WO2025231068A1 - Compositions and methods for enhancing systemic and mucosal immune responses - Google Patents

Abstract

The disclosure provides compositions and methods for improving and enhancing systemic and mucosal vaccine immune responses. In particular, the disclosure provides mucosal adjuvant for vaccine delivery and methods of using the mucosal adjuvant in a vaccination regimen to induce sterilizing immunity in a vaccinated subject.

Description

COMPOSITIONS AND METHODS FOR ENHANCING SYSTEMIC AND MUCOSAL

IMMUNE RESPONSES

RELATED APPLICATION INFORMATION

[0001] This application claims priority to U.S. Patent Application No. 63/640,639, filed on April 30, 2024, the contents of which are herein incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under All 60706, and All 76069 awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING STATEMENT

[0003] The contents of the electronic sequence listing titled

UM_43082_601_SequenceListing.xml (Size: 2,641 bytes; and Date of Creation: April 29, 2025) is herein incorporated by reference in its entirety.

BACKGROUND

[0004] Infectious diseases remain a problem throughout the world. While some progress has been made developing vaccines against some pathogens, there are many that remain a threat to human health. Examples include viruses such as smallpox, coronaviruses, Ebola, influenza and HIV, and bacterial pathogens such as B. anthracis and S', pneumonia.

[0005] While some vaccines exist, many have failed to prevent spread and/or fail to provide protective or sterilizing immunity. With regard to influenza and other seasonal viruses, vaccines have failed to provide adequate immunity.

[0006] Generating and deploying an effective vaccine and vaccine regimen relies on a combination of achievements. A useful vaccine and/or vaccine regimen must stimulate an effective immune response (e.g., a protective immune response and/or a sterilizing immune response) that reduces infection or disease by a sufficient amount to be beneficial. [0007] There remains a need for methods and compositions for inducing potent and durable immune responses against microbial pathogens (e.g., coronaviruses, smallpox, B. anthracis, S. pneumonia, etc.) responsible for infectious disease.

BRIEF SUMMARY

[0008] The disclosure provides compositions and methods for improving and enhancing systemic and mucosal vaccine immune responses. In particular, the disclosure provides mucosal adjuvant for vaccine delivery and methods of using the mucosal adjuvant in a vaccination regimen to induce protective immunity and/or sterilizing immunity in a vaccinated subject. [0009] In one aspect, the disclosure provides mucosal adjuvant for vaccine delivery and methods of using the mucosal adjuvant in a vaccination regimen to induce immunity (e.g., protective immunity and/or sterilizing immunity) in a subject. The methods and compositions disclosed are not limited to any particular setting and include but are not limited to treatment (e.g., prophylactic and/or therapeutic treatment) of a variety of diseases and conditions (e.g., methods and compositions of the disclosure can be used in combination with a broad range of vaccine types to induce protective immune responses (e.g., protective immunity and/or sterilizing immunity) to a variety of diseases and conditions including, but not limited to, infectious disease and cancer). In some embodiments, methods and compositions disclosed herein (e.g., methods of vaccination) are used with any available vaccine to improve and/or enhance a subject’s immune response thereto.

[0010] In some embodiments, the disclosure provides a method of inducing an immune response in a subject comprising administering a primary, parenterally administered immunogenic composition to the subject, wherein the primary, parenterally administered immunogenic composition induces an immune response to a pathogenic organism; and subsequently administering a secondary, mucosally administered immunogenic composition to the subject, wherein the secondary, mucosally administered immunogenic composition comprises a nanoemulsion, an agonist of retinoic acid-inducible gene I (RIG-I) and an antigen; and wherein the secondary, mucosally administered immunogenic composition induces protective immunity and/or sterilizing immunity to the pathogenic organism. The disclosure is not limited by the type or the source of the parenterally administered immunogenic composition. In some embodiments, the parenterally administered immunogenic composition is a vaccine (e.g., any vaccine known in the art and/or that is commercially available). For example, in some embodiments, the parenterally administered immunogenic composition is an inactivated virus vaccine, a live-attenuated virus vaccine, a messenger RNA (mRNA) vaccine, a subunit vaccine, a recombinant vaccine, a polysaccharide vaccine, a conjugate vaccine, a toxoid vaccine, a pseudotyped virus vaccine or a viral vector vaccine. In some embodiments, the vaccine stimulates a subject’s immune system against a particular infectious agent such as a pathogenic organism (e.g., providing acquired immunity to the particular pathogenic organism or infectious disease in the subject). The disclosure is not limited to any particular pathogenic organism or infectious disease. Indeed, the parenterally administered immunogenic composition may be a vaccine against any pathogenic organism or infectious disease disclosed herein or known in the art. In some embodiments, the primary, parenterally administered immunogenic composition comprises a coronavirus vaccine (e.g., a protein subunit vaccine, a whole virus vaccine, a live- attenuated virus vaccine, an inactivated virus vaccine, an mRNA vaccine, or a pseudotyped virus vaccine). In other embodiments, the primary, parenterally administered immunogenic composition comprises a respiratory syncytial virus vaccine. In still other embodiments, the primary, parenterally administered immunogenic composition comprises a S. pneumonia vaccine. The disclosure is not limited by the route of administration of the primary, parenterally administered immunogenic composition to the subject. Indeed, any route of parenteral administration may be used including, but not limited to, administering intramuscularly, intraperitoneally, intravenously, intracerebroventricularly, intracistemal injection or infusion, subcutaneously, or via implant. In some embodiments, the secondary, mucosally administered immunogenic composition comprises a nanoemulsion, an agonist of retinoic acid-inducible gene I (RIG-I) and an antigen selected from a protein, recombinant protein, recombinant polypeptide, lipid, carbohydrate, polysaccharide, protein extract, cell or cellular extract, tumor cell or tumor cell extract, and tissue. The disclosure is not limited by the type of nanoemulsion utilized in the secondary, mucosally administered immunogenic composition. A variety of nanoemulsions are disclosed herein and may be utilized. Likewise, the disclosure is not limited by the type of RIG-I agonist. Any one or more of the RIG-I agonists disclosed herein may be used. In some embodiments, the secondary, mucosally administered immunogenic composition comprises a nanoemulsion, an agonist of retinoic acid-inducible gene I (RIG-I) and one or more immunogens (e.g., immunogenic polypeptides) from a pathogenic organism. For example, in some embodiments, the secondary, mucosally administered immunogenic composition comprises a nanoemulsion, an agonist of retinoic acid-inducible gene I (RIG-I) and one or more immunogenic polypeptides from a respiratory pathogen (e.g., one or more immunogenic polypeptides from a coronavirus, one or more immunogenic polypeptides from repiratory syncytial virus, or one or more immunogenic polypeptides from a S. pneumonia). In some embodiments, the secondary, mucosally administered immunogenic composition comprises a nanoemulsion, an agonist of retinoic acid-inducible gene I (RIG-I) and coronavirus S protein and/or coronavirus receptor binding domain (RBD)). In some embodiments, administering the secondary, mucosally administered immunogenic composition to the subject comprises intranasal administration. In a preferred embodiment, the secondary, mucosally administered immunogenic composition induces protective immunity and/or sterilizing immunity to the pathogenic organism that is not achievable by re-vaccinating the subject using the primary, parenterally administered immunogenic composition. In some embodiments, the protective immunity and/or sterilizing immunity to the pathogenic organism obtained comprises sterilizing immunity in subjected respiratory tract (e.g., the lower respiratory tract and/or the upper respiratory tract). In some embodiments, sterilizing immunity obtained in the upper respiratory tract comprises inhibition of replication of the pathogenic organism in the lungs and/or nasal turbinates of the subject.

[0011] In another aspect, the disclosure provides a method of enhancing an immune response to a coronavirus (e.g., SARS-CoV-2) vaccine in a subject comprising providing a coronavirus vaccinated subject; and intranasally administering to the coronavirus vaccinated subject a boost vaccination, wherein the boost vaccination comprises a nanoemulsion, an agonist of retinoic acid-inducible gene I (RIG-I), and a recombinant coronavirus immunogenic polypeptide (e.g., S protein). The disclosure is not limited by the type of vaccination the coronavirus vaccinated subject received to become a coronavirus vaccinated subject. Indeed, the coronavirus vaccinated subject may have received any coronavirus vaccine available in the art. In some embodiments, the coronavirus vaccinated subject received (was vaccinated with) a coronavirus vaccine selected from mRNA-1273, BNT162b2, JNJ-78436735, and Vaxzevria. The disclosure is not limited by the type of RIG-I agonist. Any one or more of the RIG-I agonists disclosed herein may be used. For example, in some embodiments, an agonist of RIG-I is an RNA agonist. In some embodiments, the RNA agonist is a defective interfering (DI) RNA (e.g., of a Sendai virus (SeV) or an influenza virus). In some embodiments, a RIG-I agaonist/activator comprises an in vitro transcribed defective interfering (DI) RNA derived from Sendai virus comprising a 5'- triphosphate (5'-ppp) and double-stranded RNA (dsRNA) stem structure (e.g., comprising all or a portion of SEQ ID NO: 1). In some embodiments, a RIG-I activator is chemically synthesized (e.g., a chemically synthesized short stem-loop RNA molecule comprising a 5 '-triphosphate group (also referred to as a 3pRNA or SLR RNAs; e.g., SLR14)). In some embodiments, a RIG-I activator is a small molecule agonist (e.g., including, but not limited to, Inarigivir (SB 9200) or other small molecule that engages and activated RIG-I and/or NOD2). The disclosure is not limited by the type of nanoemulsion utilized in the secondary, mucosally administered immunogenic composition. A variety of nanoemulsions are disclosed herein and may be utilized. In some embodiments, the nanoemulsion comprises a poloxamer surfactant or polysorbate surfactant; an organic solvent; a halogen containing compound; oil, and water. For example, in some embodiments, the nanoemulsion comprises Tween (e.g., Tween 80); an alcohol (e.g., ethanol; cetylpyridinium chloride (CPC); oil (e.g., soybean oil); and water. In a preferred embodiment, enhancing an immune response to a coronavirus vaccine in the subject comprises generation of protective immunity in the subject that is not achievable by vaccinating the coronavirus vaccinated subject using the vaccine used to originally vaccinate the subject. In some embodiments, the protective immunity is sterilizing immunity to coronavirus. In some embodiments, enhancing an immune response to a coronavirus vaccine in the subject comprises generation of neutralizing antibodies to coronavirus in the respiratory tract (e.g., upper respiratory tract and/or lower respiratory tract) of the subject. In some embodiments, enhancing an immune response to a coronavirus vaccine in the subject comprises generation of sterilizing immunity to coronavirus in the respiratory tract (e.g., upper respiratory tract and/or lower respiratory tract) of the subject. In some embodiments, enhancing an immune response to a coronavirus vaccine in the subject comprises inhibition of coronavirus replication in the subject (e.g., in the lungs of the subject and/or in the nasal turbinates of the subject).

[0012] In another aspect the disclosure provides a method of treating, protecting against, and/or preventing infection by a coronavirus (e.g., SARS-CoV-2) in a subject in need thereof, the method comprising administering a primary, parenterally administered coronavirus mRNA vaccine to the subject; and subsequently mucosally administering a boost vaccination, wherein the boost vaccination comprises a nanoemulsion, an agonist of retinoic acid-inducible gene I (RIG-I), and recombinant coronavirus immunogenic polypeptide (e.g., S protein), wherein the boost vaccination induces protective immunity and/or sterilizing immunity in the subject. In some embodiments, the protective immunity and/or sterilizing immunity obtained in the subject is not achievable by re- vaccinating the subject with the primary, parenterally administered coronavirus mRNA vaccine. In some embodiments, the protective immunity is sterilizing immunity to coronavirus. In some embodiments, the protective immunity and/or sterilizing immunity comprises generation of neutralizing antibodies to coronavirus in the respiratory tract (e.g., upper respiratory tract and/or lower respiratory tract) of the subject. In some embodiments, the protective immunity and/or sterilizing immunity comprises generation of sterilizing immunity to coronavirus in the respiratory tract (e.g., upper respiratory tract and/or lower respiratory tract) of the subject. In some embodiments, the protective immunity and/or sterilizing immunity comprises inhibition of coronavirus replication in the subject (e.g., in the lungs of the subject and/or in the nasal turbinates of the subject). In some embodiments the protective immunity and/or sterilizing immunity inhibits transmission of the coronavirus from the subject.

BRIEF DESCRIPTION OF THE DRAWING(S)

[0013] FIG. 1 shows heterologous IM/IN prime-boost immunization induces robust S protein-specific IgG and enhances mucosal IgA production compared to homologous mRNA IM/IM prime-boost. (FIG. 1 A) C57B1/6 mice were given two immunizations 4- wks apart. Mice were primed either IM with 0.25pg of BNT162b2 mRNA or PBS, or IN with 15 pg full-length S protein with either NE or NE/IVT. Mice were then boosted IM with 0.25 pg of BNT162b2 mRNA or PBS, or IN with PBS or S protein in PBS, NE or NE/IVT as indicated. Serum antigenspecific total IgG titers against (FIG. IB) WT S protein and (FIG. 1C) WT RBD as measured by ELISA 2wks after the prime immunization, and (FIG. ID and FIG. IE) 2wks after the boost immunization at wk6. (FIG. IF and FIG. 1H) Subclass profiles for S-specific serum antibodies measured at wk6. BALF S-specific (FIG. II) IgA and (FIG. 1 J) IgG measured at wk6. (n=5/grp; *p<0.05, **p<0.01, ****p<0.0001 by Mann- Whitney U test shown only for select groups).

[0014] FIG. 2 shows heterologous IM/IN prime-boost immunization induces robust crossneutralizing antibody responses against multiple variant viruses. Serum neutralizing antibody titers from immunized C57B1/6 mice at wk 6 after both prime/boost immunizations with the indicated adjuvant/antigen regimens were measured using pseudoviruses for the (FIG. 2A) wildtype, (FIG. 2B) B.1.617.2, (FIG. 2C) B.1.351, and (FIG. 2D) B.l.1.529 (BA.l) variants. (n=5/grp; *p<0.05, **p<0.01 by Mann- Whitney U test shown only for select groups).

[0015] FIG. 3 shows antigen recall responses assessed in splenocytes isolated from IM/IN immunized mice demonstrate enhanced TH1/TH17 profiles. Splenocytes were isolated from mice given prime/boost immunizations with the indicated adjuvant/antigen regimens two weeks after the final immunization (wk 6). Splenocytes were stimulated ex vivo with 5 pg S protein for 72h, and levels of secreted (FIG. 3 A) IFN-y, (FIG. 3B) IL-2, (FIG. 3C) IP- 10, (FIG. 3D) TNF- a, (FIG. 3E) IL-5, (FIG. 3F) IL-13, (FIG. 3G) IL-6, (FIG. 3H) IL- 17 A, and (FIG. 31) IL-10 were measured by multiplex immunoassay relative to unstimulated cells. (n=4-5/grp; *p<0.05, **p<0.01 by Mann- Whitney U test shown only for select groups).

[0016] FIG. 4 shows antigen recall responses assessed in cervical lymph node isolates from IM/IN immunized mice demonstrate even greater enhancement in TH 1 /TH 17 profiles. cLN cellular isolates from mice given prime/boost immunizations with the indicated adjuvant/antigen regimens were harvested at wk 6 and stimulated ex vivo with 5 pg S protein for 72h. Levels of secreted (FIG. 4A) IFN-y, (FIG. 4B) IL-2, (FIG. 4C) IP- 10, (FIG. 4D) TNF-a, ( FIG. 4E) IL-5, (FIG. 4F) IL-13, (FIG. 4G) IL-6, (FIG. 4H) IL- 17 A, and (FIG. 41) IL-10 were measured by multiplex immunoassay relative to unstimulated cells. (n -5/grp; *p<0.05, **p<0.01 by Mann- Whitney U test shown only for select groups).

[0017] FIG. 5 shows that IN administration of antigen with NE adjuvants after IM mRNA priming effectively pulls antigen-specific immunity to mucosal sites. Single cell suspension were isolated from the spleen, cLNs, and lungs of mice given prime/boost immunizations with the indicated adjuvant/antigen regimens. Mice were given 2 pg mRNA IM, and 20pg of S protein IN in either PBS, NE, or NE/IVT. Cells were stimulated with 25pg/mL of S protein, and antigenspecific cytokine responses were quantified in CD4⁺ T cells by intracellular cytokine staining and FACS analysis. (n -5/grp with data represented as mean ± SEM; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by one-way ANOVA with Tukey post-hoc test shown only for select groups).

[0018] FIG. 6 shows that heterologous IM/IN prime-boost immunization induces robust virus-neutralizing antibody titers in 129S1 and K18-hACE mice. 129S1 and K18-hACE2 mice were vaccinated twice 4-wks apart. Mice were primed either IM with 0.25 pg of BNT162b2 mRNA or PBS, or IN with 15 pg full-length S protein with either NE or NE/IVT. Mice were then boosted 4 wks later IM with 0.25pg of BNT162b2 mRNA, or IN with 15 pg S with PBS, NE or NE/IVT as indicated. Groups receiving two immunizations with IM Advx/S or PBS were included for comparison. Serum (FIG. 6A) IgG titers against WT S-protein, and nAb titers against (FIG. 6B) WT, (FIG. 6C) B.1.351, (FIG. 6D) B.l.1.529 (BA.l), and (FIG. 6E) BA.4/5 variant PSVs were measured 2wks after the boost immunization (wk6). K18-hACE2 transgenic mice were similarly primed either IM with 0.25 pg of BNT162b2 mRNA, or PBS, or IN with 15 pg S with either NE or NE/IVT. Mice were boosted 4 wks later IM with 0.25pg of BNT162b2 mRNA or PBS, or IN with 15 pg S with PBS, NE or NE/IVT as indicated. Groups receiving two immunizations with IM Advx/S or IN PBS were included for comparison. Serum (FIG. 6F) IgG titers against WT S -protein, and nAb titers against (FIG. 6G) WT, (FIG. 6H) B.1.351, (FIG. 61) B.1.1.529 (BA.l), and (FIG. 6J) BA.4/5 variant PSVs were measured 2wks after the boost immunization (wk6). (n=4-5/grp;*p<0.05, **p<0.01 by Mann- Whitney U test shown only for select groups).

[0019] FIG. 7 shows that heterologous IM/IN prime/pull and IN/IN immunization strategies provide sterilizing immunity upon heterologous challenge in both the upper and lower respiratory tracts in contrast to IM/IM immunization with BNT162b2 mRNA or Addavax/S. 129S1 mice were primed either IM with 0.25 g of BNT162b2 mRNA or PBS, or IN with 15 pg S with either NE or NE/IVT. Mice were boosted 4 wks later IM with 0.25pg of BNT162b2 mRNA, or IN with 15 pg S with PBS, NE or NE/IVT as indicated. Groups receiving two immunizations with IM Advx/S or IN PBS were included for comparison. 3 wks post-boost immunization, mice were challenged IN with 10⁴ pfu B.1.351, and viral titers were measured at 4 d.p.i. in the (FIG. 7A) lungs and (FIG. 7B) nasal turbinates. KI 8-hACE2 mice were primed IM with 0.25pg of BNT162b2 mRNA and boosted 4 wks later IM with 0.25 g of BNT162b2 mRNA, or IN with PBS alone, or 15 pg S with PBS, NE or NE/IVT as indicated. Groups receiving two immunizations with IM Advx/S or IN PBS were included for comparison. 3 wks post-boost immunization, mice were challenged IN with 10⁴ pfu BA.5, and viral titers were measured at 4 dpi in the (FIG. 7C) lungs and (FIG. 7D) nasal turbinates. (n=4-5/grp;*p<0.05, **p<0.01 by Mann- Whitney U test).

[0020] FIG. 8 shows that cytokine/chemokine levels in lung homogenates from immunized mice post-challenge demonstrate different host response skewing depending on vaccination type and route. Cytokine and chemokine levels in lung homogenate measured by multiplex immunoassay from immunized (FIG. 8A) 129S1 mice in Figure 7 measured at 4 d.p.i. with 10⁴ pfu B.1.351 , (FIG. 8B) KI 8-hACE2 mice in Figure 7 measured at 4 d.p.i. with 10⁴ pfu BA.5. Individual cytokine/chemokine levels were normalized to the cytokine/chemokine range and then normalized based on multiplication with log2 fold changes to normalize expression changes. Heatmap shows expression changes for the mean of each group. [0021] FIG. 9 shows that cytokine production in lung homogenates from 129S1 immunized mice post-challenge demonstrate different host response skewing depending on vaccination type and route. Individual cytokine levels in lung homogenate (shown as heatmap in Figure 8) measured by multiplex immunoassay from immunized 129S1 mice in Figure 7 measured at 4 d.p.i. with 10⁴ pfu B.1.351. (A) IFN-y, (B) IL-2, (C) TNF-a, (D) IL-12p70, (E) IP-10, (F) IL-4, (G) IL-5, (H) IL-13, (I) IL-6, (J) IL-17A, (K) IL-10, (L) IL-22, (M) IL-23, (N) IL-27, (O) IL-18, (P) IL-9, (Q) IL-ip, (R) MCP-1, (S) MCP-3, (T) MIP-la, (U) MIP-ip, (V) MIP-2a, (W) RANTES, (X) GROa, (Y) GM-CSF, (Z) eotaxin («=4-5/grp; *p<0.05, **p<0.01 by Mann- Whitney U test).

[0022] FIG. 10 shows that cytokine production in lung homogenates from KI 8-hACE2 immunized mice post-challenge demonstrate different host response skewing depending on vaccination type and route. Individual cytokine levels in lung homogenate (shown as heatmap in Figure 8) measured by multiplex immunoassay from immunized K18-hACE2 mice in Figure 7 measured at 4 d.p.i. with 10⁴ pfu BA.5. (A) IFN-y, (B) IL-2, (C) TNF-a, (D) IL-12p70, (E) IP- 10, (F) IL-4, (G) IL-5, (H) IL-13, (I) IL-6, (J) IL-17A, (K) IL-10, (L) IL-22, (M) IL-23, (N) IL- 27, (O) IL-18, (P) IL-9, (Q) IL-ip, (R) MCP-1, (S) MCP-3, (T) MIP-la, (U) MIP-ip, (V) MIP- 2a, (W) RANTES, (X) GROa, (Y) GM-CSF, (Z) eotaxin (n=4-5/grp; *p<0.05, **p<0.01 by Mann-Whitney U test).

[0023] FIG. 11 shows mean fluorescent intensity (MFI) of IFN-y expressing CD4 T cells. Mean fluorescent intensity of IFN-y expression in CD4 T cells in spleen, cervical lymph node (cLN) and lung cells stimulated with 25pg/mL Spike protein for 24 hours. (n=4-5/grp; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by One way ANOVA with Tukey post-hoc test).

[0024] FIG. 12 shows IL-17A expression is highly induced in the lung by two doses of antigen with NE. Single cell suspension were isolated from the lungs of mice immunized IM with 2 pg BNT162b2 mRNA, or IN with 20 pg of Spike protein in either PBS, NE, or NE/IVT. Cells were stimulated with 25pg/mL of S protein and IL-17A responses were quantified in CD4 T cells by intracellular cytokine staining. Data was analyzed by one-way ANOVA with Tukey post-hoc test. *p<0.05, **p<0.01, ***p<0.001.

[0025] FIG. 13 shows S-specific IgG induced 2 wks post-prime immunization in (FIG. 13 A) 129S1 and (FIG. 13B) K18hACE2 mice immunized IM with 0.25pg of BNT162b2 mRNA or Advx with 15 pg S, or IN with 15 pg S with either NE or NE/IVT or PBS (n=5/grp; *p<0.05, **p<0.01, *** <0.001, ****£><0.0001 by Mann- Whitney U test).

[0026] FIG. 14 shows heterologous immunization with NE/IVT adjuvanted spike induces neutralizing antibodies and mucosal IgA with prime immunization location shaping humoral immune responses. (A) Mice were immunized with a prime/boost/boost schedule at 3 week intervals and additionally boosted at wk 17. Humoral response profiling was done at wk2, wk 5, and wk8 , and cellular and mucosal responses were characterized at wk 8. Serum levels of WT-S IgG 2 weeks post prime immunization (wk2) (B), 2 weeks post prime/boost (wk5) (C), and 2 weeks post prime/boost/boost (wk 8) immunization (D) determined by ELISA. Serum neutralization antibody titers post prime/boost/boost to WT PSV (E), B.1.351 PSV (F), OMI BA.l PSV (G) and OMI BA.4/BA.5 PSV (H) as measured by a microneutralization assay. BALF - IgA specific to WT-S (I) and OMI BA.l-S (J) in mice after prime/boost/boost immunization. Significance was determined with Mann Whitney U test (Serum) or one-way ANOVA with Tukey post-hoc test (BALF). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0027] FIG. 15 shows heterologous boosting with IN NE/IVT/S drives the formation of spike-specific T cells in mucosal tissues compared to homologous immunization and priming mucosally. Single cell suspensions from the spleens, cervical lymph nodes, lungs and NALT of immunized mice were isolated 2 weeks post prime/boost/boost. Cells were stained for viability, surface markers, and S-specific cells. (A) Representative gating pathway for CD8+Class I Tetramer+ and CD4+Class II Tetramer+ in spleen and cLN. Frequency of spike-specific effector memory (CD3+CD8+CD44+Class I Tet+CD69+CD62L-) CD8+ T cells (B,D) and spike-specific effector memory (CD3+CD4+CD44+Class II Tet+CD69+CD62L-) CD4+ T cells (C,E) in the spleen and cLN. (F) Representative gating pathway for CD8+Class I Tetramer+ in NALT and lungs. Frequency of spike-specific tissue resident memory (CD3+CD8+IV(CD45)-CD44+Class I Tet+CD69+CD103+) CD8+ T cells in the lungs (G) and NALT (H). Significance was determined with one-way ANOVA with Tukey post-hoc test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0028] FIG. 16 shows heterologous IN boost immunization with NE/IVT/S induces Thl/Thl7 cellular immune responses at systemic and mucosal locales. Single cell suspensions from the spleens, cervical lymph nodes, and lungs of vaccinated mice were isolated 2 weeks post prime/boost/boost and stimulated with 25pg/mL SARS-CoV-2 WT S protein for 24 hours. Cells were stained for viability, surface markers, and intracellular cytokines. (A). Representative gating pathway to discriminate cytokine expression in CD4 and CD8 T cells. Cytokine expression data in CD4 T cells and CD8 T cells in the spleen (B-F), cLN (G-K), and lungs (L-P). Significance was determined with one-way ANOVA with Tukey post-hoc test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0029] FIG. 17 shows Heterologous IN NE/TVT/S boosting drives a Thl/Thl7 response in spleen and cLN while boosting with IM mRNA drives a Thl/Th2 response in the spleen. Single cell suspensions from spleen (A-G) and cervical lymph nodes (H-N) of vaccinated mice were isolated 2 weeks post prime/boost/boost and stimulated with 25pg/mL SARS-CoV2 WT S protein for 3 days. Supernatants were assessed for IFN-y (A, H), IL-2 (B, I), TNFa (C, J), IL-5 (D, K), IL- 13 (E, L), IL-17A (F, M) and IL- 10 (G, N) by bead-based Luminex. Significance was determined with one-way ANOVA with Tukey post-hoc test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0030] FIG. 18 shows heterologous immunization with NE/TVT adjuvanted spike is protective in a lethal model of SARS-CoV-2. KI 8-hACE2 mice were immunized in a prime/boost/boost schedule at 3 weeks intervals and then infected with 104PFU of B.1351 SARS-CoV-2, 3 weeks post final boost. Homogenates from lungs (A), nasal turbinates (B), and brain (C) were tested for the presence of virus with plaque assay at the indicated time point post infection. (D) Weight loss in mice post-infection with B.1351 SARS-CoV-2. (E) Heatmap of cytokines and chemokines in lung homogenates at 3 days post infection. Individual cytokine/chemokines were normalized to the range and then normalized based on multiplication with log2 fold changes to normalize expression changes. Significance was determined with Kruskal-Wallis test with Dunn’s post-hoc test or two-way ANOVA with Tukey post-hoc test. *p<0.05, **p<0.01.

[0031] FIG.19 shows heterologous immunization with IN NE/IVT/S boosting induces a more durable humoral and cellular systemic and mucosal immune response than homologous immunization. Mice were immunized in a prime/boost/boost schedule at 3 week intervals with spleens and cervical lymph nodes harvested at 8 weeks (2 weeks post final boost) and at 18 weeks (12 weeks post final boost). Serum neutralization antibody titers at wk8 and wkl8 to WT PSV (A) and OMI BA.4/BA.5 PSV (B) as measured by a microneutralization assay. (C) BALF - IgA specific to WT-S at wk8 and wkl8. Single cell suspensions of spleens, cLN, and lungs were stimulated with 25pg/mL SARS-CoV2 WT S protein overnight. (D-H) Frequency of cytokine expressing CD4⁺ and CD8⁺ T cells in spleen after overnight stimulation. (I-M) Frequency of cytokine expressing CD4⁺ and CD8⁺ T cells in cLN after overnight stimulation. (N-R) Frequency of cytokine expressing CD4⁺ and CD8⁺ T cells in lungs after overnight stimulation. Durability effects significance determined by mixed-effects model with Tukey post-hoc test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0032] FIG. 20 shows Heterologous immunization with IN NE/IVT/S boosting induces a more durable immune response than homologous immunization. Mice were immunized in a prime/boost/boost schedule at 3 week intervals with spleens and cervical lymph nodes harvested at 8 weeks (2 weeks post final boost) and at 18 weeks (12 weeks post final boost). Single cell suspensions of spleens and cLN were stimulated with 25pg/mL SARS-CoV2 WT S protein. IFN-y (A), IL-5 (B), IL-10 (C), and IL-17A (D) in spleen supernatants after 3 days assessed by bead-based Luminex. IFN-y (E), IL-5 (F), IL-10 (G), and IL-17A (H) in cLN supernatants after 3 days assessed by bead-based Luminex. Durability effects significance determined by mixed- effects model with Tukey post-hoc test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0033] FIG. 21 shows Heterologous prime-pull immunization with IN NE/IVT/S is more responsive to further IM mRNA and IN NE/IVT/S boosting than homologous IM mRNA prime/boost. Mice were immunized in a prime/boost/boost schedule at 2 week intervals over 6 weeks. Mice were boosted again at week 17 either IM with mRNA vaccine or IN with NE/IVT/S. Spleens, cervical lymph nodes, and lungs were harvested at week 8 (2 weeks post boost of initial regimen) and at 19 weeks (2 weeks additional boost). Serum neutralization antibody titers to WT PSV (A) and OMI BA.4/BA.5 PSV (B) variant 2 weeks post additional boost immunization as measured by microneutralization assay. (C) BALF - IgA specific to WT-S 2 weeks post additional boost immunization. Single cell suspensions of spleens, cLN, and lung were stimulated for 25pg/mL SARS-CoV2 WT S protein for 24 hours. (D-H) Frequency of cytokine expressing CD4⁺ T cells and CD8⁺ T cells in spleen. (I-M) Frequency of cytokine expressing CD4⁺ T cells and CD8⁺ T cells in cLN. (N-R) Frequency of cytokine expressing CD4⁺ T cells and CD8⁺ T cells in lungs. Boosting effect significance determined by two-way ANOVA with Dunnett’s post-hoc test or Tukey’s post-hoc test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0034] FIG. 22 shows route of priming immunization in heterologous immunization drives antibody diversification and class switching. Mice were immunized in a prime/boost/boost schedule at 3 weeks interval. Serum levels of OMI BA.l-S IgG (A) 2 weeks post prime/boost/boost immunization. Levels of WT-S IgGl (B), IgG2b (C), IgG2c (D) 2 weeks post prime/boost/boost immunization. Significance was determined with Mann Whitney U test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0035] FIG. 23 shows homologous mRNA-LNP vaccination enhances the frequency of CD8⁺ T cells within the circulation. Single cell suspensions from the spleens, cervical lymph nodes, lungs and NALT of vaccinated mice were isolated 2 weeks post prime/boost/boost immunization. Cells were stained for viability, surface markers, and tetramer. Frequency of total CD8⁺ T cells within the spleen (A), cLN (C), lungs (E), and NALT (F). Frequency of total CD4⁺ T cells within the spleen (B) and cLN (D). Significance was determined with one-way ANOVA with Tukey post-hoc test with significance set at p<0.05. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0036] FIG. 24 shows heterologous IN NE/IVT/S boosting drives a Thl response in spleen while boosting with IM mRNA drives a Thl response only in the spleen. Single cell suspensions from the spleens of vaccinated mice were isolated 2 weeks post final immunization and stimulated with 25pg/mL SARS-CoV2 WT S protein for 24 hours with Brefeldin A for the last 6 hours. Cells were stained for viability, surface markers, and intracellular cytokines. IL-2 and TNFa expression data in CD4 T cells in spleen (A,B), cLN (C,D), and lungs (E,F). Significance was determined with one-way ANOVA with Tukey post-hoc test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0037] FIG. 25 shows Heterologous IN NE/IVT/S boosting enhances IL-6 expression in spleen and cLN. Single cell suspensions from spleens and cervical lymph nodes of vaccinated mice were isolated 2 weeks post final immunization and stimulated with 25pg/mL SARS-CoV2 WT S protein for 72 hours. Supernatants were assessed for IL-4 (A, C) and IL-6 (B, D) by beadbased Luminex. Significance was determined with one-way ANOVA with Tukey post-hoc test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0038] FIG. 26 shows heterologous immunization induces neutralizing antibodies in KI 8- hACE2 mice. KI 8-hACE2 mice were immunized in a prime/boost/boost schedule at 2 weeks interval. Serum levels of WT-S IgG (A) and OMI BA.l-S IgG (B) 2 weeks post prime/boost/boost immunization. Serum neutralization antibody titers to WT PSV (C) and B.1.351 PSV (D) variant 2 weeks post prime/boost/boost immunization as measured by microneutralization assay. Significance was determined with Mann Whitney U test. *p<0.05,

**p<0.01, ***p<0.001, ****p<0.0001. [0039] FIG. 27 shows heterologous immunization with NE/IVT/S boosting induces a more durable cellular response both systemically and in mucosal tissues. Mice were immunized in a prime/boost/boost schedule at 2 week intervals with spleen, cervical lymph nodes, and lung harvested at 8 weeks (2 weeks post final boost) and at 18 weeks (12 weeks post final boost). Single cell suspensions of spleens and cLN were stimulated for 25pg/mL SARS-CoV2 WT S protein. TNFa expression in CD4⁺ T cells in spleen (A), cLN (D), and lungs (G) after 24 hours. IL-2 (B,E) and TNFa (C,F) were assessed in spleen (B,C) and cLN (E,F) by bead-based Luminex. Significance determined by mixed-effects model with Tukey post-hoc test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0040] FIG. 28 shows spike specific T cells in mucosal tissues and systemically are more long lived in mice immunized with a heterologous prime-pull scheme. Mice were immunized in a prime/boost/boost schedule at 2 week intervals with spleen, cervical lymph nodes and lung harvested at 8 weeks (2 weeks post final boost) and at 18 weeks (12 weeks post final boost). Single cell suspensions of spleens, cLN, and lungs were assessed for antigen specific CD8 and CD4 T cells. Frequency of spike-specific CD8+ T cells expressing effector memory (CD69⁺CD62L ) or tissue resident memory (CD69⁺CD103⁺IV ) markers within the spleen (A), cLN (B), and lungs (C). Frequency of total CD8⁺ T cells within the spleen (D), cLN (E), and lungs (F). Frequency of spike-specific CD4⁺ T cells expressing effector memory (CD69⁺CD62L‘ ) markers within the spleen (G) and cLN (H). Frequency of total CD4⁺ T cells within the spleen (I) and cLN (J). Significance determined by mixed-effects model with Tukey post-hoc test.

*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0041] FIG. 29 shows additional IM mRNA boosting enhances spike-specific CD8 while additional IN NE/IVT/S boosting enhances spike-specific CD4 T cells in the cLN. Mice were immunized in a prime/boost/boost schedule at 2 week intervals. Mice were then boosted 11 weeks later (wkl7) and 2 weeks post final boost (wkl9), spleen, cervical lymph nodes and lung were harvested. Single cell suspensions of spleens, cLN, and lungs were assessed for antigen specific CD8 and CD4 T cells. Frequency of spike-specific CD8+ T cells expressing effector memory (CD69⁺CD62L ) or tissue resident memory (CD69⁺CD103⁺IV ) markers within the spleen (A), cLN (B), and lungs (C). Frequency of total CD8⁺ T cells within the spleen (D), cLN (E), and lungs (F). Frequency of spike-specific CD4⁺ T cells expressing effector memory (CD69⁺CD62L ) within the spleen (G) and cLN (H). Frequency of total CD4⁺ T cells within the spleen (I) and cLN (J). Significance determined by mixed-effects model with Tukey post-hoc test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0042] FIG. 30 shows nucleic acid sequence of Sendai Virus (SeV) IVT DI (SEQUENCE ID NO: 1), including T7 promoter, hepatitis delta virus ribozyme, and the T7 terminator; the SeV DI sequence is underlined.

DEFINITIONS

[0043] To facilitate an understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.

[0044] It is to be understood that this disclosure is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0045] As used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “an antigen” includes one or more antigens and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[0046] The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the present disclosure.

[0047] The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any nonclaimed element as essential to the practice of the disclosure.

[0048] As used herein, the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or”, a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or.” [0049] The terms “host” or “subject,” as used herein, refer to an individual to be treated by (e.g., administered (e.g., injectably and/or mucosally (e.g., intranasally) administered)) compositions and methods of the present disclosure. Subjects include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and most preferably includes humans. In the context of the disclosure, the term “subject” generally refers to an individual who will be administered (e.g., injectably and/or intranasal administered) or who has been administered one or more compositions of the present disclosure (e.g., a coronavirus vaccine and a composition comprising a nanoemulsion and an agonist of retinoic acid-inducible gene I (RIG-I)). In some embodiments, the subject is at elevated risk for infection (e.g., by a coronavirus). In some embodiments, the subject may have a healthy or normal immune system. In some embodiments, the subject is one that has a greater than normal risk of being exposed to an infectious disease and/opathogen (e.g., a coronavirus). In some embodiments, the subject is a soldier, an emergency responder or other subject that has a higher than normal risk of being exposed to a pathogen (e.g., a coronavirus).

[0050] As used herein, the terms “at risk for infection” and “at risk for disease” refer to a subject that is predisposed to experiencing a particular infection or disease (e.g., an infectious disease caused by a virus, or a disease such as cancer). This predisposition may be genetic, or due to other factors (e.g., age, immunosuppression, compromised immune system, immunodeficiency, environmental conditions, exposures to detrimental compounds present in the environment, etc.). Thus, it is not intended that embodiments of the present disclosure be limited to any particular risk (e.g., a subject may be “at risk for disease” simply by being exposed to and interacting with other people), nor is it intended that embodiments of the present disclosure be limited to any particular disease or condition.

[0051] As used herein, the term “sample” is used in its broadest sense and encompasses materials obtained from any source. As used herein, the term “sample” is used to refer to materials obtained ftom a biological source, for example, obtained from animals (including humans), and encompasses any fluids, solids, and/or tissues. In particular embodiments of the present disclosure, biological samples include blood and blood products such as plasma, serum and the like. However, these examples are not to be construed as limiting the types of samples that find use with the present disclosure.

[0052] The term “emulsion,” as used herein, includes classic oil-in-water or water in oil dispersions or droplets, as well as other lipid structures that can form as a result of hydrophobic forces that drive apolar residues (e.g., long hydrocarbon chains) away from water and drive polar head groups toward water, when a water immiscible oily phase is mixed with an aqueous phase. These other lipid structures include, but are not limited to, unilamellar, paucilamellar, and multilamellar lipid vesicles, micelles, and lamellar phases. Similarly, the term “nanoemulsion,” as used herein, refers to oil-in-water dispersions comprising small lipid structures. For example, in some embodiments, the nanoemulsions comprise an oil phase having droplets with a mean particle size of approximately 0.1 to 5 microns (e.g., about 100, 150, 200, 250, 300, 350, 400, 450, 500 nm or larger in diameter), although smaller and larger particle sizes are contemplated. The terms “emulsion” and “nanoemulsion” and “NE” may be used interchangeably herein to refer to the nanoemulsions of the present disclosure.

[0053] The term “surfactant” refers to any molecule having both a polar head group, which energetically prefers solvation by water, and a hydrophobic tail that is not well solvated by water. The term “cationic surfactant” refers to a surfactant with a cationic head group. The term “anionic surfactant” refers to a surfactant with an anionic head group.

[0054] As used herein, the term “adjuvant” refers to any substance that can stimulate an immune response (e.g., a mucosal immune response). Some adjuvants can cause activation of a cell of the immune system (e.g., an adjuvant can cause an immune cell to produce and secrete a cytokine). Examples of adjuvants that can cause activation of a cell of the immune system include, but are not limited to, saponins purified from the bark of the Q. saponaria tree, such as QS21 (a glycolipid that elutes in the 21st peak with HPLC fractionation; Aquila Biopharmaceuticals, Inc., Worcester, Mass.); poly(di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus Research Institute, USA); derivatives of lipopolysaccharides such as monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland); and Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, Wash.).

Traditional adjuvants are well known in the art and include, for example, aluminum phosphate or hydroxide salts (“alum”). In some embodiments, the disclosure provides nanoemulson adjuvants and methods of using the same to induce an immune response in a subject.

[0055] A used herein, the term “immune response” and grammatical equivalents thereof refer to a response by the immune system of a subject. For example, immune responses include, but are not limited to, a detectable alteration (e.g., increase) in Toll-like receptor (TLR) activation, lymphokine (e.g., cytokine (e.g., Thl or Th2 type cytokines) or chemokine) expression and/or secretion, macrophage activation, dendritic cell activation, T cell activation (e.g., CD4+ or CD8+ T cells), NK cell activation, and/or B cell activation (e.g., antibody generation and/or secretion). Additional examples of immune responses include binding of an immunogen (e.g., antigen (e.g., immunogenic polypeptide)) to an MHC molecule and inducing a cytotoxic T lymphocyte (“CTL”) response, inducing a B cell response (e.g., antibody production), and/or T-helper lymphocyte response, and/or a delayed type hypersensitivity (DTH) response against the antigen from which the immunogenic polypeptide is derived, expansion (e.g., growth of a population of cells) of cells of the immune system (e.g., T cells, B cells (e.g., of any stage of development (e.g., plasma cells), and increased processing and presentation of antigen by antigen presenting cells. An immune response may be to immunogens that the subject’s immune system recognizes as foreign (e.g., non-self antigens from microorganisms (e.g., pathogens), or self-antigens recognized as foreign). Thus, it is to be understood that, as used herein, “immune response” refers to any type of immune response, including, but not limited to, innate immune responses (e.g., activation of Toll receptor signaling cascade), cell-mediated immune responses (e.g., responses mediated by T cells (e.g., antigen-specific T cells) and non-specific cells of the immune system), and humoral immune responses (e.g., responses mediated by B cells (e.g., via generation and secretion of antibodies into the plasma, lymph, and/or tissue fluids). The term “immune response” is meant to encompass all aspects of the capability of a subject’s immune system to respond to antigens and/or immunogens (e.g., both the initial response to an immunogen (e.g., a pathogen) as well as acquired (e.g., memory) responses that are a result of an adaptive immune response). Accordingly, an immune response may be a humoral (B cell mediated) and/or a cellular (T cell mediated) immune response.

[0056] As used herein, the term “immunity” refers to protection from disease (e.g., preventing or attenuating (e.g., suppression) of a sign, symptom or condition of the disease) upon exposure to a microorganism (e.g., pathogen) capable of causing the disease. Immunity can be innate (e.g., non-adaptive (e.g., non-acquired) immune responses that exist in the absence of a previous exposure to an antigen) and/or acquired/adaptive (e.g., immune responses that are mediated by B and/or T cells following a previous exposure to antigen (e.g., that exhibit increased specificity and reactivity to the antigen)).

[0057] An immune response induced in a subject obtained using the compositions and methods disclosed herein may be sterilizing immunity. The term “sterilizing immunity” as used herein means elimination of a pathogen (e.g., virus, bacteria, fungi, etc.) before it can detectably replicate in a host subject. An immune response induced in a subject obtained using the compositions and methods disclosed herein may be protective immunity. As used herein, the term “protective immunity” refers to an immune response that limits pathogen (e.g., virus, bacteria, fungi, etc.) replication before signs or symptoms of infection / disease develop and/or shortly thereafter and/or are detectable (e.g., only mild signs or symptoms of disease occur). [0058] “Prevention” as used herein, is used interchangeably with “prophylaxis” and can mean complete prevention of an infection or disease, or prevention of the development of signs or symptoms of that infection or disease; a delay in the onset of an infection or disease or its signs or symptoms; or a decrease in the severity of a subsequently developed infection or disease or its signs or symptoms.

[0059] The terms "treat," "treatment," and "treating," as used herein, refer to an approach for obtaining beneficial or desired results, for example, clinical results. For the purposes of this disclosure, beneficial or desired results may include inhibiting or suppressing the initiation or progression of an infection or a disease; ameliorating, or reducing the development of, symptoms of an infection or disease; or a combination thereof. [0060] As used herein, the terms “toll receptors” and “TLRs” refer to a class of receptors (e.g., TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR 11) that recognize special patterns of pathogens, termed pathogen-associated molecular patterns (see, e.g., Janeway and Medzhitov, (2002) Annu. Rev. Immunol., 20: 197-216). These receptors are expressed in innate immune cells (e.g., neutrophils, monocytes, macrophages, dendritic cells) and in other types of cells such as endothelial cells. Their ligands include bacterial products such as LPS, peptidoglycans, and lipopeptides. TLRs are receptors that bind to exogenous ligands and mediate innate immune responses leading to the elimination of invading microbes. The TLR-triggered signaling pathway leads to activation of transcription factors including NFKB, which is important for the induced expression of proinflammatory cytokines and chemokines. TLRs also interact with each other. For example, TLR2 can form functional heterodimers with TLR1 or TLR6. The TLR2/1 dimer has a different ligand binding profile than the TLR2/6 dimer (Ozinsky et al., PNAS, 97(25): 13766-13771 (2000)). In some embodiments, a nanoemulsion adjuvant activates cell signaling through a TLR (e.g., TLR2, TLR3, and/or TLR4). Thus, methods described herein include a nanoemulsion adjuvant composition combined with one or more immunogens (e.g., a vaccine, protein antigens, or other antigen described herein)) that when administered to a subject, activates one or more TLRs and stimulates an immune response (e.g., innate and/or adaptive/acquired immune response) in a subject. Such an adjuvant can activate TLRs (e.g., TLR2, TLR3, and/or TLR4) by, for example, interacting with TLRs (e.g., NE adjuvant binding to TLRs) or activating any downstream cellular pathway that occurs upon binding of a ligand to a TLR. NE adjuvants described herein that activate TLRs can also enhance the availability or accessibility of any endogenous or naturally occurring ligand of TLRs. A NE adjuvant that activates one or more TLRs can alter transcription of genes, increase translation of mRNA, or increase the activity of proteins that are involved in mediating TLR cellular processes. For example, NE adjuvants described herein that activate one or more TLRs (e.g., TLR2, TLR3, and/or TLR4) can induce expression of one or more cytokines (e.g., IL-8, IL- 12p40, and/or IL-23).

[0061] As used herein, the term “antibody” refers to an immunoglobulin molecule that is typically composed of two identical pairs of polypeptide chains, each pair having one “light” (L) chain and one “heavy” (H) chain. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 3 or more amino acids. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, Cm and CH3- Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy/light chain pair (VH and VL), respectively, form the antibody binding site. The term “antibody” encompasses an antibody that is part of an antibody multimer (a multimeric form of antibodies), such as dimers, trimers, or higher-order multimers of monomeric antibodies. It also encompasses an antibody that is linked or attached to, or otherwise physically or functionally associated with, a non-antibody moiety. Further, the term “antibody” is not limited by any particular method of producing the antibody. For example, it includes, inter alia, recombinant antibodies, synthetic antibodies, monoclonal antibodies, polyclonal antibodies, bi-specific antibodies, and multi-specific antibodies.

[0062] The terms “fragment of an antibody,” “antibody fragment,” and “functional fragment of an antibody” are used interchangeably herein to mean one or more fragments of an antibody that retain the ability to specifically bind to an antigen (see, generally, Holliger et al., Nat. Biotech., 23(9): 1126-1129 (2005)). An antibody fragment desirably comprises, for example, one or more CDRs, the variable region (or portions thereof), the constant region (or portions thereof), or combinations thereof. Examples of antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CHI domains, (ii) a F(a’)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) a Fab’ fragment, which results from breaking the disulfide bridge of an F(ab’)i fragment using mild reducing conditions, (v) a disulfide-stabilized Fv fragment (dsFv), and (vi) a domain antibody (dAb), which is an antibody single variable region domain (VH or VL) polypeptide that specifically binds antigen.

[0063] As used herein, the term “antibody derivative” or “derivative” of an antibody refers to a molecule that is capable of binding to the same antigen that the antibody from which it is derived binds to and comprises an amino acid sequence that is the same or similar to the antibody linked to an additional molecular entity. The amino acid sequence of the antibody that is contained in the antibody derivative may be the full-length antibody, or may be any portion or portions of a full-length antibody. The additional molecular entity may be a chemical or biological molecule. Examples of additional molecular entities include chemical groups, amino acids, peptides, proteins (such as enzymes, antibodies), and chemical compounds. The additional molecular entity may have any utility, such as for use as a detection agent, label, marker, pharmaceutical or therapeutic agent. The amino acid sequence of an antibody may be attached or linked to the additional entity by chemical coupling, genetic fusion, noncovalent association or otherwise. The term “antibody derivative” also encompasses chimeric antibodies, humanized antibodies, and molecules that are derived from modifications of the amino acid sequences of an antibody, such as conservation amino acid substitutions, additions, and insertions.

[0064] In the context of the present disclosure, a “neutralizing antibody” is an antibody that binds to a virus (e.g., a coronavirus) and interferes with the virus’ ability to infect a host cell. [0065] As used herein, the term “an amount effective to induce an immune response” (e.g., of a composition for inducing an immune response), refers to the dosage level required (e.g., when administered to a subject) to stimulate, generate and/or elicit an immune response in the subject. An effective amount can be administered in one or more administrations (e.g., via the same or different route), applications or dosages and is not intended to be limited to a particular formulation or administration route.

[0066] As used herein, the term “under conditions such that said subject generates an immune response” refers to any qualitative or quantitative induction, generation, and/or stimulation of an immune response (e.g., innate or acquired).

[0067] As used herein, the terms “immunogen” and “antigen” are used interchangeably to refer to an agent (e.g., a microorganism (e.g., bacterium, virus, or fungus) and/or portion or component thereof (e.g., protein, glycoprotein, lipoprotein, peptide, glycopeptide, lipopeptide, toxoid, carbohydrate, tumor-specific antigen, etc.)) that is capable of eliciting an immune response (e.g., a specific humoral and/or cell-mediated immune response) in a subject. In preferred embodiments, immunogens elicit immunity against the immunogen (e.g., a coronavirus or a coronavirus antigen) when administered in combination with a nanoemulsion adjuvant formulation of the disclosure comprising one or more antigens/immunogens (e.g., a coronavirus antigen) together with an adjuvant formulation comprising an emulsion delivery system formulated for administration, e.g., via injectable route (e.g., intradermal, intramuscular, subcutaneously, etc.), mucosal route (e.g., nasally or vaginally), or other route, to a subject. [0068] By “epitope” is meant a sequence of an antigen that is recognized by an antibody or an antigen receptor. Epitopes also are referred to in the art as “antigenic determinants.” In certain embodiments, an epitope is a region of an antigen that is specifically bound by an antibody or a T cell receptor. In certain embodiments, an epitope may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups. In certain embodiments, an epitope may have specific three-dimensional structural characteristics (e.g., a “conformational” epitope) and/or specific charge characteristics. The immunogen or antigen can be a protein or peptide of viral, bacterial, parasitic, fungal, protozoan, prion, cellular, or extracellular origin, which provokes an immune response in a mammal, preferably leading to protective immunity. An immunogen or antigen also may be based on one or more antigenic components of a particular organism and can be generated using recombinant DNA technology.

[0069] “Nasal application”, as used herein, means applied through the nose into the nasal or sinus passages or both. The application may, for example, be done by drops, sprays, mists, coatings or mixtures thereof applied to the nasal and sinus passages.

[0070] A “portion” of a nucleic acid sequence comprises at least ten nucleotides (e.g., about 10 to about 5000 nucleotides). Preferably, a “portion” of a nucleic acid sequence comprises 10 or more (e.g., 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, or 100 or more) nucleotides, but less than 5,000 (e.g., 4900 or less, 4000 or less, 3000 or less, 2000 or less, 1000 or less, 800 or less, 500 or less, 300 or less, or 100 or less) nucleotides. Preferably, a portion of a nucleic acid sequence is about 10 to about 3500 nucleotides (e.g., about 10, 20, 30, 50, 100, 300, 500, 700, 1000, 1500, 2000, 2500, or 3000 nucleotides), about 10 to about 1000 nucleotides (e.g., about 25, 55, 125, 325, 525, 725, or 925 nucleotides), or about 10 to about 500 nucleotides (e.g., about 15, 30, 40, 50, 60, 70, 80, 90, 150, 175, 250, 275, 350, 375, 450, 475, 480, 490, 495, or 499 nucleotides), or a range defined by any two of the foregoing values. More preferably, a “portion” of a nucleic acid sequence comprises no more than about 3200 nucleotides (e.g., about 10 to about 3200 nucleotides, about 10 to about 3000 nucleotides, or about 30 to about 500 nucleotides, or a range defined by any two of the foregoing values).

[0071] A “portion” of an amino acid sequence comprises at least three amino acids (e.g., about 3 to about 1,200 amino acids). Preferably, a “portion” of an amino acid sequence comprises 3 or more (e.g., 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, or 50 or more) amino acids, but less than 1,200 (e.g., 1,000 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less, 300 or less, 200 or less, or 100 or less) amino acids. Preferably, a portion of an amino acid sequence is about 3 to about 500 amino acids (e.g., about 10, 100, 200, 300, 400, or 500 amino acids), about 3 to about 300 amino acids (e.g., about 20, 50, 75, 95, 150, 175, or 200 amino acids), or about 3 to about 100 amino acids (e.g., about 15, 25, 35, 40, 45, 60, 65, 70, 80, 85, 90, 95, or 99 amino acids), or a range defined by any two of the foregoing values. More preferably, a “portion” of an amino acid sequence comprises no more than about 500 amino acids (e.g., about 3 to about 400 amino acids, about 10 to about 250 amino acids, or about 50 to about 100 amino acids, or a range defined by any two of the foregoing values).

[0072] The term “vaccine,” as used herein, refers to a biological preparation that stimulates a subject’s immune system (e.g., against a particular infectious agent such as a pathogenic organism) and provides active acquired immunity (e.g., to a particular pathogenic organism or infectious disease) in the subject. A vaccine typically contains an agent (e.g., one or more immunogens/antigens) that resembles a disease-causing microorganism and is often made from weakened or killed forms of the microbe, its toxins, and/or one or more of its proteins. The agent stimulates a subject’s immune system to recognize the agent as a threat, destroy it, and to further recognize and destroy any of the microorganisms associated with that agent that it may encounter in the future. Vaccines can be prophylactic (to prevent or ameliorate the effects of a future infection by a pathogen), or therapeutic (to ameliorate a disease that has already occurred, such as cancer). There are multiple types of vaccines known and used in the art, including, for example, inactivated virus vaccines, live-attenuated virus vaccines, messenger RNA (mRNA) vaccines, subunit vaccines, recombinant vaccines, polysaccharide vaccines, conjugate vaccines, toxoid vaccines, and viral vector vaccines. The administration of vaccines is referred to as “vaccination.” A vaccine may, in some embodiments, contain one or more cancer specific immunogens (e.g., tumor antigens) that elicit an immune response toward cancer in a subject administered the vaccine.

[0073] The terms “homologous prime-boost vaccination,” “homologous vaccination,” and the like refer to a vaccination regimen in which the first (priming) administration and any subsequent boosting administration use the same immunogenic composition (e.g., vaccine) and route of administration as described herein. The terms “heterologous prime-boost vaccination,” “heterologous vaccination,” and the like refer to a vaccination regimen in which the first (priming) administration uses an immunogenic composition (e.g., vaccine) and route of administration that is different than a subsequent boosting administration that uses a different immunogenic composition (e.g., vaccine) and/or route of administration.

[0074] As used herein, the term “pharmaceutically acceptable” means being approved by a regulatory agency of a U.S. Federal or a state government or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.

[0075] Physiologically acceptable “carrier” and “diluents” for vaccine preparation include water, saline solution, human serum albumin, oils, polyethylene glycols, aqueous dextrose, glycerin, propylene glycol or other synthetic solvents. Carriers may be liquid carriers (such as water, saline, culture medium, saline, aqueous dextrose, and glycols) or solid carriers (such as carbohydrates exemplified by starch, glucose, lactose, sucrose, and dextrans, anti-oxidants exemplified by ascorbic acid and glutathione, and hydrolyzed proteins).

[0076] The term “virus” means viruses, virus particles and viral vectors. The term includes wild-type viruses, recombinant and non-recombinant viruses, live viruses and live- attenuated viruses.

DETAILED DESCRIPTION

[0077] The disclosure provides compositions and methods for improving and enhancing systemic and mucosal vaccine immune responses. In particular, the disclosure provides mucosal adjuvant for vaccine delivery and methods of using the mucosal adjuvant in a vaccination regimen to induce immunity (e.g., protective immunity and/or sterilizing immunity) in a subject. As disclosed herein, the methods and compositions disclosed herein find use in a variety of settings including, but not limited to, the treatment (e.g., prophylactic and/or therapeutic treatment) of a variety of diseases and conditions. For example, as detailed herein, the methods and compositions of the disclosure can be used in combination with a broad range of vaccine types to induce protective immune responses (e.g., protective immunity and/or sterilizing immunity) to a variety of diseases and conditions including, but not limited to, infectious disease and cancer. Indeed, the methods and compositions disclosed herein (e.g., methods of vaccination) may be used with any vaccine to improve and/or enhance a subject’s immune response thereto.

[0078] Multiple effective vaccines exist for a variety of diseases (e.g., infectious disease) and conditions (e.g., cancer). For example, multiple effective COVID- 19 vaccines have been developed which have played a pivotal role in overcoming the acute phase of the pandemic.

Some of the most efficacious COVID-19 vaccines have been the mRNA vaccines, which were initially administered intramuscularly (IM) as a prime/boost regimen. However, waning antibody titers and the emergence of antigenically drifted versions of the virus (i.e. Omicron variants) with mutations in the viral spike (S) protein that facilitate immune escape, have limited the duration of vaccine-induced protective immunity.^{1 4} Frequent booster immunizations along with introduction of updated vaccines containing mRNAs encoding for the S proteins of Omicron BA.4/5 and XBB1.5 have been deployed in order to address this waning immunity.^5-8 These booster vaccines are also given IM and have been shown to enhance circulating B/T cell responses and improve protection from severe disease.^2,9,10 However, even with these current vaccines and booster regimens, breakthrough infections and viral transmission continue to occur in fully vaccinated individuals, demonstrating that these vaccines do not confer sterilizing immunity. This picture is not unique to coronaviruses. Indeed, a highly similar landscape exists for a variety of respiratory viruses including influenza viruses.

[0079] For respiratory viruses, the importance of inducing protective mucosal immune responses, including secretory antibodies and tissue-resident T cells, lies in the potential to block initial infection and viral dissemination to the lower respiratory tract (LRT). Moreover, mucosal immune responses in the respiratory tract play a critical role in preventing viral shedding and transmission. As such, there has been a significant need for development of improved vaccines that induce robust cross-protective mucosal immunity in addition to systemic immunity.

Vaccines against respiratory viruses given via the IM route poorly induce mucosal immune responses. Mucosal vaccine delivery and natural infection have both been shown to induce robust mucosal immunity.¹¹ Furthermore, hybrid immunity against SARS-CoV-2, the result of vaccine-induced immunity boosted by infection, has been suggested to provide superior protection from re-infection even with antigenically drifted viruses.^12,13 This is potentially mediated in part through re-routing and boosting vaccine-induced B- and T-cell responses to mucosal sites. In view of this paradigm, it was hypothesized that a similar prime/pull strategy may be employed by utilizing mucosal vaccination to mimic events occurring during natural infection, to boost and re-route existing vaccine- (e.g., mRNA vaccine-) induced immunity to the respiratory tract. While natural infection results in robust activation of local innate mucosal immune responses, achieving such responses through intranasal (IN) immunization with subunit antigens alone has proved challenging with limited utility. As disclosed herein, the disclosure provides, in some embodiments, rationally designed adjuvants and vaccine regimens that target immune receptor pathways activated by viral infection in the mucosa (e.g., that generate/induce similar or better outcomes as hybrid immunity).

[0080] In one aspect, the disclosure provides a method of inducing an immune response in a subject comprising the subject receiving a vaccine regimen comprising a primary systemic administration of a vaccine followed by a secondary mucosal administration of the same or different vaccine. The disclosure also provides a method of inducing an immune response in a subject comprising the subject receiving a vaccine regimen comprising a primary parenteral administration of a vaccine followed by a secondary mucosal administration of the same or different vaccine. In some embodiments, the mucosal administration comprises intranasal administration. In some embodiments, the secondary mucosal administration comprises an adjuvant comprising a nanoemulsion (NE) and an agonist of retinoic acid-inducible gene I (RIG- I). In another embodiment, the secondary mucosal administration comprise a NE and an agonist of a toll-like receptor (TLR). The disclosure provides that the disclosed primary-secondary (i.e., prime-boost) vaccination regimens disclosed herein represent a new and effective way to effectively pull systemic immune responses induced by a primary vaccination to mucosal sites (e.g., resulting in protective immunity and/or sterilizing immunity).

[0081] In some embodiments, a mucosal boost vaccination comprising an adjuvant (e.g., a nanoemulsion (NE), an agonist of retinoic acid-inducible gene I (RIG-I), and immunogen/antigen) of the disclosure is used to boost, induce and/or pull (e.g., using a vaccination regiment disclosed herein) a systemic immune response primed by a primary parenteral administration of a vaccine. The disclosure is not limited to any particular adjuvant for use in the mucosal boost vaccination. In some embodiments, the mucosal boost vaccination comprises an adjuvant comprising a NE and a RIG-I agonist wherein the RIG-I agonist is based on an in vitro transcribed RNA derived from Sendai virus (strain Cantell) defective interfering RNA (IVT).^14-17 NE/IVT adjuvant displays potent immunological properties and Phase I clinical safety profiles have been established for its use as an IN adjuvant (NCT01354379, NCT04148118).¹⁸ While an understanding of a mechanism is not needed to practice the present disclosure, and while the present disclosure is not limited to any particular mechanism, in some embodiments, NE adjuvant induces mucosal and systemic immune responses mediated at least in part, through TLR2 and 4 activation and through NLRP3 activation via induction of immunogenic apoptosis.^19-21 IVT is a selective RIG-I agonist and potent inducer of type I interferons (IFN-Is).¹⁷ As a combined agonist, NE/IVT can thus activate all three major innate receptor classes (TLRs, RLRs, NLRs) important for induction of antiviral immune responses.¹⁴ The NE/IVT adjuvant platform has shown a good safety profile in preclinical models, is compatible with whole virus as well as recombinant protein vaccines, and induces potent systemic and mucosal immune responses when used for intranasal (IN) vaccination.

[0082] A mucosal boost vaccination comprising an adjuvant (e.g., a nanoemulsion (NE), an agonist of retinoic acid-inducible gene I (RIG-I)) and immunogen/antigen of the disclosure may be separately formulated as individual compositions, or may be formulated together in any combination. In some aspects, for example, the nanoemulsion, the agonist of RIG-I, and/or the agonist of a TLR, and vaccine or immunogen/antigen are present in the same composition. In other aspects, the vaccine and/or immunogen/antigen is present in a first composition, and the nanoemulsion, agonist of RIG-I, and/or agonist of a TLR are present in a second composition. In yet further aspects, each of the vaccine and/or immunogen/antigen, the nanoemulsion, the agonist of RIG-I, and/or the agonist of a TLR is present in a separate compositions.

[0083] Examples of vaccine regimens utilizing a mucosal boost vaccination comprising an adjuvant (e.g., a nanoemulsion (NE), an agonist of retinoic acid-inducible gene I (RIG-I)) and immunogen/antigen to boost, induce and/or pull a systemic immune response primed by a primary parenteral administration of a vaccine are provided herein. For example, a mucosal boost vaccination comprising recombinant SARS-CoV-2 S protein adjuvanted with NE/IVT induced strong mucosal SARS-CoV-2 spike protein-specific immune responses primed by IM mRNA vaccination with the BNT162b2 vaccine (See, Examples 2-8). The heterologous regimen of IM mRNA priming followed by IN NE/IVT/S boost resulted in mucosal IgA responses similar to homologous IN NE/IVT/S prime/boost vaccination, but also induced markedly enhanced THI polarized T cell responses in the upper respiratory tract draining lymph nodes compared to homologous IM mRNA prime/boost and IN NE/IVT/S prime/boost. The strong mucosal B- and T-cell responses that resulted from this heterologous prime/pull vaccination strategy was associated with optimal cross-protection against various divergent VoCs as reflected in protection from morbidity as well as sterilizing virus control in both the upper (URT) and lower respiratory tracts (LRT) of experimentally SARS-CoV-2 infected mice. In contrast, IM mRNA prime/boost could not impart sterilizing immunity in the URT. Accordingly, the disclosure provides new and useful methods and compositions for heterologous IN boosting, inducing and pulling (e.g., focusing) the systemic immunity imparted by primary vaccination (e.g., primary parenteral vaccination with mRNA vaccine) to drive potent mucosal immune responses (e.g., to provide protective immunity and/or sterilizing immunity not achievable with homologous vaccination schemes).

[0084] The disclosure is not limited by the means or route of systemic administration (e.g., of a primary or prime vaccination of a prime-boost vaccine regimen disclosed herein). Indeed, any means or route of administration of a vaccine that results in systemic exposure of the vaccine and/or systemic immune response to the vaccine may be used. Examples of systemic administration include but are not limited to oral administration, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracistemal injection or infusion, subcutaneous injection, or implant) administration, or by sublingual, urethral (e.g., urethral suppository) or topical administration (e.g., gel, ointment, cream, aerosol, etc.).

[0085] The disclosure is not limited by the means or route of parenteral administration. Indeed, any parenteral means or route of administration of a vaccine may be used. Examples of parenteral administration include but are not limited to intramuscular injection or infusion, intraperitoneal injection or infusion, intravenous injection or infusion, ICV injection or infusion, intracistemal injection or infusion, subcutaneous injection, and implant.

[0086] The disclosure is not limited by the means or route of mucosal administration of a vaccine (e.g., of a secondary or boost vaccination of a prime-boost vaccine regimen disclosed herein). Indeed, any means or route of mucosal administration may be used. Examples of mucosal routes of administration include but are not limited to nasal mucosa, pulmonary, intravaginal, and intrarectal.

[0087] The disclosure provides vaccination regimens comprising a boost vaccination containing an adjuvant comprising a NE (e.g., a nanoemulsion disclosed herein) and RIG-I agonist (e.g., any RIG-I agonist disclosed herein, for example, defective interfering (DI) RNA produced by Sendai virus strain Cantell). As detailed herein, experiments conducted during development of the disclosure identified new vaccine regimens that include a boost vaccination containing an adjuvant comprising NE and RIG-I agonist that are capable of targeting multiple key innate antiviral pathways thereby promoting induction of robust protective systemic and mucosal humoral (antibody) and cell mediated (T cell mediated) immune responses to multiple viruses. For example, in one embodiment, the disclosure provides that a vaccination containing an adjuvant comprising NE and RIG-I agonist together with recombinant protein can be used as a mucosal secondary (boost) vaccine with primary (prime) vaccination regimens. In further embodiments, the disclosure provides that mucosal boost vaccination with NE/ RIG-I agonist and recombinant subunit antigens after parenteral prime vaccination (e.g., with an mRNA vaccine) results in effective rerouting of the systemic immune responses to local mucosal sites (e.g., protective immunity and/or sterilizing immunity) that are not achievable with a parenteral only (e.g., homologous) prime/boost immunization regimen. In some embodiments, a vaccine regimen of the disclosure provides robust mucosal immunity resulting in optimal protection from respiratory viruses (e.g., induction of local mucosal antibody and tissue-resident T cell responses within the upper and lower respiratory tracts (e.g., that improve and/or enhance vaccine responses of currently approved parenteral vaccines)).

[0088] Currently licensed IM administered mRNA-based vaccines are efficient inducers of systemic neutralizing antibodies (nAbs) and protective T cell responses but induce poor protective mucosal immune responses. Accordingly, the disclosure provides compositions and methods for inducing, via a secondary mucosal immunization, robust humoral and cellular immune responses in both the periphery and at mucosal sites (e.g., protective immunity and/or sterilizing immunity).

[0089] Humoral responses in serum were compared between various heterologous IM/IN and homologous IM/IM and IN/IN immunization regimens. In C57B1/6 mice, priming with IM mRNA induced higher antigen-specific IgG than the IN NE or NE/IVT/S. After boost vaccination however, IM mRNA prime/boost, IN NE/IVT/S prime/boost and IM mRNA prime followed by heterologous boost with IN NE/S or NE/IVT/S resulted in equally robust S- and RBD-specific IgG titers. In contrast, minimal increase in antigen-specific IgG was observed in IM mRNA primed animals boosted IN with unadjuvanted S alone, highlighting the role of the NE adjuvants in driving an optimal boost effect for IN immunization. Similar effects were observed in mice of different genetic backgrounds. Furthermore, heterologous boosting with IN NE/S or NE/IVT/S induced robust cross-neutralizing antibody responses across WT, B.1.617.2, B.1.351, and B.1.1.529 variants which were enhanced compared to those induced by IM mRNA; IN S alone in IM mRNA primed C57B1/6 and KI 8-hACE2 mice, confirming the effectiveness of the NE adjuvants in IN booster vaccines. The same relative patterns for nAbs were maintained in 129S1 mice. Taken together, the disclosure provides methods and compositions (e.g., IN adjuvants) that improve and enhance the breadth of the humoral immune response compared to homologous mRNA prime/boost regimens.

[0090] Combination of NE and IVT results in a more THI -polarized host immune response compared to the balanced TH1/TH2 response induced with NE alone, which is reflected in the antigen-specific IgG2b/IgGl and IgG2c/IgGl ratios in serum.¹⁴ Induction of IgG2c is significant due to its role in Fc-mediated effector functions including ADCC, ADCP, and complement activation which have shown to be key contributors to SARS-CoV-2 immunity.³⁵ IN NE/IVT/S prime/boost induced higher IgG2b and IgG2c titers compared to IN NE/S prime/boost, confirming the THI skewing of the RIG-I agonist. However, IM mRNA prime followed by IM mRNA boost or IN boost with NE/S or NE/IVT/S resulted in similar IgG2b and significantly greater IgG2c as compared to prime/boost vaccination with IN NE/IVT/S, suggesting that priming with IM mRNA can enhance the THI polarization effects induced by IN NE/TVT. Indeed, T cell antigen-recall assessment in splenocytes and cLN isolates from vaccinated mice revealed dramatic enhancement of THI cytokine production by the heterologous IM mRNA; IN NE/IVT/S vaccination regimen compared to IM mRNA prime/boost, with high levels of secreted antigen-specific IFN-y, IL-2, TNF-a, and IP- 10 observed in response to S protein stimulation of splenocytes, with particularly high levels of these cytokines in the local mucosal draining lymph nodes. For example, IM mRNA; IN NE/IVT/S resulted in a nearly 20-fold enhancement in antigen-specific IFN-y production in the cLN compared to IM mRNA prime/boost, demonstrating the crosstalk between the immune responses primed in the periphery by the mRNA and those triggered within the mucosa by the NE/IVT adjuvanted boost. Notably, IN boosting of IM mRNA primed T cell responses with NE/S or NE/IVT/S induced significantly higher percentages of S-specific polyfunctional IFN-y⁺IL-2⁺TNF-a⁺CD4⁺ T cells in the cLN compared to homologous IM mRNA, IN NE/S, or IN NE/IVT/S prime/boost regimens. This is significant, as polyfunctional IFN-y⁺IL-2⁺TNF-a⁺T cells have been shown to be a strong predictive indicator of potent antiviral T-cell responses. CD4⁺ T cells in secondary lymphoid tissues are required for class switching to result in production of IgG and IgA. Therefore, the optimal induction of CD4⁺ T cell responses in cLN promotes class switching events in mucosal associated lymphoid tissues like the cLN, which correlates with the higher levels of IgA observed in heterologously boosted groups. Interestingly, while all three IM mRNA prime, IN boost groups (S alone, NE/S, NE/TVT/S) induced higher frequencies of polyfunctional CD4⁺ T cells in the lung compared to IM mRNA prime/boost, the IN S alone boost did not significantly enhance these cells within the cLN, which required the NE or NE/IVT adjuvants for optimal enhancement. These results highlight the enhanced mucosal cellular responses induced by this mucosal booster strategy and underscore the role of the NE adjuvants in potentiating these responses. While homologous IM mRNA prime/boost induced significant levels of TH2 cytokines, IL-5 and IL- 13 (mainly driven by some high responders), heterologous IN boosting with NE/S or NE/TVT/S did not enhance these responses, and reduced these responses, consistent with the strongly THI -polarizing effects of the NE/TVT adjuvant. Finally, NE/IVT is a strong inducer of TH17 responses in the context of IL-10 production. TH17 cells in the context of IL-10 have been shown to be critical in promoting high and sustained levels of IgA production at mucosal sites-particularly the lung, and in the establishment of resident memory T cells.²⁵ Induction of IL-17A is exclusive to the IN route of NE or NE/IVT administration and is a key component of NE/IVT mediated immunity. Interestingly, boosting IM mRNA primed mice with IN NE/TVT/S resulted equivalent levels of IL-17A production in the cLN as two immunizations with IN NE/TVT/S, which was higher than that of mice given only one IN NE/IVT/S immunization. Accordingly, the IN NE/S or NE/IVT/S boost of IM mRNA primed mice resulted in increased frequencies of IL-17A-secreting CD4⁺T cells in the lung. These findings highlight the ability to use parenteral prime immunizations to set the stage for induction of more robust local mucosal responses upon mucosal pull immunization.

[0091] Consistent with IL-17A production, when induction of mucosal S-specific slgA in B ALF was considered, IN immunization, either as a homologous prime/boost or as part of a heterologous IM prime/IN boost regimen, was required to obtain detectable S-specific IgA. IM mRNA prime/boost did not induce detectable S-specific IgA, while IN NE/S and IN NE/TVT/S prime/boost resulted in robust slgA responses. While single IN NE/S or IN NE/TVT/S immunizations induced only low levels of slgA, equivalently high antigen-specific IgA was induced upon boosting IM mRNA primed animals with IN NE/TVT/S to similar levels as those induced by two immunizations with IN NE/TVT/S. These results further underscore the potential of the NE/IVT adjuvanted pull approach to drive improved mucosal immune responses initially primed by IM mRNA vaccination. It is well described that IgG antibodies can exudate from the blood into the lung and indeed, groups with highest serum ELISA S-binding titers also had highest S-specific binding ELISA titers in BALF. This indicates that deep lung antibody- mediated protection during infection may also be provided by IgG and not only slgA, although aside from their shared ability to mediate viral neutralization, they can provide protection via different mechanisms. For example, IgA-mediated protection can involve removal of immune complexes through immune exclusion, as well as triggering FcaRl -mediated immune mechanisms like respiratory burst from neutrophils.³⁶ IgG-mediated protection can also occur through activation of ADCC and ADCP through Fey receptor-mediated mechanisms.³⁷ Thus, in some embodiments, the disclosure provides compositions and methods that induce protection (e.g., protective immunity and/or sterilizing immunity) in the respiratory tract provided by IgA and IgG that occur via a combination of mechanisms including direct virus neutralization, opsonization and the activation of Fc receptor dependent mechanisms.

[0092] As the N501 Y mutation of B.l .351 allows it to infect WT mice, this variant was used to challenge 129 SI mice for evaluation of the impact of different vaccination strategies on virus replication and virus-host responses. Sterilizing immunity in the lungs was only observed for IM mRNA prime/boost mice and IM mRNA primed mice receiving a boost immunization through the IN route. These results demonstrate that induction of potent antibody responses through IM, IN or a combination of both can result in sterilizing immunity in the lungs. Prime/boost with IM Advx/S did not impart sterilizing immunity in the lung, despite having similar nAb titers against the B.l.351 variant as the IM mRNA; IN S group and similar serum S-specific serum IgG titers against the ancestral S protein as groups demonstrating sterilizing immunity. Thus, additional components besides serum nAbs are key contributors to sterilizing immunity in the LRT. When viral replication in the URT (nasal turbinates) was examined, a mucosal boost vaccination was required to provide sterilizing immunity. Despite full control of viral replication in the LRT for the IM mRNA prime/boost group, viral replication was still detected in the URT. Furthermore, replicating virus was detected in the URT of all mice vaccinated with IM Advx/S prime/boost. Full control of viral replication in the URT was associated with induction of slgA and local cellular responses by the mucosal booster strategies.

[0093] Heterologous boosting of IM mRNA primed mice IN with NE/S or NE/IVT/S in 129S1 mice markedly suppressed induction of major inflammatory markers associated with severe disease upon challenge with B.l.351, particularly in the IN NE/TVT/S boosted group. These findings confirm the protection afforded by these vaccination strategies. Similarly, homologous IM mRNA and IN NE/IVT/S prime/boost groups displayed a similar cytokine/chemokine profile post-infection in the lung, with effective prevention of virally induced inflammatory responses typically associated with more severe disease³³. Boosting IM mRNA primed mice with IN/S alone or homologous IN prime/boosting with NE/S also reduced inflammatory responses to infection compared to singly vaccinated animals and PBS control mice, however, in accordance with the incomplete protection observed in these groups as assessed by viral titers, this suppression was only partial. Increases in cytokines associated with type II polarization (IL-4, IL-5 and IL- 13) in lungs post-infection were observed in mice that received IM Advx-adjuvanted vaccine. Promotion of TH2-driven vaccine responses appear to translate to type II host immune responses in the lungs of infected mice.

[0094] The KI 8-hACE2 infection model provides a good representation of mild Omicron SARS-CoV-2 infection for testing vaccine effectiveness when virus titers are considered in the URT and LRT. Similar to observations with B.1.351 infection in 129S1 mice, a mucosal boost vaccination of IM mRNA primed animals was required to obtain sterilizing immunity in both the URT and LRT in BA.5 -challenged K18-hACE2 mice. In contrast, IM mRNA given once or as a prime/boost as well as IM Advx/S prime/boost were insufficient. In the K18-hACE2 model, mice that received only a single IM mRNA immunization showed a typical macrophage inflammatory profile upon BA.5 infection, whereas groups receiving heterologous IM mRNA prime, IN NE/S or NE/IVT/S boost effectively prevented induction of this inflammatory profile, consistent with the sterilizing immunity offered by these groups. If vaccination can prevent replication of virus in the nasal turbinates, this is seen as a first step in efficient interference with virus transmission. Taken together, the disclosure provides that heterolgous IN boost vaccination induces optimal mucosal immunity that effectively controls viral replication in the URT.

[0100] The ability to induce cross-variant sterilizing immunity within the respiratory tract is critical both for limiting viral transmission and disease progression, particularly as new SARS- CoV-2 variants continue to emerge. The present disclosure provides that the compositions and methods disclosed herein are useful for inducing robust, tailored, and cross -sterilizing antibody and T cell responses both systemically and locally within the mucosa of the upper and lower respiratory tracts of a subject (See Examples 1-13). Furthermore, the disclosure further provides that heterologous prime/pulling imprints a unique cellular profile that is more responsive to further boosting and also demonstrates that cellular immunity in IM mRNA prime/boosted mice can still be pulled to mucosal sites twelve weeks post initial immunization series (See. e.g., Examples 14-20). Thus, the disclosure provides, in some embodiments, that compositions and methods disclosed herein (e.g., IN NE/IVT used as an intranasal subunit vaccine) are useful to boost IM mRNA vaccination. While an understanding of a mechanism is not needed to practice the present disclosure and while the disclosure is not limited to any specific mechanism, in some embodiments, the immunogenic compositions formulated for mucosal administration and methods disclosed herein effectively reroute parenterally primed immune responses to the mucosal surface (e.g., provide enhanced humoral a cellular mucosal immune responses), establish long-lived tissue-resident memory cells within the respiratory tract, shape downstream immune response outcomes, for example, by strongly polarizing both CD4⁺/CD8⁺ T cell responses toward a profile optimal for protection against respiratory pathogens (e.g., polarized toward a polyfunctional Thl/Thl7 profile), and/or improve the durability of the immune response and/or viral clearance induced by parenterally administered (e.g., IM administered) vaccines (e.g., mRNA vaccines) (See Examples 14-20).

[0095] Accordingly, in some embodiments, the disclosure provides immunogenic compositions (e.g., comprising a nanoemulsion, an agonist of retinoic acid-inducible gene I (RIG-I), and an immunogen (e.g., a vaccine or subunit thereof (e.g., recombinant coronavirus S protein))) for use in administration to a subject (e.g., a previously vaccinated subject) to induce robust, cross-sterilizing antibody and T cell responses in the subject (e.g., systemic and/or local immune responses within the mucosa of the upper and lower respiratory tracts). As shown in Examples 1-20, the immunogenic compositions disclosed herein (e.g., comprising NE/IVT) activate multiple pathogen recognition receptors which mimic the viral innate immune activation events that occur during natural infection, and skews adaptive immune responses to an optimal Thl/Thl7 profile. The disclosed immunogenic compositions (e.g., comprising NE/IVT) effectively promotes induction of protective immunity at mucosal surfaces as a stand-alone IN vaccine series and as part of a heterologous immunization regimen as a booster after IM mRNA vaccine priming. As a heterologous booster, the disclosed immunogenic compositions (e.g., comprising NE/IVT) given with S protein (e.g., NE/IVT/S) through the IN route effectively boosts antibody and T cell responses primed parenterally by IM BNT162b2 mRNA, uniquely inducing robust slgA in the respiratory tract and enhancing tissue-resident memory T cells (TRM) in the lungs compared to homologous IM mRNA prime/boosting and IN NE/TVT/S prime/boosting. Furthermore, heterologous IM mRNA;IN NE/TVT/S prime/boosting uniquely shaped T cell cytokine profiles, driving highly enhanced Th 1 /Th 17 polarized responses compared to the more Thl/Th2 profile of the IM mRNA alone.

[0096] Vaccines and Antigens for Primary-Boost Vaccination

[0097] In some embodiments, the disclosure provides a method of inducing an immune response in a subject comprising the subject receiving a vaccine regimen comprising a primary systemic (e.g., parenteral) administration of a vaccine followed by a secondary mucosal administration of the same or different vaccine. In a preferred embodiment, the mucosal administration comprises intranasal administration. In a further preferred embodiment, the secondary mucosal administration comprises an adjuvant comprising a nanoemulsion (NE) and an agonist of retinoic acid-inducible gene I (RIG-I) and/or an agonist of a toll-like receptor (TLR). As described in the Examples, the primary-secondary (i.e., prime-boost) vaccination regimens and compositions for use therein effectively pull systemic immune responses induced by a primary vaccination to mucosal sites (e.g., resulting in protective immunity and/or sterilizing immunity). The disclosure is not limited by the vaccine used for the primary and secondary immunizations. Any known vaccine or component thereof (e.g., one or more antigens/immunogens) may be used. In some embodiments, the primary vaccine comprises an inactivated virus vaccine, live-attenuated virus vaccine, messenger RNA (mRNA) vaccine, subunit vaccine, recombinant vaccine, polysaccharide vaccine, conjugate vaccine, toxoid vaccine, or viral vector vaccine. In some embodiments, the secondary vaccine comprises a nanoemulsion (NE), an agonist of retinoic acid-inducible gene I (RIG-I) and/or an agonist of a toll-like receptor (TLR), and an inactivated virus vaccine, live-attenuated virus vaccine, messenger RNA (mRNA) vaccine, subunit vaccine, recombinant vaccine, polysaccharide vaccine, conjugate vaccine, toxoid vaccine, or viral vector vaccine. The disclosure is not limited by the antigen/immunogen used in a vaccine. Indeed, the disclosed compositions and methods may be used with and are applicable to a wide variety of immunogens (e.g., used in a mucosal boost vaccination comprising a nanoemulsion (NE), an agonist of retinoic acid-inducible gene I (RIG-I), and the immunogen/antigen.

[0098] In some embodiments, the antigen is a protein (including recombinant proteins), polypeptide, or peptide (including synthetic peptides). In certain embodiments, the antigen is a lipid or a carbohydrate (e.g., a polysaccharide). In certain embodiments, the antigen is a protein extract, cell (e.g., tumor cell), or tissue. The compositions provided herein can contain one or more antigens (e.g., at least two, three, four, five, six, seven, eight, or more antigens). [0099] In some embodiments, an antigen is a polypeptide that induces an immune response against an infectious disease. In other embodiments, an antigen is a polypeptide that induces an immune response against cancer cells. In some embodiments, an antigen is a polypeptide that modifies or redirects an immune response against an allergen.

[00100] Examples of antigens include but are not limited to one or more antigens from a pathogen (e.g. a virus, a bacterium, a parasite, a fungus) or tumors (e.g., a tumor antigen). Other exemplary antigens include autoantigens.

[00101] Examples of antigens for use in the disclosed methods and compositions (e.g., a mucosal boost vaccination comprising a nanoemulsion (NE), an agonist of retinoic acidinducible gene I (RIG-I), and the immunogen/antigen) include, but are not limited to, any antigen derived from a pathogenic bacterial, fungal, or viral organism, Streptococcus species, Candida species, Brucella species, Salmonella species, Shigella species, Pseudomonas species, Bordetella species, Clostridium species, Norwalk virus, Bacillus anthracis, Mycobacterium tuberculosis, human immunodeficiency virus (HIV), Chlamydia species, human Papillomaviruses, Influenza virus, Paramyxovirus species, Herpes virus, Cytomegalovirus, Varicella-Zoster virus, Epstein- Barr virus, Hepatitis viruses, Plasmodium species, Trichomonas species, Ebola, sexually transmitted disease agents, viral encephalitis agents, protozoan disease agents, fungal disease agents, cancer cells, or mixtures thereof.

[00102] In some embodiments, the antigen is one that is useful for the prevention of infectious disease. Non-limiting examples of antigens include the RSV antigens (e.g., F or G antigens); coronavirus antigens (e.g., S protein SI subunit, and receptor binding domain (RBD)); hepatitis B virus antigens (e.g., HBV surface antigen or core); human immunodeficiency virus antigens (e.g., (e.g., gpl20, gpl40, and gpl60); Chlamydia antigens (e.g., major outer membrane protein (mOMP)); influenza antigens (e.g., hemagglutinin (HA), M2 protein, and neuraminidase); dengue virus antigens (e.g., type 1 to 4 envelope proteins); zika virus antigens, malaria antigens (e.g., and circumsporozoite protein), or antigenic fragments of any of the above

[00103] The disclosed methods and compositions (e.g., a mucosal boost vaccination comprising a nanoemulsion (NE), an agonist of retinoic acid-inducible gene I (RIG-I), and immunogen/antigen) can be used to treat (e.g., prophylactically and/or therapeutically) a variety of diseases and disorders including but not limited to infectious diseases, cancer, inflammatory diseases, allergic diseases, and immunologic disease or disorder. [00104] Non-limiting examples of infectious disease include, but are not limited to, viral infectious diseases, such as AIDS, Respiratory Syncytial Virus (RSV), chickenpox (Varicella), common cold, cytomegalovirus infection, Colorado tick fever, dengue fever, Ebola hemorrhagic fever, hand, foot and mouth disease, hepatitis, herpes simplex, herpes zoster, HPV, influenza, lassa fever, measles, marburg hemorrhagic fever, infectious mononucleosis, mumps, norovirus, poliomyelitis, progressive multifocal leukencephalopathy, rabies, rubella, coronavirus 229E (alpha coronavirus;) coronavirus NL63 (alpha coronavirus); coronavirus OC43 (beta coronavirus); coronavirus HKU1 (beta coronavirus); MERS-CoV (the beta coronavirus that causes Middle East Respiratory Syndrome, or MERS); SARS-CoV (the beta coronavirus that causes severe acute respiratory syndrome, or SARS); and SARS-CoV-2 (the coronavirus that causes coronavirus disease 2019, or COVID-19), smallpox (Variola), viral encephalitis, viral gastroenteritis, viral meningitis, viral pneumonia, West Nile disease and yellow fever; bacterial infectious diseases, such as anthrax, bacterial meningitis, botulism, brucellosis, campylobacteriosis, cholera, diphtheria, typhus, gonorrhea, impetigo, legionellosis, leprosy, leptospirosis, listeriosis, lyme disease, melioidosis, rheumatic Fever, MRSA infection, pertussis, plague, pneumococcal pneumonia, psittacosis, Rocky Mountain Spotted Fever (RMSF), salmonellosis, scarlet fever, shigellosis, syphilis, tetanus, trachoma, tuberculosis, tularemia, typhoid fever, typhus and urinary tract infections; parasitic infectious diseases, such as African trypanosomiasis, amebiasis, ascariasis, babesiosis, Chagas disease, clonorchiasis, cryptosporidiosis, cysticercosis, diphyllobothriasis, Ddracunculiasis, echinococcosis, enterobiasis, fascioliasis, fasciolopsiasis, filariasis, amebic infection, giardiasis, gnathostomiasis, hymenolepiasis, isosporiasis, kalaazar, leishmaniasis, malaria, metagonimiasis, myiasis, onchocerciasis, pediculosis, pinworm, scabies, schistosomiasis, taeniasis, toxocariasis, toxoplasmosis, trichinellosis, trichinosis, trichuriasis, trichomoniasis and trypanosomiasis; fungal infectious disease, such as aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, cryptococcosis, histoplasmosis, tinea pedis, and tinea cruris; and Creutzfeldt-Jakob disease.

[00105] In some embodiments, the methods and compositions (e.g., a mucosal boost vaccination comprising a nanoemulsion (NE), an agonist of retinoic acid-inducible gene I (RIG- I), and immunogen/antigen) provided herein contain an antigen associated with an infectious agent such as Adenoviridae, Picomaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papillomaviridae, Rhabdoviridae, Togaviridae or Paroviridae family. In still another embodiment, the infectious agent is adenovirus, coxsackievirus, hepatitis A virus, poliovirus, Rhinovirus, Herpes simplex virus, Varicella-zoster virus, Epstein-barr virus, Human cytomegalovirus, Human herpesvirus, Hepatitis B virus, Hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, HIV, Influenza virus, Measles virus, Mumps virus, Parainfluenza virus, Respiratory syncytial virus, Human metapneumovirus, Human papillomavirus, Rabies virus, Rubella virus, Human bocarivus or Parvovirus Bl 9. In yet another embodiment, the infectious agent is a bacteria of the Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia and Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema Vibrio or Yersinia genus. In a further embodiment, the infectious agent is Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitides, Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae or Yersinia pestis. In another embodiment, the infectious agent is a fungus of the Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis or Stachybotrys genus. In still another embodiment, the infectious agent is C. albicans, Aspergillus fumigatus, Aspergillus flavus, Cryptococcus neoformans, Cryptococcus laurentii, Cryptococcus albidus, Cryptococcus gattii, Histoplasma capsulatum, Pneumocystis jirovecii or Stachybotrys chartarum.

[00106] Non-limiting examples of cancers include, but are not limited to breast cancer; biliary tract cancer; bladder cancer; brain cancer including glioblastomas and medulloblastomas; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms including acute lymphocytic and myelogenous leukemia, e.g., B Cell CLL; T-cell acute lymphoblastic leukemia/lymphoma; hairy cell leukemia; chronic myelogenous leukemia, multiple myeloma; AIDS-associated leukemias and adult T-cell leukemia/lymphoma; intraepithelial neoplasms including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphomas including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral cancer including squamous cell carcinoma; ovarian cancer including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancer including melanoma, Merkel cell carcinoma, Kaposi's sarcoma, basal cell carcinoma, and squamous cell cancer; testicular cancer including germinal tumors such as seminoma, non-seminoma (teratomas, choriocarcinomas), stromal tumors, and germ cell tumors; thyroid cancer including thyroid adenocarcinoma and medullar carcinoma; and renal cancer including adenocarcinoma and Wilms tumor. Any antigen associated with any of the diseases or conditions disclosed herein can be used in the compositions and methods provided herein.

[00107] Any antigen associated with any of the cancers disclosed herein can be used in the compositions and methods provided herein. Non-limiting examples of cancer antigens include HER 2 (pi 85), CD20, CD33, GD3 ganglioside, GD2 ganglioside, carcinoembryonic antigen (CEA), CD22, milk mucin core protein, TAG-72, Lewis A antigen, ovarian associated antigens such as OV-TL3 and MOvl8, high Mr melanoma antigens recognized by antibody 9.2.27, HMFG-2, SM-3, B72.3, PR5C5, PR4D2, and the like. Further examples include MAGE, MART- 1/Melan-A, gplOO, Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), FAP, cyclophilin b, Colorectal associated antigen (CRC) C017-1A/GA733, Carcinoembryonic Antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, arnll, prostatic acid phosphatase (PAP), Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, pro state-specific membrane antigen (PSMA), T-cell receptor/CD3- zeta chain, MAGE-family of tumor antigens (e.g., MAGE-I or MAGE-II families) (e.g., MAGE- Al, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-CI, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-family of tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1, a-fetoprotein, E- cadherin, a-catenin, -catenin and y-catenin, pl20ctn, gplOOPmell 17, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, pl 5, gp75, GM2 and GD2 gangliosides, viral products such as human papilloma virus proteins, Smad family of tumor antigens, lmp-1, PIA, EBV-encoded nuclear antigen (EBNA)-l, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, CD20 and c-erbB-2.

[00108] The disclosure is not limited by the vaccine type used as a primary (prime) vaccine in a prime-boost vaccination regimen disclosed herein. Indeed, any commercially available vaccine known in the art may be used as a primary vaccine (e.g., for primary systemic administration). For example, any commercially available vaccine for Chickenpox (Varicella), Dengue, Diphtheria, Flu (Influenza), Hepatitis A, Hepatitis B, Hib (Haemophilus influenzae type b), HPV (Human Papillomavirus). Measles, Meningococcal. Mumps, Pneumococcal, Polio (Poliomyelitis), Rotavirus, RSV (Respiratory Syncytial Virus), Rubella (German Measles), Shingles (Herpes Zoster), Tetanus (Lockjaw), or Whooping Cough (Pertussis) may be used. [00109] In some embodiments, the primary vaccine is a vaccine that induces an immune response against SARS-CoV-2. Any type of vaccine directed against any type of coronavirus may be administered to a subject using the compositions and methods of the disclosure, such as a human that has been exposed to, or is suspected of exposure to, a coronavirus, and/or a subject at risk for coronavirus infection (e.g., the elderly and/or immunocompromised). In this regard, for example, the coronavirus vaccine may be a protein subunit vaccine (e.g., S protein or RBD), an mRNA vaccine, a DNA vaccine, a viral vector vaccine, a live-attenuated virus vaccine, an inactivated virus vaccine, a pseudotyped virus vaccine, etc. (Lin et al., Antivir Ther.

2007;12(7):l 107-13; Martin et al., Vaccine. 2008;26(50):6338-43 ; Modjarrad et al., Lancet Infect Dis. 2019;19(9):1013-22; Koch et al., Lancet Infect Dis. 2020; doi.org/10.1016/S1473- 3099(20)30248-6; and Folegatti et al., Lancet Infect Dis. 2020;20(7):816-26). The coronavirus vaccine may be one of mRNA-1273 (ModemaTX, Inc.), BNT162b2 (Pfizer, Inc., and BioNTech), and JNJ-78436735 (Janssen Pharmaceuticals, Inc.). The mRNA-1273 and BNT162b2 vaccines are mRNA vaccines, and the JNJ-78436735 vaccine is a viral vector (i.e., adenoviral vector) vaccine. Another viral vector vaccine, Vaxzevria (also referred to as “COVID- 19 Vaccine (ChAdOxl-S [recombinant])”) has been authorized for use by the European Medicines Agency (EMA) and other non-U. S. countries. Thus, any of these vaccines may be administered to a subject in accordance with the disclosed methods. Several other SARS-CoV-2 vaccines are currently in preclinical and clinical trials (see, e.g., Li et al., Journal of Biomedical Science volume 27, Article number: 104 (2020)), any of which also may be employed in the disclosed compositions and methods.

[00110] In some embodiments, the vaccine is a protein subunit vaccine, a whole virus vaccine, or a pseudotyped virus vaccine. The term “subunit vaccine,” as used herein, refers to a vaccine composed of protein or glycoprotein components of a pathogen that are capable of inducing a protective immune response, and may be produced by conventional biochemical or recombinant DNA technologies. A “whole virus vaccine” comprises an entire virus that has been killed, attenuated, or weakened so that it cannot cause disease. Whole virus vaccines can elicit strong protective immune responses. A whole virus vaccine may comprise a live cold-adapted virus, which is a virus comprising a temperature sensitive mutation that allows for replication and confers stability in nasal mucosa, but has restricted ability to replicate in the lungs.

“Pseudotyping” refers to the process of producing viruses or viral vectors using foreign viral envelope proteins. The resulting virus is referred to as a “pseudotyped virus.” In some cases, the inability to produce viral envelope proteins renders the pseudovirus replication-incompetent, which enables investigation of dangerous viruses in a lower risk setting. Indeed, pseudotyping viral systems have been widely employed to study highly infectious and pathogenic viruses, such as Ebola virus, Middle Eastern Respiratory Syndrome (MERS) virus, or SARS viruses (McWilliams et al., Cell Rep. (2019) 26:1718-26.e4. doi: 10.1016/j.celrep.2019.01.069; Liu et al., Antiviral Res. (2018) 150:30-8. doi: 10.1016/j.antiviral.2017.12.007; andFukushi et al., SARS- and Other Coronaviruses: Laboratory Protocols. Totowa, NJ: Humana Press (2008). p. 331-8). The two most commonly used pseudotyping systems are retro/lentiviruses and vesicular stomatitis virus (VSV) which lacks the VSV envelope glycoprotein (VSV G). The use of replication-restricted pseudoviruses bearing foreign viral coat proteins represents a safe and useful method that has been widely adopted by virologists to study viral entry, detection of neutralizing antibodies in serum samples, and therapeutic development under less stringent biosafety conditions (e.g., biosafety level-2 (BSL-2)). Pseudotyped viruses have been used to produce vaccine candidates against HIV (Racine et al., AIDS Research and Therapy. 14 (1): 55. doi:10.1186/s 12981 -017-0179-2); Nipah henipavirus (Nie et al., Emerging Microbes & Infections. 8 (1): 272-281; doi: 10.1080/22221751.2019.1571871); Rabies lyssavirus (Moeschler et al., Viruses. 8 (9): 254. doi:10.3390/v8090254), SARS-CoV (Kapadia et al., Virology. 376 (1): 165-172. doi:10.1016/j.virol.2008.03.002); Zaire ebolavirus (Salata et al., Viruses. 11 (3): 274. doi: 10.3390/vl 1030274), and SARS-CoV-2 (Johnson et al., Journal of Virology. 94 (21). doi: 10.1128/JVI.01062-20; and Condor Capcha et al., Front. Cardiovasc. Med., 15 January (2021)). In some embodiments, a vaccine encompassed by the present disclosure may comprise a vesicular stomatitis virus pseudotyped with SARS-CoV-2 spike protein, or a portion thereof. A pseudotyped virus may be further attenuated via the use of misrepresented mammalian codons (referred to as “codon deoptimization”), which also are within the scope of this disclosure.

[00111] In other embodiments, the vaccine may be an mRNA vaccine. An “mRNA vaccine”, like the FDA-authorized mRNA- 1273 and BNT162b2, is a nucleic acid vaccine based on messenger RNA. The mRNA typically encodes at least one pathogen-specific antigen, and complexed or formulated with carriers (e.g., lipids, polymers) that facilitate cellular uptake of mRNA and protect it from degradation. mRNA vaccine technology is further described in, e.g., Pardi et al., Nature Reviews Drug Discovery volume 17: 261-279 (2018); Schlake et al., RNA Biol. 2012 Nov 1; 9(11): 1319-1330; and Rahman et al., Vaccines (Basel). 2021 Mar 11;9(3):244. doi: 10.3390/vaccines9030244.

[00112] The vaccine may be a viral vector vaccine. A “viral vector vaccine,” like the FDA- authorized JNJ-78436735 vaccine, consists of a recombinant virus that is often attenuated to reduce its pathogenicity, in which genes encoding viral antigen(s) have been cloned using recombinant DNA techniques. Viral vector vaccines can either be replicating or non-replicating. Replicating vector vaccines infect cells in which the vaccine antigen is produced and are able to replicate and infect new cells that will then also produce the vaccine antigen. Non-replicating vector vaccines initially enter cells and produce the vaccine antigen, but no new virus particles are formed. Because viral vector vaccines result in endogenous antigen production, both humoral and cellular immune responses may be stimulated. Viral vector vaccines may be based on any suitable virus, including, but not limited to, adenovirus, adeno-associated virus (AAV), retrovirus, lentivirus, vaccinia virus, and cytomegalovirus (CMV). Viral vector-based vaccines are described in detail in, e.g., Ura et al., Vaccines (Basel). 2014 Sep; 2(3): 624-64; Lauer et al., Clin Vaccine Immunol 24:e00298-16; doi.org/10.1128/CVI.00298-16 (2017); and van Riel, D., de Wit, E., Nature Materials, 19: 810-812(2020).

[00113] Whatever type of vaccine is chosen, the vaccine desirably comprises one or more antigens, or portions or epitopes thereof. In some embodiments, the antigen is the SARS-CoV-2 spike protein (“S” protein as provided by, e.g., UniProtKB Accession Number P0DTC2) or the spike protein receptor-binding domain (RBD) (see, e.g., Wrapp (2020), Science 367: 1260-63; Walls (2020) Cell 180: 1-12). In some embodiments, the antigen is a viral transcription and/or replication protein (e.g., replicase polyprotein la (Ria) or replicase polyprotein lab (Rlab)). In some embodiments, the antigen is a viral budding protein (e.g., protein 3 a or envelope small membrane protein (E)). In some embodiments, the antigen is a virus morphogenesis protein (e.g., membrane protein (M)). In some embodiments, the antigen is non-structural protein 6 (NS6), protein 7a (NS7A), protein 7b (NS7B), non-structural protein 8 (NS8), or protein 9b (NS9B). In some embodiments, the antigen is a viral genome packaging protein (e.g., nucleocapsid protein (N or NC)). In some embodiments, the antigen is an uncharacterized protein.

[00114] In some embodiments, the antigen may comprise a protein and/or a nucleic acid, or a portion thereof, from a genetic variant of the SARS-CoV-2 virus, e.g., a SARS-CoV-2 variant of interest, variant of concern, or variant of high consequence. In some embodiments, the variant is B.1.526, B.1.525, P.2, B.l.1.7 (also known as 20I/501Y.V1 and VOC 202012/01), P.l, B.1.351 (also known as 20H/501Y.V2), B.1.427, B.1.429, XBB1.5 (or other variant of the Omicron lineage) or B.1.617. SARS-CoV-2 variants are further described in, e.g., Zhou et al., Nature (February 26, 2021); Volz et al., Cell 2021; 184(64-75); Korber et al., Cell 2021; 182(812-7); Davies et al., MedRXiv 2021; Horby et al., New & Emerging Threats Advisory Group, Jan. 21, 2021; Emary et al., Lancet (February 4, 2021); Fact Sheet For Health Care Providers Emergency Use Authorization (EUA) Of Regen-Cov (fda.gov); Wang P, Wang M, Yu J, et al. Increased Resistance of SARS-CoV -2 Variant P.1 to Antibody Neutralization. BioRxiv 2021 ; and Li et al., Innovation (NY). 2021 May 11 ;100116. doi: 10.1016/j.xinn.2021.100U6).

[00115] A vaccine may comprise one or more nucleic acid and/or amino acid sequences that is at least about 70% identical (e.g., about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to any of the aforementioned antigens/immunogens. The degree of nucleic acid and/or amino acid identity can be determined using any method known in the art, such as the BLAST sequence database.

[00116] Vaccines formulations may comprise pharmaceutically acceptable carriers, excipients and/or adjuvants. Adjuvants and carriers suitable for administering vaccines and immunogens are known in the art. Conventional carriers and adjuvants are for example reviewed in Kiyono et al. 1996.

[00117] A vaccine adjuvant is a component that potentiates the immune responses to an antigen and/or modulates it towards the desired immune responses. A vaccine may include one or more adjuvants. Exemplary adjuvants include mineral salts including but not limited to aluminium salts (such as amorphous aluminum hydroxyphosphate sulfate (AAHS), aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate (Alum)) and calcium phosphate gels; Oil emulsions and surfactant based formulations, including but not limited to MF59 (microfluidised detergent mmunoprec oil-in-water emulsion), QS21 (purified saponin), AS02 [SBAS2] (oil-in- water emulsion + MPL + QS-21), Montanide ISA-51 and ISA-720 ( mmunoprec water-in-oil emulsion); Particulate adjuvants, including but not limited to virosomes (unilamellar liposomal vehicles incorporating influenza haemagglutinin), AS04 ([SBAS4] Al salt with MPL), ISCOMS (structured complex of saponins and lipids), polylactide co-glycolide (PLG). And ; microbial derivatives (natural and synthetic), including but not limited to monophosphoryl lipid A (MPL), Detox (MPL + M. Phlei cell wall skeleton), AGP [RC-529] (synthetic acylated monosaccharide), DC Chol (lipoidal immunostimulators able to self mmunopr into liposomes), OM-174 (lipid A derivative), CpG motifs (synthetic oligonucleotides containing immunostimulatory CpG motifs), modified LT and CT (genetically modified bacterial toxins to provide non-toxic adjuvant effects); endogenous human immunomodulators, including but not limited to hGM-CSF or hlL-12 (cytokines that can be administered either as protein or plasmid encoded), Immudaptin (C3d tandem array) and inert vehicles, such as gold particles. The vaccine formulations may also comprise a stabilizer. Suitable stabilizer are known in the art and include but are not limited to amino acids, antioxidants, cyclodextrins, proteins, sugars/ sugar alcohols, and surfactants. See for example Morefield, AAPS J. 2011 Jun; 13(2): 191 — 200). [00118] A vaccine can be incorporated into liposomes, microspheres or other polymer matrices. Liposomes can consist of phospholipids or other lipids, and can be nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

[00119] In some embodiments, vaccines formulations comprise lipid nanoparticle delivery formulations of nucleic acid-based vaccines. Optionally, the lipid is cationic. Appropriate cationic lipids are known in the art. Non-limiting examples include phosphatidylcholine/cholesterol/PEG-lipid, Cl 2-200, dimethyldioctadecylammonium (DDA), 1,2- dioleoyl-3 -trimethylammonium propane (DOTAP) or l,2-dilinoleyloxy-3- dimethylaminopropane (DLinDMA). Also see for example, U.S. Patent No. 10,221,127 (incorporated by reference) and Reichmuth AM et al. (Therapeutic Delivery. 2016 ;7(5):319-334. DOI: 10.4155/tde-2016-0006). In specific embodiments, vaccines formulations comprise lipid nanoparticle delivery formulations of SAM RNA vaccines. In specific embodiments, the LNPs comprise an ionizable cationic lipid (phosphatidylcholine :cholesterol/PEG-lipid (50:10:38.5:1.5 mol/mol). In certain embodiments, the RNA to total lipid ratio in the LNP is approximately 0.05 (wt/wt). In certain embodiments, the LNPs have a diameter of ~80 nm.

[00120] In other embodiments, Charge-Altering Releasable Transporters (CARTs) are utilized as a mRNA delivery platform. Accordingly, in certain embodiments, vaccines are viral vectorbased vaccines or nucleic acid- based vaccines. In specific embodiments, the vaccines are SAM RNA-based vaccines. Optionally, the SAM vaccines are in lipid nanoparticle formulations.

Immunogenic Compositions

[00121] The disclosure provides immunogenic compositions. In some embodiments, the disclosure provides immunogenic compositions comprising a nanoemulsion, an agonist of retinoic acid-inducible gene I (RIG-I) and/or an agonist of a toll-like receptor (TLR), and/or an inactivated virus vaccine, live-attenuated virus vaccine, messenger RNA (mRNA) vaccine, subunit vaccine, recombinant vaccine, polysaccharide vaccine, conjugate vaccine, toxoid vaccine, or viral vector vaccine. The nanoemulsion, RIG-I agonist and/or TLR agonist, and inactivated virus vaccine, live-attenuated virus vaccine, messenger RNA (mRNA) vaccine, subunit vaccine, recombinant vaccine, polysaccharide vaccine, conjugate vaccine, toxoid vaccine, or viral vector vaccine may be separately formulated as individual compositions, or may be formulated together in any combination. In some aspects, for example, the inactivated virus vaccine, live-attenuated virus vaccine, messenger RNA (mRNA) vaccine, subunit vaccine, recombinant vaccine, polysaccharide vaccine, conjugate vaccine, toxoid vaccine, or viral vector vaccine, the nanoemulsion, the agonist of RIG-I, and/or the agonist of a TLR are present in the same composition. In other aspects, the inactivated virus vaccine, live-attenuated virus vaccine, messenger RNA (mRNA) vaccine, subunit vaccine, recombinant vaccine, polysaccharide vaccine, conjugate vaccine, toxoid vaccine, or viral vector vaccine is present in a first composition, and the nanoemulsion, agonist of RIG-I, and/or agonist of a TLR are present in a second composition. In yet further aspects, each of the inactivated virus vaccine, live-attenuated virus vaccine, messenger RNA (mRNA) vaccine, subunit vaccine, recombinant vaccine, polysaccharide vaccine, conjugate vaccine, toxoid vaccine, or viral vector vaccine, the nanoemulsion, the agonist of RIG-I, and/or the agonist of a TLR is present in separate compositions.

Nanoemulsions [00122] Nanoemulsion formulations described herein are simply examples to illustrate the variety of nanoemulsion adjuvants that find use in the present disclosure. The present disclosure contemplates that many variations of these formulations, as well as additional nanoemulsions, may be used in the methods of the present disclosure. Candidate nanoemulsions can be easily tested to determine if they are suitable for use in the compositions described herein.

[00123] Nanoemulsion formulations encompassed by the present disclosure generally are nontoxic (e.g., to humans, plants, or animals), non-irritant (e.g., to humans, plants, or animals), and non-corrosive (e.g., to humans, plants, or animals or the environment), and retain stability when mixed with other agents (e.g., a composition comprising an immunogen (e.g., bacteria, fungi, viruses, and spores).

[00124] The nanoemulsion can comprise an aqueous phase, at least one oil, at least one surfactant, and at least one solvent. Nanoemulsions of the present disclosure may comprise the following properties and components.

[00125] The nanoemulsion of the present disclosure may comprise droplets having an average diameter size of less than about 1000 nm, less than about 950 nm, less than about 900 nm, less than about 850 nm, less than about 800 nm, less than about 750 nm, less than about 700 nm, less than about 650 nm, less than about 600 nm, less than about 550 nm, less than about 500 nm, less than about 450 nm, less than about 400 nm, less than about 350 nm, less than about 300 nm, less than about 250 nm, less than about 200 nm, less than about 150 nm, or any combination thereof.

In one embodiment, the droplets have an average diameter size greater than about 125 nm and less than or equal to about 600 nm. In other embodiments, the droplets have an average diameter size greater than about 50 nm or greater than about 70 nm, and less than or equal to about 125 nm.

[00126] The aqueous phase of the nanoemulsion can comprise any type of aqueous phase including, but not limited to, water (e.g., H2O, distilled water, purified water, water for injection, de-ionized water, tap water) and solutions (e.g., phosphate buffered saline (PBS) solution). In certain embodiments, the aqueous phase comprises water at a pH of about 4 to 10, preferably about 6 to 8. The water can be deionized (hereinafter “DiHiO”). In some embodiments the aqueous phase comprises phosphate buffered saline (PBS). The aqueous phase may further be sterile and pyrogen free.

[00127] Organic solvents in the nanoemulsion can include, but are not limited to, C1-C12 alcohol, diol, triol, dialkyl phosphate, tri-alkyl phosphate, such as tri-n-butyl phosphate, semi- synthetic derivatives thereof, and combinations thereof. In one aspect, the organic solvent is an alcohol chosen from a nonpolar solvent, a polar solvent, a protic solvent, or an aprotic solvent. Suitable organic solvents include, but are not limited to, ethanol, methanol, isopropyl alcohol, glycerol, medium chain triglycerides, diethyl ether, ethyl acetate, acetone, dimethyl sulfoxide (DMSO), acetic acid, w-butanol, butylene glycol, perfumers alcohols, isopropanol, n-propanol, formic acid, propylene glycols, glycerol, sorbitol, industrial methylated spirit, triacetin, hexane, benzene, toluene, diethyl ether, chloroform, 1,4-dixoane, tetrahydrofuran, dichloromethane, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, formic acid, semi-synthetic derivatives thereof, and any combination thereof.

[00128] The oil in the nanoemulsion can be any cosmetically or pharmaceutically acceptable oil. The oil can be volatile or non-volatile, and may be chosen from animal oil, vegetable oil, natural oil, synthetic oil, hydrocarbon oils, silicone oils, semi-synthetic derivatives thereof, and combinations thereof.

[00129] Suitable oils include, but are not limited to, mineral oil, squalene oil, flavor oils, silicon oil, essential oils, water insoluble vitamins, Isopropyl stearate, Butyl stearate, Octyl palmitate, Cetyl palmitate, Tridecyl behenate, Diisopropyl adipate, Dioctyl sebacate, Menthyl anthranhilate, Cetyl octanoate, Octyl salicylate, Isopropyl myristate, neopentyl glycol dicarpate cetols, Ceraphyls®, Decyl oleate, diisopropyl adipate, C12-15 alkyl lactates, Cetyl lactate, Lauryl lactate, Isostearyl neopentanoate, Myristyl lactate, Isocetyl stearoyl stearate, Octyldodecyl stearoyl stearate, Hydrocarbon oils, Isoparaffin, Fluid paraffins, Isododecane, Petrolatum, Argan oil, Canola oil, Chile oil, Coconut oil, com oil, Cottonseed oil, Flaxseed oil, Grape seed oil, Mustard oil, Olive oil, Palm oil, Palm kernel oil, Peanut oil, Pine seed oil, Poppy seed oil, Pumpkin seed oil, Rice bran oil, Safflower oil, Tea oil, Truffle oil, Vegetable oil, Apricot (kernel) oil, Jojoba oil (simmondsia chinensis seed oil), Grapeseed oil, Macadamia oil, Wheat germ oil, Almond oil, Rapeseed oil, Gourd oil, Soybean oil, Sesame oil, Hazelnut oil, Maize oil, Sunflower oil, Hemp oil, Bois oil, Kuki nut oil, Avocado oil, Walnut oil, Fish oil, berry oil, allspice oil, juniper oil, seed oil, almond seed oil, anise seed oil, celery seed oil, cumin seed oil, nutmeg seed oil, leaf oil, basil leaf oil, bay leaf oil, cinnamon leaf oil, common sage leaf oil, eucalyptus leaf oil, lemon grass leaf oil, melaleuca leaf oil, oregano leaf oil, patchouli leaf oil, peppermint leaf oil, pine needle oil, rosemary leaf oil, spearmint leaf oil, tea tree leaf oil, thyme leaf oil, wintergreen leaf oil, flower oil, chamomile oil, clary sage oil, clove oil, geranium flower oil, hyssop flower oil, jasmine flower oil, lavender flower oil, manuka flower oil, Marhoram flower oil, orange flower oil, rose flower oil, ylang-ylang flower oil, Bark oil, cassia Bark oil, cinnamon bark oil, sassafras Bark oil, Wood oil, camphor wood oil, cedar wood oil, rosewood oil, sandalwood oil), rhizome (ginger) wood oil, resin oil, frankincense oil, myrrh oil, peel oil, bergamot peel oil, grapefruit peel oil, lemon peel oil, lime peel oil, orange peel oil, tangerine peel oil, root oil, valerian oil, Oleic acid, Linoleic acid, Oleyl alcohol, Isostearyl alcohol, semisynthetic derivatives thereof, and any combinations thereof.

[00130] The oil may further comprise a silicone component, such as a volatile silicone component, which can be the sole oil in the silicone component or can be combined with other silicone and non-silicone, volatile and non-volatile oils. Suitable silicone components include, but are not limited to, methylphenylpolysiloxane, simethicone, dimethicone, phenyltrimethicone (or an organo-modified version thereof), alkylated derivatives of polymeric silicones, cetyl dimethicone, lauryl trimethicone, hydroxylated derivatives of polymeric silicones (e.g., dimethiconol), volatile silicone oils, cyclic and linear silicones, cyclomethicone, derivatives of cyclomethicone, hexamethylcyclotrisiloxane, octamethylcyclotetrasiloxane, decamethylcyclopentasiloxane, volatile linear dimethylpolysiloxanes, isohexadecane, isoeicosane, isotetracosane, polyisobutene, isooctane, isododecane, semi-synthetic derivatives thereof, and combinations thereof.

[00131] The volatile oil can be the organic solvent, or the volatile oil can be present in addition to an organic solvent. Suitable volatile oils include, but are not limited to, a terpene, monoterpene, sesquiterpene, carminative, azulene, menthol, camphor, thujone, thymol, nerol, linalool, limonene, geraniol, perillyl alcohol, nerolidol, farnesol, ylangene, bisabolol, famesene, ascaridole, chenopodium oil, citronellal, citral, citronellol, chamazulene, yarrow, guaiazulene, chamomile, semi-synthetic derivatives, or combinations thereof. In some embodiments, the volatile oil in the silicone component is different than the oil in the oil phase.

[00132] Surface active agents, or surfactants, are amphipathic molecules that consist of a nonpolar hydrophobic portion, usually a straight or branched hydrocarbon or fluorocarbon chain containing 8-18 carbon atoms, attached to a polar or ionic hydrophilic portion. The hydrophilic portion can be nonionic, ionic or zwitterionic. The hydrocarbon chain interacts weakly with the water molecules in an aqueous environment, whereas the polar or ionic head group interacts strongly with water molecules via dipole or ion-dipole interactions. Based on the nature of the hydrophilic group, surfactants are classified into anionic, cationic, zwitterionic, nonionic and polymeric surfactants. [00133] The surfactant in the nanoemulsion can be a pharmaceutically acceptable ionic surfactant, a pharmaceutically acceptable nonionic surfactant, a pharmaceutically acceptable cationic surfactant, a pharmaceutically acceptable anionic surfactant, or a pharmaceutically acceptable zwitterionic surfactant. Further, the surfactant can be a pharmaceutically acceptable ionic polymeric surfactant, a pharmaceutically acceptable nonionic polymeric surfactant, a pharmaceutically acceptable cationic polymeric surfactant, a pharmaceutically acceptable anionic polymeric surfactant, or a pharmaceutically acceptable zwitterionic polymeric surfactant. Examples of polymeric surfactants include, but are not limited to, a graft copolymer of a poly(methyl methacrylate) backbone with multiple (at least one) polyethylene oxide (PEO) side chain, polyhydroxystearic acid, an alkoxylated alkyl phenol formaldehyde condensate, a polyalkylene glycol modified polyester with fatty acid hydrophobes, a polyester, semi-synthetic derivatives thereof, or combinations thereof. Exemplary surfactants are described in Applied Surfactants: Principles and Applications (Tharwat F. Tadros, Copyright Aug. 2005 WILEY- VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-30629-3).

[00134] Suitable surfactants include, but are not limited to, ethoxylated nonylphenol comprising 9 to 10 units of ethyleneglycol, ethoxylated undecanol comprising 8 units of ethyleneglycol, polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monopalmitate, polyoxyethylene (20) sorbitan monostearate, polyoxyethylene (20) sorbitan monooleate, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, ethoxylated hydrogenated ricin oils, sodium laurylsulfate, a diblock copolymer of ethyleneoxyde and propyleneoxyde, Ethylene Oxide-Propylene Oxide Block Copolymers, and tetra-functional block copolymers based on ethylene oxide and propylene oxide, Glyceryl monoesters, Glyceryl caprate, Glyceryl caprylate, Glyceryl cocate, Glyceryl erucate, Glyceryl hydroxysterate, Glyceryl isostearate, Glyceryl lanolate, Glyceryl laurate, Glyceryl linolate, Glyceryl myristate, Glyceryl oleate, Glyceryl PABA, Glyceryl palmitate, Glyceryl ricinoleate, Glyceryl stearate, Glyceryl thiglycolate, Glyceryl dilaurate, Glyceryl dioleate, Glyceryl dimyristate, Glyceryl disterate, Glyceryl sesuioleate, Glyceryl stearate lactate, Polyoxyethylene cetyl/stearyl ether, Polyoxyethylene cholesterol ether, Polyoxyethylene laurate or dilaurate, Polyoxyethylene stearate or distearate, polyoxyethylene fatty ethers, Polyoxyethylene lauryl ether, Polyoxyethylene stearyl ether, polyoxyethylene myristyl ether, a steroid, Cholesterol, Betasitosterol, Bisabolol, fatty acid esters of alcohols, isopropyl myristate, Aliphati-isopropyl n- butyrate, Isopropyl n-hexanoate, Isopropyl n-decanoate, Isoproppyl palmitate, Octyldodecyl myristate, alkoxylated alcohols, alkoxylated acids, alkoxylated amides, alkoxylated sugar derivatives, alkoxylated derivatives of natural oils and waxes, polyoxyethylene polyoxypropylene block copolymers, nonoxynol-14, PEG-8 laurate, PEG-6 Cocoamide, PEG-20 methylglucose sesquistearate, PEG40 lanolin, PEG-40 castor oil, PEG-40 hydrogenated castor oil, polyoxyethylene fatty ethers, glyceryl diesters, polyoxyethylene stearyl ether, polyoxyethylene myristyl ether, and polyoxyethylene lauryl ether, glyceryl dilaurate, glyceryl dimystate, glyceryl distearate, semi-synthetic derivatives thereof, or mixtures thereof.

[00135] Additional suitable surfactants include, but are not limited to, non-ionic lipids, such as glyceryl laurate, glyceryl myristate, glyceryl dilaurate, glyceryl dimyristate, semi-synthetic derivatives thereof, and mixtures thereof.

[00136] In other embodiments, the surfactant is a polyoxyethylene fatty ether having a polyoxyethylene head group ranging from about 2 to about 100 groups, or an alkoxylated alcohol having the structure Rs — (OCH2 CH2)_y -OH, wherein Rs is a branched or unbranched alkyl group having from about 6 to about 22 carbon atoms and y is between about 4 and about 100, and preferably, between about 10 and about 100. Preferably, the alkoxylated alcohol is the species wherein Rs is a lauryl group and y has an average value of 23. In other embodiments, the surfactant is an alkoxylated alcohol which is an ethoxylated derivative of lanolin alcohol. Preferably, the ethoxylated derivative of lanolin alcohol is laneth-10, which is the polyethylene glycol ether of lanolin alcohol with an average ethoxylation value of 10.

[00137] Nonionic surfactants include, but are not limited to, an ethoxylated surfactant, an alcohol ethoxylated, an alkyl phenol ethoxylated, a fatty acid ethoxylated, a monoalkaolamide ethoxylated, a sorbitan ester ethoxylated, a fatty amino ethoxylated, an ethylene oxide-propylene oxide copolymer, Bis(polyethylene glycol bis[imidazoyl carbonyl]), nonoxynol-9, Bis(polyethylene glycol bis[imidazoyl carbonyl]), Brij® 35, Brij® 56, Brij® 72, Brij® 76, Brij® 92V, Brij® 97, Brij® 58P, Cremophor® EL, Decaethylene glycol monododecyl ether, N- Decanoyl-N-methylglucamine, n-Decyl alpha-D-glucopyranoside, Decyl beta-D- maltopyranoside, n-Dodecanoyl-N-methylglucamide, n-Dodecyl alpha-D-maltoside, n-Dodecyl beta-D-maltoside, n-Dodecyl beta-D-maltoside, Heptaethylene glycol monodecyl ether, Heptaethylene glycol monododecyl ether, Heptaethylene glycol monotetradecyl ether, n- Hexadecyl beta-D-maltoside, Hexaethylene glycol monododecyl ether, Hexaethylene glycol monohexadecyl ether, Hexaethylene glycol monooctadecyl ether, Hexaethylene glycol monotetradecyl ether, Igepal CA-630, Igepal CA-630, Methyl-6-O-(N-heptylcarbamoyl)-alpha- D-glucopyranoside, Nonaethylene glycol monododecyl ether, N-Nonanoyl-N-methylglucamine, N-Nonanoyl-N-methylglucamine, Octaethylene glycol monodecyl ether, Octaethylene glycol monododecyl ether, Octaethylene glycol monohexadecyl ether, Octaethylene glycol monooctadecyl ether, Octaethylene glycol monotetradecyl ether, Octyl-beta-D-glucopyranoside, Pentaethylene glycol monodecyl ether, Pentaethylene glycol monododecyl ether, Pentaethylene glycol monohexadecyl ether, Pentaethylene glycol monohexyl ether, Pentaethylene glycol monooctadecyl ether, Pentaethylene glycol monooctyl ether, Polyethylene glycol diglycidyl ether, Polyethylene glycol ether W-l, Polyoxyethylene 10 tridecyl ether, Polyoxyethylene 100 stearate, Polyoxyethylene 20 isohexadecyl ether, Polyoxyethylene 20 oleyl ether, Polyoxyethylene 40 stearate, Polyoxyethylene 50 stearate, Polyoxyethylene 8 stearate, Polyoxyethylene bis(imidazolyl carbonyl), Polyoxyethylene 25 propylene glycol stearate, Saponin from Quillaja bark, Span® 20, Span® 40, Span® 60, Span® 65, Span® 80, Span® 85, Tergitol, Type 15-S-12, Tergitol, Type 15-S-3O, Tergitol, Type 15-S-5, Tergitol, Type 15-S-7, Tergitol, Type 15-S-9, Tergitol, Type NP-10, Tergitol, Type NP-4, Tergitol, Type NP-40, Tergitol, Type NP-7, Tergitol, Type NP-9, Tergitol, Tergitol, Type TMN-10, Tergitol, Type TMN-6, Tetradecyl-beta-D-maltoside, Tetraethylene glycol monodecyl ether, Tetraethylene glycol monododecyl ether, Tetraethylene glycol monotetradecyl ether, Triethylene glycol monodecyl ether, Triethylene glycol monododecyl ether, Triethylene glycol monohexadecyl ether, Triethylene glycol monooctyl ether, Triethylene glycol monotetradecyl ether, Triton CF- 21, Triton CF-32, Triton DF-12, Triton DF-16, Triton GR-5M, Triton QS-15, Triton QS-44, Triton X-100, Triton X-102, Triton X-15, Triton X-151, Triton X-200, Triton X-207, Triton® X- 100, Triton® X-l 14, Triton® X-165, Triton® X-305, Triton® X-405, Triton® X-45, Triton® X- 705-70, TWEEN® 20, TWEEN® 21, TWEEN® 40, TWEEN® 60, TWEEN® 61, TWEEN® 65, TWEEN® 80, TWEEN® 81, TWEEN® 85, Tyloxapol, n-Undecyl beta-D-glucopyranoside, semisynthetic derivatives thereof, or combinations thereof.

[00138] In other embodiments, the nonionic surfactant can be a poloxamer. Poloxamers are polymers made of a block of polyoxyethylene, followed by a block of polyoxypropylene, followed by a block of polyoxyethylene. The average number of units of polyoxyethylene and polyoxypropylene varies based on the number associated with the polymer. For example, the smallest polymer, Poloxamer 101, consists of a block with an average of 2 units of polyoxyethylene, a block with an average of 16 units of polyoxypropylene, followed by a block with an average of 2 units of polyoxyethylene. Poloxamers range from colorless liquids and pastes to white solids. In cosmetics and personal care products, Poloxamers are used in the formulation of skin cleansers, bath products, shampoos, hair conditioners, mouthwashes, eye makeup remover and other skin and hair products. Examples of Poloxamers include, but are not limited to, Poloxamer 101, Poloxamer 105, Poloxamer 108, Poloxamer 122, Poloxamer 123, Poloxamer 124, Poloxamer 181, Poloxamer 182, Poloxamer 183, Poloxamer 184, Poloxamer 185, Poloxamer 188, Poloxamer 212, Poloxamer 215, Poloxamer 217, Poloxamer 231, Poloxamer 234, Poloxamer 235, Poloxamer 237, Poloxamer 238, Poloxamer 282, Poloxamer 284, Poloxamer 288, Poloxamer 331, Poloxamer 333, Poloxamer 334, Poloxamer 335, Poloxamer 338, Poloxamer 401, Poloxamer 402, Poloxamer 403, Poloxamer 407, Poloxamer 105 Benzoate, and Poloxamer 182 Dibenzoate.

[00139] Suitable cationic surfactants include, but are not limited to, a quarternary ammonium compound, an alkyl trimethyl ammonium chloride compound, a dialkyl dimethyl ammonium chloride compound, a cationic halogen-containing compound, such as cetylpyridinium chloride, Benzalkonium chloride, Benzalkonium chloride, Benzyldimethylhexadecylammonium chloride, Benzyldimethyltetradecylammonium chloride, Benzyldodecyldimethylammonium bromide, Benzyltrimethylammonium tetrachloroiodate, Dimethyldioctadecylammonium bromide, Dodecylethyldimethylammonium bromide, Dodecyltrimethylammonium bromide, Dodecyltrimethylammonium bromide, Ethylhexadecyldimethylammonium bromide, Girard’s reagent T, Hexadecyltrimethylammonium bromide, Hexadecyltrimethylammonium bromide, N,N’ ,N’ -Polyoxyethylene( 10)-N -tallow- 1 , 3 -diaminopropane, Thonzonium bromide, Trimethyl(tetradecyl)ammonium bromide, l,3,5-Triazine-l,3,5(2H,4H,6H)-triethanol, 1- Decanaminium, N-decyl-N, N-dimethyl-, chloride, Didecyl dimethyl ammonium chloride, 2-(2- (p-(Diisobutyl)cresosxy)ethoxy)ethyl dimethyl benzyl ammonium chloride, 2-(2-(p- (Diisobutyl)phenoxy)ethoxy)ethyl dimethyl benzyl ammonium chloride, Alkyl 1 or 3 benzyl-1- (2-hydroxethyl)-2-imidazolinium chloride, Alkyl bis(2 -hydroxyethyl) benzyl ammonium chloride, Alkyl demethyl benzyl ammonium chloride, Alkyl dimethyl 3,4-dichlorobenzyl ammonium chloride (100% C12), Alkyl dimethyl 3,4-dichlorobenzyl ammonium chloride (50% C14, 40% C12, 10% Cie), Alkyl dimethyl 3,4-dichlorobenzyl ammonium chloride (55% C14, 23% C12, 20% Cie), Alkyl dimethyl benzyl ammonium chloride, Alkyl dimethyl benzyl ammonium chloride (100% C14), Alkyl dimethyl benzyl ammonium chloride (100% Cie), Alkyl dimethyl benzyl ammonium chloride (41% C14, 28% C12), Alkyl dimethyl benzyl ammonium chloride (47% C12, 18% C14), Alkyl dimethyl benzyl ammonium chloride (55% Cl 6, 20% C14), Alkyl dimethyl benzyl ammonium chloride (58% C14, 28% Cie), Alkyl dimethyl benzyl ammonium chloride (60% C14, 25% C12), Alkyl dimethyl benzyl ammonium chloride (61% C11, 23% C14), Alkyl dimethyl benzyl ammonium chloride (61% C12, 23% C14), Alkyl dimethyl benzyl ammonium chloride (65% C12, 25% C14), Alkyl dimethyl benzyl ammonium chloride (67% C12, 24% C14), Alkyl dimethyl benzyl ammonium chloride (67% C12, 25% C14), Alkyl dimethyl benzyl ammonium chloride (90% C14, 5% C12), Alkyl dimethyl benzyl ammonium chloride (93% C14, 4% C12), Alkyl dimethyl benzyl ammonium chloride (95% Ci6, 5% Cis), Alkyl dimethyl benzyl ammonium chloride, Alkyl didecyl dimethyl ammonium chloride, Alkyl dimethyl benzyl ammonium chloride, Alkyl dimethyl benzyl ammonium chloride (C1 -16), Alkyl dimethyl benzyl ammonium chloride (C12-18), Alkyl dimethyl benzyl ammonium chloride, dialkyl dimethyl benzyl ammonium chloride, Alkyl dimethyl dimethybenzyl ammonium chloride, Alkyl dimethyl ethyl ammonium bromide (90% C14, 5% Ci6, 5% C12), Alkyl dimethyl ethyl ammonium bromide (mixed alkyl and alkenyl groups as in the fatty acids of soybean oil), Alkyl dimethyl ethylbenzyl ammonium chloride, Alkyl dimethyl ethylbenzyl ammonium chloride (60% C14), Alkyl dimethyl isopropylbenzyl ammonium chloride (50% C12, 30% C14, 17% Ci6, 3% Cis), Alkyl trimethyl ammonium chloride (58% Cis, 40% Ci6, 1% C14, 1% C12), Alkyl trimethyl ammonium chloride (90% Cis, 10% Cie), Alkyldimethyl(ethylbenzyl) ammonium chloride (C12-18), Di-(Cs-io)-alkyl dimethyl ammonium chlorides, Dialkyl dimethyl ammonium chloride, Dialkyl methyl benzyl ammonium chloride, Didecyl dimethyl ammonium chloride, Diisodecyl dimethyl ammonium chloride, Dioctyl dimethyl ammonium chloride, Dodecyl bis (2-hydroxyethyl) octyl hydrogen ammonium chloride, Dodecyl dimethyl benzyl ammonium chloride, Dodecylcarbamoyl methyl dinethyl benzyl ammonium chloride, Heptadecyl hydroxyethylimidazolinium chloride, Hexahydro- 1,3, 5 - tris(2-hydroxyethyl)-s-triazine, Hexahydro-1, 3, 5-tris(2-hydroxyethyl)-s- triazine, Myristalkonium chloride (and) Quat RNIUM 14, N,N-Dimethyl-2- hydroxypropylammonium chloride polymer, n-Tetradecyl dimethyl benzyl ammonium chloride monohydrate, Octyl decyl dimethyl ammonium chloride, Octyl dodecyl dimethyl ammonium chloride, Octyphenoxyethoxyethyl dimethyl benzyl ammonium chloride, Oxydiethylenebis(alkyl dimethyl ammonium chloride), Quaternary ammonium compounds, dicoco alkyldimethyl, chloride, Trimethoxysily propyl dimethyl octadecyl ammonium chloride, Trimethoxysilyl quats, Trimethyl dodecylbenzyl ammonium chloride, semi-synthetic derivatives thereof, and combinations thereof. [00140] Exemplary cationic halogen-containing compounds include, but are not limited to, cetylpyridinium halides, cetyltrimethylammonium halides, cetyldimethylethylammonium halides, cetyldimethylbenzylammonium halides, cetyltributylphosphonium halides, dodecyltrimethylammonium halides, or tetradecyltrimethylammonium halides. In some particular embodiments, suitable cationic halogen containing compounds comprise, but are not limited to, cetylpyridinium chloride (CPC), cetyltrimethylammonium chloride, cetylbenzyldimethylammonium chloride, cetylpyridinium bromide (CPB), cetyltrimethylammonium bromide (CTAB), cetyidimethylethylammonium bromide, cetyltributylphosphonium bromide, dodecyltrimethylammonium bromide, and tetrad ecyltrimethylammonium bromide. In particularly preferred embodiments, the cationic halogen containing compound is CPC, although the compositions of the present disclosed are not limited to formulation with an particular cationic containing compound.

[00141] Suitable anionic surfactants include, but are not limited to, a carboxylate, a sulphate, a sulphonate, a phosphate, chenodeoxycholic acid, chenodeoxycholic acid sodium salt, cholic acid, ox or sheep bile, Dehydrocholic acid, Deoxycholic acid, Deoxycholic acid, Deoxycholic acid methyl ester, Digitonin, Digitoxigenin, N,N-Dimethyldodecylamine N-oxide, Docusate sodium salt, Glycochenodeoxycholic acid sodium salt, Glycocholic acid hydrate, synthetic, Glycocholic acid sodium salt hydrate, synthetic, Glycodeoxycholic acid monohydrate, Glycodeoxycholic acid sodium salt, Glycodeoxycholic acid sodium salt, Glycolithocholic acid 3 -sulfate disodium salt, Glycolithocholic acid ethyl ester, N-Lauroylsarcosine sodium salt, N-Lauroylsarcosine solution, N-Lauroylsarcosine solution, Lithium dodecyl sulfate, Lithium dodecyl sulfate, Lithium dodecyl sulfate, Lugol solution, Niaproof 4, Type 4, 1 -Octanesulfonic acid sodium salt, Sodium 1- butanesulfonate, Sodium 1 -decanesulfonate, Sodium 1 -decanesulfonate, Sodium 1- dodecanesulfonate, Sodium 1 -heptanesulfonate anhydrous, Sodium 1 -heptanesulfonate anhydrous, Sodium 1 -nonanesulfonate, Sodium 1 -propanesulfonate monohydrate, Sodium 2- bromoethanesulfonate, Sodium cholate hydrate, Sodium choleate, Sodium deoxycholate, Sodium deoxycholate monohydrate, Sodium dodecyl sulfate, Sodium hexanesulfonate anhydrous, Sodium octyl sulfate, Sodium pentanesulfonate anhydrous, Sodium taurocholate, Taurochenodeoxycholic acid sodium salt, Taurodeoxycholic acid sodium salt monohydrate, Taurohyodeoxy cholic acid sodium salt hydrate, Taurolithocholic acid 3-sulfate disodium salt, Tauroursodeoxy cholic acid sodium salt, Trizma® dodecyl sulfate, TWEEN® 80, Ursodeoxycholic acid, semi-synthetic derivatives thereof, and combinations thereof. [00142] Suitable zwitterionic surfactants include, but are not limited to, an N-alkyl betaine, lauryl amindo propyl dimethyl betaine, an alkyl dimethyl glycinate, an N-alkyl amino propionate, CHAPS, minimum 98% (TLC), CHAPS, SigmaUltra, minimum 98% (TLC), CHAPS, for electrophoresis, minimum 98% (TLC), CHAPSO, minimum 98%, CHAPSO, SigmaUltra, CHAPSO, for electrophoresis, 3-(Decyldimethylammonio)propanesulfonate inner salt, 3-Dodecyldimethylammonio)propanesulfonate inner salt, SigmaUltra, 3- (Dodecyldimethylammonio)propanesulfonate inner salt, 3-(N,N- Dimethylmyristylammonio)propanesulfonate, 3-(N,N- Dimethyloctadecylammonio)propanesulfonate, 3-(N,N- Dimethyloctylammonio)propanesulfonate inner salt, 3-(N,N- Dimethylpalmitylammonio)propanesulfonate, semi-synthetic derivatives thereof, and combinations thereof.

[00143] In some embodiments, the nanoemulsion comprises a cationic surfactant, which can be cetylpyridinium chloride. In other embodiments, the nanoemulsion comprises a cationic surfactant, and the concentration of the cationic surfactant is less than about 5.0% and greater than about 0.001%. In yet another embodiment, the nanoemulsion comprises a cationic surfactant, and the concentration of the cationic surfactant is selected from the group consisting of less than about 5%, less than about 4.5%, less than about 4.0%, less than about 3.5%, less than about 3.0%, less than about 2.5%, less than about 2.0%, less than about 1.5%, less than about 1.0%, less than about 0.90%, less than about 0.80%, less than about 0.70%, less than about 0.60%, less than about 0.50%, less than about 0.40%, less than about 0.30%, less than about 0.20%, or less than about 0.10%. Further, the concentration of the cationic agent in the nanoemulsion is greater than about 0.002%, greater than about 0.003%, greater than about 0.004%, greater than about 0.005%, greater than about 0.006%, greater than about 0.007%, greater than about 0.008%, greater than about 0.009%, greater than about 0.010%, or greater than about 0.001%. In one embodiment, the concentration of the cationic agent in the nanoemulsion is less than about 5.0% and greater than about 0.001%.

[00144] In another embodiment, the nanoemulsion comprises at least one cationic surfactant and at least one non-cationic surfactant. The non-cationic surfactant is a nonionic surfactant, such as a polysorbate (Tween), such as polysorbate 80 or polysorbate 20. In one embodiment, the non-ionic surfactant is present in a concentration of about 0.01% to about 5.0%, or the nonionic surfactant is present in a concentration of about 0.1% to about 3%. In yet another embodiment, the nanoemulsion comprises a cationic surfactant present in a concentration of about 0.01% to about 2%, in combination with a nonionic surfactant.

[00145] The nanoemulsion may further comprise additional components, including, for example, one or more solvents, such as an organic phosphate-based solvent, bulking agents, coloring agents, pharmaceutically acceptable excipients, a preservative, pH adjuster, buffer, chelating agent, etc. The additional components can be admixed into a previously emulsified nanoemulsion composition, or the additional components can be added to the original mixture to be emulsified. In certain of these embodiments, one or more additional components are admixed into an existing nanoemulsion composition immediately prior to its use.

[00146] Suitable preservatives in the nanoemulsion include, but are not limited to, cetylpyridinium chloride, benzalkonium chloride, benzyl alcohol, chlorhexidine, imidazolidinyl urea, phenol, potassium sorbate, benzoic acid, bronopol, chlorocresol, paraben esters, phenoxyethanol, sorbic acid, alpha-tocophemol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, sodium ascorbate, sodium metabisulphite, citric acid, edetic acid, semi -synthetic derivatives thereof, and combinations thereof. Other suitable preservatives include, but are not limited to, benzyl alcohol, chlorhexidine (bis (p- chlorophenyldiguanido) hexane), chlorphenesin (3-(-4-chloropheoxy)-propane-l,2-diol), Kathon CG (methyl and methylchloroisothiazolinone), parabens (methyl, ethyl, propyl, butyl hydrobenzoates), phenoxyethanol (2 -phenoxyethanol), sorbic acid (potassium sorbate, sorbic acid), Phenonip (phenoxyethanol, methyl, ethyl, butyl, propyl parabens), Phenoroc (phenoxyethanol 0.73%, methyl paraben 0.2%, propyl paraben 0.07%), Liquipar Oil (isopropyl, isobutyl, butylparabens), Liquipar PE (70% phenoxyethanol, 30% liquipar oil), Nipaguard MPA (benzyl alcohol (70%), methyl & propyl parabens), Nipaguard MPS (propylene glycol, methyl & propyl parabens), Nipasept (methyl, ethyl and propyl parabens), Nipastat (methyl, butyl, ethyl andpropyel parabens), Elestab 388 (phenoxyethanol in propylene glycol plus chlorphenesin and methylparaben), and Killitol (7.5% chlorphenesin and 7.5% methyl parabens).

[00147] The nanoemulsion may further comprise at least one pH adjuster. Suitable pH adjusters that may be used in the nanoemulsion include, but are not limited to, diethyanolamine, lactic acid, monoethanolamine, triethylanolamine, sodium hydroxide, sodium phosphate, semisynthetic derivatives thereof, and combinations thereof.

[00148] In some embodiments, the nanoemulsion can comprise a chelating agent. The chelating agent may be present in an amount of about 0.0005% to about 1%. Examples of suitable chelating agents include, but are not limited to, ethylenediamine, ethylenediaminetetraacetic acid (EDTA), phytic acid, polyphosphoric acid, citric acid, gluconic acid, acetic acid, lactic acid, and dimercaprol, and a preferred chelating agent is ethylenediaminetetraacetic acid.

[00149] The nanoemulsion may further comprise a buffering agent, such as a pharmaceutically acceptable buffering agent. Examples of suitable buffering agents include, but are not limited to, 2-Amino-2-methyl-l,3-propanediol, >99.5% (NT), 2-Amino-2-methyl-l- propanol, >99.0% (GC), L-(+)-Tartaric acid, >99.5% (T), ACES, >99.5% (T), ADA, >99.0% (T), Acetic acid, >99.5% (GC/T), Acetic acid, for luminescence, >99.5% (GC/T), Ammonium acetate solution, for molecular biology, ~5 M in H2O, Ammonium acetate, for luminescence, >99.0% (calc, on dry substance, T), Ammonium bicarbonate, >99.5% (T), Ammonium citrate dibasic, >99.0% (T), Ammonium formate solution, 10 M in H2O, Ammonium formate, >99.0% (calc, based on dry substance, NT), Ammonium oxalate monohydrate, >99.5% (RT), Ammonium phosphate dibasic solution, 2.5 M in H2O, Ammonium phosphate dibasic, >99.0% (T), Ammonium phosphate monobasic solution, 2.5 M in H2O, Ammonium phosphate monobasic, >99.5% (T), Ammonium sodium phosphate dibasic tetrahydrate, >99.5% (NT), Ammonium sulfate solution, for molecular biology, 3.2 M in H2O, Ammonium tartrate dibasic solution , 2 M in H2O (colorless solution at 20 °C), Ammonium tartrate dibasic, >99.5% (T), BES buffered saline, for molecular biology, 2x concentrate, BES , >99.5% (T), BES, for molecular biology, >99.5% (T), BICINE buffer Solution, for molecular biology, 1 M in H2O, BICINE, >99.5% (T), BIS-TRIS, >99.0% (NT), Bicarbonate buffer solution , >0.1 M Na₂CO₃, >0.2 M NaHCCh, Boric acid , >99.5% (T), Boric acid, for molecular biology, >99.5% (T), CAPS, >99.0% (TLC), CHES, >99.5% (T), Calcium acetate hydrate, >99.0% (calc, on dried material, KT), Calcium carbonate, precipitated, >99.0% (KT), Calcium citrate tribasic tetrahydrate, >98.0% (calc, on dry substance, KT), Citrate Concentrated Solution , for molecular biology, 1 M in H2O, Citric acid , anhydrous, >99.5% (T), Citric acid , for luminescence, anhydrous, >99.5% (T), Diethanolamine, >99.5% (GC), EPPS , >99.0% (T), Ethylenediaminetetraacetic acid disodium salt dihydrate, for molecular biology, >99.0% (T), Formic acid solution , 1.0 M in H₂O, Gly-Gly-Gly, >99.0% (NT), Gly-Gly, >99.5% (NT), Glycine, >99.0% (NT), Glycine, for luminescence, >99.0% (NT), Glycine, for molecular biology, >99.0% (NT), HEPES buffered saline, for molecular biology, 2x concentrate, HEPES , >99.5% (T), HEPES, for molecular biology, >99.5% (T), Imidazole buffer Solution, 1 M in H2O, Imidazole, >99.5% (GC), Imidazole, for luminescence, >99.5% (GC), Imidazole, for molecular biology, >99.5% (GC), Lipoprotein Refolding Buffer, Lithium acetate dihydrate, >99.0% (NT), Lithium citrate tribasic tetrahydrate, >99.5% (NT), MBS hydrate, >99.5% (T), MES monohydrate, for luminescence, >99.5% (T), MES solution, for molecular biology, 0.5 M in H2O, MOPS, >99.5% (T), MOPS, for luminescence, >99.5% (T), MOPS, for molecular biology, >99.5% (T), Magnesium acetate solution, for molecular biology, ~1 M in H2O, Magnesium acetate tetrahydrate, >99.0% (KT), Magnesium citrate tribasic nonahydrate, >98.0% (calc, based on dry substance, KT), Magnesium formate solution, 0.5 M in H2O, Magnesium phosphate dibasic trihydrate, >98.0% (KT), Neutralization solution for the in-situ hybridization for in-situ hybridization, for molecular biology, Oxalic acid dihydrate, >99.5% (RT), PIPES, >99.5% (T), PIPES, for molecular biology, >99.5% (T), Phosphate buffered saline, solution (autoclaved), Phosphate buffered saline, washing buffer for peroxidase conjugates in Western Blotting, lOx concentrate, Piperazine, anhydrous, >99.0% (T), Potassium D-tartrate monobasic , >99.0% (T), Potassium acetate solution, for molecular biology, Potassium acetate solution, for molecular biology, 5M in H2O, Potassium acetate solution, for molecular biology, ~1 M in H2O, Potassium acetate, >99.0% (NT), Potassium acetate, for luminescence, >99.0% (NT), Potassium acetate, for molecular biology, >99.0% (NT), Potassium bicarbonate , >99.5% (T), Potassium carbonate , anhydrous, >99.0% (T), Potassium chloride, >99.5% (AT), Potassium citrate monobasic , >99.0% (dried material, NT), Potassium citrate tribasic solution , 1 M in H2O, Potassium formate solution, 14 M in H2O, Potassium formate , >99.5% (NT), Potassium oxalate monohydrate, >99.0% (RT), Potassium phosphate dibasic, anhydrous, >99.0% (T), Potassium phosphate dibasic, for luminescence, anhydrous, >99.0% (T), Potassium phosphate dibasic, for molecular biology, anhydrous, >99.0% (T), Potassium phosphate monobasic, anhydrous, >99.5% (T), Potassium phosphate monobasic, for molecular biology, anhydrous, >99.5% (T), Potassium phosphate tribasic monohydrate, >95% (T), Potassium phthalate monobasic, >99.5% (T), Potassium sodium tartrate solution, 1.5 M in H2O, Potassium sodium tartrate tetrahydrate, >99.5% (NT), Potassium tetraborate tetrahydrate, >99.0% (T), Potassium tetraoxalate dihydrate, >99.5% (RT), Propionic acid solution, 1.0 M in H2O, STE buffer solution, for molecular biology, pH 7.8, STET buffer solution, for molecular biology, pH 8.0, Sodium 5,5-diethylbarbiturate , >99.5% (NT), Sodium acetate solution, for molecular biology, ~3 M in H2O, Sodium acetate trihydrate, >99.5% (NT), Sodium acetate, anhydrous, >99.0% (NT), Sodium acetate, for luminescence, anhydrous, >99.0% (NT), Sodium acetate, for molecular biology, anhydrous, >99.0% (NT), Sodium bicarbonate, >99.5% (T), Sodium bitartrate monohydrate, >99.0% (T), Sodium carbonate decahydrate, >99.5% (T), Sodium carbonate, anhydrous, >99.5% (calc, on dry substance, T), Sodium citrate monobasic, anhydrous, >99.5% (T), Sodium citrate tribasic dihydrate, >99.0% (NT), Sodium citrate tribasic dihydrate, for luminescence, >99.0% (NT), Sodium citrate tribasic dihydrate, for molecular biology, >99.5% (NT), Sodium formate solution, 8 M in H2O, Sodium oxalate, >99.5% (RT), Sodium phosphate dibasic dihydrate, >99.0% (T), Sodium phosphate dibasic dihydrate, for luminescence, >99.0% (T), Sodium phosphate dibasic dihydrate, for molecular biology, >99.0% (T), Sodium phosphate dibasic dodecahydrate, >99.0% (T), Sodium phosphate dibasic solution, 0.5 M in H2O, Sodium phosphate dibasic, anhydrous, >99.5% (T), Sodium phosphate dibasic, for molecular biology, >99.5% (T), Sodium phosphate monobasic dihydrate, >99.0% (T), Sodium phosphate monobasic dihydrate, for molecular biology, >99.0% (T), Sodium phosphate monobasic monohydrate, for molecular biology, >99.5% (T), Sodium phosphate monobasic solution, 5 M in H2O, Sodium pyrophosphate dibasic, >99.0% (T), Sodium pyrophosphate tetrabasic decahydrate, >99.5% (T), Sodium tartrate dibasic dihydrate, >99.0% (NT), Sodium tartrate dibasic solution ,1.5 M in H2O (colorless solution at 20 °C), Sodium tetraborate decahydrate , >99.5% (T), TAPS , >99.5% (T), TES, >99.5% (calc, based on dry substance, T), TM buffer solution, for molecular biology, pH 7.4, TNT buffer solution, for molecular biology, pH 8.0, TRIS Glycine buffer solution, 10x concentrate, TRIS acetate - EDTA buffer solution, for molecular biology, TRIS buffered saline, 10* concentrate, TRIS glycine SDS buffer solution, for electrophoresis, 10x concentrate, TRIS phosphate-EDTA buffer solution, for molecular biology, concentrate, 10x concentrate, Tricine, >99.5% (NT), Triethanolamine, >99.5% (GC), Triethylamine, >99.5% (GC), Triethylammonium acetate buffer, volatile buffer, ~1.0 M in H2O, Triethylammonium phosphate solution, volatile buffer, ~1.0 M in H2O, Trimethylammonium acetate solution, volatile buffer, ~1.0 M in H2O, Trimethylammonium phosphate solution, volatile buffer, ~1 M in H2O, Tris-EDTA buffer solution, for molecular biology, concentrate, 100x concentrate, Tris-EDTA buffer solution, for molecular biology, pH 7.4, Tris-EDTA buffer solution, for molecular biology, pH 8.0, Trizma® acetate, >99.0% (NT), Trizma® base , >99.8% (T), Trizma® base, >99.8% (T), Trizma® base , for luminescence, >99.8% (T), Trizma® base, for molecular biology, >99.8% (T), Trizma® carbonate, >98.5% (T), Trizma® hydrochloride buffer solution, for molecular biology, pH 7.2, Trizma® hydrochloride buffer solution, for molecular biology, pH 7.4, Trizma® hydrochloride buffer solution, for molecular biology, pH 7.6, Trizma® hydrochloride buffer solutio, for molecular biology, pH 8.0, Trizma® hydrochloride, >99.0% (AT), Trizma® hydrochloride, for luminescence, >99.0% (AT), Trizma® hydrochloride, for molecular biology, >99.0% (AT), and Trizma® maleate, >99.5% (NT).

[00150] In some embodiments, the nanoemulsion can comprise one or more emulsifying agents to aid in the formation of emulsions. Emulsifying agents include compounds that aggregate at the oil/water interface to form a kind of continuous membrane that prevents direct contact between two adjacent droplets. The nanoemulsion may readily be diluted with water or another aqueous phase to a desired concentration without impairing its desired properties.

RIG-I and TLR Agonists

[00151] As described herein, adjuvants function through the induction of innate immune pathways, thereby providing an optimal cytokine and chemokine environment that promotes the induction of quantitatively and qualitatively improved immune responses. For viruses that induce long-lasting immunity, natural viral infection stimulates strong innate immune responses through the activation of three main pathways involving Toll-, RIG-I-, and NOD-like receptors (TLRs, RLRs, NLRs).

[00152] TLR signaling drives T cell responses and promotes affinity maturation of antiviral antibodies. Multivalent stimulation of TLRs through combined agonists has been shown to enhance antibody responses in a SARS-CoV vaccine, and skewed responses towards a more TH1 response. Thus, in some aspects, an immunogenic composition described herein comprises an agonist of retinoic acid-inducible gene I (RIG-I) and/or an agonist of a toll-like receptor. RIG-I is an intracellular molecule that responds to viral nucleic acids and activates downstream signaling, resulting in the induction of members of the type I interferon (IFN) family. RIG-I is a member of the pattern-recognition receptors (PRRs) family of proteins, which includes toll -like receptor (TLR) proteins. RIG-I belongs to the cytosolic DExD/H box RNA helicases and is one of three members of the RIG-I-like helicases family (others include MDA5 and LGP2). RIG-I is closely related to the Dicer family of helicases of the RNAi pathway. RIG-I contains a RNA helicase domain and two N-terminal CARD domains which relay the signal to the downstream signaling adaptor mitochondrial antiviral-signaling protein (MAVS). RIG-I signaling via MAVS not only leads to the induction of type I IFN responses via TBK1 and IRF7/8, but it also activates caspase-8-dependent apoptosis, preferentially in tumor. Furthermore, RIG-I has been shown to mediate MAVS-independent inflammasome activation, specifically in the context of viral infection. RIG-I structure and function is further described in, e.g., Matsumiya T, Stafforini DM., Crit Rev Immunol. 2010;30(6):489-513. doi:10.1615/critrevimmunol.v30.i6.10; and Rehwinkel, J., Gack, M.U., Nat Rev Immunol 20, 537-551 (2020); doi.org/10.1038/s41577-020- 0288-3).

[00153] Investigations into ligand recognition by the RIG-I protein have elucidated a number of ligands that serve as agonists and antagonists for RIG-I (see, e.g., Ranjith-Kumar et al., J Biol Chem. 2009 Jan 9; 284(2): 1155-1165). The term “agonist,” as used herein, refers to a molecule, substance, or compound that binds to a receptor and activates the receptor to produce a biological response. In contrast, the term “antagonist” refers to a molecule, substance, or compound that inhibits or blocks the activity of a receptor to which it binds. Any suitable RIG-I agonist may be included in the nanoemulsion-containing compositions and methods disclosed herein. In some embodiments, the RIG-I agonist is a substance or compound that mimics the pathogen-associated molecular pattern (PAMP) induced by a natural viral infection. In some embodiments, the RIG-I agonist is an RNA agonist. Exemplary RIG-I RNA agonists include single-stranded and doublestranded RNAs, such as those described in Ranjith-Kumar et al., supra. In some embodiments, the RNA agonist is a defective interfering (DI) RNA of a Sendai virus (IVT DI) or an influenza virus 5’ triphosphate hairpin RNA (3phpRNA (InvivoGen, San Diego, CA). IVT DI is an in vitro transcribed RNA consisting of the full-length (546nt) copy-back defective interfering RNA of Sendai virus strain Cantell (see, e.g., Martinez-Gil et al., J Virol 2013, 87 (3), 1290-300; and Patel et al., EMBO reports 2013, 14 (9), 780-7, and See SEQ ID NO: 1). The hairpin structure of IVT DI, along with its dsRNA panhandle and 5’ triphosphate, make it a potent and selective RIG-I agonist, and thus, a strong inducer of type I interferons (IFN-Is, e.g., IFN-a and/or IFN-0) and interferon-stimulated genes (ISGs).

[00154] In some embodiments, the RIG-I agonist is a 5’ triphosphate hairpin RNA (3p-hp- RNA), a 5’ triphosphate double stranded synthetic RNA (5’ppp-dsRNA), a synthetic double stranded DNA (poly (dA:dT), a synthetic double stranded RNA (poly (I:C) high molecular weight (BMW)), a synthetic double stranded RNA (poly (I:C) low molecular weight (LMW)), and/or a synthetic stem-loop RNA (e.g., SLR14 or SLR10) In other embodiments, the RIG-I agonist is a small molecule. Any suitable small molecule RIG-I agonist may be used, for example, those known in the art (see, e.g., Loo et al., Cytokine, 70, Issue 1, November 2014, Page 56; and Hemann et al., J Immunol May 1, 2016, 196 (1 Supplement) 76.1). Examples of small molecule agonists of RIG-I include, but are not limited to, Inarigivir (SB 9200), KIN1400, M8 and M8 dimers. [00155] In other embodiments, compositions and methods disclosed herein comprise an agonist of a toll-like receptor (TLR). Any suitable agonist of any suitable toll-like receptor (such as those described herein) may be included in the nanoemulsion-containing composition. For example, a polyriboinosinic polyribocytidylic (pIC) adjuvant activates TLR3 and the RLR MDA5, the synthetic oligodeoxynucleotide CpG is a TLR9 agonist, and the monophosphoryl lipid A stimulates TLR4 signaling (Evans et al., Expert Rev. Vaccines 2:219-229 (2003)). In some embodiments, the TLR agonist is an agonist of TLR3. For example, the TLR3 agonist may be a synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid (also referred to as “pIC,” “poly(I:C),” “poly I:C,” and “p(I:C)”.) pIC is a double-stranded RNA that elicits an immune response by activating toll-like receptor 3 (TLR3), and has long been known as a potent inducer of type I IFN for decades (Field et al., PNAS, 58(5): 2102-2108 (1967)). pIC also has been shown to engage the cytosolic helicase MDA-5. Although originally deemed too toxic for human use, recently the potent adjuvant activity of poly I:C has been newly appreciated in vaccine formulations targeted to dendritic cells (DCs) (Trumpfheller et al., Proc Natl Acad Sci USA, 105(7):2574-9 (2008). doi: 10.1073/pnas.07119761052008). Small molecule TLR agonists also may be employed in the disclosed compositions and methods, several of which are known in the art (see, e.g., Zhang et al., J Med Chem. 2017 Jun 22;60(12):5029-5044. doi: 10.1021/acs.jmedchem.7b00419; Wang et al., Chem. Soc. Rev., 2013,42, 4859-4866; and Shukla et al., ACS Med. Chem. Lett. 2018, 9, 12, 1156-1159).

Compositions and Methods for Inducing an Immune Response

[00156] Methods and compositions disclosed herein desirably comprise pharmaceutically acceptable (e.g., physiologically acceptable) compositions, which comprises a carrier, preferably a pharmaceutically acceptable (e.g., physiologically acceptable) carrier, the nanoemulsion, the RIG-I agonist and/or the TLR agonist, and/or immunogen/antigen/vaccine. Compositions of the present disclosure may be formulated into pharmaceutical compositions that are administered in a therapeutically effective amount to a subject and may further comprise suitable, pharmaceutically-acceptable excipients, additives, or preservatives. Suitable excipients, additives, and preservatives are well known in the art.

[00157] The compositions described herein desirably comprise therapeutically effective amounts of the immimogen/antigen/vaccine, the nanoemulsion, the RIG-I agonist, and/or the TLR agonist. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. Alternatively, the pharmacologic and/or physiologic effect may be prophylactic, i.e., the effect completely or partially prevents a disease or symptom thereof. In this respect, the disclosed compositions comprise “prophylactically effective amounts” of the immunogen/antigen/vaccine, the nanoemulsion, the RIG-I agonist, and/or the TLR agonist. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result (e.g., prevention of subsequent infection and/or disease onset).

[00158] Exemplary dosage forms for pharmaceutical administration are described herein, and include, but are not limited to liquids, ointments, creams, emulsions, lotions, gels, bioadhesive gels, sprays, aerosols, pastes, foams, sunscreens, capsules, microcapsules, suspensions, pessary, powder, semi-solid dosage forms, etc. The compositions can be generated in accordance with conventional techniques described in, e.g., Remington: The Science and Practice of Pharmacy, 23rd Edition, Academic Press (2020).

[00159] The disclosed compositions can be provided in many different types of containers and delivery systems. For example, in some embodiments, the composition can be presented in unitdose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze- dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, immediately prior to use. In some embodiments, the compositions are provided in a suspension or liquid form. Such compositions can be delivered in any suitable container including spray bottles and any suitable pressurized spray device. Such spray bottles may be suitable for delivering the compositions intranasally or via inhalation. These containers can further be packaged with instructions for use to form kits (described below).

[00160] The disclosure provides methods and compositions to induce a desired immune response in a subject. As detailed herein, the compositions and methods of the disclosure comprising a systemic prime and mucosal boost vaccine regimen induce immune responses that have heretofore been unachievable with conventions vaccine strategies. In some embodiments, the desired immune response obtained is protective immunity. In another embodiment, the desired immune response obtained is sterilizing immunity. In some embodiments, the protective immunity and/or sterilizing immunity comprises neutralizing antibodies in the upper respiratory and/or lower respiratory tract. In some embodiments, the protective immunity and/or sterilizing immunity prevents viral replication in the lungs of a subject. In some embodiments, the protective immunity and/or sterilizing immunity prevents viral replication in the nasal turbinates of a subject. In some embodiments, parenteral prime vaccination (e.g., IM mRNA prime) and mucosal secondary vaccination (e.g., with IN adjuvanted booster vaccination (e.g., NE/S or NE/IVT/S)) provides sterilizing immunity in both the LRT and URT and complete absence of replicating virus in both the lungs and nasal turbinates of a subject. In further embodiments, compositions and methods of the disclosure are utilized as a strategy pull acquired, systemic immune response to mucosal sites. In some embodiments, the disclosure provides compositions and methods utilizing IN vaccination to induce sterilizing immune responses in the URT.

[00161] For example, in one aspect, the disclosure provides a method for vaccination against, or for prophylaxis or therapy (prevention or treatment) of exposure to, or infection with, a coronavirus (such as those described herein) via a parenteral (e.g., IM) prime vaccination with an mRNA vaccine (e.g., any one or more mRNA vaccines described herein or known in the art) followed by one or more mucosal (e.g., intranasal) boost/secondary vaccinations with a recombinant and/or subunit vaccine (e.g., comprising NE, RIG-I agonist, and S subunit or NE and S subunit (that provides protective and/or sterilizing immunity in the upper and/or lower respiratory tracts and/or inhibits viral replication in the lungs)). In some embodiments, the prime and mucosal boost vaccine regimen induces both humoral (e.g., development of specific antibodies) and cellular (e.g., cytotoxic T lymphocyte) immune responses (e.g., against a coronavirus). In another aspect, the disclosure provides a method for vaccination against, or for prophylaxis or therapy (prevention or treatment) of exposure to, or infection with, a respiratory pathogen (e.g., a virus, bacteria, or fungi described herein) via a parenteral (e.g., IM) prime vaccination with an mRNA vaccine (e.g., any one or more mRNA vaccines described herein or known in the art) followed by one or more mucosal (e.g., intranasal) boost/secondary vaccinations with a recombinant and/or subunit vaccine (e.g., comprising NE, RIG-I agonist, and recombinant antigen and/or vaccine subunit (e.g., that provides protective and/or sterilizing immunity in the upper and/or lower respiratory tracts and/or inhibits pathogen replication in the lungs)). In some embodiments, the prime and mucosal boost vaccine regimen induces both humoral (e.g., development of specific antibodies) and cellular (e.g., cytotoxic T lymphocyte) immune responses (e.g., against the respiratory pathogen). In still other aspects, the disclosure provides a method for vaccination against, or for prophylaxis or therapy (prevention or treatment) of exposure to, or infection with, a pathogen (e.g., a virus, bacteria, or fungi described herein) via a parenteral (e.g., IM) prime vaccination with a vaccine (e.g., an mRNA vaccine or any one or more vaccines described herein or known in the art) followed by one or more mucosal (e.g., intranasal) boost/secondary vaccinations with the same or a different vaccine (e.g., the same vaccine or a recombinant antigen and/or subunit vaccine (e.g., comprising NE, RIG-I agonist, and the same vaccine or a recombinant antigen and/or subunit vaccine (e.g., that provides protective and/or sterilizing immunity against the pathogen in the subject)). In some embodiments, the prime and mucosal boost vaccine regimen induces both humoral (e.g., development of specific antibodies) and cellular (e.g., cytotoxic T lymphocyte) immune responses (e.g., against the pathogen).

[00162] Cytokines play a role in directing the immune response. Helper (CD4+) T cells orchestrate the immune response of mammals through production of soluble factors that act on other immune system cells, including B and other T cells. Most mature CD4+T helper cells express one of two cytokine profiles: Thl or Th2. Th 1 -type CD4+ T cells secrete IL-2, IL-3, IFN-y, GM-CSF and high levels of TNF-a. Th2 cells express IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL- 13, GM-CSF and low levels of TNF-a. Thl type cytokines promote both cell-mediated immunity, and humoral immunity that is characterized by immunoglobulin class switching to IgG2a in mice and IgGl in humans. Thl responses may also be associated with delayed-type hypersensitivity and autoimmune disease. Th2 type cytokines induce primarily humoral immunity and induce class switching to IgGl and IgE. The antibody isotypes associated with Thl responses generally have neutralizing and opsonizing capabilities, whereas those associated with Th2 responses are associated more with allergic responses.

[00163] Several factors have been shown to influence skewing of an immune response towards either a Thl or Th2 type response. The best characterized regulators are cytokines. IL- 12 and IFN-y are positive Thl and negative Th2 regulators. IL-12 promotes IFN-y production, and IFN-y provides positive feedback for IL- 12. IL-4 and IL- 10 appear important for the establishment of the Th2 cytokine profile and to down-regulate Thl cytokine production.

[00164] For example, in some embodiments, the disclosed prime and mucosal boost vaccine regimens result in the skewing of a host’s immune response away ftom Th2 type immune response and toward a Thl type immune response. In other words, the disclosed prime and mucosal boost vaccine regimens induce a cellular immune response that is a Thl -biased immune response. In particular, conventional alum based vaccines for a variety of diseases, such as respiratory syncytial virus (RSV), anthrax, and hepatitis B virus, each lead to a predominant Th2 type immune response in a subject administered the vaccine (e.g., characterized by enhanced expression of Th2 type cytokines and the production of IgGl antibodies). However, administration of a coronavirus vaccine in combination with the nanoemulsion and RIG-I agonist disclosed herein is able to, in one embodiment, redirect the conventionally observed Th2 type immune response in host subjects administered conventional vaccines. Thus, in some embodiments, the present disclosure provides prime and mucosal boost vaccine regimens for skewing and/or redirecting a host’s immune response (e.g., away from Th2 type immune responses and toward Thl type immune responses) to one or a plurality of immunogens/antigens. [00165] With respect to viral infections, “humoral immunity” occurs when virus and/or virus- infected cells stimulate B lymphocytes to produce antibody that is specific for viral antigen. IgG, IgM, and IgA antibodies have all been shown to exert antiviral activity. Such neutralizing antibodies can exert antiviral activity by (1) blocking virus-host cell interactions or (2) recognizing viral antigens on virus-infected cells which can lead to antibody-dependent cytotoxic cells (ADCC) or complement-mediated lysis. IgG antibodies are responsible for most antiviral activity in serum, while IgA is the most important antibody when viruses infect mucosal surfaces. In some embodiments, the heterologous prime and mucosal boost vaccine regimens disclosed herein induce a greater neutralizing antibody response and/or a neutralizing antibody response not achievable with homologous prime boost regimens.

[00166] In some embodiments, the prime and mucosal boost vaccine regimens reduce the number of booster injections (e.g., of an antigen containing composition) required to achieve a desired immune response (e.g., a protective immune response (e.g., a memory immune response)). In some embodiments, the disclosed prime and mucosal boost vaccine regimens result in a higher proportion of recipients achieving seroconversion and/or more consistent immune responses within a population of subjects administered the immunogenic composition. [00167] The compositions of the present disclosure can be administered by any suitable route of administration. It will also be appreciated that the chosen route will vary with the condition and age of the recipient, and the disease and/or infection being treated.

[00168] For example, the compositions can be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracistemal injection or infusion, subcutaneous injection, or implant), by nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository) or topical routes of administration (e.g., gel, ointment, cream, aerosol, etc.), and can be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration. Non-limiting examples of carriers include phosphate buffered saline (PBS), saline or a biocompatible matrix material such as a decellularized liver matrix (DCM as disclosed in Wang et al. (2014) J. Biomed. Mater Res. A. 102(4): 1017- 1025) for topical or local administration. The compositions can optionally contain a protease inhibitor, glycerol and/or dimethyl sulfoxide (DMSO).

[00169] In some embodiments, compositions of the present disclosure are administered mucosally (e.g., using standard techniques; see, e.g., Remington: The Science and Practice of Pharmacy, 23rd Edition, Academic Press (2020) (e.g., for mucosal delivery techniques, including intranasal, pulmonary, vaginal and rectal techniques), as well as European Publication No. 517,565 and Ilium et al., J. Controlled Rel., 1994, 29:133-141 (e.g., for techniques of intranasal administration)). Alternatively, the compositions of the present disclosure may be administered dermally or transdermally, using standard techniques (see, e.g., Remington: The Science and Practice of Pharmacy, 23rd Edition, Academic Press (2020)). The present disclosure is not limited by the route of administration.

[00170] In some embodiments, the disclosed methods are used to protect and/or treat a subject susceptible to, or suffering from, a disease or infection by means of administering the disclosed compositions via injection (e.g., via intradermal, subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal, intrathecal, epidural, intracardiac, intraarticular, intracavemous, and/or intravitreal route). Methods of systemic administration include conventional syringes and needles, or devices designed for ballistic delivery (see, e.g., WO 99/27961), or needleless pressure liquid jet device (see, e.g., U.S. Pat. Nos. 4,596,556 and 5,993,412), or transdermal patches (see, e.g., WO 97/48440 and WO 98/28037). In some embodiments, the present disclosure provides a delivery device for systemic administration, pre-filled with a composition composition of the present disclosure.

[00171] In some embodiments, the composition is administered via a mucosal route (e.g., an oral/alimentary or nasal route). Alternative mucosal routes include intravaginal and intrarectal routes. In some embodiments, a nasal route of administration is used, which is also referred to herein as “intranasal administration” or “intranasal vaccination.” Methods of intranasal vaccination are well known in the art, including the administration of a droplet or spray form of a composition into the nasopharynx of a subject to be immunized. Intranasal administration may also include contact with the oral mucosa, bronchial mucosa, and other epithelia. In some embodiments, a nebulized or aerosolized composition is provided. Formulations for nasal delivery may include those with dextran or cyclodextran and saponin as an adjuvant. [00172] Enteric formulations such as gastro-resistant capsules for oral administration and suppositories for rectal or vaginal administration also may be employed. Compositions of the present disclosure may also be administered via the oral route. Under these circumstances, a composition may comprise a pharmaceutically acceptable excipient and/or include alkaline buffers, or enteric capsules. When the composition is administered via a vaginal route, the composition may comprise pharmaceutically acceptable excipients and/or emulsifiers, polymers (e.g., CARBOPOL), and other known stabilizers of vaginal creams and suppositories. When the composition is administered via a rectal route, the composition may comprise excipients and/or waxes and polymers known in the art for forming rectal suppositories.

[00173] Oral compositions can be prepared according to methods known in the art, and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents, and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain active ingredients in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients can be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents (e.g., com starch or alginic acid); binding agents (e.g., starch, gelatin, or acacia); and lubricating agents (e.g., magnesium stearate, stearic acid, or talc). The tablets can be left uncoated or they can be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. They may also be coated by the techniques described in U.S. Pat. Nos. 4,256,108; 4,166,452; and 4,265,874 to form osmotic therapeutic tablets for control release. The pharmaceutical compositions of the disclosure may also be in the form of oil-in-water emulsions. [00174] Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups, or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin, or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, cremophore™, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p- hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, preservatives, flavoring, coloring, and sweetening agents as appropriate.

[00175] In some embodiments, the composition may be applied and/or delivered utilizing electrophoretic delivery/electrophoresis. Further, compositions may be applied by a transdermal delivery system such as a patch or administered by a pressurized or pneumatic device (i.e., “gene gun”). Such methods, which comprise applying an electrical current, are well known in the art. [00176] The compositions described herein may be administered topically. If applied topically, the compositions may be occluded or semi-occluded. Occlusion or semi-occlusion may be performed by overlaying a bandage, polyoleofin film, article of clothing, impermeable barrier, or semi-impermeable barrier to the topical preparation.

[00177] The pharmaceutical compositions for administration (e.g., a vaccine) may be applied in a single administration or in multiple administrations. Indeed, as discussed above, following an initial administration of a composition of the present disclosure (e.g., a primary or prime vaccination), a subject may receive one or more secondary or boost administrations (e.g., around 2 weeks, around 3 weeks, around 4 weeks, around 5 weeks, around 6 weeks, around 7 weeks, around 8 weeks, around 10 weeks, around 3 months, around 4 months, around 6 months, around 9 months, around 1 year, around 2 years, around 3 years, around 5 years, around 10 years or more) subsequent to a first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, and/or more than tenth administration. The boost can be with the same formulation given for the primary immune response, or, as detailed herein, can be with a different formulation that contains the same or different immunogen, and, as also described herein, may be administered via the same or different route (e.g., a primary vaccination administered parenterally followed by one or more boost vaccines administered mucosally via intranasal administration). For example, in some embodiments, a subject is administered a prime vaccination with a vaccine (e.g., an mRNA vaccine) against a respiratory pathogen (e.g., coronavirus) followed by one or more mucosal (e.g., intranasal) boost/secondary vaccinations with a different vaccine (e.g., a recombinant and/or subunit vaccine comprising NE, RIG-I agonist, and a recombinant antigen (e.g., recombinant S protein) and/or subunit vaccine). The dosage regimen may also, at least in part, be determined by the need of the subject and be dependent on the judgment of a practitioner. [00178] Dosage units may be proportionately increased or decreased based on several factors including, but not limited to, the weight, age, and health status of the subject. In addition, dosage units may be increased or decreased for subsequent administrations (e.g., boost administrations). [00179] In some embodiments, a therapeutically effective amount of a disclosed immunogenic composition can be administered to a subject to treat or inhibit a tumor and/or a cancer in a subject. The subject can be selected for treatment that has, is suspected of having or is at risk of developing the tumor and/or cancer. In some embodiments, treating the tumor and/or cancer in the subject decreases growth and/or proliferation of the tumor. The tumor can be any tumor of interest and can be benign or malignant. Treatment of the tumor is generally initiated after the diagnosis of the tumor, or after the initiation of a precursor condition (such as dysplasia or development of a benign tumor). Treatment can be initiated at the early stages of cancer, for instance, can be initiated before a subject manifests symptoms of a condition, such as during a stage I diagnosis or at the time dysplasia is diagnosed. However, treatment can be initiated during any stage of the disease, such as but not limited to stage I, stage II, stage III and stage IV cancers. In some examples, treatment is administered to these subjects with a benign tumor that can convert into a malignant or even metastatic tumor.

[00180] Treatment initiated after the development of a condition, such as malignant cancer, may result in decreasing the severity of the symptoms of one of the conditions, or completely removing the symptoms, or reducing metastasis, tumor volume or number of tumors. In some examples, the tumor becomes undetectable following treatment. In one aspect of the disclosure, the formation of tumors, such as metastasis, is delayed, prevented or decreased. In another aspect, the size of the primary tumor is decreased. In a further aspect, a symptom of the tumor is decreased. In yet another aspect, tumor volume is decreased.

[00181] Subjects can be screened prior to initiating the disclosed therapies, for example to determine whether the subject has a tumor. The presence of a tumor can be determined by methods known in the art, and typically include cytological and morphological evaluation. The tumor can be an established tumor. The cells can be in vivo or ex vivo, including cells obtained from a biopsy. The presence of a tumor indicates that the tumor can be treated using the methods provided herein.

[00182] The therapeutically effective amount will depend upon the severity of the disease and the general state of the patient's health. A therapeutically effective amount is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer. In one embodiment, a therapeutically effective amount is the amount necessary to inhibit tumor growth, or the amount that is effective at reducing a sign or a symptom of the tumor. In another embodiment, a therapeutically effective amount is the amount necessary to inhibit infection by an infectious agent, or the amount that is effective at reducing a sign or a symptom of the infection. The therapeutically effective amount of the agents administered can vary depending upon the desired effects and the subject to be treated. In some examples, therapeutic amounts are amounts which eliminate or reduce the patient's tumor burden, or which prevent or reduce the proliferation of metastatic cells, or which reduce the load of infectious agent in the subject.

[00183] The actual dosage will vary according to factors such as the disease indication and particular status of the subject (for example, the subject's age, size, fitness, extent of symptoms, susceptibility factors, and the like), time and route of administration, other drugs or treatments being administered concurrently, as well as the specific pharmacology of the compound for eliciting the desired activity or biological response in the subject. Dosage regimens can be adjusted to provide an optimum prophylactic or therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental side effects is outweighed in clinical terms by therapeutically beneficial effects. Dosage can be varied by the attending clinician to maintain a desired concentration at a target site (for example, the lungs or nasal turbinates). Higher or lower concentrations can be selected based on the mode of delivery, for example, trans- epidermal, rectal, oral, pulmonary, intraosseous, or intranasal delivery versus intravenous or subcutaneous or intramuscular delivery.

[00184] It is contemplated that the compositions and methods of the present disclosure will find use in various settings, including research settings. For example, compositions and methods of the present disclosure also find use in studies of the immune system (e.g., characterization of adaptive immune responses (e.g., protective immune responses (e.g., mucosal or systemic immunity))). Uses of the compositions and methods provided by the present disclosure encompass human and non-human subjects and samples from those subjects, and also encompass research applications using these subjects. Compositions and methods of the present disclosure are also useful in studying and optimizing nanoemulsions, immunogens, and other components and for screening for new components. Thus, it is not intended that the present disclosure be limited to any particular subject and/or application setting. The disclosure is further described by reference to the following examples, which are provided for illustration only. The disclosure is not limited to the examples, but rather includes all variations that are evident from the teachings provided herein. All publicly available documents referenced herein, including but not limited to U.S. patents are specifically incorporated by reference.

EXAMPLES

[0101] The following examples further illustrate the disclosure but should not be construed as in any way limiting its scope.

[0102] Example 1. Materials and methods useful for homologous and heterologous vaccination to induce mucosal B- and T-cell responses.

Adjuvants and antigen. NE was produced by emulsifying cetylpyridinium chloride (CPC) and Tween 80 at a 1 :6 (w/w) ratio, with ethanol (200 proof), super refined soybean oil (Croda) and water using a high-speed homogenizer (See 40., 41). The sequence and synthesis of IVT DI RNA has been described in detail (See 17, and See FIG. 31 SEQ ID NO: 1). Briefly, SeV Cantell RNA enriched in DI RNA was obtained by growing the virus in 10-day-old chicken eggs. The virus was then purified by ultracentrifugation through a sucrose cushion, and the RNA was extracted and further cleaned up using the RNeasy kit from Qiagen. The plasmid expressing the SeV DI RNA was constructed by PCR amplification of the SeV DI sequence from A549 SeV- infected cells using a 5’ primer containing the T7 promoter and a 3 ’ primer containing the hepatitis delta virus genomic ribozyme site followed by the T7 terminator. The resulting DNA was then cloned between EcoRI/Hindlll sites in a pUC19 plasmid. The sequence of the plasmid was confirmed by Sanger sequencing. IVT DI RNA was synthesized by cutting out the IVT DI encoding DNA from the plasmid using EcoRI and Hindlll restriction enzymes, and purifying the DNA using a Monarch DNA cleanup kit (New England Biolabs) followed by in vitro transcription using a HiScribe T7 in vitro transcription kit (New England BioLabs). After transcription, DNA was removed by TURBO DNase treatment followed by clean up using the RNeasy kit. Recombinant WT SARS-CoV-2 full-length S protein and RBD (aa319-545) (derived from Wuhan-Hu- 1) with C-terminal His tags were produced in Expi293F or ExpiCHO cells, respectively, and purified by the University of Michigan Center for Structural Biology as described (See 42). Addavax (MF59 similar) was obtained from Invivogen, and the BNT162b2 mRNA vaccine (Pfizer) was obtained through the NIH SAVE program.

[0103] Cell lines. Vero E6 cells (ATCC) were maintained in DMEM supplemented with 10% heat inactivated fetal bovine serum (HI FBS) and IX non-essential amino acids (NEAA). HEK293T cells expressing hACE2 (293T-hACE2) were obtained from BEI resources and maintained in HEK293T medium: (DMEM with 4 mM L-glutamine, 4500 mg/L L-glucose, 1 mM sodium pyruvate and 1500 mg/L sodium bicarbonate, 10% HI FBS and 100 IU penicillin, and 100 pg/mL streptomycin).

[0104] Viruses. SARS-CoV-2 clinical isolate USA-WA1/2020 (BEI resources; NR-52281), and B.1.351 and BA.5 variant viruses were propagated in Vero E6 cells or Vero-TMPRSS2. All viral stocks were verified by deep sequencing. All work with authentic SARS-CoV-2 viruses were performed in certified BSL3 or ABSL3 facilities in accordance with institutional safety and biosecurity procedures.

[0105] Lentivirus pseudotyped virus. Generation of pseudotyped lentiviruses (PSVs) expressing the SARS-CoV-2 S proteins from WT, B.1.351, B.1.617.2, and B.1.1.529 (BA.l), and BA.4/5 variants harboring GFP and luciferase reporter genes was performed as described for the WT PSV (See 43). Plasmids carrying the full-length SARS-CoV-2 spike protein from each variant containing a C-terminal 19 amino acid deletion to remove the ER retention signal were used for pseudotyping (Invivogen). Viral titers (TU/mL) across variants were determined by measuring PSV transduction of GFP in 293T-hACE2 cells.

[0106] Animals. All animal procedures were approved by the Institutional Animal Care and Use Committees at the University of Michigan and Icahn School of Medicine at Mount Sinai and were carried out in accordance with these guidelines. 6-8-wk-old female C57B1/6, 129 SI (Jackson Laboratory), or KI 8-hACE2 mice (bred in-house) were housed in specific pathogen- free conditions. Mice were acclimated for 2 wks prior to initiation of each study. For challenge studies, mice were transferred to ABSL3 facilities 1 wk prior to viral challenge.

[0107] Immunization. Mice were anesthetized using isoflurane in a IMPAC6 precision vaporizer. For IN immunization, mice were given 12 pL (6 pL/nare) of each vaccine formulation, and for IM immunization, the vaccine was delivered in a 50 pL volume. Each group received prime and boost immunizations at a 4-wk interval. For C57B1/6 mice, mice were primed IM with 0.25 pg BNT162b2 mRNA (Pfizer/BioNTech). Mice were boosted either through the IM route with the same dose of mRNA, or through the IN route with PBS or 15 pg of WT S protein in PBS, 20 % NE (w/v) (NE/S) or 20% NE with 0.5 pg IVT DI (NE/IVT/S). Immune responses were compared to mice given homologous prime/boost immunizations with IN NE/S, IN NE/IVT/S, or IM 50% Addavax/S with the same amount of S protein. Comparison groups primed with IM PBS and boosted with IN NE/S or NE/IVT/S were also included. Select immunization regimens using the same adjuvant/antigen doses were chosen for evaluation in 129S1 and K18-hACE2 mice.

[0108] Serum was obtained by saphenous vein bleeding 2 and 4 wks after the prime, and by cardiac puncture at the end of the experiment at wk 8. Bronchial alveolar lavage fluid (BALF) was obtained by lung lavage with 0.8 mL PBS containing protease inhibitors at wk 8.

[0109] ELISA. Immunograde 96-well ELISA plates (Midsci) were coated with 100 ng S protein or RBD in 50 pL PBS/well overnight at 4°C, and then blocked in 5% non-fat dry milk/PBS for 1 h at 37°C. Sera (or BAL) from immunized mice were serially diluted in PBSB (PBS/0.1% BSA). Blocking buffer was removed, and serum dilutions were added and incubated for 2 h at 37°C followed by overnight incubation at 4°C. Plates were washed with PBST (0.05% Tween20), and alkaline phosphatase conjugated secondary antibodies diluted in PBSB were added (goat-anti-mouse IgG, IgGl, IgG2b, IgG2c, (IgA for BAL) Jackson Immuno Research Laboratories), and incubated lh at 37°C. Plates were washed with PBST, and developed by incubation with p-nitrophenyl phosphate (pNPP) substrate in diethanolamine (ThermoFisher) at RT. Absorbance was measured at 405 nm, and titers were determined using a cutoff defined by the sum of the average absorbance at the lowest dilution of naive serum and two times the standard deviation.

[0110] Pseudovirus microneutralization (MNT) assays. Pseudovirus (PSV) MNT assays were performed as described (See, 16). Briefly, 1.25xl0⁴HEK293T-hACE2 cells/well were seeded overnight on 96-well tissue culture plates. Sera from immunized mice were serially diluted by a factor of three, starting at a dilution of 1 : 30 in HEK293T medium. 50 pL of diluted sera was added to 50 pL of PSVs (40,000 TU/mL) and incubated for lh at 37°C. The PSV titer used across variant PSVs was selected based on the titer of WT PSV giving >100,000 RLUs above background. The virus/serum mixture was then added to the cells and incubated for 3 d at 37°C . Infection media was removed, and luminescence was measured by addition of 25 pL BrightGlo in 25 pL PBS. Neutralization titers were determined as the dilution at which the luminescence remained below the luminescence of the (virus only control-uninfected control)/2. Samples with undetectable neutralization were designated as having a titer of 10°. Neutralization assays with these PSVs have been demonstrated by us and numerous groups to be representative of authentic virus neutralization assays (See 15, 44).

[0111] Tissue isolation and single cell suspension preparation. Two weeks post-boost (wk8), the left lung lobe was harvested, and single-cell suspensions were prepared by mincing with surgical scissors, and adding minced tissue to 3 mL of digestion media (RPMI, 10% HI FBS, 2 mM L-glutamine, 1% NEAA, 1 mM sodium pyruvate, 50 pM 2 -mercaptoethanol, 100 IU penicillin, 100 pg/mL streptomycin with 1 mg/mL collagenase A (Roche), 20 U/mL of DNAse-I (Roche)) followed by incubation at 37°C for Ih with shaking. Tissue dissociation was continued by passages through an 18 gauge needle and filtering through a cell strainer. Cells were incubated in ACK lysis buffer for 5m at RT and washed with PBS. Methods for splenocyte and cLN lymphocyte preparation have been described (See 16). All cells were resuspended in T cell media (DMEM, 5% HI FBS, 2 m L-glutamine, 1% NEAA, 1 mM sodium pyruvate, 10 mM MOPS, 50 pM 2-mercaptoethanol, 100 IU penicillin, 100 pg/mL streptomycin) for further downstream analysis.

[0112] Antigen Recall Response. T cell antigen recall response was assessed by cytokine release in cell isolates from the spleen and cLN of immunized mice 2 wks post-boost (wk 8). For antigen recall, isolated cells were plated at 8xl0⁵cells/well and stimulated with 5 pg (25 pg/mL) S protein (WT) in T cell media for 72h at 37°C. Secreted cytokines (IFN-y, IL-2, IP-10, IL-5, IL- 6, IL- 13, IL- 10, IL- 17 A, and TNF-a) were measured relative to unstimulated cells in supernatants using a Milliplex MAP Magnetic Mouse Cytokine/Chemokine multiplex immunoassay (EMD Millipore).

[0113] Flow Cytometry. For CD4⁺ intracellular cytokine analysis, IxlO⁶ cells isolated from the lungs, spleen, or cLNs were stimulated with 25 pg/mL S protein (WT) in T cell media for 24 h at 37°C, with brefeldin A (Biolegend) added during the last 6 h. Cells were stained with LIVE/DEAD Fixable Yellow Dead Cell Stain in PBS/2mM EDTA for 20 m at 4C, washed, and then incubated with Fc block (Biolegend) in FACS buffer (PBS, 1% FBS, 2mM EDTA) for 10 m at 4C prior to staining for surface T cell makers (CD3, CD4, CD8) for 30 m at 4C. Cells were washed with FACS buffer, fixed in IC fixation buffer for 30 m at RT and permeabilized 30 m at 4C before staining with antibodies for intracellular cytokines in permeabilization buffer for 30 m at 4C. Cells were washed with permeabilization buffer and resuspended in PBS/2mM EDTA. FACS was performed on a Novocyte3000 flow cytometer and analyzed using FlowJo software.

[0114] Viral Challenge. Immunized mice were challenged 3 weeks post-boost (wk 7). 129S1 mice were challenged IN with 10⁴ PFU B.1.351, and K18-hACE2 mice were challenged IN with 10⁴ PFU BA.5 delivered in 30 pL PBS. Mice were sacrificed 4 days post infection (d.p.i.). Lungs were harvested in 500 pL of PBS, and nasal turbinates were collected. Homogenates were prepared for virus titration by plaque assay described, and cytokine profiling post-challenge was performed on homogenate using a Milliplex MAP Magnetic Mouse Cytokine/Chemokine multiplex immunoassay (EMD Millipore) (See 45).

[0115] Statistical Analysis. Statistical analyses were performed with GraphPad Prism 9 (GraphPad Software). Comparisons were performed by Mann-Whitney U test or one-way ANOVA with Tukey post-hoc as indicated.

[0116] Example 2. NE/IVT-adjuvanted S protein vaccination as homologous IN prime/boost or heterologous IN boost after IM mRNA prime results in strong serum and mucosal IgG as well as mucosal IgA responses.

[0117] In an effort to characterize differences in immune responses induced by homologous versus heterologous prime/boost strategies, C57B1/6 mice were given two immunizations 4-wks apart (FIG. 1 A). Mice were primed IM either with BNT162b2 mRNA or PBS, or IN with WT S protein adjuvanted with NE (NE/S) or NE/IVT (NE/IVT/S). Two weeks after the prime (wk2), antibody responses were measured against full-length WT S protein and the receptor binding domain (RBD), as RBD is the major target for virus-neutralizing antibodies. Priming with IM mRNA induced robust S protein-specific total IgG titers that were higher than those induced by priming with IN NE/S (by 0.5 log) or IN NE/IVT/S (by 1 log) (FIG. IB). However, RBD- specific total IgG titers were equivalent between the primed groups (FIG. 1C). Mice primed with IM mRNA were then boosted either IM with mRNA, or IN with PBS, S protein alone, NE/S, or NE/IVT/S as indicated in FIG. 1 A. Additionally, mice primed with IN NE/S were boosted with IN NE/S, and those primed with IN NE/IVT/S were boosted with IN NE/IVT/S as homologous IN/IN prime/boost comparison groups. Finally, to examine the effect of a single IN immunization at the boost timepoint, mice primed with PBS were immunized with IN NE/S or NE/IVT/S.

[0118] Two weeks post-boost (wk6), serum S- and RBD-specific total IgG titers increased for all groups, and were highest in animals receiving IM mRNA prime/boost and for those which received IM mRNA prime followed by IN NE/S or IN NE/IVT/S boost, which resulted in equivalently high IgG titers (GMT 2.8xl0⁶, 2.0xl0⁶, 2.8xl0⁶ against S protein, respectively). S- specific IgG titers for each of these groups increased by at least two logs after the boost, demonstrating the ability of IN NE/S and NE/IVT/S to boost systemic antibody responses induced by IM mRNA priming as effectively as an additional IM mRNA immunization (FIG. ID). In contrast, IM mRNA prime followed by unadjuvanted IN S protein boost only modestly increased S-specific IgG titers relative to levels induced by IM mRNA prime alone. Mice receiving two homologous IN immunizations with NE/TVT/S mounted comparable serum S- specific IgG titers as the IM mRNA prime/boost group, illustrating the ability to induce strong circulating antibody responses by IN immunization with NE/IVT/S. The inclusion of IVT with NE enhanced the magnitude of the induced S -specific IgG response compared to the singly adjuvanted NE/S group for the homologous IN prime/boost groups, representing a synergistic activity of the combined NE/IVT adjuvant. All singly immunized groups (IM mRNA;IN PBS, IM PBS;IN NE/S, IM PBS;IN NE/IVT/S) induced equivalent S-specific IgG titers. While RBD- specific IgG titers were lower by ~llog for all groups compared to the S-specific IgG titers, the same relative pattern between treatment groups was maintained (FIG. IE).

[0119] Example 3. IgG subclass skewing was dependent upon the vaccination regimen. [0120] IM mRNA prime/boost, heterologous IM mRNA prime followed by IN NE/S or NE/IVT/S boost, as well as IN NE/IVT/S prime/boost all resulted in high and similar antigenspecific IgGl and IgG2b titers, with the IN NE/IVT/S prime/boost inducing slightly higher levels of IgGl (FIGS. 1F-G). The presence of IVT in the IN NE/IVT/S prime/boost group enhanced IgG2b relative to the IN NE/S prime/boost group. mRNA vaccination, either as homologous prime/boost or as prime followed by IN NE/S or NE/IVT/S boost was required to induce the strongest IgG2c antibody responses, inducing equally high titers (GMT 6x10⁵) in these groups, which was enhanced by -Hog relative to the group given NE/IVT/S prime/boost via the IN route only (FIG. 1H).

[0121] While the serum antigen-specific antibody levels and subclass profiles were similar between the homologous IM mRNA prime/boost, and the heterologous IM mRNA;IN NE/S and IM mRNA;IN NE/IVT/S groups, induction of mucosal IgA responses in bronchoalveolar lavage fluid (BALF) was exclusive to groups receiving IN vaccination with NE/S or NE/IVT/S, either as a boost immunization in IM mRNA primed mice, or as a homologous IN prime/boost regimen (FIG. II). No BALF S-specific IgA was detectable in the IM mRNA prime/boost group.

Moreover, no S-specific IgA was observed for mice given IM mRNA;IN S alone, highlighting the role of the NE and NE/IVT adjuvants in driving the mucosal response after IN administration. Surprisingly, while a single IN NE/S or NE/TVT/S immunization induced low or no IgA, immunization with IM mRNA;IN NE/TVT/S resulted in equivalent IgA titers as homologous IN NE/IVT/S prime/boost. Thus, the disclosure provides, in some embodiments, the ability to use an adjuvanted IN “pull” immunization to harness parenterally primed immune responses in order to drive robust mucosal immune responses. While dimeric IgA plays the predominant role in first-line defense in the mucosa of the upper respiratory tract, mucosal IgG also contributes to protection through transudation from the blood into the lung (See 22). Indeed, in contrast to the pattern observed for IgA, B ALF IgG correlated with serum IgG titers, with IM mRNA prime/boost immunization inducing robust BALF antigen-specific IgG titers similar to the IM mRNA;IN NE/S and IM mRNA;IN NE/IVT/S treatment groups, as well as the IN NE/IVT/S prime/boost group (FIG. 1 J).

[0122] Example 4. Cross-reactive serum neutralizing antibody titers reflect serum S-specific IgG binding antibody titers.

[0123] Next, cross-reactive neutralizing antibody (nAb) titers induced in immunized C57B1/6 mice using pseudotyped viruses carrying the S proteins of SARS-CoV-2 variants of concern (VoCs) were quantified. In general, vaccination regimens that resulted in the highest IgG binding titers (IM mRNA prime/boost, IN NE/S and NE/IVT/S prime/boost or heterologous IM mRNA;IN NE/S or IM mRNA;IN NE/IVT/S) not only resulted in the highest nAb titers against vaccine matched ancestral virus (FIG. 2A), but also against the antigenically more distant B.1.617.2 (Delta, FIG. 2B), and B.1.351 (Beta, FIG. 2C) variants. These treatment groups induced similar levels of cross-neutralizing nAbs against each variant examined. While IM mRNA;IN NE/S immunization induced slightly higher nAbs against the WT and B.1.351 variants relative to homologous IM mRNA or homologous IN NE/S prime/boost, homologous IM mRNA and homologous IN NE/IVT/S prime/boost induced similar titers as heterologous EM mRNA;IN NE/IVT/S immunization. In contrast, IM mRNA primed mice boosted IN with unadjuvanted S alone induced the lowest nAb titers, giving similar or lower titers as the single IM mRNA immunization group against all four variants tested. Neutralization potential was reduced across all vaccination groups by a similar degree (~21og) towards the more antigenically distant B.1.1.529 (Omicron BA.l) variant, maintaining the same relative pattern of nAb response magnitude observed between immunization groups as against the WT virus (FIG. 2D).

[0124] Example 5. Heterologous EM mRNA prime followed by IN NE/IVT/S boost markedly enhanced THI and TH17 polarized antigen recall responses in spleen and cervical lymph nodes.

[0125] To evaluate T cell antigen recall responses, 2 wks after the boost immunization, splenocytes and cervical lymph node (cLN) isolates were harvested from immunized mice and restimulated ex vivo with S protein. Heterologous boosting of EM mRNA primed mice with IN NE/S or IN NE/EVT/S resulted in a marked enhancement of THI -polarized responses compared to homologous IM mRNA, IN NE/S, or IN NE/IVT/S prime/boost groups. High levels of S- specific IFN-y were induced in splenocytes by IM mRNA vaccination boosted with either IM mRNA or IN NE/S or NE/IVT/S, with the heterologous IN boosted groups inducing equivalent (or higher) levels of IFN-y than the IM mRNA prime/boost group. The heterologous IM mRNA;IN NE/S and IM mRNA;IN NE/IVT/S groups also demonstrated enhanced levels of IFN- y compared to the homologous IN NE/S and IN NE/TVT/S prime/boost groups (FIG. 3 A). Inclusion of IVT in the IN NE/IVT/S prime/boost significantly enhanced the IFN-y response relative to IN NE/S (FIG. 3A). Similar patterns were observed for antigen-specific IL-2 responses, with enhanced cytokine levels in animals primed with IM mRNA and then boosted with IN NE/S or NE/TVT/S compared to those given two doses of IM mRNA, IN NE/S, or IN NE/IVT/S (FIG. 3B). While significant variation was observed for IP- 10, IM mRNA primed animals boosted with IN NE/S or NE/TVT/S induced similar levels of IP- 10 in splenocytes as the IM mRNA prime/boost group, with the exception of one mouse in each of the heterologous groups showing much higher IP-10 (5-10 fold relative to the IM mRNA prime/boost) (FIG. 3C). Interestingly, singly immunized groups (IM mRNA;IN PBS, IN PBS;IN NE/S, IN PBS;IN NE/IVT/S) induced higher TP- 10 than the corresponding homologous prime/boost groups. No significant differences were observed between vaccination groups for TNF-a in stimulated splenocytes, with all immunized groups inducing similar levels of TNF-a, with the singly immunized IN NE/S and NE/TVT/S groups exhibiting slightly reduced levels compared to their respective prime/boost groups (FIG. 3D).

[0126] Example 6. Homologous TM mRNA administration induced significant TH2 responses in splenocytes as measured by IL-5 (FIG. 3E) and IL-13 (FIG. 3F) in comparison to homologous IN NE/S or IN NE/IVT/S, which did not induce detectable levels of these cytokines.

[0127] Heterologous boosting of IM mRNA primed animals with IN NE/S or NE/TVT/S did not enhance these TH2 cytokines and appeared to reduce IL-5 levels compared to the groups given one or two doses of IM mRNA. Finally, while TM mRNA prime/boost induced substantial IL-6 in the spleen (greater than IN NE/S or NE/TVT/S prime/boost), priming with TM mRNA followed by boosting with IN NE/S or NE/TVT/S resulted in significantly increased IL-6 production especially with the NE/TVT/S pull (FIG. 3G).

[0128] Example 7. High levels of IL-17A induction was observed in splenocytes for the IN NE/S prime/boost group, which was further enhanced ~2-fold in the IN NE/TVT/S prime/boost group (FIG. 3H). In contrast, no IL-17A was observed for the TM mRNA prime/boost group. Interestingly, however, while the single IN immunization groups (IM PBS;IN NE/S, IM PBS;IN NE/IVT/S) induced only low levels of IL-17A (~ 10-fold lower than the corresponding prime/boost groups), boosting IM mRNA primed animals with IN NE/S or NE/IVT/S induced high levels of IL-17A. Thus, the disclosure provides, in some embodiments, the use of the IN adjuvants to boost and shape immune responses primed by initial IM mRNA vaccination, (e.g., in some embodiments, promoting a shift in IM mRNA-primed T cell responses towards TH17). Notably, IM mRNA boosted with IN S alone did not induce significant IL-17A production. These results are significant, as TH 17 responses have been shown to be a critical component of host defense at mucosal sites (See, e.g., 23-25). While IL-17A induction has been associated with immune pathology in certain contexts, it has been shown to be non-pathogenic in the context of IL- 10 co-production (See 26-27). Indeed, all prime/boost immunization groups with an IM mRNA prime induced similarly high levels of IL- 10 in the spleen, which was enhanced relative to the single or two dose IN NE/S or IN NE/IVT/S regimens (FIG. 31). The lack of immune pathology was observed and is shown in the challenge studies discussed below.

[0129] Example 8. Cervical lymph nodes (cLNs) drain the upper respiratory tract (URT), and therefore are relevant for assessment of local protective immunity near the portal entry sites of pulmonary pathogens and for evaluating mucosal T cell responses induced by IN immunization. [0130] IN administration of vaccines as part of a boost regimen enhanced S protein-specific cytokine responses within the cLNs, demonstrating effective pulling of antigen-specific immune responses to mucosal sites (FIG. 4). In general, cytokine profiles in the cLN reflected the recall responses measured in spleen but were heavily skewed by the IN vaccination regimens, resulting in greater magnification of the TH1/TH17 polarization. Boosting with IN NE/IVT/S after IM mRNA prime induced markedly high antigen-specific IFN-y levels within the cLN (8514±794 pg/mL (mean±SEM)) compared to homologous prime/boost with IM mRNA (667±581 pg/mL), IN NE/S (303±208 pg/mL), or IN NE/IVT/S (2224±814 pg/mL) (FIG. 4A). Furthermore, administration of IN NE, or IN NE/TVT formulations either as a homologous prime/boost regimen or as part of a heterologous boost regimen after IM mRNA priming also significantly enhanced antigen-specific IL-2 and IP- 10 responses within the cLN compared to two doses of IM mRNA, with the highest levels observed with heterologous IM mRNA;IN NE/IVT/S vaccination (FIGS. 4B-4C). Notably, IM mRNA prime/boost induced only low levels of these cytokines in the cLN. Finally, enhancement in TNF-a was also observed in the cLN for the IM mRNA;IN NE/IVT/S group as compared to the IM mRNA and IN NE/IVT/S prime/boost groups (FIG. 4D). These results are of significance, as the co-production of IFN-y, IL-2, and TNF-a has been established as a strong mediator of optimal control of viral infection and a major correlate of vaccine protection. Thus, the disclosure provides that IN NE/IVT/S boost drives enhanced THI polarized T cell responses in the mucosal lymphoid tissue through ‘pulling’ of responses systemically primed by the IM mRNA.

[0131] Similar to the TH2 cytokine pattern in the spleen, two doses of IM mRNA induced the highest levels of IL-5 and IL-13 in the cLN, while two doses of IN NE/S or IN NE/IVT/S induced only low levels of IL-5 and no detectable IL- 13 (FIG. 4E-4F). While heterologous boosting of IM mRNA primed animals with IN NE/S or NE/IVT/S resulted in higher levels of IL-5 than the homologous IN NE/S and IN NE/IVT/S prime/boost groups, the levels remained similar to or lower than those induced in the IM mRNA prime/boost group, and no significant IL- 13 was detected in the heterologous prime/boost groups. These results confirm the lack of TH2 response enhancement with the adjuvanted heterologous pull immunizations. Minimal levels of spike-specific IL-6 were induced in the cLNs (FIG. 4G) as compared to splenocytes (FIG. 3G) for all vaccination groups (2 orders of magnitude lower), with the highest levels induced in animals receiving IN NE/IVT/S as part of their vaccine regimen.

[0132] Example 9. The enhancement in IL-17A production observed in the splenocytes was even more pronounced in the cLN for the heterologous IM mRNA;IN NE/IVT/S group (FIG. 4H). The combination of IM mRNA prime with IN NE/IVT/S boost resulted in similarly strong IL-17A responses (4398±142 pg/mL) as homologous IN NE/TVT/S prime/boost immunization (4657±136 pg/mL). In contrast, IM mRNA prime/boost did not induce detectable IL-17A. Heterologous boost with IN NE/S was also able to enhance IL-17A in the IM mRNA primed mice, however, inclusion of IVT in the IN boost was critical to driving maximal THI 7 responses in the cLN. IL- 10 production in the cLN followed the same pattern as IL- 17 A, with the IN adjuvanted groups inducing the highest levels of IL- 10 (FIG. 41). Overall, the disclosure provides that IM mRNA vaccination resulted in priming events that, when boosted IN with NE/IVT/S resulted in a unique antigen-specific cytokine profile in both the spleen and in the local mucosal- draining LNs. The disclosure further provides, in some embodiments, that employing an intranasal “pull” with a NE/TVT adjuvanted vaccine after IM mRNA priming drives a more robust and tailored response towards SARS-CoV-2 through enhancing the TH1/TH17 polarization. [0133] Example 10. The induction of high IgG2a, IgG2c, and IgA antibody titers requires efficient class switching in germinal center reactions which require strong CD4⁺ T cell responses.

[0134] Experiments were performed to characterize vaccine induced CD4⁺ T cell responses in the spleens, cLNs, and lungs of immunized animals. To better distinguish differences in cytokine production, C57B1/6 mice were given the same prime/boost regimens but at higher doses of mRNA (2 pg) and S protein (20 pg) (FIG. 5). Mice receiving IM mRNA prime/boost showed robust antigen-specific CD4⁺ T cell responses in the spleen, giving the highest frequencies of IFN-y⁺, IL-2⁺ and TNF-a⁺ CD4⁺ T cells (FIGS. 5A-5C), as well as polyfunctional (IFN-y⁺IL-2⁺ TNF-a⁺) CD4⁺ T cells upon stimulation with S protein (FIG. 5D). IN boost with S, NE/S or NE/IVT/S after mRNA priming induced similar frequencies of IFN-y , IL-2⁺ and TNF- a⁺CD4⁺ T cells, as well as polytunctional CD4⁺ T cells, although at a lower frequency than two doses of IM mRNA. Interestingly, comparison of CD4⁺ IFN-y⁴ responses by mean fluorescence intensity (MFI) rather than by frequency, showed equivalent, or higher MFIs for the IM mRNA prime with IN S, NE/S, or NE/IVT/S boost groups as the IM mRNA prime/boost group, demonstrating that even though the frequency of IFN-y⁺ cells was slightly lower for the IM/IN groups, these cells produced higher levels of IFN-y (FIG. 11). In the cLN, however, IM mRNA groups receiving IN NE/S or NE/IVT/S boost developed the strongest antigen-specific CD4⁺T cell responses, with significant enhancement in the frequency of polyfunctional cells compared to groups that received a second IM mRNA dose or IN S (FIGS. 5E-5H). While IN NE/S or NE/IVT/S in a prime/boost regimen induced IL-2 and TNF-a expressing CD4⁺ T cells, IM mRNA priming was necessary to induce optimal IFN-y expressing CD4⁺ T cells in the cLN, demonstrating the role of the initial IM prime immunization in driving optimal local mucosal responses after a mucosal boost. Furthermore, while IN S boost of IM mRNA primed mice showed enhanced polyfunctional CD4⁺ T cell responses in the spleen comparable to the IN NE/S and NE/IVT/S boost groups, the unadjuvanted boost group showed minimal levels of polyfunctional CD4⁺ T cells in the cLNs, highlighting the critical role of the NE-based adjuvants in driving robust local mucosal cellular responses. Similar to the cLN, in the lungs, groups primed with IM mRNA and then boosted IN with S, NE/S, or NE/IVT/S had similar levels of IFN-y expressing CD4⁺ T cells which were enhanced compared to the other vaccination regimens (FIGS. 5I-5L). These groups also displayed enhanced polyfunctional CD4⁺ T cells in the lung compared to the other vaccination regimens. Interestingly, boosting with IN S or NE/S after IM mRNA priming induced higher frequencies of polyfunctional CD4⁺T cells in the lung compared to boosting with IN NE/IVT/S, which was the reverse pattern observed in the cLN, indicating differences in trafficking and kinetics at this time point between the free antigen vs. NE/IVT adjuvanted S protein. Finally, in accordance with the cytokine secretion data, mucosal boost of IM mRNA primed animals displayed higher frequencies of IL-17A⁺CD4⁺ T cells in the lung than the IM mRNA prime/boost group which had no detectable response (FIG. 12). However, the highest frequencies of IL-17A⁺CD4⁺ T cells were induced in the IN NE/S and IN NE/IVT/S homologous prime/boost groups. Taken together, the disclosure provides that boosting with a IN NE antigen regimen represents an effective pulling strategy to mucosal sites of systemic immune responses induced by mRNA vaccination.

[0135] Example 11. Heterologous IM/IN prime-boost immunization induces robust virusneutralizing antibody titers in 129S1 and K18-hACE2 mice and results in superior protection in the upper respiratory tract.

[0136] In order to examine the effects of genetic background on induced immune responses, vaccination with the heterologous prime/boost regimens were repeated in 129S1 and KI 8- hACE2 mice. The 129S1 strain was selected for vaccination/challenge studies as they are WT mice that have shown to be more susceptible to morbidity after experimental infection with SARS-CoV-2 viruses with the N501Y mutation, such as B.1.351 (See, 28). In contrast, SARS- CoV-2 Omicron lineages do not efficiently infect WT mice but can replicate in the respiratory tract of transgenic KI 8-hACE2 mice, which overexpress human ACE2 receptor in the epithelia. Thus, this transgenic model was utilized for protective efficacy studies with Omicron BA.5 challenge.

[0137] Prime immunization with IM mRNA, IN NE/S, IN NE/IVT/S induced equivalent S- specific serum IgG in 129S1 mice, giving similar titers as primed C57B1/6 and K18-hACE2 mice (FIG. IB and FIG. 13). Boosting of IM mRNA primed animals with IM mRNA, IN NE/S or IN NE/IVT/S resulted in high serum total binding IgG against WT S protein in both genetic backgrounds (FIGS. 6A and 6F). In both 129S1 and K18-hACE2 mice, comparable induction of high S -specific IgG titers with the IM mRNA prime/boost was observed as for the heterologous IM mRNA prime with IN NE/S or NE/IVT/S boost, with these titers being enhanced compared to the IN NE/S and IN NE/IVT/S prime/boost, and IM mRNA;IN S groups. Overall, an identical pattern for S-specific IgG titers between immunization groups was observed for the 129S1, K18- hACE2, and C57B1/6 mice. Furthermore, for comparison it was assessed whether prime/boost immunization with S protein adjuvanted with the IM adjuvant, Addavax (Advx), would induce similar immune responses. IM Advx/S prime/boost resulted in similar S-specific IgG as the IM mRNA prime/boost group in both 129S1 and KI 8-hACE2 strains (FIGS 6A and 6F). Interestingly, while IM mRNA;IN NE/S and IM mRNA;IN NE/IVT/S groups induced similarly robust neutralization titers as the IM mRNA prime/boost groups in 129S1, K18-hACE2, and C57B1/6 mice against ancestral virus, a more distinct enhancement in breadth of viral neutralization with these heterologous IM/IN groups was observed in the 129 SI mice compared to the other two strains when measured against B.1.351, BA.l, and especially against BA.4/5 (FIGS. 6B-6E, and 6G-6J).

[0138] For example, in the 129S1 mice, IM mRNA;IN NE/IVT/S treatment resulted in a 1 log enhancement in B.1.351 and BA.1 neutralization compared to the IM mRNA prime/boost group. The mouse strain difference was most apparent when the difference in nAb induction efficiency against the antigenically most distant BA.4/5 variant was considered. In 129S1 mice, all vaccine groups that included a prime boost had detectable BA.4/5-specific microneutralization titers except for mice that received IM mRNA prime/boost in which half of the group failed to show detectable neutralization (FIG. 6E). In KI 8-hACE2 mice, the same vaccination regimens resulted in less efficient induction of BA.4/5-specific nAb titers, with only the group that was primed with IM mRNA followed by IN NE/IVT/S boost inducing significant nAb titers in all of the animals within the immunization group (FIG. 6J). Thus, while the general trends are the same amongst the three mouse strains, there are nuanced differences which highlight the importance of comparing responses in the context of different genetic backgrounds. Finally, while IM Advx/S prime/boost induced similar total binding S-specific IgG titers as IM mRNA prime/boost, IN NE/IVT prime/boost, and IM mRNA prime with IN NE/S or NE/IVT/S boost, the IM Advx/S group displayed lower viral neutralization titers overall compared to these treatment groups. For example, in K18-hACE2 mice, the majority of IM Advx/S prime/boost mice showed no detectable neutralization of BA.4/5, pointing to the reduced breath of the antibody response induced by this adjuvant (FIG. 6J).

[0139] Example 12. Heterologous IM/IN prime-boost immunization induces broadly protective, immune responses (e.g., sterilizing immunity).

[0140] To evaluate protection against cross-variant viral challenge, 129S1 mice were infected with 10⁴ PFU of B.1.351 (FIGS. 7A-7B) and K18-hACE2 mice with 10⁴ PFU of BA.5 (FIGS. 7C-7D) SARS-CoV-2 virus. Lungs as well as nasal turbinates (NTs) were harvested at 4 dpi for viral load determination by plaque assay. B.1.351 contains the N501Y mutation which allows it to replicate in WT 129S1 mice, causing up to 10% body weight loss (See, 28). In contrast, while SARS-CoV-2 Omicron variants, including BA.5, can replicate in the respiratory tract of transgenic KI 8-hACE2 mice, infection is mainly characterized by the absence of overt morbidity (See, 29). Nonetheless, it provides a useful model for evaluating breadth of immune protection with virus titer reduction as a surrogate of protection. In B.1.351 challenged 129S1 mice, IM mRNA prime/boost resulted in complete protection in the lungs (lower respiratory tract (LRT)) with the absence of replicating virus (FIG. 7A). However, the IM mRNA prime/boost vaccination failed to prevent viral replication in the nasal turbinates (upper respiratory tract (URT)) (FIG. 7B). In contrast, IM mRNA prime with IN adjuvanted S booster vaccination (NE/S, NE/IVT/S) demonstrated sterilizing immunity in both the LRT and URT, with complete absence of replicating virus in both the lungs and NTs of challenged mice. The IN NE/IVT/S prime/boost regimen also conferred sterilizing immunity in both the LRT and URT, highlighting the critical role of mucosal immunization in promoting URT protection. IN NE/S prime/boost immunization also conferred sterilizing immunity in the NTs, as well as significant protection in the lungs with most mice showing no viral replication in the lungs. However, 2/5 mice in this treatment group demonstrated modest breakthrough viral replication just above the limit of detection, supporting the advantage of the NE/IVT combined adjuvant. While IM Advx/S prime/boost offered a significant degree of protection as compared to the unvaccinated PBS control group, the Advx group showed the highest viral load in the lungs compared to all the other groups which received two immunizations. Furthermore, all mice in the Advx group demonstrated high viral titers in the NTs that were not significantly different from the unvaccinated control. Thus, in some embodiments, the disclosure provides compositions and methods utilizing IN vaccination in promoting sterilizing immune responses in the URT. Notably, a single immunization with IN NE/S or NE/IVT/S did not confer significant protection in the lungs, yielding similar viral titers as the unvaccinated PBS group. However, a few animals in these groups did show a lack of viral replication in the NTs, while the others had similar titers as the unvaccinated control. Thus, in some embodiments, the disclosure provides that the robust protective effects observed for the heterologous immunization groups is attributable to the synergistic effects of both the IM mRNA prime and NE-based IN pull components. [0141] Similar sterilizing immunity was observed for the IM mRNA prime, IN NE/S or IN NE/IVT/S boosted K18-hACE2 mice challenged with BA.5, with no viral replication detected in the lungs or NTs (FIGS. 7C-7D). Consistent with the results from the B.1.351 challenge in 129 SI mice, IM mRNA prime/boost also showed strong protection in the lungs after BA.5 challenge in vaccinated K18-hACE2 mice, as only 1/5 mice had detectable viral titers just above the detection limit. These mice also displayed higher levels of viral replication in the NTs compared to the IM mRNA prime, IN adjuvanted S groups showing low, but detectable viral titers in 2/5 mice. Similar to the B.1.351 challenged mice, the IM Advx/S prime/boost group also provided some degree of protection in the lungs of BA.5 challenged K18-hACE2 mice but had 2/5 mice with viral titers that were close those of unvaccinated controls. Moreover, no protection against BA.5 challenge in the NTs was observed in this group compared to unvaccinated controls. Notably, overall viral titers were lower for the BA.5 challenged KI 8-hACE2 mice even for the unimmunized control group as compared to B.1.351 challenged 129S1 mice, reflecting the poor infectivity of the Omicron variants in mouse models as observed elsewhere (See, 35- 36). Interestingly, IM mRNA prime, IN S only boost also provided sterilizing immunity to challenge in both the LRT and URT, for K18-hACE2 mice, as well as 129S1 mice, with only 1/5 129S1 mice showing viral titers at the detection limit (FIGS. 7A-7B). Thus, while the IM mRNA, IN adjuvanted S groups showed the most complete cross-variant sterilizing immune responses throughout the respiratory tract, such hybrid immunization approaches show a benefit in promoting protective mucosal immune responses even with unadjuvanted antigen alone delivered IN.

[0142] Example 13. Host immune responses upon infection reflect disease course and pathogenesis, or lack thereof due to vaccine-mediated protection.

[0143] Cytokine levels were measured in lung homogenates at 4 d.p.i. for the vaccinated and challenged 129S1 and K18-hACE2 mice (FIGS. 8A-8B; individual cytokine data are provided in FIGS. 9-10). In 129S1 mice, pro-inflammatory innate cytokines/chemokines MIP-la, MIP-ip, IP- 10, MIP-2a, MCP-1, MCP-3, RANTES, GRO-a, IL-6 and TNFa were elevated in mice that showed breakthrough infection after receiving single vaccinations with either IN NE/S or IN NE/IVT/S, as well as with IM prime/boost with Advx/S or in the negative control group that received IN PBS twice (FIG. 8A). The T cell cytokines IFN-y, IL- 18, IL-22, and IL- 10 were also increased in these groups, indicating strong immune activation to control viral infection. Similarly, IM mRNA primed mice boosted IN with S alone or with NE/S had elevated innate cytokine/chemokine responses although with reduced adaptive immune cytokines. In contrast, 129S1 mice given homologous IM mRNA or IN NE/IVT/S prime/boost immunizations as well as mice given heterologous IM mRNA prime followed by IN NE/IVT/S boost had very low levels of both innate and adaptive chemokines/cytokines in the lungs post-challenge, consistent with the effective viral control observed in these groups. Furthermore, 129 SI mice that received IM Advx/S showed elevated TH2 cytokines IL-4, IL-5, IL- 13 and eotaxin, which appeared higher than the unvaccinated group, reflecting the TH2 bias of the adjuvant. Unlike B.1.351 infection in 129 SI mice which resulted in strong host immune responses, BA.5 infection resulted in overall lower immune responses in K18-hACE2 mice, reflecting the better replication efficiency and pathogenesis of B.1.351 compared to BA.5 (FIGS. 8A-8B, and FIGS. 9-10).

[0144] In K18-hACE2 mice, breakthrough infection in mice singly vaccinated with IM mRNA resulted in strongest induction of the innate pro-inflammatory cytokines/chemokines, MIP-la, MIP-ip, IP-10, MCP-1, MCP-3, and IL-6 along with the cytokines TNF-a, IFN-y, and IL-18. In contrast, similar to the 129 SI mice, groups primed with IM mRNA, boosted either IM with mRNA, or IN with NE/S or NE/IVT/S showed minimal induction of these inflammatory chemokines and cytokines, reflecting the effective viral control in these groups. Interestingly, IM mRNA prime followed by IN S alone showed a similar pro-inflammatory chemokine/cytokine profile as the single IM mRNA immunization group, albeit slightly reduced, highlighting the importance of the IN NE and NE/IVT adjuvants in mediating optimal protection. Overall, cytokine/chemokine profiles are elevated in groups for which replicating virus could be detected in lungs or nasal turbinates at 4 d.p.i., and chemokine/cytokine responses are skewed both by adjuvant type and vaccination routes.

[0145] Example 14. Heterologous prime-pull immunization strategies for SARS-CoV-2 drives enhanced, long-lived mucosal and systemic cellular and antibody responses. Impact of prime immunization type and route of administration on shaping downstream humoral and mucosal antibody responses.

[0146] Additional experiments were performed to examine how the type and route of administration of prime immunization in heterologous immunization regimens shapes humoral, cellular, and mucosal immune responses. The durability of the induced immune responses was also tested through optimizing heterologous immunization sequences with the IM mRNA and IN NE/IVT/S vaccines. It was determined that route of administration in prime immunization drives the formation of systemic and mucosal immune responses shaped by further subsequent booster immunizations. Furthermore, prime-pull heterologous immunization strategies with IM mRNA prime and IN NE/IVT adjuvanted protein boost were observed to generate a unique cellular and humoral profile that is not only considerably more durable than homologous IM mRNA prime/boost vaccination, but also more responsive to further boosting than both homologous IM mRNA immunization and heterologous immunization regimens based on mucosal prime immunizations. Thus, the disclosure provides that incorporation of adjuvanted mucosal immunizations with parenteral vaccination series imparts more complete and durable protective immune responses.

[0147] To investigate the impact of the prime immunization on shaping responses to subsequent booster immunizations and determine whether the chronological sequence of repeated antigen exposures through heterologous routes (mucosal vs. parenteral) influences the final immune responses, mice were immunized with various heterologous prime/boost regimens containing IM mRNA or IN S -protein-based vaccines (See FIG. 14A for study design and abbreviations). A prime/boost/boost regimen was employed in which IN immunizations were performed with the S protein of Wuhan Hu-1 SARS-CoV-2 (WT S) (20 pg) with/without NE/IVT adjuvant, and IM immunizations were performed with the BNT162b2 mRNA vaccine (mRNA) (1 pg). Immunizations were performed at a three-week interval. To examine the influence of parenteral priming, mice were primed with IM mRNA and subsequently boosted twice with either IN S or IN NE/IVT/S. To examine the impact of priming mucosally, mice were primed and boosted with IN S or IN NE/IVT/S, followed by a final parenteral boost immunization with IM mRNA. Comparator homologous immunization regimens included mice given prime/boost/boost immunizations with IN NE/IVT/S, and mice given prime/boost immunizations with IM mRNA.

[0148] Two weeks post-prime (FIG. 14B), both IM mRNA and IN NE/IVT/S induced high levels of serum WT-S-specific IgG with IM mRNA inducing slightly higher titers than IN NE/IVT/S. In contrast, unadjuvanted IN S alone did not induce detectable IgG. Single boosting of IM mRNA primed mice with IN NE/IVT/S significantly enhanced serum WT S-specific IgG, whereas boosting with unadjuvanted IN S alone did not result in significant enhancement in IgG compared to single IM mRNA immunization (FIG. 14C). Homologous IN NE/IVT/S prime/boost immunization also induced robust S-specific IgG levels comparable to those induced by the heterologous IM mRNA; IN NE/IVT/S regimen. In contrast, IN S prime/boost gave only low or undetectable titers. Two weeks post third immunization (prime/boost/boost), total WT S- specific IgG was further enhanced to a comparable level between groups receiving a single IM mRNA immunization and two IN NE/IVT/S immunizations regardless of chronological sequence (IM mRNA; IN NE/IVT/S; IN NE/IVT/S vs. IN NE/IVT/S; IN NE/IVT/S; IM mRNA) (FIG. 14D). These groups produced slightly higher IgG compared to the group given homologous IN NE/IVT/S prime/boost/boost, and induced comparable titers as the homologous IM mRNA prime/boost group. Heterologous regimens with IM mRNA and two unadjuvanted IN S immunizations resulted in lower titers than those with IN NE/IVT adjuvanted regimens, highlighting the benefit of the NE/IVT adjuvant. Interestingly, mice receiving an IM mRNA prime (IM mRNA; IN S; IN S) showed higher titers than those receiving an IN S prime (IN S; IN S; IM mRNA), even though these groups received the same immunizations overall. The IN S; IN S; IM mRNA group notably showed IgG titers equivalent to the IM mRNA singly immunized mice. Upon examining the breadth of the IgG response towards the BA.l S protein, the relative pattern between groups was maintained, with the IM mRNA; IN NE/IVT/S; IN NE/IVT/S group showing equivalent titers against the WT and BA.1 S proteins (FIG. 22A). However, the IN S; IN S; IM mRNA group showed the largest reduction in IgG against the heterologous antigen, further increasing the difference between the IM mRNA; IN S; IN S group. Thus, in some embodiments, the disclosure provides that the prime immunization influences downstream immune responses.

[0149] After prime/boost/boost immunizations, all regimens induced overall balanced antigen-specific IgG subclass profiles. IgGl and IgG2b titers were nearly equivalent within each immunization group, following the relative trends observed for total IgG between groups (FIGS. 22B-22C). The highest IgGl and IgG2b titers were found in mice given homologous IM mRNA immunizations or heterologous prime/boost/boost immunizations with IM mRNA and IN NE/IVT/S regardless of the sequence of immunization, while the homologous IN NE/IVT/S immunization regimen resulted in slightly lower titers of both subclasses. Heterologous IM mRNA with two IN S immunizations induced similar titers of WT S -specific IgGl regardless of sequence of immunization, with titers lower than the groups given IM mRNA with two adjuvanted IN NE/IVT/S immunizations. The highest levels of WT S-specific IgG2c were generated in mice given homologous IM mRNA prime/boost immunizations and the heterologous IM mRNA; IN NE/IVT/S; IN NE/IVT/S regimen (FIG. 22D). Notably, while total WT S-specific IgG titers were similar between IM mRNA; IN NE/IVT/S; IN NE/IVT/S and IN NE/IVT/S; IN NE/IVT/S; IM mRNA groups, higher levels of IgG2c were generated in mice first primed with IM mRNA and subsequently boosted with IN NE/IVT/S compared to those primed with IN NE/IVT/S and boosted with IM mR A. These observations further support the far reaching influence of the prime immunization properties (type and route of initial antigen encounter) in shaping immune responses induced by subsequent booster immunizations.

[0150] The differences in the quality of the induced antibodies between immunization regimens was determined by quantifying neutralizing antibodies (nAbs) utilizing pseudovirus (PSV) reporters expressing the S proteins from the WT, B.1.351, BA.l, and BA.4/5 variants (FIGS. 14E-14H). The homologous IM mRNA immunization group, and the heterologous prime/boost/boost groups given IM mRNA with adjuvanted IN NE/IVT/S (regardless of sequence) all induced equivalent and robust titers across all four divergent variants, demonstrating the breadth of the immune responses induced by these regimens. The homologous IN NE/IVT/S group demonstrated equivalent nAb titers against WT, BA.1.351, and BA.4/5 as the heterologous IM mRNA and IN NE/IVT/S regimens, however, these titers were notably reduced against BA.l, indicating variations in epitope specificity. Furthermore, in heterologous IM mRNA with unadjuvanted IN S regimens, the dependence on immunization sequence was further accentuated, with the group given IM mRNA prime followed by IN S boost generating higher nAb titers against all four variants compared to the group given IN S prime followed by IM mRNA boost. No significant cross-neutralizing antibodies against BA.1 or BA.4/5 was detected in the IN S; IN S; IM mRNA group, while half of the mice in the IM mRNA; IN S; IN S group had substantial nAb titers (FIGS. 14G and 14H). Interestingly, while the IN S; IN S; IM mRNA group showed no difference in nAbs compared to the singly immunized IM mRNA group, boosting the IM mRNA primed mice with IN S led to enhanced responses. The inclusion of the NE/IVT adjuvant in IN boosts was important for inducing optimal cross-neutralizing Abs in heterologous regimens.

[0151] The generation of protective antibodies at mucosal surfaces is a key advantage of mucosal immunization as they play a major role in preventing infection and viral transmission. Accordingly, induction of antigen-specific secreted IgA is a major goal of heterologous prime/boost immunization strategies. Quantification of antigen-specific IgA in bronchoalveolar lavage fluid (BALF) two weeks post prime/boost/boost showed similar levels of reactivity to WT and OMI BA.l S proteins. Heterologous boosting of IM mRNA primed mice twice with IN NE/IVT/S resulted in a dramatic increase in BALF WT S and OMI S specific IgA (FIGS. 141 and 14J). In contrast, the singly immunized IM mRNA; IN PBS; IN PBS group induced no detectable S-specific IgA to either variant, consistent with the suboptimal induction of mucosal immunity inherent to parenterally administered vaccines. While some S-specific IgA was induced upon homologous boost of the IM mRNA group, levels were significantly lower than those induced by the heterologous IM mRNA prime; IN NE/IVT/S; IN NE/TVT/S regimen. Interestingly, while the homologous IN NE/IVT/S series induced higher S-specific IgA than the homologous IM mRNA series, heterologous prime-pull immunization enhanced IgA induction to levels exceeding an additive effect of the homologous IN or IM immunization regimens, indicating a synergistic effect with the IM/IN approach disclosed herein. This synergistic enhancement in IgA was not observed for the reverse immunization sequence with mucosal prime, parenteral boost (IN NE/IVT/S; IN NE/TVT/S; IM mRNA), which induced only low levels of IgA similar to homologous IM mRNA immunization. While boosting of IM mRNA primed mice with unadjuvanted IN S showed modest enhancement in IgA compared to the single IM mRNA immunization, no IgA was detected in mice given the reverse IN S; IN S; IM mRNA immunization sequence. These results demonstrate the importance of mucosal immunization in driving optimal IgA induction and highlight the utility of the disclosed compositions and methods comprising mucosal immunizations as a booster to pull immune responses primed systemically to mucosal surfaces. Conversely, systemic boosting did not boost mucosally primed mucosal antibody responses. Thus, in some embodiments, the disclosure provides compositions and methods to induce a desired immune response in a subject (e.g., enhanced and/or optimized IgA induction) that comprise an IM prime followed by one or more IN boost administrations that takes into consideration the impact of antigen exposure history (vaccine type and route of administration) in order to shape and/or obtain an optimal vaccination outcome. [0152] Example 15. Priming route in heterologous immunization shapes the formation of tissue-resident memory T cells in the nasal mucosa and draining lymph nodes

[0153] In the absence of robust neutralizing antibodies (nAbs), which wane quickly after IM mRNA immunization, protection from viral infection is afforded by T cells. Further, T cell epitopes are more highly conserved than nAb epitopes and can provide durable protection as viral escape variants continue to emerge. Tissue resident memory T cells (TRM’S) in particular, are important mediators in both protection and blocking viral transmission. Experiments were conducted to assess how the prime immunization in heterologous immunization sequences impacted the generation of S-specific effector memory and tissue resident memory T cells, and the influence of vaccine sequence on dictating the polarization of CD4⁺/CD8⁺ T cell responses within the systemic (spleen) and mucosal (mucosal draining lymph nodes, lung) immune compartments.

[0154] Antigen-specific effector memory (CD44⁺CD69⁺CD62L ) T cells (TEM) within the spleen and draining lymph nodes (Gating Strategy in FIG. 15 A) are recalled quickly to tissues after re-encountering antigen. To profile responses, splenocytes and cellular isolates from the cLN, lung, and the nasal-associated lymphoid tissue (NALT) were isolated from vaccinated mice two weeks after the final boost immunization. Within the spleen, homologous IM mRNA immunization and heterologous immunization with IM mRNA and IN NE/IVT/S were noted to be effective in generating S-specific CD8⁺ TEM’S (FIG. 15B) and CD4⁺ TEM’S (FIG. 15C) within the spleen; however, between heterologous immunization regimens, priming parenterally with IM mRNA and boosting with IN NE/IVT/S (IM mRNA; IN NE/IVT/S; IN NE/IVT/S) generated higher frequencies of S-specific CD8⁺ TEM’S and CD4⁺ TE ’S than priming mucosally with IN NE/IVT/S and boosting with IM mRNA (IN NE/IVT/S; IN NE/IVT/S; IM mRNA). Notably, heterologous immunization regimens utilizing unadjuvanted spike had little effect on the frequency of S-specific CD8⁺ TEM’S and CD4⁺ TEM’S confirming the importance of the NE/IVT adjuvant. Notably, homologous IM mRNA immunization increased the overall frequency of CD8⁺ T cells in the spleen compared to the other immunization regimens (FIG. 23A). However, none of the immunization regimens affected the overall CD4⁺ T cell frequencies in the spleen (FIG. 23B).

[0155] The cervical lymph nodes (cLNs) regulate upper respiratory tract immunity, making them an early site of viral detection and ideal for immunoprofiling the mucosal responses induced by IN immunization. Homologous IM mRNA prime/boost and heterologous prime/boost/boost with IM mRNA and IN NE/IVT adjuvanted S protein, regardless of which immunization sequence was used induced S-specific CD8⁺ TEM’S within the cLNs, with slightly higher frequencies induced in mice primed with IM mRNA and boosted with IN NE/IVT/S (FIG. 15D). An even more pronounced benefit of heterologous IM/IN immunization on the CD4⁺ response was observed. IM mRNA; IN NE/IVT/S; IN NE/IVT/S immunization resulted in robust enhancement in the frequency of S-specific CD4⁺ TEM’S within the cLNs in mice as compared to IM mRNA prime/boosting which induced minimal CD4⁺ TEM’S. Notably, heterologous immunization in which the immunization sequence was reversed to priming IN with NE/IVT/S twice and boosting with IM mRNA generated minimal or undetectable S-specific CD4⁺ TEM’S in the cLN (FIG. 15E). Thus, the data of this disclosure highlights that even though subjects receive the same vaccines overall, the chronological sequence in which systemically primed immune responses are “pulled” and boosted to mucosal sites by IN immunization is more effective for inducing robust mucosal responses as compared to systemically boosting mucosally primed immune responses. Accordingly, while significantly lower than the heterologous IM mRNA; IN NE/IVT/S; IN NE/IVT/S group, the homologous IN NE/IVT/S regimen also resulted in induction of S-specific CD4⁺ TEM’S in the cLN higher than both the IN NE/IVT/S; IN NE/IVT/S; IM mRNA group and the homologous IM mRNA groups. Utilizing unadjuvanted spike protein in immunizations did not increase S-specific CD8⁺ or CD4⁺ TEM’S in the cLN compared to PBS controls, further confirming the role of the NE/TVT adjuvant in inducing these robust cellular immune responses. In contrast to what was observed in the spleen, no notable differences in the overall frequency of total CD8⁺ T cells in the cLN were observed nor in the overall CD4⁺ T cells between treatment groups (FIGS. 23C and 23D).

[0156] Viral infection progression to the lower respiratory tract increases the likelihood of poor clinical outcomes. Therefore, the formation of tissue resident memory (CD44⁺CD69⁺CD103⁺IV ) T cells (TRM’S) within the lungs (Gating Strategy FIG. 15F) is a hallmark of a protective immunization regimen. Homologous IM mRNA prime/boost increased the overall frequency of total CD8⁺ T cells within the lungs (FIG. 23E). However, heterologous immunization with IM mRNA priming and IN NE/IVT/S boosting generated the highest frequency of S-specific CD8⁺ TR ’S (FIG. 15G), with only minimal induction of S-specific CD8⁺ TRM’S by the homologous IM mRNA prime/boost regimen. These results indicate that the increase in CD8⁺ T cells within the lungs induced by homologous IM mRNA prime/boost immunization without mucosal boosting is largely attributable to circulatory CD8⁺ T cells. Heterologous immunization using a different chronological sequence of administration consisting of IN NE/IVT/S priming with IM mRNA boost induced only low frequencies of S- specific CD8⁺ TRM’S and did not enhance the S-specific CD8⁺ TRM’S beyond levels induced by IM mRNA prime/boost. Heterologous immunizations with IM mRNA with unadjuvanted IN S protein regardless of chronological sequence did not result in detectable CD8⁺ TRM’S within the lungs, demonstrating the critical role of the NE/IVT adjuvant in driving the observed establishment of TRM’S.

[0157] The NALT is an upper respiratory tract organized lymphoid structure of defense which can support anti-viral resident memory T cells. The NALT of immunized mice for S- specific CD8⁺ TRM’S was assessed (FIG. 15H). All the immunization regimens had little effect on the overall total CD8⁺ T cell frequency within the NALT (FIG. 23F). Heterologous immunization with IM mRNA priming followed by two IN boosts with S protein with or without NE/IVT adjuvant generated markedly higher S-specific CD8⁺ T cells expressing tissue resident markers within the NALT compared to all the other immunization regimens (FIG. 15H). The ability of the unadjuvanted IN S protein boost to induce equivalent S-specific CD8⁺ TRM’S in the NALT as the IN NE/IVT/S boosts was surprising given the lack of induction of S-specific CD8⁺ TRM’S in the lungs by this regimen, indicating a difference in the mechanism of TRM induction between the upper and lower respiratory tracts. The reverse regimens in which unadjuvanted or NE/IVT adjuvanted S-protein priming through the IN route is boosted with IM mRNA induced undetectable, or minimal levels of CD8⁺ TR ’S, respectively. These results further highlight the findings that the sequence of heterologous immunization impacts the ability to foster tissueresident memory CD8⁺ T cells in mucosal sites.

[0158] Example 16. CD4/CD8 T cell cytokine profiles are shaped not only by the type and route of administration of heterologous prime/boost regimens, but also by the chronological sequence of immunizations.

[0159] While it is clear that S-specific T cells are optimally enhanced by IM mRNA; IN NE/IVT/S heterologous vaccination regimen both systemically and in mucosal tissues, tetramer staining does not give a comprehensive picture of the functionality of the T cells within the tissues. As the frequencies of IFN-y⁺ and polyfunctional CD4⁺ and CD8⁺T cells are strongly associated with viral control, cytokine recall responses of CD4⁺ and CD8⁺ T cells within these same tissues were determined in the vaccinated mice two weeks after the final boost immunization (Gating Strategy provided in FIG. 16A). Within the spleen, IM mRNA prime/boost and heterologous IM mRNA priming with IN NE/IVT/S boosting generated similar frequencies of IFN-y⁺-(FIG. 16B), IL-2⁺- (FIG. 24A), TNFa⁺- (FIG. 24B), and polyfunctional- (IFN-Y⁺IL-2⁺TNFa⁺ triple-expressing) (FIG. 16C) CD4⁺ T cells. However, only heterologous IM mRNA priming and IN NE/IVT/S boosting markedly enhanced the frequency of IL-17A⁺CD4⁺ T cells within the spleen (FIG. 16D). This is consistent with the disclosed observation that the Thl7 response is unique to the IN route of immunization. Notably all other immunization regimens were significantly lower in frequency of these cytokine expressing CD4⁺ T cells, demonstrating the importance of both the immunization route of priming and the role of the IN NE/IVT adjuvant. High frequencies of fFN-y⁺CD8⁺ T cells were also induced in mice immunized with homologous IM mRNA prime/boost and heterologous IM mRNA priming with IN NE/IVT/S boosting, while levels were undetectable for the IN NE/IVT/S primed group with IM mRNA boosting (FIG. 16E). The frequency of polyfunctional IFN-Y⁺TNFa⁺ CD8⁺ T cells within the spleen, however, was low relative to polyfunctional CD4⁺ T cells, with the highest frequences observed in the homologous IM mRNA or heterologous IM mRNA; IN NE/IVT/S immunization groups (FIG. 16F).

[0160] Within the cLN, the mucosal immunization as a boost was critical to driving CD4⁺ T cell cytokine responses in heterologous immunization. Heterologous immunization with IM mRNA priming with IN NE/IVT/S boosting markedly enhanced IFN-y*- (FIG. 16G), IL-2⁺- (FIG. 24C), TNFa⁺- (FIG. 24D), and polyfunctional- (FIG. 16H) expressing CD4⁺ T cells. Only immunization regimens with IN NE/IVT/S as a boost (IM mRNA; IN NE/IVT/S; IN NE/IVT/S and homologous IN NE/IVT/S) induced robust IL-17A⁺CD4⁺ T cells within the cLN (FIG. 161). Distinctly enhanced frequencies of IFN-y*- (FIG. 16 J) CD8⁺ T cells were observed in the cLNs from IM mRNA primed; IN NE/IVT/S boosted mice compared to all other immunization regimens. While polyfunctional IFN-Y⁺TNFa⁺ CD8⁺ T cells were low in the cLNs as well, IM mRNA primed; IN NE/IVT/S boosted mice showed the highest frequencies (FIG. 16K).

[0161] Similar to the cLN, mucosal boost immunization and the presence of the IN NE/IVT adjuvant drove the T cell cytokine response within the lungs. IM mRNA priming with IN NE/IVT/S hosting induced remarkably high frequencies of IFN-y*- (FIG. 16L), IL-2⁺- (FIG. 24E), TNFa⁺- (FIG. 24F), and polyfunctional (FIG. 16M) CD4⁺ T cells than other immunization regimens. While IL-2⁺-, TNFa⁺, and IL-17A⁺- expressing CD4⁺ T cells were generated in heterologous (IM mRNA;IN NE/IVT/S and IN NE/IVT/S; IM mRNA) and homologous IN NE/IVT/S immunizations, priming with IM mRNA was necessary to generate a significant IFN-y response within the lungs (FIG. 16N). Boosting IM mRNA primed mice with IN unadjuvanted S protein generated IL-2⁺ and TNFa⁺ expressing CD4⁺ T cells but minimal IFN-y and IL-17A responses in the lungs. Additionally, IM mRNA priming with IN NE/IVT/S boosting also generated the highest frequencies of IFN-y⁺ (FIG. 160) and IFN-y⁺TNFa⁺- (FIG. 16P) CD8⁺ T cells in the lungs compared to other immunization regimens. The results of the T cell cytokine profiling within these immune compartments demonstrate the effectiveness of the IN NE/IVT adjuvant in both redirecting systemically primed immune responses to the respiratory mucosa and optimally enhancing these responses. Further, these results indicate the importance of chronological sequence used in heterologous prime/boost immunization regimens in shaping the T cell responses and dictating their location.

[0162] Example 17. Sequence of heterologous prime/boost immunizations dramatically shifts T cell response polarization.

[0163] As sequence in heterologous immunization affects the generation of Thl and Thl7 cytokine-expressing T cells (FIG. 16), experiments were performed to profile the T cell cytokine recall response in the spleen and cLN from the vaccinated mice. Supernatants of single cell suspensions stimulated with S-protein were assessed by bead-based multiplex immunoassay. A significant Thl response to S-protein was generated in the splenocytes of the heterologous IM mRNA primed; IN NE/IVT/S boosted group and the homologous IM mRNA prime/boosted groups, as evidenced by markedly increased IFN-y production (FIG. 17 A); however, the heterologous immunization group showed greater enhancement in IL-2 and TNFa production compared to the homologous IM mRNA group (FIGS. 17B-17C). In contrast, splenocytes from the reverse heterologous immunization sequence, IN NE/IVT/S primed; IM mRNA boosted, group showed dramatically reduced IFN-y production compared to these groups. Notably, splenocytes from homologous IM mRNA prime/boosted mice also had a significant Th2 response as evidenced by increased IL-4 (FIG. 25 A), IL-5 (FIG. 17D), and IL- 13 (FIG. 17E), which was not observed in the IM mRNA primed; IN NE/IVT/S boosted group. IL-5 was significantly enhanced in the spleen of IN NE/IVT/S primed; IM mRNA boosted mice, while the singly IM mRNA immunized group and the homologous IN NE/IVT/S group showed minimal induction of IL-5. These results indicate that the IM mRNA used as a boost to the mucosal prime shifts the primed immune responses towards Th2 responses in contrast to IM mRNA priming with IN NE/IVT/S boost. Similar to the flow cytometry results, all groups receiving IN immunization with NE/IVT/S generated a significant S-specific IL-17A response within the spleen (FIG. 17F), with heterologous IM mRNA prime; IN NE/IVT/S pull immunization generating more IL-17A than homologous and heterologous priming with IN NE/IVT/S. IL- 10 was also induced by these heterologous groups, however, the highest levels of IL- 10 were induced in homologous IM mRNA prime/boosted mice (FIG. 17G). Higher levels of IL-6 were noted in the spleens of heterologous IM mRNA; IN NE/IVT/S immunized mice (FIG. 25B). Thus, the disclosure provides in some embodiments that the sequence in heterologous immunization affects the polarization of the cytokine response systemically. For example, in some embodiments, homologous IM mRNA immunization generates a systemic antigen-specific Thl/Th2 profile and homologous IN NE/IVT/S immunization generates a systemic Th 1 /Th 17 profile, heterologous IM mRNA prime; IN NE/IVT/S boost shifts the profile heavily towards a magnified Thl/Thl7 responses and heterologous IN NE/IVT/S prime; IM mRNA boost shifts the profile to a more heavily Th2/Thl7 skewed profile.

[0164] Similar to the flow cytometry data, heterologous IM mRNA prime; IN NE/TVT/S boost generated a significant IFN-y (FIG. 17H), IL-2 (FIG. 171), and TNFa (FIG. 17 J) response to S-protein within the cLNs which was not seen in mice primed IN with NE/IVT/S and subsequently boosted with IM mRNA. Minimal generation of the Th2 cytokines, IL-4 (FIG. 25C), IL-5 (FIG. 17K), and IL- 13 (FIG. 17L) was noted within the cLN in response to S-protein stimulation with minor differences between immunization regimens. Similar to the spleen, a S- specific IL-17A response (FIG. 17M) was observed in the cLNs of all mice given an IN NE/IVT/S immunization. IM mRNA; IN NE/IVT/S prime-pull heterologous immunization generated an enhanced IL-17A response compared to homologous IN NE/IVT/S immunization and heterologous immunization in which IN NE/IVT/S was utilized in priming. Furthermore, IL- 10 (FIG. 17N), and IL-6 (FIG. 25D) were also significantly produced in the cLN of heterologous IM mRNA primed; IN NE/IVT/S boosted mice compared to all other immunization regimens. These results further confirm that the sequence of adjuvanted mucosal boosting of systemically primed immune responses optimally drives localization of antigen-specific responses to the respiratory draining lymph nodes and promotes an effective Thl/Thl7 polarized cytokine profile. In contrast, systemic boosting of mucosally primed immune responses not only less effectively promote T cell responses in the mucosa, but significantly shifts the polarization of these responses towards a less ideal Th2/Thl7 profile.

[0165] Example 18. Heterologous prime/boost immunization with IN NE/IVT adjuvanted S- protein affords cross-variant sterilizing immunity in a lethal K18-hACE2 mouse model of SARS- CoV-2 infection.

[0166] As the immunization regimens shaped differing humoral and cellular profiles at mucosal and systemic sites, it was determined whether they would offer differing protection levels as well. To this end, a lethal B.1.351 SARS-CoV-2 challenge in K18-hACE2 mice was used as a model of severe disease. The B.1.351 model has the potential to induce lung and brain pathology along with chemokine/cytokine storm. Heterologous immunization regimens with IM mRNA and IN NE/IVT/S regardless of priming mucosally or systemically, induced similar serum titers of WT-S IgG (FIG. 26A) as homologous immunization regimens. Lower titers were induced by heterologous regimens with IM mRNA and unadjuvanted S-protein. However, heterologous IM mRNA prime; IN NE/IVT/S pull immunization induced higher titers of BA.1 -S IgG than homologous IM mRNA immunization and heterologous immunization when IN NE/IVT/S was used as the prime immunization (FIG. 26B). Quantification of serum nAb titers to WT (FIG. 26C) and B.1.351 (FIG. 26D) pseudoviruses reflected the results obtained in WT C57B1/6 mice, and further demonstrated the enhancement of the humoral response with utilization of the NE/IVT adjuvant over unadjuvanted S-protein. Three weeks after the final boost, mice were challenged with lxl0⁴pfu B.1.351 SARS-CoV-2. Viral titers were quantified at three and five days post infection (dpi). At 3dpi, high viral titers were present in the lungs (FIG. 18A) and nasal turbinates (FIG. 18B) of mock PBS immunized mice. By 5dpi, viral titers were somewhat reduced compared to 3 dpi, but persisted in the lungs of all mice in this group and in the nasal turbinates (NTs) of half of the mice at 5 dpi. Virus was also detected in the brain in half of the mice in this group by 5dpi but in no other vaccinated groups at either time point (FIGS. 18C and 18F). Mock immunized mice demonstrated significant weight-loss, all reaching humane endpoints by 5dpi (FIG. 18G). Significantly lower viral loads were also detected in the lungs of all the mice (FIG. 18A) and in the NTs of select mice (2/5) at 3 dpi (FIG. 18B) in the group vaccinated with heterologous IN unadjuvanted S prime;IM mRNA boost. However, by 5dpi, no viral load was detectable in the lungs or NTs, demonstrating a degree of protection and faster viral clearance in these vaccinated animals compared to mock vaccination (FIGS. 18D and 18E). Mice given IM mRNA prime followed by IN unadjuvanted S boost showed minimal viral titers detected in the lungs of 1/5 mice and none in the NTs at either time point. Both groups demonstrated no weight loss, pointing to reduction in morbidity even in the presence of viral infection. In contrast, homologous regimens with IM mRNA or IN NE/IVT/S, as well as heterologous regimens with IM mRNA and IN NE/IVT-adjuvanted S-protein (regardless of immunization sequence) imparted sterilizing immunity in both the upper and lower respiratory tracts.

[0167] While these results indicated that heterologous or homologous regimens utilizing IN NE/IVT-adjuvanted S-protein and/or IM mRNA immunization were equally protective, experiments were conducted to characterize the chemokine and cytokine response within the lungs from vaccinated mice post-viral challenge to determine how the immunization regimens shaped the early responses to infection. The cytokine response post-challenge is influenced both by the vaccination regimen as well as by the extent of productive viral infection. While both the IM mRNA prime; IN NE/IVT/S boost as well as the IN NE/IVT/S prime; IM mRNA boost groups showed minimal cytokine production in the lung homogenate due to effective blockage of viral infection, clear differences in cytokine profiles that were dependent upon immunization sequence could be observed in the heterologous immunization groups given IM mRNA and IN unadjuvanted S-protein (FIG. 18H). A clear Th2-skewed response, characterized by higher levels of IL-4, IL-5 and IL- 13 was observed in the lungs post-challenge from mice that received heterologous IN unadjuvanted S prime; IM mRNA boost. Two out of 5 mice of this group also had high levels of eotaxin (CCL11) in their lung homogenates. This group also shared enhanced expression of the following cytokines and chemokines with the unvaccinated PBS group at 3dpi, but with reduced levels compared to the PBS group by 5dpi: IL-6, GRO alpha (CXCL1), RANTES (CCL5), MIP-1 alpha (CCL3), MCP-3 (CCL7), MCP-1 (CCL2), MIP-2 alpha (CXCL2) and MIP-1 beta (CCL4). Levels of IL-18 were also higher on average in the heterologous IN unadjuvanted S primed; IM mRNA boosted mice compared to other groups at 3dpi. RANTES (CCL5) was also high in lungs of challenged mice that received homologous IM mRNA, indicating induction of a strong T cell component in these groups. Mice that received homologous IN NE/IVT/S immunization showed a Thl7 response as reflected in the IL-17A levels at 3 and 5dpi. Mice that received heterologous IN NE/IVT/S prime; IM mRNA boost also induced a Th 17 response, with somewhat slower kinetics when compared to 3x NE/IVT/S given the lower levels at 3dpi but equivalent levels by 5dpi.

[0168] Example 19. Heterologous prime-pull immunization induces a more durable cellular response than homologous immunization regimens.

[0169] As durability is a major limitation facing IM mRNA vaccines, experiments were conducted to determine whether heterologous vaccination regimens with IN NE/IVT adjuvanted S-protein platform could improve the durability of induced immune responses. Humoral, mucosal and cellular immunity was determined at 2 wks or 4 mo post-final boost (wk 8 or 18, respectively). Overall, all vaccination regimens evaluated for longevity (homologous IM mRNA or IN NE/IVT/S prime/boost, and heterologous IM mRNA prime; IN NE/IVT/S boost or IN NE/IVT/S prime; IM mRNA boost) showed largely maintained serum nAb titers against both WT (FIG. 19A) and BA.4/5 (FIG. 19B) variants over the period of 4 mo from the last immunization. While a slight decrease in BA.4/5 nAbs was observed for the homologous EM mRNA group, nAbs against BA.4/5 induced by both heterologous immunization sequences and by homologous IN NE/IVT/S immunization were maintained over this time period. Within the BALF, WT-S IgA responses were maximally induced by the heterologous immunization regimen of IM mRNA prime followed by IN NE/IVT/S boosting upon assessment at 2 wks postfinal boost (wk8). BALF WT-S-specific IgA levels decreased slightly in this treatment group after 4 mo, however, levels at this late timepoint were still higher than those induced by the homologous IM mRNA and IN NE/IVT/S groups at the early time point post-final boost (FIG. 19C). The homologous IN NE/IVT/S group also induced relatively durable IgA responses, albeit at lower levels than the heterologous group. In contrast, heterologous IN NE/IVT/S priming; IM mRNA boosting or homologous IM mRNA immunization resulted in the least antigen-specific IgA, which decreased further to baseline levels at the late timepoint.

[0170] Experiments were conducted to assess the durability of the cellular response by comparing single cell isolates from the spleen, cLNs, and lungs from immunized mice at 2 wks (wk8) and 4 mo post-final boost (wkl 8). Intracellular cytokine profiling revealed a pronounced drop in the frequencies of IFN-y⁺- (FIG. 19D) TNFa⁺-(FIG. 27 A), and IFN-y⁺IL-2⁺TNFa⁺ polyfunctional CD4⁺ T cells (FIG. 19E) within the splenocytes in response to WT-S stimulation for mice immunized with homologous IM mRNA over the 4mo interval. In contrast, the frequencies of these cytokine expressing CD4⁺ T cells within the spleen for the heterologous IM mRNA primed; IN NE/IVT/S boosted mice showed remarkable durability, giving the same (or higher) frequencies at the later timepoint as what was observed more immediately after the final booster immunization. While IL-17A⁺CD4⁺ T cells were not detected in the spleens of the homologous IM mRNA group, frequencies of IL-17A⁺CD4⁺T cells were also maintained over this time period for all groups given heterologous or homologous IN NE/IVT/S (FIG. 19F). The frequency of IFN-y⁺CD8⁺ T cells within the spleen showed a slight decrease for the homologous IM mRNA group over the 4 mo interval, but the higher responses observed with the heterologous IM mRNA prime; IN NE/IVT/S boost were highly durable and maintained (FIG. 19G). The frequencies of polyfunctional IFN-y⁺TNFa⁺CD8⁺ T cells within the spleen declined in both groups over time (FIG. 19H). Assessment of splenocyte supernatants after S-protein stimulation further confirmed the loss of cellular responses as IFN-y (FIG. 20A), IL-5 (FIG. 20B), and IL-10 (FIG. 20C) were reduced in homologous IM mRNA immunized mice at wkl 8 compared to wk8. In contrast, cellular cytokine profiles were maintained over the 4 mo in splenocytes from heterologous IM mRNA primed; IN NE/IVT/S boosted mice and even enhanced in the case of IL-17A (FIG. 20D). Strongly maintained IL-2 (FIG. 27B) and TNFa (FIG. 27C) responses were observed in the splenocytes in this group. [0171] Within the cLNs, an increase in the frequencies of IFN-y⁴- (FIG. 191), polyfunctional IFN-y⁴IL-2⁴TNFa⁺- (FIG. 19 J), and IL-17A⁺- (FIG. 19K) CD4⁺ T cells and IFN-y⁴CD8⁺ T cells (FIG. 19L) were seen in heterologous IM mRNA; IN NE/IVT/S mice at wkl 8 compared to wk8. No significant changes in the frequencies of these cytokine expressing cells for other immunization regimens over this duration were noted due to their lower initial frequencies. IL- 17 A⁺CD4⁺ T cells were also maintained over this duration in the cLNs of homologous IN NE/IVT/S immunized mice (FIG. 19K). Similar to the CD4⁺ response, heterologous IM mRNA; IN NE/IVT/S mice also demonstrated a significant increase in IFN-y⁺ CD8⁺ T cells in the cLN over the 4 mo. While polyfunctional IFN-y⁺TNFa⁺CD8⁺ T cells decreased slightly over time in the cLNs from this group, frequencies still remained at a higher level than those induced by homologous IM mRNA immunization more immediately post-final boost. Assessment of supernatants from stimulated cLNs further confirmed increases in IFN-y (FIG. 20E) production in the cLN over this duration in heterologous IM mRNA; IN NE/IVT/S immunized mice. A further increase in IL-5 (FIG. 20F) and IL- 10 (FIG. 20G) were noted within the cLN of mice primed with IN NE/IVT/S and boosted with IM mRNA; however, IL- 10 expression decreased between wk8 and wkl 8 in mice boosted with IN NE/IVT/S. Furthermore, mice immunized IN with NE/IVT/S showed a resilient or increased IL-17A (FIG. 20H) response with time. Within the cLN of heterologous immunized mice, an increases in IL-2 (FIG. 27B) expression maintained TNFa (FIG. 27C) expression and increased IL- 13 was observed with the highest increase in IL- 13 in mice primed with IN NE/IVT/S and subsequently boosted with IM mRNA. Similar to the spleen, the frequency of IFN-y - (FIG. 19N), TNFa⁺- (FIG. 27G), and triple positive⁺- (FIG. 190) CD4⁺ T cells significantly decreased in the lungs of IM mRNA prime/boosted mice between wk8 and wkl 8. While IFN-y⁺- and triple positive⁺-CD4⁺ T cells were maintained in heterologous prime-pull immunized mice, TNFa⁺- and IL- 17 A⁴- (FIG. 19P, FIG. 27G) CD4⁺ T cells decreased between wk8 and wkl 8 which was also noted in heterologous immunized mice primed with IN NE/IVT/S and boosted with IM mRNA and homologous IN NE/IVT/S immunized mice. Within the lungs, the frequency of cytokine expressing CD8⁺ T cells was low with minor changes in IFN-y⁴- (FIG. 19Q) and IFN-y⁴-TNFa⁺- (FIG. 19R) CD8⁺ T cells in heterologous prime-pull immunized mice.

[0172] Additional experiments were conducted to examine whether these changes in cytokine expression were accompanied by changes in frequency of spike-specific CD4⁺ and CD8⁺ T cells within the tissues. The frequency of spike-specific effector memory in CD8⁺ T cells decreased from wk8 to wkl8 in the spleen (FIG. 28A) and the cLN (FIG. 28B) decreased in all regimens with the largest decreases in homologous IM mRNA immunization. Within the lungs, a decrease in the frequency of spike-specific resident memory in CD8⁺ T cells (FIG. 28C) was noted with all immunization regimens with the largest decrease in mice primed with IM mRNA and boosted twice with IN NE/IVT/S. Furthermore, a decrease in the frequency of CD8⁺ T cells within the spleen (FIG. 28D), cLN (FIG. 28E) and lungs (FIG. 28F) was observed with time in homologous IM mRNA prime/boost mice indicating that changes induced by IM mRNA immunization are not durable. While the frequency of spike-specific in CD8⁺ T cells proved to be difficult to maintain, the frequency of spike-specific effector memory in CD4⁺ T cells increased in the spleen (FIG. 28G) and cLN (FIG. 28H) between wk8 and wkl8 in mice primed with IM mRNA and boosted with IN NE/IVT/S while the frequency decreased in IM mRNA prime/boosted mice. Furthermore, frequency of spike-specific effector memory in CD4⁺ T cells was maintained in IN NE/IVT/S primed/IM mRNA boosted and homologous IN NE/IVT/S immunized mice. The frequency of CD4⁺ T cells was reduced in the spleen (FIG. 281) in all groups of mice with time although this was most significant in mice boosted with IM mRNA while CD4⁺ T cell frequency in cLN (FIG. 28 J) was maintained with time.

[0173] Example 20. Heterologous prime-pulled immunization induces a cellular response more responsive to further boosting.

[0174] As humoral and cellular memory responses waned more rapidly in homologous IM mRNA prime/boosted mice, experiments were conducted to determine how the observed mucosal responses established by the heterologous IM mRNA; IN NE/IVT/S regimen would be differentially affected by further boosting through either parenteral or mucosal routes after a prolonged 3 mo interval. Additionally, experiments were conducted to determine whether IN NE/IVT/S boosting of the homologous IM mRNA prime/boosted mice after a 3 mo interval could still result in similar mucosal immune response enhancement as more proximal heterologous boosting.

[0175] Homologous IM mRNA prime/boosted and heterologous prime-pulled mice were further boosted at wkl7 and immune responses were assessed at week 19. Further IM and IN boosting of IM mRNA prime/boosted mice did not further enhance neutralizing antibodies titers to WT (FIG. 21 A) or OMI BA.4/5 (FIG. 2 IB) PSV indicating that an upper limit of nAbs had been reached. However, further IM mRNA boosting of heterologous prime-pulled mice enhanced nAbs to WT (FIG. 21 A) and OMI BA.4/5 (FIG. 21B) PSV to a titer similar to homologous IM mRNA immunized mice. Furthermore, additional boosting of heterologously IM mRNA; IN NE/TVT/S prime/boosted mice with either IM mRNA or IN NE/IVT/S both resulted in further enhancement of BALF WT-S specific-IgA to similar degrees (FIG. 21C). These results indicate that establishment of strong mucosal immune responses initially allows for optimal further boosting of these responses through either parenteral or mucosal routes.

[0176] Examination of cellular cytokine responses further demonstrated the unique differences imprinted by homologous IM mRNA prime/boost and heterologous prime-pull immunization. Within the spleen, further IM mRNA boosting enhanced the frequency of IFN-y⁺- (FIG. 21D) and IFN-y⁺IL-2⁺TNFa⁺- (FIG. 21E) CD4⁺ T cells in both IM mRNA prime/boosted and IM mRNA primed/IN NE/IVT/S boosted mice however, TFN-y⁴ CD4⁺ T cells were substantially more enhanced in the heterologous prime-pulled mice with this additional boost. Notably, changes in the frequency of IL- 17 A⁺- (FIG. 2 IF) CD4⁺ T cells in the spleen was dependent on the booster route and type in heterologous prime/pulled mice with parental boosting substantially reducing the frequency and mucosal NE/IVT/S boosting increasing the frequency. Furthermore, IM mRNA boosting enhanced the frequency of IFN-y⁺- (FIG. 21G) and IFN-y⁺TNFa⁺- (FIG. 21H) CD8⁺ T cells in the spleen with the largest increases in heterologous prime-pulled mice. While cytokine changes in splenic CD4⁺ and CD8⁺ T cells of IM mRNA prime/boosted mice were less responsive to further boosting, IN NE/IVT/S boosting of IM mRNA prime/boosted mice enhanced the frequency of IFN-y⁺- (FIG. 211), and IFN-y⁺IL- 2⁺TNFa⁺- (FIG. 21 J) CD4⁺ T cells within the cLN. Additional boosting either IM mRNA or IN NE/IVT/S of heterologous prime-pull immunized mice also enhanced the frequency of IFN-y⁺-( FIG. 211), IFN-y* IL-2⁺TNFa⁺-( FIG. 21 J) and IL-17A⁺- (FIG. 21K) CD4⁺ T cell and IFN-y⁺- (FIG. 21L) and IFN-' TNFa⁺- (FIG. 21M) CD8⁺ T cells within the cLN with IM mRNA boosting enhancing primarily Thl responses and IN NE/IVT/S boosting enhancing both Thl and Thl7 responses. Within the lung, additional IM mRNA boosting significantly shifted the response within heterologous prime-pull immunized mice with enhanced Thl and decreased Th 17 profile as evidenced by increased frequency of IFN-y⁺- (FIG. 2 IN) and IFN-y⁺IL- 2⁺TNFa⁺-( FIG. 210) and decreased frequency of IL-17A⁺- (FIG. 21P) CD4⁺ T cells. Interestingly, IM mRNA boosting markedly enhanced the frequency of IFN-y⁺- (FIG. 21Q) CD8⁺ T cells within the lungs of prime-pull immunized mice, while IN NE/IVT/S boosting had less of an impact. These results are in line with observations above that upon establishment of strong mucosal immune responses initially, optimal further boosting of these responses within the mucosa can subsequently occur through either parenteral or mucosal routes. The frequency of IFN-y⁺TNFa⁺ (FIG. 21R) CD8⁺ T cells was unchanged with further boosting.

[0177] Experiments were also conducted to assess whether boosting induced changes in cytokine expressing T cells would correlate with changes in the frequency of spike-specific T cells. The frequency of S-specific effector memory in CD8⁺ T cells was expanded in the spleen (FIG. 29 A) and cLN (FIG. 29B) with further IM mRNA and IN NE/IVT/S boosting in both IM mRNA prime/boosted and prime-pulled immunized mice although these changes were more significant with IM mRNA boosting and in IM mRNA prime/boosted mice. Within the lungs, further boosting decreased the frequency of spike-specific resident memory in CD8⁺ T cells (FIG. 29C) regardless of the initial immunization regimen. The enhancement of spike-specific effector memory in CD8⁺ T cell frequency with IM mRNA boosting was noted with a substantial increase in the frequency of CD8⁺ T cells within the spleen (FIG. 29D), cLN (FIG. 29E) and lungs (FIG. 29F). The frequency of S-specific effector memory in CD4⁺ T cells was substantially enhanced with further boosting in the spleen (FIG. 29G) and cLN (FIG. 29H). Notably, only IM mRNA boosting enhanced the frequency of spike-specific effector memory in CD4⁺ T cells in only heterologous prime-pulled mice. However, IN NE/IVT/S boosting substantially enhanced the frequency of S-specific effector memory in CD4⁺ T cells in the cLN of both IM mRNA prime/boosted and heterologous prime-pulled mice. Intriguingly, the frequency of CD4 T cells was reduced in both the spleen (FIG. 291) and cLN (FIG. 29 J) with further boosting with the largest reductions with IM mRNA boosting in IM mRNA prime/boosted mice.

[0178] For reasons of completeness, various aspects of the disclosure are set out in the following numbered clauses:

[0179] Clause 1. A method of inducing an immune response in a subject comprising the steps of:

A) administering a primary, parenterally administered immunogenic composition to the subject, wherein the primary, parenterally administered immunogenic composition induces an immune response to a pathogenic organism; and

B) subsequently administering a secondary, mucosally administered immunogenic composition to the subject, wherein the secondary, mucosally administered immunogenic composition comprises a nanoemulsion, an agonist of retinoic acid-inducible gene I (RIG-I) and an antigen; and wherein the secondary, mucosally administered immunogenic composition induces protective immunity and/or sterilizing immunity to the pathogenic organism.

[0180] Clause 2. The method of clause 1, wherein the primary, parenterally administered immunogenic composition comprises an inactivated virus vaccine, a live-attenuated virus vaccine, a messenger RNA (mRNA) vaccine, a subunit vaccine, a recombinant vaccine, a polysaccharide vaccine, a conjugate vaccine, a toxoid vaccine, a pseudotyped virus vaccine or a viral vector vaccine.

[0181] Clause 3. The method of clause 1, wherein the secondary, mucosally administered immunogenic composition comprises a nanoemulsion, an agonist of retinoic acid-inducible gene I (RIG-I) and an antigen selected from a protein, a recombinant protein, a recombinant polypeptide, a lipid, a carbohydrate, a polysaccharide, a protein extract, a cell or cellular extract, a tumor cell or tumor cell extract, and/or a tissue.

[0182] Clause 4. The method of any one of clauses 1 -3, wherein the primary, parenterally administered immunogenic composition comprises a coronavirus vaccine.

[0183] Clause 5. The method of clause 4, wherein the coronavirus vaccine is a protein subunit vaccine, a whole virus vaccine, a live-attenuated virus vaccine, an inactivated virus vaccine, an mRNA vaccine, or a pseudotyped virus vaccine.

[0184] Clause 6. The method of clause 5, wherein the coronavirus vaccine is a coronavirus mRNA vaccine.

[0185] Clause 7. The method of clause 6, wherein the coronavirus mRNA vaccine is mRNA- 1273 or BNT162b2.

[0186] Clause 8. The method of any one of clauses 1-7, wherein administering a primary, parenterally administered immunogenic composition to the subject comprises administering intramuscularly, intraperitoneally, intravenously, intracerebroventricularly, intracistemal injection or infusion, subcutaneously, or via implant.

[0187] Clause 9. The method of any one of clauses 1 -3, wherein the secondary, mucosally administered immunogenic composition comprises a nanoemulsion, an agonist of retinoic acidinducible gene I (RIG-I) and recombinant coronavirus S protein and/or recombinant coronavirus receptor binding domain (RBD).

[0188] Clause 10. The method of any one of clauses 1-9, wherein administering a secondary, mucosally administered immunogenic composition to the subject comprises intranasal administration. [0189] Clause 11. The method of any one of clauses 1-10, wherein the protective immunity and/or sterilizing immunity to the pathogenic organism comprises sterilizing immunity in the lower respiratory tract and/or the upper respiratory tract.

[0190] Clause 12. The method of clause 11, wherein sterilizing immunity in the upper respiratory tract comprises inhibition of replication of the pathogenic organism in the lungs and/or nasal turbinates of the subject.

[0191] Clause 13. A method of enhancing an immune response to a coronavirus vaccine in a subject comprising:

[0192] A) providing a subject parenterally administered a coronavirus vaccine; and

[0193] B) intranasally administering to the subject a boost vaccination, wherein the boost vaccination comprises a nanoemulsion, an agonist of retinoic acid-inducible gene I (RIG-I), and an antigenic component of coronavirus.

[0194] Clause 14. The method of clause 13, wherein the subject parenterally administered a coronavirus vaccine received a coronavirus vaccine selected from mRNA-1273, BNT162b2, JNJ-78436735, and Vaxzevria.

[0195] Clause 15. The method of clause 13, wherein the subject parenterally administered a coronavirus vaccine received a coronavirus mRNA vaccine.

[0196] Clause 16. The method of clause 15, wherein the subject parenterally administered a coronavirus vaccine received mRNA- 1273.

[0197] Clause 17. The method of clause 15, wherein the subject parenterally administered a coronavirus vaccine received BNT162b2.

[0198] Clause 18. The method of clause 15, wherein the antigenic component of coronavirus is recombinant coronavirus S protein.

[0199] Clause 19. The method of clause 15, wherein the antigenic component of coronavirus is recombinant coronavirus receptor binding domain (RBD).

[0200] Clause 20. The method of clause 13, wherein the agonist of RIG-I is an RNA agonist.

[0201] Clause 21. The method of clause 20, wherein the RNA agonist is a defective interfering (DI) RNA of a Sendai virus (SeV) or an influenza virus.

[0202] Clause 22. The method of clause 13, wherein the nanoemulsion comprises:

(a) a poloxamer surfactant or polysorbate surfactant;

(b) an organic solvent;

(c) a halogen containing compound; (d) oil, and

(e) water.

[0203] Clause 23. The method of clause 13, wherein the nanoemulsion comprises:

(a) Tween 80;

(b) ethanol;

(c) cetylpyridinium chloride (CPC);

(d) soybean oil; and

(e) water.

[0204] Clause 24. The method of clause 13, wherein the coronavirus is SARS CoV-2.

[0205] Clause 25. The method of clause 13, wherein enhancing an immune response to a coronavirus vaccine in the subject comprises generation of humoral and cellular mucosal immune responses specific for coronavirus.

[0206] Clause 26. The method of clause 13, wherein enhancing an immune response to a coronavirus vaccine in the subject comprises generation of tissue-resident memory cells within the respiratory tract of the subject.

[0207] Clause 27. The method of clause 13, wherein enhancing an immune response to a coronavirus vaccine in the subject comprises generation of secretory IgA (sig A) in the respiratory tract and/or tissue-resident memory T cells (TRM) in the lungs of the subject.

[0208] Clause 28. The method of clause 13, wherein enhancing an immune response to a coronavirus vaccine in the subject comprises generation of neutralizing antibodies to coronavirus in the upper respiratory tract and/or lower respiratory tract.

[0209] Clause 29. The method of clause 13, wherein enhancing an immune response to a coronavirus vaccine in the subject comprises generation of sterilizing immunity to coronavirus in the upper respiratory tract and/or lower respiratory tract.

[0210] Clause 30. The method of clause 13, wherein enhancing an immune response to a coronavirus vaccine in the subject comprises inhibition of coronavirus replication in the lungs of the subject.

[0211] Clause 31. The method of clause 13, wherein enhancing an immune response to a coronavirus vaccine in the subject comprises inhibition of coronavirus replication in the nasal turbinates of the subject.

[0212] Clause 32. A method of treating and/or preventing infection by a coronavirus in a subject in need thereof, the method comprising A) administering a primary, parenterally administered coronavirus mRNA vaccine to the subject; and

B) subsequently mucosally administering a boost vaccination, wherein the boost vaccination comprises a nanoemulsion, an agonist of retinoic acid-inducible gene I (RIG-I), and an antigenic component of the coronavirus, wherein the boost vaccination induces protective immunity and/or sterilizing immunity in the subject.

[0213] Clause 33. The method of clause 32, wherein the protective immunity and/or sterilizing immunity inhibits transmission of the coronavirus from the subject.

[0214] Clause 34. The method of clause 32, wherein the coronavirus mRNA vaccine is mRNA- 1273.

[0215] Clause 35. The method of clause 32, wherein the coronavirus mRNA vaccine is BNT162b2.

[0216] Clause 36. The method of clause 32, wherein the antigenic component of the coronavirus is recombinant coronavirus S protein.

[0217] Clause 37. The method of clause 32, wherein the antigenic component of the coronavirus is recombinant coronavirus receptor binding domain (RBD).

[0218] Clause 38. The method of clause 32, wherein the agonist of RIG-I is an RNA agonist.

[0219] Clause 39. The method of clause 32, wherein the RNA agonist is a defective interfering (DI) RNA of a Sendai virus (SeV) or an influenza virus

[0220] It is to be understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the biological agents, structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the disclosure, may be made without departing from the spirit and scope thereof. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. Thus, it is intended that the present disclosure cover modifications and variations thereof provided they come within the scope of the appended claims and/or their equivalents.

References [0221] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

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Claims

What is claimed is:

1. A method of inducing an immune response in a subject comprising the steps of:

A) administering a primary, parenterally administered immunogenic composition to the subject, wherein the primary, parenterally administered immunogenic composition induces an immune response to a pathogenic organism; and

B) subsequently administering a secondary, mucosally administered immunogenic composition to the subject, wherein the secondary, mucosally administered immunogenic composition comprises a nanoemulsion, an agonist of retinoic acid-inducible gene I (RIG-I) and an antigen; and wherein the secondary, mucosally administered immunogenic composition induces protective immunity and/or sterilizing immunity to the pathogenic organism.

2. The method of claim 1, wherein the primary, parenterally administered immunogenic composition comprises an inactivated virus vaccine, a live-attenuated virus vaccine, a messenger RNA (mRNA) vaccine, a subunit vaccine, a recombinant vaccine, a polysaccharide vaccine, a conjugate vaccine, a toxoid vaccine, a pseudotyped virus vaccine or a viral vector vaccine.

3. The method of claim 1, wherein the secondary, mucosally administered immunogenic composition comprises a nanoemulsion, an agonist of retinoic acid-inducible gene I (RIG-I) and an antigen selected from a protein, a recombinant protein, a recombinant polypeptide, a lipid, a carbohydrate, a polysaccharide, a protein extract, a cell or cellular extract, a tumor cell or tumor cell extract, and/or a tissue.

4. The method of any one of claims 1-3, wherein the primary, parenterally administered immunogenic composition comprises a coronavirus vaccine.

5. The method of claim 4, wherein the coronavirus vaccine is a protein subunit vaccine, a whole virus vaccine, a live-attenuated virus vaccine, an inactivated virus vaccine, an mRNA vaccine, or a pseudotyped virus vaccine.

6. The method of claim 5, wherein the coronavirus vaccine is a coronavirus mRNA vaccine.

7. The method of claim 6, wherein the coronavirus mRNA vaccine is mRNA-1273 or BNT162b2.

8. The method of any one of claims 1-7, wherein administering a primary, parenterally administered immunogenic composition to the subject comprises administering intramuscularly, intraperitoneally, intravenously, intracerebroventricularly, intracistemal injection or infusion, subcutaneously, or via implant.

9. The method of any one of claims 1-3, wherein the secondary, mucosally administered immunogenic composition comprises a nanoemulsion, an agonist of retinoic acid-inducible gene I (RIG-I) and recombinant coronavirus S protein and/or recombinant coronavirus receptor binding domain (RBD).

10. The method of any one of claims 1-9, wherein administering a secondary, mucosally administered immunogenic composition to the subject comprises intranasal administration.

11. The method of any one of claims 1-10, wherein the protective immunity and/or sterilizing immunity to the pathogenic organism comprises sterilizing immunity in the lower respiratory tract and/or the upper respiratory tract.

12. The method of claim 11, wherein sterilizing immunity in the upper respiratory tract comprises inhibition of replication of the pathogenic organism in the lungs and/or nasal turbinates of the subject.

13. A method of enhancing an immune response to a coronavirus vaccine in a subject comprising:

A) providing a subject parenterally administered a coronavirus vaccine; and

B) intranasally administering to the subject a boost vaccination, wherein the boost vaccination comprises a nanoemulsion, an agonist of retinoic acid-inducible gene I (RIG-I), and an antigenic component of coronavirus.

14. The method of claim 13, wherein the subject parenterally administered a coronavirus vaccine received a coronavirus vaccine selected from mRNA-1273, BNT162b2, JNJ-78436735, and Vaxzevria.

15. The method of claim 13, wherein the subject parenterally administered a coronavirus vaccine received a coronavirus mRNA vaccine.

16. The method of claim 15, wherein the subject parenterally administered a coronavirus vaccine received mRNA- 1273.

17. The method of claim 15, wherein the subject parenterally administered a coronavirus vaccine received BNT162b2.

18. The method of claim 15, wherein the antigenic component of coronavirus is recombinant coronavirus S protein.

19. The method of claim 15, wherein the antigenic component of coronavirus is recombinant coronavirus receptor binding domain (RBD).

20. The method of claim 13, wherein the agonist of RIG-I is an RNA agonist.

21. The method of claim 20, wherein the RNA agonist is a defective interfering (DI) RNA of a Sendai virus (SeV) or an influenza virus.

22. The method of claim 13, wherein the nanoemulsion comprises:

(a) a poloxamer surfactant or polysorbate surfactant;

(b) an organic solvent;

(c) a halogen containing compound;

(d) oil, and

(e) water.

23. The method of claim 13, wherein the nanoemulsion comprises: (a) Tween 80;

(b) ethanol;

(c) cetylpyridinium chloride (CPC);

(d) soybean oil; and

(e) water.

24. The method of claim 13, wherein the coronavirus is SARS CoV-2.

25. The method of claim 13, wherein enhancing an immune response to a coronavirus vaccine in the subject comprises generation of humoral and cellular mucosal immune responses specific for coronavirus.

26. The method of claim 13, wherein enhancing an immune response to a coronavirus vaccine in the subject comprises generation of tissue-resident memory cells within the respiratory tract of the subject.

27. The method of claim 13, wherein enhancing an immune response to a coronavirus vaccine in the subject comprises generation of secretory IgA (slgA) in the respiratory tract and/or tissueresident memory T cells (TRM) in the lungs of the subject.

28. The method of claim 13, wherein enhancing an immune response to a coronavirus vaccine in the subject comprises generation of neutralizing antibodies to coronavirus in the upper respiratory tract and/or lower respiratory tract.

29. The method of claim 13, wherein enhancing an immune response to a coronavirus vaccine in the subject comprises generation of sterilizing immunity to coronavirus in the upper respiratory tract and/or lower respiratory tract.

30. The method of claim 13, wherein enhancing an immune response to a coronavirus vaccine in the subject comprises inhibition of coronavirus replication in the lungs of the subject.

31. The method of claim 13, wherein enhancing an immune response to a coronavirus vaccine in the subject comprises inhibition of coronavirus replication in the nasal turbinates of the subject.

32. A method of treating and/or preventing infection by a coronavirus in a subject in need thereof, the method comprising

A) administering a primary, parenterally administered coronavirus mRNA vaccine to the subject; and

B) subsequently mucosally administering a boost vaccination, wherein the boost vaccination comprises a nanoemulsion, an agonist of retinoic acid-inducible gene I (RIG-I), and an antigenic component of the coronavirus, wherein the boost vaccination induces protective immunity and/or sterilizing immunity in the subject.

33. The method of claim 32, wherein the protective immunity and/or sterilizing immunity inhibits transmission of the coronavirus from the subject.

34. The method of claim 32, wherein the coronavirus mRNA vaccine is mRNA-1273.

35. The method of claim 32, wherein the coronavirus mRNA vaccine is BNT162b2.

36. The method of claim 32, wherein the antigenic component of the coronavirus is recombinant coronavirus S protein.

37. The method of claim 32, wherein the antigenic component of the coronavirus is recombinant coronavirus receptor binding domain (RBD).

38. The method of claim 32, wherein the agonist of RIG-I is an RNA agonist.

39. The method of claim 32, wherein the RNA agonist is a defective interfering (DI) RNA of a Sendai virus (SeV) or an influenza virus.