 114.15.2), anti-Ly6G PerCP / Cy5.5(1A8), anti-CD11b Pacific Blue(M1 / 70), anti-Ly6C BV711(HK1.4), anti-CD11c BV785(N418), anti-CD11c APC(N418), anti-CD86 APC / Cy7(GL-1), anti-CD86 APC(GL-1), anti-OX40L PE(RM134L), anti-CD169 APC(3D6.112), anti-CD169 Alexa Fluor 647(3D6.112), anti-CD4 APC / Fire 750(GK1.5), anti-CD45R / B220 Alexa Fluor 594 (RA3-6B2), anti-CD3 biotin (145-2C11), anti-CD19 biotin (6D5), anti-NK1.1 biotin (PK136), anti-Ter119 biotin (TER-119), TrueStain FcX (93), True-Stain Monocyte Blocker and Zombie Red Fixable Viability Kit were purchased from Biolegend; anti-Dectin-2 PE (REA1001) was purchased from Miltenyi Biotec; rat anti-Dectin-2 (D2.11E4) was purchased from GeneTex; anti-phospho-Syk (Tyr525 / 526) (C87C1) was purchased from Cell Signaling Technology; CellTrace CFSE Cell Proliferation Kit, Alexa Fluor 488 NHS Ester (succinimidyl ester) and DAPI were purchased from Thermo Fisher Scientific.

[0132] For in vitro and in vivo experiments, the following reagents were used: Iscove's modified Dubecco's medium (IMDM), phosphate-buffered saline (PBS), penicillin / streptomycin (pen / strep), and L-glutamine (L-Gln) were purchased from Lonza; fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific; collagenase from Clostridium histolyticum, deoxyribonuclease (DNase) I from bovine pancreas, and dispase II were purchased from MilliporeSigma; TLR Grade Escherichia coli LPS (serotype O555:B5, 1 μg / ml) was purchased from Enzo Life Sciences; curdlan (10 μg / ml) was purchased from Wako Chemical Industries; mannan, β-glucan, and their Alexa Fluor 488 conjugates (10 μg / ml for in vitro experiments and 500 μg / mouse for in vivo experiments) were purchased from Michael D Kruppa, Zuchao Carboxyl latex beads 3 μm were purchased from Thermo Fisher Scientific and used either directly (cell:beads ratio 1:10 for in vitro experiments) or after coating with diaminopropane-derivatized mannan, kindly provided by Michael D Kruppa, Zuchao Ma, and David L Williams (East Tennessee State University); WGP-S and WGP-D (500 μg / mouse for in vivo experiments) were purchased from Invivogen; Lipo-CpG was kindly provided by Darrell J.Diphtheria toxin (unnicked) from Corynebacterium diphtheriae (200ng / mouse for CD11-DTR mice and 500ng / mouse for CD169-DTR mice) was purchased from Cayman Chemical; ovalbumin (OVA) EndoFit (5μg / mouse) and Alhydrogel adjuvant 2% (aluminum hydroxide, 2μg / ml for in vitro experiments and 100μg / mouse for in vivo experiments) were purchased from Invivogen; recombinant prefusion stabilized SARS-CoV-2 spike trimer (1μg / mouse) and RBD were expressed and purified from plasmids generously provided by Dr. Berney S. Graham (NIH Vaccine Research Center) and Dr. Aaron G. Schmidt (Ragon Institute), respectively; SARS-CoV-2 spike peptide pool (PepTivator SARS-CoV-2 Prot_S) was purchased from Miltenyi Biotec; anti-CD62L (Mel-14, 100 μg / mouse), anti-IFNγ (XMG1.2, 200 μg / mouse), anti-Ly6G (1A8, 50 μg / mouse) and their isotype controls rat IgG2a (2A3) and rat IgG1 (HRPN) were purchased from Bio X Cell.

[0133] The AH / mannan (AH / mann) formulation was obtained by mixing AH (100 μg / 10 μl), mannan (500 μg / 25 μl) and saline (15 μl). When antigen (e.g. SARS-CoV-2 spike trimer or 2020 season Flublok) was formulated, the amount of saline was reduced to keep the total volume constant. This formulation is further described in the Vaccine Adjuvant Compendium (https: / / vac.niaid.nih.gov). The 2020 season Flublok recombinant hemagglutinin (rHA) is commercially available from Sanofi Pasteur and contains an equimolar ratio of rHA from the following influenza virus strains: influenza A / Guangdong-Maonan / SWL1536 / 2019[H1N1], influenza A / Hong Kong / 2671 / 2019[H3N2], influenza B virus B / Washington / 02 / 2019, and influenza B virus B / Phuket / 3073 / 2013.

[0134] Isolation of mannan from C. albicans. Candida albicans strain SC5314 was maintained on blood agar (Remel) plates grown at 37°C. To isolate mannan, C. albicans was inoculated into 15 liters of YPD (1% yeast extract, 2% peptone, 2% dextrose) and grown for 20 h at 37°C. Cells were harvested by centrifugation at 5000g for 5 min. This resulted in a pellet of 100 g from 15 liters of medium. Standard protocols were used for isolation and NMR characterization of mannan (Kruppa et al., 2011;Lowman et al., 2011). Briefly, the cell pellet was suspended in 200 ml of acetone, the cells were degreased for 20 min, then centrifuged at 5000g for 5 min, the acetone was removed, and the pellet was dried for 1 h. The dried pellet was ground and transferred to a bead beater. An equal volume of acid washed glass beads and 200ml of dH2O were added to the mixture. The cells were subjected to bead beating with three 30 second pulses, after which the entire mixture was transferred to a 1 liter flask. The material was autoclaved for 2 hours, allowed to cool, and then centrifuged at 5000g for 5 minutes. The supernatant was retained and the cell pellet was discarded. Pronase (500mg in 20ml of dH2O), filter sterilized and heat treated at 65°C for 20 minutes (to remove glycosidic activity), was added to the supernatant to a concentration of 1mM along with sodium azide. The mixture was then incubated at 37°C overnight (20 hours) to degrade any proteins in the solution. Mannan was extracted by adding an equal volume of Fehling's solution to the protease treated mannan solution and mixed at room temperature for 1 hour. After mixing, the solution was left for 20 minutes to facilitate precipitation of the mannan. The supernatant was decanted and the precipitate was dissolved in 10 ml of 3M HCl to release the copper from the reducing ends of the mannans. 500 ml of an 8:1 mixture of methanol:acetic acid was added to the dissolved mannan solution and the mixture was stirred to allow the mannans to precipitate overnight. After the material had settled, the supernatant was decanted, washed again with 500 ml of methanol and left for 6 hours for the mannans to settle. The supernatant was decanted and the remaining precipitate was dissolved in 200 ml of dH2O.The mannans were dialyzed against a 200-fold change of dH2O using a 2000 MW cutoff membrane for 48 hours to remove residual acid, methanol, and other compounds from the extraction process. The dialysate was then lyophilized and stored at -20°C until needed. A small sample of the material (10 mg) was subjected to NMR to confirm the purity of the N-linked mannan (Lowman et al., 2011) and to estimate the molecular weight (Kruppa et al., 2011). Prior to in vitro or in vivo use, the mannans were pyrogenated to remove residual endotoxins and filter sterilized.

[0135] Preparation of diaminopropane (DAP) derivatized mannan. Mannan (100 mg) was stirred for 1 h and then dissolved in 1 ml of dimethyl sulfoxide (DMSO) in a 4 ml vial. 1,3-diaminopropane (100 μL) was added and stirred at ambient temperature for 3 h. Sodium cyanoborohydride (100 mg) was added and the reaction mixture was stirred for 48 h, followed by sodium borohydride (50 mg) and stirring for 24 h. Acetic acid (200.0 μl) was added dropwise at 0° C. to quench the reaction, and the reaction mixture was stirred at ambient temperature for 3 h and then dialyzed against ultrapure water using a 1000 MWCO RC membrane (1000 ml×4). The retentate was collected and lyophilized to obtain DAP-attached mannan. The recovery was 88.5 mg, approximately 88%. Mannan-DAP was 1 Characterization by 1 H-NMR confirmed the identity of the compound.

[0136] For conjugation with Alexa Fluor 488 NHS Ester (succinimidyl ester), approximately 15 mg of mannan-DAP was resuspended in 1 ml of sodium borate conjugation buffer (100 mM, pH 8.5) and solvated for at least 24 hours. Then, 1 mg of Alexa Fluor 488 NHS Ester resuspended in 35 μl of DMSO was added to the solution and incubated overnight at room temperature in the dark with gentle agitation. The reaction mixture was dialyzed against saline (1000 ml x 4) using a 6000-8000 MWCO RC membrane, and the retentate was filter sterilized. For conjugation to 3 μm carboxyl latex beads, mannan-DAP was resuspended in BupH MES conjugation buffer, pH 4.5 (Thermo Fisher Scientific) at a concentration of 10 mg / 1 ml and solvated for at least 24 h. 1 ml of mannan-DAP was diluted with 50 × 10 6 100 ml of beads were added and then mixed with 4 mg / 1 ml of EDC (Thermo Fisher Scientific) resuspended in pure water. The reaction mixture was incubated for 4 hours in the dark at room temperature with gentle agitation. The beads were then washed twice with saline (4000 g for 10 minutes) and diluted with saline for 10 minutes. 8 The beads were resuspended at a concentration of 10 ...

[0137] Preparation of Candida albicans β-glucan particles. β-glucan particles were isolated from Candida albicans SC5314 as previously described in our laboratory (Lowman et al., 2014). Briefly, glucan was isolated from C. albicans using a base / acid extraction approach, yielding water-insoluble glucan particles with purity ≥95%. The structure and purity of the glucan were determined by elution of β-glucan particles in DMSO-d6. 1 H-NMR was used to determine this (Lowman et al., 2014). Prior to in vitro or in vivo use, the β-glucan particles are depyrogenated and filter sterilized.

[0138] Preparation of diaminopropane (DAP) derivatized β-glucan. β-glucan particles (20 mg) were stirred for 1 h and then dissolved in 1 ml of dimethylsulfoxide (DMSO) in a 4 ml vial. 1,3-diaminopropane (100 μL) was added and stirred at ambient temperature for 3 h. Sodium cyanoborohydride (100 mg) was added and the reaction mixture was stirred for 48 h, followed by sodium borohydride (50 mg) and stirring for 24 h. The reaction was quenched by dropwise addition of acetic acid (200 μl) at 0° C. and the reaction mixture was stirred at ambient temperature for 3 h. β-glucan particles were collected and washed five times with water by centrifugation (862 g). The recovery was >95%. Glucan-DAP was1 Characterization by H-NMR confirmed that the mannan structure was unchanged and also detected the presence of DAP. The glucan-DAP was dried by lyophilization and stored at -20 °C in a desiccator in the dark until required. For conjugation with Alexa Fluor 488 NHS Ester (succinimidyl ester), 20 mg of glucan-DAP was suspended in 1 ml of sodium borate conjugation buffer (100 mM, pH 8.5) and allowed to hydrate for at least 24 h at 4 °C. Then, 1 mg of Alexa Fluor 488 NHS Ester resuspended in 35 μl of DMSO was added to the solution and incubated overnight at room temperature in the dark with gentle agitation. The reaction mixture was centrifuged and washed five times (862 g) and 488 labeled glucan particles were collected.

[0139] Quantification of mannan in aluminum hydroxide / mannan formulations. Supernatant was collected from the AH / mannan mixture. The supernatant was lyophilized. The lyophilized supernatant and mannan not mixed with AH (4.0 mg) were dissolved in 500 μl of deuterium oxide (99.9% D) containing 0.01% (W / V) of the internal standard TMSP-2,2,3,3-D4 (98.0% D). 1 H NMR data were collected on a Bruker Avance 400 MHz Ultra Shield NMR spectrometer at 295 K using the same acquisition parameters for all samples. NMR spectra were processed using TOPSPIN 2.1 running on an Avance 400 MHz NMR. Ring proton resonances (3.25-4.50 ppm) were integrated by reference to the integrals of an internal standard (-0.02-0.02 ppm) calibrated as 1. Based on the ratio of the standard mannan mass (4.0 mg) to its ring proton integral (average 39.12), the mannan mass in the supernatant was calculated using the detected ring proton integrals and adjusted against a blank. The amount of mannan absorbed by AH was determined by the relative mass loss of mannan in the supernatant after formulation.

[0140] Analysis of skin and LN responses. To evaluate the skin and LN spontaneous responses, mice were intradermally injected with the indicated compounds in a volume of 50 μl on both sides of the back on day 0 (one side was compound and the other side was saline vehicle control). At 6 or 24 hours after injection, skin samples from the injection site and draining (upper arm) LN were collected for later analysis. The skin samples were transferred to a bead beater and homogenized in 1 ml of TRI Reagent (Zymo Research).The samples were then centrifuged at 12000g for 10 min and 800 μl of the clear supernatant was transferred to a new tube for subsequent RNA isolation. LNs were weighed on an analytical scale before being transferred to a beadbeater and either homogenized in TRI reagent as indicated for skin samples or processed by a modified version of a previously published protocol (Fletcher et al., 2011) to generate an LN cell suspension. Briefly, individual LNs were incubated in 400 μl of digestion mixture (IMDM+pen / strep+FBS 2%+collagenase 100 mg / ml+dispase II 100 mg / ml+DNase 10 mg / ml) for 20 min at 37°C. LNs were then triturated by pipetting with a 1000 μl tip and the supernatant was transferred to a new tube and kept at 4°C while 200 μl of digestion mixture was added to the pellet and incubated at 37°C for 10 min. After repeating this cycle one more time, the pooled supernatants of individual LNs were divided into two aliquots: one for flow cytometric analysis and the other was centrifuged at 300 g for 5 min, and the cell pellet was resuspended in 800 μl of TRI reagent for subsequent RNA isolation. For certain experiments, mice were treated with: anti-CD62L blocking antibody or isotype control, injected intravenously on day -1; anti-IFNγ blocking antibody or isotype control, injected intravenously on days -1 and 0; anti-Ly6G depleting antibody or isotype control, injected intraperitoneally on days -1 and 0; diphtheria toxin, injected intravenously on day -1 and intradermally on day 0 (co-injected with mannan) for CD11c-DTR mice, and intraperitoneally on day -2 for CD169-DTR mice.

[0141] In vitro stimulation of GM-CSF-differentiated bone marrow-derived phagocytes. Bone marrow-derived phagocytes were differentiated from bone marrow in IMDM + 10% B16-GM-CSF-derived supernatant + 10% FBS + pen / strep + L-Gln and used after 7 days of culture. Cells were then harvested and plated in flat-bottom 96-well plates at 10 5 Cells were plated at a density of 200 μl / well and stimulated with the indicated compounds for 18–21 h. At the end of stimulation, supernatants were harvested and TNF and IL-2 concentrations were measured by ELISA (Biolegend) according to the manufacturer's protocol. Cells were detached with 2 mM PBS+EDTA and transferred to round-bottom 96-well plates for subsequent flow cytometry staining and analysis. Alternatively, cells were stimulated for 6 h, lysed with TRI Reagent, and RNA was extracted for gene expression analysis.

[0142] In vivo quantification of fluorescently labeled β-glucan and mannan. Mice were injected intradermally with 500 μg / mouse of Alexa Fuor 488-conjugated β-glucan and mannan (in selected experiments, mannan was combined with 100 μg / mouse AH prior to injection). At the indicated time points, dLNs were collected, transferred to a beadbeater, and homogenized in 400 μl of deionized water. Samples were then centrifuged (12000 g for 10 min) and the clear supernatants were transferred to 96-well clear-bottom black plates. Fluorescence values ​​were measured with a SpectraMax i3x microplate reader (Molecular Devices) and expressed in arbitrary units after subtraction of background (deionized water).

[0143] Flow cytometry, fluorescence-activated cell sorting (FACS), imaging cytometry, and confocal microscopy. For flow cytometry analysis, cells were first stained with Zombie Red Fixable Viability in PBS for 5 min at 4°C, washed once with PBS+BSA 0.2%+NaN3 0.05% (300g for 5 min), and then stained with antibodies against surface antigens diluted in PBS+BSA 0.2%+NaN3 0.05% for 20 min at 4°C. Cells were then washed and fixed with 2% paraformaldehyde for 10 min at room temperature, washed again, and resuspended in PBS+BSA 0.2%+NaN3 0.05%. Samples were acquired on a BD LSRFortessa flow cytometer and data were analyzed using FlowJo v.10 software (BD Biosciences). Absolute cell numbers were quantified using CountBright Absolute Counting Beads. In selected experiments, after fixation with 2% paraformaldehyde, cells were permeabilized by incubation with saponin-based permeabilization buffer (BioLegend) for 10 min at 4° C. and stained with antibodies against intracellular cytokines diluted in permeabilization buffer for 20 min at 4° C. Cells were then washed with permeabilization buffer, resuspended in PBS+BSA 0.2%+NaN3 0.05%, and acquired as previously indicated.

[0144] For FACS and imaging cytometry, mice were injected intradermally with AF488-mannan and dLNs were harvested 6 hours later to obtain LN cell suspensions. For FACS, cells were stained with antibodies against surface antigens diluted in PBS+BSA 0.2% for 20 min at 4°C. Cells were then washed once, resuspended in 1 ml PBS+BSA 0.2%, filtered through a 70 μm cell strainer (Fisher Scientific), and sorted directly into 1 ml TRI Reagent on a Sony MA900 cell sorter. The following cell subsets were sorted: CD3 CD19 NK1.1 - Ter119 - CD45 + AF488-Mannan + Ly6G - (CD11b+ Ly6C + ) CD11b + CD11c + For imaging cytometry, lymphoid and erythroid cell removal from the cells was performed by sequential staining with biotinylated antibodies against anti-CD3, anti-CD19, anti-NK1.1, anti-Ter119 and streptavidin microbeads (MiltenyiBiotec) according to the manufacturer's protocol. The remaining cells were stained with anti-CD45 APC, fixed with 2% paraformaldehyde, washed once and resuspended in 60 μl of PBS+DAPI (0.2 μg / ml). Samples were then acquired with an Amnis ImageStream X Mark II (Luminex Corporation). Mannan internalization was analyzed with Amnis Ideas software and calculated as AF488 signal within the APC mask by the internalization function.

[0145] For confocal microscopy, dLNs were isolated at steady state or 1 hour after injection of AF488-mannan and fixed overnight in 4% paraformaldehyde. Tissue slides were prepared from frozen LN samples at the Beth Israel Deaconess Medical Center (BIDMC) Histology Core Facility and stained at the BIDMC Confocal Imaging Core Facility. Briefly, frozen sections were air-dried for 30 minutes and rehydrated. Sections were permeabilized with 0.05% Triton X-100 for 10 minutes at room temperature and washed three times in TBS. Sections were then incubated with 5% normal donkey serum (Jackson ImmunoResearch Lab) for 1 hour at room temperature. For dectin-2 staining of steady-state LNs, slides were incubated with rat anti-dectin-2 overnight at 4°C. Slides were washed three times and incubated with Alexa Fluor 488-conjugated donkey anti-rat secondary antibody (Jackson ImmunoResearch Lab) for 90 minutes at room temperature and washed four times. Slides were then incubated with Alexa Fluor 647-conjugated rat anti-CD169 primary antibody and Alexa Fluor 594-conjugated rat anti-CD45R / B220 primary antibody for 90 minutes at room temperature and then washed with TBS. For phospho-Syk staining of AF488-mannan-treated LNs, slides were incubated with rabbit anti-phospho-Syk (Cell Signaling Technology) overnight at 4°C. Slides were washed three times and incubated with Alexa Fluor 647-conjugated donkey anti-rabbit secondary antibody (Jackson ImmunoResearch Lab) for 90 minutes at room temperature and washed four times. Slides were then incubated with Alexa Fluor 594-conjugated rat anti-CD45R / B220 primary antibody for 90 minutes at room temperature and then washed with TBS. Rabbit anti-phospho-Syk (Cell Signaling Technology). Samples were counterstained with Hoechst 33342 (Thermo Fisher Scientific) and washed three times with TBS.Slides were mounted with Prolong Gold antifade mounting medium (Thermo Fisher Scientific) and imaged on a Zeiss 880 laser scanning confocal microscope at the Boston Children's Hospital Harvard Digestive Disease Center.

[0146] RNA isolation, qPCR, transcriptome and pathway analysis. RNA was isolated from TRI Reagent samples using phenol-chloroform extraction or a column-based extraction system (Direct-zol RNA Microprep and Miniprep, Zymo Research) according to the manufacturer's protocol. RNA concentration and purity (260 / 280 and 260 / 230 ratios) were measured by NanoDrop (Thermo Fisher Scientific). Gene expression of purified RNA was analyzed by qPCR in a CFX384 real-time cycler (Bio-rad) using predesigned KiCqStart SYBR Green Primers (MilliporeSigma) specific for Cxcl9 (RM1_Cxcl9 and FM1_Cxcl9), Gbp2 (RM1_Gpb2 and FM1_Gbp2), Ifit2 (RM1_Ifit2 and FM1_Ifit2), Rsad2 (RM1_Rsad2 and FM1_Rsad2), Il6 (RM1_Il6 and FM1_Il6), Cxcl1 (RM1_Cxcl1 and FM1_Cxcl1), or predesigned PrimeTime qPCR Primers (Integrated DNA Technologies) specific for Gapdh (Mm.PT.39a.1).

[0147] RNA isolated from LN samples was submitted to Genewiz for bulk RNA-seq analysis. RNA samples were quantified using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific) and RNA integrity was checked using an RNA Screen Tape on an Agilent 2200 TapeStation (Agilent Technologies). RNA-sequencing libraries were prepared using the TruSeq Stranded mRNA Library Preparation Kit, following the manufacturer's protocol (Illumina, Cat# RS-122-2101). Briefly, mRNA was first enriched with Oligod (T) beads. The enriched mRNA was fragmented at 94°C for 8 min. Subsequently, first- and second-strand cDNA were synthesized. The second strand of cDNA was marked by incorporating dUTP during synthesis. The 3' ends of the cDNA fragments were adenylated and indexed adapters were ligated to the cDNA fragments. The libraries were enriched using limited cycle PCR. The dUTP incorporated into the second strand cDNA suppressed second strand amplification, which helped to maintain strand specificity. Sequencing libraries were validated using a DNA Analysis Screen Tape on an Agilent 2200 TapeStation (Agilent Technologies) and quantified using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific) and quantitative PCR (Applied Biosystems). Sequencing libraries were multiplexed and clustered onto one lane of a flow cell. After clustering, the flow cells were loaded onto an Illumina HiSeq instrument according to the manufacturer's instructions. Samples were sequenced using a 2 × 150 paired-end (PE) high output configuration. Image analysis and base calling were performed by HiSeq Control Software (HCS) on the HiSeq instrument. Raw sequence data (.bcl files) generated from the Illumina HiSeq were converted to fastq files and demultiplexed using the Illumina bcl2 fastq program version 2.17. One mismatch was allowed for index sequence identification.Reads were quality controlled using FastQC. Illumina adapters were removed using cutadapt. Trimmed reads were mapped to the mouse transcriptome (GRCm38) based on Ensembl annotations using Kallisto (Bray et al., 2016). Transcript counts were imported using tximport and aggregated to gene counts (Soneson et al., 2015). Gene counts were analyzed using the R package DESeq2 (Love et al., 2014). Where applicable, batch was used as a blocking factor in statistical models. Differentially expressed genes (DEGs) were identified as passing FDR significance thresholds (0.05 in skin and 0.01 in lymph nodes, stringent thresholds thanks to greater power due to the larger number of replicates); here, the alternative hypothesis was that absolute log2 FC was greater than 0. Genes induced by mannan or glucan treatment versus saline were plotted in a heatmap using log2-normalized abundance of Z-scores with the R package ComplexHeatmap. Genes were arranged by abundance delta between glucan and mannan (aggregated from multiple time points as necessary), with a gap separating two clusters: genes more highly expressed with mannan stimulation vs. genes more highly expressed with glucan stimulation. Pathway analysis was performed using the R package hypeR (Federico and Monti, 2020) using hypergeometric enrichment tests of genes belonging to clusters of interest and the Hallmark gene set collection from the Broad Institute's MSigDB collection.

[0148] For targeted transcriptome sequencing, 25ng of RNA isolated from sorted cells was reverse transcribed into cDNA using the SuperScript VILO cDNA synthesis kit (Thermo Fisher Scientific). Barcoded libraries were prepared using the Ion AmpliSeq Transcriptome Mouse Gene Expression Kit according to the manufacturer's protocol and sequenced using the Ion S5 system (Thermo Fisher Scientific). Differential gene expression analysis was performed using the ampliSeqRNA plugin (Thermo Fisher Scientific). To quantify the number of DEGs, gene-level fold changes <-1.5 or >1.5 and gene-level p-values ​​<0.05 (ANOVA) were considered. For heatmap display, DEGs were defined as F-test FDRs <0.05 and log2 fold changes (FC) >1 (or <-1) between mutants and WT controls. Hierarchical clustering was performed using Pearson correlation and average linkage. Pathway analysis was performed on genes ranked according to log2FC using the Kolmogorov Smirnov test in the R package hypeR.

[0149] CD4 in vivo + and CD8 + T cell proliferation assay. Spleens were isolated from OT-II or OT-I mice and crushed with the plunger end of a syringe. The cell suspension of splenocytes was then treated with ACK lysis buffer (2 ml, 2 min at room temperature), washed with PBS (300 g for 5 min), and filtered through a 70 mm cell strainer. CD4 + and CD8 + T cells were purified using CD4 (L3T4) or CD8a (Ly-2) MicroBeads (Miltenyi Biotec) according to the manufacturer's protocol, respectively, and stained with CellTrace CFSE (5 mM in PBS+FBS 2.5%, 20 min in the dark). At the end of the incubation, cells were washed twice with PBS and 5 × 10 5Cells were resuspended at a concentration of cells / 100 ml saline, and 100 ml of the cell suspension was injected intravenously into each mouse. 24 hours later (day 0), mice were injected intradermally with OVA (5 mg / mouse) alone or in combination with mannan (500 mg / mouse). Mice injected with saline were used as controls. dLNs were harvested on day +3, and LN cell suspensions were stained with anti-CD19, anti-Ter119, anti-CD3, and anti-CD4 or anti-CD8 antibodies. Adoptively transferred CFSE-labeled OT-II and OT-I cells were stained with CD19, anti-Ter119, anti-CD3, and anti-CD4 or anti-CD8 antibodies, respectively. - Ter119 - CD3 + CD4 + Gating and CD19 - Ter119 - CD3 + CD8 + The results were detected by the gate. - Ter119 - CD3 + CD4 + CFSE lo or CD19 - Ter119 - CD3 + CD8 + CFSE lo The absolute number of cells (i.e., cells that have undergone at least one division cycle) or CD19 - Ter119 - CD3 + CD4 + Gate or CD19 - Ter119 - CD3 + CD8 + It is expressed as a percentage of each split peak within the gate.

[0150] Expression and purification of SARS-CoV-2 spike and RBD. Full-length SARS-CoV-2 spike glycoprotein and RBD constructs (amino acid residues R319–K529), both with HRV3C protease cleavage sites, TwinStrepTag and 8XHisTag at the C-terminus, were obtained from Dr. FBarney S. Graham (NIH Vaccine Research Center) and Dr. Aaron G. Schmidt (Ragon Institute), respectively. These expression vectors were used to transiently transfect Expi293 cells (Thermo Fisher Scientific) using polyethylenimine (Polysciences). Proteins were purified from filtered cell supernatants using StrepTactin resin (IBA) or Cobalt-TALON resin (Takara). The affinity tag was cleaved from the eluted protein samples by HRV3C protease, and the tag-free proteins were subjected to further purification by size-exclusion chromatography using a Superose 6 10 / 300 column (GE Healthcare) or a Superdex 75 10 / 300 Increase column (GE Healthcare) in PBS (pH 7.4) buffer.

[0151] Immunization and antibody quantification. CB6F1 mice were immunized on days 0 and +14 by intradermal injection of spike (1 mg / mouse) alone or in combination with AH (100 mg / mouse), β-glucan (500 mg / mouse), mannan (500 mg / mouse), AH / mannan (AH / mann), AddaS03 or AH / PHAD. Alternatively, C57BL / 6 mice were immunized on days 0 and +14 by intradermal injection of Flublok vaccine (1 μg Flubok2020; 0.25 μg per rHA) alone or in combination with AH (100 μg AH), AH / mann (100 μg AH, 500 μg mannan), AddaVax or AH / PHAD. Saline-injected mice were used as controls. Blood samples were collected by retro-orbital bleeding on days +14 (before boost) and +28, and serum samples were separated after centrifugation at 1500g for 10 min twice. In selected experiments, blood samples were collected on days +98 or 7 after challenge. Spike, RBD, Flublok, and rPR8-specific IgG, IgG1, and IgG2c antibody levels in serum samples were quantified by ELISA using a modified protocol previously described (Borriello et al., 2017). Briefly, high-binding flat-bottom 96-well plates were coated with 0.5 μg / ml spike, 1 μg / ml RBD, 1 μg / ml Flublok, or 1 μg / ml rPR8 in PBS, incubated overnight at 4°C, washed once with PBS+0.05% Tween-20 (PBST), and blocked with PBS+BSA 1% for 1 h at room temperature. Serum samples were then added at an initial dilution of 1:100 and serial dilutions of 1:4 in PBS+BSA 1% to generate 11-point curves and incubated for 2 h at room temperature. Plates were then washed 3 times with PBST and incubated with HRP-conjugated anti-mouse IgG, IgG1 or IgG2c (Southern Biotech) antibodies for 1 h at room temperature.At the end of the incubation, plates were washed five times with PBST and developed with tetramethylbenzidine (BD OptEIA substrate solution for spike, rHA, and rPR8; Thermo Fisher Scientific 1-Step Ultra TMB-ELISA substrate solution for RBD) for 5 min and then stopped with 2N H2SO4. Optical density (OD) was read at 450 nm using a SpectraMax iD3x microplate reader (Molecular Devices) and endpoint titers were calculated using 3 times the background optical density as a cutoff. Values ​​below 100 were reported as 25.

[0152] Splenocyte restimulation assay. Immunized mice were sacrificed on day 35 and spleens were harvested. To isolate splenocytes, spleens were crushed through a 70 μm cell strainer and the resulting cell suspension was washed with PBS and incubated with ACK lysis buffer (2 ml, 2 min at room temperature) to lyse red blood cells. Splenocytes were washed again with PBS and plated into flat-bottom 96-well plates (2 × 10 per well). 6 Then, SARS-CoV-2 spike peptide (PepTivator SARS-CoV-2 Prot_S, Miltenyi Biotec) was added at a final concentration of 0.6 nmol / ml (total cell culture volume, 200 μl per well). After 96 h, supernatants were harvested and IFNγ levels were measured by ELISA (Thermo Fisher Scientific) according to the manufacturer's protocol.

[0153] SARS-CoV-2 surrogate virus neutralization test. The surrogate virus neutralization test was performed with a modification of a previously published protocol (Tan et al., 2020). Briefly, high-binding flat-bottom 96-well plates were coated with 2 μg / ml recombinant human ACE2 (hACE2, MilliporeSigma) in PBS, incubated overnight at 4 °C, washed three times with PBST, and blocked with PBS+BSA 1% for 1 h at room temperature. Meanwhile, each serum sample (final dilution 1:160) was preincubated with 3 ng of RBD-Fc (R&D Systems) in PBS+BSA 1% for 1 h at room temperature and then transferred to the hACE2-coated plates. As a positive control, RBD-Fc was also added to the hACE2-coated wells without preincubation with serum samples. After 1 h at room temperature, the plates were washed three times with PBST and incubated with HRP-conjugated anti-human IgG Fc antibody (Southern Biotech) for 1 h at room temperature. At the end of the incubation, the plates were washed five times with PBST and developed with tetramethylbenzidine (BD Biosciences) for 5 min, then stopped with 2N H2SO4. Optical density was read at 450 nm using a SpectraMax iD3x microplate reader (Molecular Devices). The inhibition rate of RBD binding to hACE2 was calculated by the following formula: Inhibition (%) = [1-(sample OD value-background OD value) / (control OD value-background OD value)] x 100.

[0154] Determination of SARS-CoV-2 neutralization titers. All serum samples were heat inactivated at 56 °C for 30 min to remove complement and equilibrated to room temperature before processing for neutralization titers. Samples were diluted in duplicate to an initial dilution of 1:5 or 1:10, followed by serial dilutions of 1:2, resulting in a 12-step dilution series with each well containing 100 μl. All dilutions were performed in DMEM (Quality Biological) supplemented with 10% (v / v) fetal bovine serum (heat inactivated, MilliporeSigma), 1% (v / v) penicillin / streptomycin (Gemini Bio-products) and 1% (v / v) L-glutamine (final concentration 2 mM, Thermo Fisher Scientific). The dilution plates were then transported to a BSL-3 laboratory, where 100 μl of diluted SARS-CoV-2 (WA-1, courtesy of Dr. Natalie Thornburg / CDC) inoculum was added to each well to result in a multiplicity of infection (MOI) of 0.01 when transferred to the titration plates. Untreated virus-only controls and mock-infected controls were included on each plate. The sample / virus mixtures were then transferred to 96-well titer plates with confluent VeroE6 cells after 1 h of incubation at 37°C (5.0% CO2). After incubating the titer plates at 37°C (5.0% CO2) for 72 h, cytopathic effect (CPE) was determined for each well of the plate. The first sample dilution showing CPE was reported as the minimum sample dilution required to neutralize >99% of the SARS-CoV-2 concentration tested (neut99).

[0155] Viral Scan. Phage IP and sequencing were performed as previously described (Xu et al., 2015a) with minor modifications. A sublibrary encoding a 56-mer peptide library tiling every 28 amino acids through the proteomes of six HCoVs and three bat coronaviruses most closely related to SARS-CoV-2 (Shrock et al., 2020) was mixed with the original VirScan library to allow mapping of SARS-CoV-2 epitopes. Each VirScan reaction contained 0.6 μl of mouse serum, or approximately 2 μg of IgG. Immunoprecipitation was performed using Protein A and Protein G magnetic Dynabeads (Thermo Fisher Scientific) as previously described (Xu et al., 2015a).

[0156] Statistics. When necessary, data were log-transformed before statistical analysis to approximate a normal distribution. One-sample t-tests were used to compare each group with a value of 1 (or 0 after log-transformation, representing the contralateral control sample by fold expression). Statistical differences between groups in datasets with one categorical variable were assessed by two-sample t-tests (two groups) or one-way ANOVA corrected for multiple comparisons (three or more groups). Statistical differences between groups in datasets containing two categorical variables were assessed by two-way ANOVA corrected for multiple comparisons. # or *, and ** or ## indicate p ≤ 0.05 and 0.01, respectively.

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[0158] Equivalents and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the embodiments described herein. The scope of the present disclosure is not limited to the above description, but rather is as set forth in the appended claims. Articles such as "a," "an," "the," and the like, mean one or more, unless indicated to the contrary or clear from the context. A claim or description containing "or" between two or more members of a group is deemed to be satisfied when one, more than one, or all of the group members are present, unless indicated to the contrary or clear from the context. A disclosure of a group containing "or" between two or more group members provides embodiments in which exactly one member of the group is present, embodiments in which more than one member of the group is present, and embodiments in which all members of the group are present. For the sake of brevity, these embodiments are not individually detailed herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

[0159] It should be understood that the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, or descriptive terms from one or more claims or from one or more relevant parts of the description are introduced into another claim. For example, a claim that is dependent on another claim can be amended to include one or more limitations found in other claims that are dependent on the same base claim. Furthermore, if a claim describes a composition, it should be understood that it includes the method of making or using the composition by any of the methods of making or using disclosed herein, or by any method known in the art, if any, unless otherwise stated or where a contradiction or inconsistency would occur.

[0160] When elements are presented as a list, for example in Markush group format, it is understood that all possible subgroups of elements are also disclosed, and any element or subgroup of elements can be removed from the group. It is also noted that the term "comprising" is intended to be open, allowing for the inclusion of additional elements or steps. In general, when an aspect, product, or method is said to include a particular element, feature, or step, it is understood that an aspect, product, or method consisting of or consisting essentially of such element, feature, or step is also provided. For the sake of brevity, these aspects are not individually detailed herein, but it will be understood that each of these aspects is provided herein and may be specifically claimed or disclaimed.

[0161] When ranges are specified, endpoints are included.Furthermore, unless otherwise indicated or clear from the context and / or the understanding of a person skilled in the art, values ​​expressed as ranges may in some embodiments assume any particular value within the stated range to the tenth of the unit of the lower limit of the range, unless otherwise clearly indicated by the context.For the sake of brevity, the values ​​of each range are not detailed individually herein, but it will be understood that each of these values ​​is provided herein and can be specifically claimed or disclaimed.It should also be understood that unless otherwise indicated or clear from the context and / or the understanding of a person skilled in the art, values ​​expressed as ranges may assume any subrange within the given range; where the endpoint of the subrange is expressed to the same degree of precision as the tenth of the unit of the lower limit of the range.

[0162] When websites are provided, the URL addresses are provided as code that cannot be executed by a browser, and the periods in each web address are shown in parentheses - the actual web address does not contain the parentheses. Furthermore, it should be understood that any particular aspect of the present disclosure may be expressly excluded from any one or more claims. Where a range is specified, values ​​within the range may be expressly excluded from any one or more claims. Any aspect, element, feature, use, or aspect of the compositions and / or methods of the present disclosure may be excluded from any one or more claims. For the sake of brevity, not all of the aspects in which one or more elements, features, purposes, or aspects are excluded are explicitly described herein.

Claims

1. An adjuvant system comprising a viral antigen and a fungal polysaccharide for use in a method of inducing an immune response against a virus in a subject in need thereof.

2. An immunogenic composition comprising a viral antigen and an adjuvant system including a fungal polysaccharide.

3. The fungal polysaccharide, (i) is a soluble polysaccharide; (ii) is a mannan; and / or (iii) isolated from Candida albicans, 2. An adjuvant system comprising a viral antigen and a fungal polysaccharide for the use according to claim 1, or a composition according to claim 2. (i) the adjuvant system further comprises alum, optionally wherein the fungal polysaccharide is adsorbed to the alum; (ii) the virus is a betacoronavirus selected from Middle East Respiratory Syndrome coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome (SARS)-related coronavirus (SARS-CoV)-1, and SARS-CoV-2; (iii) a) the viral antigen comprises a betacoronavirus protein or polypeptide, optionally wherein the betacoronavirus protein or polypeptide comprises a betacoronavirus spike protein or spike protein receptor binding domain (RBD), further optionally wherein the betacoronavirus spike protein is a MERS-CoV spike protein, a SARS-CoV-1 spike protein, or a SARS-CoV-2 spike protein, or wherein the betacoronavirus spike protein RBD is a MERS-CoV spike protein RBD, a SARS-CoV-1 spike protein RBD, or a SARS-CoV-2 spike protein RBD; or b) the viral antigen comprises a nucleic acid encoding a betacoronavirus protein or polypeptide, optionally wherein the nucleic acid is DNA or RNA, further optionally wherein the RNA is messenger RNA (mRNA); (iv) the viral antigen comprises a MERS-CoV, SARS-CoV-1, or SARS-CoV-2 viral particle; (v) the viral antigen comprises killed or inactivated MERS-CoV, SARS-CoV-1, or SARS-CoV-2; or (vi) the viral antigen comprises killed or live attenuated MERS-CoV, SARS-CoV-1, or SARS-CoV-2; 4. An adjuvant system comprising a viral antigen and a fungal polysaccharide for the use according to claim 1 or 3, or a composition according to claim 2 or 3. (i) the virus is an influenza A virus or an influenza B virus; (ii) a) the viral antigen comprises an influenza A virus or an influenza B virus protein or polypeptide, optionally wherein the influenza A virus or influenza B virus protein or polypeptide is a hemagglutinin (HA) protein, a neuraminidase (NA) protein, or a polypeptide thereof; or b) the viral antigen comprises a nucleic acid encoding an influenza A virus or influenza B virus protein or polypeptide, optionally wherein the nucleic acid is DNA or RNA, further optionally wherein the RNA is mRNA; (iii) the antigen comprises an influenza A virus or an influenza B virus virus particle; (iv) the antigen comprises a killed or inactivated influenza A virus or influenza B virus; or (v) the antigen comprises a killed or attenuated live influenza A virus or influenza B virus; 1. An adjuvant system comprising a viral antigen and a fungal polysaccharide, or a composition according to claim 2, 3 or claim 4(i), for the use according to claim 1, 3 or claim 4(i).

6. The subject matter comprises: (i) a) is a human, optionally wherein the human is a human newborn, a human infant, an adult, or an elderly person; or b) is a companion animal or a research animal; and / or (ii) is immunocompromised and has chronic lung disease, asthma, cardiovascular disease, cancer, obesity, diabetes, chronic kidney disease, and / or liver disease; An adjuvant system comprising a viral antigen and a fungal polysaccharide for use according to any one of claims 1, 3 to 5.

7. (i) The viral antigen and the adjuvant system are administered simultaneously or separately; (ii) the viral antigen and adjuvant system are administered intramuscularly, intradermally, orally, intravenously, topically, intranasally, or sublingually; (iii) administration is prophylactic; (iv) administration induces a type 1 immune response in the subject; (v) the adjuvant system promotes activation of dendritic cell-associated C-type lectin 2 (Dectin 2) in the subject; (vi) the adjuvant system elicits an innate immune response in the subject; (vii) the adjuvant system enhances B cell immunity; and / or (viii) the adjuvant system enhances the production of antigen-specific antibodies compared to administration of the viral antigen alone, optionally wherein the anti-specific antibodies are of the IgG2c type; An adjuvant system comprising a viral antigen and a fungal polysaccharide for use according to any one of claims 1, 3 to 6.

8. The adjuvant system comprising: (i) enhances the production of antigen-specific antibodies that target a broader range of epitopes compared to administration of the viral antigen alone; (ii) enhances cytokine production compared to administration of the viral antigen alone, optionally where the cytokine comprises IFNγ; (iii) polarizing the innate immune response towards T follicular helper (Tfh) cell immunity; (iv) polarizing the innate immune response towards T helper 1 (Th1) cell immunity; (v) prolonging the protective effect against a viral antigen in a subject compared to administering the viral antigen alone; and / or (vi) increasing the rate of immune response compared to administering the viral antigen alone; An adjuvant system comprising a viral antigen and a fungal polysaccharide for use according to any one of claims 1, 3 to 7.

9. An adjuvant system comprising a viral antigen and a fungal polysaccharide for use as described in any one of claims 1, 3 to 8, wherein the viral antigen generates the same level of immune response to the antigen in the presence of the adjuvant system at a lower dose compared to administering the viral antigen alone.

10. An adjuvant system comprising a viral antigen and a fungal polysaccharide for use as described in any one of claims 1, 3 to 9, wherein the likelihood of antibody-mediated disease enhancement (ADE) is reduced in a subject compared to administering the viral antigen alone.

11. An adjuvant system comprising a fungal polysaccharide for use in inducing an immune response against a virus in a subject in need thereof.

12. An adjuvant system comprising a fungal polysaccharide and alum for use in inducing an immune response against a virus in a subject in need thereof.

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